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This is to certify that the
dissertation entitled

INDUCTION OF SOUAMOUS CELL CARCINOMA IN THE
MANDIBLE AND MAXILLA, AND REPRODUCTIVE
DYSFUNCTION IN MINK (MUSTELA VISON) CAUSED BY
3,3’,4,4’,5-PENTACHLOROBIPHENYL (PCB 126)

presented by
Kerrie J. Beckett

has been accepted towards fulfillment
of the requirements for the

Ph.D degree in Animal Science

 

 

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2/05 czfilfiwateoueindd-pjs

 

INDUCTION OF SQUAMOUS CELL CARCINOMA IN THE MAN DIBLE AND
MAXILLA, AND REPRODUCTIVE DYSFUNCTION IN MINK (M US TELA
VISON) CAUSED BY 3,3’,4,4’,5—PENTACHLOROBIPHENYL (PCB 126)

By

Kerrie J. Beckett

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Science
and
Environmental Toxicology

2005

ABSTRACT

INDUCTION 0F SQUAMOUS CELL CARCINOMA IN THE MANDIBLE AND
MAXILLA, AND REPRODUCTIVE DYSFUNCTION IN MINK (MUSTELA
VISON) CAUSED BY 3,3’,4,4’,5—PENTACHLOROBIPHENYL (PCB 126)

By

Kerrie J. Beckett

The hypothesis of this dissertation is that the lesion induced by 3,3’,4,4’,5—
pentachlorobiphenyl (PCB 126), identified as squamous epithelial proliferation in the
maxilla and mandible of mink (Mustela vison), is oral squamous cell carcinoma. The
objectives of this study were to determine the progression of the lesion after removal of
the stimulus (PCB 126), and the ability of the cells comprising the lesion to form a
tumor after being transplanted or injected into nude, athymic mice. The lesion was
histologically detectable in mink exposed to 24 ug PCB 126/kg feed for one to six
weeks, and was clinically detectable in the PCB 126—exposed mink of the three-, four-,
and five-week groups, by 12-weeks post-treatment. The lesion worsened during the six-
or eight-month post-exposure period in 100% of the mink in all exposure groups, thus
meeting the first criteria of lesion progression after withdrawal of the PCB 126
stimulus. In the athymic mouse tn'al, nodular growths were observed in two of the 18
mice that were implanted with gingiva from the oral lesion of PCB 126-exposed mink.
Although the tumors in the mice were classified as non-malignant, the origin was non-
host (mink) stratified squamous epithelium, demonstrating consistency with neoplastic

criteria. Another objective of the study was to determine if the oral neoplasia caused

systemic osteolysis in addition to local osteolysis ofthejawbones in PCB 126-exposed
mink. A series ofradiographs evaluated dental alignment, bone lysis, nail growth, organ
integrity and seven skull and subcutaneous fat measurements of PCB 126-exposed and
control mink. In addition, calcium and phosphorus content of femurs were assessed.
Radiographs revealed that PCB 126 caused severe localized mandibular and maxillary
osteolysis that aggressively progressed into the zygoma and nasal turbinates. However,
systemic effects of PCB 126 on bone were not observed in this study, and there were no
significant changes in femur calcium or phosphorus content. In addition to the oral
lesion assessments, a study was conducted to determine the reproductive effects of PCB
126 in female mink. No reproductive effects were observed at 0.24 pg PCB 126/kg
feed. However, total reproductive failure occurred at dietary concentrations as low as
2.4 pg PCB 126/kg feed. A number of changes in clinical, hematological and serum
chemistry parameters were observed in both the adult female and juvenile mink exposed
to PCB 126.

Wild mink naturally exposed to environmental contaminants were also assessed
for the prevalence of the oral neoplasia. Four of nine mink trapped in a PCB-
contaminated, Superfund site exhibited histological evidence of the oral lesion. There
was a significant correlation between the severity of the lesion and hepatic
concentrations of total PCBs and 2,3,7 ,8-tetrachlorodibenzo-p-dioxin toxic equivalents
in these mink. Results from these experiments indicate that PCB 126 induces an
extremely invasive and destructive form of oral squamous cell carcinoma, as well as

other adverse effects in mink.

Copyright By
KERRIE J. BECKETT
2005

DEDICATION

~ To Mom ~
&
~ My Co-Pilots ~

~

“If I could wish for anything,
I should not wish for wealth and power,
but for the passionate sense of what can be,
for the eye, which, ever young and ardent sees the possible.”

- Soren Kierkegaard

~ Thank You ~

For seeing in me what others never did, and
Instilling in me the Passion for Life,
and
Following my Dreams and the Possibilities.

ACKNOWLEDGMENTS

I would like to take this opportunity to acknowledge those that have been with
me and supported me in so many different facets on this journey of graduate school. So
to my committee: to my advisor, Dr. Steve Bursian, I express my appreciation for your
guidance and friendship. Drs. Ben Yamini, Mike Orth, Diana Rosenstein and Jim
Sikarski, the team that has offered their support and expertise during my program at
MSU. Dr. Richard Aulerich deserves special thanks as an honorary committee member
and mentor. It goes without saying that he has been a rock of support and knowledge for
me and the kindest of friends, and also the man that introduced me to mink.

Before I go any further, this research would not have been possible without the
mink over the years (most of whom were named, and listing them would take up several
pages in this acknowledgment). And to those of you who know me best, it would be
most appropriate for the animals to come first.

The Michigan State University (MSU) Fur Farm has been a fabulous
Opportunity for toxicology research, and a special thanks goes out to the crew at the
farm for their help, even when we got sprayed by the mink: Angelo Napolitano, Jeff
Greenlee, and especially Dianne Karsten. In addition, thanks to Bob Kriegel, Jackie
Christie, and Betty Walther, your help and friendship have been immeasurable.

With that said, my pillar of support: my mom, you’re the best. Thank you for
always being there for me, and giving me the strength and courage to do whatever it
was that I set out to do. I would also like to thank my sister Kris, (and not necessarily

for those annoying little lectures along the way). My beloved pets: Harley, my co-pilot -

vi

always my calm in the storm. The dogs, cats, and Petie are always the ones who
understand without expectation or judgment, and who have always greeted me warmly
after an unspeakably stressful day of graduate school (to say the least).

Learning as a graduate student only begins at the book level. Learning becomes
significant when we are excited by what we learn, and we become creative in our
learning. Struggling, cOping, stressing-out, and remembering how to laugh are lessons
that are vital to learning in graduate school. It is how you put them together in a
package that is important. Thanks to ALL of you who have been there for me, as I have

also learned a part of my education from you.

vii

TABLE OF CONTENTS

LIST OF TABLES .................................................... xi
LIST OF FIGURES ................................................... xiii
LIST OF ABBREVIATIONS .......................................... xvii
INTRODUCTION ..................................................... 1
CHAPTER 1
Literature Review ............................................... 4
Environmental Contaminants ................................ 4
History of PCBs ...................................... 4
PCB Classification .................................... 5
PCB Configuration .................................... 6
PCB Mechanism of Toxic Action ......................... lO
Toxic Responses of PCBs ............................... 1 1
Exposure to PCBs .................................... 12
Toxic Differences Between Species ...................... l3
Mink (Mustela vison) ....................................... 14
Description ......................................... l4
Mink and Toxicology ................................. 15
Reproduction ....................................... 16
Mink Skeletal System (Axial Skeleton) ....................... ' . . 18
Skull .............................................. 18
Periodontium ....................................... 18
Incisors ............................................ 21
Canines ............................................ 21
Premolars .......................................... 21
Molars ............................................ 22
Gingiva ............................................ 22
Oral Diseases ............................................. 23
Periodontal Disease .................................. 23
Neoplasia .......................................... 24
Types of Neoplasms .................................. 24
References ................................................ 27

viii

CHAPTER 2

Female Mink (Mustela vison) Fed Diets Containing 3,3’,4,4’,5-
Pentachlorobiphenyl (PCB 126). 1: Reproductive Performance and Kit

Survivability
Abstract .................................................. 34
Introduction .............................................. 35
Methods ................................................. 37
Results .................................................. 4 1
Discussion ............................................... 47
Conclusions .............................................. 52
References ............................................... 54
CHAPTER 3

Adult Female and Juvenile Mink (Mustela vison) Fed Diets Containing
3,3’,4,4’,5-Pentachlorobiphenyl (PCB 126). II: Clinical, Hematological, and
Serum Chemistry Parameters

Abstract .................................................. 58
Introduction ............................................... 59
Methods .................................................. 61
Results ................................................... 64
Discussion ................................................ 70
Conclusions ............................................... 89
References ................................................ 90

CHAPTER 4

PCB 126-Induced Oral Squamous Cell Carcinoma of the Mandible and
Maxilla in Mink (Mustela vison)

Abstract .................................................. 94
Introduction ............................................... 95
Methods ................................................. 100
Results .................................................. 108
Discussion ............................................... 1 27
Conclusions .............................................. 139
References ............................................... 141

ix

CHAPTER 5

Bone Lysis Associated with PCB 126-Induced Neoplastic Oral Lesion of the
Mandible and Maxilla in Mink (Mustela vison)

Abstract ................................................. 147
Introduction ............................................. 148
Methods ................................................. 150
Results .................................................. 156
Discussion ............................................... 160
Conclusions .............................................. 178
References ............................................... 179

CHAPTER 6

Squamous Epithelial Lesion of the Mandiwa and Maxillae of Wild Mink
(Mustela vison) Naturally Exposed to Polychlorinated Biphenyls

Abstract ................................................. 184
Introduction .............................................. l 85
Methods ................................................. 187
Results .................................. g ................ 190
Discussion ............................................... 190
Conclusions .............................................. 194
References ............................................... 196

LIST OF TABLES

CHAPTER 2

Table 2-1. Composition and nutrient analysis of the basal
diet used for the PCB 126 experiments .......................... 39

Table 2-2. Reproductive performance of female mink fed diets
containing PCB 126 at a dietary concentration of 0, 0.24,
2.4, or 24.0 pg PCB 126/kg feed .............................. 44

Table 2-3. The effects of dietary PCB 126 on kit survivability from
whelping through weaning at 6-weeks Of age .................... 45

Table 2—4. The effects of dietary PCB 126 on kit body weights at
birth, three, six and 28 weeks of age ........................... 46

CHAPTER 3

Table 3-1. Chart of daily observations for signs of toxicity in mink
(Mustela vison) fed PCB 126 to monitor general health and
physical condition .......................................... 65

Table 3-2. Body weight and mean organ weights of female mink
fed diets containing 0, 0.24, 2.4, or 24.0 pg PCB 126/kg
feed ..................................................... 7 1

Table 3-3. Body weight and mean organ weights of juvenile mink
fed diets containing 0, 0.24, 2.4, or 24.0 pg PCB 126/kg
feed ..................................................... 72

Table 3-4. Effects of exposure to dietary PCB 126 on serum
chemistry parameters in adult female mink ...................... 73

Table 3-5. Effects of exposure to dietary PCB 126 on
hematological values for adult female mink ..................... 74

Table 3-6. Effects of exposure to dietary PCB 126 on

hematological parameters on hematological parameters for
juvenile mink ............................................. 75

xi

Table 3—7. Effects of dietary PCB l26 on serum chemistries in
juvenile mink exposed in utero through seven months of
age ...................................................... 76

CHAPTER 4

Table 4-]. Liver analysis Of percent lipid and PCB 126
concentrations were determined on a wet weight and
lipid weight basis in livers from female mink (Mustela
vison) exposed to 24.0 pg PCB 126/kg feed ..................... 124

CHAPTER 5

Table 5-1. Measurements (mm) of skull and subcutaneous fat of
female mink exposed to PCB 126 through their diet (24.0
pg PCB 126/kg feed) ....................................... 165

CHAPTER 6

Table 6-1. Incidence of mandibular and maxillary squamous
hyperplasia, and hepatic concentrations of total
polychlorinated biphenyls (PCBs), toxic equivalents
(TEQs) and percent contribution toward toxic equivalents
by 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126) in wild mink
trapped in the Kalamazoo River Basin .......................... 193

xii

LIST OF FIGURES

*Nole: Images in this dissertation, including H&E photomicrographs. are presented in
color.

CHAPTER 1

Figure 1-1 a. The basic PCB structure: Two phenyl rings are
connected, with chlorine atoms distributed around
the biphenyl at the ortho, meta, and/or meta positions ............... 8

Figure l-lb. A PCB has the basic formula of C12 Hm- n C] n,
where n = 1 - 10 ............................................ 8

Figure 1-2. The chemical structure of 3,3’,4,4’,5-
pentachlorobiphenyl (PCB 126) ................................ 9

Figure 1-3. The dental formula for the permanent dentition of
mink (Aulerich and Swindler, 1968a, 1968b) ..................... 19

Figure 1—4. Sub-gross photomicrograph depicting normal
permanent mandibular dentition of a standard dark mink
(Mustela vison). H&E ...................................... 20

CHAPTER 2

Figure 2-1. Implantation sites within a uterine horn of a bred
female receiving a diet that contained 24.0 pg PCB
126 /kg feed. The female did not whelp ......................... 43

CHAPTER 3

Figure 3-1. Increased toenail growth induced by dietary
exposure to 24.0 pg PCB 126/kg feed in female mink 16
weeks into the trial was characterized by thickening,
elongation, and deformity of the nails in the front feet.
Nail growth in PCB 126-exposed mink was considered
moderate and continued throughout the trial period, including
the period while mink were on clean feed post-PCB 126
exposure ................................................. 68

xiii

CHAPTER 4

Figure 4- l. Week One: Oral squamous epithelial
proliferation in mink exposed to PCB 126 for one week,
and histological evidence at six months after the removal
of the stimulus (PCB 126 diet). Tooth (T); Alveolar Bone
(AB); Gingiva (Gin); Periodontal Ligament (PDL). Arrows
on right point to squamous epithelial cysts that have invaded
either the PDL and/or the AB. H&E .......................... 1 11

Figure 4-2. Week Two: Histological evidence of oral squamous
epithelial proliferation in mink exposed to PCB 126 for two
weeks exposure, and six months after the removal of the
stimulus (PCB 126 diet). H&E ............................... 112

Figure 4-3. Week Three: Histological evidence of oral
squamous epithelial proliferation in mink exposed to PCB
126 for three weeks, and six months after the removal of
the stimulus (PCB 126 diet). Squamous epithelial cysts
and/or nests depicted in the histology slide on the right are
throughout the entire tissue. H&E ............................. 114

Figure 4—4. Week Four: Invasive oral squamous epithelial
proliferation in mink exposed to PCB 126 for four weeks,
and six months after the removal of the stimulus (PCB
126 diet). In the histology slide on the right, there was no
AB that remained in this section to support tooth structures,
cysts and/or nests and cords destroyed and replaced almost
all maxilla and mandible bone tissue. H&E .................... 115

Figure 4-5. Week Five: Development of oral squamous
epithelial lesion with evidence of osteolysis in mink exposed
to PCB 126 for five weeks, and six months after the removal
of the stimulus (PCB 126 diet). Cysts and/or nests and cords
have invaded the PDL of most of the teeth of this mink on the
left. On the right, cysts and/or nests and cords have expanded
and occupied almost all of the periodontium. .H&E ............... 117

Figure 4-6. Week Six: Evidence of an osteolytic oral
squamous epithelial proliferation in mink exposed to PCB
126 for six weeks, and eight months following the removal
of the stimulus (PCB 126 diet). The image on the right
consists of teeth and lesion, all other structures have been
destroyed and/or replaced by the lesion. H&E .................. 120

xiv

Figure

4—7. Proliferation with substantial thickening of the
gingival tissue forming nodular growths along the inside
of the lower palate (along the dental arches), reaching
almost to the central midline*. A) Nodular growths were
observed in the mandible of a mink exposed to PCB 126

via the diet; B) the mandible from a control mink ................ 121

Figure 4-8. Athymic, nude mice implanted or injected

CHAPTER 5

Figure

Figure

Figure

with gingival tissue harvested from mink exposed to
24.0 pg PCB 126/kg feed via their diet. *Staple over

incision ................................................ 126

5-1. Skull measurements of the mink (Mustela vison) to
evaluate PCB 126 induced osteolysis (left lateral view

of the skull) ............................................

5-2. Dental malalignment and increased interdental
spacing in mink induced by consumption of a diet
containing 24.0 pg PCB 126/kg feed. A) Notice
inflammation of gingival tissue and moderate spreading
of incisors. B) Canines appear extended and incisors
were missing (mandibular and maxillary); gums were

bloody and swollen .....................................

5-3. Radiographic images of female mink (Mustela vison)
showing dental malalignment and increased interdental
spacing induced by dietary exposure to 24.0 pg PCB 126/kg
feed. A control mink (left) depicts normal lucency in

jaws and alignment of teeth compared to a PCB 126 exposed
mink (right), at Week 12 (6-week PCB-exposure period

..155

.16]

followed by six weeks of being on control feed) ................. 162

Figure 5-4. Extensive osteolysis of mandibular and maxillary

alveolar bone in a female mink (Mustela vison) exposed
to 24 pg PCB 126 /kg feed. Note: no bony support
surrounding dentition, resulting in loss of teeth. (Skull

cleaned by dermestid beetles.) ............................

Figure 5-5. Bone lysis and tooth loss observed in the mandible,

maxilla, and nasal bones in mink exposed to 24.0 pg PCB

...163

126/kg feed. (Skulls cleaned by dermestid beetles.) .............. 164

XV

Figure 5-6. Osteolysis of the zygoma and zygomatic arch in a
female mink (Mustela vison) exposed to a diet that
contained 24.0 pg PCB 126/kg feed ............................ I66

Figure 5-7. Radiographic image of a female mink (Mustela vison)
exposed to 24 pg PCB 126 /kg feed via the diet for six weeks,
and then on control feed for three weeks. Radiograph depicts
decreased subcutaneous fat (arrow), and increased interdental
spacing of the incisors, as well as increased lucency in the
jawbones ................................................ 167

CHAPTER 6

Figure 6-1. Mandibular and maxillary squamous epithelial
hyperplasia in wild mink exposed to polychlorinated
biphenyls in the Kalamazoo River area of concern
(KRAOC). A: A section from a control mink is shown
for reference. Note the solid-appearing alveolar bone
(AB) surrounding the teeth (T), which is above the gingiva
(GIN). B: KRAOC Mink #03 had a lesion rating of mild,
with one focus (arrow). C: KRAOC Mink #06 had a lesion
rating of mild-moderate, with a large invasive cyst (arrow)
and small multi-focal diffuse nests. D: KRAOC Mink #08
had a lesion rating of moderate, with diffuse squamous cysts
throughout the jaw (arrows). Extensive infiltration by
large islands of squamous epithelial cells has occurred and
formed cysts that resulted in the loosening of teeth. These
cysts may contain centers of exfoliated epithelia and
sometimes keratin H&E (Black and White) ..................... 192

xvi

AB

AG
AHH
AhR
ALB
ALK PHOS
ALT
AMYL
ARNT
AST
bHLH
BUN

BW

Ca
CHOL
CK

C1

C02
CREAT

CYP1A1

LIST OF ABBREVIATIONS

alveolar bone

anion gap

aryl hydrocarbon hydrolase

aryl hydrocarbon receptor
albumin concentration

alkaline phosphatase activity
alanine aminotransferase activity
amylase activity

aryl hydrocarbon receptor nuclear translocator
aspartate aminotransferase activity
basic helix-loop-helix

blood urea nitrogen concentration
body weight

canine teeth

calcium concentration

cholesterol concentration
creatinine kinase activity

chloride concentration

carbon dioxide

creatinine concentration

cytochrome P-450 1A protein/enzyme; the major hydrocarbon
inducible form of the P-450 biotransformation enzyme

xvii

DDT

DNA

DRE

DV

Eagle’s ASP
EDTA

EGF

ELISA

FA

FCRA

Fe
gamma-GTP
Gin; GIN
GJIC

GLOB
GLUC

GPx

GR

GSH

H&E
HCT

HGB

1,1, l -trichloro—2,2-bi.s'( p-chloropheny|)ethane
deoxyribonucleic acid
dioxin-response-element

dorso-ventral view

Eagle’s minimum essential solution
ethylenediaminetetraacidic acid
epidermal growth factor
enzyme-linked-immunosorbent-assay
fluctuating asymmetry

Fort Custer Recreation Area

iron concentration, serum
gamma-glutamyl transpeptidase activity
gingiva

gap junctional intercellular communication
globulin concentration

glucose concentration

glutathione peroxidase activity

GSH reductase activity

glutathione concentration

height

hematoxylin and eosin

hematocrit

hemoglobin concentration,

xviii

hsp90

IARC

KRAOC

kVp

LDso

LOAEL

mAs
MCHC
MCV
MEM
Mg
mRNA

MSU

NA
Na

N OAEL

heatshock proteins

incisor teeth

internal diameter

International Agency for Research on Cancer
potassium concentration
Kalamazoo River area of concern
kilivolt peak

length

median lethal dose

lowest observed adverse effect level
molar teeth

rnilliamps per second

mean corpuscular hemoglobin concentration
mean corpuscular volume

Modified Eagle’s Medium solution
magnesium concentration
messenger ribonucleic acid
Michigan State University

number

not available

sodium concentration

no Observed adverse effect level

National Toxicology Program

xix

 

 

OC

PAHs

PBS

PCB 30

PCB 77

PCB 81

PCB 126

PCB 136

PCB 153

PCB 169

PCB 204

PCDDs

PCDFs

PDL

PLT

PM

rpm
SAS

SCC

osteocalcin

level of significance, based on Type I error rate of or = 0.05
(p S 0.05)

pol yhalogenated aromatic hydrocarbons
buffering solution

2,4,6-triphenyl

3 ,3 ’ ,4,4 ’ -tetrachlorobiphenyl

3 ,4,4’ ,5-tetrachlorobiphenyl

3 ,3 ’ ,4,4’ ,5-pentachlorobiphenyl
2,2’,3,3’,6,6’-hexachlorobiphenyl

2,2’ ,4,4’ ,5 ,5 ’ -hexachlorobiphenyl

3 ,3 ’ ,4,4’ ,5 ,5 ’ -hexachlorobipheny1
2,2’,3,4,4’,5,6,6’-octaphenyl
polychlorinated dibenzodioxins
polychlorinated dibenzofurans
periodontal ligament

platelet volume

premolar teeth

coefficient of determination

red blood cell count

red cell distribution width

rotations per minute (centrifuge)
Statistical Analysis Systems software, version 8.0

squamous cell carcinoma

SDH
SE
Se

SOT

TCDD
TC02
TEF
TNF-a
TRVs

U.S.EPA

WBC

ZB-S

I18

CID

dL

sorbitol dehydrogenase activity

standard error of the mean

Selenium concentration

squamous odontogenic tumor

tooth

2,3,7,8-tetrachlorodibenzo-p-dioxin

total carbon dioxide concentration, serum
toxic equivalency factor, or toxicity equivalency factor
tumor necrosis factor

toxicity reference values

US. Environmental Protection Agency
width

white blood cell count

5% phenyl polysiloxane

Abbreviations for Units of Measure:

microgram
microliter
centimeter
deciliter
femtoliter
gram

hour

xxi

 

lU International Units

kg kilogram
km kilometer
mg milligram
millisec milliseconds
min minute

mL milliliter
mm millimeter
mM millimolar
mos milliosmole; unit used for osmolality, also written mOsm
ng nanograrn
pg picogram

xxii

 

 

INTRODUCTION

The mink (Mustela vison) is a relevant wildlife species, and an important species
in toxicology research. The mink is an indicator species of environmental contaminant
exposure and ecosystem health, and has been approved by the United States

Environmental Protection Agency (US. EPA) and recognized by the National Academy

 

of Science as a preferred toxicological model (Calabrese et a1. 1992). The mink is a
highly sensitive species to environmental contaminants, especially polychlorinated
biphenyls (PCBs). Mink are found throughout most of the northern US. and Canada
and associated with semi-aquatic environments, as well as being apex predators. Being
at the top of the food web, the mink has the potential to bioaccumulate environmental
contaminants through the prey‘items it consumes. Polychlorinated biphenyls are
ubiquitous environmental contaminants that are resistant to biological and chemical
degradation, and thus can be encountered via prey items. The toxicity of these
contaminants can give rise to serious and significant effects on animal and human
health. The most toxic PCB congener is considered to be 3,3’,4,4’,5-
pentachlorobiphenyl (PCB 126) and is known to be prevalent in the environment.

Our laboratory originally identified an oral lesion in mink kits and juvenile mink
induced by PCB 126 and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The lesion was
initially identified as squamous epithelial proliferation of the mandible and maxilla, and
was described as clumping of the incisors and thickening and nodular growths of the
gingiva. Further studies indicated that kits and juvenile mink were not uniquely

sensitive to the toxicity of PCB 126 in terms of the development of maxillary and

mandibular squamous proliferation as the endpoint, as the lesion was induced in adult
female mink fed a diet containing PCB 126.

The oral lesion was the focus of this research. Therefore, a complete description
of the lesion is important to understanding its progression. Initially, small islands of
atypical squamous epithelial cells form within the gingiva in no particular location.
Subsequently, these stratified squamous epithelial cells form large islands and develop
into cysts. Some of these cysts may contain centers of exfoliated epithelia and possibly
keratin. Nests and/or cOrds of cells infiltrate into the periodontal ligament and/or
alveolar bone destroying the underlying tissue. This leads to periodontoclasia
(loosening and displacement of teeth), and in severe cases, tooth loss. In addition,
pathologic changes were observed as swollen, inflamed, and bleeding gingiva,
including recession of the gums. Aphagia, defined as the inability to eat, often ensued at
this stage, leading to a subsequent rapid weight loss.

Commonly accepted criteria for malignancy are: 1) tissue invasion (referred to
as a hallmark of malignant neoplasia); 2) the lack of dependence on the continued
presence of the stimulus (irreversible); 3) increased rate of tissue growth; 4)
differentiation and anaplasia (with higher proliferative activity); and 5) metastasis (also
considered a hallmark of malignancy). Metastases are not observed as frequently in
animals as they are in humans, either due to the shorter life span of animals, biological
reasons, or the fact that they are not screened for and thus, detected as a practice.

The mink is an important animal model for studying PCB 126-induced oral
squamous cell carcinoma (SCC), and the pathological effects this PCB congener has on

bone and possibly other biological systems. The overall hypothesis of the research

 

described in this dissertation is that the oral lesion that is induced by PCB 126 in the

maxilla and mandible of mink is aggressive oral squamous cell carcinoma. The

objectives of this research were to:

1)

2)

3)

4)

5)

determine the reproductive effects of PCB 126 in female mink and its effects
on kit survival;

determine the biological effects, as assessed by clinical, hematological, and
serum chemistry parameters, in adult female and seven-month-old mink
caused by consumption of PCB 126;

determine if the oral lesion in mink was malignant, by meeting two
additional criteria of carcinogenicity, (a) the progression of the lesion after
removal of the stimulus (PCB 126); and (b) the ability of the cells
comprising the lesion to grow and form a tumor after being transplanted
and/or injected into athymic mice;

determine if the oral neoplasia caused systemic osteolysis in addition to the
observed local (maxillary and mandibular) osteolysis; and

determine the prevalence of the oral neoplasia in wild mink that were
naturally exposed to environmental contaminants, and were trapped in the

Kalamazoo River Basin, a designated Superfund site.

 

 

CHAPTER I

Literature Review

A. Environmental Contaminants

History of PCBs:

Polychlorinated biphenyls (PCBs) are recognized as environmental
contaminants that are of concern. The history of PCBs started in 1929 when they were
commercially produced because of their heat-insulating and physical stabilizing
properties. Production of PCB industrial mixtures continued under the trade name
Aroclor® by the major US. producer, Monsanto Corporation (St. Louis, MO). A
variety of uses for PCBs included dielectric fluids in capacitors and transformers,
hydraulic lubricants, organic diluents, plasticizers, pesticide extenders, adhesives, dust—
reducing agents, cutting oils, flame retardants, sealants, printing inks, paints, and
pigment in carbonless copy paper (Hutzinger et al. 1974; Safe 1990; Erickson 1997).
Polychlorinated biphenyls also occurred as unintentional byproducts through synthesis
of compounds, such as vinyl chloride, chlorinated benzenes, chloroform, and adhesives
(Safe 1990; Erickson 1997). Production of PCBs was banned in North America and
Western Europe in 1977, and Eastern Europe and Russia in the 19905 (Giesy and
Kannan, 1998).

Disposal regulations were not implemented until after the impacts of PCBs had
been recognized. Legal and illegal uses, as well as disposal, spills, and leaking of PCBs

have contributed to their entry into the environment. Gradual advection of PCBs near

 

 

the source has contributed to low background level contamination in the environment
(Safe 1990; Erickson 1997). The transport of PCBs in the environment is complex and
global (Erickson 1997). The properties that made PCBs so appealing for industry are
also the characteristics that are responsible for PCBs being a ubiquitous environmental
contaminant.

Polychlorinated biphenyls are considered to be one of the most widespread and

 

persistent anthropogenic contaminants in the global ecosystem giving rise to serious

environmental contamination and creating a potential health hazard (Safe 1990;

 

Nesaretnam et a1. 1996; Wilson and Safe, 1998). The stability of PCBs has made them
highly persistent in the environment, where degradation depends on the structural
arrangement of the chlorines. Polychlorinated biphenyls have a tendency to
bioaccumulate and biomagnify in the food chain, due to the lipophilicity of the
chemicals (Safe 1990; Giesy and Kannan, 1998). Thus, PCBs are detected in every
environmental compartment of the global ecosystem, including the air, water,
soil/sediment, and biota such as fish, wildlife, and human adipose tissue, milk, and

serum (Safe 1990; Safe 1993; Wilson and Safe, 1998).

PCB Classification:

Polychlorinated biphenyls belong to a larger class of chemicals known as
polyhalogenated aromatic hydrocarbons (PAHs), which include polychlorinated
dibenzo-p-dioxins, polychlorinated dibenzofurans, polychlorinated biphenyls,

polychlorinated diphenyl ethers, and polychlorinated naphthalenes. Polychlorinated

biphenyls are formed through the process of chlorination, in which substitution
reactions attach chlorines to the biphenyl rings.

The group of PCBs consisting of 209 discrete chemical compounds can exhibit
different physiochemical and biochemical (e. g. metabolism and biodegradation)
properties that result in environmental and biological breakdown. These molecular
qualities of PCBs contribute to the analytical properties that can affect environmental
transport and fate, bioconcentration factors, persistence and bioaccumulation. These
physical properties of PCBs include molecular weight, vapor pressure, melting point,
partition coefficient, aqueous solubility, sediment sorption properties, partitioning
among gases (fugacity), and Henry’s Law constants. Chemical properties can also
include such parameters as density, viscosity, flash point, specific gravity, vaporization
rate, dielectric constant, and solubility (in 25°C water) (Safe 1990; Erickson 1997).

These 209 individual PCB chemical compounds are called congeners. The term
homolog is used when PCBs are subdivided by their degree of chlorination (the number
of chlorines, which can range from one to ten, are distributed around the biphenyl). The
chlorines are attached at one of the ten positions of the phenyl ring, identified as ortho-,
meta-, and para- positions (Figures 1a and lb). Polychlorinated biphenyls within a
given homolog that present different positions of chlorine substitution are called

isomers (Erickson 1997).

PCB Configuration:
Within the PCB structure, the two-phenyl moieties can rotate about the single

‘bridge-bond’. Chlorine atoms in the ortho positions create more steric hindrance to the

 

 

rotation. PCB congeners with zero (or one) chlorine substitution in the ortho position
lack steric hindrance, and therefore can assume a coplanar configuration (phenyl rings
lying in the same plane). A few PCB congeners are capable of this coplanar
configuration, but only four PCB congeners are non-ortho and are considered to be

most like TCDD. These include 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126),

 

3,3’,4,4’,5,5’-hexachlorobiphenyl (PCB 169), 3,3’,4,4’-tetrachlorobiphenyl (PCB 77), P
L
and 3,4,4’,5-tetrachlorobiphenyl (PCB 81). These congeners induce toxic responses

similar to those reported for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Studies on

 

the structure-function relationships have confirmed the similarities between TCDD
toxicity and coplanar, non-ortho substituted PCB congener toxicity (Safe 1993;
Erickson 1997).

The PCB congener PCB 126 is non-ortho substituted and is considered to be the
most toxic PCB congener (Figure 2) (Van den Berg et al. 1998). PCB 126 is structurally
similar to TCDD, and is one-tenth as toxic as TCDD (Van den Berg et al. 1998). PCB
126 assumes a coplanar configuration and induces toxic responses similar to those
reported for TCDD, and PCB 126 is known to be prevalent in the environment (Safe et
al. 1985; Safe 1990; Peterson et a1. 1993). This coplanar configuration may be
necessary for these PCB congeners and TCDD to competitively bind to a common
receptor known as the aryl hydrocarbon receptor (AhR) that has been suggested as the

mechanism of toxic action for these chemicals.

 

 

Figure 1-1 a. The basic PCB structure: Two phenyl rings are connected, with chlorine

atoms distributed around the biphenyl at the ortho, meta, and/or meta positions.

 

meta ortho

para

 

meta ortho

 

Figure 1-1b.A PCB has the basic formula of C 12 H .0." Cl n, where n = l - 10.

 

 

 

 

Figure 1-2. The chemical structure of 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126).

 

 

3,3 ’,4,4’,5-Pentachlorobiphenyl
(PCB 126)

 

PCB Mechanism of Toxic Action:

2,3,7,8-Tetrachlorodibenzo-p-dioxin and TCDD-like PCB congeners may act
via a common mechanism of action, the Ah receptor. Initial studies by Poland et al.
(1979), and a variety of studies that followed, including genetic, biochemical, and
toxicological studies, lend support to the role that the AhR protein may play in
mediating the toxic effects of TCDD and TCDD-like PCB congeners (Safe 1985; Safe
1988; Safe 1990; Wilson and Safe, 1998).

The AhR belongs to a group of proteins that contains a basic helix-loop-helix
(bHLH) domain near the N-terrninus, and thus can undergo dimerization and DNA-
binding (Wilson and Safe, 1998). The AhR is a ligand-activated transcription factor that
has no known endogenous, or naturally occurring ligand (Safe 1990; Josephy 1997;
Giesy and Kannan, 1998). The AhR is the only known protein in its superfamily that
requires an exogenous ligand for activation (Safe 1990; Wilson Safe, 1998). 2,3,7,8-
Tetrachlorodibenzo-p-dioxin binds to the AhR with high affinity, and has been
identified as the “prototype ligand” of the Ah receptor. In addition, the AhR appears to
bind several different structural classes of chemicals that are considered to be AhR
agonists, including halogenated aromatics, in addition to TCDD (Safe 1990; Yamaguchi
et al. 1997).

Prior to binding of the ligand (e.g. TCDD), the unoccupied cytosolic AhR is
normally complexed in a 1:2 ratio with heatshock proteins (hsp90). The hsp90
dissociate when the ligand binds to the AhR, enabling the receptor to be phosphorylated
by tyrosine kinase. The activated AhR then enters the nucleus and forms a heterodimer

complex with the Ah-receptor-nuclear translocator (ARNT). Inside the nucleus, the

10

 

 

AhR-ARNT complex binds to a regulatory sequence, known as the dioxin-response-
element (DRE), and enhances transcription of the CYP1A1 gene and other genes with a
DRE or DRE-like sequence in the upstream enhancer region (i.e. glutathione-S-
transferase). The DRE is only a small segment of DNA (the consensus sequence is 5’-
TXGCGTG-3’ [X=T or A], in the 5’-flanking regions of the responsive genes), which
can be located more than 1000 bases from the initiation site for transcription. The
enhancer region of the CYP1A1 gene contains multiple DREs, which account for the
marked increase (<100 fold) in CYP1A1 mRNA and protein levels following exposure
to ligands for AhR (Safe 1988; Safe 1990; Safe 1993; Josephy 1997; Wilson and Safe,
1998).

The ARNT was initially thought to be a cytosolic protein that simply facilitated
the translocation of the ligand-bound AhR into the nucleus. It is now recognized as an
important component of the receptor complex that binds to DNA and activates
transcription of genes under the control of AhR. The AhR complex has the ability to act
as a ligand-dependent DNA-binding transactivator of gene expression (Schmidt and

Bradfield, 1996; Rowlands and Gustafsson, 1997; Wilson and Safe, 1998).

Toxic Responses of PCBs:

A plethora of adverse effects have been attributed to PCBs. Studies have
reported enzyme induction, thymic involution and atrophy, immunotoxicity,
hyperkeratosis, dermal toxicity, cleft palate, hydronephrosis, hepatotoxicity,

teratogenesis, developmental and reproductive toxicity, endocrine effects, neurotoxicity,

ll

 

 

carcinogenicity, and lethality (Kociba 1978; Safe 1993; Abbott et al. 1994; Safe 1994;
Erickson 1997; Giesy and Kannan, 1998).

As stated above, the toxic effects of a PCB congener are directly related to its
structure. Therefore, the substitution and arrangement of chlorines around the biphenyl
dictate the toxicity of the chemical. The toxic effects of a chemical are also mediated

through a mechanism of action, which may or may not be common to subclasses of

 

congeners. The structural class of PCB congeners that are not TCDD-like based on their
chlorine substitution, appear to exhibit adverse effects including neurotoxicity,
estrogenicity, and protein-binding activities (Erickson 1997; Giesy and Kannan, 1998).
PCBs have been extensively studied in both in vivo animal studies and in vitro assays
(Erickson 1997). The majority of the studies have used commercial mixtures of PCBs,

and only a minimal number have been conducted using individual PCB congeners.

Exposure to PCBs:
As a result of toxicity potential, widespread distribution, persistency, and

potential for bioaccumulation, PCBs are considered a health concern. Polychlorinated

 

biphenyls are lipophilic and resistant to biochemical degradation, therefore they have
the ability to biomagnify in the food chain. This can result in an increase in the
exposure potential for apex predators including humans. Polychlorinated biphenyl
concentrations in food (prey), and therefore, tend to correlate with the fat content of that

food source (Erickson 1997).

12

Toxic Differences Between Species:

Species, strain, as well as age, sex, reproductive status, and conditions of
exposure (concentration, duration, and frequency) can influence the toxic effects of
PCBs. When comparing species typically used in PCB toxicology studies, i.e. rats,
guinea pigs, hamsters, and mink, a pronounced sensitivity to PAHs varies over a 5000-
fold range between these species and strain, with guinea pigs being the most sensitive
and the hamster being the least sensitive (Poland and Knutson 1982; Safe 1990; Josephy
1997). For example, the reported oral LDso for TCDD in the guinea pig is
approximately 1.0 pg/kg body weight (BW). The oral LDSO for the rat is reported at 10
to several 1000-fold higher than the guinea pig, depending on sex and strain (Josephy
1997).

The biological response an animal will have following exposure to PCBs that is
measured as an endpoint is influenced by these characteristics, i.e., sex, age,
reproductive status, strain, and species (Josephy 1997). A specific endpoint in one
species may be a good indicator of a toxic response, but not a good indicator in another
species. Thus, based on species specificity and species differences, not all species
respond the same. For example, Hori et al. (1997) reported on the different species
mechanisms by which PCB 126 induced toxicity in rats. Selenium (Se) and glutathione
(GSH) concentrations, and glutathione peroxidase (GPx), GSH reductase (GR) and
gamma-glutamyl transpeptidase (gamma—GTP) activities were significantly decreased
by PCB 126 treatment in rat liver (Hori et al. 1997). In hepatic tissue of the guinea pig,
GPx activity was significantly increased by PCB 126 exposure. No effect on Se, GSH

or the enzyme activities was observed in guinea pigs (Hori et al. 1997).

13

B. Mink (Mustela vison):

Description:

Mink (Mustela vison) belong to the order Carnivora and the family Mustelidae,
which also includes four subfamilies: Mephitinae (skunks), Taxidinae (badgers),
Lutrinae (otters), and Mustelinae (martens, polecats, ferrets, weasels, and mink),
comprised of 25 genera (Calabrese et al. 1992; Aulerich et al. 1999).

Wild mink are dark brown in color, sometimes with a white throat patch. Their

fur is extremely dense and silky-soft. They have elongated bodies that are “weasel-

 

like”, with relatively short legs. Their tail measures about one-third of the body length
and is somewhat bushy. Females are approximately one-half to two-thirds the size of
the males. Females may weigh 600 g and males weigh about 1000 g (wild mink). A
feature common to the Mustelidae is the presence of anal glands, which produce a

distinct odor. These glands are derived from apocrine glands, and are important in

 

defense, recognition, territorial marking, and courtship behavior (Smith and Schenk,
2000). The mink is very agile and quick, an excellent swimmer, and has powerful jaws.
These are characteristics that enable the mink to be an efficient predator.

Mink are a semi-aquatic species, inhabiting the margins of aqueous habitats, i.e.
rivers, ponds, marshes, lakes, and estuarines that extend throughout most of the northern
parts of North America. Mink are considered solitary by nature and are generally
nocturnal. Mink are piscivorous mammals and are considered to be true predators.

Thus, mink are apex predators and placed at the peak of the food chain. Therefore, mink
are potentially exposed to a wide variety of environmental contaminants through

consumption of contaminated prey.

14

Mink were trapped for their fine quality of fur. Concern that the wild stock was
being depleted prompted people to attempt raising mink by the end of the nineteenth
century (Bowness 1996). However, successful mink ranching as a commercial practice
is a relatively recent development and has occurred only within the last 80 years
(Calabrese et al. 1992; Bowness 1996). The information that exists on the mink
(biology, health, and husbandry) is more or less a direct result of the commercial
ranching of mink. For example, bringing solitary animals into close contact with one
another presented health risks, and as a result, vaccinations have been developed to
reduce the incidence of diseases in mink (Freeman 1996). Because mink are one of the
most recent species to be domesticated, their nutritional requirements needed to be
identified and met in a commercial setting (NRC 1982; Atkinson 1996). Meeting
nutritional requirements, especially during vulnerable periods of growth and
development, and during high demand periods such as lactation, is essential for the
health and well-being of the mink (Atkinson 1996; Aulerich et al. 1999). Without
proper balancing of nutrition, deficiencies may lead to additional health problems such
as wet belly, urolithiasis (with associated uroliths), steatitis (Vitamin E deficiency),
cotton fur (iron deficiency), fur loss and depigmentation (biotin deficiency) and
convulsions with severe neurological signs and potential death (thiamin deficiency)

(Atkinson 1996; Aulerich et al. 1999).

Mink and Toxicology:
The mink, as an indicator species of environmental contaminant exposure and

ecosystem health, is approved by the US. Environmental Protection Agency (US.

15

EPA) and recognized by the National Academy of Science as a preferred toxicological
model (Calabrese et al. 1992; Aulerich et al. 1999). A ranch-raised strain of American
mink represent a wildlife species that occupies a high trophic level and consumes prey
affected by environmental contaminants, such as PCBs. In addition, the mink can be an
important mammalian model for wildlife toxicity studies because of its presence in
threatened habitats, toxicity studies can be done in a controlled laboratory situation, and
there exists a common body of knowledge on mink husbandry and biology (Joergensen
1985; Calabrese et al. 1992; Lipscomb et al. 1996; Aulerich et al. 1999).

Mink are among the most sensitive mammals to environmental contaminants,
especially PCBs (Aulerich and Ringer, 1977; Ringer et a1. 1981; Aulerich et al. 1985,
1987). Studies have shown a wide range of sensitivities to commercial PCB mixtures
and to the limited number of individual PCB congeners that have been investigated
(2,2’,3,3’,6,6’- hexachlorobiphenyl [PCB 136], 2,2’,4,4’,5,5’-hexachlorobiphenyl [PCB
153], 3,3’,4,4’,5,5’-hexachlorobiphenyl [PCB 169], and TCDD (Chapter 2; Aulerich et
al. 1985, 1987; Hochstein et al. 1988; Patnode and Curtis, 1994). Reproductive
functions in mink are often disrupted as a result of exposure to PCBs (mixtures and
congeners) due to the sensitivity of mink to such compounds (Aulerich and Ringer

1977; Aulerich et al. 1985, 1987; Backlin and Bergman, 1992; Kihlstrom et al. 1992).

Reproduction:
The mink is defined as a seasonal breeder. The annual breeding season is
regulated by the length of day (photoperiod) (Aulerich et al. 1999). Unlike some of the

other members of this order, mink are short-day breeders, meaning that the shortening

l6

 

 

0)]

test
mid-
phat
Man
0th

(Orig;

of day length triggers the reproductive season (Boissin-Aggase et al. 1986; Aulerich et
al. 1999). The female mink has induced ovulation and delayed implantation, which are
characteristics shared by other members of the family Mustelidae. Ovulation is induced
by copulation, and it is believed to induce the release of luteinizing hormone from the
pituitary gland, which then acts on mature follicles to induce their rupture (Murphy
1996). Delayed implantation is also known as embryonic diapause, which is a variable
period in the mink terminated by implantation. Implantation follows the initial increase
in progesterone by five to 10 days, and is influenced by the photoperiod, where an
increase in photoperiod decreases the length of embryonic diapause (Murphy 1996;
Aulerich et al. 1999). The mink also possesses unique reproductive characteristics,
which include an abbreviated mating season and a curious pattern of repetition of
ovulation in the presence of fertilized embryos (Murphy 1996).

In the male mink, as in other mustelids, testes size and spermatogenic activity
varies during the year, and is positively correlated with elevated circulating testosterone
concentrations (Onderka 1996; Aulerich et al. 1999; Wolf et a1, 2000). The onset, as
well as the regression, of testicular development in mink is triggered by an increase in
testosterone beginning in October-November, and another increase in late March to
mid-April (Aulerich et al. 1999; Wolf et al. 2000). This cycle is mediated through the
photoperiod, resulting in the testes descending only during the breeding season in
March (Aulerich et al. 1999). The mink penis contains a penile bone called the os penis
or baculum. This feature is shared with other species of mammals, such as the walrus

(Odobenus rosmarus) and rodents.

l7

 

 

C. Mink Skeletal System (Axial Skeleton):

Skull:
The skull is part of the axial division of the skeleton. The skull is comprised of
several bones fused together by sutures, part of which compose the neurocranium. The

neurocranium forms a rigid, protective enclosure for the brain and sensory organs (eyes,

 

nose, and ears) (Radke and Chiassom, 1998; Smith and Schenk, 2000).
The anterior of the skull, called the splanchnocranium, is comprised of facial

bones that support the digestive and respiratory systems (Radke and Chiassom, 1998). i '1

 

The general bones appropriate for this study include the nasal bones, palatines, the
premaxilla (or incisive bones), and specifically the maxillae and the mandibles. The
premaxillae contain the sockets for the incisor teeth and articulate caudally with the

maxillary bones. The maxillae are large paired dentary bones that are caudal to the

 

premaxilla and have sockets for the upper canines, premolars, and molars. The
mandibles are a pair of fused dentary bones that articulate with each other in the rostral

midline by the mandibular symphysis. The mandible also has sockets for all of the

lower teeth are in the portion of the mandible called the body (Radke and Chiassom,

1998).

Periodontium (Teeth):

Mink are true predators, therefore requiring specialized dentition to shear tough
tissue, such as flesh, tendons, cartilage, and bone. The term palatal dentition is used to
refer to teeth in the upper jaw, and lingual is used for teeth in the lower jaw (Radke and

Chiasson, 1998). Kits are born without teeth, but ridges for future tooth eruption are

18

present (Hunter and Barker, 1996). Mink have both deciduous teeth and permanent
teeth, with permanent teeth eruption being complete by approximately 2.5 months of
age (Aulerich and Swindler, 1968a, 1968b). As with most mammals, there are four
types of teeth that make up the permanent dentition of the mink: incisors, canines,
premolars, and molars (Radke and Chiasson, 1998; Aulerich and Swindler, 1968a,
1968b)

The Tritubercular Theory in dental nomenclature was used. The number of teeth
presented in the numerator, for example, is for a single quadrant. Incisor (l), canines
(C), premolar teeth (PM), and molar teeth (M) are represented in each quadrant of the
mink jaw for a total of 34 permanent teeth as designated by the formula. Superscript

designates teeth located in the maxilla, subscript denotes teeth in the mandible.

 

Figure 1-3. The dental formula for the permanent dentition of mink (Aulerich and

Swindler, 1968a, 1968b).

 

Maxilla 3 l 3 l
I—; C—; PM—; M— =34
Mandible 3 1 3 2

 

l9

 

 

 

Figure 1-4. Sub-gross photomicrograph depicting normal permanent mandibular

dentition of a standard dark mink (Mustela vison). H & E.

 

 

 

 

"‘ The teeth are differentiated into four categories: Incisors (1)3/3; Canines (C)1/1;
Premolars (P)3/3; and Molars (M)1/2. Lower incisors are irregularly placed in the
mandible with the 12 situated lingual to 11 and 13. The premolars have two roots per
tooth. The mandible has two molars, with M2 being a single-rooted tooth. H & E.

Bar=5mm.

20

Incisors:

The six incisors of each jaw are simple teeth, each with a single cusp and root.
The permanent incisors are larger, and erupt lingual to the deciduous incisors. Size does
vary noticeably, with the third incisor (13) being largest of the three in the upper jaw.
The incisors of the premaxilla are set transversely. In the mandible, the incisors are
irregularly placed, with the second incisor (I2) lingual to the other two (I. and I3)

(Aulerich and Swindler, 1968a, 1968b).

Canines:

The canines are large, cone-shaped teeth that are slightly recurved, with a
substantial root anchoring then to the mandible or maxilla. The upper canines are larger
than the lower canines, and there is a space between the upper canine and 13. This

interval receives the canines of the mandible and thus allows the mouth to close.

Premolars:

There are three upper and three lower premolars, and the deciduous and
permanent premolars are very similar. The premolars in general are more complex in
their structure, as they are sectorial in nature (Aulerich and Swindler, 1968a, 1968b).
The large cusps of the premolars meet to form the camassials, which are used to shear

tissues (Radke and Chiasson, 1998).

21

 

 

 

Molars:

The dentition of the mink reflects that the species is a true predator and
specialized flesh-eating diet. Therefore, there has been a reduction in the post-camassial
molars because grinding of plants is not required for the diet (Aulerich and Swindler,
1968a; Radke and Chiasson, 1998). The unique buccolingual dimension of the first
upper molar of the mink and other Mustelidae is a characteristic that distinguishes this

subfamily from all other carnivores (Aulerich and Swindler, 1968a).

 

Gingiva:

A complex relationship is necessary to maintain a healthy periodontium that
supports adequate function of teeth (Jones and Boyde, 1999). This includes four
primary components, periodontal ligament, alveolar bone (lamina dura), the gingiva,
and cementum (Bartold et al. 2000). The periodontal ligament supports the tooth, and
through these fibers functioning teeth are linked to each other, the gingiva, and the
alveolar bone (Jones and Boyde, 1999). Normal gingiva covers the alveolar bone and
tooth root to a level just coronal to cementoenamel junction (Bartold et al. 2000). The
gingiva is comprised of two tissue types, epithelial tissue (overlying tissues) and
connective tissues (fibrous underlying tissues) (Bartold et al. 2000). Both tissue types
are responsible for regulation of normal function, repair or regeneration following

injury, and/or protection associated with inflammatory responses (Bartold et al. 2000).

22

h:

Pt

$01

bill

D. Oral Diseases:

Description of oral diseases described in mink are relatively limited. The most
common oral “disease” in mink is the lodging of foreign objects, such as fish or chicken
bones. If sharp fragments penetrate the oral cavity, it causing local abscesses behind the
teeth, under the tongue, or may even penetrate the esophagus, leading to severe
infection (Hunter and Barker, 1996).

Inflammatory responses have been reported as tooth abscesses and gingivitis
affecting older mink. Cellulitis is seen in mink kits with erupting teeth and is associated
with bacteria (Hunter and Barker, 1996). In addition, congenital abnormalities have
been reported for the oral cavity in the mink. These include cleft palate, bradygnathia
(abnormal shortening of the mandible) and prognathia (abnormal protrusion of the
mandible) (Hunter and Barker, 1996). Older ranch mink have been observed with a

heavy plaque load, but no confirmed periodontitis has been reported.

Periodontal Disease:

Periodontitis is a very common chronic inflammatory disease in humans and
some animal species. The manifestation of periodontitis requires the presence of oral
bacterial flora and the host inflammatory response, as well as environmental factors
(Bartold et al. 2000). Studies now suggest that changes in local bacterial density and
species composition may contribute to periodontal diseases, i.e. nearly 500 bacterial
strains have been identified in the subgingival crevice of humans (Kroes et al. 1999).

Calculus build-up results from an accumulation of bacteria on the tooth surfaces

23

 

 

adjacent to the supragingival and subgingival tissues and contributes to the recession of
the gingiva and the initiation of the inflammatory response (Jeffcoat 1999; Bartold et al.
2000). The response then spreads to the tooth socket and deeper into the periodontal
tissues, until the alveolar bone is lost. At this stage the lesion is termed periodontitis.
Destruction of periodontal tissues including periodontal ligament and alveolar bone, is

followed by subsequent tooth loss.

Neoplasia:

Neoplasms are differentiated by morphologic and behavioral characteristics.
Benign tumors are non-cancerous, and are generally localized, slow growing, composed
of cells that are well differentiated, do not invade surrounding tissue, have limited
potential for growth and do not metastasize to distant sites. Criteria for malignant
neoplasia include higher proliferative activity, invasiveness of atypical cell type and
diffuse infiltration, and tissue destruction. Malignant neoplasms tend toward rapid
growth, may contain anaplastic cells, and can metastasize to other organ sites.
Malignant neoplasms tend to be more aggressive and characteristically invade
surrounding tissue, therefore are often more osteolytic than benign neoplasia (Myer

1994; Thompson and Pool, 2002).

Types of Neoplasms:
Various proliferative lesions of the oral cavity (especially in canines) have been

reported that included epulides, malignant melanomas, fibrosarcomas, osteosarcomas,

24

 

 

squamous odontogenic tumor, pseudo-carcinomatous hyperplasia, and squamous cell
carcinoma (SCC) (Berg 1998; Yoshida et al. 1999b).

Epulides are localized tumor-like lesions of periodontal origin, which can be
categorized as both non-neoplastic reactive and neoplastic lesions (Yoshida et al.
1999a). Epulis have also been described as a non-specific, localized growth of the
gingiva (Gardner 1996). Some acanthomatous epulides have exhibited bone invasion
characteristics that resemble SCC (Yoshida et al. 1999b). Based on the literature,
acanthomatous epulides, peripheral ameloblastoma and basal cell carcinoma of the
gingiva are most likely the same lesion (Gardner 1996).

The squamous odontogenic tumor (SOT) is a benign lesion of the periodontium
that arises from the rest of Malassez in the periodontal tissue. Therefore, the SOT is
most likely not from oral squamous epithelium (Tatemoto et al. 1989).

Pseudo-carcinomatous hyperplasia can be caused by an inflammatory process
that originated in the dental lamina in a mandibular osteitis case reported by Penneau et
al. (1980). Pseudoepitheliomatous hyperplasia, may be a similar diagnostic lesion to
pseudo-carcinomatous hyperplasia. Histopathologically, pseudoepitheliomatous
hyperplasia is an inflammatory lesion in juveniles that can become highly proliferative
and assume neoplasm-like characteristics (Bill et al. 2001). Irregular infiltration and
extension of atypical cells into the submucosa is uneven, accompanied by the
development of jagged epidermal cell masses, leading to the development of SCC, and
demonstrate no evidence of metastasis (Bill et al. 2001). Thus, SCC in adolescents can

present itself as pseudoepitheliomatous hyperplasia that initiated as an ulcerative lesion

25

 

 

with no palpable nodules, and radiographic examination revealed erosion of the alveolar
bone (Bill et al. 2001).

Squamous cell carcinoma of the oral cavity in cats and dogs is an aggressive
disease, which frequently invades the periosteum of the underlying bone in up to 77%
of canine cases. Often the early lesions of gingival squamous cell carcinoma are

relatively small nodular masses along the dental arcade. These nodules do not always

 

form fleshy masses, and rarely metastasize to distal sites, often at a rate of less than 3%.
Lymphatic drainage from the gums is more restricted and therefore limits potential for
metastases (Konde 1994; Thompson and Pool, 2002). Frequently, lesions referred to in
the literature may have been reported elsewhere under a different lesion or name, but

truly being the same diagnosis.

 

26

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Environmental Health Perspective, 106(12):775-792.

32

CHAPTER 2

Female Mink (Mustela vison) Fed Diets Containing
3,3’,4,4’,5-Pentachlorobiphenyl (PCB 126).
1: Reproductive Performance and Kit Survivability

33

Female Mink (Mustela vison) Fed Diets Containing
3,3’,4,4’,5-Pentachlorobiphenyl (PCB 126). 1: Reproductive Performance and
Kit Survivability

ABSTRACT

This study was conducted to determine if dietary exposure to 3,3 ’,4,4’,5-
pentachlorobiphenyl (PCB 126) would affect reproductive success of female mink
(Mustela vison). Standard dark, female mink were fed diets containing PCB 126 at
concentrations of 0, 0.24, 2.4, and 24.0 pg PCB 126/kg feed from 21 days prior to
breeding until weaning of their kits at six weeks of age. There were no significant
differences in the number of females that whelped or the average litter size between
dams that were fed the diet containing 0.24 pg PCB 126/kg feed and the control group.
In addition, kit body weights at birth, three weeks and six weeks, and kit survivability
through weaning (six weeks of age) were also similar between these two groups.
Females fed 2.4 and 24.0 pg PCB 126/kg feed had confirmed matings, but failed to
whelp. Histological examination of their uterine horns verified fetal implantation sites,
or placental scars indicating partial embryo development. These results demonstrate that
PCB 126 causes severe reproductive dysfunction in the mink at dietary concentrations

as low as 2.4 pg PCB 126/kg feed.

34

 

INTRODUCTION

Polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDS)
and polychlorinated dibenzofurans (PCDFs) are ubiquitous environmental contaminants
that are resistant to biological and chemical degradation. The toxicity of these
contaminants can give rise to serious and significant effects on animal and human
health. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is recognized as the most toxic
congener of these chemicals (Van den Berg et al. 1998). The structurally similar, non-
ortho substituted 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126) is considered to be the
most toxic PCB congener (Van den Berg et al. 1998). PCB 126 assumes a coplanar
configuration that elicits toxic responses similar to those reported for TCDD, and PCB
126 is known to be prevalent in the environment (Safe 1990; Peterson et al. 1993).

Mink have been shown to be highly sensitive to PCB and TCDD exposure,
which has resulted in disrupted reproductive fimction at relatively low concentrations
(Aulerich and Ringer 1977; Aulerich et al. 1987; Hochstein et a1. 1988). Reproductive
dysfunction has been demonstrated in mink that have been exposed to commercial PCB
mixtures, as well as PCB ‘cocktails’ comprised of selected PCB congeners (Aulerich
and Ringer, 1977; Aulerich 1985; Backlin and Bergman, 1992; Kihlstrom et al. 1992;
Jones et al. 1997). However, exposure to PCB mixtures has precluded the ability to
ascertain specific PCB congeners associated with the observed adverse effects. Mink
reproductive studies assessing the effects of single PCB congeners are limited. Two
ortho-substituted congeners, 2,2’,3,3’,6,6’- hexachlorobiphenyl (PCB 136) and

2,2’,4,4’,5,5’-hexachlorobiphenyl (PCB 153), had no effect on reproduction in exposed

35

mink (Aulerich et al. 1985). Only one non-ortho PCB congener, 3,3’,4,4’5,5’-
hexachlorobiphenyl (PCB 169), has been investigated in regard to its toxic effects on
reproduction in mink (Aulerich et al. 1985, 1987; Patnode and Curtis, 1994). Mink fed
diets containing 0.01 mg PCB 169/kg feed had average litter size and kit survivability
that was similar to controls. However, when mink were fed a diet containing 0.05 mg
PCB 169/kg feed, 50% of the females died prior to whelping.

The toxic equivalency factor (TEF) is an estimate of the toxicity of a compound
relative to TCDD based on individual toxicity reference values (TRVs). These TRVs
are derived from in vivo studies that assess a number of endpoints (body weight loss,
thymic atrophy, and endocrine dysruption) that are suggested to be mediated by the aryl
hydrocarbon receptor (AhR), or from in vitro assays that utilize aryl hydrocarbon
hydrolase [AHH] induction as an endpoint (Safe 1990; Giesy and Kannan 1998; Van
den Berg et al. 1998). The individual TRVs can be highly variable; thus a TEF value for
a particular congener may not accurately reflect the magnitude of its effects on a
specific endpoint in a specific species.

The congener PCB 126 is considered to be a highly potent PCB congener, and
has been assigned a mammalian TEF value of 0.1 (Van den Berg et al. 1998). Yet
limited data are available on its effects in animals, and no information exists on the
effects of this congener on reproductive function in mink. Thus, this study was designed
to assess reproductive function in female mink fed diets containing PCB 126 and

subsequent survivability of their offspring.

36

METHODS

Twenty-eight standard dark, primiparous, female mink were randomly selected
from the Michigan State University (MSU) Experimental Fur Farm herd, weighed, and
assigned to treatment groups so that each group had a similar average body weight.
Siblings were not placed in the same group to minimize sensitivity to treatment
attributable to genetic pre-disposition. All mink were housed individually in wire mesh
breeder cages (61 cm L x 76 cm W x 46 cm H) with a wooden nest boxes (30 cm L x
22.5 cm W x 25 cm H) bedded with aspen shavings that attached to the outside of the
cages. These cages were located within the MSU Experimental Fur Farm containment
facility. Lighting in the room simulated the natural light/dark cycle for the Eastern
Standard Time zone. Mink were started on their designated treatment diets on 05
February 2001, after a one-week acclimation period. Mink were observed daily for the
duration of the study for any signs of toxicity including refiisal to eat, changes in

physical appearance, and behavior alterations.

Diet:

A standard ranch diet (Table 2-1), which was supplemented with 0.24, 2.4, or
24.0 pg PCB 126/kg feed, was used throughout the study. The standard ranch diet
without supplemental PCB 126 was used as the control diet. A PCB 126 premix was
prepared by first dissolving 5000 pg of the congener (Ultra Scientific, North Kingstown
R1; 99% purity) in 2 mL hexane. The appropriate quantity of PCB 126 was then added

to 80 mL corn oil and mixed well. The corn oil containing PCB 126 was then added to

37

1 kg dry mink cereal, followed by thorough mixing. The PCB 126-cereal premix was
added to the remaining ingredients of the standard diet (Table 2-1) and thoroughly
mixed. Samples of each diet were collected for PCB analysis in I-Chem® jars (l-Chem,
New Castle, DE). Additionally, samples of each diet for nutrient analysis were collected
in Whirl-Pak® bags (Nasco, Fort Atkinson, WI). All feed samples were stored in an
ultra-cold freezer (~74°C) until analysis. Diets were then packed in 2-liter plastic
containers and stored in a walk-in freezer (-7°C). As feed was needed, containers were
transferred to a walk-in cooler (4°C) for thawing. Each two-liter container provided a
two-day supply of feed. During the maintenance period and breeding season, all mink

were fed to condition them for optimum reproduction. Water was available ad libitum.

Reproduction:

Breeding of females began during the third week of February, which was
approximately one week earlier than usual for mink housed in open-sided sheds,
because the females showed signs of estrus by pronounced vulvar swelling. All females
were bred according to the MSU Experimental Fur Farm standard operating procedures.
Non-exposed males were used for breeding. Mating was attempted once early in the
21-day breeding period, and then again eight days later. All potential matings were
verified by the presence of normal motile spermatozoa in the postcoital vaginal
aspirations collected immediately after mating. An initial positive mating was followed
by a second breeding attempt on the following day with a different male, as well as
another attempt on the eighth and ninth days afier the first successful mating. Females

not having a verified mating were designated as non-breeders.

38

 

Table 2-1. Composition and nutrient analysis of the basal diet used for the PCB 126

 

 

 

 

 

experiments.

n ==
Feed Composition "/o
Ingredients

Mink cereal a 30.00
Duck by-products b 32.00
Water 21.00
Menhaden fish meal c 7.00
Eggs (spray—dried) d 2.00
Liver (freeze-dried) C 2.00
Corn oil 1.00
Phosphoric acid (85%) f 0.80
Vitamin premix g ' 1.5 %
d-Biotin/kg h 0.07 mg
Larvadex ' 28L 62.0 ml.
Proximate AnalysisJ (on a dry weight basis) %
Moisture 45.65
Fat 24.16
Protein 35.02
Crude fiber 2.79
Ash 9.74
Calcium 2.03
Phosphorus 1 .78
Potassium 0.86
Magnesium 0.19
Sodium 0.65
Total digestible nutrients (T DN) 96.90
Iron 367 ppm
Manganese 47 ppm
Copper 7 ppm
Zinc 96 ppm

 

 

 

a XK-40 mink cereal, XK Mink Foods, Inc., Plymouth. WI
b United Feeds Inc., Plymouth, WI

c Omega Protein, Inc., Hammond, LA

d Van Elderen, Inc., Martin, Ml

c Van Elderen, Inc., Martin, MI

fAlexander Chemical Corp., Kingsbury, IN

8 MSU Vitamin PMX, Akey, Lewisburg, OH

{1 Archer Daniel Midland, Des Moines, [A

f Larvadex ® 25L, Novartis, Greensboro, NC

1 Litchfield Analytical Services, Litchfield, M1

 

 

39

Females were checked daily during the whelping period (mid-April to mid-May)
for evidence of whelping. Gestation length was defined as the number of days from the
last successful mating to parturition, a period that encompasses a variable period of
delayed implantation (diapause) as well as true pregnancy. Livebirths and stillboms
were counted, sexed and weighed on the day of birth. Kits, as well as their dams, were
weighed again at three and six weeks of age. Kits were weaned at six weeks of age

(mid-June) and maintained on their respective treatment diets.

Necropsies:

In May 2001, a sub-group of nine non-whelping females was euthanized using
CO2 to determine reproductive status. These females included two control females, three
females from the 2.4 pg PCB 126/kg feed group, and four females from the 24.0 pg
PCB 126/kg feed group. A gross examination was performed on each dam and
reproductive tissues were collected and fixed in 10% buffered formalin (pH 7.4) for
subsequent histopathological assessment using hematoxylin and eosin-stained sections.
Uteri were grossly and histologically examined for implantation sites, as well as for
signs of early embryonic death or late fetal death. In addition, ovaries were
histologically examined for signs of ovulation and degree of follicular development. All

female mink that died on trial were necropsied as described above.

Statistical Analysis:
Data were analyzed using SAS® software (Statistical Analysis Systems, version

8.0, Cary, NC) by analysis of variance, and least square means of treatment parameters.

40

The mixed model with repeated measures was used, and data were log transformed to
meet the assumption of the mixed model for kit body weights. Residual plots were used
to determine normal probability distribution and to check outlier status. The Tukey’s
method was used for adjustment of p-value in multiple tests. The Chi-square test was
used to analyze kit survival. The level of significance was based on a Type I error rate

of or = 0.05 (p S 0.05).

RESULTS

Reproduction:

The number of females with confirmed matings per group was similar (Table
2-2). The average number of confirmed matings per group ranged from 1.28 to 1.71.
Only females from the control group and the 0.24 pg PCB 126/kg feed group whelped,
also noting that all females with confirmed matings in these two groups whelped (Table
2-2). The length of gestation between the control group and the 0.24 pg PCBlZ6/kg
feed group did not differ significantly (Table 2-2) and ranged from 46 to 55 days and 46
to 59 days for the control and 0.24 pg PCB 126/kg feed groups, respectively.

Of the nine non-whelping females that were examined, one control female, two
females from the 2.4 pg PCB 126/kg feed group and one female from the 24.0 pg PCB
126/kg feed group did not have evidence of placental scars. The remaining females had
implantation sites located throughout the uteri (Figure 2-1), indicating fetal death in the
uterine horns. In addition, ovaries collected from non-whelping females from the 2.4

and 24.0 pg PCB 126/kg feed groups contained corpora lutea in different stages of

41

inv
we]
Ad.

“3:

line

5301

mum
and .
rt‘Sp:
PC B

“til

Clint

mom
anore
3101361

after 4

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involution, indicating active ovaries. Changes in mucosal thickness of uterine horns
were also apparent in females from the 2.4 and 24.0 pg PCB 126/kg feed groups.
Additionally, the presence of hemosiderin-laden macrophages in the mucosal stroma
was noted in one female from the 2.4 pg PCB 126/kg feed group.

There were no significant differences in the total number of kits whelped per
litter (livebirths plus stillboms) between the control and the 0.24 pg PCB 126/kg feed
groups (Table 2-3). Of the whelped kits, 28.6% (control) and 33% (0.24 pg PCB 126
/kg diet) were stillborn or died within the first 24 hours postpartum. The average
number of liveborn kits per whelping female was not significantly different with 3.75
and 4.0 livebirths per female for the control and 0.24 pg PCB 126/kg feed groups,
respectively. Survivability and growth of kits whelped by females exposed to 0.24 pg
PCB 126 /kg feed were not adversely affected when compared to controls through

weaning or at 28 weeks of age (Tables 2-3 and 2-4).

Clinical Signs of Toxicity:

Animals exposed to the two greater concentrations of PCB 126 exhibited
mortality as well as showed clinical signs of toxicity, including hind-quarter paralysis,
anorexia, “wasting syndrome”, poor coat condition, increased shedding with possible
alopecia, eye infections and nasal discharge, bloody and tarry feces. Three females died
afier 42, 47 and 74 days of consuming the diet containing 24.0 pg PCB 126 /kg feed. In

addition, one control female, one female in the 0.24 pg PCB 126 /kg feed group, and

two females in the 24.0 pg PCB 126 /kg feed group died from non-treatment related

42

 

Figure 2-1. Implantation sites that were seen within a uterine horn of a bred female

receiving the diet that contained 24.0 pg PCB 126 /kg feed, that did not whelp.

 

 

* Arrows (4) depict implantation sites at states of resorption.

 

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46

causes. Squamous epithelial hyperplasia of the mandibles and maxillae was observed
grossly in all of the females receiving the 2.4 and 24.0 pg PCB 126 /kg feed diet.

In general, kits whelped to dams in both the control group and the group
receiving 0.24 pg PCB 126 /kg feed appeared normal, and few clinical manifestations
of toxicity were observed. However, one litter in the 0.24 pg PCB 126 /kg feed group
had general signs of unthrifiiness and poor health, with a preliminary diagnosis of
aplastic anemia or red cell aplasia. These are typically considered genetic disorders, and

thus were considered to be non-treatrnent related.

DISCUSSION

The results of the present study demonstrate that PCB 126 adversely affects
reproduction in mink. Dietary exposure to concentrations as low as 2.4 pg PCB 126/kg
feed induced complete reproductive failure in mink. This concentration of PCB 126 is
an order of magnitude greater than environmentally relevant concentrations with the
toxic equivalent (TEQ) concentration being approximately three times greater than
environmental TEQs being previously reported (Heaton et al. 1995; Tillitt et al. 1996;
Bursian et al. 2003). In this study, the lowest observed adverse effect level (LOAEL)
based on reproductive failure induced by PCB 126 was 2.4 pg /kg feed, while the no
observed adverse effect level (NOAEL) was 0.24 pg PCB 126/kg feed.

The toxic equivalent method relates the toxicity of TCDD-like chemicals

(specific PCDD, PCDF and PCB congeners) to the toxicity of TCDD. The TEF

47

constitutes an estimate of the toxicity of a compound relative to TCDD, and applies to
aryl hydrocarbon receptor (Ah-R) mediated responses (Van den Berg et al. 1998). Each
congener is assigned a TEF value, for instance, the TEF of PCB 126 is 0.1 (Van den
Berg et al. 1998). Toxic equivalents are derived by multiplying the TEF of a particular
congener by the concentration of the chemical in the sample of interest (Safe 1990; Van
den Berg et al. 1998). In a sample containing a number of different PCDD, PCDF and
PCB congeners, the total TCDD-like activity can be calculated by summing the TEQs
contributed by each congener.

The TEQ concentration of the 0.24 pg PCB 126/kg feed diet was 24.0 ng
TEQ/kg feed, while the dietary TEQs at the LOAEL was 240.0 ng TEQs/kg. Heaton et
al. (1995) and Tillitt et al. (1996) reported that diets containing 10, 20, and 40 %
Saginaw River fish contained 19.4, 40.0, and 80.8 ng/kg dietary TEQs, respectively. If
the currently accepted TEF values reported by Van den Berg et al. (1998) are used for
the above mentioned study, the corrected TEQ concentrations are 22.2, 43.0, and 85.0
ng/kg feed TEQs for the 10, 20, and 40 % Saginaw River fish, respectively (Heaton et
al. 1995; Tillitt et al. 1996; Van den Berg et al. 1998).

In the present study, the estimated total PCB 126 consumption for an average
54-day exposure period was 19.1 pg PCB 126/kg BW (2.4 pg PCB 126/kg diet), and
232.6 pg PCB 126/kg BW (24.0 pg PCB 126/kg dietary exposure). Females that
received the diet containing 0.24 pg PCB 126/kg feed had an average total PCB 126
consumption of 4.47 pg PCB 126/kg BW during their 133 days of exposure for dams

rearing litters through weaning.

48

 

The TEQ concentration of the 0.24 pg PCB 126/kg feed diet for the NOAEL
was 24.0 ng /kg feed. The LOAEL (2.4 pg PCB 126/kg feed) TEQ concentration was
240.0 ng/kg diet at which concentration complete reproductive failure was observed. In
the Heaton et al. (1995)/Tillitt et al. (1996) study, the LOAEL, based on kit
survivability, was the lowest PCB concentration fed (0.72 mg total PCB/kg diet) or 22.2
ng TEQs/kg feed using the TEF values presented in Van den Berg et al. (1998). Thus,
the NOAEL TEQ concentration in the present study was equivalent to the LOAEL TEQ
concentration in the Heaton et al. ( 1995)/1' illitt et al. (1996) study. It is of interest to
point out that kit survivability at 24 ng TEQs/kg feed in the present study is quite
comparable to kit survivability at 22.2 ng TEQs/kg feed in the Heaton et al.
(1995)/Tillitt et al. (1996) study, but the relatively poor whelping and survivability in
the control animals in the present study preclude statistical significance.

Other parameters of reproductive performance that were measured were
comparable between the current study and Heaton et al. (1995) for the two exposure
groups (24.0 ng TEQs/kg feed and 22.2 ng TEQs/kg feed, respectively). In fact, Heaton
et al. (1995) reported a higher number of livebirths per litter, with 3.8 livebirths, as
compared to 2.8 livebirths observed in the present study. Average litter size was smaller
in the present study with an average of 4.0 kits per litter as compared to 5.3 kits per
litter reported by Heaton et al. (1995), as well as birth weight (8.16 g) was lower in the
present study as compared to 9.76 g from Heaton et al. (1995).

Litter size indicates the capability of the dam to carry fetuses to parturition. The
number of kits whelped per litter was recorded within the first 24 hours of whelping.

The number of living kits per mated female (3.75 and 4.00 kits for the control and 0.24

49

pg PCB 126/kg feed groups, respectively) was not influenced by exposure to 0.24 pg
PCB 126 in the feed. Net natality, or the number of young weaned per female, was not
significantly different between the control group at 2.25 and the mink exposed to 0.24
pg PCB 126/kg feed which weaned 2.83 kits per female.

Aulerich et al. (1985, 1987) reported 50% mortality in female mink following 88
days of dietary exposure to 0.05 mg PCB 169/kg feed, thus reproductive function could
not be assessed. The TEF value of PCB 169 is 0.01 (Van den Berg et al. 1998).
Therefore, the TEQ concentration at which 50% mortality in mink was reported was
500 ng TEQs/kg feed. The ortho-substituted congener PCB 136 had no reproductive
effects at a dietary concentration of 2.5 mg PCB 136/kg feed (Aulerich et al. 1985).

Kit body weights reported by Heaton et al. (1995) were substantially greater,
and kit productivity was higher in all groups except the “40% carp” group as compared
to the present study. This may be explained by the greater than average length in
gestation that was reported by Heaton et al. (1995) (64.5, 58.7, 55.2, and 58.7 days for
control, 22.2, 43.0, and 85.0 ng TEQs/kg feed groups, respectively), which allowed the
kits to develop to a greater extent prior to parturition. The average reported gestation
length for standard dark, female mink in the MSU Fur Farm herd during the same time
period, as the present study was 49.1 days. For single-mated dark mink, the average
gestation period reported is 51.2 days (Bowness 1968). In the study by Heaton et al.
(1995), the overall average length in gestation was 59.3 days.

Several studies have indicated that exposure to commercial PCB mixtures,
environmentally-derived PCBs, and selected PCB congeners cause adverse reproductive

impairment in mink although apparently ovulation, fertilization and implantation are

50

apparently not disrupted (Aulerich and Ringer, 1977; Aulerich et al. 1985; Backlin and
Bergman, 1992; Jones et a1. 1997). Mink are among the most sensitive species to the
PCB congeners that are chemically similar to TCDD and mink are naturally exposed to
many of these compounds in the environment via their diet (Aulerich et a1 1985). The
effects of TCDD exposure in mink have been investigated, but the effects on
reproduction could not be clearly determined (Hochstein et al. 2001).

It is evident, based on histological examination of reproductive tracts, that PCB
126 at 2.4 and 24.0 pg PCB 126/kg feed did not prevent ovulation, fertilization and
implantation in mated female mink. Females from both of these dietary groups had
implantation sites located throughout both uterine horns, and ovaries were active and
producing follicles. The implantation sites examined appeared as dark colored bands,
also termed placental scars. These pigmented areas of uterine tissue are marking sites of
previous placental attachment, which occur in species with deciduous placentae,
including the family Mustelidae. These scars are interpreted as signs of resorption of
conceptus, or early fetal stage death, and are commonly used indices that provide
accurate determination and confirmation of pregnancy that can also be used to
approximate litter size (Harder and Kirkpatrick, 1994).

The reproductive disruption induced by PCB 126 at the two higher doses (2.4
and 24.0 pg PCB 126/kg feed) may have been caused by PCB-mediated action on
female reproductive hormones, or altered endocrine cycling as was observed in Backlin
et al. (1997). Post-implantation embryo mortality may occur in female mink as a result
of interference with hormones required for maintenance of pregnancy (Backlin et al.

1997). Earlier studies do indicate hormonal involvement as a mechanism by which PCB

51

exposure induces fetal death (Backlin et al 1997). The effects of PCBs and dioxins on
the endocrine system are pronounced (Bimbaum 1994), including the reproductive
dysfirnction observed in mink exposed to PCBs. The endocrine system plays an
essential role in the preparation and maintenance of the reproductive system for
pregnancy. Estrogen is necessary for normal uterine development and maintenance, as
well as for the continuation of pregnancy (Peterson et al. 1993). TCDD exposure caused
a decrease in plasma concentrations of estrogen and progesterone in rhesus monkeys
(Barsotti et a1. 1979). Results from chronic dietary exposure to TCDD suggested
suppression or inhibition of the estrous cycle in rats (Kociba et a1. 1976). Impairment of
progesterone receptor function has been observed in uteri of pregnant mink after a
single dose of the coplanar PCB 169 (Patnode and Curtis, 1994; Backlin et al.1997).
Clinical signs of the animals that died on-test included wasting syndrome,
adipose depletion, alopecia, ascites, and bloody and tarry feces. These signs were seen
throughout all groups of adult females receiving PCB 126. The female mink receiving
24.0 pg PCB 126/kg feed also had notably thickened, deformed, and elongated toenails,
which compares with other studies exposing mink to TCDD and PCB 126 (Hochstein et
al. 1998; Render et al. 2000a, 2000b; Beckett et al. 2002). Clinical signs will be
discussed in detail in Chapter 3. A lesion, initially identified as squamous epithelial
proliferation of the mandibles and maxillae, was observed clinically in all of the females
receiving the 2.4 and 24.0 pg PCB 126 /kg feed diet and will be discussed in detail in
Chapter 4. One female that died on-test from the control group became agalactic, as
well as had uroliths, two factors that contributed to the decrease in growth and survival

of her five kits.

52

CONCLUSIONS

In conclusion, dietary administration of PCB 126 produced complete
reproductive failure in mink at concentrations of 2.4 and 24.0 pg PCB 126/kg feed.
Ovulation, fertilization and implantation in mated female mink did not appear to be
altered by PCB 126 exposure. Females from both of these dietary groups had placental
scars where implantations had failed. These placental scars were located throughout
both uterine horns, and ovaries were active in these females. Based on the results from
the current study, PCB 126 is highly toxic to mink, and caused severe impairment of
reproductive function. Therefore, potential concentrations of PCB 126 in the
environment may be high enough in certain areas to cause adverse effects in wild mink

and other wildlife populations.

53

REFERENCES

Aulerich, R]. and R.K. Ringer 1977. Current status of PCB toxicity to mink, and effect
on their reproduction. Arch. Environ Contam T oxicol, 6:279-292.

Aulerich, R.J., SJ. Bursian, WJ. Breslin, B.A. Olson and R.K. Ringer. 1985.
Toxicological manifestations of 2,4,5,2’,4’,5’-, 2,3,6,2’,3’,6’-, 3,4,5,3’,4’,5’-,
hexachlorobiphenyl and Aroclor 1254 in mink. J T oxicol and Environ] Health, 15:63-
79.

Aulerich, R.J., SJ. Bursian, M.G. Evans, J.R. Hochstein, K.A. Koudele, B.A. Olson and
AC. Napolitano. 1987. Toxicity of 3,4,5,3’,4’,5’-hexachlorobiphenyl to mink. Arch
Environ Contam T oxicol, 16:53-60.

Backlin, B.M. and A. Bergman. 1992. Morphological aspects on the reproductive
organs in female mink (Mustela vison) exposed to polychlorinated biphenyls and
fractions thereof. AMBIO, 21(8):596-601.

Backlin, B.M., A. Madej and M. Forsberg. 1997. Histology of ovaries and uteri and
levels of plasma progesterone, oestradiol-17B and oestrone sulphate during the
implantation period in mated and gonadotrophin-releasing horrnone-treated mink
(Mustela vison) exposed to polychlorinated biphenyls. J Applied Toxicol, 17(5):297-
306.

Barsotti, D.A., L.H. Abrahamson and J .R. Allen. 1979. Hormonal alterations in female
rhesus monkeys fed a diet containing 2,3,7,8-tetrachlorodibenzo-p-dioxin. Bull Environ

Contam T oxicol, 21:463-469

Beckett, K.J., B. Yamini, RJ. Aulerich and SJ. Bursian. 2002. PCB 126 induces
mandibular and maxillary pseudocarcinomatous hyperplasia and reproductive
dysfunction in mink (Mustela vison). Proceedings of 23rd Annual Society of
Environmental Toxicology and Chemistry (SETAC) Conference, Salt Lake City, UT.

Bimbaum, LS. 1994. Endocrine effects of prenatal exposure to PCBS, dioxins, and
other xenobiotics: implications for policy and research. Environ Health Perspect,
102:676-679.

Bowness, ER. 1968. The variable pregnancy period in mink. Fur Trade J Can,
46(1):4-7.

Giesy, J .P. and K. Kannan. 1998. Dioxin-like and non-dioxin-like toxic effects of

polychlorinated biphenyls (PCBs): implications for risk assessments. Critical Reviews
in T oxicol, 28(6):511-569.

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Harder, J .D. and R.L. Kirkpatrick. 1994. Chapter 11: Physiological Methods in Wildlife
Research. Q2: Research and Management Techniques for Wildlife and Habitats, (Fifth
Edition). T.A. Bookhout (Ed.), Allen Press, Inc., Lawrence, KS.

Heaton, S.N., SJ. Bursian, J.P. Giesy, D.E. Tillitt, J.A. Render, P.D. Jones, D.A.
Verbrugge, T.J. Kubiak and R.J. Aulerich. 1995. Dietary exposure of mink to carp from
Saginaw Bay, Michigan. 1. Effects on reproduction and survival, and the potential risks
to wild mink populations. Arch Environ Contam T oxicol, 28:334-343.

Hochstein, J .R., R.J. Aulerich and SJ. Bursian. 1988. Acute toxicity of 2,3,7,8-
tetrachlorodibenzo-p-dioxin to mink. Arch Environ Contam T oxicol, 17:33-37.

Hochstein, J .R., SJ . Bursian, and R.J. Aulerich. 1998. Effects of dietary exposure to
2,3,7,8-tetrachlorodibenzo-p-dioxin in adult female mink (Mustela vison). Arch Environ
Contam T oxicol, 35:348-353.

Hochstein, J.R., J .A. Render, SJ. Bursian and R.J. Aulerich. 2001. Chronic toxicity of
dietary of 2,3,7,8-tetrachlorodibenzo-p-dioxin to mink. Veterinary and Human T oxicol,
43(3): 134-139.

Jones, J .P., B.M. Backlin, R.W. Stoddart and V. Dantzer. 1997. Environmental
pollutants as aetiological agents in female reproductive pathology: placental glycan
expression in normal and polychlorinated biphenyl (PCB)-exposed mink (Mustela
vison). Placenta, 18:689-699.

Kihlstrom, J E M. Olsson, S. Jensen, A. Johansson, J. Ahlbom and A. Bergman. 1992.
Effects of PCB and different fractions of PCB on the reproduction of the mink (Mustela
vison). AMBIO, 21(8):563-569.

Kociba, R.J., P.A. Keeler, C.N. Park and PJ. Gehring. 1976. 2,3,7,8-
Tetrachlorodibenzo-p-dioxin (TCDD): results of a 13-week oral toxicity study in rats.
T oxicol Appl Pharmacol, 35:553-574.

Patnode, K.A. and LA. Curtis. 1994. 2,2’,4,4’,5,5’-Hexachlorobiphenyl alteration of
uterine progesterone and estrogen receptors coincides with embryotoxicity in mink
(Mustela vison). T oxicol Appl Pharmacol, 127:9-18.

Peterson, R.E., H.M. Theobald and G.L. Kimmel. 1993. Developmental and
reproductive toxicity of dioxins and related compounds: cross-species comparisons.
Critical Reviews Toxicology, 23(3):283-335.

Render, J .A., J .R. Hochstein, R.J. Aulerich and SJ. Bursian. 2000a. Proliferation of

periodontal squamous epithelium in mink fed 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD). Veterinary and Human Toxicology, 42(2):85-86.

55

 

Render, J.A., R.J. Aulerich, SJ. Bursian and RF. Nachreiner. 2000b. Proliferation of
maxillary and mandibular periodontal squamous cells in mink fed 3,3’,4,4’,5-
pentachlorobiphenyl (PCB 126). J Vet Diagn Invest, 12:477-479.

Safe, S. 1990. Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs),
dibenzofurans (PCDFs), and related compounds: environmental and mechanistic
considerations which support the development of toxic equivalency factors (TEFS).
CRC Crit Rev T oxicol, 21:51-88.

Tillitt, D.E., R.W. Gale, J.C. Meadows, IL. Zajicek, P.H. Peterman, S.N. Heaton, P.D.
Jones, SJ. Bursian, T.J. Kubiak, J .P. Giesy and R.J. Aulerich. 1996. Dietary exposure
of mink to carp from Saginaw Bay. 3: Characterization of dietary exposure to planar
halogenated hydrocarbons, dioxin equivalents, and biomagnification. Environ Sci

T echnol, 30:283-291.

Van den Berg, M., L. Bimbaum, A.T.C. Bosveld, B. Brunstrom, P. Cook, M. Feeley,
J.P. Giesy, A. Hanberg, R. Hasegawa, S.W. Kennedy, T. Kubiak, J .C. Larsen, F.X.R.
van Leeuwen, A.K.D. Liem, C. Nolt, R.E. Peterson, L. Poellinger, S. Safe, D. Schrenk,
D. Tillitt, M. Tysklind, M. Younes, F. Waern and T. Zacharewski. 1998. Toxic
equivalency factors (TEFS) for PCBS, PCDDS, PCDFs, for humans and wildlife.
Environmental Health Perspective, 106(12):775-792.

56

 

 

CHAPTER 3

Adult Female and Juvenile Mink (Mustela vison) Fed Diets
Containing 3,3’,4,4’,5-Pentachlorobiphenyl (PCB 126).
II: Clinical, Hematological and Serum Chemistry Parameters

57

 

Adult Female and Juvenile Mink (Mustela vison) Fed Diets Containing
3,3’,4,4’,5-Pentachlorobiphenyl (PCB 126). II: Clinical, Hematological and
Serum Chemistry Parameters

ABSTRACT

Polychlorinated biphenyls (PCBS) are ubiquitous environmental contaminants,
and wildlife species are potentially exposed to these contaminants through their prey
items. In addition, PCBs and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are known to
cause adverse effects in many species. The purpose of the current study was to
determine the biological effects, as assessed by clinical, hematological, and serum
chemistry parameters, in adult female and seven-month-old mink (Mustela vison)

caused by the dietary consumption of 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126).
Standard dark, female mink were fed diets containing PCB 126 at concentrations of 0,
0.24, 2.4, and 24.0 pg PCB 126/kg feed three weeks prior to breeding and continuing
post-weaning of kits. Kits whelped to females fed diets containing PCB 126 were
continuously exposed in utero and through lactation via the dam, and then by
consuming the diet containing the same concentration of PCB 126 as their dam. Clinical
signs induced by PCB 126 exposure in the females included bloody and black tarry
feces, poor coat condition, eye infections, nasal discharge and wasting syndrome.
Excessive toenail growth was observed in the front feet of the adult females. There was
a significant decrease in mean absolute heart weight in the adult females fed diets
containing 24.0 pg PCB 126/kg feed. Mean liver weight was significantly increased in
the adult females receiving 24.0 pg PCB 126/kg feed as compared to controls.

Significant decreases in hematological parameters were observed as RBC, HGB, HCT

58

and eosinophils in adult females. Increases in neutrophils, WBC and MCV were
observed in dams fed PCB 126. Juvenile mink fed diets containing PCB 126 exhibited
significant decreases in RBC, HGB and HCT. Serum chemistry parameters that were
significantly decreased in dams exposed to PCB 126 included albumin, calcium, total
protein, and TCO2 concentrations, and anion gap. A substantial trend in reduced
cholesterol concentrations was observed in a dose-related manner in adult mink,
although not statistically significant. Juvenile mink fed diets containing PCB 126
expressed significant decreases in alkaline phosphatase activity, and cholesterol and
glucose concentrations. Potassium concentrations were significantly increased in
juvenile mink exposed to PCB 126 via their diet. This study correlated clinical,
hematological and serum chemistry parameters to evaluate the adverse effects of PCB
126 exposure in mink. Results indicate that PCB 126 induced adverse effects in clinical,

hematological and serum chemistry parameters in mink.

INTRODUCTION

Polychlorinated aromatic chemicals like polychlorinated biphenyls (PCBS),
polychlorinated dibenzodioxins (PCDDS) and polychlorinated dibenzofurans (PCDFS)
are ubiquitous contaminants in the environment. These contaminants are resistant to
biological and chemical degradation, and their toxicity and bioaccumulation give rise to
serious and significant impacts on wildlife and human health. Polychlorinated aromatic

chemicals are highly lipid soluble and partition into fatty tissues of the animals that

59

consume them, with the assumption that these fat stores or bioaccumulated reserves are
passed to the next highest level in the food web (Borlakoqlu and Haegele, 1991).

The individual congeners that comprise this group of polychlorinated aromatic
chemicals can differ markedly in relation to their toxicity to a Species, as well as the
response(s) elicited from specific species. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)
is recognized as the most potent congener of this class of chemicals. The structurally
similar 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126), the PCB congener that has
stereochemical and toxicological properties most similar to those of TCDD, is
considered to be the most toxic PCB congener. It exhibits “TCDD-like” activities, and
is prevalent in the environment (Safe 1990; Erickson 1997).

Mink are naturally exposed to many of these compounds in the environment via
their diet. Being at the peak of the food web, a wide variety of prey items that mink
incorporate as part of their diet have been exposed to environmental contaminants, and
thus, mink are exposed to potentially bioaccumulated contamination through dietary
sources. Mink are among the most sensitive mammalian Species known to chemical
compounds such as PCBs and TCDD (Aulerich and Ringer, 1977; Aulerich et al. 1987;
Hochstein et al. 1988). Thus, mink are an important model species that can be used as a
biological indicator of environmental contaminant exposure and ecosystem health.

Polychlorinated biphenyls are known to adversely affect basic biological
functions and physiological homeostasis. A plethora of biological effects can occur in
association with ingestion and uptake of PCBS, including “wasting syndrome”,
chloracne, edema, hepatic hypertrophy, porphyria, estrogenic activity,

immunosuppression, and neOplaSia (Bleavins et al. 1983; Aulerich et al. 1987; Safe

60

1990; Silberhorn et al. 1990; Hochstein et al. 2001). Biochemical, hematological, and
morphological markers may help to characterize and assess exposure to PCBS, as well
as to assess the health condition of the animal. In a chronic toxicity study, Hochstein et
al. (2001) reported significant changes in hematology and biochemical parameters in
mink fed diets containing 0.04 to 5.0 pg TCDD /kg feed. Edqvist et al. (1992) reported
changes in biochemical blood parameters in mink as a result of exposure to commercial
PCBs and individual or combinations of individual PCB congeners. The purpose of the
current study was to determine the biological effects, as assessed by clinical,
hematological, and serum chemistry parameters, in adult female and seven-month-old

mink caused by the dietary consumption of PCB 126.

METHODS

This study was conducted at the Michigan State University (MSU) Experimental
Fur Farm from February, 2001 through December, 2001. The methods used in this
study are presented in detail in Chapter 2. However, they will be briefly described
below.

Twenty-eight, standard dark, breeding female mink were randomly selected and
assigned to one of four dietary treatments. All mink were housed individually at the
MSU Experimental Fur F arm containment facility. Mink were started on their
designated treatment diets on 05 February 2001, after a one-week acclimation period

and 21 days prior to breeding. A standard ranch diet (Table 2-1) was used as the control

61

diet. For the treatment diets, the standard diet was supplemented with 0.24, 2.4, or 24.0
pg PCB126/kg feed. Water was available ad libitum. Mink were observed daily for the
duration of the study for any Signs of toxicity including, refusal or inability to eat
(aphagia), changes in physical appearance, and behavior alterations (Table 3-1). The
kits that were evaluated in this study were offspring that were whelped from dams
exposed to 0.24 pg PCB 126/ kg feed (Chapter 2). No other females in the study were

successful in whelping any kits.

Blood Collection:

Adult female mink were anesthetized with a 10:1 mixture of ketamine
hydrochloride (25mg/mL; Ketaset®, Fort Dodge Animal Health, Fort Dodge, IA) and
Xylazine-20 (20 mg/mL; Butler®, Ben Venue Laboratories, Inc., Bedford, OH) at 0.4
mL/kg BW that was administered via intramuscular injection. Approximately 5 mL of
blood was collected from each mink via cardiac puncture using a sterile 5 mL syringe
with an 18 gauge needle. Approximately 2 mL of blood were put in a clot tube for
serum chemistry, and 2.5 mL of blood was put in an ethylenediaminetetraacidic acid
(EDTA)-coated tube for hematology. Following collection, the blood samples were
submitted to the MSU Clinical Pathology Laboratory for analyses.

Hematology parameters analyzed for each sample included red blood cell (RBC)
count, white blood cell (WBC) count, hemoglobin (HGB) concentration, hematocrit
(HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration
(MCHC), red cell distribution width (RDW), platelet volume (PLT) and leukocyte

differential cell counts (neutrophils, lymphocytes, eosinophils, basophils, and

62

monocytes). Blood samples were analyzed using an Advia Model 120 analyzer (Bayer
Corporation, Tarrytown, NY).

Serum biochemical analyses included concentrations of calcium (Ca), chloride
(Cl), cholesterol (CHOL), creatinine (CREAT), albumin (ALB), total protein, total
carbon dioxide (TCOz), pOtassium (K), sodium (Na), glucose (GLUC), magnesium
(Mg), serum iron (Fe), blood urea nitrogen (BUN), globulin (GLOB), anion gap (AG),
and osmolality. Serum enzyme analyses included measurements of aspartate
aminotransferase (AST), amylase (AMYL), alanine aminotransferase (ALT), alkaline
phosphatase (ALK PHOS), creatinine kinase (CK), and sorbitol dehydrogenase (SDH)
activities. Serum biochemical analyses were performed on an Abbott Spectrum Series 11

Analyzer (Abbott Laboratories, Irving, TX).

Necropsies:

Following blood collection, the adult females were euthanized using CO2. A
gross examination was performed prior to collecting tissues. Organs (adrenal glands,
brain, heart, kidneys, liver, reproductive tract, spleen, and thyroids) were examined and
weighed. A section of each tissue was then fixed in 10% buffered formalin (pH 7.4).
Tissues were trimmed, embedded in paraffin, and then sectioned to 5 pm. Following
this preparation, tissues were mounted on microscopic Slides, and stained with

hematoxylin and eosin for subsequent routine histopathological assessment.

63

Statistical Analysis:

Data were analyzed using SAS® software (Statistical Analysis Systems, version
8.0, Cary, NC) by one-way analysis of variance and least square means of treatment
parameters. The mixed model procedure was used and the normality assumption was
tested accordingly. Data were log transformed to meet the assumption of the mixed
model. Residual plots were used to determine normal probability distribution and to
check outlier status. The level of significance was based on a Type I error rate of 0.05
(p S 0.05). The Tukey’s method was used for adjustment of p-value in multiple tests.
Organ weights for the mink are presented as absolute weights (mean 3: SE of the mean).
No thyroid glands were collected from the adult females receiving 24.0 pg PCB 126/kg
feed. Statistics for all parameter comparisons involving male and female kits were run
in a sex by treatment comparison to determine any significant differences. If no
differences were detected, males and females were grouped together by treatment,

otherwise they are reported separately.

RESULTS

Mortality:

Three females on the diet containing 24.0 pg PCB 126 /kg feed died after 42,
47, and 74 days of being exposed to the PCB 126 diet, resulting in 29% mortality. One
of these mink (24.0 pg PCB 126 /kg feed, 47-d) was euthanized due to hind-quarter
paralysis in addition to decreased BW, it was also noted that the skull bones were

brittle, and the front nails were splitting and also brittle. In addition, one control female

64

 

Table 3-1. Chart of daily observations for signs of toxicity in mink (Mustela vison) fed

PCB 126 to monitor general health and physical condition.

 

Changes in Physical Condition:
Weight changes

Coat condition
Stool characteristics
Urination check

Injury

Level of Activig:

Increase
Normal
Decrease

Behavioral Alterations:
Alertness
‘Increased’ aggression
‘lncreased’ reclusion
‘Increased/decreased’ fearfulness
Self-mutilation (fur and/or tail chewing)
Feed wastage
General uncleanliness

 

65

(264 days), one female from the 2.4 pg PCB 126 /kg feed group (128 days), and one
female in the 24.0 pg PCB 126 /kg feed group (109 days) died from complications of
uroliths in the kidney, which was considered non-treatment related.
Clinical Signs of Toxicity:

Clinical signs observed throughout all dietary groups receiving PCB 126 bloody
and tarry feces, poor coat condition, eye infections and nasal discharge. Shedding was

increased in PCB-126 exposed females as compared to control females. Noticeable

 

shedding was first observed in the group of females receiving the 24.0 pg PCB 126 /kg
feed diet. The patterns of shedding were not correlated with natural patterns of a
seasonal molt.

Mink exposed to the two greater concentrations of PCB 126 showed signs of
toxicity that included ascites (one mink, 24.0 pg PCB. 126 /kg), anorexia and “wasting
syndrome”, resulting in depleted adipose tissue. Squamous epithelial proliferation of the
mandibles and maxillae was clinically observed in all of the females receiving the 2.4
and 24.0 pg PCB 126 /kg feed diet.

As stated in Chapter 2, one litter whelped to a dam receiving the 0.24 pg PCB
126 /kg diet showed general signs of unthriftiness and poor health, with a preliminary

diagnosis of aplastic anemia or possibly red cell aplasia. These are typically considered

genetic disorders, and thus could be considered non-treatment related.

66

Toenails:

The adult female mink on PCB 126 diets had notably thickened, deformed, and
elongated toenails (Figure 3-1). Increased nail growth was first noted in animals
receiving the diet containing 24.0 pg PCB 126/kg feed within approximately five weeks
of exposure. Nail growth was continuous and became pronounced in most mink in the
two higher PCB 126 treatment groups after approximately 12 to 16 weeks of dietary
exposure. Not all mink showed the same rate of growth, and growth rate was not
measured in this study. Only the front nails had significant growth, as no unusual
growth was seen in the toenails of the hind feet in any of the animals. Minimal changes

in nail growth were noted in one juvenile male mink:

Necropsy Data:

The observed decrease in body weight (32%) of adult females exposed to 24.0
pg PCB 126/kg feed compared to controls was not significant (Table 3-2); although,
this was considered a biologically significant decrease in body weight.

Heart weights were significantly decreased (24.3%) in dams in the 24.0 pg PCB
126 /kg feed group as compared to the control group (Table 3-2). Mean liver weight of
the adult females in the 2.4 pg PCB 126 /kg feed group was significantly increased
compared to the control group. No other significant changes in mean organ (adrenal
glands, brain, kidneys, spleen, or thyroid glands) weights were observed in adult
females. There were no significant differences in any organ weights for juvenile mink in

this study (Table 3-3).

67

 

Figure 3-1. Increased toenail growth induced by dietary exposure to 24.0 pg PCB
126/kg feed in female mink 16 weeks into the trial was characterized by thickening,
elongation, and deformity of the nails in the front feet. Nail growth in PCB 126-exposed
mink was considered moderate and continued throughout the trial period, including the

period while mink were on clean feed post-PCB 126 exposure.

 

 

 

68

Hematology:

Adult female mink fed diets containing PCB 126 had significant changes in
hematological parameters (Table 3-4). Dams receiving the diet containing 24.0 pg PCB
126 /kg feed had significant decreases in RBC, HGB and HCT. Additionally, dams in
this same dose group had significant changes in leukocyte differential counts. A
significant increase in neutrophils, and a significant decrease in eosinophils and
lymphocytes was observed in these adult females as compared to all other treatment
groups. In addition to these changes, there were significant increases in WBC count and
MCV in dams fed the 24.0 pg PCB 126 /kg diet. Although there was a trend-increase
observed in platelet counts in the PCB 126-exposed adult females, these changes were
not statistically significant. Juvenile mink in the PCB 126 treatment group (0.24 pg
PCB 126 /kg feed) had significant decreases in RBC, HGB and HCT as compared to the

control group (Table 3-6).

Serum Chemistries:

Several of the serum chemistry parameters in dams were significantly affected
following exposure to PCB 126 (Table 3-5). A Significant decrease in ALB was
observed in the mink fed the diet containing 24.0 pg PCB 126 /kg feed compared to the
other groups. Calcium and AG were significantly decreased in the 24.0 pg PCB 126 /kg
group as compared to the 0.24 and 2.4 pg PCB 126 /kg feed group for Ca, and to the
0.24 pg PCB 126 /kg feed group for anion gap. Total CO2 was significantly decreased
in the 24.0 pg PCB 126 /kg group compared to the 2.4 pg PCB 126 /kg feed group, and

total protein was significantly decreased in the 24.0 pg PCB 126 /kg group when

69

compared to the control group. Serum CHOL in the adult female mink was substantially
reduced in a dose-related manner in all groups exposed to PCB 126, but it was not
statistically significant (Table 3-5).

In the juvenile mink that consumed 0.24 pg PCB 126/kg feed, there were
significant decreases in alkaline phosphatase activity, as well as CHOL and glucose
concentrations as compared to the control group (Table 3-7). Potassium concentration
was Significantly increased in the juvenile mink exposed to 0.24 pg PCB 126 compared

to control mink.

DISCUSSION

3,3’4,4’5-Pentachlorobiphenyl (PCB 126) is considered to be the most toxic of
the PCB congeners. The results from this study and the previous reproductive study
(Chapter 2) demonstrate the highly toxic potency of this PCB congener to mink. This
supports previous reports that mink are among the most sensitive mammalian species to
PCB toxicity. In the current study, mortality was reported in the treatment group
receiving 24.0 pg PCB 126/kg feed, in which three females died during the course of
the trial resulting in 29% mortality. However, based on the reported jaw lesion in this
study (to be discussed in Chapter 4), there would have been 100% mortality if animals
were allowed to continue on treatment. Hochstein et al. (2001) reported 16.7% mortality

in mink exposed to a diet containing 5.0 pg TCDD/kg feed during a 125 day feeding

70

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76

trial. This equates to 5.0 pg TEQ/kg feed since TCDD is used as the standard chemical
to which all other TCDD-like PCB congeners are compared, and thus, has a toxic
equivalency factor of 1.0 (Van den Berg et al. 1998). Aulerich et al. (1987) reported
50% mortality in mink exposed to PCB 169 for 135 days at a concentration of 50 pg/kg
feed, whereas no deaths occurred at 10 pg PCB 169/kg feed in the same study. Since
PCB 169 has a TEF value of 0.01 , mortality occurred at a concentration of 5.0 pg
TEQ/kg feed, compared to the current study in which mink were exposed to 2.4 pg

TEQ/kg feed via the diet containing 24.0 pg PCB 126/kg feed.

Clinical Signs of Toxicity:

The clinical signs induced by PCB 126 were similar to signs previously reported
for PCB toxicity in mink, such as “wasting syndrome” and disrupted molting patterns.
Mink exposed to the two greater concentrations of PCB 126 showed signs of “wasting
syndrome”, a PCB-induced toxicity that has been well documented (Aulerich et al.
1987; Hochstein et al. 2001). Aphagia, or the inability to eat, appeared to be associated
with the jaw lesion, described as squamous epithelial proliferation in the mandibles and
maxillae (Chapter 4). This condition was followed by clinical detection of
periodontoclasia first observed in mink in the highest dose group. This condition
hindered feed consumption because the animals could not chew the food.

Animals in all PCB 126 dietary treatments expressed anorexia, melena, poor
coat condition, increased shedding (inconsistent with natural patterns and timing of a
seasonal molt), eye infections, and nasal discharge. The latter two conditions are

suggestive of chronic low-grade infection, possibly resulting from immunosuppression

77

as suggested by the changes in the leukocyte differentials described below. The immune
system is a target for PCB toxicity, and PCBs have been reported to cause

immunosuppression (Bleavins et al. 1983; Safe 1990; Faustman and Omenn, 2001).

Nail Growth Abnormalities:

Dietary exposure to PCB 126 resulted in orthokeratotic hyperkeratosis of the
toenails of mink. Clinical changes of the nails, including marked thickening, elongation,
and deformity, were observed during daily monitoring. Growth appeared to vary
between individuals, but was continuous. Only the front nails had abnormal growth.
The nails of two mink were so long that they hindered the animals’ ability to climb
around the cage because the nails would get hooked on the wires. The growth
characteristics (marked thickening, elongation, and deformity) of the toenails are
comparable with results from other studies where mink were exposed to TCDD or PCB
126 (Hochstein et al. 1998, 2001; Render et al. 2000a, 2000b; Beckett et al. 2002).

Similar findings of abnormal nail grth in mink and European ferrets (Mustela
putoriusfuro) exposed to PCBs have been reported in other studies. Mink exposed to
3,3’,4,4’,5,5’-hexachlorobiphenyl (PCB 169) at a dietary concentration of 50 pg/kg for
135 days, displayed pronounced nail growth, in addition to delayed molting patterns and
altered guard hair growth and/or retention of underfur (Aulerich et al. 1987). Bleavins et
al. (1982) reported that European ferrets (Mustela putoriusfuro) fed diets containing 20
mg/kg feed of the commercial PCB mixture Aroclor® 1242 for 266 days developed
similar clinical signs of excessive nail growth. Histological examination of the feet and

nails of ferrets indicated keratin debris adherent to the ventrum of the nail and in the

78

hair follicles, perivascular accumulation of mononuclear cells in the footpad dermis,
nuclear indentations and prominent nucleoli in the stratum basale, and vacuolization of
degenerating epidermal cells.

The toenail growth observed in the current study occurred within a shorter time
span as compared to the aforementioned studies, suggesting that PCB 126 induced a
more rapid and pronounced effect. Toenail growth reported for PCB 169 was observed
at 19 weeks of exposure (Aulerich et al. 1987). Aroclor® 1242 induced nail growth in
ferrets following 38 weeks of exposure (Bleavins et al. 1982). In the current study,
excessive nail growth was first reported at five weeks of exposure to PCB 126, and

growth continued throughout the study.

Body Weight Loss:

The decrease in body weight observed in the dams receiving 24.0 pg PCB
126/kg feed supports the occurrence of the characteristic PCB-induced “wasting
syndrome”. PCB-induced “wasting syndrome” was associated with toxicity signs in
mink, which often occurred within the first month of the trial. Body weight loss
associated with aphagia tended to occur after approximately two months of feed
consumption. Body weight (BW) loss related to the inability to eat also correlated to the
animal’s interest in food and activity level during daily monitoring, as well as clinical
signs of dental misalignment or gingival swelling or bleeding. Females with
periodontoclasia were offered soft feed to overcome chewing difficulties. However,
although there was not a statistically significant decrease in mean BW in PCB 126

exposed females compared to controls, dams in the 24.0 pg PCB 126/kg feed group lost

79

an average of 36% of their BW during the study period (initial BW to terminal BW), as
compared to the mean BW loss in the control (8.7%), 0.24 pg PCB 126 (9.6%), and 2.4
pg PCB 126/kg feed (5.9%) groups over the same time period. The current study
compares well with the study by Hochstein et al. (2001) that reported a mean BW loss
of 26% in mink exposed to 5.0 pg TCDD/kg feed (Hochstein et al. 1998). Aulerich et
al. (1987) also reported a significant decrease in BW in mink exposed to PCB 169, as a

result of PCB-induced “wasting syndrome”.

Organ Weights:

Adult females in the 24.0 pg PCB 126/kg feed group expressed a significant
reduction in mean absolute heart weight compared to the control group (Table 3-2).
Similarly, Hochstein et al. (1998) also reported a significant decrease in mean heart
weight in animals fed 1.0 pg TCDD/kg feed compared to the control mink.

The liver comprises 4.9 to 5.1% of the total body weight of the adult mink
(Aulerich et al. 1999). In addition to the significant increase in mean absolute liver
weight observed in this study in the dams receiving 2.4 pg PCB 126/kg feed, there was
a dose-related increase in liver weight expressed as percent of the total body weight
(3.2, 3.6, 4.8, and 5.4% for the control, 0.24, 2.4, and 24.0 pg PCB 126/kg feed groups,
respectively) (Table 3-2). Increased liver weight is a relatively common toxic response
due to induction of hepatic cytochrome P-450 enzymes and possibly changes in fat
metabolism in the liver of mink exposed to PCBs and TCDD (Aulerich et al. 1985;
1987; Bergman et al. 1992; Heaton et al. 1995; Hochstein et al. 2001). PCB- and

TCDD-induced hepatocellular injury have been previously reported in mink (Platonow

8O

and Karstad, 1973; Aulerich and Ringer, 1977; Bergman et al. 1992; Edqvist et al.
1992; Heaton et al. 1995), guinea pigs (reviewed in Safe 1990), and rats (Chu et al.
1994). Liver injury caused by toxic compounds is often due to the disruption of the
metabolic capabilities of the hepatocytes that can lead to the following diseases, i.e.,
fatty liver, direct cytotoxic damage, cholestatic damage, cirrhosis, vascular lesions and

hepatic neoplasms (Timbrell 1991).

Kidney Uroliths:

Kidney uroliths were observed during necropsy in one dam from each of the
following groups: control, 2.4 and 24.0 pg PCB 126/kg feed groups. Uroliths are
commonly referred to as (kidney) stones or calculi in the urinary tract, bladder and/or
kidneys, and are associated with urolithiasis, a condition that is relatively common in
ranch mink (Zellen 1996). As reported in the literature, stones are most frequently
composed of magnesium ammonium phosphate (struvite) and occasionally calculi of
calcium oxalate have been reported (Zellen 1996). Thus, it appears that PCB 126 does
not alter the composition of kidney uroliths, and PCB 126 did not increase the incidence
of acute urolithiasis. Mink frequently suffer from urinary tract infections, and may die
without showing any signs of this condition, or they may have difficulty urinating
(Aiello 1998).

Calculi recovered from PCB 126—exposed mink were analyzed for mineral
content by the Minnesota Urolith Center (University of Minnesota, College of
Veterinary Medicine, Department of Small Animal Clinical Sciences, St. Paul, MN).

All specimens consisted of 90 to 100% magnesium ammonium phosphate hexahydrate

81

as the core of the urolith, and 0 to 10% of the shell consisted of calcium phosphate

carbonate.

Hematology:

Exposure of mink to PCB 126 resulted in significant changes in hematological
parameters in this study (Tables 3-4 and 3-6). In addition to our study, decreases in
RBC counts, HGB, and HCT in mink have been reported in studies as a result of
TCDD-exposure (Hochstein et al. 2001), as well as exposure to contaminated fish
(Heaton et al. 1995) and rats (Kociba et al. 1976; Chu et al. 1994). An absolute decrease
in RBC count and HGB and HCT concentration are indicative of anemia, where HCT is
the most accurate measure (Duncan et al. 1994). Therefore, this would suggest that the
dams and juvenile mink in the current study suffered from PCB 126-induced anemia.
Mild to moderate anemia has been reported as a toxic response of PCB exposure in
several species (Bergman et al. 1992).

Red blood cell indices may be used to help classify the type of anemia, which
would suggest primary or secondary PCB-induced toxicity. Based on the RBC indices,
the anemia observed in the current study may be suggestive of regenerative anemia
based on the bone marrow response of the production and thus the presence of
reticulocytes in circulation. Macrocytosis (a significant increase in MCV), hypochromia
(a significant decrease in MCHC, but not MCH), and an increase in red cell distribution
width (RDW) were observed in the current study, which are suggestive of this
regenerative type of anemia (Tips 1993; Duncan et al. 1994). Macrocytic anemia is

generally only associated with severe, chronic, progressive liver disease, and not

82

typically seen in acute (even when severe) liver disease (Ravel 1995). Mean corpuscular
volume was significantly increased by PCB 126 exposure in the dams. In the juvenile
mink, there was a non-significant (p = 0.07) increase in MCV in the PCB 126-treated
group when compared to the control group. In addition, serum iron concentrations of
the adult females in all of the PCB 126-treated groups were decreased as compared to
the control group, but not significantly. This suggests that PCB 126 could cause
hemolysis, but the mechanism is not understood. However, hemolytic states have not
previously been reported as a toxic response following PCB exposure in mink or other
species (Bergman et al. 1992; Edqvist et al. 1992).

Increases in total WBC counts were observed in a dose-related manner in adult
(significant) (Table 3-4) and juvenile (non-significant) (Table 3-6) mink exposed to
PCB 126 in the present study. This observation compares favorably with previous
findings suggesting that leukocytosis is a common observation following PCB treatment
in different species including mink (Gillette et al. 1987; Bergman et al. 1992).
Neutrophilia was observed in the dams exposed to PCB 126 in this study. A change in
the presence of neutrophils is an indication that the immune system may be having
difficulty in overcoming a latent infection (Tips 1993).

A dose-related increase in platelets was observed in both the dams and the
juvenile mink in this study, however, the increase was not statistically significant
(Tables 3-4 and 3-6). Contrary to this finding of increased thrombocytes in the current
study, Osweiler (1996) suggested that a toxicity-induced hematopoietic depression
would cause thrombocytopenia. As stated above, rats exposed to PCB 126 also

exhibited decreased thrombopoiesis, which is indicative of abnormal activity of the

83

bone marrow supported by histological findings (Chu et al. 1994). This suggests that
there may be differences between species and their responses to PCB 126 exposure.
Platelet number also can be affected be splenic activity, and biologically, there is a

greater risk of hypercoagulation in animals with thrombocytosis (Duncan et al. 1994).

Serum Chemistries:

Several significant changes in serum chemistries were observed in the current
study in both adult and juvenile mink exposed to PCB 126. Enzyme activity is an
important measure of function and potential cellular injury, especially when it involves
the hepatic system.

Serum ALT activity in juvenile mink was increased in the PCB 126-exposed
group as compared to the control group (non-significant) (Table 3-7). In a study where
mink were exposed to various commercial PCBs and PCB fractions, Edqvist et al.
(1992) reported elevated ALT activity only in mink exposed to the commercial PCB
compared to the control group. Hochstein et al. (2001) reported that the group of mink
fed a diet containing 5.0 pg TCDD/kg feed had significantly increased ALT activity.
Contrary to the ALT increases observed in mink, a study exposing guinea pigs to
TCDD found decreased ALT activity (reviewed in Safe 1990), which might be
suggestive of a decreasing number of hepatocytes due to the liver undergoing fibrosis,
or because there are fewer hepatocytes in general (Duncan et al. 1994). The enzyme
ALT is a more sensitive indicator of chronic progressive hepatic disease, and
concentrations remain elevated in serum longer compared to AST (Tips 1993; Duncan

et al. 1994). Changes in ALT activity are indicative of liver function, and more

84

specifically may reflect hepatocellular injury as a result of chronic hepatic disease or an
altered state of hepatocellular integrity as well as a decrease in hepatocyte number (Tips
1993; Duncan et al. 1994; Ravel 1995).

A significant decrease in alkaline phosphatase (ALP) was observed in the
juvenile mink receiving the PCB 126 diet. Similar findings were also reported for mink
fed commercial PCBs (Edqvist et al. 1992). Hochstein et al. (2001) reported a numerical
decrease in ALP activity in mink fed TCDD. However, it was reported that alkaline
phosphatase activity in rats significantly increased following exposure to PCB 126 (Chu
et al. 1994). Alkaline phosphatase is an enzyme that is primarily found in liver, bone,
placenta, and intestinal mucosa. Thus, when these tissues are damaged, increased
amounts of ALP are released into the bloodstream. When this increase of ALP is due to
liver disease, it is usually associated with cholestasis, or obstruction of bile flow
(Duncan et al. 1994).

However, in agreement with Edqvist et al. (1992), cholestasis is probably not
extensive in the current study based on the decreased ALP activity observed in mink.
As aforementioned, some degree of cholestasis is invariably associated with
hepatocellular damage or injury. For example, based on swelling that can accompany
hepatocellular injury, compression of the bile canaliculi can lead to some degree of
cholestasis (Tips 1993; Duncan et al. 1994). Decreases in ALP activity, as observed in
the present study and in the study by Edqvist et al. (1992), can also be suggestive of low
adrenal function, which would indicate impaired phosphate liberation during utilization

of ATP for energy (Tips 1993).

85

 

Serum cholesterol was significantly reduced in juvenile mink exposed to PCB
126. In addition, cholesterol was substantially decreased (non-significantly) in a dose-
related manner in adult females exposed to dietary PCB 126. Edqvist et al. (1992)
reported that the non-ortho PCB fractions were most active in reducing the
concentration of cholesterol in mink, which corresponds with the coplanar structure of
PCB 126 used in the current study that significantly reduced serum cholesterol observed
in both juvenile and adult mink. Hochstein et al. (2001) also reported significant
reductions in serum cholesterol in mink that had been treated with TCDD. Contrary to
the decreases in cholesterol observed in mink, increases in serum cholesterol have been
reported in rats, presumably due to enhanced cholesterol production (Chu et al. 1994).
In mink, a reduced synthesis of cholesterol may be due to decreased functional mass of
the liver leading to hypocholesterolemia, and this decrease could be viewed as a health
concern, particularly since it can indicate poor digestion of fats. The effects of PCBs on
cholesterol may be indicative of altered fat metabolism as a result of PCB-induced
hepatic dysfiinction.

Albumin, which comprises 35-50% of the total serum protein (Duncan et al.
1994), was significantly reduced in the dams receiving the diet with 24.0 pg PCB
126/kg feed in the present study. A significant decrease in albumin was also reported in
mink exposed to TCDD (Hochstein et al. 2001). Chronic cachectic or “wasting
diseases” may contribute to hypoalbuminemia, although it is unclear whether the
mechanism leading to the decrease in albumin is due to impaired synthesis or other

factors (Ravel 1995).

86

The decreases in BUN observed in mink were not significant (p = 0.07) for
dams, and no changes in BUN levels were observed in juvenile mink fed PCB 126 in
this study. However, BUN is an important indicator of hepatic function and injury, and
the reduced BUN concentrations in the adult mink may indicate hepatic insufficiency,
and may be viewed as a precursor to hepatic injury. Contrary to the current study,
Edqvist et al. (1992) reported azotemia in mink treated with PCBs. The cause(s) for this
difference is unknown but may correlate with previous pathological observations in
mink is postrenal (obstructional) azotemia caused by ureteral or urethral obstruction by
uroliths.

Significant changes were observed in various electrolytes (Na, K, Cl, and TCOz)
and AG in the dams and the juvenile mink exposed to PCB 126 in the present study.
Hyperkalemia was significant in the juvenile mink in the PCB 126 group, and
increased, but not significantly, in the dams receiving 24.0 pg PCB 126/kg feed.
Potassium concentration can increase in the blood as a result of RBC hemolysis, as in
the case of severe hemolytic anemia (Ravel 1995). The observation of anemia in mink
exposed to PCB 126 in this trial is supported by the hematological data previously
presented. Clinical signs for hyperkalemia may include muscle weakness, nausea,
anorexia, and mental changes (tend toward drowsiness and lethargy) (Ravel 1995).

Significant hypocalcemia was observed in dams consuming the PCB 126 diet.
Mink exposed to Aroclor 1254 were also reported as having decreased concentrations of
serum calcium (Edqvist et al. 1992). Hypocalcemia is often associated with
hypoalbuminemia and alkalosis in most species, as well as hypoproteinemia (Duncan et

al. 1994).

87

Increased chloride concentration was observed in the females on the highest
PCB 126 diet as compared to the other PCB 126 dose groups. Generally,
hyperchloridemia follows the direction of changes in sodium ion gradient (Duncan et al.
1994; Ravel 1995). Biologically, maintaining the chloride concentration is essential, as
chloride is an important component of secretions (saliva and gastric secretions) (Duncan
et al. 1994). In addition, the significant decrease in serum TCOz that was observed in
dams on the PCB 126 diet suggesting metabolic acidosis correlates with the observed
increase in chloride ion concentration in the dams.

Anion gap significantly decreased in dams fed the PCB 126 diet. Anion gap
changes were not significantly changed in the juvenile mink in this study, as was also
reported in mink exposed to TCDD (Hochstein et al. 2001).

Serum glucose concentrations were significantly reduced in the juvenile mink
fed the PCB 126 diet, whereas the dams on the PCB 126 diets had no changes in
glucose concentrationsas compared to controls. In other studies, glucose concentrations
were decreased in mink exposed to PCBs (Edqvist et al. 1992), and TCDD (Hochstein
et al. 2001), and in rats exposed to PCB 126 (Chu et al. 1994). Glucose metabolism can
become disrupted through various mechanisms: the liver fails to breakdown glycogen,
the absorption of glucose in the intestines is impaired, insulin receptors are reduced, or
insulin production is reduced as a result of a decline in pancreatic beta cells (Tips 1993).

Other serum chemistry changes included decreased creatinine levels in both
dams and juvenile mink on the PCB 126 diets, which could be suggestive of muscle
wasting (Ravel 1995). Bilirubin was unchanged in females, and mildly lowered in

juvenile mink on the PCB 126 diet.

88

Due to changes observed in various blood clinical parameters that can be
associated with pancreatic function, it would prove highly valuable to histology
evaluated pancreatic tissue in future studies. Changes observed in this study included
mild hyperamylasemia (females, p = 0.09), neutrophilia, increased ALT,

hyperglycemia, and mild hypocalcemia.

CONCLUSIONS

In conclusion, this study reports that the dietary consumption of PCB 126
produced a multitude of toxic effects in adult female and juvenile mink including
clinical, hematological, and biochemical changes. Although the majority of the effects

occurred at a dietary concentration of 24.0 pg PCB 126/kg feed, effects were seen in all

dose groups, including the juvenile mink receiving the diet containing 0.24 pg PCB
126/kg feed.

A review of biological parameters correlated several of the observed
hematological and serum chemistry parameters from this study and others with the
pathologic and clinical changes that have been reported for PCB-induced toxicity. In
doing so, data have also shown distinct species differences in the biochemical responses
to PCB-induced toxicity. The results from this study strongly suggest that these changes
in hematology and serum chemistry are biologically relevant effects of PCB 126, and
are consistently observed in mink exposed to PCBs and TCDD. This demonstrates the

highly toxic potential of PCB 126 and supports the sensitivity of the mink to PCBs.

89

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Duncan, J .R., K.W. Prasse and EA. Mahaffey. 1994. Veterinary Laboratory Medicine:
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Edqvist, L.E., A. Madej and M. Forsberg. 1992. Biochemical blood parameters in
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Erickson, MD. 1997. Chapter 2: Physical, Chemical, Commercial, Environmental, and
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Hochstein, J .R., J .A. Render, SJ. Bursian and R.J. Aulerich. 2001. Chronic toxicity of
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Render, J .A., J .R. Hochstein, R.J. Aulerich and SJ. Bursian. 2000a. Proliferation of
periodontal squamous epithelium in mink fed 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD). Veterinary and Human Toxicology, 42(2):85-86.

Render, J .A., R.J. Aulerich, S.J. Bursian and RF. Nachreiner. 2000b. Proliferation of
maxillary and mandibular periodontal squamous cells in mink fed 3,3’,4,4’,5-
pentachlorobiphenyl (PCB 126). J Vet Diagn Invest, 12:477-479.

Safe, S. 1990. Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs),
dibenzofurans (PCDFs), and related compounds: environmental and mechanistic
considerations which support the development of toxic equivalency factors (TEFS).
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Silberhom, B.M., H.P. Glauert and L.W. Robertson. Carcinogenicity of
polyhalogenated biphenyls: PCBs and P385. Crit Rev T oxicol, 20(6):439-496.

Timbrell, J .A. 1991. Chapter 6: Toxic Responses to Foreign Compounds. I_n: Principles
of Biochemical Toxicology, (Second Edition). Taylor & Francis, Washington, DC.

Tips, J. 1993. Blood Chemistry and Clinical Nutrition. Apple-A-Day Press, Austin TX.

Van den Berg, M., L. Bimbaum, A.T.C. Bosveld, B. Brunstrom, P. Cook, M. Feeley,

J .P. Giesy, A. Hanberg, R. Hasegawa, S.W. Kennedy, T. Kubiak, J .C . Larsen, F.X.R.
van Leeuwen, A.K.D. Liem, C. Nolt, R.E. Peterson, L. Poellinger, S. Safe, D. Schrenk,
D. Tillitt, M. Tysklind, M. Younes, F. Waern and T. Zacharewski. 1998. Toxic
equivalency factors (TEFs) for PCBs, PCDDs, PCDFs, for humans and wildlife.
Environmental Health Perspective, 106(12):775-792.

Zellen, G. 1996. Chapter 16: Urinary System of Mink. in: Mink: Biology, Health, and

Disease. D.B. Hunter and N. Lemieux (Eds), Graphic and Print Services, University of
Guelph, Guelph, ON.

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CHAPTER 4

PCB 126-Induced Oral Squamous Cell Carcinoma of the
Mandible and Maxilla in Mink (Mustela vison)

93

PCB 126-Induced Oral Squamous Cell Carcinoma of the Mandible and Maxilla
in Mink (Mustela vison)

ABSTRACT

Mandibular and maxillary squamous epithelial proliferation was induced
experimentally in mink (Mustela vison) by 3,3’4,4’,5-pentachlorobiphenyl (PCB 126), a
ubiquitous contaminant, which is structurally similar to 2,3,7,8-tetrachlorodibenzo-p-
dioxin (TCDD). The histopathological appearance of this lesion suggests squamous cell
carcinoma (SCC). The characteristics of the lesion observed in mink from the current
study were consistent with some of the criteria for malignant neoplasia, including
invasiveness of atypical cell type, tissue destruction, increased proliferative activity, and
diffuse infiltration. However, additional criteria needed to be satisfied in order to
classify the lesion as malignant neoplasia. The current study was designed to determine
if a PCB 126-induced lesion in the gingival tissue of mink was cancerous by satisfying
two additional criteria of carcinogenicity. These included (1) the progression of the
lesion after removal of the stimulus (PCB 126); and (2) the ability of the cells
comprising the lesion to grow and form a tumor after being transplanted into athymic
mice. The lesion was histologically present in mink euthanized after only two weeks of
consuming a diet containing 24.0 pg PCB 126/kg feed. The lesion was clinically
detectable in mink exposed to the PCB 126 diet for three, four, and five weeks
following 12 weeks post-exposure. In addition, the lesion progressively worsened
during the six or eight months post-exposure period (classified the non-PCB period) in

animals fed diets containing PCB 126 for one to six weeks, thus meeting the first

94

criteria of progression after withdrawal of the PCB 126 stimulus. In the nude, athymic
mouse trial, nodular growths were observed in two of the 18 mice that were implanted
with gingival tissue from the oral lesion of mink exposed to PCB 126. Although the
nodules in the mice were classified as non-malignant, the origin was non-host (mink)
stratified squamous epithelium, demonstrating consistency with neoplastic criteria. Both
objectives of this study were met, therefore meeting additional criteria for

carcinogenicity. This is the first report of PCB 126-induced oral SCC in mink.

INTRODUCTION

Chemically-induced carcinogenesis, caused by exposure to chemicals such as
polychlorinated biphenlys (PCBS) and 2,3,7,8-tetrachorodibenzo-p-dioxin (TCDD), is a
multi-stage process that can lead to subsequent neoplasia (Safe 1989; Whysner and
Williams, 1996; Gregus and Klaassen, 2001; Klaunig and Kamendulis, 2001; Cockerell
and Cooper, 2002). Chemical carcinogens may affect multiple cellular and molecular
mechanisms that alter normal function of cells at various levels, leading to neoplastic
changes (Pelkonen and Nebert, 1982; Silberhom et al. 1990; Klaunig and Kamendulis,
2001). Carcinogens can be classified into two broad categories, genotoxic and
epigenetic chemicals, based on their chemical attributes in the process of
carcinogenesis.

These terms “genotoxic” and “epigenetic” chemicals were identified by

Williams and Weisberger (1983) to recognize the apparent differences of these

95

chemicals that induced neoplasia and to describe their possible mode of action (Klaunig
and Kamendulis, 2001). Genotoxic chemicals generally cause direct damage to genomic
DNA, and are activated in the target cell. Genotoxic compounds are capable of
producing a preneoplastic change following a single administration, which is generally
at or close to the site of chemical exposure (Klaunig and Kamendulis, 2001). Much less
is known about the mechanism(s) of epigenetic carcinogens, although they do not
appear to cause damage to DNA directly (Klaunig and Kamendulis, 2001). However,
these non-genotoxic agents appear to modulate cell proliferation and apoptosis, and may
act by producing an early proliferative response (mitotic activity) that is reversible
following removal of the test chemical stimulation (Klaunig and Kamendulis, 2001;
Boatman et al. 2004). To elicit neoplasia, exposure to epigenetic chemicals must be
chronic and there is a threshold dose (Klaunig and Kamendulis, 2001; Boatman et al.
2004)

Neoplasia can be categorized as benign or malignant neoplasia. However, a
neoplasm can defy categorization (Cotran et al. 1999). Benign and malignant neoplasms
are differentiated by morphological and behavioral characteristics. Benign neoplasms
are non-cancerous and generally localized, with limited potential for growth and slow
growing in nature. These tumors are composed of cells that are well differentiated, do
not invade surrounding tissue, and do not metastasize to distant sites. Benign tumors
adjacent to bone may cause cortical erosion in a response to local pressure, without
actually invading the bone (Rosenberg 1999; Thompson and Pool, 2002). Malignant
neoplasms tend to grow rapidly, may contain anaplastic cells, and can metastasize to

other organ sites depending to their site of origin. Malignant neoplasms

96

characteristically invade surrounding tissue, and tend to be more aggressive and
destructive, therefore they are often more osteolytic than benign neoplasms (Konde
1994; Myer 1994; Aiello 1998; Cotran et al. 1999; Thompson and Pool, 2002).

Malignant soft tissue neoplasms of the oral cavity often involve adjacent bone
(Dennis 1991; McGlennon 1991; Myer 1994; Cotran et a1. 1999; Cockerell and Cooper,
2002; Thompson and Pool, 2002). Osteolysis may be focal or diffuse, and may involve
the mandible, maxilla, premaxilla, or hard palate (Dennis 1991). In the oral cavity, the
lamina dura is a sheet of compact alveolar bone that lies adjacent to the periodontal
membrane, which is the lining of the tooth socket. Tissues adjacent to alveolar bone,
such as the gingiva and the periodontal ligament, can be invaded by epithelial cells,
which can lead to osteolysis (Birek et al. 1983). Invasiveness is a hallmark of malignant
neoplasia, along with metastases, and it is the most reliable feature that differentiates
malignant from benign tumors (Cotran et al. 1999).

Carcinogenicity of PCBs has been demonstrated in numerous rodent studies
reviewed by Safe (1989), Erickson (1997), Smith (1997), and Cogliano (1998).
However, animal-feeding studies that investigated the carcinogenic potential of PCBs
focused primarily on commercial PCB mixtures (Erickson 1997; Cogliano 1998).
Commercial mixtures with higher chlorine content, such as Aroclor 1260 that contains
60% chlorine, are suggested to have a higher neoplastic potential (Cogliano 1998).
Therefore, it is not known if a specific PCB congener in the commercial mixtures is
responsible for these effects or if there are additive effects, decomposition products,
contaminants, or metabolites that are involved in the toxic response (Erickson 1997).

Very few studies have been conducted with specific PCB congeners (Cogliano 1998).

97

In fact, only one PBC congener, 2,2’,3,3’,6,6’-hexachlorobiphenyl, had been studied
and the results indicated no significant neoplastic effect (Cogliano 1998).

Studies that investigated the carcinogenic potential of PCBs and TCDD have
also examined their activity as initiators and/or as tumor promoters (Safe 1989). 2,3,7,8—
Tetrachorodibenzo-p-dioxin has carcinogenic potential by acting as a promoter of
neoplastic activity, and this may have resulted in an increased incidence of squamous
cell carcinoma in rats and hamsters (Kociba et al. 1978; Whysner and Williams, 1996).
Studies have shown that PCB 126 has tumor-promoting activity in liver and lung tissues
(Safe 1989; Silberhorn et al. 1990; Cogliano 1998). Thus, PCB 126 could be a tumor-
promoting agent in other tissues, including the gingiva (only liver, brain, mammary
gland, and male thyroid gland were investigated in the cited group of studies) (Cogliano
1998)

Our laboratory has reported a TCDD- and PCB 126-induced lesion in juvenile
mink (Mustela vison) identified as mandibular and maxillary squamous epithelial
proliferation (Render et al. 2000a, 2000b, 2001). The lesion is characterized by
proliferation of squamous cells that form nests and/or cords that infiltrate into the
periodontal ligament and alveolar bone causing osteolysis of the mandible and maxilla.
The histological appearance of this jaw lesion is suggestive of squamous cell carcinoma
(SCC). Standard dark, female mink (Mustela vison) were used to determine if the jaw
lesion observed in mink was in fact oral carcinogenicity induced by dietary exposure to
PCB 126.

Specific criteria of malignant neoplasia, including higher proliferative activity,

tissue destruction, and invasiveness of atypical cell type and diffuse infiltration (Cotran

98

et al. 1999), are conditions that are consistent with characteristics of the jaw lesion
observed in mink exposed to PCB 126. Thus, this study was designed to determine if
the jaw lesion in mink would meet additional criteria of malignant neoplasia, including
progression or regression after withdrawal of the stimulus (the PCB 126-contaminated
diet), and the formation of a tumor from a tissue transplant or injection of cells into a
suitable host animal (nude, athymic mice). No prior carcinogenic studies in mink using
PCB 126 studies have been conducted. Therefore, it was hypothesized that PCB 126
induces squamous cell carcinoma in the maxilla and mandible of mink. The first
objective was to determine if the histologically detectable lesion in the maxilla and
mandible of mink would progress or regress in exposed animals afier withdrawal of the
diet containing 24.0 pg PCB 126/kg feed. Female mink were provided one, two, three,
four, or five weeks of exposure to the PCB 126 diet followed by a 6-month withdrawal
period. A separate experiment group was exposed to the PCB 126 diet for six weeks
followed by an 8-month withdrawal period. The second objective was to determine if
the squamous epithelial hyperplasia previously reported in mink exposed to 24.0 pg
PCB 126/kg feed was in fact SCC, by extracting gingival tissue from the oral lesion and
transplanting or injecting the pathologic cells into athymic mice, thus producing a tumor

growth.

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METHODS

Standard dark, female mink were used as an animal model to assess the oral
carcinogenicity of PCB 126. All mink were housed individually in wire mesh cages (61
cm L x 76 cm W x 46 cm H) with an attached wooden nest box (30 cm L x 22.5 cm W
x 25 cm H) at the Michigan State University (MSU) Experimental Fur Farm
containment facility. Lighting in the room was maintained to simulate the natural
light/dark cycle, of the Eastern Standard Time zone. Mink were acclimated for one
week afier being moved into the containment facility. Females were randomly assigned
to a designated treatment group, except that litter-mates were not placed within the
same treatment group. Mink were monitored daily for the duration of the study for any
signs of toxicity (lethargy, inappetence, body weight loss, behavior alterations, and
teeth displacement) and weighed weekly. If the jaw lesion progressed to a stage where a
mink was unable to eat, or the mink was clinically unhealthy, the animal was euthanized
and the jaws and major organs (adrenal glands, brain, heart, liver, kidneys, reproductive
tract, spleen, and thyroid glands) were collected and processed for subsequent

histopathological examination.

Diet - PCB 126-Supplemented Feed:

Complete methods for diet preparation are previously described in Chapter 2.
Briefly, a standard ranch mink diet (Table 2-1) was supplemented with 24.0 pg PCB
126/kg feed and was designated the treatment diet. The standard ranch diet without

supplemental PCB 126 was used as the control diet. The diets were aliquoted into two-

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liter, plastic containers that contained enough feed for one to two days per diet. Feed
was stored frozen and thawed in a cooler when required. Fresh thawed food was feed

each day at approximately 140 g per female per day. Water was available ad libitum.

Lesion Classification:

Observations were used to clinically classify the jaw lesion in mink. Initially,
swelling of the muzzle was observed, and gingival tissue appeared red and inflamed.
Mild changes in dentition alignment included increased interdental space between the
incisors and incisor unevenness within the mandibles and/or maxillae. Periodontoclasia,
or the loosening of permanent teeth due to the breakdown and absorption of the
supporting bone, and tooth displacement varied from subtle to severe with complete
tooth loss. Occasionally, the incisors appeared to be very thin, suggesting a decrease in
the enamel layer. Upper canine teeth appeared extended probably due to the receding of
the gums and the loss of support from around the root of the tooth, including the
periodontal ligament and the alveolar bone, allowing the tooth to loosen and extend out
of the socket. With severe progression of the lesion, mink had bleeding and swollen
gums, and nodular growths that were evident along the dental arches.

Histologically, jaw lesions were rated from mild to severe based on the
following criteria: mild was characterized by one or a few small, focal cyst(s) that are
considered to be the initial stage of the lesion. A lesion that was classified as moderate
in severity consisted of either several foci of cysts and islands of squamous epithelial
cells, or fewer large cysts that invaded the periodontal ligament and disrupted the teeth.

A severe lesion consisted of several cysts of increasing size and invasiveness and has

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progressed throughout the mandible and/or maxilla, destroying all surrounding tissues
including the underlying alveolar bone that supports the teeth.

Two experiments were conducted to determine if the histologically detectable
jaw lesion progressed or regressed after withdrawal of mink from the diet containing
PCB 126. The two experimental procedures differed by number and age of animals, as
well as exposure regimen. Both trials took place at the MSU Experimental Fur Farm in
the containment facility, and mink were fed the same PCB 126 dietary concentration,
24.0 pg PCB 126/kg feed.

In Experiment 1, four young (8-months of age) female mink were randomly
assigned to each of the five exposure groups, with no group having more than one
animal from the same litter. Mink were fed a diet containing 24.0 pg PCB 126/kg feed
for one, two, three, four, or five weeks. At the end of each exposure period, two mink
from each group were placed on clean ranch feed for six months to assess lesion
development, and the other two mink from each group were euthanized to examine the
jaws histologically for evidence of the jaw lesion.

In Experiment 2, 20 female mink comprised of juvenile (6-months of age) and
adult (IS-months of age) were assigned to either the control group (five young and five
adult mink) or the treatment group, which were fed a diet containing 24.0 pg PCB
126/kg feed (five young and five adult mink). The mink that were randomly assigned to
the treatment group receiving the PCB 126/kg feed were fed the diet for a 6-week
period. Following the period of exposure to the PCB 126-supplemented diet, two
control (one young, one adult) and two treatment (one young, one adult) mink were

euthanized to determine the histopathological prevalence of the lesion. The remaining

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eight PCB 126-exposed mink were taken off contaminated feed and maintained on a
clean ranch diet for an additional eight months (38 weeks) to determine progression or

regression of the lesion.

Necropsies:

At the end of six (Experiment 1) or eight (Experiment 2) months, mink were
euthanized using C02, and a gross examination was performed. Organs (adrenal glands,
brain, heart, kidneys, liver, reproductive tract, spleen, and thyroid glands) were
examined, collected and weighed, and sections were fixed in 10% buffered formalin
(pH 7.4). Approximately 10 g of the liver were frozen (—74°C) immediately after
extraction for subsequent PCB 126 analysis. Organ tissues were trimmed, embedded in
paraffin, sectioned to 5 pm, mounted on microscopic slides, and stained with
hematoxylin and eosin (H & E) for subsequent histological assessment.

At necropsy, the maxilla and mandible were collected from each female and
fixed in 10% buffered formalin (pH 7.4). The jaws were decalcified in Surgipath®
Decalcifier II (hydrochloric acid) (Surgipath Medical Industries, Inc., Richmond, IL)
and trimmed. The decalcified tissues were then processed for histological assessment,
which included paraffin embedding, sectioning, and staining with H & E for light
microscopic examination. Photomicrographs of jaw histopathology are presented in

color in the Results Section of this chapter and other sections of this dissertation.

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PCB 126 Analysis of Mink Livers:

Following necropsies of mink, one or two liver samples were pooled from each
exposure period and placed in I-Chem® jars (New Castle, DE). Approximately 8 g of
liver (wet weight) were homogenized with anhydrous sodium sulfate and Soxhlet
extracted with dichloromethane and hexane (3:1, 400 mL) for 16 hr. PCB-30 (17.5 ng)
was added as an internal standard prior to extraction, for the first five samples and a
blank, and PCB 30 and PCB 204 (17 .5 ng each) were added as internal standards for the
second set of six samples. Extracts were then concentrated to 6 ml and 0.5 mL was used
for the analysis of lipid content by gravimetry. The remaining extract was then treated
with concentrated sulfuric acid for the removal of lipids. Extracts were then passed
through a multi-layer silica gel column (2 g 100-200 mesh, 2 g of 40% acidic silica,
followed by another 2 g of 100-200 mesh silica gel; Davisil (Sigma, St. Louis, MO).
The congener PCB 126 was eluted with 5% dichloromethane in hexane (100 mL), and
the extract was concentrated to 1 mL, treated with concentrated sulfuric acid and
injected into an Agilent 6890N gas chromatograph with 7683 autosampler, equipped
with a 63Ni electron capture detector (Boston, MA). A fused silica capillary column (30
m x 0.25 mm id.) coated with ZB-5 (5% phenyl polysiloxane) at 0.25 pm film
thickness was used for the separation. The column oven was programmed from an
initial temperature of 50°C (1 min hold) to 180°C at a rate of 20°C/min, held for 3 min,
and then ramped at a rate of 2.5°C/min to 280°C with a final hold time of 8 min. The
injector and detector temperatures were maintained at 250°C and 300°C, respectively.

Helium and nitrogen were the caniers and the make-up gases, respectively.

104

3,3 ’,4,4’,5—Pentachlorobiphenyl was quantified using an external, standard
prepared at a calibration range of 1 ng/mL to 100 ng/mL. Recoveries of PCB 30 and
PCB 204 through the analytical method ranged from 60 to 88% (means: 68 and 70%,
respectively). Concentrations of PCB 126 were corrected for the recoveries of PCB 30
and PCB 204. The detection limit for PCB 126 was 50 pg/ g, wet weight (K. Kannan,
personal communication).

In Experiment 3, four standard dark, juvenile, female mink (6-months of age)
were fed a diet supplemented with 24.0 pg PCB 126/kg feed to induce neoplasia of
squamous epithelium in the mandible and maxilla, while two mink that served as
controls were fed a standard ranch mink diet. Procedures for Experiment 3 (housing,
animal assignment, acclimation and mixing of diets) followed the methods as stated
above. Fresh thawed feed was provided every day at approximately 140 g per female
per day. Water was provided ad libitum. Mink were observed daily. Mink remained on
treatment until clinical determination of the oral lesion.

Once the lesion was clinically classified as severe, mink were euthanized using
C02. The gingival tissue was harvested using sterile razor blades. Following gingival
harvest, the mandible and maxilla were collected and processed for histological
examination as previously described. Extraction of oral tissue from the two control
mink and implantation of tissue followed the same protocols as for the exposed mink.

Extracted tissue was placed into Modified Eagle’s Medium (MEM) solution
(Eagle’s minimum essential [Eagle’s ASP] medium supplemented with L-aspartic acid
[0.2 mM], L-serine [0.2 mM], and pyruvate [1.0 mM]). Gentamicin (GIBCO-Invitrogen

Corp., Carlsbad, CA) (0.05 mg/mL) was then added to the MEM solution. After

105

complete extraction of the mink lesion, all tissue pieces were placed in three
independent washes of MEM. Tissue was then placed in a fresh vial of MEM solution
for approximately 20 minutes. All surgical instruments were sterilized at 250°C for
procedures using an Ionotec® Bead Sterilizer (Runcom, Cheshire, England).

Nude, athymic mice (both sexes) were approximately 5-weeks of age at the time
of the procedure. Three mice per affected mink were used for the transfer of gingival
cells. Two methods were used to deposit the gingival cells from the mink lesion into the
right and left flank of each mouse. The first procedure involved transplanting sections
of gingival tissue, which were approximately two to three mmz. The second procedure
involved injection of a single cell suspension derived from the oral lesion cells. Both the
transplant and the injection procedures were carried out under a laminar flow hood to
maintain a sterile environment. Mice were first anesthetized using 0.5 to 1.0%
isoflurane with oxygen at 0.3 liter/min (Bickford Vapomatic Anesthetic Vaporizer for
Isofluorane Company,Wales Center, NY). The mouse’s back was prepared for the
surgical procedure by a 3-step cleaning process that involved swabbing with a sterile
ethanol swab, swabbing with a sterile cotton swab with Novassan, and then wiping it
with a sterile cotton bud with sterile water.

The toe reflex test that checks for muscle relaxation was conducted to ensure
that the animal was maintained under deep anesthesia. An incision approximately 1.0
cm in length over the lower spinal column was made using shears and holding with
forceps through the skin layer only. The skin was dissociated from the muscle layer to

create a pocket, one on each flank. The lesion tissue was placed into the skin pockets

106

using sterile forceps. The incision was then stapled using a 9 mm autoclip, and swabbed

with a sterile alcohol pad.

Single Cell Suspension Preparation:

A MedimachineTM (Becton Dickinson, Franklin Lakes, NJ) was used to
disaggregate tissue into individual cells. Briefly, the single cell suspension was prepared
by inserted four or five pieces of gingival tissue, approximately 2.5 mm2 in size, into the
MediconsTM (Becton Dickinson, Franklin Lakes, NJ) disposable capsule with 1 to 1.5
mL of buffering solution (PBS) with antibiotics. The MediconsTM was then inserted into
the MedimachineTM housing and run for one minute. In standby mode, tissue was
checked, and the MedimachineTM run for an additional 1 min. The MediconsTM was then
removed from the machine. A sterile F ilconsm was applied to a 3.0 mL syringe in the
housing of the MedimachineTM, withdrawing the cellular suspension and placing it in a
test tube. The cell suspension was centrifuged for five minutes at 800 rpm, forming a
pellet of cells. The pellet was resuspended in 500 pL in solution containing 10 pg of
metronidazole. The resuspended pellet was washed three times in metronidazole at 20
mg/mL. Following the washes, 1 mL of the cellular suspension was drawn into a
syringe, and using a 25-gauge needle 500 pL was injected into sponges that had been
surgically inserted subcutaneously on each flank of the mouse, one week prior to the
injection.

Following both the cell injection and the tissue transplant procedures, the
recovering mouse was returned to a clean, autoclaved cage with irradiated food and

autoclaved water. All mice were monitored daily to ensure the surgical sites were

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healing properly and that each mouse was in good health by monitoring robustness,
pinkness of nose and skin color, alertness and active, and having no signs of morbidity
or illness. Also, each mouse was individually tagged for identification with an
individual ear notch. The staples were removed seven days after surgery, and the mice
were monitored twice a week for tumor formation. The skin was palpated to check for
small nodules, and any growths were measured to monitor growth rate. If a growth was
present, the mouse was euthanized when the tumor reached 1 cm3. The mice were
euthanized at six months following transplant or injection if no growths developed or

became apparent during this period.

RESULTS

The PCB 126-induced oral lesion in mink was histologically identified by
specific characteristics. Nests and/or cords of proliferating stratified squamous epithelial
cells destroyed gingival tissue with enlarging cysts and would also infiltrate into the
adjacent periodontal ligament as well as the underlying alveolar bone. Some cysts had
centers of exfoliating epithelia with possible keratin.

The lesion rating system that was developed for these studies is defined in the
methods section. Briefly, a minimal to mild lesion consisted of one or a few small, focal
nests of squamous epithelial cells that characterized the initial phase of the lesion.
Invasion of alveolar bone had been detected, but not caused clinical signs of

periodontoclasia. As the lesion progressed to a moderate rating, the number and size of

108

these islands of squamous cells generally increased, forming large islands of squamous
epithelial cells or cysts. At this stage, the islands generally were more diffuse, and often
several foci were found in more than one quadrant of the jaw. Also at this stage, nests
and/or cords of squamous epithelial cells had invaded the periodontal ligament (PDL)
and alveolar bone, resulting in changes to dental support structures and osteolysis.
Clinical signs were visible at this stage. A severe rating based on histology was
determined by the aggressive invasion of the cysts causing destruction of gingival
tissue, loss of the soft-tissue attachment to the tooth (PDL) and severe osteolysis of the
underlying alveolar bone. Clinical signs of periodontoclasia were visible, including
spreading of incisors and often missing dentition. Also, individual variation appears to
play a role in the susceptibility and the presentation of the lesion. Some family groups
(siblings), even though on different treatment groups exhibited signs more quickly and
more severely that other mink.

Clinical signs were evaluated in addition to the histological lesions, and thus
used to rate the lesion and health status of the PCB 126-exposed mink. Although
histological changes occur prior to clinical changes, clinical signs were observed
visually. Clinically, swelling of the muzzle was observed, and gingival tissue appeared
red and inflamed. Mild changes in dentition alignment included increased interdental
spreading of the incisors, as well as periodontoclasia to tooth loss. With severe
progression of the lesion, mink had swollen and bleeding gums, and nodular growths
that were evident along the dental arches.

Nodular growths were observed above the oral palette in one mink exposed to

PCB 126 for six weeks and then on clean feed for eight month, and in mink exposed

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continuously for Experiment 3: the athymic mouse trial. Also noted was severe
osteolysis of the nasal turbinates of mink in both of these treatment groups.

The histological results for exposure to PCB 126 in mink are organized by week,
and include the weekly exposures (weeks one through six), and the post-exposure

evaluation following the removal of the stimulus (PCB 126 diet) for six or eight months

(Figures 4-1 through 4-6).

A) PCB 126 Exposure for One Week (Figure 4-1). 1) Weekly exposure: The two mink
that were euthanized after one week of exposure to a PCB 126 diet exhibited no
histological evidence of the lesion. 2) Six months post-exposure: following removal
of the contaminated diet for six months, both mink were euthanized after receiving
only one week exposure to the PCB 126 diet. One mink exhibited one focal cyst
near the incisors of the mandible, thus, a minimal rating was assigned. The second
mink exhibited moderate changes in all four quadrants of the jaw. In addition,

multiple foci were cystic and diffuse in the gingival tissue.

110

 

Figure 4-1. Week One: Oral squamous epithelial proliferation in mink exposed to PCB
126 for one week, and histological evidence at six months after the removal of the
stimulus (PCB 126 diet). Tooth (T); Alveolar Bone (AB); Gingiva (Gin); Periodontal
Ligament (PDL). Arrows on right point to squamous epithelial cysts that have invaded

either the PDL and/or the AB. H&E

 

 

 

B) PCB 126 Exposure for Two Weeks (Figure 4-2). 1) Weekly exposure: One of the
two mink that was euthanized following two weeks of dietary exposure to PCB 126
showed no histological evidence of lesion formation, while the other mink exhibited
multiple cysts throughout the right maxilla deeming a rating of moderate. 2) Six
months post-exposure: One mink died prior to the six months from wasting

syndrome, therefore only having one month on clean feed following the two of PCB

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126 diet. However, this mink exhibited a severe lesion rating even without the
grow-out period. Foci were multi-diffuse, and severe including invasive into the
PDL. All quadrants were affected, and all apparent gingiva had been replaced with
stratified squamous epithelial cysts. Periodontoclasia had become apparent during
necropsy. The other mink that was euthanized at six months following removal of
the contaminated diet after two weeks of exposure to the PCB 126 diet, exhibited
multi-diffuse changes in the gingival tissue throughout all quadrants. Some
increased space between incisors had become apparent. Because of the diffuse foci

and clinical changes, a moderate rating was assigned to this mink.

 

Figure 4-2. Week T wo: Histological evidence of oral squamous epithelial proliferation
in mink exposed to PCB 126 for two weeks exposure, and six months after the removal

of the stimulus (PCB 126 diet). H&E

 

 

 

 

112

C) PCB 126 Exposure for Three Weeks (Figure 4-3). 1) Weekly exposure: Both mink
euthanized after three weeks of exposure to 24.0 pg PCB 126/kg feed had
histological evidence of mild lesion formation. Squamous cysts were small and few
in number in both mink. Some cysts had exfoliated centers, but no osteolysis was
apparent. One mink appeared to have islands and/or cords that had developed in or
near the PDL. 2) Six months post-exposure: One mink (T510) euthanized at six
months post-three weeks PCB 126-exposure had a moderate lesion rating. The
beginning clinical signs of the lesion were evident as spreading of the incisors,
heavy plaque build up on the mandibular teeth, and histologically, several small foci
dispersed throughout the gingiva (all quadrants), and. The jaw lesion was clinically
apparent in another female (T560), as spreading of the maxillary incisors.
Additionally, there was histological evidence of a severe lesion rating throughout
the jaw, in which cystic formation was diffuse and multiple cysts were apparent in
the PDL in this mink. This mink (T560) also exhibited signs of PCB toxicity,
including enlarged liver (73.134 g), enlarged and discolored (mottled) kidneys,
enlarged adrenal glands (0.1 132 g), and highly vascularized and altered, lobular

lymph nodes.

113

 

Figure 4-3. Week Three: Histological evidence of oral squamous epithelial proliferation
in mink exposed to PCB 126 for three weeks, and six months afier the removal of the
stimulus (PCB 126 diet). Squamous epithelial cysts and/or nests depicted in the

histology slide on the right are throughout the entire tissue. H&E

 

 

 

D) PCB 126 Exposure for Four Weeks (Figure 4-4). 1) Weekly exposure: Both mink
that were exposed to the PCB diet for four weeks had mild or moderate histological
evidence of the squamous epithelial cysts. One mink (T344) had mild changes only
in the mandible. The other mink (T574) had minimal foci in the right maxilla, and
no foci were observed in the lefi maxilla. Multiple diffuse foci were observed in the
mandible, therefore warranting a rating of mild to moderate. 2) Six months post-
exposure: Mink euthanized following four weeks on the PCB 126 diet and then six

months on clean feed had histological evidence of a moderate of the lesion. The

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lesion was detected in all quadrants in both mink, but the severity did differ between
quadrants in one mink. There was moderate to severe destruction to the gingival
tissue, referring to replacement of gingival by stratified squamous epithelial cysts.
In addition, the mink had clinical signs of displaced and loose teeth, as well as
bleeding of the gingival tissue. One mink (T632) exhibited clinical signs of
spreading of the incisors, as well as a heavy bacteria load evident by the plaque
accrual. The other mink in this group (T586) also exhibited clinical signs of the
lesion based on the mobility of teeth and receding of the gums, but to a lesser degree

than the other female in this group.

 

Figure 4—4. Week Four: Invasive oral squamous epithelial proliferation in mink
exposed to PCB 126 for four weeks, and six months afier the removal of the stimulus
(PCB 126 diet). In the histology slide on the right, there was no AB that remained in
this section to support tooth structures, cysts and/or nests and cords destroyed and

replaced almost all maxilla and mandible bone tissue. H&E

 

 

 

115

E) PCB 126 Exposure for Five Weeks (Figure 4-5). 1) Weekly exposure: Both mink
euthanized at five weeks of exposure to the PCB 126 diet had histological evidence
of the lesion, which was detectable in all quadrant of the jaw. The lesions in both
mink were rated as severe, based on the diffuse nature and multiple foci throughout
all the quadrants. The development of this lesion in one mink (T506) consisted
mainly of cords, as opposed to cysts as in most of the mink. The lesion in the other
mink (T552) was considered very severe. All gingival tissue and alveolar bone had
been invaded and completely replaced by multiple stratified squamous epithelial
cysts around dentition. Also, this mink had initial signs of interdental spreading of
incisors. 2) Six months post-exposure: Six months follbwing the five weeks on the
PCB 126 diet, mink had severe diffuse lesions based on histological evidence of
invasion of squamous epithelial cells throughout both the maxillae and mandibles.
The mink exhibited clinical signs by loose and/or missing teeth as a result of loss of
PDL attachment, osteolysis, and receding gums. Heavy plaque build-up was also
noted in these mink, and is most likely a result of an increased bacterial load that is
fi'equently observed in PCB 126 treated mink. One mink (T310) exhibited clinical
signs of the lesion, including marked spreading of the incisors, increased plaque
accrual, and visual thickening of gingival tissue. The other mink (T672) expressed
the same clinical characteristics of the lesion including separation and mobility of the

teeth, tartar and plaque build-up, and noticeable thickening of the gingival tissue.

116

 

Figure 4-5. Week Five: Development of oral squamous epithelial lesion with evidence
of osteolysis in mink exposed to PCB 126 for five weeks, and six months after the
removal of the stimulus (PCB 126 diet). Cysts and/or nests and cords have invaded the
PDL of most of the teeth of this mink on the lefi. On the right, cysts and/or nests and

cords have expanded and occupied almost all of the periodontium. H&E

 

 

 

F) PCB 126 Exposure for Six Weeks (Figure 4-6). 1) Weekly exposure: The two PCB
126 treated mink (one 6-month-old and one 18-month old) that were euthanized
following six weeks of dietary exposure to PCB 126 exhibited lesion development
compared to controls. The adult mink expressed a mild lesion that consisted of small
foci, somewhat diffuse. Infiltration into the PDL had not occurred, but some invasion

into the alveolar bone was apparent. The yearling mink expressed a moderate lesion

117

that included infiltration of the PDL and invasion into the alveolar bone. Minimal
periodontoclasia was observed in this mink. An additional mink (R42) that was
euthanized at six weeks due to “wasting syndrome” already had moderately
increased interdental spacing of the incisors and bloody gums; depleted adipose
tissue and ascites were also observed during necropsy. 2) Eight months post-
exposure: Clinically, one mink (T302) displayed extensive hyperplasia of gums on
the inside of the lower palate and lips resulting in nodular growth formations (Figure
4-7). In addition, there was massive tissue destruction of the periodontal ligament
and support around the incisors and cuspids (molars), as well as receding of the gums
from around the teeth, externally. The mandible had succumbed to extensive
osteolysis, which contributed to bi-lateral tooth loss of premolars and incisors. Mink
“R290” had histological evidence of the oral lesion in all quadrants of the jaw,
expressing squamous epithelial cells invading gingival tissue as well as PDL and
alveolar bone. This mink also exhibited clinical signs including spreading of the
incisors, and receding of the gums that made the canine teeth appear extended.
Following necropsy, it was determined that the loss of PDL attachment, as well as
receding gums and osteolysis around the teeth, caused the canines to both appear
longer and actually protrude from the periodontal sockets. Another mink (R216)
exhibited presence of the oral lesion by moderate histological changes that included
multi-diffuse foci comprised of small islands of stratified squamous epithelium, as
well as clinical changes observed as increased spreading of the incisors. The lesion
rating in another mink (T624) in this exposure group was considered severe based on

histological evaluation, as well as the observed clinical signs. In this mink, only two

118

maxillary incisors remained, and of the other remaining teeth (canines, premolars and
molars), all of the teeth were very loose with minimal or no ligament or bony support
remaining. This mink (T624) also had thickening of the gingiva of the sofi and hard
palate areas, but receding of the gingiva along the gum-line. Additionally, adverse
effects of PCB toxicity were observed in this mink (T624) that included enlarged
spleen (10.1258 g), enlarged liver (62.4531 g), and enlarged adrenal glands (0.1128
g). One mink that was exposed to PCB 126 for six weeks and then on clean feed for
eight months had nodular growths above the oral palette. Histological examination
indicated that these were potentially preneoplastic tumors, as they were an extension
of the stratified squamous epithelial cells in combination with squamous cyst
development originating from the gingiva. In one mink (T302), the tumor growth had
developed on the dorsal side of the palate and measured 5.78 mm x 3.42 mm x 4.23
mm). Also noted was severe osteolysis of the nasal turbinates of mink in this
treatment group that had extended far into the nasal cavity and frontal bone. This had
also caused detachment of the nasal support structures. Osteolysis of the zygoma and
zygomatic arches was also observed in this treatment group and is reported in
Chapter 5. In addition, one mink in this group (R184) had an unusually brittle skull

noted during necropsy.

119

 

Figure 4-6. Week Six: Evidence of an osteolytic oral squamous epithelial proliferation
in mink exposed to PCB 126 for six weeks, and eight months following the removal of
the stimulus (PCB 126 diet). The image on the right consists of teeth and lesion, all

other structures have been destroyed and/or replaced by the lesion. H&E

 

 

 

Mink from Experiment 3 with histologically and clinically induced lesion:

T596 (70 days of exposure, or 10 weeks): Histologically, massive tissue
invasion and destruction in the gingiva, PDL, and the alveolar bone was observed. The
lesion was extensive, multi-diffuse with large and smaller cysts encompassing all
visible tissues (except for teeth), thus appearing to be severe to very severe. Clinical
observations revealed recession and deterioration of the gingival tissue along the gum

line, resulting in the teeth and roots being exposed and unsupported. There was

120

 

Figure 4-7. Proliferation with substantial thickening of the gingival tissue forming
nodular growths along the inside of the lower palate (along the dental arches), reaching
almost to the central midline“. A) Nodular growths were observed in the mandible of a

mink exposed to PCB 126 via the diet; B) the mandible from a control mink.

 

 

 

proliferation with substantial thickening of the gingival tissue forming nodular growths
along the soft palate of the mandible (along inside of teeth), reaching almost to the
center. All of the connective tissue along gums and the lips of the maxilla, as well as
under nose and the nasal septum, was necrotic and detached from deterioration.
Osteolysis was extensive throughout both the mandible and maxilla, and was noted into

the nasal turbinates and frontal bones.

121

T600 (85 days of exposure, 12 weeks): Histologically, the lesion appeared
similar to other mink with a severe to very severe rating. The mandible exhibited some
proliferation forming nodular growths (Figure 4-7), however, not as extreme as the
previous mink (T596). The mandible was literally “crumbling”, with only the very
distal section of the mandible remaining intact, and no other intact bone, and little
gingival tissue remained. Because teeth had no remaining bone or tissue support, most
had fallen out. The upper lip was still attached near the septum of the nose. However,
the gums of the upper lip above the incisors were mostly necrotic.

T5901: (86 days of exposure, 12 weeks): Grossly in this mink, tooth loss had
occurred in the incisors, and premolars and molars were loose due to loss of PDL
ligament support, osteolysis of alveolar bone and recession of the gums. Gums were
bloody and swollen, and thickening of the gingiva was apparent along the dental arches
of the mandible (Figure 4-7). Histologically, there was severe invasion of gingival
tissue and the alveolar bone throughout the mandible and maxilla by nests and cords of
squamous epithelial cells causing substantial tissue destruction.

T420 (140 days of exposure, 20 weeks): Grossly, gums frequently appeared
bloody and swollen. Periodontoclasia occurred prior to loss of incisors. Two small
nodules were observed in the upper palate cavity, consisting of stratified squamous
epithelial cells in combination with squamous cyst development originating from the
gingiva. In addition, one nodule was located in the right maxillary sinus, just off center
of mid-line (location was 9.95 mm caudally of the canine), and measured 3.10 mm x
3.54 mm. The left maxillary nodule was located 16.08 mm caudally of the canine, and

measured 3.23 mm x 4.10 mm. Histology of the nodules was similar to those reported in

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the mink (T302) exposed to PCB 126 for six weeks and then on clean feed for eight
months. The oral lesion was histologically classified as severe, with loss of alveolar

bone due to invasion of squamous epithelial cells that caused osteolysis.

Results for PCB 126 analysis of mink livers:

Concentrations of PCB 126 in mink liver samples are presented in Table 4-1.
The concentrations of PCB 126 in the liver tend to be elevated during exposure
(consumption of the PCB 126 diet), and increasingly so with duration of exposure.
These concentrations then noticeably decrease following the removal of the
contaminated diet and animals are provided clean feed. As reported in Table 4-1 , the
longer the duration of exposure the higher the concentrations of PCB 126 in the livers
of exposed mink. In addition, a gradual decrease in the PCB 126 concentration is
observed in mink over time; the longer the mink have been provided a clean source of
feed, the lower the liver concentration of PCB 126.

Two samples had higher lipid content than the rest of the samples (Table 4-1).
Although lipid content could be associated with fatty liver syndrome following
exposure to PCBs, artifacts due to the portion of the liver that is sampled for analysis
could not be ruled out. The PCB 126 concentrations in the two samples with high lipid
content were also high. One sample contained the highest concentration of PCB 126
although this individual mink had the lowest fat content in the liver. Another sample did
not contain PCB 126 at the detection limit of 50 ng/kg on a wet weight basis. The

weekly exposures and sacrifices evaluate PCB 126 accumulation in mink liver, and the

123

residual PCB 126 in the livers of the exposed mink can then be evaluate by the 6- or 8-

month withdrawal data when the mink were on control feed.

 

Table 4-1. Liver analysis of percent lipid and PCB 126 concentrations were determined

on a wet weight and lipid weight basis in livers from female mink (Mustela vison)

exposed to 24.0 pg PCB 126/kg feed.

 

 

 

 

1D Exposure/ % Lipid PCB 126 PCB 126
(Mink) Withdrawal ' (pg/g wet wt) (pg/g lipid)
(Week/Month)
NA2 1/0 NA NA NA
T7003 2/0 17.1 28,990 169,600
T484 + T536 3/0 3.5 20,930 598,000
T574 + T344 4/0 10.9 100,500 922,300
T552 + T506 5/0 2.8 179,400 6,408,000
NA2 a0 NA NA NA
(Week/Month)
T582x + T550 1/6 5.1 137 2,699
T6024 2/6 4.4 <50 <1,140
T560 + T510 3/6 4.9 805 16,430
T632 + T586 4/6 5.7 574 10,070
T672 + T310 5/6 3.8 1,635 43,030
R216 + R290 6/8 3.9 380 9,784
T624 + T302 6/8 5.2 455 8,742

 

' Exposure is expressed in weeks, withdrawal is expressed in months.

2 Data for Week 1 and Week 6 exposures are not available (NA).

3 Mink T700 died on 2-13-03.

4 Mink T602 was chronically on and off feed (refusal to eat), therefore ingesting an
overall lesser concentration of PCB 126.

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Experiment 3 : Carcinogenic Study with Athymic Mice:

All mice developed lesions over the tissue implant sites within two to 12 days
after implantation or injection of the cell suspension. The lesions initiated with redness
of the skin, and what appeared to be an inflammatory response. These lesions generally
healed within 10 days after formation, leaving signs of scar tissue. Two control mice
died during the trial, that was not treatment related. All other mice remained healthy
throughout the experiment.

Two mice developed measurable nodular growths at the transplant sites of the
mink oral tissue. One mouse developed a round nodule that measured 5.11 mm x 4.92
mm x 2.92 mm within 14 weeks (98 days). The second mouse that developed a firm
nodular growth received the gingival tissue from a different exposed mink, and the
tumor measured 5.56 mm x 5.23 mm x 1.13 mm at 17 .5 weeks (122 days) after
implantation of the tissue. A third mouse had a small grth develop, but was not
measurable during the week it was noticed, or palpable at necropsy. This resulted in an
incidence of 12% for tumor growth in mice that received gingival tissue from mink with

probable SCC.

125

 

Figure 4-8. Nude, athymic mice implanted or injected with gingival tissue harvested

from mink exposed to 24.0 pg PCB 126/kg feed via their diet. *Staple over incision.

 

 

A) Lesion B) Tumor

A) All mice developed a lesion over the transplant sites, including mice injected with
the single cell suspension. It was determined that the lesion was caused by bacterial
overload from the non-host oral mucosa. Thus, the viability of any potentially
tumorigenic cells following the bacterial insult was questioned.

B) Two mice developed nodular growths at the transplant sites. These tumors were
classified as non-malignant. However, they were identified as non-host (mink)

squamous epithelial cells, and were considered extremely aggressive and robust.

 

126

DISCUSSION

Lesion Description:

The jaw lesion observed in the present study indicated that PCB 126 induced an
aggressive form of oral SCC in mink. Histologically, small nests of stratified squamous
epithelial cells characterized the initial stages of the lesion. As the lesion progressed, the
number and size of these islands of cells generally increased. Subsequently, large
islands of cells developed and formed cysts. These cysts often contained centers of
exfoliated epithelia and possibly keratin. Nests and/or cords of proliferating squamous
epithelial cells infiltrated into the adjacent periodontal ligament as well as the
underlying alveolar bone. The alveolar bone is the portion of the maxilla and mandible
that forms the dental arch, which serves as a bony investment for the teeth. The
aggressive invasion of the cysts reported in this study, destroyed the gingival tissue and
caused osteolysis of the alveolar bone. In general, osteolysis that is observed as loss of
lamina dura, also known as the alveolar bone, tend to be aggressive malignant
neoplasias that have a tendency to invade, expand, and perforate the cortex of the
maxillae and mandibles (www.dentistrv.vcu.edu). A benign lesion, on the other hand,
would be more inclined to resemble compression, or even displacement, of the lamina
dura (www.dentistrv.vcu.edu).

In addition, clinical changes were observed in the condition of the gingiva and
jaws (i.e. mandibles and maxillae) of PCB 126 exposed mink in the present study.
Recession of the gingiva, destruction of the periodontal ligament and loss of alveolar

bone support, are factors that were consistently observed with the oral lesion. These

127

conditions are characteristics of periodontoclasia, which results in the loosening of
permanent teeth due to the breakdown and absorption of the supporting bone, and the
teeth eventually fall out (Anderson et al. 1998). Additional changes included swollen,
inflamed, and bleeding gingiva, and dental plaque buildup, which are predominantly
external characteristics of severe periodontitis (Bartold et al. 2000). Heavy plaque
build-up was also noted in PCB 126 treated mink, which is most likely the result of an
increased bacterial load that was frequently observed in PCB 126 treated mink in this
study.

A healthy periodontium, made up of four components including the gingiva,
periodontal ligament, alveolar bone and cementum, is necessary to support and maintain
the teeth and oral environment (Bartold et al. 2000). In general, gingival epithelia have
important roles in the overall health of the oral environment, including protection
against disease and cellular protection (in regard to nutrient balance, as well as
toxicants) (Bartold et al. 2000). As observed in mink from this study, the overall health
of the periodontium, including at least three of its four principal components (and
possibly cementum, although it was not investigated in this study) has been adversely
affected by dietary exposure to PCB 126, resulting in invasion and massive destruction
of these tissues.

The lesion was histologically apparent following PCB 126 exposure as short as
two weeks in mink on the PCB 126 diet. After this time interval, all animals exposed to
the PCB 126 diet had evidences of the lesion. Week two mink expressed a lesion that
was rated minimal to mild. Following week two, 100% of the remaining mink exposed

to PCB 126 for weeks three through six expressed the lesion, with a lesion rating that

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ranged from mild to moderate. Thus, as exposure time to the PCB 126 diet increased
through week six, so did the severity of the lesion expressed histologically in the mink
jaws. This indicated that the length of initial exposure to the chemical stimulus (PCB
126) does influence the length of time in which the lesion is detected histologically.
Although mink in group “week one” did not have histologically evidence of the lesion
at the week one necropsy, the mink in that same group that were grown out for six
months on clean feed following that one week exposure'did express the lesion
histologically. In all of the weekly exposure groups, the lesion progressed in severity
following the removal of the stimulus (PCB 126 diet). There was 100% incidence of the
lesion in mink following six or eight months on clean feed, demonstrating that removal
of the source does not allow the lesion to regress, but in fact the lesion continued to
progress and worsen with no stimulus.

In mink that were exposed to PCB 126 for six weeks and then on clean feed for
eight months, mink were giving watered-down feed because of periodontoclasia and/or
aphagia. Nodular cysts above the oral palette were observed, and histological
examination indicated the tumors to be an extension of the stratified squamous cells in
combination with squamous cyst development. Also noted was severe osteolysis of the
nasal turbinates of mink in this treatment group. This indicated the severity and
destructiveness of this oral lesion as it progressed even at the duration of eight months
post-exposure, suggesting that if these mink could naturally survive the toxicity of PCB

126 and the destruction of the lesion, malignancy and possibly metastasis might ensue.

129

Liver Analysis:

Analysis of liver PCB 126 concentrations from exposed mink indicated that
when the PCB 126 feed was withdrawn, PCB 126 concentrations in the liver decreased
over time. However, one could argue that the PCB 126 source had not been removed
entirely due to the possibility of l) the lipophilicity of PCBs, and/or 2) the high
concentration that had accumulated in mink over the exposure period, which decreases
slowly over time. In it possible that the mechanism(s) by which PCB 126 induces SCC
acts through its physiochemical properties, i.e. lipophilicity, and thus, its
bioaccumulation potential. However, based on the data presented in Table 4-1, the PCB
126 concentration in exposed mink decreased over time as depicted by the weekly

exposure groups.

Nude, Athymic Mice:

The first group of mice that were transplanted with gingival tissue from the
mink lesion developed lesions over the transplant sites. It was suspected that the lesions
in the mice were caused by bacterial overload from the oral cavity of the mink, even
though the tissue was treated with antibiotics. The antibiotic was changed to a broad-
based spectrum penicillin and metronidazole combination to process the gingival tissues
prior to implantation. In addition to the altered antibiotic combination, the gingival
tissue was processed into a single cell suspension (pelleted cells) to eliminate or reduce
the bacteria. This way the bacteria cells were exposed to the antibiotics as opposed to
large tissue pieces on which only the surface was exposed. However, mice injected with

the single cell suspension still developed the same lesions over the sites of injection.

130

Based on these caveats, the viability of any potentially tumorigenic cells was
questioned. However, development of nodular growths despite the insult from the
overload of oral bacteria and development of cutaneous lesions eluted to the potency of
these gingival cells. In two mice that had the tissue transplants, nodular growths
developed at the transplant sites were the lesions had been. These tumors were
classified as non-malignant, but were identified as originating from non-host, foreign
(mink) squamous epithelia. The results from this phase of the study were somewhat
equivocal based on the limited number of mice in the study and the complications with
mice developing lesions over the tissue sites. However, there are substantial
implications to the growth of tumors, especially based on these complications with
lesions in nude, athymic mice, even though growths were not classified as malignant.
Therefore, the significance of these data cannot go unnoticed.

A study by the National Toxicology Program (NTP) in rats supports the results
of SCC in mink from the present study, and supports previous indications that mink are
more sensitive than rodents based on the incidence of confirmed lesions (NTP 1996a,
1996b). The NTP conducted a toxicologic and carcinogenic study with PCB 126 and
TCDD in female Harlan Sprague-Dawley rats (exposure by gavage). PCB 126 showed
clear evidence of carcinogenic activity based on the increased incidence of liver and
lung neoplasias in rats (NTP 1996b). In addition, neoplastic lesions of the oral cavity, as
gingival squamous cell carcinoma were observed in the oral mucosa of the rats, with an
incidence of 0/53, 1/53, 1/53, 2/53, 2/53, 7/53, and 2/50 for doses of 0, 30, 100, 175,
300, 550, and 1000 ng PCB 126/ kg BW, respectively. TCDD was found to cause non-

neoplastic lesions of the oral cavity of rats, as gingival squamous hyperplasia in

131

generally the same percentage, although carcinogenic activity was not reportedly caused
by TCDD (NTP 1996a).

However, an additional study from our laboratory was conducted to determine if
the jaw lesion that was observed in mink could be induced in a sensitive strain of rats,
the Long Evans (Aulerich et al. 2001). The weanling (28 days old) rats did not exhibit
any evidence of the lesion grossly or histologically following 101 days of exposure to
20 or 100 pg of PCB 126, or 1.0 or 10 pg of TCDD via the diet, as was seen in mink
exposed to 0.24 to 24.0 pg of PCB 126/kg feed.

Several characteristics of the lesion observed in mink from this study were
consistent with criteria for malignant neoplasia, including increased proliferative
activity, invasiveness of atypical cell type and diffuse infiltration, and tissue destruction.
Morphological and behavioral characteristics of neoplasia can be used to differentiate
between benign and malignant neoplasms (Cotran et al. 1999). However, a neoplasm
can defy categorization, and ultimately, the morphological diagnosis of neoplasia is
subjective, and based on criteria that are guidelines, not absolutes (Cotran et al. 1999).

Malignant neoplasms arising from soft tissues of the oral cavity often involve
focal destruction of bone adjacent to teeth (Dennis 1991; McGlennon 1991; Myer 1994;
Thompson and Pool, 2002). Osteolysis may be focal or diffuse, and may involve the
mandible, maxilla, premaxilla, or hard palate (Dennis 1991). These oral malignancies
adjacent to the alveolus tend to more lytic, whereas a benign process would tend to have
a well-demarcated border that is reflective of its slow growth (Myer 1994).

Squamous cell carcinoma of the oral cavity in cats and dogs is an aggressive

disease, which frequently invades the periosteum of the underlying alveolar bone in up

132

to 77% of canine cases (Thompson and Pool, 2002). Often the early lesions of gingival
squamous cell carcinoma are relatively small nodular masses along the dental arcade.

These nodules do not always form fleshy masses, and rarely metastasize to distal sites,
usually at a rate of less than 3%. Lymphatic drainage from the gums is more restricted,

and therefore limits potential for metastases (Head et al. 2002).

PCB-Induced Carcinogenesis - Pathways and Possible Mechanisms:

The definitions of genotoxic and epigenetic chemicals do vary somewhat, and it
is recognized that some chemicals fit neither, and chemicals may in fact have attributes
from both categories. However, there are apparent differences in chemicals that induce
neoplasia, as well as their possible mechanisms of action (Klaunig and Kamendulis,
2001). PCB 126 may be considered a chemical carcinogen that has attributes from both
categories. It does not require chronic exposure at threshold doses, it is irreversible, it
may cause direct DNA damage, and it may have the potential to cause multi-organ
tumorigenicity (genotoxic). It is unknown if PCB 126 exhibits species specificity
because mink is a sensitive species. However, the study in rats by NTP demonstrates
that PCB 126 causes multi-organ tumors in more than just one species, suggesting that
PCB 126 is possibly a genotoxic agent. Reversibility was not observed following
removal of the PCB 126 diet. In fact, the lesion continued to progress in all PCB 126-
exposed mink. Therefore, a non-genotoxic classification that is reversible following
removal of the test chemical stimulation does not seem accurate. In addition, epigenetic
chemicals require chronic exposure and a threshold dose to elicit neoplasia (Klaunig.

and Kamendulis, 2001; Boatman et al. 2004). The existence of a threshold dose for PCB

133

126 has not been established, and chronic exposure is not required for the induction of
SCC in mink.

Exposure to chemical carcinogens leading to the development of neoplasia can
result from the imbalance between cell proliferation and apoptosis (Williams 2000;
Boatman et al. 2004). Hyperplasia induced by nongenotoxic carcinogens is threshold-
based and reversible following removal of the test chemical (Boatman et al. 2004).
Chronic cell proliferation in preneoplastic cells, in turn, may result in papillomas and
eventually leading to carcinoma (Boatman et al. 2004). However, the association
between chemicals and carcinogenicity and cellular and molecular mechanisms, such as
epidermal growth factor (EGF) receptors, tumor necrosis factor (TNF-or), interleukins,
estrogen, and glucocorticoids, all appear to be mechanistic possibilities (Whysner and
Williams, 1996; Klaunig and Kamendulis, 2001). Neoplasias developing in soft tissues
(such as the gingiva) may have mutations that target oncogenes that regulate
transcription factors or cell cycle proteins, causing this dysfunction or imbalance in cell
proliferation (Cotran et al. 1999).

The PCB congeners that maintain a TCDD-like configuration apparently interact
with the cytosolic protein, the aryl hydrocarbon receptor (AhR) (Chapter 1). Following
activation and entry into the nucleus, and formation of the AhR-ARNT complex results
in up-regulation of a battery of AhR-regulated genes (Rinaldi et al. 2002). At least six
of the AhR-responsive genes are associated with carcinogen metabolism, including the
potential carcinogen bioactivator CYP1A1 (Rinaldi et al. 2002). Due to the vigorous
xenobiotic metabolism that occurs within the oral cavity (Rinaldi et al. 2002), AhR-

activating agents, such as PCB 126, are characterized as potential intraoral carcinogens.

134

Bioactivation represents a valid strategy to reduce. CYP1A1 is one of the primary
carcinogen-activating enzymes in oral mucosa that is suggested in initiation and
promotion of oral SCC (Rinaldi et al. 2002).

The PCB 126-induced SCC mechanistic pathway responsible for its toxicity
needs to be elucidated. The mechanism(s) by which PCB 126 induces SCC may be
through the activation of transcription factors that we have not studied. A proposed
epigenetic mechanism of carcinogenicity is cell cycling such as an increase in the cells
entering the S-phase. A proliferating cell marker, Ki-67, can be used to determine the
proliferative state, but we know there is an increase in cell proliferation in mink
expressing the lesion. Additional analyses to investigate these pathways would include
proliferating-cell-nuclear-antigen and immunohistochemistry. However, a study using
the proliferative index and Ki-67 as markers of cell proliferation found no significant
difference between dysplasia and carcinoma (Macluskey et al. 1999). Based on these
results, it is suggested that epithelial proliferation may continue to increase during the
transition from dysplasia to carcinoma, but this is likely to occur at a slow rate and Ki-
67 expression is not a good indicator of neoplastic transformation (Macluskey et al.
1999).

Chemical carcinogens known to be tumor promoters have been shown to affect
gap junctional intercellular communication (GJIC) structure and/or function and to
inhibit cell-cell communication (Weis et al. 1998; Trosko et al. 2000). Dysfunctional
GJIC plays a crucial role in the tumor promotion phase of carcinogenesis, which may
involve the regulation of cellular growth, or more specifically down-regulation of GJIC

(Weis et al. 1998; Trosko 2000). Inhibition of gap junction-mediated intercellular

135

communication has been proposed as a potential mechanism by which a wide variety of

chemical and biological agents may act as promoters of neoplasia.

Species Comparisons:

Apparently, PCB 126 does not act similarly in mink as it does rats and mice.
This may be due to species differences in aryl hydrocarbon receptor (AhR)
characteristics, the target receptor for TCDD and dioxin-like compounds including PCB
126. Also, rodents may be less susceptible to the toxic effects of TCDD and similar
compounds due to the rodents’ ability to rapidly metabolize these chemicals (Huff et al.
1991). For example, the half-life of TCDD in rodents is between 12 and 30 days,
depending on the rodent species (and strain). In contrast, the TCDD half-life in humans
has been estimated at 7.1 years (Whysner and Williams, 1996).

Carcinogenicity, xenobiotic metabolism, as well as the responses that may be
induced by exposure to chemicals, can be affected by species and strain differences, as
well as inter-individual variation (Pelkonen and Nebert, 1982; Safe 1990). Additional
conditions that affect the development or determination of a neoplasm in an animal
include sex, contaminant, dose, duration (single, continuous, repeated, or withdrawal)
and overall length of exposure/experiment (grow-out period or percentage of lifetime),
targeted tissue(s) or complete organism evaluation, and complete neoplastic screening
of tissues (Safe 1990).

The jaw lesion that was observed in mink exposed to PCB 126 in the current
studies and earlier reports of TCDD appears to have some similarities to lesions

reported in humans accidentally exposed to PCB contaminants, as well as marine

136

mammals inhabiting areas contaminated with high concentrations of organochlorine
compounds. Following accidental PCB exposures in Japan through the consumption of
contaminated rice oil (the Yusho incident), and Taiwan via the consumption of
contaminated fish (the Yu-Cheng incident), a number of changes were noted in both the
victims and their offspring. These changes included alveolar bone resorption,
periodontal disease, oral pigmentation and increased depth of periodontal pockets
(Shimizu et al. 1992; Hashiguchi et al. 1995). In addition, early eruption of teeth,
gingival hyperplasia, spotted calcification of the parieto-occipital area of the skull, and
poor skeletal development in human neonates were also reported as a result of exposure
to these PCB contamination (Yamashita and Hayashi, 1985; Rogan et al. 1988). The
lesions that resulted from humans exposed to PCBs through the ingestion of
contaminated rice bran oil, the accidental spills of Yusho or Taiwan, is similar to the
lesion reported in this study.

Beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary have
been reported as having a high incidence of unnatural pathology including unusual
cancers and periodontitis with necrosis and loss of teeth, believed to be caused by
exposure to environmental contaminants (Beland et al. 1993; DeGuise et a1. 1995).
Harbor seals (Phoca vitulina) and gray seals (Halichoerus grypus) from the Baltic Sea
exhibited exostosis and osteolysis of the mandibular alveolar bone, as well as loss of
teeth (Bergman et a1. 1992; Mortenson et al. 1993). The pathology of the PCB
126/TCDD-induced proliferation of maxillary and mandibular squamous epithelia may

be a lesion that is commonly occurring in a number of different species exposed to

137

environmentally relevant concentrations of contaminants suggesting that there may be

an interspecies correlation.

Review of Carcinogenic Potential of PCB:

Cancers induced by PCB mixtures and TCDD have been focused primarily in
the hepatic system, without mention of any oral carcinogenesis. The studies that have
investigated the carcinogenic potential of PCBs have been with PCB mixtures except
for one study that looked at the PCB congener 2,2’,3,3’,6,6’-hexachlorobiphenyl. It
appears that these studies were based on constant exposure of the animals to the
stimulus (the contaminant). Commercial PCBs have been reported to induce
preneoplastic lesions and hepatocellular carcinomas in rats and mice when administered
at concentrations of mg/kg generally per feed for extended periods of time (reviewed by
Silberhom et al. 1990). PCB mixtures that contained higher chlorine contents such as
Kanechlor 500 and Aroclor 1260 induce neoplastic nodules and hepatocellular
carcinomas more frequently. Studies have found that the liver is the primary target
organ for PCB-induced tumorigenicity and carcinogenicity in rats and mice, although
there is some evidence that the gastrointestinal tract may be affected (Silberhom et al.
1990).

In regard to PCB-induced carcinogenicity, the US. Environmental Protection
Agency (US. EPA) has concluded that there are sufficient data and evidence of the
carcinogenicity of PCBs to animals. In addition, US. EPA holds a position on the
toxicity of PCBs (commercial preparations in general), emphasizing the importance of

analyzing all biological information as opposed to relying solely on tumor findings

138

(US. EPA 1996; Erickson 1997). The International Agency for Research on Cancer
(IARC) has concluded that there is sufficient evidence that TCDD is a carcinogen in

animals (Whysner and Williams, 1996).

CONCLUSIONS

This study was designed to evaluate the carcinogenic potential of a PCB 126-
induced jaw lesion previously identified as mandibular and maxillary squamous
epithelial proliferation. The invasiveness of squamous epithelial cysts and severity of
osteolysis that resulted in periodontoclasia indicate that PCB 126 is a potent inducer of
squamous cell carcinoma (SCC) in mink. Following only one week of exposure to
PCB 126 and six months post-exposure on clean feed, SCC was histologically evident
in mink. The present study met several criteria of neoplasia, including invasive
behavior of atypical cells, tissue destruction and replacement with a new cell/tissue
type, progression after stimulus removal, and tumor growth in non-host species. Also,
individual variation appears to play a role in the susceptibility and the presentation of
the lesion. Some family groups (siblings), even though they were assigned to different
treatment groups, exhibited signs of the oral/jaw lesion more quickly and more
severely that other mink. Although histological changes occur prior to clinical changes,
clinical signs were observed visually prior to other changes. Thus, the value of this
study in determining a minimal time necessary to induce the lesion histologically was

important in identifying the onset of the clinical signs and progression of this lesion.

139

The accuracy of the histological classification of SCC in mink significantly contributes
to the recognition of PCB 126 as a chemical carcinogen.

These results would suggest, to some extent that PCB 126 possesses
characteristics that are consistent with attributes of a genotoxic carcinogen: it is
irreversible, the lesion occurs at or near the site of exposure, it is potentially a complete
carcinogen and probably causes direct DNA damage. However, PCB 126 may also
possess attributes that are consistent with epigenetic carcinogens such as species
specificity.

As aforementioned, there are apparent species differences, especially between
mink and rodents, thus it is imperative when performing comparative studies to
investigate all possibilities and data on these species differences especially when
applying studies to other species, such as humans in a risk assessment situation. The
US. EPA confirms that there are sufficient data and evidence of the carcinogenicity of
PCBs (commercial mixtures) to animals (rodents). The results of the present study
indicate that PCB 126 is carcinogenic by inducing oral squamous cell carcinoma in

mink.

140

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CHAPTER 5

Bone Lysis Associated with PCB 126-Induced
Neoplastic Oral Lesion of the Mandible and Maxilla
in Mink (Mustela vison)

146

Bone Lysis Associated with PCB 126-Induced Neoplastic Oral Lesion of the
Mandible and Maxilla in Mink (Mustela vison)

ABSTRACT

Polychlorinated biphenyls (PCBS) are ubiquitous contaminants in the
environment that are resistant to biological and chemical degradation. The toxicity and
bioaccumulation of PCBs give rise to serious and significant health effects. Because
mink (Mustela vison) are sensitive to the toxicity of PCBs, the mink is an important
animal model for studying 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126)-induced effects
on bone, as well as the neoplastic processes of PCB 126-induced oral squamous cell
carcinoma (SCC) in the mandible and maxilla that causes aggressive osteolysis. Mink
were fed a diet containing 24.0 pg PCB 126/kg feed for six weeks and then placed on
clean feed to determine if PCB-induced oral SCC caused local (maxillary and
mandibular) osteolysis, as well as systemic osteolysis. Radiographs were taken at weeks
0, 3, 6, 9, and 12 of the trial, and evaluation of radiographs included objective criteria
(dental alignment, bone lysis, nail growth, and organ system evaluation) and seven skull
measurements. Body condition was also assessed radiographically by measurement of
body wall soft tissue, including subcutaneous fat. In addition, post-mortem skeletal
mineral analyses of femurs were performed. Dental malalignment was observed as early
as the third week of exposure in one treatment mink, and was apparent as incisor
interdental spreading. Osteolysis was observed at six weeks of exposure in radiographs
of four out of 10 treatment mink. Of the surviving mink at week 12, six out of seven

PCB 126-exposed mink exhibited moderate spreading and/or movement of the teeth. No

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malaligned teeth were observed in any of the control mink at any time, or had signs of
osteolysis during the trial. The width of the zygomatic arch was significantly decreased
between mink within the treated group over time. Investigation of the skulls revealed
osteolysis had progressed up the maxilla and upward along the zygoma. The
measurement of subcutaneous fat was significantly decreased in the mink receiving the
PCB-treated diet as compared to the control mink. Thus, PCB 126 caused localized
mandibular and maxillary osteolysis that aggressively progressed through the facial
bones (i.e., the zygoma and nasal turbinates) until death ensued. However, the effects of

PCB 126 were not manifested systemically on bone in mink.

INTRODUCTION

A number of neoplastic processes are associated with bone lysis. These
mechanisms of osteolysis can include the replacement of normal bone cells with
neoplastic cells (primary bone tumors), growth of adjacent soft tissue neoplasm into
bone and/or pressure necrosis, tumor mass destruction of blood supply to bone,
endocrine mediators, and paraneoplastic syndromes, such as hypercalcemia. Some of
these neoplastic processes may be linked to the tumor-promoting activity and endocrine
disruption of PCBs, demonstrating cancer potency and/or clear evidence of the
carcinogenic potential of contaminants, including PCBs (Cogliano 1998).

Primary bone tumors (and secondary, such as metastatic carcinomas or

sarcomas) develop when healthy bone cells are replaced with diseased cells, such as

148

facial and mandibular osteosarcomas, which can be characterized by osteolysis (Myer
1994; Whyte 1999; Thompson and Pool, 2002). Soft tissue tumors that are adjacent to
bone can cause osteolysis by the cancer cells directly invading the bone by infiltration,
or by causing erosion of the bone due to localized pressure (Rosenberg 1999;
Thompson and Pool, 2002). Expanding tumor mass can cause ishemic necrosis of bone
due to compression of vessels, which also is an important mechanism of osteolysis
(Cramer et al. 1981). Neoplasms can alter endocrine homeostasis (Nelson and F eldman,
1991; Cotran et al. 1999), and can release mediators that cause a direct osteolytic effect
through the local release of proteolytic enzymes by either tumor cells or host stromal
cells, changing cellular function (Cramer et al. 1981; Body 1999; Cotran et al. 1999;
Cockerell and Cooper, 2002). Paraneoplastic syndromes can often result in a systemic
effect from neoplastic associated diseases, of which the most common are of endocrine
origin (Cotran et al. 1999). Hypercalcemia, the most common of this syndrome, is
frequently associated with malignant neoplasia as a result of invasion of bone (Dobson
and Gorman, 1991; Cotran et al. 1999).

Neoplasia can be categorized as benign or malignant neoplasia, and are
differentiated by morphological and behavioral characteristics. Benign tumors are non-
cancerous, and are generally localized, slow growing, consist of well-differentiated
cells, have limited potential for growth, do not invade surrounding tissue and do not
metastasize to distant sites. Malignant neoplasms tend toward rapid growth, may
contain anaplastic or poorly differentiated cells, characteristically invade surrounding
tissue and can metastasize to other organ sites. Malignancies tend to be more aggressive

and have a tendency to be more destructive than benign neoplasia, and if left untreated

149

may result in death (Myer 1994; Aiello 1998; Cotran et al. 1999; Cockerell and Cooper,
2002; Thompson and Pool, 2002). Invasiveness is a hallmark of malignant neoplasia,
along with metastases, and it is the most reliable feature that differentiates malignant
from benign tumors (Cotran et al. 1999).

It was hypothesized in this study that the oral squamous cell carcinoma induced
by dietary PCB 126 caused systemic osteolysis, in addition to local (maxillary and
mandibular) osteolysis in mink. To test this hypothesis, serial radiographs and post-
mortem skeletal mineral analyses were performed in mink fed diets containing 24.0 pg

PCB 126/kg feed.

METHODS

Five juvenile female (six months of age) mink and five adult (18 months of age)
female mink were assigned to each of the two treatment groups (control and PCB 126-
exposed) on day one of the trial. The exposed group of mink was fed a diet that
contained 24.0 pg PCB 126/kg feed for the first six weeks of the 38-week trial. This
group then received the standard ranch mink diet for the remaining 32 weeks of the
trial. The other group, which served as the control group, was fed a standard ranch mink
diet (Table 2-1). The standard ranch mink diet also served as the basal feed into which
the supplemental PCB 126 was added. Complete methods of dietary mixing of PCB 126

are described in Chapter 2.

150

Mink were housed individually in wire-mesh cages (61 cm L x 76 cm W x 46
cm H) with a wooden nest box (30 cm L x 22.5 cm W x 25 cm H) attached to the
outside of each cage at the Michigan State University (MSU) Experimental Fur Farm
containment facility. Lighting in the room was maintained to simulate the natural
light/dark cycle, Eastern Standard Time. Mink were observed daily for any signs of
toxicity including decreased feed and/or water intake, refusal to eat, changes in physical
appearance, activity level, and behavior alterations (Table 3-1). Mink were also
weighed on day one of the trial, and body weights of mink were recorded on a weekly
basis thereafter throughout the study. Food was provided fresh each day at

approximately 140 g per female per day. Water was provided ad libitum.

Radiographs:

Radiographs of the mink were taken at the MSU Small Animal Veterinary
Teaching Hospital. A 10:1 mixture of ketamine hydrochloride (25 mg/mL; Ketaset®,
Fort Dodge Animal Health, Fort Dodge, IA) and Xylazine-20 (20 mg/mL; Butler®, Ben
Venue Laboratories, Inc., Bedford, OH) was given at 0.4 mL/kg BW via intramuscular
injection to anesthetize each mink for radiographs.

Two radiographic views of each mink were taken, which included the dorso-
ventral (DV) view of the whole body and the lateral projection of the head. The
radiographs were taken at weeks 0, 3, 6, 9, and 12 for all treatment animals and
representative control animals. A tabletop technique with settings at 55 kVp, 2.5 mAs
(400 mAs at 6.4 millisec) and 30 cm focal-film-distance was used (UltraVision® x-ray

film/screen system, DuPont, Wilmington, DE). Evaluation criteria for radiographs

151

included subjective and empirical measurements of the skull and skeleton. Skull
measurements are identified on Figure 5-1. Body condition was assessed
radiographically by measurement of thoracic body wall soft tissues (Figure 5-7). This
measurement included subcutaneous fat, and was taken on the right side of the animal,
at the level of the ninth rib on the DV view of the radiograph. All measurements were
acquired using a digital, sliding caliper (Digimatic Caliper, Mitutoyo, Japan) and
recorded to the nearest 0.01 mm. One individual performed all measurements for
consistency. Subjective evaluation of the radiographs including observations of
alterations in bone opacity, dental alignment, and overall systemic bone condition was
also conducted. In addition, nail growth and organ systems were evaluated from

radiographs of the mink during the trial.

Necropsies:

At the end of the trial, mink were anesthetized with 0.4 mg/kg BW of the
ketamine/xylazine mixture, and approximately 10 mL of blood were collected from
each mink via cardiac puncture using a sterile 18 gauge needle, a 5 mL syringe, and a
10 mL serum collection tube. Blood samples were centrifuged (3000 x g, for 15 min),
and aliquoted into 1.2 mL plastic cryogenic vials using disposable Pasteur pipettes.
Serum aliquots were frozen at —74°C until analysis for osteocalcin.

Mink were euthanized with C02 gas following blood collection. A final body
weight was taken and a gross examination was performed. Organs (adrenal glands,

brain, heart, kidneys, liver, reproductive tract, spleen, and thyroid glands) were

152

examined, collected, weighed, and fixed in 10% buffered formalin (pH 7.4) for

subsequent evaluations.

Osteocalcin Assay:

Serum samples were analyzed for osteocalcin (OC), an indicator of systemic
bone formation, using an enzyme-linked-immunosorbent-assay (ELISA). The samples
were analyzed for OC within 26 weeks of collection using an ELISA assay (Metra
Osteocalcin EIA kit, Quidel Corp., San Diego, CA). Metra TM Osteocalcin assay is a
competitive immunoassay that quantitatively measures intact (de novo) OC in serum,
using a 96-well plate. Plates were read at 405-nm optical density on a Spectra Max 340
plate reader (Molecular Devices Corp., Sunnyvale, CA). Serum samples were run in

duplicate following manufacturer’s instructions.

Mineral Content Analysis of F emurs:

At termination of the study, femurs were removed and frozen. Bones were
cleaned of all adhering tissue, weighed and length was measured prior to initial
processing. F emurs were then tagged for identification, and extracted with ethyl ether
using a Soxhlet apparatus for 24 hr. Bones were air dried under a hood for 72 hr and
weighed to determine the dry fat-free weight. F emurs then were placed into acid-
washed, porcelain crucibles and ashed in a muffle furnace (Thermolyne 30400,
Bamstead/Thermalyne, Dubuque, IA) at 500°C for 16 hr. Ash was then weighed, and

expressed as a percentage of the dry fat-free femur weight.

153

The ashed femurs were prepared for calcium and phosphorus analyses by nitric
acid wet digestion. Samples were transferred to digestion vessels with two 5 mL rinses
of 70% nitric acid (Omni Trace, EMD Chemicals, Inc., Gibbstown, NJ) and microwave
digested (MARS 5, CEM Corp., Matthews, NC). Following digestion, 2 mL of 30%
hydrogen peroxide (Sigma-Aldrich, St. Louis, MO) was added to the warm digest to
complete sample oxidation. Digests were then transferred to volumetric flasks and
brought to constant volume (50 mL) for analysis. Calcium concentration was
determined by atomic absorption spectroscopy (Unicam 989, Thermo Electron Corp.,
Franklin, MA). Phosphorus was determined spectrophotometrically by the method of
Gomori (1942).

Three skulls were retained intact from one PCB-exposed mink and one control
mink at necropsy. These skulls were cleaned of all soft tissue by dermestid beetles for

gross examination of the osseous tissue.

Statistical Analysis

Data were analyzed using Statistical Analysis System (SAS) program version
8.0 by one-way analysis of variance, and Tukey’s method was used for adjustment of p-
value in multiple tests. The mixed model procedure was used and the normality
assumption was tested accordingly, and data was log transformed to meet the

assumption of the mixed model. The level of significance was based on a Type I error

rate of 0.05 (p S 0.05).

154

 

Figure 5-1. Skull measurements of the mink (Mustela vison) to evaluate PCB 126

induced osteolysis (left lateral view of the skull).

 

 

 

 

 

 

 

Footnote: A) Skull Length; B) Parietal Height; C) Nasal to Mandible Height (jaw was
closed); D) Nasal to Palate; E) Mandible Length; F) Zygomatic Arch Width 0

(measured dorsally); G) Temporal Width I (measured dorsally).

Mink skull diagram was modified from W.J. Radke and RB. Chiasson (1998) and

Beckett, unpublished drawings.

 

155

RESULTS

Objective Criteria:

Dental malalignment (Figure 5-2) was observed on the second set of radiographs
taken at three weeks of PCB 126 exposure in one animal (R42) of 10 PCB 126 exposed
mink, in which the left maxillary incisor interdental spaces were increased. At six
weeks of PCB 126 exposure, an additional five animals (totaling six out of 10) on
treatment had mild changes in dentition alignment that included spreading of maxillary
incisors and incisor unevenness. One animal had increased interdental space between
the incisors, as well as a possible decrease in the enamel layer of the incisors.
Radiographs taken during the 12‘h week of the trial (six weeks post-PCB exposure)
revealed further dental malalignment in five of the remaining six exposed mink. In mink
that had previously exhibited interdental spreading of the incisors, radiographs
displayed progressive increases in the spreading of incisors and the dental malalignment
(Figure 5-3). Three of these mink had considerably increased interdental spacing of the
incisors that was categorized at a moderate level.

Jaw osteolysis was observed at six weeks of PCB-exposure in four animals out
of the 10 mink receiving the PCB-treated diet, with radiographic changes consisting of
loss of the maxillary and mandibular lamina dura around the canines and incisors, and
increased lucency around the canine tooth roots. At nine weeks (three weeks post-PCB
126 exposure) radiographs showed that the degree of osteolysis had increased in all of
the mink that had previously expressed lysis. At 12 weeks (six weeks post-PCB 126

exposure), all treated mink had progressive maxillary osteolysis that ranged from mild

156

to moderate. A progressive and marked increase in maxillary and mandibular osteolysis
that was considered severe in the exposed mink was observed at week 15 of the study

(nine weeks post-PCB 126 exposure) in the radiographs.

Intact Skull:

Osteolysis was observed on the intact skulls that were cleaned by dermestid
beetles and preserved (Figures 5-4 and 5-5). The destruction of bone had penetrated the
hard palate and progressed into the nasal turbinates, the frontal lobes, and the zygomatic

arches.

Radiographic Measurements (Skull and Subcutaneous Fat):

The overall change in width of the zygomatic arch significantly decreased (p =
0.03) within the PCB 126 treatment group over time (treatment-by-day effect) as
measured on the sets of radiographs (Table 5—1). The zygomatic arch width within the
control group did not change, and this measurement was not significantly different
between control mink and mink exposed to PCB 126 via their diet. Osteolysis of the
zygomatic arch, as well as the zygoma, was observed in the skulls cleaned by the
dermestid beetles (Figure 5-6). No other significant differences were observed in skull
measurements in the PCB 126-exposed mink (i.e. skull length, parietal height, nasal to

mandible height, nasal to palate height, mandible length, or temporal width).

157

Subcutaneous Fat:

Subcutaneous fat was significantly decreased (p = 0.03) in the PCB 126-exposed
mink as compared to the control mink (Figure 5-7). The subcutaneous fat measured in
the mink within the PCB 126 treatment group had significantly decreased (p = 0.0004)
over the 15-week radiographic series, (as was statistically measured as the treatment-by-

day effect).

Skeletal Changes:
No skeletal malformations or abnormalities, including osteolysis of long bones
or bone fractures, were observed in any of the mink, at any of the time points in the

radiographs.

Osteocalcin:
The bone formation marker, osteocalcin, which was analyzed in serum, did not
appear to show any cross-reactivity of the antibody with mink. Therefore, no data was

obtained from this assay.

Mineral Analyses of F emur Bones:

There were no significant differences in the parameters measured for the mink
femurs between the control mink and the mink receiving 24.0 pg PCB 126/kg feed.
Weight of femurs after bone ashing was not significantly different between the two
groups of mink with an average group weight of 0.74 i- 0.05 g and 0.66 i 0.04 g for

control and PCB-exposed groups, respectively. Mean calcium content was 245.16 i

158

4.97 and 245.95 i 4 mg Ca+ lg bone for control and PCB 126 group, respectively. Mean
phosphorus content of 107.94 3: 1.79 and 106.64 i 1.46 mg P /g bone, for control and
PCB 126 exposed mink, respectively, which were not statistically significantly

different.

Other Pathologic Changes:

Nail Growth Abnormalities: No abnormal growth of the toenails was detected
on any of the sets of radiographs. Increased toenail grth was observed visually during
daily observations in one mink exposed to PCB 126 at 16 weeks, and in several animals
from this same exposure group by 20 weeks, but only in the front feet. However, nails

were not histologically examined in this study.

Kidney Uroliths: Uroliths were detected in one control animal in week 12 of the
radiographic series. No other mink on trial, either exposed to PCB 126 or in the control

group, had any signs of urinary calculi.

Organ Changes: Other conditions that were observed on the radiographs
included cardiomyopathy (T402) and enlarged aortic arch (T600) that appeared normal
thereafter. These cardiac conditions were observed on the initial set of radiographs,
suggesting a pre-existing condition in these two animals. Cardiomegaly (R216) and
microcardia (T402) (the initial radiograph taken of T402 showed cardiomyopathy) were

observed at 12 weeks of the study. Both of these mink were receiving the PCB 126 diet.

159

DISCUSSION

Localized bone resorption of the maxillae and mandibles of female mink was
due to direct invasion of gingival squamous cell carcinoma (SCC) induced by
consumption of PCB 126 mixed into the diet at a concentration of 24.0 pg PCB 126/kg
feed. With the extent of the destruction of the lesion in the oral cavity, including the
gingiva, mandible and maxilla, assessing the aggression of the lesion in bone was
important in understanding the effects of PCB 126 and other contaminants and the
potential systemic effects they may have.

The dental malalignment expressed in one animal occurred as early as three
weeks of exposure to the PCB 126 diet. In subsequent radiographs of this same animal,
the incisors appeared to be very thin suggesting a decrease in the enamel layer, making
them appear to be floating in the maxilla. This has been explained by the destruction of

the lamina dura, which can cause the teeth to appear as if they are floating in the

160

 

Figure 5-2. Dental malalignment and increased interdental spacing in mink induced by
consumption of a diet containing 24.0 pg PCB 126/kg feed. A) Notice inflammation of
gingival tissue and moderate spreading of incisors. B) Canines appear extended and

incisors were missing (mandibular and maxillary); gums were bloody and swollen.

 

 

 

 

A) B)

 

161

 

Figure 5-3. Radiographic images of female mink (Mustela vison) showing dental
malalignment and increased interdental spacing induced by dietary exposure to 24.0 pg
PCB 126/kg feed. A control mink (left) depicts normal lucency in jaws and alignment
of teeth compared to a PCB 126 exposed mink (right), at Week 12 (6-week PCB-

exposure period followed by six weeks of being on control feed).

 

 

Control PCB 126

 

162

 

Figure 5-4. Extensive osteolysis of mandibular and maxillary alveolar bone in a female
mink (Mustela vison) exposed to 24 pg PCB 126 /kg feed. Note: no bony support

surrounding dentition, resulting in loss of teeth. (Skull cleaned by dermestid beetles.)

 

 

 

163

 

Figure 5-5. Bone lysis and tooth loss observed in the mandible, maxilla, and nasal
bones in mink exposed to 24.0 pg PCB 126/kg feed. (Skulls cleaned by dermestid

beetles.)

 

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Figure 5-6. Osteolysis of the zygoma and zygomatic arch in a female mink (Mustela

vison) exposed to a diet that contained 24.0 pg PC8126/kg feed.

 

 

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166

 

Figure 5-7. Radiographic image of a female mink (Mustela vison) exposed to 24 pg
PCB 126 /kg feed via the diet for six weeks, and then on control feed for three weeks.
Radiograph depicts decreased subcutaneous fat (arrow), and increased interdental

spacing of the incisors, as well as increased lucency in the jawbones.

 

 

 

 

 

*Same radiograph (of the same mink) shown in positive and negative film exposure.

 

167

demineralized maxilla and mandible, thus allowing teeth to become displaced (Myer
1994).

The number of affected mink and the severity of the dental malalignment
continued to increase as the trial progressed, and led to periodontoclasia. Tooth
loosening and displacement varied from subtle to complete tooth loss over the course of
the trial, even following removal of the PCB 126 after week six. The radiographs taken
during the twelfth week of the trial revealed further dental malalignment in exposed
mink as compared to the control mink. In several of the PCB 126 treated mink, bone
support was completely lost around the roots of one or more of the canine teeth in
addition to increased interdental spacing of the incisors that was observed. During
regular observations and monitoring, several of the treatment mink displayed canine
teeth that appeared to be “growing” longer, or extended from previous observations.
Two possible explanations were considered for this observed change in canine length.
Firstly, the canine could have appeared longer due to the recession of the gums or
gingiva from around the base of the tooth. Secondly, this apparent lengthening could
have been due to the loss of connective support of the teeth, including the loss of
periodontal ligament and the alveolar bone, that allowed the tooth to actually loosen and
extend out of the socket.

Maxillary and mandibular lysis in the mink was first observed in the radiographs
as a mild loss of the lamina dura around the canines and incisors of the maxilla, and also
increased lucency around the roots of the canines that eventually led to increased
mobility of the teeth. This lucency was detected in mink as early as six weeks following

exposure to the PCB 126 diet. Osteolysis around the teeth was observed as both focal

168

 

and diffuse in the mink jaws. However, as the trial continued, the loss of osseous tissue
became diffuse through the jaws. Bone resorption became so severe, that no bony
support remained around the incisors, canines, or the premolars in some of the mink that
were exposed to the PC8126 diet (Figure 5-4).

Four tissue structures comprising the periodontium, the periodontal ligament,
gingiva, alveolar bone, and cementum (Jones and Boyde, 1999) appear to be adversely
affected in mink exposed to PCB 126. Receding gingiva, loss of periodontal ligament,
and loss of alveolar bone support were factors that contributed to the spreading and
malalignment of teeth noted in the radiographs in this study.

Oral gingiva and the periodontal ligament appear to be adversely affected by
invasion of squamous epithelial cells prior to osteolysis of the lamina dura. Thus, tooth
support is decreased by the loss of fibrous connective tissue provided by the periodontal
ligament, as well as the surrounding gingival tissue. However, in the radiographs,
lucency around teeth was observed prior to the gingival and periodontal effects. The
loss of tooth support from the periodontal ligament, the gingival tissue, and alveolar
bone resulted in the teeth spreading, loosening, and becoming displaced, and often
falling out. Previous studies by Render et al. (2000a, 2000b, 2001) originally described
the oral lesion induced by dietary exposure to 2,3,7,8-tetrachlorodibenzo—p-dioxin
(TCDD) and 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126) as squamous epithelial
proliferation. Dental clumping and mandibular and maxillary osteolysis in mink kits
exposed to PCB 126, including loose and displaced dentition following the PCB 126
exposure for one to two months. In addition, the mink kits had nodular proliferation and

gingival thickening of the oral mucosa (Render et al. 2000b).

169

Because osteolysis of the lamina dura was observed, this indicated a malignant
neoplasm, as opposed to a benign lesion that would more resemble compression, and/or
displacement of the lamina dura (Rosenberg 1999; Thompson and Pool, 2002;
www.dentistry.vcu.edu). In addition, aggressive malignant neoplasms have a tendency
to invade, expand, and perforate the cortex of the maxillae and mandibles
(www.dentistrv.vcu.edu). The destruction and aggressiveness of this oral SCC caused
partial lysis of the cortex of the maxillae and mandibles, as well as partial to complete
lysis of several tooth sockets. Bone lysis and dental displacement continued to worsen
following removal of the PCB 126 stimulus. In mink from this study that exhibited
severe oral neoplasia and osteolysis, the osteolysis progressed into the nasal turbinates
and caused destruction throughout the nasal passage and frontal bones over time.

The width of the zygomatic arch significantly decreased during the course of the
trial in PCB 126 exposed mink within the treatment group. The aggressive osteolysis
affecting the maxilla had progressed upward into the zygoma, compromising its
structural integrity and allowing the zygomatic arches to deviate axially, or fold in upon
itself. This effect may also be related to, or explain, the visual effects of flattening and
broadening of skulls that were noted in a prior trial of mink exposed to 24.0 pg PCB
126/kg feed.

No other skull measurements were significantly different between control and
treatment mink, or within the treatment group over time. However, in a study by Allen
and Leamy (2001), the authors used fluctuating asymmetry (FA) to assess the effects of
low doses of TCDD on size and shape of the mandibles in offspring from adult mice

exposed during pregnancy. The results from that study showed that TCDD decreased

170

the size of mandibles in mice. The largest effect was on the mandible landmark in close
proximity to the teeth, particularly the molar tooth row area of the mandible. Allen and
Leamy (2001) suggest that FA has become an important diagnostic tool in areas such as
environment and conservation biology, and ecotoxicology. However, based on the
results from the current study with mink, skull measurements were not affected by
exposure to PCB 126, with the exception of zygomatic arch width, which was the result
of osteolytic changes.

The results from various mineral analyses such as percent bone ash, and femur
Ca and P concentrations indicated that there were no differences between control mink
and mink that received the diet containing PCB 126. These data, in addition to the fact
that there were no observed changes on the radiographs, indicated that a systemic effect
on bone resorption was not occurring in mink consuming the PCB 126 diet. Contrary to
our study, Lind et al. (1999) demonstrated pronounced effects in rats by PCB 126 that
resulted in decreased length of tibiae, as well as increased bone mineral density and
cortical thickness. Exposure to PCB 126 impaired the mineralization process of tibiae as
indicated by significant increases in organic content and in osteoid surface (Lind et al.
1999). In another exposure study, Andrews et al. (1989) reported that commercial PCBs
(Aroclor 1254) caused changes in the structural morphometry of the femur in young,
growing rats. F emur weight, volume, and length were significantly decreased, and
femur density was significantly increased in rats exposed to 25 mg/kg Aroclor 1254
(Andrews et al. 1989). Jamsa et al. (2001) reported TCDD-induced inhibition of tibial
grth in Han/Wister rats at a concentration of 170 pg/kg BW, and the same effect

observed in Long-Evans rats at a concentration of 1.7 pg/kg BW.

171

Although osteocalcin (OC) was analyzed in this study, the antibody did not
appear to show cross-reactivity in mink. Based on the use of the OC assay in various
carnivorous species, the ELISA kit was believed to show positive cross-reactivity of the
antibody with mink protein. Osteocalcin (bone Gla protein) is one of the principal non-
collagen proteins found in bone and dentin (Gundberg and Nishimoto, 1999; Jones and
Boyde, 1999), and contains y—carboxyglutamate residues that bind to hydroxyapatite
(Gundberg and Nishimoto, 1999). Osteocalcin is secreted by osteoblasts (bone-forming
cells), odontoblasts, and hypertrophic chondrocytes and is therefore known as a bone
formation marker. Decreases in OC concentrations in serum have been reported as a
result of dietary exposure to PCB 126 (Lind et al, 2000). Although no data were
obtained for this study, OC has proved to be a valuable marker in bone research (Body
1999; Gundberg and Nishimoto, 1999).

Osteolysis affecting the skulls and jaws of other wildlife species has been
reported, but not frequently. A disease complex of the skull reported in beluga whales
(Delphinapterus leucas) (Beland et al. 1993) and Baltic grey seals (Halichoerus grypus)
(Bergman et al. 1992; Lind et al. 2003) may be a similar lesion to that observed in
laboratory studies with mink (Render et al. 2000a, 2000b, 2001), and now reported in
wild populations of mink (Beckett et al. 2005). Similar to the lesion reported in mink,
characteristics of the skull disease complex reported in the Baltic seals and belugas
included loss of alveolar bone, widening of the teeth sockets, tooth loss, osteolysis, and
frequent severe erosions and perforations of masticatory bones. High PCB and DDT
concentrations in belugas from the St. Lawrence Estuary (Beland et al. 1993) and high

organochlorine compounds in Baltic seals from the Baltic Sea (Lind et al. 2003) may

172

have contributed to the lesions that developed into the skull disease complex. A skull of
a mountain sheep exhibited some characteristics that appear similar to the lesion
presented in this study (Bleich et al. 1990). In addition to lateral asymmetry of the
cranium and rostrum, the skull exhibited areas of osteoporosis in the mandible and the
alveolar process of the maxilla, as well as malocclusion of the molars (Bleich et al.
1990). However, the authors eventually concluded that the lesion was caused by
malnutritional osteoporosis.

Following accidental PCB exposures in Yusho, Japan and Taiwan, some human
newborns of victims that ingested PCB contaminated rice oil were noted to have poor
skeletal development (Yamashita and Hayashi, 1985; Rogan et al. 1988). These skeletal
changes in neonates included spotted calcification of the parieto-occipital area of the
skull, early eruption of teeth, and gingival hyperplasia (Yamashita and Hayashi, 1985;
Rogan et al. 1988). These skeletal and oral changes in humans that were caused by the
PCB exposure suggest some similarity to the neoplastic lesion that is being reported in
mink.

Body weights of the female mink were taken weekly, and the significant
differences in measurements of subcutaneous fat that were observed on the radiographs
corresponded with loss in body weights that were recorded during the same three-week
intervals (data not shown). As seen in other studies with mink, animals exposed to this
class of compounds experience significant weight loss, often termed “wasting
syndrome”, that may not be entirely due to a decrease in food consumption. The weight
loss observed in the PCB-exposed mink may have been complicated by conditions of

neoplastic disease as well.

173

Five female mink from the PCB treatment group expressed classic signs of
cachexia, which is the indirect result of malignant neoplasia. Animals often exhibit
anorexic, generalized wasting, and overall weakness (Cotran et al. 1999). Several of
these PCB exposed females either died or were euthanized during week six of the trial,
and others were euthanized between the 9th and 12th weeks of the trial, due to their poor
condition and deteriorating health. The loss of these females may account for the
statistical increase reported for the radiographic measurement of subcutaneous fat that
was taken during the 12th week of the trial (Table 5-1). In addition, several of the mink
that expressed the lesion also exhibited aphagia. This resulted from the presence of the
lesion, and was due to gums that were swollen and often bloody, and the mobility of the
teeth. Often the osteolysis was so extensive throughout the jaw that several of the PCB
exposed mink exhibited the loss of teeth. Mink that expressed any difficulty in eating
were offered additional feed that had been watered down to minimize their need to

chew food.

Neoplastic Bone Lysis:

The bone lysis reported in the present study may have occurred as a result of a
type of soft tissue neoplasia that caused a direct effect on the adjacent bone.
Histologically, lysis occurred as a result of invasion of the bone by the cancer cells.
Alternatively, neoplasms can also release mediators that cause a direct osteolytic effect
through the local release of proteolytic enzymes by either tumor cells or host stromal
cells, or both, to break barriers such as basement membranes, cell-cell junctions, and the

extra-cellular matrix (Cramer et al. 1981; Body 1999; Cockerell and Cooper, 2002).

174

Bone resorption can be affected by neoplastic cells through mediators of
osteoclast-activity, causing an increase in the stimulation and proliferation of
osteoclasts (the bone cells responsible for resorption) (Body 1999). Osteoclast-
activating factor can be mediated by either tumor cells or normal host cells that act
systemically and distant to the tumor (Sasaki et al. 1998; Cockerell and Cooper, 2002).
One of the primary cellular mechanisms of osteolysis associated with metastatic cancer
may be osteoclast-mediated (Sasaki 1998).

A form of paraneoplastic syndrome may be occurring in mink exposed to PCB
126 in this study. However, in mink observed with the bone lysis and oral lesion, the
duration between lesion progression and the demise of the mink was rapid and thus,
prohibiting systemic changes. Hypercalcemia, the most common paraneoplastic
syndrome, is frequently associated with malignant neoplasia, including squamous
carcinoma, and results from the invasion of bone (Dobson and Gorman, 1991).

The mink exposed to PCB 126 in this study may have succumbed to death
before the cancerous state of the lesion had spread systemically. Neoplastic diseases are
reported less frequently in mink. This is because mink are generally maintained on
commercial fur farms, and pelted before they reach old age. However, because mink
and ferrets (Mustela putoriusfuro) are both mustelids and closely related, one would
expect the potential for a similar occurrence in tumor rate. In the domestic ferrets,
lymphoma is the most common type of neoplasm encountered, with both solid and
leukemia types being reported (Williams 1991). Therefore, if the mink in this study had
survived to old age it is possible that the neoplastic lesion would have progressed

systemically.

175

The specific mechanism of action of PCBs or TCDD on bone has not been fully
elucidated. There may be more than one mechanism, and potentially a complex of
integrated pathways that is required for specific effects on bone, either locally or
systemically. Species differences may exist in the mechanism(s) of action responsible
for the bone lysis observed between mink in the present study and rats and mice from
studies referenced herein. Also, rodents may be less susceptible to the toxic effects of
PCBs and TCDD due the rodents’ ability to rapidly metabolize the chemicals (Huff et
al. 1991). Because the mechanism inducing this neoplastic lesion in mink in this study
has not been elucidated, it is suggestive that PCB 126 may interact through several

different and complex pathways, thus altering or interacting with the endocrine system.

Other Changes:

Orthokeratotic hyperkeratosis of the toenails occurred in mink exposed to PCB
126 over the length of the study. Radiographs did not reveal any osteolytic changes in
the nails. However, clinical changes of the nails, including marked thickening,
elongation, and deformity were observed during daily monitoring. Only the front nails
had abnormal growth, and nails on the hind feet appeared normal. Minimal grth in
the nails was first noticed at approximately 16 weeks of the trail in mink exposed in the
PCB 126 diet. The observed growth in nails varied between mink in the PCB 126-
exposure group. Although two mink exposed to PCB 126 had minimal changes in nail

growth, two other mink in this same group had extreme nail growth that continued until

176

termination of the project. The nail growth in these two mink often hindering their
mobility to move around their cages, because the nails would get caught (hooked) on
the wires.

The one mink that radiographically displayed uroliths, or stones, was a control
mink that had not previously been a part of the selected control group for films. Most
likely, based on the size of the stones, this was a pre-existing condition prior to the start
of this study. No other calculi were observed during this trial. Thus, it indicates that
PCB 126 does not increase the incidence of acute urolithiasis with the presence of
urinary calculi. A description of calculi and urolithiasis in mink was presented
previously (Chapter 3).

Radiographs showed different cardiac conditions in the mink such as
cardiomegaly and microcardia. One mink had an enlarged right aortic arch on the initial
radiograph, which appeared normal thereafter. However, adult female mink receiving
the diet containing PCB 126 showed a significant reduction in mean heart weight
compared to the control group (Chapter 3). In support of that finding, Hochstein et al.
(1998) also observed a significant decrease in mean heart weight in surviving mink
exposed to 1 pg TCDD/kg feed in their diet compared to the control mink. The

mechanism resulting in this effect is unclear.

177

CONCLUSIONS

Mink exposed to PCB 126 exhibited a neoplastic bone lesion characterized by
osteolysis of the mandible and maxilla. However, the effects of PCB 126 on bone were
not manifested systemically in mink, and no changes in mineral analyses were
observed. Based on the current mink studies, the observed osteolysis of maxillae and
mandibles is similar to the lesion that has been reported in other wildlife species
exposed to environmental contaminants. This neoplastic lesion in mink exposed to PCB
126 may also be similar to the oral lesions that have been reported in humans from
Yusho, Japan and Taiwan. However, PCB 126 apparently does not act similarly in mink
as it does in rats and mice (Lind 1999). Therefore, when performing comparative
studies it is imperative to consider the possibilities of species differences, especially
when extrapolating studies to other species, such as humans in a risk assessment

situation.

178

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Zellen, G. 1996. Chapter 16: Urinary System of Mink. I_n: Mink: Biology, Health, and

Disease. D.B. Hunter and N. Lemieux (Eds), Graphic and Print Services, University of
Guelph, Guelph, ON.

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CHAPTER 6

Squamous Epithelial Lesion of the Mandibles and Maxillae of
Wild Mink (Mustela vison) Naturally Exposed to
Polychlorinated Biphenyls.

K.J. Beckett, S.D. Millsap, A.L. Blankenship, M.J. Zwiernik, J .P. Giesy
and SJ. Bursian

2005

Environmental Toxicology and Chemistry, 24(3):674-677

183

Squamous Epithelial Lesion of the Mandibles and Maxillae of Wild Mink
(Mustela vison) Naturally Exposed to Polychlorinated Biphenyls.

ABSTRACT

Approximately 125 km of the Kalamazoo River, located in southwestern
Michigan, USA, are designated as a Superfund site with polychlorinated biphenyls
(PCBS) as the contaminant of concern. Mink (Mustela vison) are a naturally occurring
predator in this area, and also a species of concern due to their known sensitivity to
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and structurally similar compounds such
as PCBs. Four of nine mink trapped from the Kalamazoo River area of concern
(KRAOC) exhibited histological evidence of a jaw lesion previously identified in ranch
mink. The jaw lesion, proliferation of squamous epithelium in the mandible and
maxilla, is known to be caused by 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126) and
TCDD. Mink trapped from an upstream reference area (Fort Custer Recreation Area,
FCRA) did not exhibit the lesion. Mean concentrations of total PCBs were 2.8 and 2.3
mg/kg, wet wt in the livers of mink from the KRAOC and F CRA, respectively, and
TCDD toxic equivalent (TEQ) concentrations were 0.30 and 0.11 pg TEQs/kg, wet wt,
respectively. There were significant correlations between the severity of the lesion and
hepatic concentrations of total PCBs and TEQs This is the first published report of the

lesion occurring in wild mink.

184

INTRODUCTION

The Kalamazoo River Superfund site is located in southwestern Michigan, USA
and encompasses approximately 125 km (80 miles) of the Kalamazoo River, from
Morrow Dam in Kalamazoo County to Lake Michigan. The Kalamazoo River area of
concern (KRAOC) became contaminated with polychlorinated biphenyls (PCBs) by
waste discharged from the recycling and processing of carbonless copy paper where the
primary source of the PC88 was Aroclor 1242 and to a lesser degree Aroclor 1254
(Camp et al. 1999).

Mink are a naturally occurring predator in the Kalamazoo River area, and also a
species of concern due to their known sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) and compounds of similar structure such as PCBs (Aulerich and Ringer, 1977;
Aulerich et al. 1985; Hochstein et al. 1988; Heaton et al. 1995; Hochstein et al. 1998;
Pastva 2003). To assess the possible effects of PCBs on wildlife residing in the
Kalamazoo River basin, wild mink were trapped from two areas, the KRAOC and the
Fort Custer Recreation Area (F CRA), a reference area with lesser concentrations of
PCBs (Pastva 2003).

The potential for adverse effects of PCBs on mink in the KRAOC was estimated
using two approaches, by applying dietary exposure models and by measuring liver
concentrations of TCDD toxic equivalents (TEQs) and total PCBs in trapped mink
(Pastva 2003). The concentrations of total PCBs and TEQs measured in the livers of the

wild mink were in the range of those of ranch mink that had expressed proliferation of

185

 

squamous epithelium in the mandibles and maxillae when exposed to PCBs in the diet
(Bursian et al. 2003).

Previous studies have indicated that ranch mink of different ages exposed to
24.0 pg 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126)/kg feed or 2.4 pg TCDD/kg feed
developed clinical signs of mandibular and maxillary squamous epithelial proliferation
that in severe cases resulted in the loss of teeth (Render et al. 2000a; 2000b; 2001). In
an unpublished study (K. Beckett, Michigan State University, East Lansing, MI), we
have shown that mink fed diets containing concentrations as low as 0.24 pg PCB
126/kg developed the clinical lesion, although the required period of exposure was
longer. The lesion develops when squamous epithelial cells form infiltrating nests and
cords into the periodontal ligament and alveolar bone, which results in osteolysis.
Infiltration of squamous cells into the periodontal ligament eventually causes loose and
displaced teeth. The maxillae and mandibles became markedly porous due to loss of
alveolar bone, with concomitant loss of teeth, leading, in severe cases, to aphagia (the
inability to eat).

The purpose of this study was to determine if wild mink inhabiting a PCB-
contaminated environment exhibited squamous epithelial lesions of the mandibles and
maxillae that until now have been reported only in ranch mink exposed to PCB 126 or

TCDD under laboratory conditions.

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METHODS

Wild mink were trapped throughout the KRAOC and the upstream reference
area, F CRA, during the winters of 2000, 2001, and 2002 (Pastva 2003). Nine male mink
were collected within the KRAOC from D Avenue downstream to the former
Trowbridge impoundment, a distance of approximately 25 river km. Three mink (two
males and one female) were collected from the upstream reference area at Fort Custer,
approximately 25 river km upstream of D Avenue, in Kalamazoo, MI, USA. Frozen
mink carcasses were brought to the laboratory and thawed. A gross necropsy was
performed by a board-certified veterinary pathologist. The weight (without pelt) and
length (nose to base of tail) of each mink were determined. Organ condition was
assessed during necropsy to determine if there were gross abnormalities. Samples of the
livers were collected for histological assessment, as well as for determination of total
PCB and TEQ concentrations.

For chemical analysis, polychlorinated biphenyls were extracted from liver
tissue based on US. Environmental Protection Agency method 3540 (Soxhlet
extraction) (US. EPA 1996). An aliquot of the extract was analyzed for total PCBs with
a gas chromatograph (Perkin Elmer Autosystem, Boston, MA, USA) equipped with a
63 Ni electron capture detector according to Nakata et al. (1998). A second aliquot of the
extract was subjected to carbon column chromatography for the separation of non-
ortho-substituted (coplanar) PCB congeners. The resulting extracts were analyzed by

gas chromatography-mass spectroscopy on a Hewlett Packard 5890 Series 11 gas

187

chromatograph (Rolling Meadows, IL, USA) equipped with a Hewlett Packard 5972
series mass selective detector (Pastva 2003).

The toxic equivalency method relates the toxicity of TCDD-like chemicals
including certain polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated
dibenzofurans (PCDFs) and PCBs to the toxicity of TCDD, which is considered the
most toxic of this group of chemicals. In this study, TEQs were calculated by
multiplying the concentration of an individual PCB congener by its toxicity equivalency
factor (TEF) as presented in Van den Berg et al. (1998). The TEF denotes a congener’s
toxicity relative to TCDD, which has a toxicity value of 1.0. Total TEQ concentrations
in liver samples were determined by summing the concentrations of TEQs of individual
congeners detected in each sample. PCB congeners 77, 81, 105, 118, 126, 156, 157, 167
and 169 were used for the calculation of TEQs (Pastva 2003).

In all cases, heads were removed from carcasses and fixed in 10% neutral-
buffered formalin. Heads were then decalcified in Surgipath® Decalcifier II (Surgipath
Medical Industries, Richmond, IL, USA) and trimmed, and the mandibles and maxillae
collected. Jaws were processed for histological examination, sectioned to 5 pm, stained
with hematoxylin and eosin, and the slides examined using light microscopy.

Lesions, the development of which is a dynamic process, were rated from mild
to severe based on the following criteria: a rating of “mild” is characterized by one or a
few small, focal cyst(s), which is the initial phase of the lesion; a rating of “moderate” is
characterized by the presence of several foci of cysts or islands of squamous epithelial
cells, or a few larger cysts, that invade the periodontal pockets and disrupt the teeth; a

rating of “severe” is assigned when cysts, increasing in size and invasiveness, infiltrate

188

throughout the mandible and/or maxilla, destroying all surrounding tissues including the
alveolar bone that supports tooth structure. Correlation between total PCB and TEQ
concentrations in the liver and the severity of the lesion (numerically scored from one to
three based on the above rating description) was assessed by the Pearson correlation
analysis using SAS PROC CORR (Statistical Analysis System, Release 8.0, Cary, NC,

USA).

RESULTS

Mink from both the KRAOC and F CRA areas appeared to be healthy based on
body weights, gross examination and histological assessment of liver tissue. However,
four of the nine mink from KRAOC had histological evidence of mandibular and
maxillary squamous epithelial proliferation (Figure 6-1), while none of the mink in the
F CRA had the lesion. The severity of the lesion ranged from mild to moderate (Table
6-1).

Total concentrations of PCBs in the livers of the four mink from the KRAOC
that expressed the lesion ranged from 2.9 to 6.0 mg/kg, wet wt. Total PCB
concentrations in livers of the other five mink from the KRAOC ranged from 0.05 to
3.4 mg/kg, wet wt. Concentrations of TEQs in the livers of the four mink from KRAOC
that exhibited the lesion ranged from 0.21 to 1.3 pg/kg, wet wt and from 0.02 to 0.25
pg/kg, wet wt in the five mink not expressing the lesion (Table 6-1). Concentrations of

total PCBs in the livers of the three mink from the FCRA ranged from 1.6 to 3.7 mg/kg,

189

wet wt, while concentrations of TEQs ranged from 0.05 to 0.20 pg/kg, wet wt (Table
6-1). PCB 126 is known to be a potent inducer of squamous proliferation of the
mandibles and maxillae (Render et al, 2000a; 2000b; 2001). In this study, the primary
PCB congener contributing to the TEQ concentrations in livers of the mink trapped in
the Kalamazoo River Basin was PCB 126 with an average contribution of 77% at
KRAOC and 69% at F CRA (Table 6-1).

Lesion severity was significantly and positively correlated with both
concentrations of total PCBs and TEQs in livers of mink collected from the KRAOC.
Correlation coefficients (r2) were 0.88 (p < 0.001) and 0.89 (p < 0.001) for total PCBs

and TEQs, respectively.

DISCUSSION

When six- and 12-week-old ranch mink kits were exposed to 2.4 pg TCDD or
24.0 pg PCB 126/kg feed, they displayed gross displacement of incisor and canine teeth
with nodular swelling of the mandibular and maxillary gingiva within four weeks of
exposure. The PCB 126- and TCDD-induced lesion reported in these initial studies was
characterized by proliferation of squamous epithelial cells that formed infiltrating nests
and cords into the periodontal ligament and the alveolar bone causing osteolysis. The
maxillae and mandibles became markedly porous due to loss of alveolar bone, with

concomitant loss of teeth, which led to aphagia and rapid weight loss (Render et al.

2000a;2000b;2001)

190

The lesion subsequently was reported to develop by 24 weeks of age in ranch
mink kits exposed to 0.24 pg PCB 126/kg feed in utero through sexual maturity (K.
Beckett, Michigan State University, East Lansing, MI). Histological evidence of
proliferation of squamous epithelial cells in the mandibles and maxillae was also
observed in adult mink fed a diet containing 0.24 pg PCB 126/kg feed (K. Beckett,
Michigan State University, East Lansing, MI), which is an environmentally relevant
concentration (Hochstein et al. 1988; Tillitt et al. 1996; Bursian et al. 2003). Hepatic
concentrations of PCB 126 and TCDD were not measured in the initial jaw lesion
studies by Render and associates (Render et al. 2000a; 2000b; 2001).

Concentrations of total PCBs and TEQs in the liver were measured in a recent
study where ranch mink fed diets containing PCB-contaminated fish collected from the
Housatonic River in Massachusetts, USA developed the jaw lesion (Bursian et al.
2003). In that laboratory study in which mink were exposed to 1.6 mg total PCB/kg
feed (0.02 pg TEQs/kg feed) from conception through 31 weeks of age, the lesion was
detected at a similar frequency as reported in the present study. The average
concentrations of total PCBs and TEQs in the livers of mink fed the diet containing
Housatonic River fish were 3.5 mg/kg, wet wt and 0.10 pg/kg, wet wt, respectively. Of
the mink trapped in the KRAOC, 44% had histological evidence of mandibular and
maxillary squamous epithelial proliferation. The average concentrations of total PCBs
and TEQs in the livers of KRAOC mink exhibiting the lesion were 4.3 mg/kg, wet wt
and 0.60 pg/kg, wet wt, respectively. The average total PCB concentration in the livers

of the KRAOC mink is comparable to the value reported by Bursian et al. (2003) in

191

 

Figure 6-1. Mandibular and maxillary squamous epithelial hyperplasia in wild mink
exposed to polychlorinated biphenyls in the Kalamazoo River area of concern
(KRAOC). A: A section from a control mink is shown for reference. Note the solid-
appearing alveolar bone (AB) surrounding the teeth (T), which is above the gingiva
(GIN). B: KRAOC Mink #03 had a lesion rating of mild, with one focus (arrow). C:
KRAOC Mink #06 had a lesion rating of mild-moderate, with a large invasive cyst
(arrow) and small multi-focal difiuse nests. D: KRAOC Mink #08 had a lesion rating of
moderate, with diffuse squamous cysts throughout the jaw (arrows). Extensive
infiltration by large islands of squamous epithelial cells has occurred and formed cysts
that resulted in the loosening of teeth. These cysts may contain centers of exfoliated

epithelia and sometimes keratin.

 

 

 

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Table 6-1. Incidence of mandibular and maxillary squamous hyperplasia, and hepatic
concentrations of total polychlorinated biphenyls (PCBs), toxic equivalents (TEQs) and
percent contribution toward toxic equivalents by 3,3’,4,4’,5-pentachlorobiphenyl (PCB

126) in wild mink trapped in the Kalamazoo River Basin“.

 

 

Mink # Trapping PCBs TEQs PCB 126 Lesion
Location (mg/kg, wet wt) (pg/kg, wet wt) (%) Rating
08 KRAOC 6.0 l .3 91 Moderate
06 KRAOC 5.0 0.58 90 Mild-moderate
02 KRAOC 2.9 0.33 83 Mild
Ol KRAOC 3.4 0.25 86 No lesion
03 KRAOC 3.3 0.21 72 Mild
07 KRAOC 1.0 0.03 68 No lesion
09 KRAOC 0.05 0.03 99 No lesion
1 l KRAOC 1.6 0.03 0 No lesion
25 KRAOC 1.1 0.02 26 No lesion

2.8107 0.301014

1002 FCRA 3.68 0.20 80 No lesion
1003 FCRA 1.55 0.08 59 No lesion
1001 FCRA 1.59 0.05 68 No lesion

2.31-0.7 0.11 10.04

 

a KRAOC refers to the contaminated Kalamazoo River area of concern. F CRA refers to
the Fort Custer recreation area, which is the upstream reference site. Data for total PCB
and TEQ concentrations in the liver are presented for individual animals and as the

mean i standard error of the mean (in italics) for each of the two sites.

193

 

mink exposed to PCBs through consumption of fish collected from the Housatonic
River (3.5 mg PCB/kg, wet wt). Concentrations of TEQs in the livers of mink from the
KRAOC were six-fold greater than those in the livers of mink fed diets containing fish
from the Housatonic River (0.10 ug/kg, wet wt). Hepatic concentrations of total PCBs
and TEQs in livers of mink fed Housatonic River fish were determined in a pooled
sample that was comprised of tissue from all animals in the group, regardless of lesion
status. This could explain the difference in TEQ concentrations between these two
studies. It is possible that hepatic concentrations of TEQs in only those mink on the
feeding study exhibiting the lesion were greater than the group average (0.10 ug/kg, wet .
wt) and closer to the average reported for the KRAOC mink that had the lesion.

There was one mink each from the KRAOC and F CRA that did not develop the
lesion (Table 6-1), yet had concentrations of total PCBs and TEQs in the liver that
would be expected to be associated with the expression of mandibular and maxillary
squamous epithelial hyperplasia. One explanation is individual variation in sensitivity to
TCDD-like chemicals. In addition, the duration of exposure, the age during exposure
and/or the time required for the lesion to develop could have been suboptimal for its

manifestation in these individuals.

CONCLUSIONS

In conclusion, the data presented here indicate that wild mink trapped in the

Kalamazoo River area of concern exposed to environmentally-derived PCBs developed

194

 

the lesion characterized as mandibular and maxillary squamous epithelial hyperplasia.
The average concentrations of total PCB and TEQ in livers of mink trapped from
KRAOC that expressed the lesion were 4.3 mg/kg, wet wt and 0.60 ug/kg, wet wt,
respectively. There was a positive correlation between hepatic total PCB and TEQ
concentrations and the presence and severity of the jaw lesion observed in wild mink.
Therefore, mink exposed to relevant concentrations of TCDD-like compounds could
experience increased severity of the lesion leading to erosion of the mandibles and
maxillae with concomitant loss of teeth, and eventually aphagia. This jaw lesion, now
identified in a wild mink population, poses a threat to wildlife health and survival. The
results of this study are consistent with the predicted hazard quotients based on
concentrations of both total PCBs and TEQs in the liver and effects on survival and
weight gain in mink kits whelped of mink exposed to these compounds in the diet.
However, even though the lesion was observed in this population of mink, the body
conditions of the mink did not indicate that it was resulting in effects on body weight
(Pastva 2003). These results indicate that the occurrence of the lesion in mink is a
sensitive, functional measure of exposure to PCBs in the diet, but may not translate into
population level effects. This may be due to several compensatory factors, including
sufficient reproductive capacity to offset adverse effects on the longevity of individual
mink or the fact that the lesion does not become sufficiently severe during the normal

lifetime of mink living in the wild.

195

 

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