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MSU In An mum Action/Emil Oppommuy IMRWOH
MOMS-9.1

AN ASSESSMENT OF THE NEUROTOXIC POTENTIAL
OF SPECIFIC PCB CONGENERS

By

Lee Lipsitz

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

MASTER OF SCIENCE

Department of Anatomy

1996

ABSTRACT

AN ASSESSMENT OF THE NEUROTOXIC POTENTIAL
OF SPECIFIC PCB CONGENERS

By

Lee Lipsitz

PCBs have been an ideal industrial Chemical due to their stable nature and
resistance to' heat but over time they have become significant environmental
contaminants. The main emphasis in these studies has been to define the effects of PCBs
on the morphology of the developing chicken brain using histological techniques and
computed tomography. Experiments were also designed to assess the neurocytotoxicity
of PCB congeners using the Fink-Heimer stain for degenerating axons and terminals.
Experiments involved injecting domestic chicken (Gallus domesticus) eggs with individual
PCB congeners. There were no significant differences in the amount of asymmetry
present between treatment groups and the non-injected control group (p 2 0.05). It was
found that the normal development of asymmetry in avian neural systems as well as
external forces involved in the development of the chick in the egg were likely significant
enough to overshadow subtle changes brought on by exposure to environmental

contaminants.

To Dale and my parents, without whom this work would not have been possible.

iii

ACKNOWLEDGMENTS

This thesis would not have been possible without the guidance and encouragement
of Dr. Duke Tanaka Jr. His support throughout has been invaluable and has made it
possible for me to accomplish this thesis. I would like to also thank Drs. Steve Bursian,
Sharleen T. Sakai, and James Sikarskie for critical review of this thesis, Dr. John Gill
for his guidance in statistical analysis of the data as well as Dr. Diana Rosenstein for
technical assistance with computed tomography. Funding for this project was provided
in part by a Biomedical Research Support Grant awarded to the College of Veterinary

Medicine, Michigan State University.

iv

TABLE OF CONTENTS

LIST OF ABBREVIATIONS .............................
INTRODUCTION ....................................

Chapter 1

ASSESSMENT OF CEREBRAL HEMISPHERIC SYMMETRY IN HATCH-

LING CHICKENS EXPOSED IN 0V0 TO POLYCHLORINATED BI-

PHENYL CONGENERS ...............................
Materials and Methods ...............................
Results .........................................
Discussion .......................................

Chapter 2

COMPUTED TOMOGRAPHY (CT) AS A TOOL IN DETERMINING THE

PRESENCE OF CEREBRAL HEMISPHERIC ASYMMETRY IN LIVE

BIRDS ...........................................
Materials and Methods ...............................
Results and Discussion ...............................

Chapter 3

ASSESSMENT OF THE NEUROCYTOTOXIC POTENTIAL OF BOTH A

COPLANAR AND NON-PLANAR PCB CONGENER USING THE FINK-

HEIMER STAINING TECHNIQUE ........................
Materials and Methods ...............................
Results and Discussion ...............................

Chapter 4
CONCLUSIONS .....................................

12
13

18
19
21

26
27
29

32

APPENDIX A

TABLES 1-3

Table 1.

Table 2.

Table 3.

APPENDIX B

Mean values (mm) for the absolute differences in width and
height measurements between left and right cerebral hemispheres
of hatchling domestic chickens exposed at day 0 of incubation to
individual PCB congeners ......................

CT numbers obtained from brain images of double-crested
cormorants exposed to 100 ,ug PCB congener 126/kg egg at day
0 of incubation .............................

Mean values (mm) for the absolute differences in width and
height measurements between left and right cerebral hemispheres
of hatchling domestic chickens exposed at day 0 of incubation to
PCB congener 52 ...........................

FIGURES 1-13

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Structure of PCB congeners and comparison of structure to
TCDD ..................................

Photomicrograph of a 40 pm thick section of a hatchling
domestic chicken brain from the non-injected control group
illustrating a cereme hemisphere stained with cresyl violet . .

Line drawing of a parasagittal section through the chick brain
illustrating the levels from which each of the 8 sections were
taken for hemispheric measurements ................

Line drawing of coronal sections at the eight section levels from
which measurements for asymmetry were obtained from the
brains of all PCB treatment and control groups of hatchling
domestic chickens ...........................

Line drawing of section level 1 showing how width and height
measurements were obtained from a hatchling domestic chicken
brain for all PCB treatment and control groups .........

Histogram of width and height measurements by section level for
all PCB treatment groups and the non-injected control group of
hatchling domestic chickens .....................

Comparison of bird brain positioning on computed tomography
(CT) scanning table and histological sectioning .........

vi

35

36

36

37

38

39

4O

41

42

43

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Illustration of CT gantry and patient scanning table .......

Printed CT image showing the cursor on the midline of the
cerebral hemisphere of a hatchling double-crested cormorant
chick exposed in ova to PCB congener 52 ............

Printed CT image with cursor placement depicting width
measurement of the cerebral hemisphere of a hatchling double-
crested cormorant chick exposed in avo to PCB congener 52 .

Printed CT image with cursor placement depicting height
measurement of the cerebral hemisphere of a hatchling double-
crested cormorant chick exposed in ovo to PCB congener 52 .

Printed CT images accompanied by line drawings depicting
symmetrical positioning on the scanning table of the hatchling
double-crested cormorant chick exposed in ovo to PCB congener
52 .....................................

Printed CT images accompanied by line drawings depicting
asymmetrical positioning on the scanning table of the hatchling
double-crested cormorant chick exposed in ovo to PCB congener
52 .....................................

LIST OF REFERENCES ...............................

vii

45

46

47

48

50

52

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Illustration of CT gantry and patient scanning table .......

Printed CT image showing the cursor on the midline of the
cerebral hemisphere of a hatchling double-crested cormorant
chick exposed in ovo to PCB congener 52 ............

Printed CT image with cursor placement depicting width
measurement of the cerebral hemisphere of a hatchling double-
crested cormorant chick exposed in ovo to PCB congener 52 .

Printed CT image with cursor placement depicting height
measurement of the cerebral hemisphere of a hatchling double-
crested cormorant chick exposed in ova to PCB congener 52 .

Printed CT images accompanied by line drawings depicting
symmetrical positioning on the scanning table of the hatchling
double—crested cormorant chick exposed in ovo to PCB congener
52 .....................................

Printed CT images accompanied by line drawings depicting
asymmetrical positioning on the scanning table of the hatchling
double-crested cormorant chick exposed in ovo to PCB congener
52 .....................................

LIST OF REFERENCES ...............................

vii

45

46

47

48

50

52

Bas
BK
BO
CA
CB
CH
CO

FA
FB
FPL
HA

HV
IN P
LF S
LFSM

LMD
LPO

NB

LIST OF ABBREVIATIONS

Archistriatum anterior
Nucleus basalis

Beak

Bulbus olfactorius
Anterior commissure
Cerebellum

Cerebral hemispheres
Chiasma opticum
Ectostriatum

Eye

Tractus fronto-archistriaticus
Frontal bone

Lateral forebrain bundle
Hyperstriatum accessorium
Hyperstriatum dorsale
Hippocampus
Hyperstriatum ventrale
Nucleus intrapeduncularis
lamina frontalis

lateral frontalis suprema
Lamina hyperstriatica
Lamina medullaris dorsalis
Lobus paraolfactorius
Neostriatum

Nasal bone

viii

nCPa

PA
PP
SL
TSM

Nucleus commissurae pallii
Neck

Pineal gland

Paleostriatum augmentatum
Paleostriatum primitivum
Nucleus septalis lateralis

Tractus septomesencephalicus

ix

INTRODUCTION

In this thesis I will present the results of three studies that attempted to assess the
potential neurotoxic effects of polychlorinated biphenyls (PCBs). The introduction will
discuss PCBs, the history of their use, and the importance of determining their effects
on the central nervous system (CNS). The subsequent chapters will present different
approaches used to define the potential effects of PCBs on the CNS.

Polychlorinated biphenyls are industrial products that were first synthesized in
1881 by reacting chlorine gas with biphenyls. They became commercially available in
1930 and were produced in the US. primarily by Monsanto Chemical Co. of St. Louis,
Missouri. Polychlorinated biphenyls were sold in mixtures based on their chlorine
content under the trade name Aroclor". There are 209 individual PCB congeners, each
given an International Union of Pure and Applied Chemistry (IUPAC) number from 1
to 209. Each congener is comprised of a biphenyl ring with a different number of
chlorine atoms attached at the ortho, para, and/or meta positions (Erickson 1986).

Polychlorinated biphenyls are members of a larger class of compounds, the
halogenated aromatic hydrocarbons. Both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) are members of this class. Both of these
compounds are known environmental contaminants and considered to be highly toxic

(Safe 1984). Of the 209 PCB congeners, there are four that do not have chlorine atoms

2

at the ortho positions but have chlorine atoms at the para position and at least two meta
positions enabling them to attain a coplanar form similar enough in structure to mimic
the highly toxic behavior of both TCDD and PCDF (Safe 1984, Tanabe 1988, Rice and
O’Keefe 1995) (Figure 1). It is important to clarify coplanar versus planar congeners
as these terms are used interchangeably in the literature. The coplanar configuration of
a PCB congener means that each phenyl ring can lie in the same plane because there are
no chlorine atoms at the ortho position preventing such rotation (Rice and O’Keefe
1995). The term planar, although used by some in the literature in place of coplanar,
does not describe the position of the two phenyl rings relative to one another and is
therefore not accurate. The coplanar aromatic hydrocarbons are believed to exert their
primary biologic effects through the AH-receptor (Swanson and Bradfield 1993, Nebert
1989). The AH-receptor has been implicated in the regulation of xenobiotic metabolism,
chemical carcinogenesis, and the mechanism of toxicity of halogenated aromatic
hydrocarbons (Swanson and Bradfield 1993). One of the ligands for the AH-receptor is
TCDD (Nebert 1989). Any compound that can mimic the structure of TCDD, such as
planar PCB congeners, can also act as a ligand for the AH-receptor.

Polychlorinated biphenyls are highly stable and heat resistant, making them ideal
for use in industry. They have been used in paints, printing inks, coolants for motors,
transformer fluids, hydraulic fluids, plasticizers and plastics, in caulking compounds,
adhesives, and carbonless copy paper, and as extenders for pesticides (Peterle 1991).
Due to their widespread use and stability they have become ubiquitous in our environ-
ment. In the early 19708 it became evident that PCBs were becoming environmental
contaminants and that chronic exposure could result in hazards to human health. This

lead Monsanto Chemical Co., the primary U.S. producer to voluntarily ban the sale of

3
PCBs. In 1976 with the development of the new US. Toxic Substances Control Act the

manufacture of PCBs was banned. The ban, which became effective after implementation
by the US Environmental Protection Agency in 1979, prohibited the manufacture,
processing, commercial distribution and use of PCBs except in totally closed systems (
Rice and O’Keefe 1995). Despite this ban, PCBs still enter the environment from
approved former use, incineration, illegal dumping in landfills, and atmospheric
deposition. PCBs can also be produced unintentionally as by-products during the
processing of other chemicals (Erickson 1986).

Polychlorinated biphenyls are highly lipophilic and show low biodegradability,
which enables them to persist in fish, bird, non-human mammal, and human populations
(Peakall and Fox 1987, Tanabe 1988). The persistence of PCBs is a concern due to their
bioaccumulative and biomagnification potentials. PCBs tend to accumulate in adipose
tissue (Safe 1984). As PCBs are consumed by an individual organism, their preferential
storage in adipose tissue creates an accumulation of the compound that may not be
excreted at the same rate at which the organism has been exposed. In a study by Bush
at al. (1974), PCBs were added to the drinking water of adult hens to reproduce low-
dose chronic exposure. Bush found that PCB levels built up faster than they declined and
that at a mean of 18.7 weeks, the toxicity was five times greater than at a mean of 1.6
weeks of exposure.

The biomagnification of PCBs in the food chain is also of concern (Colburn et al.
1990). Organisms found at higher levels of the food chain will have higher concentra-
tions of toxicants than organisms present at lower levels. This creates a situation where

a small concentration of a toxicant can enter an ecosystem low in the food chain and be

4

found in several-fold higher concentrations in organisms such as fish-eating birds,
mammals, or humans residing at higher levels of the food chain (Safe et al. 1987).
Toxicant levels in the Great Lakes are high enough that there are provisional warnings
for humans not to eat various fish species that have a high lipid content (Baumann and
Whittle 1988). There is evidence to suggest that exposure to PCBs in fish-eating birds
from the Great Lakes may result in decreased reproductive success due to embryotoxicity
and teratogenicity (Kubiak et al. 1989, Peakall and Fox 1987, Baumann and Whittle
1988, Elliot et al. 1989).

When environmental samples such as water, soil, or fish are analyzed for their
PCB content, the standard method employed is gas chromatography (Burse er al. 1983,
Sawyer et al. 1995). However, these methods for measuring levels of PCBs do not take
into account individual congeners and their individual potencies. Safe (1994) emphasizes
the importance of studying individual PCB congeners and suggests that risk assessments
not rely solely on the toxicity of known commercial mixtures of PCBs. When the
concentration of individual PCB congeners found in environmental samples is compared
to the concentration of those same congeners in commercial PCB mixtures, the
concentrations are more varied than would be expected. It is for this reason that
isolating individual congeners and assessing their potential toxicity is more important than
studying PCBs in commercial mixtures.

PCBs have been shown to affect the liver, integument, and immune, reproductive,
and nervous systems (Hansen 1987, Hoffman 1994, Safe 1994). Several investigators
have focused on the nervous system with an emphasis on defining the behavioral and

chemical effects of PCBs (Agrawal er al. 1981, Ericksson 1988, Seegal and Shain 1992).

5

However, relatively few studies have investigated structural changes that may occur due
to PCB exposure. This thesis is comprised of three separate but related studies, each of
which attempts to define the toxicological effects of individual PCB congeners by
assessing structural changes in the central nervous system that may occur as a result of
exposure.

In the first study, morphometric techniques were used to assess the effects of
PCBs on the morphology of the cerebral hemispheres of hatchling domestic chickens
(Gallus domesticus). Previous investigators (Henshel et al. 1995) have reported that
exposure to a mixture of environmental contaminants including PCBs results in
morphologic asymmetry of the cerebral hemispheres in hatchling great blue herons
(Ardea herodias) and have suggested that this asymmetry may be a useful biomarker for
contamination. The present study was designed to determine whether two environmentally
relevant non-ortho substituted PCB congeners, 3 ,3 ’ ,4,4’-tetrachlorobiphenyl (IUPAC
#77) and 3,3’,4,4’,5-pentachlorobiphenyl (IUPAC #126), could cause morphologic
asymmetry in the cerebral hemispheres.

The second study was a preliminary investigation into the potential usefulness of
computed tomography (CT) to address the same issue of cerebral hemispheric
asymmetry. Eggs of double-crested cormorants (Phalacrocorax auritus) from the Great
Lakes were removed from the wild and injected with congener 126. This study was
designed to determine if CT imaging could replace the need to sacrifice the bird in order
to obtain information about the morphology of the cerebral hemispheres. If this were
possible, the brains of wild birds could be measured in situ and the birds then returned

to their habitats.

6
The third study employed the Fink-Heimer silver impregnation stain for

degenerating axons and terminals to determine whether exposure to an ortho-substituted
PCB congener, 2,2’,5,5’—tetrachlorobiphenyl (IUPAC #52), or a coplanar PCB
congener, such as congener 126, would result in axonal neuropathy. Work by Seegal and
Shain (1992) has suggested that the ortho-substituted congeners, which are non-planar
and therefore do not mimic the structure of TCDD, are responsible for the neurotoxic
effects that have been observed in their research. They found that in non-human
primates only the ortho-substituted congeners were detectable in the brain after sub-
chronic exposure to a commercial mixture of PCB congeners. The same ortho-
substituted congeners were responsible for reducing dopamine levels in both non-human
primate brains as well as in pheochromocytoma cell cultures (PC12). In the present
study, the Fink-Heimer silver stain was used to determine whether reductions in

dopamine levels may be accompanied by signs of axonal and terminal degeneration.

Chapter 1
ASSESSMENT OF CEREBRAL HEMISPHERIC SYMMETRY IN HATCH-
LING CHICKENS EXPOSED IN 0V0

TO POLYCHLORINATED BIPHENYL CONGENERS

PCBs have been shown to affect the liver, integument, and immune, reproductive,
and nervous systems of both avian and mammalian species (Hansen 1987, Hoffman 1994,
Safe 1994). Many of the studies directed toward the central nervous system (CNS) have
defined several behavioral and chemical effects of PCB exposure (Agrawal et al. 1981 ,
Ericksson 1988, Seegal and Shain 1992). Ericksson (1988) suggested that PCB congener
77 (3,3’,4,4’-tetrachlorobiphenyl) had an effect on the muscarinic cholinergic receptors
in the mouse brain during development. Agrawal et al. (1981) reported that mice
exposed in utero to PCB congener 77 showed a decrease in the number of dopamine
receptor binding sites as well as low dopamine concentrations in the corpus striatum. A
series of studies by Seegal and colleagues indicated that certain PCB congeners reduced
dopamine concentration in the hypothalamus and basal ganglia of nonhuman primates as
well as in pheochromocytoma (PC12) cell cultures (Seegal et al. 1991 , Shain et al. 1991,
Seegal and Shain 1992).

Recently, Henshel et al. (1995) reported that exposure to a mixture of

environmental toxicants including PCBs may have an impact on the morphological

8

development of the CNS. They examined the brains of great blue heron hatchlings
(Ardea herodias) from colonies in the Strait of Georgia, British Columbia, which were
contaminated with a mixture of toxicants including polychlorinated dibenzo-p—dioxins
(PCDD), polychlorinated dibenzofurans (PCDF), and PCBs. They reported a grossly
detectable asymmetry of the cerebral hemispheres and suggested a causal relationship
between cerebral asymmetry and exposure to PCDDs and related compounds. They
proposed that this asymmetry could be a useful biomarker for assessing the effects of
PCDDs and related compounds on central nervous system development.

One of the contaminants that may have contributed to the cerebral asymmetry V
.found in great blue heron chicks from these contaminated sites was PCBs. We wished
to determine if PCBs alone might also induce a morphological asymmetry of the cerebral
hemispheres. Accordingly, we initiated a study on the effects of individual PCB
congeners on the morphology of the developing chicken brain. We chose to examine the
effects of PCB congeners 77 (3,3’,4,4’-tetrachlorobiphenyl) and 126 (3,3’,4,4’,5-penta-
chlorobiphenyl) based on the results of a study by Yamashita et al. (1993) who reported
that these two congeners accounted for the majority of 2,3,7,8-tetrachlorodi-
benzo-p-dioxin (TCDD)-toxic equivalents in eggs of double-crested cormorants (Phala-
crocorox auritus) and caspian terns (Hydroprogne caspia) collected from the Great Lakes
basin. Similarly, Sanderson et al. (1994) reported that these PCB congeners were the
largest contributors to the TCDD-toxic equivalents present in double-crested cormorant
eggs from five colonies nesting on the Great Lakes.

The domestic chicken (Gallus domesticus) was chosen as an experimental subject
because much of the research dealing with in ovo exposure to environmental contami-

nants has been done with this species (Brunstrom and Orberg 1982, Brunstrom and

9
Darnerud 1983, Brunstrom and Andersson 1988, Brunstrom and Lund 1988) and it is

known to be very sensitive to PCBs, having a higher death rate upon exposure than
several other species of ducks, geese, and gulls. In addition, the domestic chicken is

very sensitive to the PCB coplanar congeners 77 and 126 (Brunstrom and Andersson

1988, Brunstrom and Lund 1988).

Materials and Methods

White Leghorn chicken eggs were obtained from the Michigan State University
Poultry Science Teaching and Research Center. PCB congeners 77 and 126 (99.9%
pure) were obtained from AccuStandard (New Haven, CT), and dissolved in an emulsion
of egg yolk, lecithin (Merck; Darmstadt, Germany), peanut oil, and water (Brunstrom
and Orberg, 1982). Eggs were divided into three treatment groups and two control
groups. Eggs in two of the treatment groups received single injections into the yolk of
either congener 77 (3.0 or 9.0 jig/kg egg) or congener 126 (0.3 or 0.9 jig/kg egg) in a
volume of 1 ml/kg egg on day 0 of incubation. Eggs from the third treatment group
were injected on day 0 with a combination of congener 77 (0.9 ug/kg egg) and congener
126 (0.8 pg/kg egg) in a volume of l m1/kg egg. The dosages used for the single
injection groups were based on the LDso values of the congeners (congener 77 = 8.8
ug/kg egg and congener 126 = 0.6 ug/kg egg) as determined in another study (Powell
et al. 1996,). The dosages in the combination treatment group corresponded to the
concentrations detected in double-crested cormorant eggs collected on Tahaquamenon
Island, the least contaminated site in a study by Yamashita et al. (1993). An additional

group of eggs served as the non-injected control, whereas the last group received an

10
injection of vehicle only (1 ml/kg egg) made up of lecithin, peanut oil, and water

(Brunstrom and Orberg, 1982).

The eggs were incubated in a Petersirne incubator (Gettysburg, OH) at 37.5—37.7 °
C and approximately 65% relative humidity. The eggs were candled on days 4 and 11
of incubation to check for viability. Chicks hatched or were in the process of pipping
by day 21, at which time they were removed from the incubator. They were weighed
and their heads examined for signs of gross abnormalities. Chicks that had not
completely pipped were removed from their eggs, weighed, and examined.

Following examination, all chicks were overdosed with an intraperitoneal injection
of sodium phenobarbital (150 mg/kg body wt; 1 ml/kg body wt) and perfused
transcardially with a heparinized 10% formalin-saline solution using a 60cc syringe and
25-gauge hypodermic needle. All procedures related to brain removal, sectioning, and
mounting were standardized in order to minimize variation caused by differences in
processing methodologies. Immediately following perfusion of each chick, the calvaria
were incised sagittally along the midline of the skull. This was followed by a cut made
perpendicular to the midline at the level of the tentorium cerebelli. The skull was then
peeled back to allow for maximum exposure to fixative. The brains were removed from
the calvaria 24 hours after perfusion, weighed, and transected at the level of the rostral
midbrain. The rostral part of the brain containing the hemispheres was weighed and
placed in 10% formalin-saline followed by a 30% sucrose-formalin solution to allow for
better sectioning of the neural tissue. Brains were then embedded in gelatin and placed
in 30% sucrose-formalin prior to sectioning. The brains were serially sectioned on a

freezing microtome in the coronal plane at a thickness of 40 um. One out of every five

11

sections (approximately 200 um apart) was stained with cresyl violet, a basophilic stain
for N issl substance (Figure 2).

The anterior commissure (CA) was chosen as an initial reference point to begin
measurements of symmetry. For the brain section to be useful for subsequent
measurements, the CA had to be visible crossing the midline in a single section and be
I evenly distributed within both hemispheres. This criterion was used to ensure that the
brains had been sectioned perpendicular to the midline. Once the caudal-most section
containing the CA had been identified, a series of 8 sections approximately 400 um apart
and located rostral to the CA were chosen for hemispheric measurements (Figures 3 &
4). The brain image was enlarged 20x and the hemispheres were outlined on graph
paper with the aid of a microprojector (Figure 5). A line was drawn vertically between
the enlarged hemispheres to highlight the true midline of the brain. A line perpendicular
to the midline was drawn to mark the widest point of the hemisphere on both the left and
right sides of the brain. The height of each hemisphere at its greatest extent was marked
by drawing a line parallel to the vertical midline. Once these width and height points
had been established, the actual distances were measured using a digital tablet and the
Bioquant Image Analysis System.

To determine whether there was a difference in the amount of asymmetry present
in the treatment brains when compared to the control brains, the absolute difference
between the widths and heights of the right and left sides of the brain was used to
perform a univariate split plot analysis of variance (Gill 1986). The least square mean
values were computed from a general linear model procedure in the statistical software

SAS (SAS Institute Inc., 1990). These data were then used to compare the amount of

12

asymmetry between treatment and control groups (p s 0.05). A first degree orthogonal
polynomial contrast was performed to assess differences in width and height
measurements at each individual section level. The level of statistical significance for

this analysis was also p _<_ 0.05.

Results

The mean values for the left-right differences for both the width and height
measurements of each group are presented in Table 1.

When width measurements of the cerebral hemispheres in PCB treatment groups
were compared to those of the non-injected control group, there were no statistically
significant differences in the amount of asymmetry present (p 2 0.05). However, width
measurements for the vehicle-injected control group were significantly different from
those of the non-injected control group (p s 0.05). There were no significant width
differences in the amount of asymmetry present between any of the PCB treatment
groups and the vehicle-injected control group (p 2 0.05).

When height measurements of the cerebral hemispheres in PCB treatment groups
were compared to those of the non-injected control group, there were no statistically
significant differences in the amount of asymmetry present (p 2 0.05). However, when
height measurements of the cerebral hemispheres in PCB treatment groups were
compared to those of the vehicle-injected control group, there were significant differences
in brains from chicks exposed in ovo to 0.9 pg congener 126/kg egg and in brains from
chicks exposed in ovo to 0.9 ug congener 77 plus 0.8 pg congener 126/kg egg (p s

0.05). No significant differences were found between the other treatment groups and the

13

vehicle-injected control group (p 2 0.05), although height measurements for the
non-injected control group were significantly different from those of the vehicle-injected
control group (p S 0.05).

A significant linear trend was apparent in the amount of asymmetry present in
each brain. The amount of asymmetry increased linearly as measurements were taken
from the most caudal end of the brain at section 1 to the most rostral end of the brain at
section 8 (p s 0.05). In testing the validity of this trend, data from all groups with the
exception of the vehicle-injected group were pooled to increase the sample size [width
n = 56, height I: = 50] (Figure 6). Data were pooled since there were no statistically
significant differences in the amount of asymmetry present in the PCB treatment groups
and the non-injected control group. The vehicle-injected control group was not included
in this analysis because the amount of asymmetry present in this group was significantly

different from the amount present in non-injected controls.

Discussion

The results of this study indicate that a degree of asymmetry may normally be
present in the cerebral hemispheres of newly hatched chicks and that in ovo exposure to
individual environmentally relevant PCB congeners from day 0 of incubation does not
appear to significantly exacerbate this asymmetry. In this respect, our results differ from
those reported by Henshel et al. (1995) in which they found a dose-related difference in
brain measurements from birds originating in colonies exposed to varying degrees of
contamination. The difference between our results and those of Henshel et al. (1995)

may be due to the type and concentration of neurotoxicants present. Henshel examined

14

eggs contaminated with a mixture of several environmentally relevant toxicants including
PCDDs and PCDFs; PCBs comprised only a small part of the entire complement of
toxicants present. It is possible that exposure to other toxicants such as the PCDDs may
have resulted in the reported enhanced asymmetrical development or that the various
toxicants acted synergistically to affect cereme development. The two PCB congeners
used in our study are, nevertheless, significant components in mixtures of Great Lakes
environmental toxicants and were chosen based on data that indicated that congeners 77
and 126 were two of the three predominant coplanar congeners present in double-crested
cormorant eggs obtained from contaminated sites along the Great Lakes (Yamashita et
al. 1993).

The presence of asymmetry in brains of control chicks is not surprising since
several structures in the avian brain, including the hypoglossal nucleus, the nucleus
mesencephalicus lateralis pars dorsalis, and the thalamofugal visual pathway, have been
shown to be anatomically asymmetrical (Bradshaw and Rogers 1993). Furthermore, this
asymmetry has been shown to be an essential feature of avian embryogenesis, persisting
into adulthood (Bradshaw and Rogers 1993). One of the systems that has been shown
to develop asymmetrically, the thalamofugal visual projection, is one of two major visual
pathways in the avian brain. Several investigators have found that in the domestic
chicken, there are a greater number of thalamofugal projections in the right forebrain
than in the left forebrain (Boxer and Sanford 1985, Rogers and Sink 1988, Rogers and
Bolden 1991) and that this asymmetry extends from hatching through the first 21 post-
hatch days (Rogers and Sink 1988). Whether this finding would translate into a gross

morphological asymmetry is presently undetermined. Although a greater number of

15

thalamofugal projections in the right forebrain than the left forebrain suggests an
underlying cellular substrate for the asymmetry noted in the Henshel et al. (1995) study
and the present study, we did not test for sidedness with respect to the observed
asymmetry, whereas Henshel reported a greater area of convexity associated with the left
hemisphere than with the right.

In addition to intrinsic structural asymmetry, there are also extrinsic factors that
may play a role in the formation of brain asymmetry. A chick embryo in the egg is
positioned so that the left side of its head is tucked against its body, occluding the left
eye, while the right side of its head is turned toward the shell (Bradshaw and Rogers
1993). As the chick continues to develop in its egg, it rapidly outgrows the available
space and begins to press against the confining shell. The unossified skull may be
susceptible to compression during this period, creating compression of the cerebral
hemispheres as well. In addition, at hatching, the skulls of birds are not yet fully
ossified, with complete ossification taking up to at least three months in some species
(Per et al. 1987). This compression, along with the substantial amount of pressure
exerted against the hard shell during pipping, may account for an initial asymmetric
appearance of the cerebral hemispheres. Our finding of a trend of increasing asymmetry
from the caudal end of the brain to the rostral end is consistent with the rostral parts of
the skull being pressed against both the Chick’s body and the shell while in the egg. Both
the intrinsic structural asymmetry and extrinsic molding forces may account, at least in
part, for any significant differences in asymmetry found in our study.

It is also important to note that the domestic chicks used in this study are

precocial and thus are further along in their development at hatch than are the altricial

16

species examined by Henshel et al. (1995). Accordingly, it might be expected that
extrinsically induced forces brought on by a hard shell alongside a thinner and less
ossified skull might result in a greater degree of cerebral hemispheric asymmetry in
altricial species than in precocial species. In either case, it is apparent that an
examination of the cerebral hemispheres for symmetry should extend beyond the newly
hatched stage to the point when the hemispheres have had a chance to regain their normal
configurations within partially or fully ossified skulls. Furthermore, an additional
examination of symmetry beyond the newly-hatched stage may be useful in assessing the
effects of any transient developmental asymmetries within the CNS, such as those
induced by thalamofugal visual projections, on hemispheric measurements.

Finally, it should be noted that although every effort was made to standardize the
methodology employed across chicks and extreme care was taken to ensure the
orientation and accuracy of the measurements, it is possible that the structure used as the
starting point for measurements was itself situated asymmetrically within the brain. We
made the assumption that the anterior commissure develops in a symmetrical manner,
crossing the midline in a plane precisely perpendicular to the midline in every brain. If
this initial plane is itself asymmetrical, then subsequent perpendicular lines used for
measuring will also be asymmetrical. This uncertainty would also apply to the use of
surrounding bony or soft tissue structures for taking measurements.

In summary, the inherent asymmetry of the hatchling avian brain should be
considered as a possible confounding factor when attempting to use cerebral asymmetry
as a biomarker for environmental contamination. The normal development of

asymmetrical CNS systems, as well as the presence of external molding forces, may

l7

overshadow any subtle changes brought on by exposure to environmental neurotoxicants.
The transient nature of both of these phenomena, as well as the accuracy of the
measuring technique itself, should be taken into consideration when assessing the

usefulness of asymmetry as a biomarker.

Chapter 2
COMPUTED TOMOGRAPHY (CT) AS A TOOL IN DETERMINING THE
PRESENCE OF CEREBRAL HEMISPHERIC ASYMMETRY

IN LIVE BIRDS

The present study was initiated to determine whether or not computed tomography
(CT) would be a useful tool for investigating the question of cerebral asymmetry in birds.
CT is an advanced imaging technique that uses x-rays and computers and can produce
cross-sectional images of individual birds. In human medicine, CT has been used for
diagnosing brain lesions caused by intracranial masses or fluid filled cavities, vascular
lesions, and changes in ventricle size, ventricle:brain ratio, cortical atrophy, brain
volume, and brain asymmetries associated with schizophrenia (Rubin et al. 1993).

In the previous study (Chapter 1), histological sections were used to measure
asymmetry. One problem related to examining neural tissue is that birds must be killed
in order to obtain measurements. This requirement may limit the scope of avian species
examined since it is not always easy to obtain specimens from the wild and species of
particular interest may be listed as threatened or endangered. The use of domestic
chickens as surrogates for wild species is problematic for two main reasons. First,
chickens may have different sensitivities to environmental toxicants than wild species.

Second, as indicated in the previous chapter, chickens are precocial species, while a large

18

19

number of wild avian species are altricial relative to brain development. The differences
in these two rates of CNS development may make comparisons between species difficult.
There is also no risk that the process of histologically sectioning the brain could cause
any physical alterations because CT sectioning does not cause any disruption of the
tissues. CT-generated sections may also be more instructive than histologic sections in
analyzing brain symmetry because the computer is able to reformat images in other
planes (dorsal, sagittal) which could be used to determine further the symmetry of the
entire brain. Based on these reasons, a preliminary study designed to assess the potential
of CT as a useful technique in establishing the presence of brain symmetry was

performed.

Materials and Methods

The eggs used in this study were from double-crested cormorants obtained from
Lake Winnipegosis in Manitoba, Canada, a site considered to have relatively low levels
of environmental contamination. Five eggs were injected with 100 pg PCB congener
126/kg egg in a vehicle of triolein. In a previous study (Powell et al. 1996,), it was
determined that the LD50 for congener 126 is 2.3 pg/kg egg in the domestic chicken.
The higher concentration of congener 126 used in the present study was chosen because
the double-crested cormorant is considerably less sensitive than the chicken. The higher
dose was also used to maximize possible toxic effects on hemispheric symmetry. Five
eggs were used as non-injected controls. The eggs were incubated in a Petersime
incubator (Gettysburg, OH) at 37.5-37.7°C and approximately 65% relative humidity.

The eggs were candled on days 4 and 11 of incubation to check for viability.

20

At hatching, the chicks were sedated with intraperitoneal injections of sodium
phenobarbitol (75 mg/kg body wt; 0.5 ml/kg body wt) to allow examination with the CT
scanning procedure, which took approximately five minutes for each bird. A GE 9800
CT scanner was used to obtain images. The birds were placed on their dorsa with their
beaks tilted back in an attempt to obtain images in the same plane as the histological
sections that would subsequently be produced from the same birds (Figure 7).

The CT procedure involves placing the bird on a table that advances into a gantry
containing the x-ray tube, x-ray collirnators, and x-ray detectors. The x-ray tube rotates
360° around the patient on the table and is the source of the x-rays. The x-ray
collimator, which is located between the tube and the patient, determines the volume of
tissue that will be represented in each image produced. The x-ray detector absorbs the
x-ray photons emitted from the patient and converts them to an electrical signal. The
computer uses this signal to produce a number that creates an image on the screen
(Figure 8).

Each image produced by CT incorporated a volume of tissue 1.5 mm in thickness,
the smallest interval obtainable with this machine. The computer screen on which the CT
image was displayed had an underlying matrix. The matrix was composed of 512
columns and 512 rows, with each column or raw designated as a consecutive number
from 1 to 512. The columns comprised the x-axis and the rows made up the y-axis.
Using this matrix, the midline of the brain section was associated with a number on the
x-axis (Figure 9). The same point on the x-axis was assumed to be at the midline of all
sections of the same brain. Once the midline was identified, the cursor was moved to
the widest point of the left hemisphere. The computer measured the distance between

those two points in centimeters (Figure 10). The same procedure was repeated for the

21

right hemisphere. The absolute difference between the left and right hemispheres was
recorded. For the height measurement, the cursor was placed along the y-axis at a point
on the dorsal-most surface of the left hemisphere. The cursor was then placed along the
ventral-most surface of the left hemisphere and the computer measured the distance
between those two points in centimeters (Figure 11). The same procedure was repeated
for the right side of the brain. The absolute difference in height measurements between
the left and right hemispheres was recorded.

A range of Hounsfield units or CT numbers was chosen to differentiate bone and
connective tissue from neural tissue. The cursor was placed over what was obviously
neural tissue, bone, or connective tissue, and the respective CT numbers generated by
the computer were recorded. Since there are no predetermined CT numbers for the
immature neural tissues of an altricial avian species, a range of numbers had to be
chosen. Any number that was generated while the cursor was overlying neural tissue but
not generated when overlying non-neural tissue was used in the chosen range.

Once the hatchlings had been scanned they were euthanized with sodium
phenobarbitol (150 mg/kg body wt; 1 ml/kg body wt), perfused, and their brains

prepared for histological analysis as described in the previous study (Chapter 1).

Results and Discussion

The results of the CT measurements are presented in Table 2. These data were
not statistically analyzed due to the discovery of several limitations that affected the
reliability and usefulness of CT as a method for assessing cerebral symmetry. The first
issue that arose was the range of CT numbers chosen to designate neural tissue. In order

to understand the numerical designation, it is necessary to provide a brief background on

22
CT concepts and methodology (Hathcock and Stickle 1993). The CT image is composed

of several tiny squares called pixels. Each individual pixel is a two-dimensional
representation of a three—dimensional block of tissue. The color assigned to each pixel,
black, white, or shades of gray, is based on the type of tissue that is being represented
in that pixel. The color in the pixel is determined by how much radiation is attenuated
by each volume of tissue. Each tissue type will attenuate the x-ray beam differently
based on its atomic number and electron density. The x-ray attenuation of each tissue
type is quantitatively measured and termed the linear attenuation coefficient. The
computer calculates the linear attenuation coefficient of the tissues in each pixel and
assigns a CT number to that pixel. CT numbers range from +1000 to -1000, with
cortical bone being + 1000, air being -1000, and water being zero. All other tissues are
assigned numbers relative to the densities of bone, air, and water.

There is normal variation in the densities of both bone and soft tissue within the
same bone or soft tissue organ (Owens 1982). Variations in the density of bone are due
to differing ratios of compact to spongy bone, trabecular bone to intertrabecular spaces,
and cortex to medullary canal. The differences in soft tissue densities arise from
variations in volume, thickness, or the degree of compactness of the tissue in question.
It is due to these reasons that CT numbers for any given species may not apply to the
same tissue in a different species. The present study has extended this finding of
variability by suggesting that CT numbers assigned to developing tissues within a single
species may also vary significantly from those reported for adult tissues in the same
species. This finding results in several problems associated with interpretation of
numerical CT data. In an altricial avian species, such as the double-crested cormorant,

the birds hatch at a very early developmental stage, whereas precocial species hatch at

23

a significantly later stage of development. In either situation, the brain at these early
stages of development has not yet completed its neural growth. The overall density of
the brain is constantly changing until maturity. Structures which are not mature at hatch
and contribute to the overall density of the brain, include axons, dendrites, myelin
sheaths, and glial cells. Thus the neural tissue being measured will differ in density
based on the different rates of maturation for altricial and precocial species. Likewise,
the structures surrounding the brain such as connective tissue and bone have not reached
their full density and cannot be compared with adult CT numbers.

Another problem that became apparent during the course of this study is that
surrounding immature non-neural tissues, such as the non-ossified skull, may attenuate
the x-ray beam to the same degree as the immature neural tissue. In this case, it was
difficult to differentiate with any certainty the CT numbers for brain from those for
connective tissue or bone. When the cursor was placed over an area in the image that
was clearly neural tissue, the CT number was the same as when the cursor was placed
over an area that was clearly not neural tissue. Because of this, a range of CT numbers
had to be defined. The range chosen was between 55 and 70 and was based on CT
numbers most consistently found in an area that was obviously neural tissue. Difficulty
arose when the cursor was placed over an area that could have either been neural tissue
or connective tissue and the CT number fell both in and out of the chosen range on
different instances. Although a standard index of CT numbers should be developed so
that specific tissue types can be delineated from one another at specific developmental
stages, it is unclear how best to deal with differentiating dissimilar tissue types when the

CT numbers overlap.

24

Another critical point is that the measurements of the cerebral hemispheres can
be artificially skewed if the birds are not positioned correctly on the scanning table.
Improper positioning resulted in only four of the ten brains being useable for measure-
ments. Part of the problem was that CT images were not viewed while the bird was still
on the table. Viewing the images immediately as they were produced, while the birds
were still on the scanning table, would have provided the information needed for correct
positioning. Another way to ensure symmetric positioning would be to place the birds
in a stereotactic apparatus on the scanning table. The use of extracranial structures such
as the frontal bone, nasal bone, and eyes could be used as guides during CT scanning to
determine if the birds have been positioned symmetrically. In this study, extracranial
structures were used after the images were taken to determine whether or not positioning
was symmetrical (Figures 12 & 13). Since the goal of this study was the investigation
of asymmetry, proper symmetrical positioning was critical for success.

The birds were sacrificed after CT scanning and perfused so that their brains
could be measured using the morphometric technique described in Chapter 1. It was
found after the brains had been sectioned that they would not be suitable for
morphometry. The fixation protocol used in the previous study was appropriate for a
precocial avian species (domestic chicken) whose neural development is at a more
advanced stage at hatching than an altricial species (double-crested cormorant). It was
apparent that for an altricial species the fixation period should have been extended. The
brains were too soft when they were sectioned and therefore led to deformed sections
when they were placed on glass slides. This created a grossly artificial asymmetry.

These preliminary studies have revealed several important considerations and

limitations concerning the use of CT in evaluating the symmetry of the developing brain.

25

First, in order for CT imaging to be useful in addressing the issue of symmetry, standard
CT numbers have to be defined for the neural tissue under consideration so that there is
confidence that only neural tissue is being measured. Measurements may have to be made
at different times during development to take advantage of changes in density occurring
during maturation and differentiation of neural and adjacent structures. Second, proper
positioning is necessary. The advantage of CT over the histologic technique is that
proper positioning can be corrected while the bird is on the scanning table if the images
are viewed immediately. Third, assessment of histological sections in altricial species
requires longer post-fixation times to allow for better preservation of neural tissue.
Finally, CT cannot differentiate specific nuclei or tracts undergoing proliferation or
degeneration. If determination of effects at the cellular level is required, it would still
be necessary to sacrifice the birds to obtain histological sections. Until these points are
addressed, CT may not be as effective a tool in defining developmental abnormalities of

the CNS as initially hoped.

Chapter 3
ASSESSMENT OF THE NEUROCYTOTOXIC POTENTIAL OF BOTH A
COPLAN AR AND NON-PLANAR PCB CONGENER

USING THE FINK-HEIMER STAINING TECHNIQUE

In the previous two studies (Chapters 1 and 2) the possible effects of coplanar
PCB congeners on the gross morphometry of the cerebral hemispheres in two avian
species were examined. The experiments in the present chapter were designed to
determine the possible cytotoxic changes caused by exposure to either a coplanar or non-
planar ortho-substituted PCB congener.

There is growing evidence to indicate the existence of two classes of PCBs differentiated
by the presence or absence of neurologic effects. In 1986, Shain and colleagues exposed
animals to complex mixtures of PCBs in their feed and analyzed the brain tissue, using
a congener specific gas chromatography technique, to determine which congeners
accumulate in the brain. They found that several ortho-substituted, non-planar congeners
were found in the brain at concentrations as much as 100—fold greater than their
concentrations in the adulterated feed, while there were no detectable concentrations of
coplanar congeners in the brain. In a comprehensive study, Seegal and Shain (1992)
demonstrated that ortho-substituted congeners such as congeners 28, 52, and 47 (2,4,4’-

trichlorobiphenyl, 2,2’,5,5’-tetrachlorobiphenyl, and 2,2’,4,4’-tetrachlorobiphenyl,

26

27

respectively), caused a reduction in dopamine content in both non-human primate brains
and pheochromocytoma (PC12) cells. Reductions in brain concentrations of dopamine
have been found in the caudate nucleus and substantia nigra and it has been suggested
that the reduction in dopamine levels was caused by an inhibition of tyrosine hydroxy-
lase, the rate-limiting enzyme in dopamine synthesis (Seegal et al. 1991). The coplanar
congeners such as those used in the two previous studies of this thesis had no effect on
dopamine content.

The specific aim of this study was to determine whether an ortho-substituted, non-
planar PCB congener shown to result in a reduction in dopamine content would also
cause detectable signs of axonal and terminal degeneration in pathways that utilize
dopamine as a neurotransmitter. In addition, brains exposed to congener 126 were
analyzed to determine if congener 126 had cytotoxic effects as well and if the brain
regions affected were the same as for congener 52. Finally, measurements were made
of cereme hemispheres in birds exposed to congener 52 to determine whether exposure

to a non-planar congener might result in cerebral asymmetry at the time of hatch.

Materials and Methods

White Leghorn chicken eggs were obtained from the Michigan State University
Poultry Science Teaching and Research Center. PCB congener 52 (2,2’,5,5’-
tetrachlorobiphenyl) and congener 126 (3,3’,4,4’,5-pentachlorobiphenyl), both 97-99%
pure, were obtained from ULTRA Scientific (North Kingstown, RI). The congeners
were dissolved in an emulsion of egg yolk, lecithin (Merck; Darmstadt, Germany),
peanut oil, and water (Brunstrom and Orberg, 1982). Eggs were divided into two

treatment groups and two control groups. Eggs in one of the treatment groups received

28

single injections of congener 52 at three different dosages (12.6, 6.3, or 3.1 pg/kg egg).
The dosages for congener 52 were based on a study by Seegal and Shain (1992) in which
they conducted a long-term feeding trial using Aroclor 1016, at dosage levels of 0.8, 1.6,
or 3.2 mg/kg per day in non-human adult primates. They found that of the 27 congeners
present in Aroclor 1016, only three were detected in the brain (ortho-substituted
congeners 52, 28, and 47). In the Aroclor 1016 mixture, congener 52 comprised 2% of
the total PCBs present, whereas in the brains, congener 52 comprised 14% of the total
congeners detected. Furthermore, the ECSO, or effective concentration at which 50% of
the dopamine content was reduced in PC12 cell cultures, was found to be 86 pM for
congener 52. Using these numbers, the dosage required for 14% of congener 52 to
accumulate in the brain at a concentration of 86 pM (22.2 mg/L) was calculated. The
calculated dose was 6.3 pg/kg egg. The low dose used for egg injection was half the
calculated dose (3.2 pg/kg egg) and the high dose used for egg injection was twice the
calculated dose (12.6 pg/kg egg). Another group of eggs received a single injection of
congener 126 at the highest dose used previously in the asymmetry study in Chapter 1
(0.9 pg/kg egg). One control group received single injections of the vehicle alone,
whereas a second control group consisted of non-injected eggs. Table 3 lists the sample
sizes and dosages for congener 52. All injections were made into the yolk on day 0 of
incubation. Eggs were incubated in a Petersirne incubator (Gettysburg, OH) at
37.5-37.7°C and approximately 65% relative humidity, and candled on days 4 and 11
of incubation to check for viability.

The chicks hatched or were in the process of pipping by day 21, at which time

they were removed from the incubator. They were weighed and their heads examined

29

for signs of gross abnormalities. Chicks that had not completely pipped were removed
from their eggs, weighed, and examined. All procedures related to brain removal and
sectioning were exactly as described in Chapter 1. Once the brains were sectioned (40
pm thick), one out of every five sections, 200 pm apart, was stained with the Pink-
Heimer silver stain for degenerating axons and terminals (Tanaka 1976). Adjacent
sections in brains exposed to congener 52 were stained with cresyl violet and the sections
handled exactly as described in Chapter 1 for the determination of symmetry in the
cerebral hemispheres. The statistical procedures used in Chapter 1 to assess asymmetry

were repeated for the congener 52 brains.

Results and Discussion

Results of hemispheric measurements indicate that no significant differences in
hemispheric symmetry were present between the congener 52 treatment groups and the
vehicle-injected or non-injected control groups (p 2 0.05) (Table 3). The same
significant linear trend was apparent in the congener 52 injected groups with the amount
of asymmetry increasing linearly as measurements were taken from the most caudal end
of the brain at section 1 to the most rostral end of the brain at section 8 (p s 0.05).

All brains from chicks exposed to either congener 52 or 126 were negative for
the presence of axonal or terminal degeneration. In the avian brain, areas such as the
paleostriatum prirnitivum, paleostriatum augmentatum, and lobus paraolfactorius are
believed to be homologous to the mammalian dopaminergic centers. Therefore,
particular attention was directed toward these sites as previous investigators have reported

a significant reduction of dopamine levels in homologous areas in mammals (Seegal et

lei

su‘

Chi

30

al. 1991). There are several possible explanations for these negative results, the simplest of
which is that the dosages chosen may have been too low. Another possibility is that the
exposure time was not long enough. In a study by Seegal et al. (1994), it was found that
after exposure of Wistar-derived rats to 30 ppm of Aroclor 1254 from gestational day 8
until post-natal day 300, the number of tyrosine hydroxylase-containing neural cells was
reduced by 46%. In the present study, exposure to PCBs extended only through 21 days of
development in the egg and not beyond hatching. This period may not be long enough or
perhaps exposure during the post-hatch period may be necessary for the full effects of PCBs
as a neurotoxicant to be detected. Similarly, work by Tanaka et al. (1994) with triphenyl
phosphite (TPP) and the development of the visual system in European ferrets has
suggested that immature neurons are initially not susceptible to the effects of the
neurotoxicant and neuropathy can be found only after there has been some degree of
maturation of visual system neurons and connections. In the present study, chicken brains
were not assessed beyond hatching. Whether a neuropathy would have been detectable
later during CNS maturation is unknown.

Several investigators, using congener specific gas chromatography, have found that
many PCB congeners accumulate in the brain (Seegal and Shain 1992, Ness et al. 1994).
However, an accumulation of a specific congener does not necessarily translate into a
cytotoxic effect. In this study, the intent was to determine the presence of cytotoxicity as a
result of exposure to individual PCB congeners. The dosages for congener 52, an ortho-
substituted non-planar congener, were determined based on information obtained from
studies in non-human primates that had been exposed through feed. In this study, the

chicks were exposed during their development in the egg.

31

Obviously there are differences related to how the PCB compounds are absorbed and
metabolized by a developing chick and a non-human primate. It was not determined in
this study whether or not the PCB congener accumulated in the brain to the concentration
that had been calculated based on the study by Seegal and Shain (1992). Seegal et al.
(1991) also reported that, when several congeners were mixed together prior to
administration, a synergistic effect was produced that reduced dopamine content in non-
human prirnate brains and in PC12 cells to a greater degree than when each congener
was administered individually. This finding suggests that it is important not only to
assess the neurotoxic potential of individual PCB congeners, but also to examine the
neurotoxic potential of complex PCB mixtures found in the environment. Since an
individuals exposure to environmental PCBs is usually as a mixture of congeners, the
potentially neurotoxic effects of synergism should be taken into account when discussing
the toxicity of individual congeners. It may be misleading to state that individual
congeners do not have substantial toxic effects when synergistic toxic effects have not

been taken into account.

Chapter 4

CONCLUSIONS

The experiments described in this thesis have attempted to define the potential
neurotoxic effects of individual PCB congeners on the developing brain using both
histologic and computer-imaging techniques. A protocol was developed to address the
morphometric changes in the cerebral hemispheres that had been hypothesized to occur
as a result of exposure to environmental contamination (Henshel et al. 1995). Computed
tomography (CT) and the Fink-Heimer staining technique designed to reveal the presence
of neurotoxicant-induced axonal and terminal degeneration were two other methodologies
employed to determine the neurotoxicity of specific PCB congeners.

The major rationale for these experiments was based on a study by Henshel et al.
(1995) in which it was found that exposure to certain environmental contaminants
correlated with the development of asymmetrical cerebral hemispheres in hatchling great
blue heron chicks. They proposed that this hemispheric asymmetry could be useful as
a biomarker for environmental contamination. Unfortunately they did not consider other
explanations for their findings. The normal development of asymmetrical CNS systems,
as well as the presence of external molding forces, such as an unossified skull at hatching
and a hard-shelled egg in which to develop, may overshadow any subtle changes brought

on by exposure to environmental neurotoxicants. The transient nature of these events

32

33

should have been taken into consideration when assessing the usefulness of asymmetry
as a biomarker.

The protocol developed in this thesis to assess the symmetry of the cerebral
hemispheres was more exacting than that used in the Henshel study. First, the brains
used in the Henshel study had been stored for varying periods of time and were not all
processed in the same manner. In our study, all procedures related to euthanasia, brain
removal, fixation, tissue sectioning, mounting, and staining were standardized in order
to minimize variation caused by differences in processing methodologies. The brains
were measured grossly with an engineering ruler in the Henshel study, whereas we used
a digitizing morphometry computer system to obtain our brain measurements. The
absolute differences in the right and left hemisphere measurements for both of these
studies ranged from 0.05 m up to 1 mm. Since these numbers are so small relative to
the overall dimensions of the brain, it is critical that measurements be made with as
much accuracy as possible. Based on the results of this study, it appears that cerebral
hemispheric asymmetry is a complex multi-factorial phenomenon, which may not be as
strongly correlated with environmental contamination as had been previously suggested
(Henshel et al. 1995). As Bradshaw and Rogers (1993) so eloquently stated: “Given the
growing list of both functional and structural asymmetries in birds, one begins to wonder
whether it is not asymmetry but symmetry which, when it occurs, needs to be
explained.”

Based on our results, more preliminary research into the applicability of CT
technology to address the issue of symmetry in cerebral hemispheres should be done.
The major problem that developed during this study was the inability to positively

identify neural tissue from other surrounding tissues in the immature bird. It was not

34

apparent at the time of the study that differentiating tissues based on CT numbers would
be so difficult and that the CT numbers given to different tissues would overlap to such
a degree. One solution to this problem is to develop standard CT numbers for the tissue
type of the particular species involved. However, this may not be realistic, as it might
be cost prohibitive. More importantly, it may be that the use of CT is more appropriate
as an initial screen for neurotoxicity and that detailed neuropathological changes might
still require the use of standard neurohistological techniques. At this time, use of CT as
a diagnostic tool is best reserved for gross pathology such as that caused by intracranial
masses or fluid filled cavities.

Finally, the last set of experiments involved the use of one PCB congener from
both the neurologic and non-neurologic classes of PCBs. Using the Fink-Heimer staining
technique the brains were evaluated for the presence of axonal and terminal degeneration.
Although the results were negative, a closer examination of dosages, exposure times, and
ages at which exposure is present is necessary before any definitive statements can be

made regarding PCBs and morphologically characterized cytotoxicity.

APPENDICES

APPENDIX A

Tables 1—3

APPENDIX A

Table 1. Mean values (mm) for the absolute differences in width and height
measurements between left and right cerebral hemispheres of hatchling domestic chickens
exposed at day 0 of incubation to individual PCB congeners

 

Mean values (mm) for measurements :1: SE

 

Treatmenta

(pg/kg egg) Width (n)b Height (11)

NIC 0.19 :1: 0.04 (19) 0.14 :t 0.02 (16)
VIC 0.32 :I: 0.04 (l3)* 0.23 j; 0.02 (ll)*
3.0 #77 (L050 = 8.8) 0.31 :t 0.08 (4) 0.13 i 0.04 (3)
9.0 #77 0.15 :l: 0.09 (3) 0.18 :t: 0.04 (3)
0.3 #126 (L050 = 0.6) 0.27 i 0.05 (8) 0.17 i 0.03 (7)
0.9 #126 0.25 i 0.04 (12) 0.16 :l: 0.02 (13)**
0.9 #77 & 0.8 #126 0.23 :1: 0.06 (6) 0.13 :1: 0.06 (5)**

 

“ NIC = non-injected control eggs, VIC = vehicle-injected control eggs, #77 = PCB
congener 77 (3,3’ ,4,4’-tetrachlorobiphenyl) and #126 = PCB congener 126 (3,3’,4,4’ ,5’-
pentachlorobiphenyl).

b Differences in sample sizes for each measurement are due to the presence of cutting
artifact. In order for the brain to be used for measurements, all cortical layers must have
been intact. Loss of cortical tissue occurred more frequently in the height plane than the
width plane.

"‘ The amount of asymmetry present was significantly different when compared to that
of the non-injected control groups (p s 0.05).

** The amount of asymmetry present was significantly different when compared to that
of the vehicle-injected control groups (p s 0.05).

35

36

Table 2. CT numbers obtained from brain images of double-crested cormorants exposed
to 100 pg PCB congener 126/kg egg at day 0 of incubation

 

Mean values (cm) for the absolute difference in measurements

 

Birda width height
1 .04 .02
2 .02 .05
3 .03 .02
4 .08 .03

 

‘ All birds scanned were from the PCB congener 126 injected group. None of the non-
injected birds were positioned correctly, therefore measurements were not taken

Table 3. Mean values (mm) for the absolute differences in width and height
measurements between left and right cerebral hemispheres of hatchling domestic
chickens exposed at day 0 of incubation to PCB congener 52

 

Mean values for measurements :1: SE

 

Treatment

(pg/kg egg) Width (n)" Height (n)

NIC 0.19 :I: 0.04 (19) 0.14 :1: 0.02 (16)
VIC 0.32 i 0.04 (13)* 0.23 d: 0.02 (ll)*
3.1 #52 0.41 :1; 0.05 (8) 0.20 i 0.03 (7)**
6.3 #52 0.27 j; 0.05 (8) 0.21 :1: 0.21 (7)*
12.6 #52 0.22 :t 0.05 (10) 0.17 i 0.02 (9)

 

‘ NIC = non-injected control eggs, VIC = vehicle-injected control eggs, #52 = PCB
congener 52 (2,2’,5,5’-tetrachlorobiphenyl)

b Differences in sample sizes for each measurement are due to the presence of cutting
artifact. In order for the brain to be used for measurements, all cortical layers must have
been intact. Loss of cortical tissue occurred more frequently in the height plane than the
width plane.

* The amount of asymmetry present was significantly different when compared to that
of the non-injected control groups (p S 0.05).

*"‘ The amount of asymmetry present was significantly different when compared to that
of the vehicle-injected control groups (p s 0.05).

APPENDIX B

Figures 1—13

APPENDIX B

Figure 1. Structure of PCB congeners and comparison of structure to TCDD

Chlorine atoms can be distributed on the ortho (o, 0’), meta (m, m’), or para (p, p’)
positions of the biphenyl structure. Those congeners that do not have chlorine atoms on
the ortho (0,0’) positions but do have chlorine atoms at the para positions and at least
two meta positions attain a structure similar to TCDD. This enables them to mimic the
highly toxic behavior of TCDD.

m o o' m'
p..—
p \ / P'
m 0 0| ml
PCB
Cl 0 Cl
Cl , Cl

TC DD

37

38

Figure 2. Photomicrograph of a 40 pm thick section of a hatchling domestic chicken
brain from the non-injected control group illustrating a cerebral hemisphere
stained with cresyl violet

A. This section is taken through the cerebral hemispheres of a newly hatched chick at
the level of the anterior commissure (level 1) magnification x 41. The boxed area
indicates the area shown in plate B. B. A higher power photomicrograph of the boxed
area in A illustrating the symmetrical orientation of the anterior commissure (between
arrowheads). Magnification x 127. Note abbreviations on page viii.

 

 

39

Figure 3. Line drawing of a parasagittal section through the chicken brain illustrating
the levels from which each of the 8 sections were taken for hemispheric
measurements

The numbers correspond to sections 1 to 8. Note abbreviations on page viii.

: 12345070
Hp \
#L‘ H
c, Q \ 3
{a

(iiggz' 1§;~,

 

 

 

     

  

 

 

CO

 

 

 

 

 

 

 

40

Figure 4. Line drawing of coronal sections at the eight section levels from which
measurements for asymmetry were obtained from the brains of all PCB
treatment and control groups of hatchling domestic chickens

Section 1 is located most caudally at the level of the anterior commissure and section 8
is located most rostrally. Nuclei and tracts were identified with the aid of an atlas
(Kuenzel 1988). Note abbreviations on page viii.

 

41

Figure 5. Line drawing of section level 1 showing how width and height measurements
were obtained from a hatchling domestic chicken brain for all PCB treatment
and control groups

Using an enlarged image of the cerebral hemispheres, this drawing illustrates a section
at the level of the anterior commissure. The line drawn between the hemispheres
indicates the true midline. Line AB, drawn perpendicular to the midline, denotes the
widest point of the hemisphere. Line CD, drawn parallel to the midline, illustrates the
maximum height of the hemisphere.

 

 

 

 

E,

‘III
‘

42

Figure 6. Histogram of width and height measurements by section level for all PCB
treatment groups and the non-injected control group of hatchling domestic

chickens

The absolute differences in width and height measurements at each section level for all
groups except the vehicle-injected control groups. At each section level n=56 for the
width measurements (total measurements = 448), and n=50 for the height measurements
(total measurements = 400). Note the trend toward an increase in the amount of
asymmetry as the sections move from the most caudal end to the most rostral end of the

brain.
0.35 V

0.3 ‘F

025 <-

02 4»

     

 

lwidth
El height

 

 

 

L-H means (mm)

0.1 v

0.05 ‘-

 

 

 

 

 

 

 

 

 

—4

 

12 3 4 5 6 7 a

section level

43

Figure 7. Comparison of bird brain positioning on computed tomography (CT)
scanning table and histological sectioning

The bird’s beak was pulled rostrally and the brain oriented in a position so that the CT
images produced would be in the same plane as the histologic sections would be cut on
the microtome.

 

 

 

 

 

 

 

 

 

44

Figure 8. Illustration of CT gantry and patient scanning table

CT GANTRY

 

 

45

Figure 9. Printed CT image showing the cursor on the midline of the cerebral
hemisphere of a hatchling double-crested cormorant chick exposed in ovo to
PCB congener 52

The white line is the scanning table which the bird was placed on. EY = eye, CH =
cerebral hemispheres, FB = frontal bone, NB = nasal bone.

 

46

Figure 10. Printed CT image with cursor placement depicting width measurement of the
cerebral hemisphere of a hatchling double-crested cormorant chick exposed
in ovo to PCB congener 52

The line marked with the number 1 depicts the distance measured between the two
cursors on the computer image. The cursor placement to obtain this measurement was
based on the determined CT number range which corresponded to neural tissue.

 

47

Figure 11. Printed CT image with cursor placement depicting height measurement of the
cerebral hemisphere of a hatchling double—crested cormorant chick exposed
in ovo to PCB congener 52

The line marked with the number 1 depicts the distance measured between the two
cursors on the computer image. The cursor placement to obtain this measurement was
based on the determined CT number range which corresponded to neural tissue.

 

48

Figure 12. Printed CT images accompanied by line drawings depicting symmetrical
positioning on the scanning table of the hatchling double-crested cormorant
chick exposed in ovo to PCB congener 52

Symmetrical positioning of the bird can be assumed using the symmetry of extracranial
structures as a guide. NK = neck, BK = beak, NB = nasal bone, FB = frontal bone,
EY = eye, CH = cerebral hemisphere

A. Skyline view of the bird on the scanning table. The horizontal line through the
bird’s head is the level at which the image in Plate B is taken.

B. Coronal image of the bird head taken at the level of the horizontal line in Plate A.
C. Coronal image of the bird head taken one section level rostral (0.15 mm) to that
illustrated in Plate B.

 

 

Figure 12. Printed CT images accompanied by line drawings depicting symmetrical
positioning on the seaming table of the hatchling double—crested cormorant
chick exposed in ovo to PCB congener 52

50

Figure 13. Printed CT images accompanied by line drawings depicting asymmetrical
positioning on the scanning table of the hatchling double-crested cormorant
chick exposed in ovo to PCB congener 52

Symmetrical positioning of the bird can be assumed using the symmetry of extracranial
structures as a guide. NK = neck, BK = beak, NB = nasal bone, FB = frontal bone,
EY = eye, CH = cerebral hemisphere

A. Skyline view of the bird on the scanning table. The horizontal line through the
bird’s head is the level at which the image in Plate B is taken.

B. Coronal image of the bird head taken at the level of the horizontal line in Plate A.
C. Coronal image of the bird head taken one section level rostral (0.15 mm) to that
illustrated in Plate B.

 

 

 

Figure 13. Printed CT images accompanied by line drawings depicting asymmetrical
positioning on the scanning table of the hatchling double-crested cormorant
chick exposed in ovo to PCB congener 52

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