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This is to certify that the

thesis entitled

LONG-TERM EFFECTS OF GREAT LAKES FISH DIET
' ' 0N REPRODUCTION IN MICE

presented by

Chang-Vi Lin

has been accepted towairds fulﬁlhnent
of the requirements for

Master of Science degree in Animal Science

Major professor

Date December 11, 1997

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DATE DUE DATE DUE

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LONG-TERM EFFECTS OF GREAT LAKES FISH DIET
ON REPRODUCTION IN MICE

By

Chang-Yi Lin

A THESIS

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

MASTER OF SCIENCE

Department of Animal Science

1 997

ABSTRACT

LONG-TERM EFFECTS OF GREAT LAKES FISH DIET
ON REPRODUCTION IN MICE

By

Chang-Yi Lin -

Pregnant C57BL/6J mice, bred to DEA/2] males, were randomly
divided into three treatment groups: C (82% lab chow with 18% ﬁsh oil),
I (60% Iowa carp with 40% lab chow) and G (60% Great Lakes carp with
40% lab chow). The treatment diets were provided from parturition through
lactation. The offspring (F-l) were fed their respective diets from weaning
to termination. High neonatal mortality was observed in the G treatment
group. Decreases in body weights were also observed in F-l males and
females of the same treatment group. F-l males at l-year of age were
paired with non-treated females, low fecundity was observed in the G
treatment group. At 53 weeks of age, sperm concentration and in vitro
sperm fertilizing ability of mice in the G treatment group were signiﬁcantly
lower than in the C and I treatment groups. F-l female mice in the G

treatment group had a delay in vaginal opening.

ACKNOWLEDGMENT

I would like to thank the following members of my committee for
their advice and guidance: Drs. Robert Cook, Steven Bursian, Michael
Kamrin, and Karen Chou. I am especially grateful to Dr. Karen Chou for
believing in me and giving me the opportunity to pursue a master of Science

degree.

Special thanks to Amy Barber, Barbara Salem, Chai-Ching Lin,
Debbie Powell, Jeng-Fung Huang, Li Chen, Mark Dow, Rochelle Inglis,
and Yu—Min Kuo who made my years at MSU enjoyable. Their
contributions were essential to the completion of this research. I would like

to also thank Dr. Pau Ku for his advice on diet preparation.

Finally I would, express deepest thanks and gratitude to my wife,
Sharon, my parents, Te-tao and Kuei-fung Lin, and my son Andy. Without
their support and encouragement, the completion of this thesis would not

have been possible.

iii

TABLE OF CONTENTS

LIST OF TABLES ............................... vi
LIST OF FIGURES .............................. xiii
LIST OF ABBREVIATIONS ......................... ix
INTRODUCTION ................................ 1
LITERATURE REVIEW ........................... 5
The Great Lakes ............................. 5
Contaminants of the Great Lakes ................... 5
Polychlorinated biphenyls (PCBs)
-General ............................. 8
-PCBs and Reproduction ................... 13
Dichlorodiphenyltrichlorethane (DDT)
-General ............................. 19
-DDT and Reproduction .................... 21
Mercury
~General ............................. 25
-Mercury and Reproduction .................. 28
HYPOTHESIS .................................. 31
MATERIALS AND METHODS ....................... 32
Animals .................................. 32
Diet Preparation ............................. 32
Chemicals and Culture Medium .................... 34
Experimental Design .......................... 34
Data Collection .............................. 35
Body Weight ........................... 35
Feed Intake ............................ 35

iv

Organ Weight ........................... 35

Vaginal Opening ......................... 35

Natural Breeding ......................... 35

Survival .............................. 36

In Vitro Fertilization Assay ...................... 36
Sperm Concentration and Motion Analysis ............. 38
Statistical Analyses ........................... 39
RESULTS ....................... ' .............. 40
DISCUSSION .................................. 44
Body Weight ............................... 44
Organ Weight ............................... 45
Female Reproduction .......................... 49

Male Reproduction and In Vitro Fertilizing Ability ........ 51
SUMMARY ........... ’ ........................ 75
APPENDIX I ................................... 76
APPENDIX 11 .................................. 77
APPENDIX HI .................................. 78
APPENDIX IV ................................. 79
APPENDIX V .................................. 84
BIBLIOGRAPHY ................................ 86

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

Table 9.

LIST OF TABLES

Nutrient analysis of treatment diets .............

Body weight and relative organ weights of F—O
female mice fed diets containing lab chow (C),

Iowa carp (I), or Great Lakes carp (G) ..........

Body weights of F—l female mice fed diets
containing lab chow (C), Iowa carp (I),

or Great Lakes carp (G) ...................

Body weights of F-l male mice fed diets
containing lab chow (C), Iowa carp (I),

or Great Lakes carp (G) ...................

Feed intake of F-l male mice ................

Body and relative organ weights of 53-week-old
F-l male mice fed diets containing lab chow (C),

Iowa carp (I), or Great Lakes carp (G). ..........

Body and relative organ weights of 64-weeks-old
F-l male mice fed diets containing lab chow (C),

Iowa carp (I), or Great Lakes carp (G). ..........

Body weight and relative organ weights of F-l
female mice fed diets containing lab chow (C), Iowa

carp (I), or Great Lakes carp (G) at 4 weeks of age. . . .

Body weight and relative organ weights of El
female mice fed diets containing Iowa carp (I),

or Great Lakes carp (G) at 5 weeks of age. .......

vi

.53

.54

.55

.56

.58

.59

. 60

61

.62

Table 10.

Table 11.

Table 12.

Table 13.

Table 14.

Table 15.

Table 16.

Table 17.

Table 18.

Table 19.

Body weight and relative organ weights of F-l female
mice fed diets containing lab chow (C), Iowa
carp (I), or Great Lakes carp (G) at 6 weeks of age

Body weight and relative organ weights of F-l female
mice fed diets containing lab chow (C), Iowa
carp (I), or Great Lakes carp (G) at 8 weeks of age

Survival of offspring from F—O females fed diets
containing lab chow (C), Iowa carp(I),

or Great Lakes carp (G) ...................

Survival of F-l male mice fed diets containing
lab chow (C), Iowa carp (I), or Great Lakes carp (G)

Effect of treatment diets on vaginal opening
in F—l mice fed diets containing lab chow (C),

Iowa carp (I), or Great Lakes carp (G) ..........

Reproductive performance of 1-year-old F-l
males fed diets containing lab chow (C), Iowa

carp (I), or Great Lakes carp (G) ..............

Epididymal sperm concentration (million/m1)
in El male mice fed diets containing
lab chow (C), Iowa carp (I), or Great Lakes carp (G)

Sperm motion analysis in 53-week-old F-l male
mice fed diets containing lab chow (C),

Iowa carp (I), or Great Lakes carp (G) ..........

Sperm motion/analysis in 64-week-old F—l male
mice fed diets containing lab chow (C),

Iowa carp (I), or Great Lakes carp (G) ..........

Fertilizing ability of epididymal sperm from
53-week-old F-l males fed diets containing
lab chow (C), Iowa carp (I), or Great Lakes carp (G)

vii

.63

65

.66

67

68

.69

70

71

.72

LIST OF FIGURES

Figure 1. Body weight of F—l female mice ............... 73

Figure 2. Body weight of F-l male mice ................ 74

viii

ALHIMS
BWI
ppﬁDDE
ppﬁDDT
HCG
Lp

Ll
PCBs

PMSG
ppb

PPm
TCB

TCDD

LIST OF ABBREVIATIONS

amplitude of lateral head displacement

body weight

1 ,1—dichloro-2,2-bis-[p-chlorophenyl]ethylene
1 ,1 ,1-trichloro—2,2-bis[p-chlorophenyl]ethane
human chorionic gonadotropin
intraperitoneal

intravenous

polychlorinated biphenyls

pregnant mare’s serum gonadotropin

parts per billion

parts per million

3 ,3 ’ ,4,4’-tetrachlo robiphenyl

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

ix

INTRODUCTION

Industrial contaminants in the Great Lakes have been implicated as
causative agents in the decline of wildlife populations as well as in the
increase of some human health effects (Mineau et al., 1984; Gilbertson,
1988; Government of Canada, 1991; Jacobson and Jacobson, 1993). The
Virtual Elimination Task Force of the International Joint Commission (II C)
identiﬁed 11 persistent toxic substances in 1985 (IJC, 1993). The use of six
of these major contaminants, polychlorinated biphenyls (PCBs), DDT,
dieldrin, mirex, toxaphene, and alkylated lead, have been restricted or
banned (Appendix D. Some of these substances are naturally occurring
[mercury, benzo(a)pyrene] while others, such as 2,3 ,7 ,8-tetrachlorodibenzo-
p-dioxin (TCDD) and polychlorinated dibenzofurans (PCDFS), are
byproducts of combustion and some industrial processes involving the use
of chlorine. In spite of measures taken to reduce the input of these
contaminants into the environment, these compounds and their metabolites
persist in the Great Lakes ecosystem (T anabe, 1988). Signiﬁcant amounts

are still present in the air, water, and soil allowing them to traverse through

2

the food chain to upper trophic level organisms (Weaver et al., 1965;
Tanabe et al., 1984; Waid, 1987; Evans et al., 1991). Therefore, human
and wildlife consumption of Great Lakes ﬁsh has become a signiﬁcant health
and ecological concern.

Adverse effects have been observed in laboratory animals exposed to
Great Lakes carp. Offspring of adult ranch mink fed diets containing Great
Lakes carp from Saginaw Bay, Michigan, have shown a signiﬁcant reduction
in birth weight and litter size and an increase in mortality (Hartsough, 1965;
Heaton et al., 1995; Restum et al., 1997). Similar ﬁndings were reported
by Summer et al. (1996) who observed a dose- and time-dependent effect on
hatchability and the occurrence of teratogenic effects in chicken embryos
from hens fed diets containing Great Lakes carp.

Polychlorinated biphenyls and DDT are two major contaminants in the
Great Lakes (U C, 1993). Some of the biological effects related to the Great
Lakes contaminants have been observed in laboratory studies when animals
are exposed to PCBs, as commercial mixtures or individual congeners, and
PCB analogs; other effects have also been related to the toxicity of DDT and
its metabolites. Increased embryo mortality was observed in chicken eggs
injected with commercial PCB mixtures (McLaughlin et al., 1963; Carlson

and Duby 1973). Hansen et al. (1992) reported that the ortho-substituted

3

PCBS have estrogenic activities as indicated by an increase in uterine weight
in immature rats and that the non-ortho substituted PCB congener, 3,3’ ,4,4’-
tetrachlorobiphenyl (TCB), produces anti-estrogenic effects like TCDD.
Male rats exposed to Aroclor 1254 during lactation were less successful in
mating than control animals; non-treated females mated with treated males
had a lower implantation rate and a decrease in the number of live fetuses
(Sager, 1983; Sager et al., 1991). Female rats lactationally exposed to
Aroclor 1254 also experienced ‘a'reduction in implantation rate (Sager and
Girard 1994). Exposure of both male and female mice to PCBS has been
shown to decrease the frequency of implanted ova (Kihlstrom et al., 1975).
DDT has also been shown to have reproductive effects in various species.
Exposure of both Sexes to DDT has been shown to lower the frequency of
implantation in mice (Kihlstrom et al. , 1975). DDT has been shown to have
estrogenic activity which is manifested by an acceleration in vaginal
opening, persistent vaginal estrus, and anovulation in female rats (Gellert et
al., 1972, 1974; Heinrichs et al., 1971). DDE, a major metabolite of DDT,
is known to cause eggshell thinning in birds (Peakall et al., 1973). DDE
has also been demonstrated to have antiandrogenic effects in exposed male
rats such as decreased ventral prostate weights, a delay in the onset of

puberty, and a decrease in the anogenital distance (Kelce et al., 1995).

4

While exposure to PCBS and DDT has been shown to cause adverse effects
on reproduction, animals in the wild are rarely exposed to a Single type of
compound. In addition to PCBS, DDT, and their metabolites, a variety of
other chemicals and ecological metabolites are present in the Great Lakes
(IJC, 1993). Humans and animals, therefore, are exposed to a mixture of
contaminants which may elicit different effects than individual compounds.
Thus, it is important to study the effects of mixtures of contaminants as they
exist in the environment.

Our laboratory has been Studying the reproductive effects of these
mixtures of contaminants on reproduction in mice. Great Lakes carp was
used as a model carrier for mixtures of contaminants in the Great Lakes.
In a previous study, in which mice were exposed to Great Lakes ﬁsh via
lactation and diet from birth through 34 weeks of age, no adverse effect on
reproductive performance in pubertal and mature male mice was observed
(Kuo, 1994). The present study expands on our earlier study. The effects
of Great Lakes contaminants on male reproduction were examined in post-
maturation mice (through 15 months of age) and the effects of these

contaminants on the onset of puberty in the female were also examined.

LITERATURE REVIEW

The r L

The Great Lakes (Lake Superior, Lake Michigan, Lake Huron, Lake
Erie, and Lake Ontario) and their connecting channels are the largest system
of fresh surface water on the earth. They contain approximately 18% of the
world’s fresh water (about 5,500 cubic miles of water), span 750 miles from
east to west, and cover a total area of 94,000 square miles. The Great
Lakes are of sizeable economic and ecological importance to both the United
States and Canada. Over 35 million residents (one-tenth of the population
of the United States and one-quarter of the population of Canada) live
around the Great Lakes basin (EPA, 1994). The Great Lakes provide
drinking water, transportation, power production, recreation, and sporting

and commercial ﬁshery.

Wm
Since 1940, increased commercial production and widespread use and

release of agricultural and industrial compounds have contaminated the Great

6

Lakes ecosystem. Tens of thousands of chemicals are created, used and
released into the Great Lakes each year from a variety of sources. These
sources include atmospheric deposition and the dumping of polluted
industrial discharge, rural sewage, urban sewage and runoff pollutants.
Sources of pollution are generally classiﬁed as two types: One type
is point sources which are single sources such as industrial or municipal
discharges. The other type of pollution is nonpoint, from a diffuse source
which includes both urban and rural runoff from the land, as well as
atmospheric deposition which originates thousands of miles from the Great
Lakes. Atmospheric deposition occurs in the following manner: 1)
Pollutants are released into the air from man-made or natural sources. Man-
made sources include vehicle exhaust and pesticide Spray. Natural sources
include volcanic eruptions and particles from forest ﬁres. 2) Pollutants are
carried by continental winds away from their original source. 3) Pollutants
are deposited on lakes when particles settle out of the air by gravity or are
removed from the atmosphere by falling rain or snow. 4) Air pollutants can
also be deposited on land and runoff into lakes. Polychlorinated biphenyls
(PCBS) and dichlorodiphenyltrichlorethane (DDT) have been detected in the
polar areas (Atlas et al. , 1986) and in marine mammals from Antarctica

(Tanabe et al., 1983). Much of the atmospheric contribution in the Great

7

Lakes basin is believed to result from long range transport to the region
(including distances as far away as Central America). Rice (1985) reported
that atmospheric loading is responsible for almost 76-98% of PCBS found
in Lake Superior, 69-84% for Lake Michigan, 57-83 % for Lake Huron, 45-
67% for Lake Erie, and 43-63% for Lake Ontario. Nearly 100 tons/year
of mercury are released into the Great Lakes from anthropogenic sources in
the US, and another 31 tons/year from Canadian sources (IJC, 1993b).
In the past 20 years, Great Lakes’ contaminants have declined
consistently due to restrictions on chemical use and bans on the use of DDT,
chlordane, dieldrin and PCBS (State of the Great Lakes, 1993 Annual report;
Bishop and Weseloh, 1990). Numerous studies have shown that the
concentrations of organochlorine pesticides and PCBS are declining in Great
Lakes ﬁsh (Schmitt et al., 1985; DeVault et al., 1986; Schmitt et al., 1990;
Borgrnann et al., 1991). There was a rapid decrease in DDE concentrations
in Lake Ontario trout from 1977 to 1980. Since this time, DDE
concentrations have remained relatively constant (Borgmann et al., 1991).
DeVault et al. (1986) reported that mean total DDT concentrations in coho
salmon ﬁllets from Lakes Michigan and Eric declined from 19.19 mg/kg in
1970 to 2.74 mg/kg in 1982. In a ﬁve-year human study, DDT levels of

115 Great Lakes ﬁsheaters were compared to the levels of 95 non-ﬁsheaters,

8
DDT levels were signiﬁcantly decreased between 1982 and 1989 (Hovinga

et al. , 1992). Dieldrin concentrations in Lake Ontario trout have also
declined between 1978 and 1988 (Borgmann et al., 1991). PCB
concentrations have ﬂuctuated over the years. They have decreased from
1977 to 1981, increased from 1982 until 1984, and decreased again in 1985
(Borgmann et al., 1991). Studies Show that PCB concentrations in coho
salmon ﬁllets from Lakes Michigan and Eric have decreased from 22.91
mg/kg in 1974 to 5.63 mg/kg in 1982 (DeVault et al., 1986). Hovinga et
al. (1992) reported that between 1982 and 1989, there was no change in

serum PCB concentration in humans who consumed Great Lakes ﬁsh.

P 1 hi rin i hen B

Polychlorinated biphenyls are chlorinated organic compounds with
biphenyl as the basic structural unit. These environmentally persistent
chemicals were ﬁrst described in 1881 by Schmidt and Schultz (Peakall and
Lincer, 1970). PCBS have a low dielectric constant. They are stable in
both acidic and basic conditions and are resistant to oxidation and reduction.
PCBS have a high heat capacity and are nonﬂammable. In 1929, PCBS were
ﬁrst commercially produced and used for industrial purposes (Tanabe,

1988). PCBS were widely used in industry as hydraulic ﬂuids, heat transfer

9

ﬂuids, lubricants, capacitors, plasticizers, protective coatings, and cardboard
cartons. PCBS were also used in commercial products like carbonless
carbon paper, organic diluents, wax extenders, printing ink, dust-reducing
agents, ﬂame retardants, and adhesives (Safe et al., 1987).

Chlorination of the biphenyl ring can produce 209 possible congeners
from monochloro- to decachloro-. The most popular brand of PCBS in
North America was "Aroclor”, manufactured by Monsanto Ltd. (U.S.).
Aroclor products include 1242, 1248, 1254, and 1260 which are complex
mixtures of different congeners. For example, Aroclor 1254 consisted
primarily of tetra, penta-, and hexachlorobiphenyls. The ﬁrst two digits
represent the number of carbon atoms in the biphenyl group and the last two
digits describe the approximate percent chlorine content (Sawhney, 1986).

Since 1929 it is estimated that 1,200,000 tons of PCBS were produced
in the world. There are an estimated 370,000 tons of PCBS in the global
environment which makes up 30% of the world PCB production, 65 %
(783,000 tons) of the world’s production is still in use in electrical
equipment. In 1988, over 280,000 tons of PCBS were still in use in the US
(1] C, 1993). In spite of precautions, accidental leaking from industrial
facilities and landﬁlls, improper disposal practices, and industrial ﬁres

(Phaneuf et al., 1995) have led to signiﬁcant environmental contamimtion.

10

PCBS are known to produce a spectrum of toxic responses in humans
and laboratory animals including lethality, reproductive and developmental
toxicity (Allen et al., 1979), porphyria, body weight loss, dermal toxicity
(Tryphonas et al., 1991), irnmunosuppressive effects (Loose et al., 1978;
Tryphonas et al., 1991), hepatotoxicity (Bruckner et al., 1974),
neurotoxicity (Loose et al., 1978), thyrnic atrophy, carcinogenesis, and
endocrine disruption (Safe, 1994).

Because of the low concentration of PCBS in the atmosphere, the
majority (80-90%) of animal exposure to organochlorine compounds such
as PCBS and DDT comes from the food pathway, a lesser amount (5-10%)
from air, and trace amounts from water (IJC, 1993). Through oral
exposure, PCBS, like other lipophilic compounds, rapidly cross the
membranes of the gastrointestinal track. More than 84% of the total dose
of PCBS was absorbed from the gastrointestinal tract, when a 3 mg
commercial PCB mixture, containing Kanechlor-300, 400, 500, and 600
(1:1:1:1 by weight) in corn oil, was administered daily by gavage to
irnmmure male Wistar rats for 5 days (Tanabe, 1981).

After PCBS cross the membranes of the gastrointestinal tract, they are
transported in the blood to the liver as well as to other organs and tissues.

In the body, PCBS are associated with lipids; the highest concentrations are

11

found in fat-rich tissues such as adipose tissue and milk (Jensen, 1989). A
study conducted in Michigan demonstrated that sport anglers who ate Great
Lakes ﬁsh (especially trout and sahnon) had higher blood and tissue levels
of PCBS than people who never ate Great Lakes ﬁsh (Humphrey, 1988).
The toxicity of PCBS are highly dependent on the degree of
chlorination of the biphenyl nucleus and the position of the chlorine atoms
(Parkinson and Safe, 1987). It is generally known that the acute toxicity
(LD50) of commercial PCB formulations increases with increasing chlorine
content, however, the highly chlorinated Aroclors 1260, 1262, and 1268 are
less toxic than Aroclor 1254 (Safe, 1984). Individual congeners are
classiﬁed into 3 groups: non-ortho, mono-ortho and di-ortho chlorinated
biphenyls. The term non-ortho is applied to congeners with no substitutes
at the ortho positions. Several non-ortho-and mono-ortho-congeners may
attain a coplanar conformation and have been found to induce
nonneurological effects (e.g., skin lesions, thymus atrophy, a wasting
syndrome, liver damage, immunotoxicity, and increased mortality) similar
to those caused by TCDD. These effects of PCBS and T CDD are mediated
through the same receptor, the Ah (cytosolic aryl hydrocarbon) receptor
(Safe, 1990). Non-ortho PCBS bind to the Ah receptor and form a receptor-

ligand complex which translocates into the nucleus and alters gene

12
expression (Spies and Gandolﬁ, 1991).

The toxicity of PCBS is decreased by addition of chlorine substituents
at ortho positions which decreases the coplanarity between the phenyl rings.
The reduced coplanarity of PCB decreases its capacity to bind to the Ah
receptor and decreases induction of cytochrome P-450 (Parkinson and Safe,
1987).

PCBS are slowly eliminated from the body. The most important route
of elimination is in tandem with lipids like milk. Lactating dams transfer
most of their body burden of PCBS to their nursing pups, whereas the body
burden of non-lactating animals remains essentially constant (Vodicnik and
Lech, 1982). Kodama et al. (1980) reported that lactational transfer of
PCBS to infants resulted in PCB blood concentration levels which exceeded
the mothers’ at 3 months postpartum and tended to increase until 1 year of
age. However, the PCB levels in the blood of bottle-fed infants remained
at a low concentration during these same time periods.

Because of their lipophilic property, PCBS require biotransfonnation
to polar molecules before they can be excreted via feces and urine (Lutz and
Dedrick, 1987). The cytochrome P—450—dependent monooxygenase enzyme
systems involved in phase I metabolism of PCBS are located in the

endoplasmic reticulum membranes of liver cells (Tirnbrell, 1991). These

13

enzyme systems require NADPH cytochrome P-450 reductase which consists
of two ﬂavoproteins that sequentially transfer electrons to hemoprotein
pigments termed cytochrome P—450. These cytochromes catalyse, via redox-
reactions, the insertion of a single oxygen atom into PCBS thereby
converting them to compounds that are more polar, thus enhancing their
excretion. In phase 11 reactions, PCBS induce epoxide hydrolases,
glucuronyl transferases, and glutathione-S-transferases. These enzymes are
catalyze conjugation reactions involving phase I products. The resulting
metabolites are more water soluble and easily excreted (Borlakoglu and
Haegele, 1991). Derivatives of gluthathione conjugates are major metabolic
products of PCBS after biotransfonnation (Matthews and Dedrick, 1984).
PCBS can also be metabolized by conjugation with cysteine and methionine,
and then excreted in the bile.

Factors that affect the rate of biotransformation of PCBS include the
number of chlorines on the biphenyl nucleus, the position of these chlorines,

animal species, age, and sex (Parkinson and Safe, 1987; Sipes and

Schnelhnann, 1987).

r in

In the past 20 years, a wealth of information has become available on

14

the effects of PCBS on animal reproduction. The effects of PCBS on
reproduction was ﬁrst demonstrated by Hartsough (1965). Hartsough
reported a higher mortality of kits when ranch mink were fed Great Lakes
ﬁsh. Later, a series of studies by Aulerich (1971, 1973, 1977) indicated
that PCBS and other environmental contaminants found in Great Lakes ﬁsh
are related to the decrease in reproduction of mink.

Heaton et al. (1995) examined the reproductive performance of adult
ranch mink fed diets containing 10, 20, and 40% of Great Lakes carp from
Saginaw Bay, Michigan. Birth weights of the offspring were signiﬁcantly
reduced in the groups fed 20 and 40% carp in the diet. Female mink fed
40% carp had the fewest number of kits and the highest mortality. No
offspring survived beyond 24 hours in this treatment group.

In a laboratory study, sexually mature female mice were treated with
0.025 mg of Clophen A-60 per day for 10 weeks. The oestrous cycle was
2 days longer and the frequency of implanted ova was 7.5 % lower than in
the control animals (Orberg and Kihlstrbm, 1973). Kihlstrom et al. (1975)
conducted a study where dams were injected with 50 mg/kg of Clophen A-
60, on the day of parturition and once a week for 4 weeks. N on—exposed
mice were bred with exposed mature offspring. No effects on frequency of

implantation in both male and female offspring were observed. However

15

when exposed animals were bred with each other, the frequency of
implantation decreased to 75 % compared to 94% in control animals. Gellert
(197 8) reported that 24 hours after a single subcutaneous injection of 1000
rig/kg b.w. of Aroclor 1221 in 22-day-old Sprague-Dawley rats, the uterine
weight was Signiﬁcantly increased by about 20 % when compared with the
control group. This effect was not observed with treatment by Aroclor
1242, 1254 and 1260. Female rat pups were injected subcutaneously with
1 and 10 mg of Aroclor 1221 on the 2nd and 3rd days of life. There was
precocious puberty, persistent vaginal estrus and anovulation by 6 months
of age in the 10 mg Aroclor 1221 treatment group (Gellert, 1978).

Male Holtzman rats exposed to 8, 16, 32, or 64 mg/kg Aroclor 1254
during lactation were mated to untreated females at 120 days of age. Lower
implantation rates were observed in the 16, 32 and 64 mg/kg treatment
groups (Sager et al., 1991). Eight, 32 or 64 mg/kg of PCB (Aroclor 1254)
were dissolved in peanut oil and administered orally to lactating dams on
days 1, 3, 5, 7, and 9. Male offspring exposed to PCBS during lactation,
were mated to untreated females at 130 day-of-age. Implantation rates were
decreased 22, 59 and 64%, respectively (Sager et al., 1987). N 0 effect on
sperm production, morphology, or motility was observed in these two

studies. Adult male white mice fed 50 and 200 ppm Aroclor 1254 for 15

16

days exhibited reduced testicular spermatozoa concentrations of about 13
percent (Sanders, 1977). Treating adult male white-footed mice
(Peromyscus leucopus) with 400 ppm of Aroclor 1254 for 2 weeks reduced
testicular spermatozoa numbers by 40% when compared to the control group
(Sanders, 1975). Pregnant Holtzman rats were treated on day 15 of
gestation with a single oral dose of TCDD (1.0 jig/kg). Pups were fostered
and cross-fostered to produce the following groups: pups not exposed to
TCDD (control), pups exposed to TCDD in utero (IU), pups exposed via
lactation (L), and pups exposed both in utero and via lactation (IUL).
Epididymal sperm concentrations from 63-day-old rats were decreased 64,
48, and 49%, in the IU, L, and [UL groups respectively, when compared
to the control (Bjerke and Peterson, 1994). Thirty-one—day-old male Fisher
rats were treated with 0, 0.1, 1, 10, or 25 mg/kg/day Aroclor 1254 by
gavage for 5, 10, or 15 weeks. Cauda epididymal sperm storage was
reduced by approximately 20% in the 25 mg/kg/day, 15 week exposure
group. No effect was observed on sperm motility and daily sperm
production in other treatment groups (Gray et al., 1993). In another study,
dams were fed a diet of 60% Great Lakes carp, from parturition through
lactation. Offspring of these dams received the same diet until termination.

No alteration in Sperm concentration, motility or reproductive performance

17
were observed at 6, 7, 8, 15, 23, 32 or 34 weeks (Kuo, 1994). Huang

(1995) performed a study where dams received 0, 3, or 30 ppm TCB
through mating, gestation, and lactation. Male offspring received the same
diet through the entire study. No differences were observed in epididymal
sperm concentration and sperm motility in any of the treatment groups at 9
and 19 weeks. However, sperm fertilizing ability decreased in the 30 ppm
treatment group at 19 weeks.

Kholkute et al. (1994) reported that Aroclor 1221, 1254 and 1268,
added to in vitro fertilization medium at various concentrations (0.01, 0.1,
1, and 10 ng/ml), inhibited fertilization and increased the incidence of
degeneration of oocytes and abnormalities in the early mouse embryos at
doses of 1 and 10 ug/ml. Mousa et al. (1996) examined the effects of
Aroclor 1242 and 1254 on 36D2F1 mouse sperm fertilizing ability. In this
study, 0, 10, and 20 ppm Aroclor 1242 and 1254 were added to the in vitro
fertilization medium, BMOC-3. In the absence of PCBS, few eggs (1-7%)
degenerated and most eggs (83-86%) were fertilized. At the high dose (20
ppm), Aroclor 1242 and 1254 caused all eggs to degenerate and completely
eliminated fertilization. At the low dose (10 ppm) of Aroclor 1242,
fertilization rates were 35 % and few eggs degenerated. Exposure to 10 ppm

of Aroclor 1254 resulted in 21% fertilization and 66% egg degeneration.

18

In Rhesus monkeys, disturbances in the menstrual cycle, reduced
conception rates, diminished ability to maintain pregnancy and low birth
weights were observed when adult animals consumed diets containing 2.5
and 5.0 ppm of Aroclor 1248 for 4 and 7 months. The total PCBS
consumed ranged from 60 to 120 mg for the 4 months treatment group, and
105 to 210 mg for the group treated for 7 months (Barsotti et al., 1976).

In human female "Yusho" patients who inadvertently consumed PCBS
through contaminated rice oil, 60% showed abnormalities of menstrual cycle
length, 55% had menstrual cycles of abnormal duration, and 85% had
abnormal basal body temperature patterns. Urinary estrogen and
pregnanediol concentrations in these exposed women were lower than the
non-exposed women (Truelove et al. , 1990). During the "Yusho" epidemic,
11 women with Yusho and 2 wives whose husbands had Yusho, but who
themselves did not show the disease, delivered 10 livebom and 2 stillborn
babies. Most of the fetuses born were smaller than the national standards
(Kimbrough, 1974). Similar exposure to PCBS occured in the "Yu-Cheng"
patients in Taiwan. One hundred and ﬁfty-nine pregnant women were
exposed to PCBS during this outbreak. Three were pregnant when the
surveyed was given, 5 miscarried, 8 were aborted, 6 were stillborn, 5 were

born live and later died, and 132 infants lived (Rogan et al., 1988).

19

Women who ate an average of 6.7 kg of ﬁsh from Lake Michigan
every year for an average of 16 years are reported to have had shorter
gestational periods, and gave birth to children with low birth weights and
small head circumferences. The exposed infants were 4.9 days less in
gestational age, 160 to 190 gm lighter than controls, and their heads were
0.6 to 0.7 cm smaller (Fein et al., 1984). These children showed
discernible cognitive motor and behavioral deﬁcits when tested at 7 months
and 4 years compared to infants born to women who had not consumed
Lake Michigan ﬁsh prior to or during their pregnancies (Jacobson and
Jacobson, 1993; IJC, 1993).

Wassermann et al. (1982) compared 17 cases of premature delivery
to 10 women with normal pregnancy. Seventeen of the premature
deliveries, 8 were associated with high serum PCBS concentration (128.0
ppb versus 19.25 ppb in the control group) and 5 cases were associated with
high serum DDT concentration (119.6 ppb versus 26.5 ppb). In some of

these cases, high levels of heptachlor epoxide and dieldrin were also

found.

i hl lri hloreth e D

Dichlorodiphenyltrichlorethane (DDT) is another environmentally

20
persistent pollutant in the Great Lakes. It was ﬁrst synthesized by Zeidler

in 1874. In 1939, Paul Muller discovered the insecticidal properties of
DDT. It became the most popular pesticide for agriculture and household
use from the late 1940s until the early 19703. Based on ecological
considerations, DDT has been banned in the US. since 1972.

DDT is an organochlorine insecticide. It exists as a crystalline solid
and is tasteless and almost odorless. The term DDT is generally understood
throughout the world and refers to p,p’-DDT (1,1,1-trichloro-2,2-bis[p-
chlorophenyl]ethane). The compound structure permits different forms,
such as o, p ’- and 0,0 ’- isomers (WHO, 1989). The major DDT metabolite,
p, p ’-DDE (1 , 1-dichloro-2,2-bis-[p-chlorophenyl] ethylene) is more persistent
than the parent compound, and is therefore the form. normally found in
foods and humans tissues. Although DDT has been banned in the US.
Since 1972, DDT and its metabolites are still found in the Great Lakes.
These products probably arise from the lake bottom sediments, contaminated
tributary sediments, runoff from sites of historical-use, leaking landﬁll Sites,
illegal use of old stocks, and long range transportation through the
atmosphere.

DDT enters organisms through inhalation, ingestion, and dermal

absorption. Although the dermal absorption of powder DDT is small, it is

21

easily absorbed by the skin when dissolved in oil or an organic solvent
(Murphy, 1980; Wester and Noonan, 1980; Kaloyanova and E1 Batawi,
1991). Variations are observed in the acute oral toxicity of DDT in
different species of animals. For example, the acute oral LD50 in rats is
250 mg/kg, mice have a value of 400 mg/kg and monkeys and humans have
a value of 200 mg/kg (Hrdina et al., 1975). In mammals, DDT mainly
affects the nervous system causing hyperexcitability, tremors, ataxia,
seizures, and convulsions (Kaloyanova and El Batawi, 1991). Death from
DDT poisoning is most often attributed to respiratory arrest (Hayes, 1971).
DDT also affects the liver, by increasing P450 enzymes or producing
necrosis and tumors. In acute poisoning, liver glycogen is mobilized
quickly and later exhausted resulting in an increase of circulating lactic acid
and a compensated acidosis (Hayes, 1971).

In mammals, DDT is believed to be metabolized to DDE (1,1-
dichloro-2,2-bis- [p-chlorophenyl] ethylene) without the formation of
intermediary metabolites or after several stages of biotransformation to DDA
(2,2-bis[4-chlorophenyl] acetic acid). Both are excreted in the urine

(Kaloyanova and El Batawi, 1991).

DD! and Reproduction

22

DDT, or more speciﬁcally, its degradation product DDE, can lower
the reproductive rate of birds by causing eggshell thinning that leads to egg
breakage during incubation which causes embryo deaths. Laboratory
experiments established that DDE inhibits calcium adenosine triphosphatase
(Ca-ATPase) in the eggshell gland, thereby reducing the transport of calcium
to the site of eggshell formation (Kolaja and Hinton, 1977). DDT also
inhibits carbonic anhydrase (CA). Carbonic anhydrase is believed to be a
necessary source of carbonate ions required for calcium carbonate deposition
(Bitman et al., 1970). Bitman et al. (1970) observed that when CA activity
was low, soft-shelled eggs or no eggs were observed.

When environmental levels of DDE decreased to less than the critical
concentration for eggshell thinning, the population of some of the ﬁsh-eating
water birds increased (Giesy et al., 1994; Burger et al., 1995). However,
different species of birds are different in their sensitivity to these chemicals.
For example, in the bald eagle (Haliaeetus leucocephalus) eggshells
containing 4.0 and 16.0 ug/g DDE (wet weight) were associated with 10
and 15% thinning, respectively (Wiemeyer et al., 1993). Bald eagle
eggshells collected from 15 states in the United States in 1980-84 showed
that production of young was normal when eggs contained less than 3.6 ug/ g

DDE (wet weight), production decreased nearly 50 % when DDE

23
concentrations were between 3.6 to 6.3 ng/g, and decreased 75% when

concentrations exceeded 6.3 ng/g (Wiemeyer et al., 1993). Captive black
ducks (Anas rubripes) fed dietary DDE at 10 ppm for two breeding seasons
produced eggs with approximately 20 % thinner shells than untreated
controls. The mean DDE concentration in the eggshell was 64.9 ppm
(Longcore and Stendell, 1977). Robson et al. (1976) did not observe any
effects of feeding 100 or 300 ppm DDE for eight consecutive 28 day periods
on eggshell thickness of Japanese quail.

The 0,p’-DDT is a weakly estrogenic compound. Immature Sprague-
Dawley rats intraperitoneally (ip) injected with 500 pg o,p’-DDT daily for
27 days had early vaginal opening and increased ovary and uterus weights
(Gellert et al., 1972). When mature ovariectomized rats were injected ip
with 10 mg/day of o,p’-DDT or p,p’-DDA for 7 days, increased uterine
weight, corniﬁed vaginal smears, and reduced serum LH concentrations
were observed (Gellert et al., 1972). Mason and Schulte (1980) reported
that treatment of ovariectomized adult rats with 10 mg 0,p’-DDT or 1 ug
estradiol for 3 days doubled the number of progesterone receptors in the
uterine cytosol and increased uterine weight when compared to the control
group. N MRI mice treated with 40 mg/kg DDT by intraperitoneal injection

showed a signiﬁcant increase in the length of the estrous cycle. The

24

prolongation of the estrous cycles appeared to decrease with time and the
lengths returned to normal after three cycles (Orberg et al., 1972). Ireland
et al. (1980) showed that 0,p’-DDT (250 mg/kg) increased uterine DNA
content and DNA synthesis in ovariectomized immature rats to the same
extent as 40 ug/kg of estradiol.

Kihlstrdm et al. (1975) injected dams with 50 mg/kg of p,p’-DDT,
beginning on the day of parturition once a week for 4 weeks. N on—exposed
offspring were bred with exposed animals. No effects on frequency of
implanted ova were reported. When exposed animals were bred with each
other, the frequency of implantation decreased to 79% compared with 94%
in the control group.

Eight-week-old male mice received an oral dose of 2x150 mg/kg of
body weight DDT over 2 days. Lethal mutations were induced in early
spermatid and spermatocyte stages. An increase in spermatocyte
chromosome breakage, stickiness and precocious separation of the X and Y
bivalent were observed (Clark, 1974). Chronic oral doses of DDT (2x100
mg/kg/BW/week) for 10 weeks caused changes in seminiferous tubule
morphology and degeneration of B-type spermatogonia in 8-week—old mice
(Clark, 1974).

The major and persistent DDT metabolite, p,p’-DDE, has little ability

25

to bind to the estrogen receptor, but has been shown to have antiandrogenic
effects. Weanling (21—day-old) male rats treated with 100 mg/kg/day of
p, p’-DDE until puberty (age at preputial separation) had a signiﬁcant 5-day
delay in the onset of puberty compared to control rats (Kelce et al., 1995).
In additional studies, l20—day-old male rats were castrated and implanted
with testosterone-containing Silastic capsules to maintain constant serum
androgen levels. The animals were then treated with 200 mg/kg/day p, p ’-
DDE by gavage for 4 days. Signiﬁcant reductions in androgen-dependent

seminal vesicle and prostate weights were observed (Kelce et al., 1995).

Mmm

Mercury is widely used within the industrial, medical, agricultural,
and consumer sectors; over 2,000 application have been identiﬁed. The
major source of mercury is the natural degassing of the earth’s crust,
including land areas, rivers and the ocean. Other sources of mercury
include mining, burning of fossil fuel, smelting, paper pulp, fungicides, and
electrical and industrial discharge. Global anthropogenic sources of mercury
have reached about 8,000 to 10,000 tons per year since 1973.

Nonanthropogenic sources are still the predominant sources and contribute

26
25,000 to 150,000 tons per year (WHO, 1976). Natural sources of mercury

may contribute from one quarter to one half of the total load of mercury to
the Great Lakes (IJC, 1993). The atmosphere is an important pathway for
the mercury load to the Great Lakes. The estimated mercury load to Lake
Superior, Lake Michigan, Lake Huron, Lake Erie, and Lake Ontario from
the atmosphere is 2,181, 1,568, 1,584, 723, and 568 kg/year, respectively
(IJ C, 1993).

Mercury can exist in three forms: elemental, inorganic and organic.
All three forms are toxic. Elemental mercury (Hgo) may be absorbed by
biological systems as a vapor and is relatively lipid soluble. It is readily
absorbed from the lungs following inhalation and is oxidized in the red
blood cells to mercuric ion (Hg2+) (Tirnbrell, 1991). Before oxidation to
Hg“, elemental mercury is rapidly transported in red blood cells, across the
blood-brain barrier and into the central nervous system (CNS) and may
seriously damage the CNS (Mitra, 1986). Elemental mercury also rapidly
crosses the blood-placenta barrier into the fetus (Mitra, 1986). However,
the elemental form of mercury is poorly absorbed from the gastrointestinal
tract. Inorganic mercury exists as monovalent or divalent ions and is also
poorly absorbed from the gastrointestinal tract. Absorption from food is less

than 15% in mice and about 7% in humans (Goyer, 1991).

27
Organic mercury is readily absorbed (90-95%) from the

gastrointestinal tract (Neathery and Miller, 1975.; Timbrell, 1991). Organic
mercury can cross the blood-brain and blood-placenta barriers. One of the
most signiﬁcant effects of mercury pollution is that aquatic organisms can
biotransform the inorganic mercury ions into methyl and dirnethyl mercury.
These forms of mercury are lipid soluble. They can enter the food chain
through the lowest trophic levels to the top predators.

Methyl mercury is the most important form of mercury in terms of
health effects from environmental exposures (Goyer, 1991). It is mutagenic,
teratogenic, and carcinogenic (Eisler, 1987). Mercury concentrations in
large carnivorous ﬁsh, such as pike and swordﬁsh, have been found to
exceed 1,000 ug/kg (EPA, 1984). Therefore, ﬁsh consumption is the
most important source of general population exposure to mercury.

Mercury distribution in tissues is dependent upon chemical form and
entry route. Metallic mercury is slowly absorbed by the gastrointestinal
tract at a rate of 0.01 percent when compared to elemental Hg vapor
through inhalation (Goyer, 1991). In orally dosed calves, absorption of
radioactive methyl mercury (CH3203HgCl) was 100 times higher than
absorption of the inorganic form (””HgClQ (Ansari et al., 1973). The

highest levels of methyl mercury were found in the kidneys and the second

28

highest levels are found in the liver. Fourteen days following oral
administration of 10 nmol 203HgCl2 to female mice, the residual body burden
in the kidneys, liver carcass, and brain were 33.76% , 21.27%, 34.33 % , and
1.36%, respectively (Nielsen and Andersen, 1990).

Mercury is eliminated from the body in the urine and feces, with the
latter being the major route. Methyl mercury is 90% excreted in the feces
via the bile as a cysteine conjugate, which undergoes extensive enterohepatic
recirculation with a mean half-life in the blood of approximately 70 days.

The major human health effects of mercury are neurotoxic effects in
adults (Bakir et al., 1973) and toxicity to the fetuses of mothers who are
exposed to methyl mercury during pregnancy (Cox et al., 1989). Clinical
manifestations of neurotoxic effects in adults include parathesia, ataxia,

vision and hearing loss, tremor, and ﬁnally coma and death (Goyer, 1991).

W

Mercury has been shown to alter reproduction in rodents. Single
intraperitoneal injections of 2.0 mg/kg of mercuric chloride to female mice
one-half to four days before mating, increased the incidence of dead
implants (Suter, 1975). Oral treatment of 30 day old female rats with 1 ml

of 100 mg/ 100 ml mercury as mercuric chloride (HgClz) solution daily for

29

12 weeks has been reported to disturb the estrus cycle. The disturbances
were manifested by the prolonging of the diestrus phase up to 10 days,
compared to the normal 4 to 5 days (Stadnicka, 1980). When golden
hamsters (Mesocricetus auratus) were subcutaneously injected with 1 mg of
mercuric chloride per day during one estrous cycle, 60% of the animals did
not ovulate by day 1 of the third cycle when the total amount of mercuric
chloride injected was 3 or 4 mg (Lamperti and Printz, 1974). Lee and
Dixon (1975) observed that CDFl male mice receiving a single
intraperitoneal injection of 1 ppm mercuric chloride had decreased fertility
and thymidine incorporation by spermatogonia.

Methylmercury is easily transported to the fetus via the placenta. The
concentration of mercury in the fetal blood is about 20% higher than the
maternal concentration (Report of an International Committee, 1969).
Pregnant Swiss-Webster mice exposed to methylrnercuric chloride
(CH3HgCl) 5 mg/kg/day orally as a single daily dose from days 6 to 17 of
gestation showed no clinical symptoms of mercury poisoning. However,
this resulted in 100 % stillbirth or neonatal death (Khera and Tabacova,
1973).

Male Wistar rats were administered single daily doses of 0, l, 2.5 and

5 mg methylrnercuric chloride/kg/day for 7 consecutive days. After dosing

30

was discontinued, the reproductive performance was examined by mating
treated rats with nontreated adult virgin female rats. A dose-related
reduction in litter size per pregnancy was observed during days 5-20
following treatment (Khera, 1973). In the same study, male Wistar rats
were dosed daily with 0.5 mg/kg or 1 mg/kg methylmercuric chloride.
Serial mating trials were programmed together. After dosing for 25-30 days
at 1 mg/kg and after 85-90 days at 0.5 mg/kg, a reduction in average litter
size due to preirnplantation losses was noted.

An epidemiologic study was conducted with people living in Sweden
who were exposed to methylmercury via the consumption of ﬁsh. All
subjects in the exposed group had more than three meals a week of
contaminated ﬁsh (0.5-7 mg mercury as methylmercury/kg ﬁsh) for more
than 3 years. A correlation was found between the mercury levels of blood
cells and the frequency of chromosome breakage in circulating lymphocytes

cultured in vitro (Skerfving et al., 1970, Skerfving et al., 1974).

HYPOTHESIS

While a number of studies have examined the effects of PCBS, DDT,
and mercury on reproduction, it is not clear what effects long-term exposure
of male mice to low-doses of environmental mixtures of contaminants will
have on reproduction. In a previous study, mice were exposed to Great
Lakes ﬁsh via lactation and diet from birth through 34 weeks of age. No
adverse effects on reproductive performance in pubertal and mature male
mice were observed (Kuo, 1994). The present study expands on our earlier
research. This study examines the effects of Great Lakes contaminants on
post-maturation mice (through 15 months of age) and the effects of these
contaminants on the onset of puberty in female mice. The hypothesis of the
present study is that long-term exposure to Great Lakes contaminated ﬁsh
will decrease sperm production, compromise sperm fertilizing ability, and

delay female puberty.

31

MATERIAL AND METHODS

Animals

C57BL/6J females (F-0) and DBA/2J (F—O) male mice were mated to
produce BﬁDz-Fl offspring (F—l). The breeders were purchased from
Jackson Labs, Bar Harbor, Maine. Mice were housed in the University
Laboratory Animal Resources facilities at Michigan State University in
groups of four or fewer per cage. Room temperature was maintained at
23°C, with 50%-70% relative humidity, and a light/dark cycle of 12 hours

light and 12 hours dark.

Diet Preparation

There were three treatment diets, prepared on a wet weight basis: C
(82% ground lab chow with 18% ﬁsh oil), I (60% Iowa carp with 40%
ground lab chow), and G (60% Great Lakes carp with 40% ground lab
chow). Both diets C and I served as control diets in this study. Lab Mouse
Chow #5015 was purchased from Purina Mills Inc. (St. Louis, MO). For

the ﬁsh control diet, carp (Cypinus carpio) ﬁllets and ﬁsh oil, intended for

32

33

human consumption, were purchased from an Iowa ﬁsh farm (Stoller
Fisheries, Spirit Lake, IA). Great Lakes carp (Cypinus carpio) were caught
from the mouth of the Saginaw River in Michigan. Fish were cooked at
77°C for 30 minutes to inactivate thiaminase (Gnaedinger, 1963; Gnaedinger
and Krzeczkowski, 1966). Bones were removed from the Great Lakes carp
before diet preparation.

Forty percent ground lab chow was added to 60% Great Lakes carp
or Iowa carp to make diets G and 1, respectively. The addition of ﬁsh oil
to diet C was intended to adjust the caloric content to match that of diets I
and G (T able 1). Major nutrients and minerals were balanced to meet NRC
requirements (NRC, 1978). The mineral sources of P, K, Mg, Na, Fe, Mn,
Cu, and Zn were from CaHPO4, KCl, MgSO4.7HZO, NaCl, FeSO4.7HzO,
MnSO4.HzO, CuSO4.5HzO and ZnSO4.7H20. Calcium was calculated based
on two sources: CaCO3 and CaHPO4. Vitamins A, D, E, K,, B6, biotin,
choline, folacine, niacin, pantothenate, riboﬂavin, and thiamine were also
used in the diet formulation. Nutrient analysis of the ﬁnal diets was
conducted by Litchﬁeld Analytical Services (Litchﬁeld, MI. Table 1).
Analysis of organochlorine pesticides and PCBS in the diets was conducted
by the Environmental Laboratory, Michigan Department of Natural

Resources (Lansing, MI) using gas chromatography (HP5089)/ECD

34
(Electron capture detector), according to EPA SW-846 method 8081.

WWW

Pregnant mare serum gonadotropin (PMSG), human chorionic
gonadotropin (HCG), CaCO3, CaHPO4, MgSO4.7H20, NaCl, FeSO4.7H20,
and pyridoxine hydrochloride were purchased from Sigma Chemical Co. (St.
Louis, MO). Fiber (alphacel non-nutritive bulk), vitamins A, D, E, K1, 8,,
biotin, choline, folacin, niacin, pantothenate, riboﬂavin, and thiamine were
obtained from ICN (Aurora, OH). Brinster’s medium for oocyte culture
(BMOC-3), sperm capacitation, and in vitro fertilization was purchased from

Gibco Life Technologies, Inc. (Gaithersburg, MD).

E rim n D i

Female (F-O) mice were randomly assigned to one of three treatment
groups upon birth of the F-1 offspring. Litter size was adjusted to eight or
nine pups per litter one day after birth, before exposure to the treatment
diets. Dams were fed the treatment diets from parturition through lactation.
F-l mice were fed the same treatment diet as their dams from weaning until
the termination of the study. Prior to weaning, F—l mice also had access to

the dams’ diet. At weaning, mice were sexed, weighed, and transferred to

35

cages which housed four or fewer mice per cage.

D II i n

Bﬂy weight Body weights of lactating F—O dams were recorded 3
weeks after parturition. Body weights of F-l male mice was recorded at 3,
5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40 and 44 weeks of age. F-l female
body weight was recorded at 3, 5, 6, 7 and 8 weeks of age.

W Feed intake was recorded for 5 consecutive days at 4, 8,
and 23 weeks of age. Actual feed consumption was calculated daily by
subtracting the amount of feed left in the feed jars from the total feed
provided on the day before and dividing by the number of mice per cage.

Organ weight Liver and thymus weights of lactating F—O dams were
recorded 3 weeks after parturition. Liver, testis and thymus weights of the
treated F-l males were recorded at 53 and 64 weeks of age. In F—l
females, liver and thymus weights were recorded at 4, 5, 6, and 8 weeks of
age.

Vaginal epeniag Beginning at 27 days of age, F-l females were
examined daily for vaginal opening.

Natural breﬂing Fecundity, litter size, sex ratio, were recorded for

both F-0 and F—l males. Forty-eight and 53-week-old treated F-l males

36

were individually paired with non-treated mature BGDz-F1 females for 7 days
between 7:30 PM to 7:30 AM, during which time feed was withheld to
avoid exposing females to the treatment diets. After breeding, females were
housed individually throughout gestation and lactation. Natural breeding
data for 48 and 53 week old mice were combined and reported as one-year
old natural breeding data, individual data for 48 and 53-week-old mice are
reported in appendix V.

Sauival Four day survival and 21-day survival were recorded for
F—l and F-2 mice. F—l male mice survival was recorded at 10, 20, 30, and

44 weeks.

I V' F . . . 11

After natural breeding, male mice were sacriﬁced by cervical
dislocation for in vitro fertilization studies. Sperm were collected from the
caudal epididymis. Both of the epididymides were placed in the inside well
of a Falcon Organ Tissue culture dish (60x15 mm style with a center well,
#3037, Becton Dickinson Labware, Lincoln Park, New Jersey) containing
1.0 ml Brinster’s medium (BMOC-3) (Gibco Life Technologies,
Gaithersburg, MD), a capacitation supporting medium. The outside well

contained 3 ml BMOC-3 without bovine serum albumin (BSA) for

37

stabilizing moisture and temperature. The epididymides were pierced with
a 25 g PrecisionGlide needle to release the sperm into the medium. The
culture dish was then incubated at 375° C, in 5% C02 in air, and 100%
humidity for one and a half hours before sperm were used for insemination.
Oocytes were collected from non-treated female mice which were
superovulated by intraperitoneal injection of 10 IU pregnant mare serum
gonadotrOpin (PMSG) followed 48 to 50 hours later by 10 IU human
chorionic gonadotropin (HCG). Twelve to fourteen hours after HCG
injection, oviducts were removed from mice and, transferred to a Falcon
Organ Tissue culture dish, containing 1 ml BMOC-3 medium. The ampulla
portions of the oviducts were teased open to allow the cumulus mass to be
released into the medium. Cumulus masses were transferred to another
culture dish and placed in the center well containing 1 ml BMOC-3 medium;
the outer well contains BMOC-3 without BSA. The pre-incubated sperm
concentration was adjusted to the ﬁnal concentration of 30,000 sperm/ml
and transferred to the oocyte-containing well, incubated for 24 hours before
examination of fertilization. The inseminated oocytes were then Stained
with 50 uM biSBenzimide for at least 30 minutes to allow for uptake and
intercalating with DNA. Oocytes were transferred to glass slides for

assessing fertilization using a Nikon Optiphot microscope equipped with a

38
100w mercury bulb, 365/10 nm excitation ﬁlter, 400 nm dichroic mirror,

and 400 nm barrier ﬁlter. Oocytes at the two cell stage or at the one cell
stage containing a second polar body were considered fertilized.

Degenerated eggs were considered non-fertilized.

Smrm Coneentratj on and Motion ﬂaiysis

Sperm concentrations were measured either manually or by computer.
Sperm concentration was measured manually by placing 20 ul sperm
suspension in a Petroff Hausser Counting Chamber (Hauser Scientiﬁc
Co.,Horsham, PA) containing 16 cells. Sperm were counted in 5 randomly
chosen cells and the total was used to calculate the ﬁnal concentration
(Appendix 11). Computer assisted sperm concentration and motion analysis
was performed at the same time of insemination using the CellSoft
computer-assisted digital image analysis system (CRYO Resources Inc.,
New York, 1986). Twenty pl of the sperm suspension (approximately 2.4
x 105 sperm cells) were placed in a CellSoft 20pm chamber and a minimum
of 100 sperm cells were analyzed to obtain measurements of concentration,
motility, velocity, linearity, mean amplitude of lateral head (ALH)

displacement, and beat/cross frequency (Appendix III).

39
Statistical Analysis

Quantitative data (body weight, organ weight, fertilization rate, feed
intake, and ALH displacement) were tested with One-Way Analysis of
Variance (ANOVA). Qualitative data (fecundity, survival index, and sperm
motility) were evaluated with the Chi-square test. Log transformations were
made for the discrete quantitative parameters and ratio data (litter size,
sperm concentration, beat/cross frequency, sperm linearity and sex ratios)
before ANOVA. These analyses were performed with Sigmastat' (Kuo et

al., 1992). All statements regarding signiﬁcance are based on p<0.05.

RESULTS

Of all the contaminants analyzed for in the diets, only p,p’-DDE and
PCBS were present at levels above the detection limits (240 and 2400 ppb,
respectively). Diet G contained 300 ppb p,p’-DDE and 2500 ppb PCBS.
These compounds were not detected in diets C or I (Appendix IV).

There were no signiﬁcant differences in the body weights of F—O
lactating females (Table 2). Decreases in the body weights of F-l females
on diet G were observed at three, ﬁve, six, and eight weeks of age (Table
3, Figure 1). In F-l males on diet G, there were signiﬁcant decreases in
body weights at all ages examined when compared to mice on either diet C
or I (Table 4, Figure 2). Body weight differences were also observed
between the two control groups; body weights of F—l males on diet I were
less than those on diet C from 10 weeks of age through 44 weeks of age
(Table 4). Feed intake of F-l males on diet G was less than that of mice on
diet C at four and 23 weeks of age, but not at eight weeks of age (Table 5).
F-l males on diet G at 53 and 64 weeks of age also had lower body weights

than those on diet C (Tables 6, 7). As the mice aged, the body weights of

40

41

mice on diet G decreased about 10% from 53 to 64 weeks of age, while
those on diets C and I were unchanged.

There were no Signiﬁcant differences among treatment groups in the
organ weights of F-O lactating females (Table 2). There were, however,
signiﬁcant differences in the organ weights of F-l females among treatment
groups (Table 8, 9, 10, 11). Relative organ weights (percentage of body
weight) are reported here because of the differences in body weights.
Relative liver weights were greater in mice on diet G compared with those
from mice on diet C at four weeks of age, but not when compared with
those on diet I (Table 8). Relative thymus weights of mice on diet G were
less than those of mice on diet I at ﬁve weeks of age (values for diet C were
not available) (Table 9). At six weeks of age, relative thymus weights of
mice on diet G were also less than those on diet 1, although not different
from those on diet C (Table 10) . In .F—l males, differences in organ
weights were also observed at 53 and 64 weeks of age (Tables 6, 7).
Relative liver weights of 53-week-old F-l males on diet G were greater than
those on diet I which, in turn, were greater than those on diet C (Table 6).
The same trend in liver weights was also observed in 64—week-old F—l males
(Table 7). Relative testes weights of 53-week and 64-week-old F—l mice on

diet G were similar to those of mice on diet C, but 45% less than those on

42
diet I (Tables 6, 7).

The survival of offspring from F-O females fed different treatment
diets is shown in Table 12. The four-day survival was lower in pups on diet
G when compared with pups on diet 1. However, survival of pups on diet
G was not different from that of pups on diet C. The 21-day survival was
less for pups of dams on diet G than those on either diets C or 1. After
weaning, male mice in all treatment groups survived through 20 weeks of
age. Between 20 and 40 weeks of age, 56, 91, and 100% survival was
observed in mice on diets G, I, and C, respectively (Table 13).

The age of vaginal opening in F-l females was signiﬁcantly delayed
by exposure to diet G (Table 14). Vaginal opening was observed on day
28.4 i 0.5 and 30.4 i 1.1 in diet C and I treatment groups, respectively.
In the diet G treatment group, a 16-day delay in vaginal opening was
observed when compared with that on diet I, and there was an 18—day delay
in vaginal opening when compared with that of diet C.

The treatment diets had a Signiﬁcant effect on F-l male reproductive
performance, sperm quality, and sperm fertilizing ability when assessed at
53 and 64 weeks of age. At one year of age, while there were no
differences in litter size and sex ratio of the offspring, the fecundity was

lower when males on diet G were paired with non-treated females (Table

43

15, Appendix V). Epididymal sperm concentration was signiﬁcantly lower
in 53- and 64—week-old F—l mice on diet G when compared with mice on
either diet I or C, assessed either manually or with the computerized image
analyzer (Table 16). A trend towards decreased sperm motion parameters
was observed at both 53 and 64 weeks of age, although only those at 15
months of age showed statistical signiﬁcance (Tables 17, 18). In addition,
the in vitro fertilizing ability of sperm from mice on diet G, when adjusted
to the same concentration in the fertilization medium, was only about 10%

of that from mice on diet C and I (Table 19).

DISCUSSION

Body Weigm

While there were no differences among the treatment groups in the
body weights of lactating F—O females, there was a 10 to 40% decrease in
post-weaning body weights of F—l males and females on diet G. This
decrease may, in part, be due to decreased feed intake, as observed in F-l
males at four and 23 weeks of age. In a previous study in our laboratory
(Kuo, 1994), in which mice were also fed Great Lakes carp, no differences
in body weights were observed despite identical experimental designs. In
other rodent studies, conﬂicting results were also reported when either PCBS
or DDT were examined for their effects on body weight. Reduced body
weight gain was observed in rat offspring following daily oral maternal
exposure to 30 mg Aroclor 1254/kg body weight for one month (Brezner et
al., 1984), while no body weight changes were observed in E1 mice
exposed to 3 or 30 ppm TCB in, the diet (Huang, 1995). Decreases in body
weights were observed in the offspring of mice receiving 5 or 10 ppm DDT

(Shabad et al., 1973; Ledoux et al., 1977). In contrast, increased body

44

45

weights were observed in weanling male rats gavaged daily with 100 mg/kg
body weight p,p’-DDE until after puberty (Kelce et al., 1995). N 0 changes
in body weight were observed in either newborn or young rats injected with
DDT (Gellert et al., 1972, 1974). Thus, no consistent pattern can be

elicited to explain the various results in body weights.

Qrgg Weights

As reported in the present study, Cleland et al. (1987) observed an
increase in liver weights in two strains of male mice (C57BL/6 and DBA/2)
fed diets containing 33% coho salmon from Lake Michigan and Lake
Ontario for four months following weaning. These mice also had elevated
ethoxyresoruﬁn O-deethylase activity compared to mice fed a control mouse
chow diet and mice fed a diet containing Paciﬁc coho salmon. Male mink
fed Great Lakes carp diet also had greater liver weights than controls
(Restum et al., 1997). A 25% increase in liver weight was observed in
female C57BL mice given weekly ip injections of 100 mg TCB/kg body
weight for four weeks beginning at ﬁve to seven weeks of age (Brouwer and
van den Berg, 1984) and in female rats given a daily oral dose of 5 mg/kg
body weight for three weeks (Clarke et al., 1984). Female C57BL/6J mice

fed 30 ppm TCB in feed for eight weeks, two weeks pre-breeding and

46

during pregnancy and lactation, had a 15% increase in liver weight (Huang
et al. , in press [b]). Furthermore, in the same study, liver weights increased
19% in six-week-old female offspring and 27% in 19—week—old male
offspring when compared to controls (Huang et al., in press [a,b]).
Offspring of female rats treated orally with a single dose of 1 pg TCDD/kg
on gestation day eight had greater liver weights than controls (Gray and
Ostby 1995). As for DDT and its metabolites, male rats receiving 100 or
300 mg o,p’-DDD/kg body weight had greater liver to body weight ratios
than controls (Straw et al., 1965) as did male monkeys given 10 or 20 mg
DDT/kg/day for seven days (Juchau et al., 1966). Therefore, increases in
liver weights are frequently observed in many species of animals exposed
to PCBS and DDT as well as to Great Lakes contaminants.

The decrease in thymus weights in female offspring reported here was
also observed when mice were exposed pre— and post-natally to 30 ppm TCB
in feed (Huang, 1995). However, in the dams and male offspring, no
decreases in thymus weights were observed in the present study or in mice
exposed to TCB as reported by Huang (1995). PCBS have been shown in
other studies to reduce thymus weights in adult female rats and mice
(Brouwer and van den Berg, 1984; Clarke et al. , 1984). PCB exposure has

also been shown to decrease cortical lymphocytes of the thymus and

47

suppress cytotoxic T cell generation (McConnell and Moore, 1979; Clarke
et al., 1984). Male and female mice exposed to 1.5 to 3.0 ug TCDD/kg
body weight during gestation also had signiﬁcant thymic atrophy (Blaylock
et al., 1992). In addition, male mice (44-dayS-old) exposed to 15 - 60 ng
TCDD/kg on gestation day 14 had decreased thymus weights when
compared to the controls (Theobald and Peterson, 1997). Thymus weights
have been reported to be reduced in third generation rat pups (males and
females) exposed to 0.01 pg TCDD/kg/day in the diet (Murry et al., 1979).
Based on these observations, it appears that for PCBS females are more
susceptible than males to thymus effects, although both males and females
appear to be equally affected by TCDD.

Changes in testes weights after PCB exposure have been observed in
many studies. These alterations, whether an increase or decrease in testes
weights appear to be dependent on the time of exposure. It has been
hypothesized that the increase in testes weight resulting from PCB exposure
is mediated through hypothyroidism when it is induced neonatally between
four and eight days after birth (Cooke et al., 1992; Stone, 1995 ; Cooke et
al., 1996). Rats injected with PCBS from birth to 25 days of age had
increased testes weights while those injected with PCBS on days 12 to 37 of

age showed no change in testes weights (Cooke et al., 1996). Increased

48

testes weights were also observed in six-month-old rats whose dams were
gavaged with Aroclor 1260 (30 mg/kg body weight) during gestation
(Gellert and Wilson, 1979) and in 23-week-old rats whose dams were dosed
with Aroclor 1254 (32 and 64 mg/kg body weight) during lactation (Sager
et al., 1983, 1991). Mice exposed to 20 and 40 mg/kg 2,2’,4,4’,5,5’-
hexachlorobiphenyl on gestation day 13 through post natal day 24 had
increased testes weights when examined at 10 to 12 weeks of age
(J ohansson, 1987). N 0 increase in testes weights have been observed when
the treatment occurs after the window of time (post-natal day four to eight)
when the animals appear to be susceptible. In addition, the increase in
testes weights are only observed when there is a recovery period,
approximately two months or longer, between exposure and when the testes
are examined. When four-week-old male rats were gavaged daily with
Aroclor 1254 (25 mg/kg body weight) for ﬁve, 10, or 15 weeks and
examined immediately at the end of treatment, no changes in testes weights
were observed (Gray et al., 1993). Likewise, testes weights were
unaffected in adult rats fed ﬁve, 50, or 500 ppm Aroclor 1254 for two,
three, or ﬁve weeks (Garthoff et al., 1977), nor were there any changes in
testes weights in adult mice fed diets of containing 62.5 to 4000 ppm

Aroclor 1254 for two weeks (Sanders et al., 1974, 1977; Sanders and

49
Kirkpatrick 1975).

In the present study, when lactating dams and the offspring were
treated continuously with diet G, no changes in testes weights were observed
in the offspring. While these mice were exposed during the sensitive
period, the continuous exposure did not allow for recovery. In addition, the
mice in this study were exposed to a complex mixture of contaminants
including compounds that are known to decrease testes weight. For
example, DDT has been shown to decrease testes weights in laboratory
animals. Eight—week—old mice orally dosed with 200 mg DDT/kg/week for

10 weeks had decreased testes weights (Clark, 1974).

F m e Re r i 11

Consistent with our ﬁndings, decreases in neonatal survival have been
observed in several other species exposed to Great Lakes contaminants.
Similar effects have also been observed after PCB or dioxin exposures.
Restum et al. (1997) reported reduced survival in mink kits of dams fed
Great Lakes carp containing 1.0 ppm PCBS. Decreased offspring survival
was also observed in female mice fed 30 ppm TCB (Huang, 1995), in white—
footed mice fed 10 ppm Aroclor 1254 (Linzey, 1987), and in rats fed 0.01

ng TCDD/kg/day (Murry et al., 1979). Continuous dietary exposure of

50

both sexes to 100 ppm DDT also resulted in decreased neonatal survival
between day four and day 30 (Del Pup et al., 1978).

In this study, female offspring on diet G exhibited a delay in the
onset of vaginal opening. This delay may, in part, be related to the lower
body weights. Nonetheless, PCBS alone could delay vaginal opening
without changing body weight. Inglis et a1. (1997) observed a signiﬁcant
delay in vaginal opening in El mice of dams orally gavaged with 32 mg
Aroclor 1254/kg body weight when there were no signiﬁcant differences in
the body weights. Others have also shown that lactational exposure to 8 or
32 pg Aroclor 1254/g delays puberty in female rats (Sager and Girard,
1994). Restum et al. (1997) reported that both the parental and El female
mink exposed to 0.25, 0.5, or 1.0 ppm PCBS via consumption of Great
Lakes carp exhibited a delay in the onset of estrous as measured by vulvar
swelling scores and the time of mating. On the contrary, existing studies in
laboratory rodents have shown that DDT and its metabolites cause
accelerated puberty as indicated by early vaginal opening, persistent vaginal
estrus, and anovulation when animals are exposed either during gestation or
neonatally (Gellert et al., 1972; 1974). Therefore, the delayed female
puberty observed in this Study is most likely a result of the PCB components

in the Great Lakes carp.

51
Mate Reprdductien add In Vm' Fegtilizing Ability

In the two control treatment groups (diets C and I), the reproductive
ability of one—year-old F-l males was less than that of younger mature mice,
based on comparisons of these data with historical breeding data from our
laboratory. Exposure to Great Lakes carp further reduced the breeding
ability of these post-maturation F-l mice. Only one of 11 non-treated
females gave birth when paired with one-year—old F-l mice on diet G. This
impaired breeding ability may be explained by a decrease in the epididymal
sperm concentration and reduced sperm fertilizing ability. This observation
seems to be age speciﬁc, since in a similar study with younger mice fed
Great Lakes carp (six to 34 weeks of age) there were no changes in sperm
concentration, motility, or reproductive performance (Kuo, 1994). In rats,
although no effect on sperm production, morphology, or motility was
observed in 120-day-old offspring when dams were orally gavaged with
Aroclor 1254 (8, 16, 32, and 64 pg/g) on days one, three, ﬁve, seven, and
nine of lactation, this postnatal exposure did reduce the percent of fertilized
eggs after mating with non-treated females (Sager et al., 1991).. Huang
(1995) reported no effect on epididymal sperm concentration or sperm
motility in nine or 19-week-old F-l mice exposed to TCB from gestation

through maturation. However, a decrease in sperm fertilizing ability was

52

observed in these 19-week-old mice, while sperm from younger mature mice
had normal fertility. DDT has also been shown to alter the male
reproductive function in laboratory animals. Although exposure of mice to
feed containing 10 to 50 ppm DDT (11—14 weeks of age) did not induce
abnormalities in sperm morphology (Wyrobeck and Bruce, 1975), eight-
week-old mice given an oral dose of DDT (200 mg/kg/wk) for 10 weeks
exhibited a reduction in sperm viability as determined by the eosin Y
staining method (Clark, 1974).

The existing information on male reproductive function and the
toxicity of Great Lakes contaminants does not provide a biological
mechanism to explain the reduced reproductive function observed in post-
maturation males and not in younger males. This could be due to any one
or a combination of three possibilities: increased susceptibility of older mice
to Great Lakes contaminants, increased body burdens of contaminants with
age, and/or an effect of neonatal exposure to Great Lakes contaminants

manifested with the aging process.

53

 

 

 

 

 

Table 1. Nutrient analysis of treatment dietsa
Treatment diet
C I G

Moisture % 4.73 53.75 50.91
Dry Matter % 95.27 46.25 49.09
Nutrientsb

Fat % 26.25 22.15 20.25
Crude Protein % 15.31 39.88 41.50
Crude Fiber % 2.70 1.75 2.00
Crude Carbohydrates % 51.04 28.82 28.05
Calcium % 0.86 1.46 1.42
Phosphorus % 0.52 0.75 0.86
Potassium % 0.72 0.94 1.17
Magnesium % 0.15 0.25 0.27
Sodium % 0.41 0.62 0.65
Iron ppm 217.00 232.00 229.00
Manganese ppm 132.00 188.00 191.00
Copper ppm 15.00 25.00 25.00
Zinc ppm 110.00 213.00 216.00
Ash % 4.70 7.40 8.20

 

“ Analysis was performed by Litchﬁeld Analytical Services,

Litchﬁeld, MI.

Units expressed on dry matter basis.

54

Table 2. Body weight and relative organ weights of F-O“ female
mice fed diets containing lab chow (C), Iowa carp (I), or
Great Lakes carp (G)

 

 

Weightb Treatment

C I G
Body Wt. (g) 26.5 i 2.1 26.8 i 2.7 25.2 i- 2.1
Liver Wt. (g) 1.5 i 0.2 1.8 i 0.3 1.8 i 0.2

Liver/B.W % 5.82 i 0.43 6.80 i 0.59 7.05 i 0.51
Thymus Wt. (mg) 0.049 i 0.006 0.036 i 0.010 0.038 i 0.008
Thymus/B.W % 0.19 i 0.03 0.13 i 0.03 0.15 :t 0.03
Sample size 8 7 8

 

F-0 females were provided treatment diets immediately after
parturition for the subsequent 3 weeks. F—0 females was terminated
on the day of weaning.

Liver/B.W, Thymus/B.W represents liver weight as percentages of
body weights and thymus weight as percentages of body weights
respectively. Values represent mean organ weights or organ weight
as percentages of body weights i Standard deviation.

55

 

 

 

Table 3. Body weights of F—la female mice fed diets containing lab
chow (C), Iowa carp (I), or Great Lakes carp (G)
Treatment diet

Age C I G

3 weeks 8.4 i 1.5A 7.1 i 1.1B 6.3 i 1.3C
(44) (39) (23)

5 weeks 16.5 i 1.1A 15.9 :1; 1.5A 9.4 -i_— 2.2'3
(32) (25) (7)

6 weeks 17.3 : 1.3A 17.1 i: 0.9A 12.3 i 3.2.3
(32) (22) (7)

7 weeks 18.9 i 1.4 18.2 i 0.9 15.7 i 2.6
(17) (11) (3)

8 weeks 19.7 i 1.6A 19.3 i 1.1A 16.7 : 0.5B
(17) (11) (3)

 

a Treatment diets were provided to the dams (F-0)

immediately after parturition through lactation and were
fed to the F—1 females from weaning through the end of
the study. Prior to weaning, F—l mice also had access to

the dams’ diet. Data presented mean i standard

deviation. Numbers in parentheses refer to sample size.

same row (p<0.05).

Different superscripts represent signiﬁcant difference within the

56

 

 

Table 4. Body weights of F-l” male mice fed diets containing lab
chow (C), Iowa carp (I), or Great Lakes carp (G)

C I G

3 weeks 8.0 i 1.7" 7.8 i 1.1A 6.4 i 1.1B
(54) (58) (37)

5 weeks 17.5 i 1.7A 18.1 i 2.0A 11.4 :1; 2.6B
(54) (58) (36)

6 weeks 20.3 i 1.8A 20.5 i 1.7A 15.0 i 3.3B
(53) (58) (32)

7 weeks 22.9 i 1.5A 22.0 i 1.5A 17.5 i 3.8B
(41) (47) (32)

8 weeks 23.9 i 1.7A 23.5 i 1.9A 19.5 i 4.1B
(41) (47) (32)

10 weeks 27.1 i 2.6A 25.3 i 1.4B 22.0 i 4.1C
(39) (47) (30)

15 weeks 35.6 i 3.6A 30.6 : 2.0B 24.9 i 3.4C
(35) (47) (27)

20 weeks 41.8 i 3.9” 34.2 i 3.2B 24.8 i 3.4C
(35) (47) (27)

25 weeks 47.8 i 3.0A 38.4 i 3.9B 25.0 i 2.2C
(32) (44) (25)

30 weeks 50.5 i 3.1A 39.9 :1; 5.3B 24.1 i 3.1C
(32) (42) (22)

35 weeks 51.8 i 3.3A 38.0 :1; 6.9B 24.9 -_i_- 3.0C
(32) (42) (14)

40 weeks 52.7 -_i- 3.6A 35.9 i 6.9B 26.3 i 1.6C
(32) (42) (13)

44 weeks 51.5 i 6.5A 35.8 i 7.6B 24.9 i 3.5C
(32) (36) (13)

 

57

Table 4. (cont’d)

a

Treatment diets were provided to the dams (F-0)
immediately after parturition through lactation and were

fed to the F-1 males from weaning through the end of

the study. Prior to weaning, F-l mice also had access to
the dams’ diet. Data presented mean i standard

deviation. Numbers in parentheses refer to sample size.
Different superscripts represent signiﬁcant differences within
the same row (p<0.05).

58

Table 5. Feed intake“ of F-l male mice

 

 

C I G

4 weeks 1.5 :1; 0.4A 1.3 i 0.5A 0.6 i.0.7B
(20) (15) (5)

8 weeks 1.4 :1; 0.2" 2.0 i 0.5B 1.3 i 0.3A
(10) (10) (10)

23 weeks 3.0 i 0.3A 3.0 : 0.3A 2.3 i 0.5B
(24) (24) (20)

 

Mice were fed diets containing lab chow (C), Iowa carp (I), or Great
Lakes carp (G) for 5 consecutive days at 4, 8 and 23 weeks of age.
Actual feed consumption was calculated daily by subtracting the
amount of feed left in the feed jars from the total feed provided on
the day before and dividing by the number of mice per cage. Values
represent mean :1; standard deviation. Units expressed as
g/mouse/day (dry weight).

Different superscripts represent signiﬁcant differences within the same
row (p <0.05).

59

 

 

 

Table 6. Body and relative organ weights of 53-week-old F-l“ male
mice fed diets containing lab chow (C), Iowa carp (I), or
Great Lakes carp (G)
Treatments
C I G
Body Wt. (g) 48.6 i 5.7A 29.9 j: 2.4B 26.1 :L- 4.0B
Liver Wt. (g) 1.99 i 0.12A 1.51 :t 0.30B 1.51 i 0.21B

Liver/B.W % 4.14 i 0.59A 5.01 :i: 0.71B 5.78 i 0.27C
Thymus Wt.(mg) 25.2 :L- 10.5A 14.0 i 4.7'3 16.5 :t 0.1AB
Thymus/B.W % 0.05 :t 0.02 0.05 i 0.01 0.06 -_l: 0.02

Testis Wt.

(mg) 206.0 i 30.6A 214.0 i 27.0A 106.9 :1: 36.9B

Testis/B.W % 0.43 i 0.06A 0.71 i- 0.07B 0.40 -i_- 0.10A

Sample size

8 8 6

 

Treatment diets were provided to the dams (F-0) immediately after

parturition through lactation and were fed to the F-1 males from
weaning through the end of the study. Prior to weaning, F-l mice
also had access to the dams’ diet.
b Liver/B.W, Thymus/B.W, Testis/B.W represents liver weight as
percentages of body weights, thymus weight as percentages of body
weights and testis weight as percentages of body weight respectively.
Values represent mean 1 standard deviation.
ABC Different superscripts represent signiﬁcant differences within the same
row (p <0.05).

60

Table 7. Body and relative organ weights of 64-weekS-old F-la
male mice fed diets containing lab chow (C), Iowa carp
(I), or Great Lakes carp (G)

 

Weightb Treatments

C I G
Body Wt. (g) 48.5 :t 8.5" 30.3 -I_- 2.8AB 23.6 : 1.5B
Liver Wt. (g) 1.89 :I: 0.37A 1.34 i 0.17AB 1.23 i 0.08'3
Liver/B.W % 3.91 i 0.27A 4.99 :l: 1.20AB 5.23 + 0.10B

 

 

Thymus Wt. (mg) 21.1 i 8.5. 15.4 i 3.6 14. 3+ _10. 7
Thymus/B.W % 0.04 -I_- 0.02 0.05 i 0.01 0.06 i 0.04

Testis Wt. 213.4 1- 19.4A 214.4 i 10.0A 83.1 i 8.8B

(mg)

Testis/B.W % 0.45 i 0.08A 0.71 :i: 0.04B 0.39 i 008"
Sample size 6 4 4

 

" Treatment diets were provided to the dams (F-0) immediately after
parturition through lactation and were fed to the F-1 males from
weaning through the end of the study. Prior to weaning, F-l mice
also had access to the dams’ diet.

b Liver/B.W, Thymus/B.W, Testis/B.W represents liver weight as
percentages of body weights, thymus weight as percentages of body
weights and testis weight as percentages of body weight respectively.
Values represent mean + standard deviation.

Different superscripts represent signiﬁcant differences within the same -.
row (p < 0. 05).

61

Table 8. Body weight and relative organ weights of F-la female mice fed

diets containing lab chow (C), Iowa carp (I), or Great Lakes
carp (G) at 4 weeks of age

 

Weightb Treatment diet
C I G

Body Wt. (g) 13.7 i 2.0“ 12.4 -_i- 2.0“ 8.8 i 2.8B
Liver Wt. (g) 0.76 i 0.14 0.74 i 0.21 0.61 i 0.18
Liver/B.W % 5.5 0.3“ 5.9 i 0.9“B 6.6 :t 0.4B
Thymus Wt. (mg) 57.6 i 15.8“ 49.8 i 8.8“ 32.9 -I_- 8.4B
Thymus/B.W % 0.42 i 0.09 0.40 i 0.02 0.31
Sample Size 6 6 6

|+

Treatment diets were provided to the dams (F-0) immediately after
parturition through lactation and were fed to the F-1 females from
weaning through 4 weeks of the age. Prior to weaning, F-l mice also
had access to the dams’ diet.

Liver/B.W, Thymus/B.W represents liver weight as percentages of
body weights and thymus weight as percentages of body weights
respectively. Values represent mean organ weights or organ weights
as percentages of body weights i standard deviation.

Different superscripts represent signiﬁcant differences within the same
row (p < 0.05).

62

Table 9. Body weight and relative organ weights of El“ female mice fed
diets containing Iowa carp (I), or Great Lakes carp (G) at 5
weeks of age

 

 

 

Weightb Treatment diet

C I G
Body Wt. (g) --- 14.7 i 1.4 11.9 :1: 2.5
Liver Wt. (g) 0.90 :i: 0.12 0.77 i 0.10
Liver/B.W % --- 6.04 i 0.30 6.63 i 0.81
Thymus Wt. (mg) --- 72.5 i 6.0“ 43.4 : 18.2B
Thymus/B.W % 0.49 :t 0.01A 0.35 i 0.10B
Sample size --- 5 5

 

“ Treatment diets were provided to the dams (F-0) immediately after
parturition through lactation and were fed to the F—1 females from
weaning through 5 weeks of the age. Prior to weaning, F-l mice also
had access to the dams’ diet.

" Liver/B.W, Thymus/B.W represents liver weight as percentages of

body weights and thymus weight as percentages of body weights

respectively. Values represent mean organ weights or organ weight
as percentages of body weights :1: standard deviation.

Different superscripts represent signiﬁcant differences within the same

row (p < 0.05).

63

Table 10. Body weight and relative organ weights of F-l’ female mice fed
diets containing lab chow (C), Iowa carp (I), or Great Lakes

carp (G) at 6 weeks of age

 

 

 

Weightb Treatment diet

C I G
Body Wt. (g) 18.3 :i: 0.8“ 19.1 : 0.1A 12.2 i- 3.1B
Liver Wt. (g) 1.04 i 0.13“C 1.22 i 0.09A 0.78 i 0.25BC
Liver/B.W % 5.68 i 0.52 6.36 i 0.46 6.29 i 0.51
Thymus Wt. (mg) 57.3 i 8.6“ 80.1 i 13.2B 32.9 i 90‘:
Thymus/B.W % 0.31 :1: 0.05“ 0.42 i 0.07'3 0.27 i 0.01“
Sample Size 5 3 3

 

“ Treatment diets were provided to the dams (F—O) immediately after
parturition through lactation and were fed to the F-1 females from
weaning through 6 weeks of the age. Prior to weaning, F-l mice also

had access to the dams’ diet.

b Liver/B.W, Thymus/B.W represents liver weight as percentages of
body weights and thymus weight as percentages of body weights
respectively. Values represent mean organ weights or organ weight
as percentages of body weights i standard deviation.

“BC Different superscripts represent signiﬁcant difference within the same

row (p < 0.05).

64

Table 11. Body weight and relative organ weights of F—la female mice fed
diets containing lab chow (C), Iowa carp (I), or Great Lakes
carp (G) at 8 weeks of age

 

Weightb Treatment diet

C I G
Body Wt. (g) 19.9 i 0.5“ 20.1 i 0.9“ 16.4 i 1.6B
Liver Wt. (g) 1.06 i 0.08“ 1.07 :i: 0.05“ 0.92 i 0.07B
Liver/B.W % 5.32 -i_- 0.29 5.34 i 0.42 5.62 i 0.20
Thymus Wt. (mg) 38.5 i 10.4' 44.3 :1; 8.4 46.0 i 13.5
Thymus/B.W % 0.19 i 0.06 0.22 i 0.05 ~ 0.28 i 0.07
Sample size 5 4 4

 

 

 

“ Treatment diets were provided to the dams (F-0) immediately after
parturition through lactation and were fed to the F-1 females from
weaning through the 8 weeks of the age. Prior to weaning, F-l mice
also had access to the dams’ diet.

Liver/B.W, Thymus/B.W represents liver weight as percentages of
body weights and thymus weight as percentages of body weights
respectively. Values represent mean organ weights or organ weight
as percentages of body weights i standard deviation.

Different superscripts represent signiﬁcant difference within the same
' row (p <0.05).

65

Table 12. Survival of offspring from F-O femalesa fed diets
containing lab chow (C), Iowa carp(I), or Great Lakes

carp (G)

 

Treatment diet

 

0

Parameter I G

 

4-day survivalb 96.0 i 11.7“B 98.7 _+_ 4.9“ 89.6 i— 12.2B
21-day survivalb 97.4 j; 5.2“ 84.9 i 31.0“ 56.1 :1: 37.2B
Sample size 14 14 16

 

8‘ F—0 female were provided treatment diets immediately after
parturition for the subsequent 3 weeks.

Mean i standard deviation of the ratio of the number of live
pups on day one after birth to the number of live pups on day
4 and day 21, respectively.

Different superscripts represent signiﬁcant differences within
the same row (p<0.05).

66

 

 

 

Table 13. Survival of F-la male mice fed diets containing lab
chow (C), Iowa carp (I), or Great Lakes carp (G)
Treatment diet
Weeks C I G
3 100% 100% 100%
10 100% 100% 100%
20 100% 100% 100%
30 100% 100% 87.5%
44 100%“ 90.7%“ 56.3%B
Sample size 37 43 32

 

" Treatment diets were provided to the dams (F-0)
immediately after parturition through lactation and were
fed to the F-1 males from weaning through the end of the
study. Prior to weaning, F-l mice also had access to the
dams’ diet.

“3 Different superscripts represent signiﬁcant differences
within the same row (p<0.05).

67

Table 14. Effect of treatment diets on vaginal opening in F-l“
mice fed diets containing lab chow (C), Iowa carp (I),
or Great Lakes carp (G)

 

Treatment diet

 

C I G

Age of vaginal 28.4 i 0.5“ 30.4 i 1.1B 46.0 : 3.9C
opening (days)b

Body weight on 12.8 i 0.6 13.3 i 1.0 11.4 i 1.7
day of vaginal

Opening” (g)

Number of mice 5 5 5

 

’ Treatment diets were provided to the dams (F-O)
immediately after parturition through lactation and were fed
to the F-1 females from weaning through the end of the
Study. Prior to weaning, F-l mice also had access to the
dams’ diet.

Data represents mean j; standard deviation.

Different superscripts represent signiﬁcant differences within
the same row (p<0.05).

68

Table 15. Reproductive performance of 1-year—old F-la males fed
diets containing lab chow (C), Iowa carp (I), or Great
Lakes carp (G)

 

Treatment diet

 

 

Parameters C I G
Fecundityb 10/ 15“ 1 1/ 15“ 1/11B
Litter sizec 8.0 i 2.7 8.1 i 2.3 12 i 0
(10) (11) (1)
Sex ratio 0.92 0.95 0.33
(male/female) (38/41) (42/44) (3/9)

 

’ Treatment diets were provided to the dams (F-0) immediately
after parturition through lactation and were fed to the F—1 males
from weaning through the end of the study. Prior to weaning, F-
1 mice also had access to the dams’ diet.

Fecundity refers to the number of females that gave birth/number
of female paired.

Litter size was recorded the day after birth. Data presented as
mean i standard deviation. Numbers in parentheses refer to
sample size.

Different superscripts represent signiﬁcant differences within the
same row (p<0.05).

69

Table 16. Epididymal sperm concentration (million/ml) in F-l“

male mice fed diets containing lab chow (C), Iowa carp
(I), or Great Lakes carp (G)

 

Treatment diet

 

 

Age(Method) C I G
53-week- 26.4 i 6.9“ 20.9 i 8.4“B 6.1 i 8.8B
old(Manual)b (6) (6) (6)
64-week- 27.5 i 13.9“ 22.7 i 10.7“ 0.01 i 0.02B
old(Manual) (6) (4) (4)
53-week— 24.1 i- 8.8“ 21.7 i 6.9“ 5.4 i 6.63
old(CellSoft)“ (6) (6) (6)
64—week- 43.2 i 16.0“ 36.7 i 9.4“ 1.5 i 0.6B
old(CellSoft) (6) (4) (4)

 

Treatment diets were provided to the dams (F-0) immediately
after parturition through lactation and were fed to the F-1
males from weaning through the end of the study. Prior to
weaning, F-1 mice also had access to the dams’ diet.

Manual sperm concentration was measured by placing 20 p1
sperm suspension on a Petroff Hausser Counting Chamber
(Hausser Scientiﬁc Co.,Horsham, PA) containing 16 cells.
Sperm were counted in 5 randomly chosen cells and the total
was used to calculate the ﬁnal concentration. Data presented
as mean i standard deviation. Numbers in parentheses refer
to sample size. "

Sperm concentration was measured by CellSoft computer-
assisted digital image analysis system (CRYO Resources Inc.,
New York). Data presented as mean -1_- standard deviation.
Numbers in parentheses refer to sample size.

Different superscripts represent Signiﬁcant differences within
the same row (p <0.05).

70

Table 17. Sperm motion analysis in 53-week-old“ F—l male mice fed
diets containing lab chow (C), Iowa carp (I), or Great
Lakes carp (G)

 

Treatment diet

 

Parameters C I G

 

Percent motile (%) 55.2 :t 11.8 59.0 i 10.5 24.6 i 25.1
Velocity (pm/sec) 122.2 :1: 7.5 124.8 i 8.9 106.0 i 98.5

Linearity 4.0 i 0.6 4.3 i 0.7 3.9 :1; 3.5
A.L.H 5.1 i 1.4 4.8 i 0.8 2.1 i- 2.5
displacement (pm)b ’

Beat/cross 11.0 i- 5.6 11.5 i 3.2 8.2 -_t 9.2

frequency (Hz)
Sample size 6 6 6

 

“ Treatment diets were provided to the dams (F-0)
immediately after parturition through lactation and were
fed to the F-1 males from weaning through the end of the
study. Prior to weaning, F-l mice also had access to the
dams’ diet. Data presented as mean i standard deviation.
A.L.H. displacement refers to amplitude of lateral head
displacement.

71

Table 18. Sperm motion analysis in 64-week-olda F-l male mice
fed diets containing lab chow (C), Iowa carp (I), or
Great Lakes carp (G)

 

Treatment diet

 

Parameters C I G

 

Percent motile (%) 65.1 -_i- 22.2“ 54.2 i 17.6“ 9.0 : 14.3B
Velocity (pm/sec) 98.4 i 13.4“ 98.2 :1: 11.5“ 16.6 i 33.3B

Linearity 5.4 i 0.3“ 5.5 i 0.8“ 1.1 : 2.2B
A.L.H 3.8 f 0.5“ 3.5 -i_- 0.8“B 0.1 i: 0.2B
displacement (pm)b

Beat/cross 12.2 i 1.2,. 10.8 :t 1.3 4.4 i 8.8
frequency (Hz)

Sample size 6 4 4

 

“ Treatment diets were provided to the dams (F-0)
immediately after parturition through lactation and were
fed to the F-1 males from weaning through the end of the
study. Data presented as mean i standard deviation.
A.L.H. displacement refers to amplitude of lateral head
displacement.

Different superscripts represent signiﬁcant differences within
the same row (p<0.05).

AB

72

Table 19. Fertilizing ability of epididymal sperm from 53-week-
old F—l‘l males fed diets containing lab chow (C), Iowa
carp (I), or Great Lakes carp (G)

 

Treatment diet

 

Parameters C I G

 

Percent of 74.0 i 8.7“ 70.4 i 16.8“ 7.1 i 7.0B
oocytes
fertilizedb

Number of 158 279 255
oocytes
observed

 

‘ Treatment diets were provided to the dams (F-0)
immediately after parturition through lactation and
were fed to the F-1 males from weaning through the
end of the study.
b Data presented as mean i standard deviation.
“3 Different superscripts represent Signiﬁcant differences within the
same row (p<0.05).

Body Weight (g)

73

25 I i T l T l

 

20- -

 

 

 

 

 

 

 

 

,/ ..
‘ “r

15 .— .-
T

10 - -»
.L

5 I L l 1 l

2 3 4 5 6 7 8 9

Age (weeks)

Figure 1. Body weight of F-1 female mice.
DietC(.O ), dietl( v ), and dietG( v )
were provided to the dams (F-O)

immediately after parturition through lactation
and were fed to F-1 from weaning through
the end of the study.

Body Weight (g)

60

50

4O

30

20

10

 

 

 

 

 

 

 

 

l I l I

 

 

10 20 30 40 50

Age (weeks)

Figure 2. Body weight of F-1 male mice.
Diets containing lab chow (C). Iowa carp
(I), or Great Lakes carp (G) were provided
to the dams (F—O) immediately after
parturition through lactation'and were fed
to F-1 from weaning through the end

of the study.

SUMMARY

Lactational and dietary exposure to the contaminants in Great Lakes
carp had an adverse effect on reproduction in mice. High neonatal mortality
and decreased body weight were observed in F-l mice on the Great Lakes

treatment diet. Liver enlargement was also observed in 4-week-old F-l
females and 53, 64-week-old F—l male mice. In the Great Lakes treatment
group, ninety-one percent of the one-year-old mice failed to breed with the
non-treated females. Lower sperm fertilizing ability, motility, velocity,
linearity, and amplitude of lateral head (ALH) displacement were also
observed. Delayed vaginal opening was observed, in female mice on the
Great Lakes treatment diet.

It is not clear why the Great Lakes ﬁsh diet adversely effected the
reproduction of old mice in present study but not young mature male mice
in a previous study. In future studies, the effects of individual PCB
congeners and DDT metabolites, the major contaminants in Great Lakes,

should be investigated in post-maturation male mice.

75

APPENDICES

APPENDIX I

Appendix 1. Pesticides banned, suspended, or severely restricted in the

Appendix I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

U.S.A.
Action Year
Aldrin All uses canceled except termite control 1974
Chlordane Cancellation of most uses, except termite 1978
control
All uses canceled 1988
DDT All agricultural use canceled 1972
Use only for public health emergencies
Diazinon Use on golf courses and sod farms 1986
canceled
Dieldrin Cancellation of most uses 1974
Ethylene All uses canceled 1984
dibromide
(EDB)
Mirex All use canceled except pineapples in 1977
Hawaii
N itrofen (T OK) Voluntary cancellation 1983
2,4,5-T/Silvex Emergency suspension 1979 ll
All use canceled 1985
Toxaphene All use canceled except sheep and cattle 1982
dip, bananas and pineapple in Puerto
Rico and Virgin Island

 

 

 

76

APPENDIX 11

APPENDIX II

Appendix II. Calculation of sperm concentration:

Concentration/ml = (Number sperm count x dilution factor x 2 x 107)/80

77

APPENDIX III

APPENDIX III

Appendix 111. Deﬁnition of sperm motion parameter:

Percent motility:
The percentage of sperm that travel more than 20
pm/sec.

Velocity (pm/sec):
Average of the distance traveled by motile sperm in one
second.

Linearity (1-10):
A measure of the straightness of a sperm track on a scale
of 0 to 10. Ten indicates a perfectly straight line and 0
indicates a circular track. It is calculated by dividing the
length of the straight line distance by the actual track
distance.

Amplitude of lateral head displacement (ALI-I) (pm):
A measure of the displacement of the sperm head from
a computer-calculated curval mean of its track.

Beat/Cross Frequency (Hz):
The beat/cross frequency is reported as the number of
beats (cross) per second. Every time the sperm cell
crosses the computer-calculated curval mean, the
computer counts that crossing as one beat.

78

APPENDIX IV

Appendix IV: Analysis of organochlorine pesticides and PCBS in the diet

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83

APPENDIX V

Appendix V. Reproductive performance of 48- and 53-week—old F—la
males fed diets containing lab chow (C), Iowa carp (I),
or Great Lakes carp (G)

Reproductive performance of 48-week-old F-lal males fed diets
containing lab chow (C), Iowa carp (I), or Great Lakes carp (G)

 

 

 

Treatments
Parameters C I G
Fecundityb 8/ 10 7/ 10 1/6
4-day survivalc 94.4 i 8.7 95.7 :l: 7.9 100 :t 0.0
21-day‘ survivalc 97.9 i 5.9 ., 100 :1: 0.0 100 i 0.0
Sex ratio 29/31(0.94) 25/29(O.86) 3/9(0.33)

male/ female

_
g

Treatment diets were provided to the dams (F-O) immediately after
parturition through lactation and were fed to the F-1 females from
weaning through 4 weeks of the age. Prior to weaning, F-l mice also

had access to the dams’ diet.

Fecundity refers to the number of females that gave birth/number of

female paired.

Mean :1: standard deviation of the ratio of the number of live pups on
day one after birth to the number of live pups on day 4 and day 21,

respectively.

84

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