AN ABSTRACT OF THE DISSERTATION OF
Kathleen A. Patnode for the degree of Doctor of Philosophy in Toxicology presented on
May 4, 1995. Title: Alterations in Mink Reproductive Physiology following Exposure to
Coplanar and Noncoplanar Polychlorinated Biphenyls (PCBs).
Abstract approved:
Redacted for Privacy
Lawrence R. Curtis
Female mink (Mustela vison) are highly sensitive to polychlorinated biphenyl
(PCB)-induced reproductive impairment. The goal of this research was to develop a
better understanding of the mechanism of PCB-induced effects on female mink, mink
embryos, and surviving mink kits. Anestrous, juvenile female mink and pregnant, adult
mink were exposed to 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB), a coplanar PCB, or
2,2',4,4',5,5'-hexachlorobiphenyl (2HCB), a noncoplanar PCB. During anestrus or at the
onset of implantation, female mink received intraperitoneal injections of 0.4 or 0.8 mg
3HCB/kg, 20 mg 2HCB/kg, corn oil (CO), or no treatment. Anestrous females were
treated with 1713-estradiol for 24 h prior to analysis. In the second experiment, COexposed animals received empty silastic implants, while 3HCB-treated mink received
either empty or progesterone (P)-filled implants.
Weights were similar between treatment groups in the first experiment, but in the
second study, 3HCB-treated females lost as much as 10 % of their body weight. Hepatic
not 2HCB. Both congeners impaired 170-estradiol-stimulated up-regulation ofuterine
nuclear estrogen receptors (ER) in anestrous mink.
Embryotoxicity and reduced embryo growth were observed 14 days after
exposure to 0.4 mg 3HCB/kg > 0.8 mg 3HCB/kg > 20 mg 2HCB/kg. In pregnant mink,
all 3 HCB treatments decreased progesterone receptor (PR) affinity. ER concentration
and cytosolic PR total receptor number (Rt) were significantly increased by 20 mg
2HCB/kg > 0.8 mg 3HCB/kg. No significant difference was observed in either the total
number or affinity of PR between in vitro systems with or without 3HCB. From day 10
forward, implantation sites from 3HCB-treated mink contained necrotic tissue from
resorbing embryos. Progesterone treatment did not prevent resorption, but increased
uterine prolactin receptor concentration. Length of rib, ulna, tibia, and fibula and
diameter of humerus were found to be significantly reduced in day 1 kits exposed to
2HCB in ?hero. We conclude that PCBs are antiestrogenic in anestrous females and
antiprogestational in pregnant mink, but reduced PR affinity is not the proximate cause of
embryo resorption. In addition, 2HCB produces estrogenic changes in bones of mink
embryos.
Alterations in Mink Reproductive Physiology following Exposure to Coplanar and
Noncoplanar Polychlorinated Biphenyls (PCBs).
by
Kathleen A. Patnode
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed May 4, 1995
Commencement June 1996
Doctor of Philosophy dissertation of Kathleen A. Patnode presented on May 4, 1995
APPROVED:
Redacted for Privacy
Major Professor, representing Toxicology Program
Redacted for Privacy
Chair of Toxicology Program
Redacted for Privacy
Dean of Graduate School
I understand that my dissertation will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my dissertation to
any reader upon request.
Redacted for Privacy
DEDICATION
This dissertation is dedicated to my parents, Richard and Eleanor, and to my
husband, Lou Reynolds. My parents instilled in me a love of learning and the work ethic
needed to achieve farsighted goals. In my mind, these tools are the most important gifts
a child can receive. My husband provided tireless support during my degree program.
Without his encouragement, many of the professional and personal hurdles would have
been insurmountable.
ACKNOWLEDGMENTS
This research was assisted by many willing and capable associates. Dr. Larry
Curtis, my major professor, collaborated in all parts of this work and I benefited greatly
from his experience. Dr. Fred Stormshak shared with me his knowledge of
endocrinology and mink physiology and provided invaluable guidance in the research.
Dr. Tony Frank, Colorado State University, and Dr. Jack Rose, Idaho State University,
provided advice on experimental design and conducted analyses that were critical to my
research. The assistance of Dr. Ov Slayden of Oregon Regional Primate Research Center
was integral in modifying the Center's steroid receptor assay for use in mink. This thesis
and the experiments described within were also improved by the recommendations of
Drs. Tom Murray, Hillary Carpenter, Dan Selivonchick, and Paul Reno.
Many others assisted in various aspects of the work. Ron Scott and Cliff
Thompson of OSU's Experimental Fur Farm cared for and handled animals and cheerfully
adjusted their schedules to accommodate my needs. Assistance with tissue collection and
assays was enthusiastically provided by Beth Siddens, Carol Forsyth, and Lou Reynolds.
Much of this work could not have been completed were it not for the cooperative
attitude within the Toxicology Program. I am particularly grateful for access to
equipment and expertise in the laboratories of Drs. Donald Buhler, Dan Selivonchick, and
David Williams. Don Griffin and Gene Johnson shared their expertise in contaminant
analysis and gave me access to and assistance with analytical equipment.
Funding for this research was generously provided by grants from The Mink
Farmers Research Foundation and National Institute of Environmental Health Sciences
grants ES07060 and ES005543. I performed this research as a predoctoral trainee
under a program of the National Institute of Environmental Health Sciences.
CONTRIBUTION OF AUTHORS
Dr. Larry Curtis was involved in the design, analysis, and writing of each
manuscript. Dr. Tony Frank performed histological evaluations and analyzed the
resulting data at his laboratory at Colorado State University for the second manuscript.
Dr. Jack Rose conducted the prolactin receptor assays for the second manuscript at his
laboratory at Idaho State University.
TABLE OF CONTENTS
Page
INTRODUCTION
Polychlorinated Biphenyl (PCB) Toxicity
Nonortho-substititued congeners
Monoortho-substituted congeners
Diortho- substituted congeners
Metabolites
Reproductive Toxicity
1
1
3
4
4
5
7
Mink Reproductive Physiology
11
PCB-Induced Reproductive Toxicity in Mink
15
2,2',4,4',5,5'- and 3,3',4,4',5,5'-HEXACHLOROBIPHENYL ALTERATION
OF UTERINE PROGESTERONE AND ESTROGEN RECEPTORS
COINCIDES WITH EMBRYOTOXICITY IN MINK (Muste la visor)
18
Abstract
19
Introduction
20
Methods
22
Anestrous mink
Pregnant mink
Statistical Analysis
Results
Anestrous mink
Pregnant mink
Discussion
3,3',4,4',5,5'-HEXACHLOROBIPHENYL (3HCB)-INDUCED CHANGES
IN UTERINE PHYSIOLOGY IN MINK (MUSTELA VISON)
23
25
26
27
27
27
40
46
Abstract
47
Introduction
48
TABLE OF CONTENTS (Continued)
Page
Methods
49
Results
52
Discussion
54
IN UTERO EXPOSURE TO 2, 4, 5, 2', 4',5'-HEXACHLOROBIPHENYL
(2HCB) PRODUCES SKELETAL CHANGES IN NEONATAL MINK
(MUSTELA VISON)
59
Abstract
60
Introduction
60
Methods
62
Results
64
Discussion
68
CONCLUSIONS
71
BIBLIOGRAPHY
76
APPENDICES
89
Appendix A
Appendix B
Embryo Resorption and Growth Impairment in Mouse
Strains with Normal and Reduced Aryl Hydrocarbon
Receptor Concentrations
90
Nonesterified Fatty Acid Concentrations in Plasma and
Uterine Tissue of Pregnant and Nonpregnant Female
Mink Following Exposure to 3,3',4,4',5,5'­
Hexachlorobiphenyl
97
LIST OF FIGURES
Page
Figure
Figure 1-1
Figure 1-2
Figure 1-3
Figure 2-1
Figure 3-1
Figure 3-2
Saturation binding curves for [31-1]-R5020 specifically bound
to progesterone receptors at 14 days for pregnant mink treated
with 0.4 or 0.8 mg 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB)/kg
or 20 mg 2,2',4,4',5,5'-hexachlorobiphenyl (2HCB)/kg ip at
implantation
36
2,2',4,4',5,5'-hexachlorobiphenyl (2HCB) concentrations (Y±SE)
in liver over time in anestrous and pregnant female mink
exposed to 20 mg 2HCB/kg. n=3 per treatment-time group
38
3,3',4,4',5,5'-hexachlorobiphenyl (3HCB) concentrations
(k+SE) in liver over time in anestrous mink exposed to
0.4 mg 3HCB/kg and pregnant female mink exposed
to 0.4 or 0.8 mg 3HCB/kg
39
Uterine prolactin receptor concentrations in pregnant and
nonpregnant mink exposed to corn oil (CO),
3,3',4,4',5,5'-hexachlorobiphenyl (3HCB) at 0.4 mg/kg bw
with or without progesterone (P)-filled silastic implants
55
Humerus length by sex for day 1 neonatal mink from
females treated with corn oil (CO) or 2,4,5,2',4',5'­
hexachlorobiphenyl (2HCB) ip at time of implantation
66
Humerus diameter by sex for day 1 neonatal mink from
females treated with corn oil (CO) or 2,4,5,2',4',5'­
hexachlorobiphenyl (2HCB) ip at time of implantation
67
LIST OF TABLES
Page
Table
Table 1-1
Table 1-2
Table 1-3
Table 1-4
Table 1-5
Table 1-6
Table 2-1
Table 3-1
Uterine nuclear estrogen receptor (ERn) in fmol/Kg DNA
±SE) in anestrous and pregnant mink over time following
exposure to 20 mg 2,2',4,4',5,5'- hexachlorobiphenyl (2HCB)/kg
or 0.4 or 0.8 mg 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB)/kg
29
Uterine cytosolic estrogen receptor (ERc) in fmol/[tg DNA
±SE) in anestrous and pregnant mink over time following
exposure to 20 mg 2,2',4,4',5,5'- hexachlorobiphenyl (2HCB)/kg
or 0.4 or 0.8 mg 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB)/kg
30
Total cytochrome P450 (P450) in nmol/mg protein (.5? ±SE) in
livers from anestrous and pregnant mink over time following
exposure to 20 mg 2,2',4,4',5,5'- hexachlorobiphenyl (2HCB)/kg
or 0.4 or 0.8 mg 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB)/kg
31
Ethoxyresorufin-O-deethylase (EROD) activity in nmol/min/mg
±SE) in livers from anestrous and pregnant mink over
protein
time following exposure to 20 mg 2,2',4,4',5,5'-hexachloro­
biphenyl (2HCB)/kg or 0.4 or 0.8 mg 3,3',4,4',5,5'-hexachloro­
biphenyl (3HCB)/kg
32
Numbers of implantation sites (IP) and embryos and embryo
characteristics (f ±SE) in mink at 14 days after ip injection on
March 30 with 20 mg/kg 2,2',4,4',5,5'-hexachlorobiphenyl (2HCB)
or 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB) at 0.4 or 0.8 mg/kg
33
Uterine progesterone receptor concentrations (Rt) in fmoUpg DNA
±SE) and dissociation constants (Kd) in nmol (.5-c ±SE) from
pregnant mink over time following exposure to 20 mg 2,2',4,4',5,5' ­
hexachlorobiphenyl (2HCB)/kg or 0.4 or 0.8 mg 3,3',4,4',5,5'
hexachlorobiphenyl (3HCB)/kg
35
Histological evaluation of implantation sites from pregnant mink
treated with 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB) or corn oil
(CO) with empty or progesterone (P)-filled silastic implants
53
Bone length (cm) and diameter (cm) on body weight (g) basis
(mean ± SE) for day 1 mink of females exposed to corn oil
(control) or 2,4,5,2',4',5'-hexachlorobiphenyl (2HCB) ip at
time of implantation
65
LIST OF APPENDIX FIGURES
Figure
Figure A-1
Figure A-2
Page
Percent of litter resorbed in female mice with normal (AhR+)
and reduced (AhR-) aryl hydrocarbon receptors exposed to
80 mg 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB)/kg, 300 mg
2,2',4,4',5,5'-hexachlorobiphenyl (2HCB)/kg, or corn oil (CO)
via ip injection on gestation day 5
92
Embryo weights from female mice with normal (AhR+) and
reduced (AhR -) aryl hydrocarbon receptors exposed to 80 mg
3,3',4,4',5,5'-hexachlorobiphenyl (3HCB)/kg, 300 mg
2,2',4,4',5,5'-hexachlorobiphenyl (2FICB)/kg, or corn oil (CO)
via ip injection on gestation day 5
94
LIST OF APPENDIX TABLES
Table
Table B-1
Table B-2
Page
Nonesterified fatty acid (NEFA) concentrations in plasma of
pregnant and nonpregnant female mink treated with 0.4 mg
3,3',4,4',5,5'-hexachlorobiphenyl (3HCB) /kg or corn oil
(CO) via ip injection at the putative time of implantation
101
Nonesterified fatty acid (NEFA) concentrations in uterine tissue
of pregnant and nonpregnant female mink treated with 0.4 mg
3,3',4,4',5,5'-hexachlorobiphenyl (3HCB) /kg or corn oil (CO)
via ip injection at the putative time of implantation
102
Alterations in Mink Reproductive Physiology following Exposure to Coplanar and
Noncoplanar Polychlorinated Biphenyls (PCBs)
INTRODUCTION
Polychlorinated biphenyls (PCBs) are ubiquitous contaminants in aquatic
ecosystems of the world. While manufacture of these compounds has been banned in
many countries, they continue to enter the environment and will remain a contaminant
problem for many more decades. The impact of environmental exposure to PCBs on
mammalian populations needs to be investigated. This objective is best accomplished by
using a sensitive model species in which the mechanisms of PCB toxicity are well
understood.
The mink (Mustela vison) is a carnivorous mammal in the family Mustelidae.
Although mink are not a traditional laboratory animal, they are highly valued as a wild and
domestic fur-bearing species and are unique among North American mammals in their top
predator position in both aquatic and terrestrial food webs. These factors have resulted in
substantial information regarding their reproductive physiology, contaminant exposure,
and toxicological responses, particularly responses to PCBs.
Polychlorinated Biphenyl (PCB) Toxicity
PCBs constitute a major group of environmental contaminants. The characteristics
of chemical stability, lipophilicity, and inflammability made PCBs valuable as plasticizers,
dielectric fluids, hydraulic lubricants, and flame retardants. They have been, and continue
2
to be, released into the environment as a result of improper disposal, leaching, or
atmospheric deposition. Those same properties that made PCBs valuable to industry are
responsible for their persistence in abiotic compartments such as sediments and their
bioaccumulationlbiomagnification in aquatic food chains (Safe 1994). Aside from
occupational and accidental exposures, trophic transfer of these compounds through
aquatic food chains is the primary route of exposure for wildlife, livestock, and humans.
Although manufacture of PCBs has been banned in the United States for more than 20
years, these contaminants continue to occur in environmental samples and animal tissues
(McFarland and Clarke 1989). With 209 possible congeners (Ballschmiter and Zell 1980),
these extracts contain complex PCB mixtures that differ significantly from the commercial
mixture due to selective degradation and absorption (Safe 1994).
Exposure of animals to commercial PCB mixtures produces a broad spectrum of
effects including mortality, inhibition of body weight gain or body weight loss, porphyria,
immunotoxicity, hepatotoxicity, neurotoxicity, thymus atrophy, dermal toxicity,
carcinogenicity, endocrine disruption, and reproductive toxicity (Safe 1994). This
pleiotropic response pattern is logical given the understanding that the toxicity of
commercial PCB mixtures is the summation of effects produced by numerous, individual
congeners. Investigations of PCBs, individually and in groups, indicate that the congeners
fall into the following 3 structural categories with characteristic of toxic effects.
3
Nonortho-substititued congeners
Chlorine located at both para positions (4 and 4') and at least one meta position (3,
3', 5,or 5') are conformationally unrestricted to the extent that the biphenyl rings
commonly lay in a single plane. Studies of structure-activity relationships in birds,
mammals, and fish have determined that these PCBs are structurally and toxicologically
similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These planar congeners induce
hepatic cytochrome P450 isozymes CYP IA1 and CYP1A2 (Brunstrom 1992), glutathione
S-transferases (Aoki et al. 1992), and epoxide hydrolase (Ahotupa 1981); produce thymic
atrophy (Brunstrom et al. 1991), hepatomegaly (Aulerich et al. 1987), and fatty livers
(Borlakoglu et al. 1990); and cause porphyria (Sinclair et al. 1990), reproductive
impairment (Aulerich et al. 1987), developmental malformations (Marks et al. 1980,
1981), wasting syndrome (Leece et al. 1985), and immunosuppression (Mayura et al.
1993). As with TCDD, these effects are thought to be mediated by high affinity, low
capacity binding to the cytosolic aryl hydrocarbon receptor (AhR) (Kafafi et al. 1993).
The mechanism of AhR action is believed to parallel that of other cytosolic
transcription factors. Upon binding of the ligand, the cytosolic AhR/PCB complex
undergoes transformation, translocation to the nucleus and DNA binding to xenobiotic
response elements (XREs) resulting in increased mRNA synthesis (Whitlock 1993). These
steps have been clearly demonstrated for CYP1A1 induction (Whitlock 1993) and XREs
have been identified upstream of DNA coding regions for human and mouse estrogen
receptor (ER) (White and Gasiewicz 1993). However, the application of this mode of
action to all toxic effects of TCDD-like PCB congeners is based on indirect evidence using
4
models with high and low AhR concentrations and in vitro structure-activity relationships
(Safe 1994). While these congeners are the most potent AhR agonists, only congener
3,3',4,4'-tetrachlorobiphenyl occurs in large enough quantities in commercial mixtures and
environmental samples to contribute substantially to dioxin-like PCB toxicity (Leonards et
al. 1995).
Monoortho-substituted congeners
The steric hindrance of a single ortho chlorine on either biphenyl ring (2 or 2')
decreases the probability that the PCB will be in the planar conformation. As a result,
monoortho PCBs have lower binding affinities for the AhR than do the nonortho
congeners (Kafafi et al. 1993). They still exert TCDD-like effects such as hepatic
CYP1A1, CYP1A2, and epoxide hydrolase induction (Parkinson et al. 1983); liver toxicity
and thymic atrophy (Brunstrom et al. 1991); reduced body weight gain (Leece et al.
1985); immunosuppression (Davis and Safe 1990); and reproductive impairment
(Kihlstrom et al. 1992). Since they also induce hepatic CYP2B1, CYP2B2, and CYP2A1,
these monoortho PCBs are considered to be mixed inducers (Safe 1994). In contrast to
the nonorotho PCBs, these congeners often comprise a large proportion of commercial
and environmental mixtures. Thus, they are considered to contribute substantially to
TCDD-like toxicity of PCB mixtures (Leonards et al. 1995).
Diortho-substituted congeners
The steric hindrance of diortho substitution (2 and 2') virtually eliminates planar
conformation of the biphenyl rings. These congeners have negligible binding affinities for
5
the AhR (Kafafi et al. 1993). They do not induce CYP1A 1 or CYP1A2 (Brunstrom et al.
1991), but are even more potent inducers of CYP2B1 and CYP2B2 than the monoortho
PCBs (Parkinson et al. 1983). They produce hepatomegaly and promote liver tumor
growth (Buchmann et al. 1991). Along with some of the monoortho congeners, they
reduce dopamine concentrations and are neurotoxic (Shain et al. 1991). Some of these
congeners are estrogenic (Jansen et al. 1993) and cause embryotoxicity without inducing
malformations (Marks and Staples 1980). Furthermore, PCB 153 partially antagonizes
TCDD-mediated cleft palate formation in prenatally-exposed mice (Biegel et al. 1989).
Concentrations of diortho PCBs in commercial mixtures and environmental
samples are varied. Concentrations of the lower chlorinated congeners are commonly
low, while the more highly chlorinated ones can predominate the total PCB mixture
(McFarland and Clark 1989, Mason and Ratford 1994, Leonards et al. 1994). However,
their role in environmental toxicity of PCBs is not known because their toxicity has not
been as thoroughly studied as that of TCDD-like congeners.
Metabolites
Like toxicity, the rate of metabolism and metabolite structure depend upon the
chlorine-substitution pattern of the individual congener. In general, lower chlorinated
congeners with at least 2 adjacent unsubstituted carbons are more readily metabolized than
more completely substituted PCBs (Borlakoglu et al. 1990). Metabolism also varies with
species as demonstrated by the much higher rate of 2,2',4,4',5,5'-hexachlorobiphenyl
metabolism in dog as compared to rodents (Duignan et al. 1988).
6
Several metabolic pathways exist for PCBs. The only known direct metabolism
results in the formation of phenols which may then be further metabolized to catechols and
phenol conjugates (Safe 1994). All other metabolic pathways proceed through an arene
oxide intermediate which is highly reactive and can form protein, RNA, and DNA adducts
(Schnellman et al. 1985). The arene oxide can also be reduced to phenol. Thus, phenols
can be produced by 2 distinct pathways and have been documented to occur in animal
tissues (Bergman et al. 1994a). The intermediate can also be acted upon by epoxide
hydrolase to form dihydrodiols and subsequently by dehydrogenase to yield catechols.
The reactive nature of catechols is likely to be responsible for their absence in tissue
samples.
Alternatively, the arene oxide can be conjugated to glutathione by glutathione S­
transferases. These conjugates can then be eliminated in the feces. However, another
pathway for these conjugates can lead to the formation of methylsulfones (Bergman et al.
1978). Cleavage by intestinal microbial lyases produces thiols. If these thiols are
methylated, their lipophilicity increases facilitating their resorption. Subsequent Soxidation of the resorbed methyl metabolites yields methylsulfones. These metabolites
have been documented in human (Haraguchi et al. 1984) and wild mammal tissues
(Bergman et al. 1994b).
PCB metabolites exert several unique toxic effects compared to the parent
compounds. PCB phenols inhibit mitochondrial oxidative phosphorylation (Narasimhan et
al. 1991) and certain P450 activities (Schmoldt et al. 1977), as well as bind to various
hormone receptors and carrier proteins (Korach et al. 1988, Rickenbacher et al. 1986,
7
Brouwer et al. 1990). Methylsulfones bind with high affinity to several hormone (Gillner
et al. 1988, Norlund-Moller et al. 1990) and fatty acid binding proteins (Larsen et al.
1992) and inhibit P4501A1 activity (Kiyohara et al. 1992). These actions have not been
thoroughly investigated, so the role that metabolites play in PCB toxicity is unknown.
Reproductive toxicity
Current knowledge indicates that PCB-induced reproductive toxicity is likely to
occur via multiple mechanisms. Structurally diverse congeners from all 3 categories
(non-, mono-, and diortho-substituted) impair reproduction at environmentally-relevant
exposures. Multiple reproductive tissues are targets for PCBs and diverse alterations
including endocrine disruption, malformations, and embryo mortality occur (Peterson et al.
1993). Furthermore, species differences in dose/response relationships for acute toxicity
and liver enzyme induction do not correlate with sensitivity to reproductive impairment
(Lubet et al. 1990, Olson et al. 1990).
As mediators of gestation, hormones are likely targets for contaminant-induced
reproductive impairment, but effects vary with species, sex, age, and tissue. Some
congeners rapidly accumulate and remain concentrated in steroidogenic corpora lutea
(CLs) and adrenals of pregnant mice (Brandt 1975). Barsotti et al. (1979) demonstrated
that TCDD-treated female rhesus monkeys had suppressed progesterone (P) and estrogen
(E) plasma concentrations throughout the menstrual cycle. In pregnant rats, TCDD had
no effects on plasma E despite decreased hepatic E metabolism (Shiverick and Muther
1983). Steroidogenesis was inhibited by TCDD in rat testes via decreased mobilization of
cholesterol by P450scc (Moore et al. 1991). Aroclor 1254 interferes with P metabolism in
8
male guinea pig adrenals, but not testes (Goldman and Yawetz 1990). Serum PRL
concentration was transiently reduced (Russell et al. 1988) and PRL/PRLR function
suppressed (Jones et al. 1987) following TCDD exposure in male rats. The mechanism by
which TCDD induces these hormone changes is unknown.
Alternatively, reproductive toxicity may involve changes in hormone receptor
function. TCDD caused dose-dependent decreases in uterine ER and progesterone (PR)
in immature rats (Romkes and Safe 1988) and mice (De Vito et al. 1992a), but failed to
down-regulate uterine ER concentrations in mature mice even at 3 times the effective dose
in immature animals (De Vito et al. 1992b). In addition to age-specificity, TCDD-induced
changes in receptors vary between species. Acutely toxic doses of TCDD in prepubertal
guinea pigs produced no change in uterine ER, while hamster ER concentrations were
elevated (Hruska and Olson 1989). Gestation-specific effects of TCDD or PCBs on
uterine steroid receptors have not been described; therefore, their potential role in
embryotoxicity and teratogenicity is unknown.
The mechanism of PCB-induced changes in receptors is also unknown. Recent
identification of an AhR response element on human and mouse DNA upstream from the
ER coding region suggests that TCDD-like PCBs down-regulate ER concentrations by
blocking transcription (White and Gasiewicz 1993). However, Nelson et al. (1989)
proposed that TCDD modifies the steroid binding properties of cytosolic glucocorticoid
receptors without altering quantities of immunoreactive receptor protein. While binding
of TCDD to ER or PR is not known to be competitive in rodent uteri (Romkes and Safe
9
1988), PCBs and their metabolites bind to steroid binding proteins in both the rat testes
(Soderkvist et al. 1986) and the rabbit uterus (Gillner et al. 1988).
PCB metabolites may cause other toxic effects. Alterations in vitamin A and
thyroid hormone metabolism, PCB binding to lung and adrenal proteins, and altered
energy metabolism occur only after increased PCB metabolite formation (Brouwer 1991).
While some congeners are metabolized at a slower rate than others (Millis et al. 1985),
limited metabolism of even the most highly chlorinated PCBs occurs via direct
hydroxylation, chlorine-shift hydroxylation, dechlorination, and sulfation in rodents (Kato
et al. 1980, Kurachi 1983, Duignan et al. 1988).
Differences in PCB metabolite formation, excretion, and tissue disposition also
exist between pregnant and nonpregnant animals (Darnerud et al. 1986). Hydroxy- and
methylsulfonyl-PCB metabolites are enriched in uterine luminal fluid in early gestation,
yolk sac placenta in midgestation, and fetuses in late gestation in mice (Brandt et al.
1982). Some hydroxy-PCB metabolites bind to murine ER; but effective concentrations
range from 42 to 5000 times that of E (Korach et al. 1988). However, steroid receptors
may possess species-specific binding characteristics as demonstrated for hamster PR (Gray
and Leavitt 1987) or alterations in receptor number and affinity in response to different
physiological states (Chilton and Daniel 1987, Kawaguchi et al. 1991). In addition,
methylsulfonyl metabolite binding kinetics have not been evaluated.
Although TCDD and other AhR ligands are considered antiestrogens, some PCBs
exert estrogenic effects on the rat uterus (Gellert 1978). PCB mixtures, diortho
congeners, and some hydroxy metabolites of diortho congeners increase uterine weight
10
(Jansen et al. 1993, Soontornchat et al. 1994). Estrogenic effects of PCB congeners in
female rats are hypothesized to be caused by metabolites based on the following
observations: 1) efficacy increases with multiple doses and with doses that maximally
induce pentoxyresorufin-O-deethylase activity (Li et al. 1994), 2) differences in congener
efficacy parallel relative metabolism rates (Ecobichon and MacKenzie 1974), and 3) the
structural similarity between hydroxy metabolites and E (Korach et al. 1988).
Alternatively, PCBs may indirectly affect reproduction via their influence on other
endogenous substances. PCB exposure causes significant weight loss and mobilization of
fat stores in mink (Gillette et al. 1987). In anestrous mink, lipoprotein lipase activity
increases and circulating triacylglycerol levels decrease (Brewster and Matsumura 1989).
These changes may expose uterine ER and PR to elevated concentrations of nonesterified
fatty acids (NEFAs) which are known inhibitors of steroid receptor binding (Kato 1989,
Viscardi and Max 1993). Direct binding at a site distinct from the hormone and heat
shock protein binding sites is the proposed mechanism of reduced receptor binding by
NEFAs. This mechanism of action is compatible with observations that the quantity of
immunoreactive receptor protein is unchanged by TCDD exposure (Nelson et al. 1989).
In addition to regulating female reproductive tissues, hormones control growth and
differentiation in developing embryos and neonates. ER has been documented in bone cell
cultures (Eriksen et al. 1988), and intestine (Prince 1994). Both prenatal and neonatal
exposure of mice to E results in shorter femurs without changes in body weight or
reproductive tract development indicating that bone development is highly sensitive to
estrogenic substances (Migliaccio et al. 1992). Research indicates that E complexes with
11
ERs on bone cells to decrease longitudinal and radial growth of the tibia resulting in
shorter and narrower bones in female rodents compared to males (Turner et al. 1994).
PCBs effects on growth and development are structure-dependent. PCB
congeners that bind to AhR produce TCDD-like malformations such as cleft palate and
hydronephrosis. However, some diortho congeners are embryotoxic without producing
cleft palate or hydronephrosis in surviving embryos (Marks and Staples 1980). First-born
children of PCB-exposed mothers where significantly shorter than siblings and children
from unexposed mothers (Guo et al. 1994) and had reduced head size and
neurodevelopmental deficits (Jacobson et al. 1990).
Mink Reproductive Physiology
Mink normally mate during a 4-5 week period in late winter producing just one
litter per year (Enders 1952). Mating induces ovulation with approximately 10 eggs
released per mating (Adams 1973). Fertilization occurs in the oviduct and transport to the
uterus requires approximately 8 days (Hansson 1947). Once in the uterus, the blastocysts
enter an obligate, embryonic diapause that varies in duration from 5-55 days depending on
the date of mating (Enders 1952). During diapause, cell division ceases, and cell growth is
minimal. The blastocysts become equally distributed throughout the bicornate uterus, but
do not attach to the uterine wall (Enders 1952).
During embryonic diapause, CLs formed after ovulation remain small and relatively
inactive and systemic concentrations of P are low compared with those during postimplantation pregnancy (Moller 1973, Pilbeam et al. 1979, Stoufflet et al. 1989).
12
Systemic concentrations of E increase during the breeding season, decline to below
detection levels during pregnancy, and remain low during anestrus (Pilbeam et al. 1979,
Slayden and Stormshak 1992). Uterine concentrations of ER and PR are elevated during
embryonic diapause and at the time of implantation, but are low during anestrus (Slayden
1990, Slayden and Stormshak 1992). Treatment of anestrous mink with E induces an
increase in the uterine concentrations of ER and PR, as well as increasing uterine
metabolic activity (Slayden and Stormshak 1992).
Embryonic diapause in mink appears to be controlled by changes in photoperiod
and shortly after the vernal equinox (March 21), blastocyst development resumes and
embryos implant into the endometrium (Mead and Wright 1983). Implantation in
carnivores involves the trophoblast adhesion to the uterine wall followed by penetration of
the endometrium (Enders 1976). Cells hypertrophy and smooth and rough endoplasmic
recticulum proliferate as the endometrium enters the secretory phase (Schlafke et al.
1981). A significant increase in mucopolysaccharide occurs (Murphy and James 1974). A
uteroglobin-like protein occurs in mink uterine fluid during gestation (Daniel 1968). The
remaining secretory products in mink uterine fluid are unknown.
Progestational support is critical to the maintenance of the developing embryos.
CLs undergo pronounced morphological and physiological changes after the vernal
equinox (Mead 1981). Increasing P concentrations, which coincide with the time of
renewed embryo development, reach a peak at mid gestation and then decline gradually in
late gestation (Stoufflet et al., 1989). Reductions in P concentrations during gestation by
ovariectomy (Moller 1974) or antiprogesterone antibody exposure (Rider and Heap 1986)
13
arrest development of embryos. In contrast, E peaks during the mating season, then
declines to below detectable levels during gestation (Stoufflet et al. 1989). Thus, E
appears to have very little regulatory influence in P-dominated mink gestation.
E and P are believed to exert their actions at target tissues by binding to nuclear
receptors (Truss and Beato 1993). Occupied receptors undergo conformational changes,
release of companion proteins, complexing with transcription factors, and ultimately,
binding to hormone response elements of DNA to initiate transcription of hormonespecific mRNA.
Concentrations of steroid receptors are used to assess the responsiveness of a
tissue to steroid exposure (Tate and Jordan 1983). Unoccupied receptors are quantified
by exposing tissues to increasing quantities of [3H]- labeled ligand in the presence or
absence of non-labeled ligand. Dissociation from endogenous ligand is prerequisite to
quantifying occupied receptors. Commonly, this is accomplished by manipulating the
assay temperature to increase dissociation or using a synthetic ligand with greater affinity
to displace the endogenous compound. Using these techniques, it has been shown that a
finite population of receptors exists at a given time in a specific tissue type, but this
population exceeds the number of receptors necessary to produce a hormone-induced
biological response. Receptor binding of steroid follows Michaelis-Menten saturation
kinetics. Affinity and specificity of receptors are high to facilitate binding of a specific
hormone at physiological concentrations.
P and E binding to their receptors stimulate a variety of responses. These
responses vary with target tissues as a result of tissue-specific cofactors required for
14
binding to response elements on DNA (Truss and Beato 1993). E/ER responses in the
uterus include increased glucose metabolism, protein and lipid biosynthesis, and increased
activity of RNA polymerase, cellular hypertrophy and hyperplasia (Tate and Jordan 1983).
Included in E/ER-induced protein biosynthesis is the upregulation of ER and PR.
Both receptors, maintained at low constitutive levels, are synthesized in response to E
resulting in an increased responsiveness to subsequent E or P exposures (Kraus and
Katzenellenbogen 1993). P in the absence of E inhibits recruitment of both ER and PR
(Hsueh et al 1975). Thus, tissue responsiveness to E and P is linked to E-priming of
receptors (Kraus and Katzenellenbogen 1993). This priming occurs as part of the normal
reproductive cycle. With E concentrations peaking during estrus, ER and PR
concentrations are elevated preparing the uterus for control by P produced by the CL
during gestation. P stimulates differentiation of target cells thus lowering its
responsiveness to subsequent exposures of both E and P.
Mink uterine PR may also be under the direct control of prolactin (PRL). With
increasing daylength following the vernal equinox, melatonin levels decline and circulating
PRL concentration increase (Martinet and Main 1985, Slayden 1990). Increasing PRL is
prerequisite for activation of the CL (Papke et al. 1980), for the cessation of diapause,
and, subsequently, for embryo implantation (Murphy et al. 1981). At this time, a
significant increase in PR occurs without any change in ER concentration in the uterus
(Slayden 1990). Prolactin receptors (PRLR) occur in the mink ovary (Rose et al. 1986)
and uterus (Rose et al. 1983). Thus, PRL may be directly responsible for increased P
15
synthesis by the CL and PR upregulation in the uterus which both coincide with the end of
the embryonic diapause.
Although the duration of diapause varies widely with date of mating, the interval
from implantation to parturition is approximately 30 days (Enders 1952). While diapause
synchronizes birth with optimum conditions for survival, these arrested embryos are
incredibly sensitive to changes in the uterine environment and as many as 60% of them can
die (Enders 1952). Thus, litter size varies from 2 to 10 kits (avg = 4), which are born in
late April or early May.
Kits are altricial and are unable to maintain their own body temperature until 35
days of age (Travis and Schaible 1961). Cold temperatures are stressful and can reduce
survival in combination with other stressors (Wren et al. 1987a). Kits are weaned in early
summer at which time adult females become anestrus until the onset of the next breeding
season (Enders 1952). Kits born in May of one year are sexually mature by February of
the following year at approximately 10 months of age (Enders 1952).
PCB-Induced Reproductive Toxicity in Mink
Mink are an excellent model for PCB-induced reproductive toxicity because of
their high sensitivity and ecological equivalence to humans as high trophic level consumers
in both aquatic and terrestrial ecosystems. In the 1960s, ranch mink experienced
reproductive failure when fed diets containing fish byproducts from the Great Lakes
ecosystem. Effects ranged from no live young to reduced size, growth, and survival of
kits (Aulerich and Ringer 1977) Ovulation, fertilization, and implantation were not
16
impaired, but gestation was arrested prior to term (Platonow and Karstad 1973). As a
result of an intensive research effort, PCBs were identified to be the causative agents
(Aulerich et al. 1973). Experiments comparing mink to ferrets (Bleavins et al. 1980, Shull
et al. 1982) and rats (Gillette et al. 1987) have clearly demonstrated the exceptional
sensitivity of mink to PCBs. Recent investigations have shown that reduced litter size
occurs as low as 656 pg TCDD equivalents/g liver (Heaton et al. 1991) which is
significantly lower than most laboratory and domestic species (Golub et al. 1991).
In addition to sensitivity, several other differences in the responses of mink to PCB
exposure have been noted. Liver enlargement does not occur at exposures which produce
complete reproductive failure (Aulerich et al. 1973). Toxicity in mink is not related to
hepatic P450 CYP1A1 or CYP1A2 induction as is seen in rats (Gillette et al. 1987). PCB
exposure which caused complete reproductive failure induced EROD activity only 2.2-fold
(Brunstrom et al. 1991). P450 isozymes in the 2B family are either unchanged or inhibited
by diortho congener exposure (Shull et al. 1982).
Mink also appear to have a significantly different metabolic capacity in comparison
to other domestic animals (Platonow and Karstad 1973). Highly chlorinated diortho
congeners do not have the long half lives in fat deposits observed for rodents (Hornshaw
et al. 1983). Both penta- and hexa-PCB metabolites occur in mink experimentally
exposed to a 2-4 ortho-substituted PCB fraction (Bergman et al. 1992), as well as wild
mink (Bergman et al. 1994b) which feed on aquatic organisms with PCB burdens
dominated by highly chlorinated congeners (Maack and Sonzogni 1988, McFarland and
Clarke 1989).
17
Wild mink are opportunistic predators consuming prey from both highly
contaminated aquatic and relatively uncontaminated terrestrial food webs. As a result of
their feeding habits, wild mink contaminant exposures are not as high as those in ranch
mink fed PCBs in their diet. However, PCB exposure in prey items is more toxic to mink
than exposure to equivalent concentrations of commercial PCB mixtures (Aulerich et al.
1986) due to biomagnification of the more highly chlorinated congeners. In addition,
other environmental stresses to which wild mink are exposed can exacerbate the toxicity
of PCBs. Contaminant toxicity is further exacerbated in adults by exposure to cold
temperatures (Wren et al. 1987a). Simultaneous exposure to mercury, which is also
widespread in aquatic ecosystems, synergizes the toxic effects of PCBs on kit growth and
survival (Wren et al. 1987b).
18
2,2',4,4',5,5'- and 3,3',4,4',5,5'-HEXACHLOROBIPHENYL ALERATION OF UTERINE
PROGESTERONE AND ESTROGEN RECEPTORS COINCIDES WITH
EMBRYOTOXICITY IN MINK (Mustela visor)
Kathleen A. Patnode and Larry R. Curtis
Published in Toxicology and Applied Pharmacology
volume 127, pages 9-18, 1994
copyright Academic Press, Inc.
19
Abstract
Female mink (Mustela visor) are highly sensitive to organochlorine (0C)-induced
reproductive impairment. However, mechanisms of this reproductive toxicity are
unknown. We have investigated the possible role of steroid receptors in embryotoxicity
and reduced neonate weights. Anestrous, juvenile female mink and pregnant adult mink
were exposed to 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB), a coplanar polychlorinated
biphenyl (PCB) or 2,2',4,4',5,5'-hexachlorobiphenyl (2HCB), a noncoplanar PCB
congener. Both congeners impaired 1713-estradiol-stimulated (24 hr after ip administration
of 1001Ag E2f3 ip) up-regulation of uterine nuclear estrogen receptors (ERn) in anestrous
mink. Embryotoxicity and reduced embryo growth were first observed 14 days after
exposure to 0.4 mg 3HCB/kg > 0.8 mg 3HCB/kg > 20 mg 2HCB/kg. In pregnant mink,
all 3 HCB treatments significantly increased progesterone receptor dissociation constants
(PR Kd). ER concentration and PR total receptor number (Rt) were increased by 20 mg
2HCB/kg > 0.8 mg 3HCB/kg, but were unaffected by 0.4 mg 3HCB/kg. Serum E213 was
below assay detection limits. Progesterone (P) concentrations were increased by 2HCB,
decreased by 0.8 mg 3HCB/kg, and unchanged by 0.4 mg 3HCB/kg. Hepatic cytochrome
P450 (P450) was induced 1.8-fold in anestrous and 2.2- fold in pregnant mink by 3HCB.
Ethoxyresorufin-O-deethylase (EROD) was induced 13-fold and 4-fold in anestrous and
pregnant mink, respectively. 2HCB exposure resulted in decreased P450 concentration in
anestrous juveniles, but had no effect on P450 during gestation or EROD activity at any
time. We propose that embryotoxicity and retarded embryo growth result from
impairment of PR function and that differences in the efficacy of HCB treatments are a
result of their dose-dependent, partial estrogenic actions which increase PR Rt via upregulation of ER.
20
Introduction
Persistent organochlorines (OCs) tend to accumulate in animal tissues and
constitute a major class of environmental contaminants. Reproductive function can be
impaired by environmental exposure to these structurally-diverse OCs (Tanabe, 1988).
Mink are an appropriate mammalian model for 0C-induced reproductive toxicity because
of their high sensitivity and ecological equivalence to humans as high trophic level
consumers in both aquatic and terrestrial ecosystems. Since the early 1960s, impaired
reproduction has been reported in ranch mink fed diets including marine and freshwater
fish byproducts. Researchers have documented effects of these 0C-contaminated fish
ranging from decreased kit growth to complete reproductive failure (Sundqvist et al.,
1989). Reduced litter size occurs as low as 656 pg 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) equivalents/g liver (Heaton et al., 1991).
Species differences in sensitivity to reproductive dysfunction are not correlated
with dose/response relationships for acute toxicity and cytochrome P450 lA 1 induction.
Despite 80- and 40-fold differences in LD50 values and ethoxyresorufin-O-deethylase
(EROD) induction response, respectively, between hamsters and rats (Lubet et al., 1990),
reproductive toxicity occurs at similar doses (Olson et al., 1990). Mink responsiveness to
TCDD isostereomers corresponds most closely to that of the guinea pig. Both species
have high susceptibility to acute (Hochstein et al., 1988; Kociba and Schwetz, 1982) and
reproductive toxicity (Kihlstrom et al., 1992; Olson and McGarrigle, 1992), yet exhibit
low hepatic enzyme induction (Gillette et al., 1987; Holcomb et al., 1988).
21
As mediators of gestation, hormones are likely targets for contaminant-induced
reproductive impairment; however, correlations have not been documented. Aulerich et
a/. (1987) observed no changes in plasma concentrations of 1713-estradiol (E213) or
progesterone (P) in 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB)-exposed mink. In pregnant
rats, TCDD had no effects on plasma E20 despite decreased hepatic E213 metabolism
(Shiverick and Muther, 1983).
Alternatively, reproductive toxicity may involve changes in steroid hormone
receptor concentration or ligand affinity. TCDD down-regulation of uterine estrogen
receptor (ER) concentration has been documented in immature Long-Evans rats (Romkes
et al., 1987) and immature CD1 mice (DeVito et al., 1992a). However, uterine ER
concentrations in ovariectomized, immature CD1 mice (DeVito et al., 1992a) and mature
CD1 mice (DeVito et al., 1992b) are unaffected by TCDD exposure. Hruska and Olson
(1989) found that TCDD treatment increased maximum ER binding in uteri of immature
hamsters and Sprague-Dawley rats, but not in uteri of immature guinea pigs. TCDDinduced down-regulation of ER is thought be responsible for decreases in progesterone
receptor (PR) concentration (Romkes and Safe, 1988). Lundholm (1988) documented
decreased PR binding in rabbit endometrium following polychlorinated biphenyl (PCB)
exposure, but did not distinguish between changes in PR concentration and affinity.
Effects of TCDD or PCBs on mink uterine steroid receptors have not been described;
therefore, their potential role in 0C-induced reproductive failure is unknown.
The objectives of our research were to identify possible HCB-induced changes in
uterine steroid receptors in mink and investigate their involvement in the course of events
22
leading to fetal resorption. Experiments were performed on both anestrous juvenile and
pregnant adult mink to facilitate comparison of HCB uptake, hepatic cytochrome P450
enzyme induction, and uterine steroid horomone receptor responsiveness at different
physiological states. Both coplanar and noncoplanar congeners were used to indirectly
investigate AhR involvement in the mechanism of action.
Methods
2,2',4,4',5,5'-Hexachlorobiphenyl (2HCB) was chosen as the noncoplanar congener
because it is a major contaminant in wild mink (Larsson et al., 1990) and humans (Bordet
et al., 1993). 3HCB was selected for its equivalence as a HCB with a coplanar structure.
We based our 3HCB doses on total doses which produced embryo resorption in feeding
studies (Aulerich et al., 1985). 2HCB dose was set at 50 times the 3HCB dose to reflect
approximate ratios of these congeners in wild mink (Larsson et al., 1990). HCB
congeners (>99% purity) were obtained from AccuStandard, New Haven,CT. Moxestrol
(R2858) and promegestone (R5020) from Dupont-NEN (Boston, MA) were the synthetic
ligands used in ER and PR binding assays, respectively.
Mink were housed individually in wire cages in open-sided sheds under natural
changes in photoperiod and temperature. Anestrous mink were fed approximately 125 g
of growth ration with 22% fat and 38% protein once daily. Following mating, females
were fed approximately 125 glday of a gestation ration containing 20% fat and 45%
protein. Water was provided ad libitum.
23
Anestrous mink
In this experiment a combination of HCB and estrogen treatments were used: 1) to
approximate uterine estrus physiology, and 2) to test HCBs ability to inhibit estrogen
stimulation. Anestrous, immature female mink were injected (ip) with either 0.4 mg
3HCB/kg (n=9), 20.0 mg 2HCB/kg (n=9), or corn oil (vehicle; n=18). Treatments were
scheduled so that livers and uteri were collected within a 6-day period from mink at 6 hr, 3
days, or 14 days after HCB exposure. Twenty-four hours prior to tissue collection, HCBtreated animals received 100 lig E213 injections sc and corn oil treated animals were
injected with E213 (n=9) or corn oil (n=9).
Steroid receptor binding assays were performed in triplicate after Slayden et al.
(1993). Briefly, uteri were homogenized on ice in 10 volumes (w/v) of cytosolic buffer
(10 nM Tris-HCI, pH 7.4, 1.5 nM EDTA, 1 nM dithiothreitol, 10% (v/v) glycerol, and 25
mM Na2Mo04) We separated uterine cytosolic and nuclear fractions by differential
centrifugation (1000 x g for 10 min) of homogenates. Nuclear pellets were washed 3
times with cytosolic buffer and then resuspended in nuclear buffer (10 nM Tris-HCI, pH
7.4, 1.5 nM EDTA, 1 nM dithiothreitol, 0.8 M KC1) and incubated for 1 hr. Samples (300
IA1) of nuclear fraction were frozen for DNA determination after Burton (1956). Nuclear
and cytosolic fractions were purified by ultracentrifugation (100,000 x g for 1 hr). To
determine total binding, we incubated nuclear supernatant for 30 min at 37°C and then
both nuclear and cytosolic supernatants for 18 hr at 4°C with 10 nM [3H] -R2858 which
was determined to be a saturating concentration in preliminary binding assays (data not
shown). Replicate samples including 200 fold concentrations of unlabelled 82858 were
24
used to determine nonspecific binding. We separated receptor bound ligand on
minicolumns of Sephadex LH-20 (1.5 ml packed volume) with 0.7 ml of the appropriate
buffer and quantified ER by liquid scintillation counting.
Hepatic microsomes were prepared from 5 g liver after Guengerich (1989). Total
cytochrome P450 (P450) enzymes were quantified following Omura and Sato (1964) and
EROD activity was determined after Prough et al. (1978). Protein concentration in
microsomes was determined according to Bradford (1976).
From remaining liver tissue, we extracted and purified HCB residues according to
Gundersen and Pearson (1992) with the following modifications. Briefly, frozen tissue
was homogenized with a Sorvall Omni Mixer. We dried 1 g subsamples of whole liver
homogenate to a powder by macerating each with 7 g of anhydrous Na2SO4 with a
mortar and pestle. Powdered samples were extracted in 100 ml redistilled acetone for 8 hr
using a Soxhlet apparatus. We removed acetone by vacuum evaporation using a Biichi
Rotavapor-RE and redissolved the lipid mixture in 5 ml redistilled hexane. We
quantitatively transferred the solutions to 25 g partially-deactivated alumina columns for
liquid chromatography cleanup. HCBs were eluted with 75 ml hexane and elutant was
reduced to a known volume of less than 5 ml to concentrate HCBs. We determined
concentrations by gas chromatography-mass spectrometry analysis on a Saturn II ion trap
detector with a Varian 3400 GC equipped with a 30 m by 0.25 mm db5 capillary column.
Helium at 30 cm/sec served as the carrier gas. We used a splitless injection with injector
and detector temperatures of 200 and 350 °C, respectively. The column program was 80
°C for 1 min, 3 min rise to 200 °C, 8 min rise to 280 °C, and 280 °C for 8 additional min.
25
Ten percent of the samples were spiked with 2HCB or 3HCB to determine percent
recovery for each congener.
Pregnant mink
Our objective in this experiment was to investigate the role of P and PR, the
dominant steroid complex in mink gestation, in HCB-induced embryotoxicity. E213 and
ER were quantified in light of changes observed in anestrous mink On March 30, average
date of implantation (Stoufflet et al., 1989), pregnant female mink received either 0.4 or
0.8 mg 3HCB/kg, 20 mg 2HCB/kg, or corn oil (vehicle) via ip injection. At 3, 7, or 14
days after HCB exposure, blood samples were collected and uteri, ovaries, and livers were
removed. Implantation sites were removed from uterine horns, measured, and weighed.
Embryos were removed and weighed. We measured crown-rump length and head length
with a micrometer. Corpora lutea on each ovary were counted. ERn and ERc
concentrations, P450 concentrations, EROD activities, and liver HCB concentrations were
determined as in anestrous animals.
Blood samples were allowed to clot overnight at 4°C and sera were obtained by
centrifugation (500 x g for 15 min). Steroids were extracted from 1 ml of serum using 1
ml reversed phase C18 silica gel columns (J.T. Baker Inc., Phillipsburg, NJ) and eluted
with 5 ml of methanol. For 10% of the samples, replicates were spiked with E213 or P to
determine the percent recovery for each steroid. We determined concentrations of E213
and P using enzyme immunoassay kits according to manufacturer's protocol (Caymen
Chemical Co., Ann Arbor, MI).
26
Since it was unknown if PR binding parameters varied during gestation in mink,
saturation binding analyses with R5020 were conducted on each sample. We incubated
supernatant with 0.13 nM to 16.67 nM [3H] -R5020 for total binding determinations.
Replicate samples with 200 fold concentration of unlabelled R5020 were used to estimate
nonspecific binding. Uterine PRn were not measurable due to high concentrations of
endogenous P. Otherwise, receptor assays were conducted as described for ER for
anestrous mink.
Statistical analysis
We analyzed log transformed data for hepatic P450 and EROD, uterine ER, and
serum E213 and P and normal data for HCB liver concentrations with 2-way analysis of
variance (ANOVA). For ANOVAs with significant F values, Duncan's multiple
comparisons (DMC) were used to test for differences among means. We used 2-way
ANOVA and DMC nested on litter (Healy, 1972) to identify differences in embryo
weights, lengths, and head sizes. A contingency table was constructed to compare
numbers of resorbed and unresorbed embryos. Paired comparisons were made with Fisher
exact tests (Seigel, 1956). PRc total receptor concentration (Rt) and PRc dissociation
constant (Kd) were estimated using Marguardt-Levenberg non-linear least squares curve
fitting algorithm (Probst and Hermkes, 1991) where specific binding = (total ligand added)
(Rt) / (total ligand added + Kd). Log transformed estimates were then compared across
treatments using 2-way ANOVA and DMC.
27
Results
Anestrous mink
After 24 hr, exogenous E213 alone increased nuclear estrogen receptor (ERn)
concentrations (Table 1). Concentrations were comparable to those previously reported
for E213-treated anestrous mink (Slayden and Stormshak, 1992). Treatment with HCBs
significantly impaired E2f3's effect on ERn at 6 hr and 3 days, but not at 14 days after
exposure. Cytosolic estrogen receptor (ERc) concentrations were unchanged by E2f3
(Table 2) suggesting the dose and/or exposure time were insufficient to induce ER
synthesis. ERc concentrations were increased 29 to 42 % by HCB treatment after 6 hr
and 3 days, but differences were not significant.
E213 treatment alone had no significant effect on total cytochrome P450
concentrations (Table 3). P450 concentrations were reduced by 2HCB; reductions of 40
and 48% at 6 hr and 14 days, respectively were significant. Treatment with 0.4 mg
3HCB/kg also suppressed P450 initially, but induced a 1.5-fold increase in concentrations
at 14 days. E2f1 and 2HCB treatments had no significant effect on EROD activity (Table
4). In 3HCB-treated mink, EROD activity was significantly induced 13-fold at 14 days.
Pregnant mink
Reproductive impairment was not evident after 3 or 7 days, but was observed 14
days after treatment with both congeners. Implantation sites did not differ significantly in
number across treatments (Table 5), yet they were 80%, 70%, and 50% smaller in weight
in 0.4 mg 3HCB/kg, 0.8 mg 3HCB/kg, and 2HCB-treated animals, respectively than in
28
corn oil treated mink (data not shown). Only amorphous necrotic tissue masses were
present within implantation sites of 0.4 mg 3HCB/kg treated mink (Table 5). Significant
increases in numbers of resorbed embryos also occurred in 0.8 mg 3HCB/kg and 2HCB­
treated animals. Embryo weight, crown-rump length, and head length were significantly
reduced by both congeners with 3HCB causing greater growth impairment.
29
Table 1-1. Uterine nuclear estrogen receptor (ERn) in fmol/pg DNA (7 ±SE) in
anestrous and pregnant mink over time following exposure to 20 mg 2,2',4,4',5,5'­
hexachlorobiphenyl (2HCB)/kg or 0.4 or 0.8 mg 3,3',4,4',5,5'-hexachlorobiphenyl
(3HCB)/kg.
TIME AFTER HCB EXPOSURE
6 hr
3 days
7 days
14 days
corn oil
0.447±0.029
0.410±0.092
NE,a
0.448±0.033
corn oilb
1.799±0.187*
1.922±0.147*
ND
1.676±0.233*
1.248±0.077**
1.114±0.191**
ND
1.388±0.115*
1.350±0.194**
1.176±0.088**
ND
1.352±0.070*
corn oil
ND
0.128±0.014
0.108±0.016
0.049±0.010
2HCB
ND
0.150±0.031
0.053±0.009
0.104±0.021
0.4 mg/kg
ND
0.158±0.024
0.099±0.022
0.044±0.008
0.8 mg/kg
ND
0.103±0.016
0.075±0.007
0.072±0.013
TREATMENT
Anestrous
Mink
2HCBb
3HCBb
0.4 mg/kg
Pregnant Mink
3HCB
a Parameter was not determined (ND) for this time point
b 100 pg estrogen injected sc 24 hr prior to tissue collection
* differs from corn oil (p<0.05)
** differs from corn oil and corn oil/estrogen (p<0.05)
30
Table 1-2. Uterine cytosolic estrogen receptor (ERc) in fmol/p,g DNA CV ±SE) in
anestrous and pregnant mink over time following exposure to 20 mg 2,2',4,4',5,5'­
hexachlorobiphenyl (2HCB)/kg or 0.4 or 0.8 mg 3,3',4,4',5,5'-hexachlorobiphenyl
(3HCB)/kg.
TIME AFTER HCB EXPOSURE
6 hr
3 days
7 days
14 days
corn oil
30.100±0.841
22.277±1.573
Npa
25.919±1.938
corn oilb
31.087±0.708
26.647±3.106
ND
21.535±2.548
2HCBb
40.079±4.436
36.259±3.414
ND
28.604±2.510
41.345±1.644
37.834±4.714
ND
22.967±1.958
corn oil
ND
4.526±0.496
2.992±0.642
1.268±0.227
2HCB
ND
5.748±0.369
2.119±0.193
4.169±0.405*
0.4 mg/kg
ND
3.103±0.632
3.410±0.853
1.290±0.118
0.8 mg/kg
ND
3.040±0.148
1.660±0.204
1.779±0.081*
TREATMENT
Anestrous
Mink
3HCBb
0.4 mg/kg
Pregnant Mink
3HCB
a Parameter was not determined (ND) for this time point
b 100 pg estrogen injected sc 24 hr prior to tissue collection
* differs from corn oil (p<0.05)
31
Table 1-3. Total cytochrome P450 (P450) in nmol/mg protein (.)7 ±SE) in livers from
anestrous and pregnant mink over time following exposure to 20 mg 2,2',4,4',5,5'­
hexachlorobiphenyl (2HCB)/kg or 0.4 or 0.8 mg 3,3',4,4',5,5'-hexachlorobiphenyl
(3HCB)/kg.
TIME AFTER HCB EXPOSURE
TREATMENT
6 hr
3 days
7 days
14 days
corn oil
0.342±0.046
0.315±0.028
NDa
0.227±0.019
corn oil b
0.304±0.050
0.272±0.021
ND
0.254±0.017
2HCBb
0.156±0.007*
0.213+0.016
ND
0.137±0.035*
0.152±0.023*
0.311±0.030
ND
0.399±0.036*
corn oil
ND
0.389±0.101
0.269±0.013
0.259±0.034
2HCB
ND
0.301±0.043
0.315±0.056
0.281±0.022
0.4 mg/kg
ND
0.455±0.077
0.559±0.053*
0.581+0.097*
0.8 mg/kg
ND
0.658±0.118
0.527±0.042*
0.667±0.125*
Anestrous Mink
3HCBb
0.4 mg/kg
Pregnant Mink
3HCB
a Parameter was not determined (ND) for this time point
b 100 pg estrogen injected sc 24 hr prior to tissue collection
* differs from corn oil (p<0.05)
32
Table 1-4. Ethoxyresorufin-O-deethylase (EROD) activity in nmoUmin/mg protein (.f ±
SE) in livers from anestrous and pregnant mink over time following exposure to 20 mg
2,2',4,4',5,5'- hexachlorobiphenyl (2HCB)/kg or 0.4 or 0.8 mg 3,3',4,4',5,5' ­
hexachlorobiphenyl (3HCB)/kg.
TIME AFTER HCB EXPOSURE
6 hr
3 days
7 days
14 days
corn oil
0.089±0.008
0.085±0.011
ND"
0.115±0.021
corn oil°
0.062±0.008
0.075±0.026
ND
0.082±0.034
2HCB°
0.057±0.034
0.357±0.124
ND
0.115±0.030
0.163±0.067
0.486±0.167
ND
1.262±0.167*
corn oil
ND
0.016±0.001
0.012±0.003
0.008±0.005
2HCB
ND
0.018±0.001
0.023±0.005
0.011±0.004
0.4 mg/kg
ND
0.020±0.001
0.020±0.003
0.030±0.006*
0.8 mg/kg
ND
0.023±0.005
0.024±0.003*
0.018±0.001
TREATMENT
Anestrous Mink
3HCB°
0.4 mg/kg
Pregnant Mink
3HCB
a Parameter was not determined (ND) for this time point
100 [ig estrogen injected sc 24 hr prior to tissue collection
* differs from corn oil (p<0.05)
33
Table 1-5. Numbers of implantation sites (IP) and embryos and embryo characteristics
±SE) in mink at 14 days after ip injection on March 30 with 20 mg/kg 2,2',4,4',5,5'­
hexachlorobiphenyl (2HCB) or 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB) at 0.4 or 0.8
mg/kg.
EMBRYO
TREATMENT
corn oil*
2HCB*
#IP
SITES
EMBRYOS
Weight(mg)
23"
21"
406.80±
69.19"
14.25±
1.68a
5.79±
101.62±
6.29+
22.88°
1.096
2.63+
0.476
0.00+
0.00c
6.05+
2.31c
0.00+
0.00c
25"
#
166
3HCB*
0.4mg/kg
22a
Oc
0.8mg/kg
27"
7d
*
CR
Length(mm)
1.21+
0.41c
n =3 per treatment group
abcd dissimilar letters within columns denote differences (p<0.05)
Head
Length(mm)
0.62a
0.00+
0.00c
0.57±
0.21c
34
PRc Kd and PRc Rt estimates in corn oil treated animals varied during gestation
reaching a peak at 7 days (Table 6). PRc Kds were increased as early as 3 days after HCB
exposure. However, these increases were not significant until 14 days after HCB
treatment when PRc Kd in corn oil treated mink declined. Significant increases were seen
in Rt estimates at 14 days for 2HCB and 0.8, but not 0.4 mg 3HCB/kg (Fig 1). It is
unlikely that PR Kd changes reflect alterations in PR Rt as increases in Kd occur at times
and HCB exposures when Rt is uneffected.
ERn and ERc concentrations in pregnant mink were markedly reduced compared
to those in anestrous mink (Tables 1 and 2). In pregnant mink, uterine ERn
concentrations were unchanged by either congener. During gestation, ERc was increased
significantly at 14 days by 2HCB and 0.8 mg 3HCB/kg exposure, while 0.4 mg 3HCB/kg
had no effect.
In corn oil treated animals, P concentrations (ng/ml) rose from 15.21 ± 2.01 at 3
days to a peak of 24.05 ± 2.67 at 7 days before falling to 10.23 ± 2.51 at 14 days.
E213
was not measurable at any time. These determinations were comparable to those
previously reported in pregnant mink (Stoufflet et al., 1989). HCB treatment did not
produce significant differences in P concentrations at any particular sampling time.
However, when data were pooled across time, 2HCB significantly elevated (19.24 ng/ml ±
1.56) and 0.8 mg 3HCB/kg significantly depressed (8.00 ± 0.90) serum P concentrations,
while 0.4 mg 3HCB/kg-treated mink (13.73 ± 1.74) had P concentrations similar to corn
oil treated animals (14.27 ± 1.85). High individual animal variability, which may obscure
35
Table 1-6. Uterine progesterone receptor concentrations (Rt) in frnol/pg DNA (-.f ±SE)
and equilibrium dissociation constants (Kd) in nM
±SE) from pregnant mink over time
following exposure to 20 mg 2,2',4,4',5,5'- hexachlorobiphenyl (2HCB)/kg or 0.4 or 0.8
mg 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB)/kg.
TIME AFTER HCB EXPOSURE (days)
PARAMETER
Rt
TREATMENT
3
7
14
corn oil
1.472±0.360
1.703±0.982
0.761±0.257
2HCB
2.162±0.465
1.258±0.328
2.779±0.211*
0.4 mg/kg
1.548±0.133
1.324±0.495
0.960±0.060
0.8 mg/kg
1.341±0.166
1.071±0.053
1.509±0.113*
corn oil
1.935±0.068
2.926±0.254
0.981±0.243
2HCB
2.578±0.359
2.682±0.591
1.973±0.019*
0.4 mg/kg
4.475±1.491
4.400±1.564
3.587±0.518*
0.8 mg/kg
4.005±1.383
3.550 ±0.664
2.617±0.489*
3HCB
Kd
3HCB
* differs from corn oil (p.0.05)
4
3
2
1
0
I
0
33
c14
6
9
12
total [3H] -R5020 added (nM)
s0.4hcb14 © s0.8hcb14
15
A
18
s2Ohcb14
Figure 1-1. Saturation binding curves for [3H] -R5020 specifically bound to progesterone receptors at 14 days for pregnant
mink treated with 0.4 or 0.8 mg 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB)/kg or 20 mg 2,2',4,4',5,5'-hexachlorobiphenyl
(2HCB)/kg ip at implantation. n=3 per treatment group. Total and nonspecific curves omitted for clarity.
37
differences at individual sampling times, is most likely due to imperfect synchronization of
gestation.
Constitutive P450 levels were not affected by gestation (Table 3) as previously
demonstrated in rats and rabbits (Neale and Parke, 1973). P450 induction was 2.1 and
2.2-fold for 0.4 mg 3HCB/kg and 2.0 and 2.6-fold for 0.8 mg 3HCB/kg at 7 and 14 days,
respectively. 2HCB treatment had no effect on P450 concentrations. Pregnancy did
suppress constitutive EROD activity (Table 4). Similar reductions in hepatic microsomal
xenobiotic metabolism during gestation have been reported for rats (Symons et al., 1982)
and mice (Sinjari et al., 1993). EROD activity did not differ significantly at any time in
2HCB-treated mink. Maximum induction was 3.8- and 2.0-fold after 0.4 and 0.8 mg
3HCB/kg, respectively in pregnant mink.
For both congeners, disposition differed between pregnant and anestrous mink
(Figs 2 and 3). Hepatic 2HCB concentrations were approximately 7- and 5-fold greater in
pregnant than anestrous mink at 3 and 14 days, respectively (Fig 2). Treatment of
pregnant mink with 0.4 mg 3HCB/kg resulted in approximately 5-fold higher liver
concentrations at 3 days than in anestrous females (Fig 3). This difference was no longer
evident after 14 days of exposure. Doubling the dose of 3HCB in pregnant mink
produced significantly higher liver concentrations only at 3 days. While ratios of 20 mg
2HCB/kg and 0.4 mg 3HCB/kg doses were 50:1, maximum ratios in liver were 65:1 for
anestrous females and 20:1 for pregnant mink suggesting greater 3HCB accumulation
during pregnancy. Time course patterns indicate a slower 3HCB accumulation relative to
2HCB in anestrous, but not pregnant animals. Background concentrations of 2HCB were
0.006378 [tg/ g liver, while 3HCB was undetectable in corn oil-treated animals (detection
25
0.125
Loanestrous
0 pregnant
20
- 0.100
15-
- 0.075
10-
0.050
0.025
0.000
4
8
12
16
exposure time (days)
Figure 1-2. 2,2',4,4',5,5'-hexachlorobiphenyl (2HCB) concentrations (k±SE) in liver over time in anestrous and pregnant female mink
exposed to 20 mg 2HCB/kg. n=3 per treatment-time group. * Significantly differs from anestrous mink (p<0.05).
2.4
0.6
0 anestrous
2.0 ­
0pregnant
A pregnant
0.5
0.04 mg 3HCB/kg
0.08 mg 3HCB/kg
1.6 ­
0.4
1.2 ­
0.3
0.8 ­
0.2
0.4 ­
0.1
0.0
0.0
0
4
8
12
16
exposure time (days)
Figure 1-3. 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB) concentrations (k±SE) in liver over time in anestrous mink exposed to 0.4 mg
3HCB/kg and pregnant female mink exposed to 0.4 or 0.8 mg 3HCB/kg. n=3 per treatment-time group. abc Dissimilar letters at
exposure times denote differences (p<0.05).
40
limit of 0.0001 p.g). Percent recovery did not differ between congeners with an average
and standard error of 72.8 ± 2.3 %.
Discussion
Embryo resorption at 14 days after implantation without reductions in number of
implantation sites confirms evidence present at parturition (Aulerich et al., 1985; Backlin
and Bergman, 1992) that PCB-induced embryotoxicity occurs following implantation.
Impaired growth in surviving embryos is undoubtedly responsible for reductions in
neonate weights from females fed PCBs (Aulerich et al., 1985; Kihlstrom et al., 1992). At
the time of these toxicities, the mink uterine environment is controlled by P (Sundqvist et
al., 1988). Reductions in functional P concentration by ovariectomy or P-antibody
treatment produce this same spectrum of effects in mink and other mustelids (Moller,
1974; Rider and Heap, 1986). Yet, our results agree with earlier findings (Aulerich et al.,
1985) that HCB-induced embryotoxicity is not correlated with changes in serum P.
Embryo resorption and growth impairment can also occur when P binding to PR is
impaired by antiprogestins. All 3 HCB treatments resulted in significant decreases in PRc
affinity for ligand. While binding of TCDD to PR is not known to be competitive in
immature rodent uteri (Romkes and Safe 1988), certain PCBs and PCB methyl-sulfonyl
metabolites bind to P binding proteins in the rat lung (Lund et al., 1985) and the rabbit
uterus (Gillner et a/., 1988). Methyl-sulfonyl metabolites have recently been documented
to occur in pregnant mink exposed to coplanar, noncoplanar, and mixed PCB fractions
(Bergman et al., 1992). Competitive PR binding assays with 2HCB, 3HCB, and tissue
41
specific metabolites of both congeners in mink are necessary to determine if these
compounds are responsible for the observed decrease in PR binding affinity during
gestation. Quantitation of cell-specific PR expression products will establish whether or
not alterations in PR Kd are sufficient in magnitude to impair P/PR function.
Alternatively, PCBs may act indirectly on the PR via their influence on other
endogenous substances. Madej et al. (1992) found that urinary cortisol was significantly
higher in PCB-treated pregnant mink beginning at implantation and continuing for 25
days. Cortisol causes increased dissociation rates of R5020 in non-estrogen primed,
uterine tissue from rats (Moguilewsky and Deraedt, 1981). They propose that cortisol
binds to sites other than the ligand binding site on PR and induces an allosteric change
which reduces the receptor's affinity for its ligand. Replicating this work in mink would
determine if elevated cortisol levels are responsible for altered PR binding affinity.
While all 3 treatments decreased PR affinity, embryotoxicity varied with congener
and dose. This observation suggests that a differential effect on some other reproductive
end point also influences embryotoxicity. In anestrous, juvenile mink when uterine ER
concentrations are high, both HCBs impaired E213-induced up-regulation of ERn in
agreement with findings in immature rodents (Romkes et al., 1987; DeVito et at., 1992a).
However, during mink gestaton when E213 concentrations are negligible and uterine ER
and PR concentrations are declining, ERc and PR Rt were increased at 14 days after
2HCB and 0.8 mg 3HCB/kg, but not 0.4 mg 3HCB/kg. This suggests that both HCBs
exert estrogenic effects which are capable of attenuating the normal physiological decline
in uterine ER and PR.
42
Some PCBs and PCB metabolites exert estrogenic effects on mammalian uteri in
the absence of exogenous E213 treatment (Jansen et al., 1993). In vitro binding assays
indicate that hydroxy metabolites of some PCBs can bind to mouse uterine ER (Korach et
al., 1988). Although their affinities are at least 40-fold lower than E2f3 (Korach et al.,
1988), their occupancy of uterine ER may reach effective proportions during gestation in
mink when endogenous estrogens are suppressed (Stoufflet et al., 1989). 2HCB, which
induced the largest increase in uterine ERc and PRc, caused fewer resorptions than either
3HCB dose; 0.8 mg 3HCB/kg produced intermediate alterations in ER and PR
concentrations and embryotoxicity; while 0.4 mg 3HCB/kg had no effect on ER or PR
concentrations while producing complete litter failure. Based on these observations,
HCB-induced embryotoxicity in the mink appears to be inversely related to the partial
estrogenic effects of HCBs or their metabolites.
HCB impaired P binding to uterine PR could lead to reduced synthesis of proteins
essential to embryo growth. As plasma P concentrations rise , uterine protein secretions in
mink increase (Daniel and Krishnan, 1969). One of these P/PR expression products is
similar to rabbit uteroglobin (Daniel 1968), which increases cell division in cultured mink
embryos (Daniel and Krishnan 1969). Reductions in this or other P/PR induced
embryonic growth regulators could be responsible for HCB-induced growth impairment.
Severe growth impairment may result in embryo inviability and activate maternal
resorption.
Alternatively, embryo resorption may also be triggered by antiprogestin treatment
independent of growth impairment. Szekeres-Bartho et al. (1990) identified gestation­
43
specific lymphocyte PR in the mouse through which P induces synthesis of an
immunosuppressive protein involved in preventing the female's immune system from
recognizing embryos as foreign bodies. The presence of gestation-specific PRs on mink
lymphocytes must be substantiated and the effects of in vivo HCB exposure on PR kinetics
by cell type determined.
2HCB had not previously been shown to cause reproductive failure in mink.
Although we derived our doses from earlier studies, our experimental design may have
produced different patterns of disposition and target organ concentrations. Contaminant
exposure in feeding studies may be lower than computed total dose due to variables such
as feed consumption (Auletta, 1988) and absorption (Houston et al., 1974). In addition,
Spindler-Vomachka and Vodicnik (1984) demonstrated differences in 2HCB distribution
among lipoprotein fractions with reproductive state. Thus, in longterm reproductive
studies where dosing spans more than one reproductive state, lower target organ
concentrations can result because of differential distribution during anestrus and gestation
periods. This hypothesis is supported by 7- and 6-fold greater 2HCB concentrations in
livers of pregnant mink than anestrous juveniles. Further support comes from teratogenic
evaluations in mice in which 2HCB exposure during gestation was embryotoxic without
producing malformations (Marks et al., 1980). Single gavage exposures to 2HCB in mice
decreased fetal weights (Morrissey et al., 1992) and increased fetal resorptions (Biegel et
al., 1989) while simultaneously antagonizing TCDD-induced fetal cleft palate. These
results with 2HCB and the work of Kihlstrom et al., (1992) with a 2-4 ortho PCB fraction
in mink demonstrate that embryotoxicity of PCBs is not mechanistically limited to
44
coplanar PCB actions and argue for the inclusion of all PCB congeners in reproductive
risk assessments.
Mink possess unique hepatic P450 characteristics. Previous studies have
demonstrated relative resistance of mink to prototypic inducers (Shull et al., 1983), PCB
mixtures (Shull et al., 1982), and planar and noncoplanar tetrachlorobiphenyl congeners
(Gillette et al., 1987). Sensitivity to inducers of EROD activity appears to be greatest in
kits (30-fold), moderate in juveniles (13-fold), and minimal in pregnant and postpartum
adults (2-4-fold) (Brunstrom et al., 1991; this study). These characteristics are in contrast
to those of mice where EROD induction potential increases with age and does not differ
between pregnant and nonpregnant adult females (Sinjari et al., 1993). These interspecies
differences are likely due to a combination of species-specific physiological parameters
and incidental induction in all mink as a result of contaminated fish and meat byproducts in
rations.
Most effects of TCDD and coplanar PCBs are thought to be mediated by AhR.
Induction of P450 1A1 is the best understood action of OC/AhR activation and
correlation with P450 induction is taken to be indicative of AhR-mediated toxicosis (Safe,
1990). With less than a 4-fold EROD induction, 3HCB exposure caused complete
reproductive failure. 2HCB caused no P450 or EROD induction in agreement with
findings that 2HCB has virtually no affinity for AhR (Biegel et al., 1988), yet 2HCB
increased embryo resorption and retarded embryo growth. All 3 treatments produced
equivalent reductions in PR affinity. KihlstrOm and coworkers (Kihlstrom et al., 1992;
BrunstrOm 1992) demonstrated increased embryotoxicity without EROD induction in
45
pregnant mink fed a 2-4 ortho PCB fraction. Collectively, these results indicate that
reproductive dysfunction in mink is not correlated with P4501A1 induction. Events
leading to changes in ER and PR must be elucidated to determine the mechanism of HCBinduced embryotoxicity and to identify appropriate biomarkers for this mode of action.
46
3,3',4,4',5,5'41EXACHLOROBIPHENYL (3HCB)-INDUCED CHANGES IN
UTERINE PHYSIOLOGY IN MINK (MUSTELA VISON)
K. A. Patnode, L. R. Curtis, W.J. Rose, and A.A. Frank
Submitted to Fundamental and Applied Toxicology (1995)
47
Abstract
Embryo resorption and coincident reductions in uterine cytosolic progesterone
receptor (PRc) affinity occurred between days 7 and 14 following maternal exposure to
3,3',4,4',5,5'-hexachlorobiphenyl (3HCB) (Patnode and Curtis, 1994). Our objectives
were to determine if 3HCB competitively binds to uterine PRc from pregnant mink,
evaluate the histological changes in mink implantation sites, and test the efficacy of
exogenous progesterone (P) treatment in inducing uterine prolactin receptors (PRLR) and
overcoming 3HCB-induced embryo resorption. At the onset of implantation, mated
female mink received 0.4 mg/kg 3HCB ip, corn oil (CO) ip, or no treatment. CO-exposed
animals received empty silastic implants, while 3HCB-treated mink received either empty
or P-filled implants. At 8, 10, and 12 days following exposure, implantation sites from
treated mink were histologically evaluated and uterine PRLR concentrations were
determined. Uteri from untreated mink at day 10 were used to evaluate the in vitro
binding capacity of 3HCB to PRc. No significant difference was observed in either the
total number or affinity of PRc between in vitro systems with or without 3HCB. From
day 10 forward, implantation sites from 3HCB-treated mink contained necrotic tissue
from resorbing embryos, while uteri from CO treatment group had no necrosis.
Exogenous P treatment was ineffective in preventing resorption, but produced significant
increases in uterine PRLR concentration. We conclude from these investigations that
decreased affinity of the PRc was not the proximate cause of 3HCB-induced embryo
resorption.
48
Introduction
Previous work has demonstrated that embryo resorption and reduced uterine
cytosolic progesterone receptor (PRc) affinity occur between days 7 and 14 following
maternal exposure to 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB) (Patnode and Curtis 1994).
Progesterone action at uterine myometrial and stromal cells is critical to normal
reproduction. Rising concentrations of P coincide with reactivation of embryonic growth
and embryo implantation in mink (Stoufflet et al. 1989). Reductions in P concentrations
by either ovariectomy (Moller 1974) or antiprogesterone antibody (Rider and Heap 1986)
cause embryonic death in mustelids. With only a small fraction of circulating progesterone
free (Louw et al. 1992), the modest changes we observed in PRc affinity may be sufficient
to impair P/PR function leading to embryo resorption.
The mechanism of PCB-induced changes in receptor affinity is unknown. While
binding of TCDD to PR is not known to be competitive in rodent uteri (Romkes and Safe
1988), PCBs and their metabolites bind to steroid binding proteins in both the rat testes
(Soderkvist et al. 1986) and the rabbit uterus (Gillner et al. 1988). In vitro assays to
evaluate potential binding of 3HCB to uterine PR are lacking.
In combination with P/PR, PRL complexing with prolactin receptor (PRLR)
regulates gestation in the mink (Rose et al. 1983). Suppression of circulating P
concentrations in mink at midgestation reduces uterine PRLR (Rose and Stormshak
1993). In ferrets, progesterone also induces increases in uterine PRLR in the presence of
estrogen (Rose et al. 1993). In ovariectomized rabbits, progesterone treatment increases
prolactin receptor (PRLR) concentrations nearly 5-fold (Chilton et al. 1988). These
49
studies suggest that uterine PRLR is an expression product of P binding to uterine PR.
Thus, analysis of uterine PRLR concentrations at the time of embryo resorption could be
used to determine if depressed PR affinity results in reductions in an expression product
critical to sustaining gestation.
Failure to maintain a progestational environment during early to mid gestation
leads to embryo resorption in mink and other mammals. Histological evaluation of
implantation sites has been used to determine the onset of embryo resorption (Duclos et
al. 1994). Identification of the onset is necessary to elucidate the steps in 3HCB-induction
of the resorption process.
The overall goal of this research was to develop a better understanding of the
mechanism of 3HCB-induced embryo resorption. Our first objective was to determine if
3HCB competitively binds to the cytosolic progesterone receptor of uteri from pregnant
mink. A second objective was to evaluate the histological changes in mink implantation
sites. Finally, we wanted to test the efficacy of exogenous P treatment in inducing PRLR
concentrations and in overcoming 3HCB-induced embryo resorption.
Methods
Mink were housed individually in wire cages in open-sided sheds under natural
changes in photoperiod and temperature. Anestrous mink were fed approximately 125 g
of growth ration with 22% fat and 38% protein once daily. Following mating, females
were fed approximately 125 g/day of a gestation ration containing 20% fat and 45%
50
protein. Water was provided ad libitum. Mink were weighed at the beginning and end of
each treatment period.
At the onset of implantation (March 30), 27 mated female mink received 0.4
mg/kg 3HCB (n = 18) or corn oil (CO; n = 9) via ip injection. Silastic implants (30mm x
0.5mm) were injected sc at the nape of the neck using a modified syringe. CO-treated
animals received empty implants, while 3HCB-treated mink received either empty (n = 9)
or progesterone-filled (n = 9) implants. At 8, 10, and 12 days following exposure, blood
samples were collected. Animals were killed by CO2 asphyxiation and uteri were
removed. Implantation sites were dissected free and fixed in neutral buffered formalin for
histological evaluation. The remaining 3HCB-exposed uterine tissue and uteri from
untreated mink were frozen in liquid nitrogen for PRLR and PR analyses, respectively.
Histological evaluations were conducted on randomly selected implantation sites
from each animal. Serial sections were generated to assure that a mid-implantation site
section was evaluated. The following grading system was used to evaluate each site:
GRADE DEFINITION
1
2
3
implantation site present; normal endometrium (minor superficial glandular
dilation & modest mucous accumulations); no necrosis
implantation site present; hyperplastic endometrium (basilar glandular dilation
& abundant mucous accumulations); ± necrosis
no implantation site present
Results of these histological evaluations were then analyzed using Fisher exact probability
testing across treatment groups and gestation day.
Uterine homogenates from 7 pregnant, untreated female mink at day 10 post-
implantation were used to conduct in vitro binding assays. Aliquots of uterine
51
homogenate were incubated for 30 min. at 4°C in duplicate in uncoated or 3HCB-coated
tubes at a final 3HCB concentration of 100pM. Steroid receptor binding assays were then
performed in triplicate after Patnode and Curtis (1994). Briefly, uteri were homogenized
on ice in 10 volumes (w/v) of cytosolic buffer. We separated uterine cytosolic and nuclear
fractions by differential centrifugation of homogenates. Nuclear pellets were washed 3
times with cytosolic buffer and then resuspended in nuclear buffer and incubated for 1 hr.
Samples (300 pl) of nuclear fraction were frozen for DNA determination after Burton
(1956). Cytosolic fractions were purified by ultracentrifugation and saturation binding
analyses with R5020 were conducted on each sample. Uterine PRn were not measurable
due to high concentrations of endogenous P.
We incubated supernatant with 0.13 nM to 16.67 nM [3H] -R5020 for 18 hr at 4°C
to determine total binding. Replicate samples with 200-fold concentration of unlabelled
R5020 were used to estimate nonspecific binding. We separated receptor bound ligand on
minicolumns of Sephadex LH-20 (1.5 ml packed volume) with 0.7 ml of the appropriate
buffer and quantified bound PRc by liquid scintillation counting. PRc total receptor
concentration (Rt) and PRc dissociation constant (Kd) were estimated using MarguardtLevenberg non-linear least squares curve fitting algorithm (Probst and Hermkes, 1991)
where specific binding = (total ligand added) (Rt) / (total ligand added + Kd). Log
transformed estimates were then compared between treatments using students t-test.
To investigate possible effects of 3HCB on PRLR concentration and the function
of P/PR complexing in 3HCB-treated mink, PRLR point assays were performed on uterine
samples on 8, 10 and 12 days following treatment in CO, 3HCB-treated, and 3HCB+P­
52
treated pregnant mink after Rose et al. (1993). Briefly, uterine homogenate was
centrifuged at 1500 x g for 10 min. The resulting supernatant was centrifuged at 15,000 x
g for 20 min. This second supernatant was centrifuged at 50,000 x g for 4 hr. The
resulting pellet was resuspended in buffer and stored at -80°C until assayed.
Homogenates were incubated with a saturating concentration of125I-PRL (ovine)
with or without a 500-fold excess of competing nonradioactive PRL (saturating
concentration was previously determined by incubating a constant concentration of uterine
microsomal protein with increasing concentrations of125I-PRL for 18 hr at 25°C). At the
end of the incubation period, 3 ml of cold buffer (Tris -HCI 0.025M, CaC12 0.025M, 0.1%
bovine serum albumin, pH 7.6) were added to each tube to terminate the reaction. Bound
and free hormone were separated by centrifugation at 1140 x g for 30 min. Bound PRL
hormone was then quantified in the pellets by scintillation counting.
Results
Mink treated with CO increased in weight as gestation progressed with percent
weight changes from day 0 of 4.36±1.51 on day 8, 7.13±1.12 on day 10, and 8.67±0.92
on day 12. In contrast, 3HCB-treated animals gained weight only between days 0 and 8
(2.35±1.91). From day 0 to day 10, they lost 2.95±4.40 % of their body weight and from
day 0 to day 12, the loss increased to 10.75±5.98 %. Body weight loss was not previously
observed at this dose of 3HCB in pregnant mink (Patnode and Curtis 1994).
Progesterone receptor concentration and dissociation constant from uteri of
pregnant, untreated mink were 3.926±0.989 fmol/i.ig DNA and 3.992±0.827 nmol.
53
Addition of 1001AM 3HCB to the in vitro assay did not significantly change either PR
parameter (PRc concentration = 2.367±0.732 fmol/pg DNA and 3.758±0.962 nmol).
Histological evaluation of the implantation sites showed no detectable differences
between corn oil- and 3HCB-treated animals through day 8 (Table 1). However, at day
10 and beyond, implantation sites from 3HCB-treated mink contained necrotic tissue from
resorbing embryos, while CO-exposed uteri had no necrosis. In 3HCB exposed uteri, the
endometrium was hyperplastic with basilar glandular dilation and abundant mucous
accumulations compared to the endometrial tissue of CO mink. Implantation sites could
not be detected macroscopically or histologically in two 3HCB-treated and five 3HCB+P­
treated mink. These animals were considered nonpregnant in all subsequent analyses. The
proportion on nonpregnant females in the 3HCB+P treatment group was significantly less
than for control and 3HCB groups (p<0.03).
Table 2-1. Histological evaluation of implantation sites from pregnant mink treated with
3,3',4,4',5,5'-hexachlorobiphenyl (3HCB) or corn oil (CO) with empty or progesterone
(P)-filled silastic implants.
TREATMENT
CO
3HCB on day 8
3HCB on days 10/12
3HCB+P
NUMBER OF SITES
Necrosis/Hyperplastic
Normal
Endometrium
8
3
0
0
* significant difference @ p<0.05
1
0
4*
2*
54
Results of PRLR binding assays are presented in Fig. 1. Data were pooled across
time because we detected no significant differences between 8, 10 and 12 day exposures.
3HCB treatment did not decrease uterine PRLR concentration (Figure 1). Exogenous P
treatment significantly increased in PRLR concentration in both pregnant and nonpregnant
mink.
Discussion
In vitro assay data indicate that 3HCB-induced changes in PRc affinity previously
observed (Patnode and Curtis, 1994) are not due to direct binding of the parent PCB to
uterine PR. However, it does not eliminate the possibility that a 3HCB metabolite is a PR
ligand. Hydroxy-PCBs compete with estrogen for the steroid binding site on the estrogen
receptor (Korach et al. 1998) and methylsufones bind to progesterone-binding proteins in
the rabbit uterus (Gillner et al. 1988) and rat lung (Andersson et al. 1991).
Methylsulfones occur in ranch mink exposed to nonortho-substituted PCB fractions
(Bergman et al. 1992), as well as in wild mink exposed to environmental mixtures of PCBs
(Bergman et al. 1994). In vitro binding assays with identified 3HCB metabolites are
needed to determine if competition with uterine PRc occurs in pregnant mink.
Histological evaluation indicates that resorption is not microscopically detectable
on day 8, but has progressed to a highly necrotic stage by day 10. This sequence is the
same as that observed in resorption-prone crosses of mouse strains (Duclos et a. 1994).
In these mice, nonspecific immune cells invade decidua on day 6, increase sharply on days
7 and 8, and plateau on days 8 to 10. Thus, the onset of resorption
0
27
b
-r
24
E
21
0c
18
Om.
5
4.0
CIS
4
15
ON.
a
12
a
9
9
T
a
0
7
tr.
6
am.
PREGNANT
4.
a.4
g
NONPREGNANT
3
CO
3HCB
3HCB+P
1
3HCB
3HCB+P
Figure 2-1. Uterine prolactin receptor concentrations in pregnant and nonpregnant mink exposed to corn oil (CO),
3,3',4,4',5,5'-hexachlorobiphenyl (3HCB) at 0.4 mg/kg bw with or without progesterone (P)-filled silastic implants
(different letters denote significant differences at p<0.05; numbers denote sample sizes).
56
precedes histological evidence first detectable in day 10 mouse embryos by as much as 4
days. Given the extent of necrosis in day 10 implantation sites of mink, we suspect that
immune cells infiltrated the microscopically-normal day 8 decidua. This chronology
indicates that the resorption process is triggered prior to the time when PRc affinity is
significantly decreased (Patnode and Curtis 1994).
As a means of evaluating the biological significance of reduced PRc affinity, we
exposed 3HCB-treated mink to exogenous P. We hypothesized that boosting the
circulating P concentration could overcome decreased PRc affinity to maintain a
progestational environment. However, this treatment was ineffective at preventing
embryo resorption. Similar results were observed in P treatment of pregnant rats exposed
to an antiprogestational synthetic hormone where P reversed anti-P effects with the
exception of embryo resorption (Kabir et al. 1992). These observations indicate that once
the resorption process is triggered, P treatment is ineffective at halting its progress.
The exogenous P treatment also assessed whether functional P/PR complexing did
occur as measured by increases in uterine PRLR, a putative P/PR expression product. We
found that PR was still capable of binding P and producing a statistically significant
response. The magnitude of the increase in PRLR that we observed was comparable of
that produced by a 10-fold decrease in P in mink early in gestation (Rose and Stormshak
1993). Greater than 5-fold increases in uterine PRLR are observed with similar P
treatments in ovariectomized rabbits (Chilton et al. 1988). In addition to species-specific
differences, mink may have control mechanisms for uterine PRLR during gestation which
maintain their concentration within a critical range.
57
We conclude that decreased affinity of the PRc is not the proximate cause of
3HCB-induced embryo resorption. The observation that the resorption must be initiated
prior to significant alterations in PRc affinity combined with P-induced upregulation of
PRLR suggest that both PR and embryo resorption may be effects of an earlier 3HCB­
induced change.
PRL, the other endocrine mediator of mink gestation, appears to be a susceptible
target. Exposure to 3HCB (DeKrey et al. 1994) and TCDD (Jones et al. 1987, Moore et
al. 1989) reduces circulating PRL concentration in rodents. Russell et al. (1988) identify
increases in dopamine, the inhibitor of PRL synthesis, as the cause of reduced PRL
secretion. Although PRL has not been measured in pregnant mink undergoing embryo
resorption, Aulerich et al. (1985) demonstrated significant increases in dopamine
concentrations in brain tissue of reproductively impaired, post-partum mink exposed to
either Aroclor 1254 or 3HCB.
In combination with P/PR, PRL/PRLR complexing mediates mammalian gestation.
PRL regulates uterine PR in mink (Slayden and Stormshak 1993) and induces 5-fold
increases in uterine estrogen receptor affinity in rabbits (Chilton and Daniel 1987). Thus,
decreased PRc affinity may be due to PCB-induced reductions in PRL from high
concentrations normally present in mink pregnancy (Rose et al. 1986). PRL is also known
to stimulate immune function (Gala 1991). It may be the stimulus for mobilizing small
lymphocytes which secrete a soluble factor that suppresses cytotoxic T lymphocyte
generation and prevents embryo resorption (Daya et al. 1985). We are currently assessing
58
the concentrations of serum and uterine fluid PRL in CO and 31-ICB-treated pregnant mink
to determine if alterations in this hormone trigger embryo resorption.
59
IN UTERO EXPOSURE TO 2, 4, 5, 2', 4',5'-HEXACHLOROBIPHENYL (2HCB)
PRODUCES SKELETAL CHANGES IN NEONATAL MINK (MUSTELA VISON)
K.A. Patnode and L.R. Curtis
Submitted to Journal of Toxicology and Environmental Health (1995)
60
Abstract
During mink gestation when circulating endogenous estrogens are below detectable
concentrations, 2HCB produced estrogenic changes in uterine steroid receptors and retarded
embryo growth (Patnode and Curtis 1994). This pattern of embryotoxicity differed from that
which followed exposure to a coplanar PCB congener. Our objective was to determine if growth
impairments observed in day 14 mink embryos exposed to 2HCB in zitero persisted in day 1
neonates and assessed potential effects of 2HCB on estrogen-sensitive bone growth. Female mink
received intraperitoneal injections of 20 mg 2HCB/kg at the time of implantation. Day 1 neonates
were examined for external, visceral, and skeletal abnormalities and bone measurements were
performed. Weights were similar between treatment groups. Skeletal deformities were observed
in 2HCB-exposed neonates, but not in the corn oil-treated group. No differences were detected
in body length, head size, or urogenital-anal distance. Average length of rib, ulna, tibia, and fibula
bones and average diameter of humerus were significantly reduced in kits exposed to 2HCB in
utero (p<0.05). Malformations such as cleft palate and hydronephrosis were not detected and
surviving neonates recovered from growth impairments observed in day 14 embryos exposed to
2HCB in utero. Skeletal defects indicated estrogenic effects of 2HCB persisted at birth and
reflected significant changes in nonreproductive estrogen-sensitive tissues in mink.
Introduction
Polychlorinated biphenyls (PCBs) accumulate in animal tissues and constitute a
major class of environmental contaminants known to cause reproductive impairment.
61
Mink are an appropriate mammalian model for PCB-induced reproductive toxicity because
of their high sensitivity and ecological equivalence to humans as high trophic level
consumers in both aquatic and terrestrial ecosystems. Researchers have documented
effects of feeding mink PCB-contaminated fish ranging from decreased kit growth to
complete reproductive failure (Sundqvist et al. 1989). Although reproductive toxicity in
most species has been attributed to PCB congeners with high binding affinity to the aryl
hydrocarbon receptor (AhR; Safe 1994), embryotoxicity and reduced embryo growth
occur in mink exposed to 2-4 ortho-substituted PCB congeners (Kihlstrom et al. 1992;
Patnode and Curtis 1994) with low affinity for the AhR (Kafafi et al. 1993).
As regulators of uterine tissue, steroid hormones and their receptors are likely
targets reproductive toxins. 2,3,7,8-Tetrachloro-dibenzo-p-dioxin (TCDD) exposure in
immature rodents (Romkes et al. 1987; DeVito et al. 1992) and PCB treatment in
anestrous mink antagonized estrogen-induced upregulation of uterine nuclear ER (Patnode
and Curtis, 1994). In contrast, PCB mixtures (Aroclor 1242), noncoplanar congeners
(2HCB and PCB 47), and some hydroxy metabolites of noncoplanar congeners increase
uterine weight, albeit less potently than estradiol (Jansen et al. 1993; Soontornchat et al.
1994). During mink gestation when circulating endogenous estrogens are below
detectable concentrations, 2HCB treatments at the time of implantation significantly
increased estrogen receptor (ER) concentration and progesterone receptor (PR) total
receptor number within 14 days of exposure (Patnode and Curtis, 1994). These
observations suggest that noncoplanar PCBs or their metabolites exert both estrogenic and
antiestrogenic effects in mink uterine tissue.
62
In addition to regulating female reproductive tissues, steroid hormones control
growth and differentiation in developing embryos and neonates. ER has been detected in
bone cell cultures (Eriksen et al. 1988), and intestine (Prince 1994). Research indicates
that estrogens complex with ERs on bone cells to decrease longitudnal and radial growth
of the tibia, resulting in shorter and narrower bones in female rodents compared to males
(Turner et al. 1994).
The objective of our research was to determine if growth impairments observed in
day 14 mink embryos exposed to 2HCB in utero (Patnode and Curtis, 1994) persist in day
1 neonates. Since we had observed both estrogenic and antiestrogenic effects in mink
reproductive tissues following exposure to 2HCB, we examined endpoints indicative of
in utero exposure to estrogens and AhR agonists.
Methods
On March 30, the average date of implantation (Stoufflet et al. 1989), pregnant
female mink received 20 mg 2HCB/kg (n = 3), or corn oil (CO, n = 6) via ip injection.
The animals were housed individually in wire cages in open-sided sheds under natural
changes in photoperiod and temperature. They were fed approximately 125 g gestation
ration/day of a containing 20 % fat and 45% protein. Water was provided ad libitum.
On the day of whelping, kits were asphyxiated with CO2 gas. Each kit was sexed
and weighed. External examinations for gross deformities were performed on all kits.
Following standard teratology guidelines (Taylor 1986), approximately half of the kits
63
from each litter were used for visceral examinations and the other half for skeletal
examinations.
Visceral exams were conducted using the sectioning technique described by Taylor
(1986). Briefly, whole bodies were fixed in Bouin's solution until decalcification was
complete (2 weeks). We then replaced Bouin's with 70% alcohol for 1 week. We began
by examining the palate for closure and then sectioning the head into transverse slices 1-3
mm thick. Following a midline incision, orientation and position of internal organs was
assessed. We compared urogenital and genital systems with the external sex
determination. Heart and kidneys were removed, sectioned, and examined for
abnormalities. Liver and lungs were removed and examined for abnormal lobulation.
For skeletal assessments (Taylor 1986), whole bodies were fixed in 95% alcohol
for at least 1 week followed by clearing with 2% KOH for 24 hours. After removing skin
and internal organs, we stained calcified bone with 0.0025% Alizarin red S in 2% KOH.
Kits were then exposed to increasing concentrations (30, 50 and 80%) of glycerin for 24
hours each and stored in 100% glycerin with thymol crystals until examinations were
conducted. Presence of calcified bones and abnormal ossification or fusion patterns were
determined. Skull length, width, and depth; body length; tail length; and urogenital-anal
distance were measured with calipers. We then measured the length and diameter of all
long bones and the 14th rib using a dissecting microscope with an ocular micrometer.
A contingency table was constructed to compare numbers of deformed and normal
embryos. Paired comparisons were made with Fisher exact tests (Seigel, 1956). Skeletal
measurements were expressed on a per gram body weight ratio to control for differences
64
in kit body sizes. We tested for differences by treatment and sex using the analysis of
variance option in Systat (version 5.03, 1993). If the sex-treatment interaction was
significant, we examined the sex-specific differences graphically.
Results
External, visceral, and skeletal examinations identified 5 kits out of 14 from
2HCB-treated females with developmental abnormalities, while none were found in
control kits. Deformities included missing digits, vestigial digits, an extra rib, and a
missing sternum segment. Although no malformations were found in CO-exposed kits, we
were unable to detect a significant difference between the 2 treatments. It is possible that
small sample sizes limited the power of our analysis.
Average long bone and rib measurements, expressed as a per gram body weight
ratio, are presented in Table 1. Rib, ulna, tibia, and fibula bones were found to be
significantly shorter in kits exposed to 2HCB in way) (p<0.05). Diameter was
significantly reduced only in the humerus. Both diameter and length changes in humerus
bones were sex-dependent. In female kits from 2HCB-treated females, humerus length
(Fig. 1) and diameter (Fig. 2) were reduced. However, 2HCB irz utero exposure had no
effect on length and may have slightly increased humerus diameter in male kits. No
difference in neonatal weight was observed between treatment groups.
65
Table 3-1. Bone length (cm) and diameter (cm) on body weight (g) basis (mean ± SE) for
day 1 mink of females exposed to corn oil (control) or 2,4,5,2',4',5'-hexachlorobiphenyl
(2HCB) ip at time of implantation.
BONE
rib
humerus
radius
ulna
femur
tibia
fibula
LENGTH/BODY WEIGHT
RATIO
Control (n=6)
2HCB (n=14)
1.007 ± 0.048
0.868 ± 0.042 a
1.030 ± 0.040
0.941 ± 0.036"
0.873 ± 0.025
0.825 ± 0.022
0.755 ± 0.025
0.676 ± 0.022a
0.742 ± 0.036
0.706 ± 0.031
0.857 ± 0.024
0.711 ± 0.022a
0.750 ± 0.025
0.649 ± 0.022a
DIAMETER/BODY WEIGHT
RATIO
Control (n=6)
2HCB (n=14)
0.095 ± 0.011
0.096 ± 0.009
0.207 ± 0.009
0.180 ± 0.008ab
0.131 ± 0.006
0.137 ± 0.005
0.114 ± 0.007
0.121 ± 0.006
0.157 ± 0.009
0.143 ± 0.008
0.147 ± .0008
0.143 ± 0.007
0.063 ± 0.006
0.072 ± 0.005
a signicantly different from control at p_.5_0.05
treatment-sex interaction significant (see Figures 1 and 2)
males
females
1.3
1.2
T
1.1
4
12
1.0
2
0.9
2
0.8
0.7
CO
2HCB
CO
2HCB
Figure 3-1. Humerus length by sex for day 1 neonatal mink from females treated with corn oil (CO) or
2,4,5,2',4"5'-hexachlorobiphenyl (2HCB) ip at time of implantation.
males
females
0.26
T
0.24
4
12
0.22
0.20
0.18
2
0.16
0.14
2
0.12
CO
2HCB
CO
2HCB
Figure 3-2. Humerus diameter by sex for day l neonatal mink from females treated with corn oil (CO) or
2,4,5,2',4',5'-hexachlorobiphenyl (2HCB) ip at time of implantation.
68
Discussion
The objective of this study was to determine if 2HCB-induced growth impairments
observed in day 14 mink embryos (Patnode and Curtis 1994) persisted to produce
abormalities in neonates. Despite reduced weights in day 14 embryos, day 1 neonates did
not weigh significantly less than control kits. Similarly, Aulerich et al. (1985) found no
weight reduction in neonates of female mink chronically exposed to 2HCB in their feed.
Differences in body length and head size between control and 2HCB exposed embryos no
longer existed in day 1 neonatal mink. These data indicate that between day 14 of
gestation and birth, surviving embryos are able to compensate for previous growth
impairment. This may result from a growth advantage to surviving embryos when litter
size is reduced by embryo resorption.
In contrast to 100 % embryo resorption produced by a coplanar PCB (3, 3', 4, 4',
5, 5'-hexachlorobiphenyl), 2HCB exposure resulted in only 24% embryonic loss
(Patnode and Curtis 1994). Given these observed potencies and the relative binding
affinities of these 2 congeners to the AhR (Kafafi et al. 1993), it is possible that
embryotoxicity of both PCBs could occur via mechanisms similar to TCDD. If 2HCB is
acting through the AhR, malformations such as cleft palate and hydronephrosis should be
present in surviving embryos. However, we did not observe either of these malformations
in day 1 neonatal mink at a dose which causes embryo resorption. A similar pattern of
embryotoxicity without cleft palate or hydronephrosis in late term fetuses was observed in
CD-1 mice exposed to 2HCB in utero (Marks and Staples 1980). Collectively, these
results suggest that the mechanism of 2HCB embryotoxicity is independent of the AhR.
69
Concurrent with embryo resorption, 2HCB exerted estrogenic effects on uterine
ER and PR (Patnode and Curtis 1994). Both prenatal and neonatal exposure of mice to
estrogens results in shorter femurs without changes in body weight or reproductive tract
development indicating that bone development is highly sensitive to estrogenic substances
(Migliaccio et al. 1992). We found that 2HCB exposure in Wen) resulted in shorter ulna,
tibia, fibula, and rib bones independent of kit sex. We also observed a female-specific
reduction in humerus length and diameter. All of the gross deformities we observed were
also skeletal alterations. These data indicate that estrogenic effects of 2HCB are not
limited to reproductive tissue, but include developing bone and potentially other estrogensensitive tissues. Similar effects on developing human embryos may explain the decreased
height of first-born children of PCB-exposed Yu-Cheng mothers (Guo et al. 1994).
The mechanism of 2HCB's estrogenicity is undetermined. Li et al. (1994)
hypothesize that estrogenic effects of 2HCB in female rats is attributable to a metabolite
based on observations that efficacy increases with multiple doses and with doses that
maximally induce pentoxyresorufin-O-deethylase activity. This phenomenon is even more
likely to occur in mink for several reasons and may account for the high sensitivity of
mink. The mustelids, including the mink, are more closely related to the dog which
possesses a P450 isozyme with a much greater capacity than the rodent isozyme for
converting 2HCB to multiple metabolites (Duignan et al. 1988). In addition, both penta­
and hexa-PCB metabolites occur in mink experimentally exposed to a 2-4 ortho­
substituted PCB fraction (Bergman et al. 1992), as well as wild mink (Bergman et al.
1994) which are known to have high 2HCB tissue concentrations (Bergman et al. 1994).
70
Alternatively, the initial dose of 2HCB could be necessary to occupy binding sites
of P450 isozymes and induce ER concentrations for interaction with a higher circulating
concentration of 2HCB molecules. Binding of PCB congeners to the ER is thought to be
limited to hydroxy metabolites due to presence of a phenolic ring (Korach et al. 1988).
However, 2HCB has been shown to bind to a steroid binding protein in the rodent lung
(Gillner et al. 1988). In vitro binding assays with parent and metabolites extracted from
mink tissues are necessary to determine the mechanism of 2HCB estrogenicity in
reproductive and other estrogen-sensitive tissues.
71
CONCLUSIONS
Embryo resorption after implantation without reductions in number of implantation
sites confirms evidence present at parturition (Aulerich et at, 1985; Backlin and Bergman,
1992) that PCB-induced embryotoxicity occurs following implantation. Histological
evaluation indicates that the resorption process is not microscopically detectable on day 8,
but has progressed to a highly necrotic stage by day 10. Given the extent of necrosis in
day 10 implantation sites of mink, it is likely that immune cells have infiltrated day 8
decidua.
All PCB treatments in these experiments significantly decreased cytosolic
progesterone receptor (PR) affinity for ligand. However, in vitro assay data indicate that
3HCB-induced changes in PR affinity are not due to direct binding of the parent PCB to
uterine PR. Metabolites occur in ranch mink exposed to PCB fractions (Bergman et al.
1992), as well as in wild mink exposed to environmental mixtures of PCBs (Bergman et al.
1994).
Exogenous progesterone (P) treatment was ineffective at preventing embryo
resorption. PR was still capable of binding P and producing a statistically significant
response. The magnitude of the increase in prolactin receptor (PRLR) that we observed
was comparable of that produced by a 10-fold decrease in P in mink early in gestation.
Similar results were seen in P treatment of pregnant rats exposed to an antiprogestational
synthetic hormone where P reversed anti-P effects with the exception of embryo
72
resorption (Kabir et al. 1992). These observations indicate that once the resorption
process is triggered, P alone is ineffective at halting its progress.
The observation that the resorption process must be initiated prior to significant
alterations in PR affinity combined with P/PR responsiveness suggest that alterations in
both PR and maternal immune cells may be effects of an earlier 3HCB-induced change.
Prolactin (PRL), the other endocrine mediator of mink gestation, is a susceptible target.
Exposure to 3HCB (DeKrey et al. 1994) reduces circulating PRL concentration in
rodents. Although PRL has not been measured in pregnant mink undergoing embryo
resorption, Aulerich et al. (1985) demonstrated significant increases in dopamine, an
inhibitor of PRL release, in brain tissue of reproductively impaired, post-partum mink
exposed to either Aroclor 1254 or 3HCB.
In combination with P/PR, PRL/PRLR complexing mediates mammalian gestation.
PRL regulates uterine PR in mink (Slayden and Stormshak 1993) and induces 5-fold
increases in uterine estrogen receptor affinity in rabbits (Chilton and Daniel 1987). Thus,
decreased PRc affinity may be due to PCB-induced reductions in PRL from high
concentrations normally present in mink pregnancy (Rose et al. 1986). PRL is also known
to stimulate immune function (Gala 1991). It may be the stimulus for mobilizing small
lymphocytes which secrete a soluble factor that suppresses cytotoxic T lymphocyte
generation and prevents embryo resorption (Daya et al. 1985).
Induction of P450 1A1 is the best understood action of AhR activation and
correlation with P450 induction is taken to be indicative of AhR-mediated toxicosis (Safe,
1990). Sensitivity to inducers of EROD activity appears to be greatest in kits (30-fold),
73
moderate in juveniles (13-fold), and minimal in pregnant and postpartum adults (2-4-fold)
(Brunstrom et al., 1991; this study). With less than a 4-fold EROD induction, 3HCB
exposure caused complete reproductive failure. 2HCB caused no P450 or EROD
induction, yet increased embryo resorption and retarded embryo growth. Increased
embryotoxicity without EROD induction occurred in pregnant mink fed a 2-4 ortho PCB
fraction (Kihlstrom et al., 1992; Brunstrom 1992). Collectively, these results indicate that
reproductive dysfunction in mink is far more sensitive than P4501A1 induction.
This research was the first to demonstrate 2HCB-induced reproductive failure in
mink. Embryos were resorbed and growth of surviving embryos was significantly
impaired. Characteristic AhR malformations such as cleft palate and hydronephrosis were
not present in neonatal mink at a 2HCB dose which causes embryo resorption. A PCB
fraction of 2-4 ortho congeners was also embryotoxic in mink without causing
malformations (KihIstrom et al. 1992). Collectively, these results suggest that the
mechanism of 2HCB embryotoxicity is independent of the AhR and argue for the inclusion
of all PCB congeners in reproductive risk assessments.
Steroid receptor responses to PCB exposure vary with reproductive status in mink.
In anestrous, juvenile mink when uterine ER concentrations are high, 2HCB antagonized
E's effects on ER. However, during mink gestaton when E concentrations are negligible
and uterine ER and PR concentrations are declining, ER and PR Rt were increased by
2HCB. In vitro binding assays indicate that hydroxy metabolites of some PCBs can bind
to mouse uterine ER (Korach et al., 1988). Although their affinities are at least 40-fold
lower than estradio1-1713 (Korach et al., 1988), their occupancy of uterine ER may reach
74
effective proportions during gestation in mink when endogenous estrogens are suppressed
(Stoufflet et al., 1989). Thus, 2HCB is antiestrogenic when serum estrogen
concentrations are elevated, yet is estrogenic during gestation.
Despite reduced weights in day 14 embryos, day 1 neonates did not weigh
significantly less than control kits. Similarly, Aulerich et al. (1985) found no weight
reduction in neonates of female mink chronically exposed to 2HCB in their feed.
Differences in body length and head size between control and 2HCB-exposed embryos no
longer existed in neonatal mink. These data indicate that between day 14 of gestation and
birth, surviving embryos compensate for previous growth impairment.
2HCB exposure in utero resulted in specific skeletal growth impairments. Ulna,
tibia, fibula, and rib bones were shorter in 2HCB-exposed neonates independent of sex.
We also observed a female-specific reduction in humerus length and diameter. Both
prenatal and neonatal exposure of mice to estrogens results in shorter femurs without
changes in body weight or reproductive tract development indicating that bone
development is highly sensitive to estrogenic substances (Migliaccio et al. 1992). These
data indicate that estrogenic effects of 2HCB are not limited to reproductive tissue, but
include developing bone and potentially other estrogen-sensitive tissues.
The mechanism of 2HCB's estrogenicity is undetermined. Li et al. (1994)
hypothesize that estrogenic effects of 2HCB in female rats is attributable to a metabolite
based on observations that efficacy increases with multiple doses and with doses that
maximally induce pentoxyresorufin-O-deethylase activity. Alternatively, the initial dose of
2HCB could be necessary to occupy binding sites of P450 isozymes and induce ER
75
concentrations for interaction with a higher circulating concentration of 2HCB molecules.
Binding of PCB congeners to the ER is thought to be limited to hydroxy metabolites due
to presence of a phenolic ring (Korach et al. 1988). However, 2HCB has been shown to
bind to a steroid binding protein in the rodent lung (Gillner et al. 1988).
In conclusion, this research has identified critical timepoints in PCB-induced
embryo resorption in mink. Changes in steroid receptors occur, but do not appear to the
proximate cause of embryotoxicity. Both nonortho and diorth congeners are toxic to mink
embryos and diortho congeners can exert both estrogenic and antiestrogenic effects.
76
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89
APPENDICES
90
APPENDIX A
Embryo Resorption and Growth Impairment
in Mouse Strains with Normal and Reduced
Aryl Hydrocarbon Receptor Concentrations
K.A. Patnode and L.R. Curtis
Submitted to Fundamental and Applied Toxicology (1995)
91
Summary
Both coplanar and noncoplanar polychlorinated biphenyl (PCB) congeners induce
embryo resorption in mink (Patnode and Curtis 1994). This observation suggests a
common mechanism of action despite known differences in congener binding affinity to
the aryl hydrocarbon receptor (AhR) (Kafafi et al. 1993). To distinguish between AhR­
dependent and AhR-independent mechanisms, we exposed C57 Black (AhR+) mice to 80
mg 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB)/kg (n=10) or corn oil (CO; n=10) via ip
injection at the time of implantation (gd 5). We also treated B6N:D2N (Ahd)
N13F13:B6JN28F28F3 (AhR) mice with 80 mg 3,3',4,4',5,5'-hexachlorobiphenyl
(3HCB)/kg (n=10), 300 mg 2,2',4,4',5,5'-hexachlorobiphenyl (2HCB) (n=10), or corn oil
(CO; n=10) using the same protocol. After 8 days (gd 13), we asphyxiated mice with
CO2, removed uteri, and dissected implantation sites. Resorption was documented
macroscopically and surviving embryos were weighed. Fisher exact probability test was
used to analyze the numbers of resorptions by treatment. Embryo weights were compared
across treatments and strains using analysis of variance.
The percent of litter resorbed was similar in CO-treated mice of both strains
indicating no strain-specific differences in spontaneous resorption rates (Fig Al.).
Treatment of AhR+ mice with 3HCB did not increase the proportion of resorptions. In
contrast, AhR mice exposed to either 3HCB or 2HCB had increased resorptions
compared to CO-treated animals. This finding suggests that AhR does not mediate
embryotoxicity and may, in fact, be protective of embryos. Binding to AhR in non-target
tissues may reduce the amount of PCB reaching the target tissue.
50
AhR+
AhR­
40
b
30
20
10
ii
CO
3HCB
CO
3HCB 2HCB
Figure A-1. Percent of litter resorbed in female mice with normal (AhR+) and reduced (AhR-) aryl hydrocarbon receptors
exposed to 80 mg 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB)/kg, 300 mg 2,2',4,4',5,5'-hexachlorobiphenyl (2HCB)/kg, or
corn oil (CO) via ip injection on gestation day 5. Dissimilar letters denote significant differences (p<0.05).
93
Embryo weights were similar for CO-treated mice of both strains indicating similar
rates of development (Fig. A2.). 3HCB treatment of AhR+ mice significantly reduced
embryo weights. In AhR mice, 3HCB-exposed embryo weights were between those of
CO-treated AhR and 3HCB-treated AhR+ and did not differ significantly from either
treatment-strain group. The order of treatments from CO to 3HCB-treated AhR to
3HCB-treated AhR+ data suggest that embryo growth impairment may be a AhR­
mediated effect. The lack of impairment in 2HCB exposed mice may reflect an insufficient
dose in a strain deficient in AhR.
The combination of AhR-mediated growth impairment and AhR-independent
embryo resorption are only compatible if the target tissues differ for these two
reproductive effects. The embryo resorption process may be triggered within the
hypothalamus. Jones et al. (1987) observed decreased serum prolactin concentrations and
attributed these to changes in brain dopamine levels. Aulerich et al. (1985) documented
decreased dopamine concentrations in mink that experienced PCB-induced embryo
resorption.
In contrast, growth impairment may be due to a direct action of PCBs on the
uterus or embryos. PCBs and their metabolites concentrate in the uterus and embryos of
pregnant mice (Darnerud et al. 1986, Lucier et al. 1978). Decreases in body weight in
children exposed to PCBs in utero are associated with decreased capacity of epidermal
growth factor to stimulate autophosphorylation of placental growth factor receptor
(Sunahara et al. 1987). Glucocorticoid receptor binding capacity in fetal livers is also
300
AhR+
AhR­
250
a
20
200
ab T
47
150
ab_
100
be T
33
T
24
39
50
0
CO
3HCB
CO
3HCB
2HCB
Figure A-2. Embryo weights from female mice with normal (AhR+) and reduced (AhR-) aryl hydrocarbon receptors exposed to
80 mg 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB)/kg, 300 mg 2,2',4,4',5,5'-hexachlorobiphenyl (2HCB)/kg, or corn oil (CO)
via ip injection on gestation day 5. Dissimilar letters denote significant differences (p<0.05).
95
decreased by dioxin-like contaminants (Ryan et al. 1989) which may reduce nutrients
available for embryo growth.
These results must be interpreted with caution as timed mating of these mouse
strains was problematic. We observed numerous matings which failed to produce a
vaginal plug. In addition, these mice had a low conception rate. The staff veterinarian at
a laboratory animal supplier indicated that C57 Black males experience poor fertility and
in commercial operations only proven breeders are used for timed mating studies. These
complications reduced our sample sizes to 6 and 5 for CO- and 3HCB-treated animals of
each strain and to 4 for 2HCB-treated AhR mice. Due to the difficulties in accurately
documenting gestation day 1 using vaginal plugs, it is possible that not all of these
embryos are gestation day 13. We recommend repeating this experiment to increase the
sample size of females and embryos accurately aged with the vaginal plug technique.
96
Literature Cited
Aulerich, R.J., Olson, B.A., Ringer, R.K., Bursian, S.J. and Breslin, W.J. 1985.
Toxicological manifestations of 2,4,5,2',2',4',5'-hexachlorobiphenyl, 2,3,6,2',3',6'­
hexachlorobiphenyl, and 3,4,5,31,4',5'-hexachlorobiphenyl and Aroclor 1254 in
mink. J. Toxicol. Environ. Health 15:63.
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and Sperber, G.O. 1986. 3,314,4'-tetrachloro[14C]biphenyl in pregnant
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alterations in prolactin, corticosterone, and thyroid hormone levels and downregulation of prolactin receptor activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin.
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Kafafi, S.A., Afeefy, H.Y., Ali, A.H., Said, H.K. and Kafafi, A.G. 1993. Binding of
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alteration of uterine progesterone and estrogen receptors coincides with
embryotoxicity in mink (Mustela vison). Toxicol. Appl. Pharmacol. 127:9.
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97
APPENDIX B
Nonesterified Fatty Acid Concentrations
in Plasma and Uterine Tissue of
Pregnant and Nonpregnant Female Mink
Following Exposure to 3,3',4,4',5,5'-Hexachlorobiphenyl
K.A. Patnode and L.R. Curtis
Submitted to Lipids (1995)
98
Summary
Both coplanar and noncoplanar polychlorinated biphenyl (PCB) congeners induce
embryo resorption in mink (Patnode and Curtis 1994). In addition to potential direct
actions of PCBs on hormones and their receptors, PCBs may act indirectly via their
influence on other endogenous substances. Aroclor 1254 increases heptic membrane
concentration of arachidonic acid (AA) (Borlakoglu et al. 1990) via induction of
microsomal fatty acid desaturases (Borlakoglu et al. 1991) in rats and pigeons. Agents
which block AA release from the membrane or subsequent steps in the cascade leading to
prostaglandin synthesis reduce the toxicity of 2,3,7,8-tetrachloro-dibenzo-p-dioxin
(TCDD) in rat livers (Al-Bayati and Stohs 1991) and chick hearts (Rifkind et al. 1984).
AA and other nonesterified fatty acids (NEFAs) are modulators of numerous
physiological processes and their liver concentrations are reduced during gestation (Feuer
1979). They inhibit progesterone receptor (PR), glucocorticoid receptor (GR), estrogen
receptor (ER) binding by direct binding at .a site distinct from the hormone and heat shock
protein binding sites (Kato et al. 1987, Kato 1989, Vallette et al. 1991). Low
concentrations of saturated NEFAs also suppress lymphocyte proliferation (Buttke 1984).
Increasing desaturation of NEFAs reverses the inhibition of DNA synthesis and leads to
increased B and T lymphocyte populations.
PCB exposure causes significant weight loss and mobilization of fat stores in mink
(Gillette et al. 1987). In anestrous mink, adipose lipoprotein lipase activity increases and
circulating triglyceride levels decrease (Brewster and Matsumura 1989). These PCBinduced changes in lipid metabolism could produce changes in NEFAs that may be the
99
proximate causes of embryo resorption in mink. Our objective was to determine if
circulating NEFAs or uterine tissue NEFAs in pregnant mink were altered by exposure to
3,3 ',4,4' ,5,5 ' -hexachlorobiphenyl.
On March 30, 1994, we injected 16 mated mink with 0.4 mg/kg 3HCB or corn oil
ip. After 10 d, a 10 ml blood sample was collected via cardiac puncture and sera obtained
by centrifugation. The uterus was removed and homogenized. Lipids were extracted
from 5 ml serum and whole uterine homogenate after Bligh and Dyer (1959). Extracts
were desiccated under nitrogen, weighed, dissolved in benzene, and stored at -20°C.
Methyl esters were prepared by methanolysis (Selivonchick et al. 1977). NEFA
composition was determined with a Varian aerograph 1200 series gas chromatograph. A
183-cm stainless steel column packed with GP 10% SP-2330 on 100/120 mesh
Chromosorb W AW was used. Column temperatures were 160°C for the first 8 min.,
increased to 190°C in steps of 4°C per min., and then held at 190°C for 20 min. The flow
rate of nitrogen was 30 ml/min. Injection port and detector temperatures were 250°C.
Fatty acid esters were identified by comparison to standard mixtures of commercially
available known methyl mixtures and quantified by a Hewlett-Packard 3380 integrator.
Analysis of variance was used to detect differences in the relative percentages of
NEFAs. Three mated females in each treatment group were not pregnant and were
analyzed as separate treatment groups. Three plasma and six uterine samples were
damaged and were not suitable for analysis.
NEFA profiles for plasma and uterine tissue are presented in Tables B1 and B2,
respectively. We did not detect significant differences between pregnant and nonpregnant
100
control animals. 3HCB treatment did not significantly alter plasma or uterine tissue NEFA
concentrations in either pregnant or nonpregnant mink. Caution should be used in
interpreting these results because small sample sizes have severely reduced the power of
our statistical analysis. In light of the known changes in lipid metabolism in mink, this
experiment should be repeated before concluding that NEFAs are not a causative factor in
3HCB-induced embryo resorption in mink.
101
Table B-1. Nonesterified fatty acid (NEFA) concentrations in plasma of pregnant and
nonpregnant female mink treated with 0.4 mg 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB)
/kg or corn oil (CO) via ip injection at the putative time of implantation.
NEFA
14:0
16:0
16:10)7
18:0
18:10)9
18:1co7
18:2(1)6
18:3co6
18:3co3
20:3(06
20:4(06
20:5(o3
22:40)6
22:5co3
22:6(0
Non-Pregnant
CO (n=2)
3HCB (n=3)
0.561±0.124
1.002±0.250
22.958±3.360
23.302±0.809
1.407±0.124
1.760±0.239
14.846±1.692
15.378±0.562
11.108±0.773
12.619±0.905
1.695±1.198
2.506±1.037
22.803±0.310
22.373±0.839
0.352±0.056
0.113±0.092
0.126±0.089
0.877±0.065
0.454±0.191
14.378±2.056
13.535±0.522
4.614±0.980
2.723±0.868
0.135±0.096
0.896±0.185
0.223±0.182
3.244±1.149
4.011±1.647
Pregnant
CO (n=3)
3HCB (n=5)
0.705±0.107
1.062±0.382
20.642±0.363
22.514±2.669
1.581±0.127
1.946±0.612
12.684±0.580
15.947±1.892
12.649±0.619
11.936±1.184
3.569±0.111
3.063±0.747
23.710±0.702
17.689±1.992
0.436±0.052
2.270±1.767
0.313±0.050
0.155±0.138
0.902-1-0.017
1.274±0.344
13.744±0.821
12.081±2.296
4.764±0.306
4.228±0.851
0.230±0.096
0.435±0.312
0.808±0.046
0.794±0.178
3.264±0.004
4.606±1.000
102
Table B-2. Nonesterified fatty acid (NEFA) concentrations in uterine tissue of pregnant
and nonpregnant female mink treated with 0.4 mg 3,3',4,4',5,5'-hexachlorobiphenyl
(3HCB) /kg or corn oil (CO) via ip injection at the putative time of implantation.
NEFA
14:0
16:0
16:10)7
18:0
18:1(1)9
18:16)7
18:2(1)6
18:30)6
18:30)3
20:36)6
20:46)6
20:5(03
22:46)6
22:50
22:6(1)3
Non-Pregnant
CO (n=2)
3HCB (n=3)
3.069±0.014
3.749±0.463
18.531 ±0.166
19.097±1.535
5.966±0.013
7.724±0.687
7.812±0.089
7.382±1.294
30.611±0.951
33 236±0. 903
2.737±1.935
2.272±1.855
16.564±0.240
15.611±0.438
0.605±0.022
0.161±0.079
2.770±1.453
0.726±0.020
0.564±0.017
0.426±0.045
5.585±0.117
4.819±0.703
0.926±0.044
0.797±0.079
0.861±0.091
0.493±0.218
0.926±0.006
0.858±0.041
2.473±.0.187
2.651±0.227
.
Pregnant
CO (n=4)
3HCB (n=1)
2.563±0.211
1.650±0.000
19.944±0.576
19.322±0.000
5.976±0.701
3.842±0.000
8.312±0.489
13.945±0.000
30.772±1.745
20.748±0.000
3.589±1.185
8.870±0.000
15.884±0.451
12.517±0.000
0.247±0.075
0.049±.0.000
0.605±0.062
0.225±0.000
0.607±0.063
0.781±0.000
6.176±0.732
11.393±0.000
1.192±0.095
0.967±0.000
0.927±0.093
1.338±0.000
0.866±0.055
1.099±0.000
2.339±0.096
3.254±0.000
103
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