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This is to certify that the
thesis entitled
CARBON TETRACHLORIDE EFFECTS ON FERTILIZATION
AND PREIMPLANTATION DEVELOPMENT IN MICE

presented by

GARY PETER HAMLIN

has been accepted towards fulfillment
of the requirements for

 

M . S . degree in Zoology

 

[yfiw MW

Major professor

Date June 18. 1991

 

0.7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

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LIBRARY
Michigan State

l University
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TO AVOID FINES return on or before date due.

DATE DUE DATE DUE ‘ DATE DUE

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MSU Is An Affirmative ActioNEqual Opportunity Institution
cmmmh.‘

 

 

 

 

CARBON TETRACHLORIDE EFFECTS ON FERTILIZATION AND

PREIMPLANTATION DEVELOPMENT IN MICE

BY

Gary Peter Hamlin

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Zoology

1991

ABSTRACT

CARBON TETRACHLORIDE EFFECTS ON FERTILIZATION AND
PREIMPLANTATION DEVELOPMENT IN MICE

BY

Gary Peter Hamlin

Carbon tetrachloride (CCl4) is a: ubiquitous
environmental contaminant. Estimates state that as many as
10 million people are occupationally exposed to CC14 every
day. There is a prodigious amount of information on the
hepatotoxicity and bioactivation of CCl4, yet little datum
exist on the reproductive hazard.

In this study female BGDZFl mice were gavaged with 1/10

L050 and 1/100 L050 of CC14 in corn oil once a day from day

1 through 5 of pregnancy (day 1 - vaginal plug). No
biologically significant effect on any maternal or neonatal
parameters studied throughout gestation and twenty-two days
postpartum were seen.

Further studies investigated the effect of CC14 on in
vitro fertilization. Emulphor was found to be too toxic for
use as a vehicle. Concentrations of CC14 in 1% t-butanol of
1 - 10 mM caused a significant decrease in fertilization and

a significant increase in abnormal ova forms.

This thesis is gratefully dedicated to Dr. Linda Crane
for standing by through thick and thin, for her
companionship, care, love and ever improving fishing skills.

Special thanks to my parents and family for maintaining
the safety line to home.

iii

Acknowledgements

I wish to thank my committee members Drs. L.G. Clemens
and L.J. Fischer as well as my committee chairman and major
advisor Dr. W.R. Dukelow.

Special thanks to all members of the Endocrine Research
Center for their friendship and guidance.

iv

TABLE OF CONTENTS

LI ST OF TABLES I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O Vii
LIST OF FIGURES O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 Vi
INTRODUCTION 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O I O O 1

LITERATUREREVIEW OOOOOOOOOOOOOO 0000......00.000.000.000...5

Overview of Carbon Tetrachloride Toxicity............ 5
Bioactivation and Cytotoxic Mechanism of

Carbon Tetrachloride.................................12
Essential Aspects of Early Mouse Pregnancy...........24
Reproductive Toxicology..............................26
In Vivo and In Vitro Systems for Reproductive
Toxicity.............................................28
Reproductive Effects of CC14.........................35

MATERIALS ANDMETHODS.00....O..0.00......0.00.00.00.00000048

Animals.‘OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.00.48

In Vivo Experiments..................................48
Mating procedure...................................48
CCl4 solutions.....................................49
Experimental protocol..............................49

Gavage protocol..................................49
Maternal parameters..............................50
Neonatal paramters...............................50
Statistical analysis.............................51

In Vitro Experiments.................................51
Culture medium.....................................51
Superovulation.....................................52
Emulphor...........................................52
CC14...............................................53
Gamete recovery and IVF............................53
Statistical analysis...............................54

RESULTS..00..OOOOOOOOOOOOOOOOOOOOOO00......0.0.00.0000000055

In Vivo Experiments 0 O O O O O O O O O O O O O O O O O O O O O I O O O O O O O O O O O 55
Maternal parameters 0 O 0000000000000 O O O O O O O 0 O O O O O O O O O 55
Neonatal parameters................................57

In Vitro Experiments..... ...... ..... ....... ..........59
EmulphorOOOOOOOOOOOOOOOOO...00......0......00......59

CC14000OOOOOOOOOOOOOOOOOOOOOO0.0.0.0.....000000000059

DISCUSSIONOOOOOOOO00.000.00.00...O...0.00.00.00.00...0.0.064
SWYAND CONCLUSIONSOOOOOOOOOOOOOOOOOOOOO0.00.00.00.00072

BIBLIOGRAPHYOOOOOOOOOOOOOOOOIOOOOOO000......0.0.0.0000000074

vi

Table

LIST OF TABLES

Page

Physiochemical characteristics of CCl4 ................ 6

Components of two culture media (BMOC and
BMOC-B) used for IVFOOOOOOOOOOOOOOOOOOOOO0.0.0.00.0..52

Maternal body weight of BéDZFl mice during
pregnancy after oral exposure to CC14 on
days 1 - 5 of pregnancy....................... ..... ..56

Maternal body, liver and kidney weights of
BGDZFI mice on days 22 postpartum after oral
exposure to CCl4 on day 1 - 5 of pregnancy...... ..... 56

Litter size and percent of males in litters
exposed to CC14 by gavage to the dam on
days 1 - 5 of pregnancy ........ .......... ............ 57

Mean pup weight of BéDZFl mice on days 1 -
22 postpartum after exposure to CC14 by
gavage to the dam on days 1 - 5 of pregnancy ...... ...58

Crown-rump length of pups on days 1 - 22
postpartum after exposure to CCl4 by gavage
to the dam on days 1 - 5 of pregnancy.......... ..... 58

Percentage of pups exposed to 1/100 and 1/10

LD50 CC14 by gavage to the dam on days 1 - 5

of pregnancy showing incisor eruption on

day 11 and eye opening on day 14. ..... .. .......... ...59

vii

Table Page

9. Effect of emulphor on mouse in vitro

fertilization.............................. .......... 60

10. Effects of CCl4 (mM) and t-butanol (v/v) on
mouse in vitro fertilization.........................60

11. Effect of CC14 on ovum morphology in mouse in
Vitro fertilizationOOOOOOOOO0.00.0000...00....0.0.0.062

viii

LIST OF FIGURES

Figure Page

1. Appearance of normal two cell embryos (a),
and abnormal forms seen at CC14 concentrations
abovelmn(b).0.00..OOOOOOOOOOOOOOOOOOOOOOO0.00.0.0.63

ix

INTRODUCTION

Carbon tetrachloride is commonly referred to by its
chemical structure CC14. Technical and chemically pure
grades of this colorless, highly volatile, dense liquid are
produced by six companies in America. CCl4 is a halogenated
hydrocarbon of low molecular weight and is included in the
group of environmental contaminants referred to as volatile
organic compounds (VOC’s).

CC14 has been widely used as an industrial solvent, a
component of fire extinguisher solutions and a fumigant,
however the major use of CC14 is in the manufacture of
fluorocarbons. In 1980 it was estimated that 3.22 x 108 kg
of CC14 were produced in the United States and only a
moderate 1% decline in CCl4 production was expected during
the following decade (EPA, 1987).

CC14 has been detected in ground and municipal water,
rivers and oceans, aquatic organisms, soil, the atmosphere,
human breath and human tissue. A recent study in Italy
(Gilli et al., 1990) and a TEAM (Total Exposure Assessment
Methodology) study conducted in North Carolina, New Jersey
and North Dakota (Wallace et a1. , 1987) discovered that
indoor concentrations of CC14 were greater than outdoor

concentrations.

2

Average adult exposure to CC14 has been estimated at 26
ug/day (EPA, 1987). Absorption of CCl4 occurs rapidly across
the intestinal mucosa and the epithelial lining of the lung
and more slowly through the skin. The major route of
excretion of both oral and inhaled doses is exhalation in
the breath. Estimates of the amount of CC14 exhaled range
from 30 - 60%. The major site of metabolism of CC14 is in
the liver where reductive dehalogenation, catalyzed by
cytochrome P450 leads to the production of electrophilic
chlorine (CL") and the trichloromethyl free radical
('CC13). Ahad et al. (1987) detected formation of free
radicals after CCl4 exposure in liver, kidney, heart, lung,
testis, brain. and. blood. Cytochrome P450 catalyzed
bioactivation of CC14 results in cell death, and has been
extensively studied but the exact mechanism is still
unclear.

Evidence for a genotoxic or mutagenic action of CC14
has been largely negative, although CC14 adducts of DNA have
been reported in mammalian cell culture systems and positive
mutagen tests have been reported with yeast (International
Agency for Research on Cancer (IARC), 1987a). CC14 has been
classified as a class 2-B mutagen with a carcinogenic effect
verified in three animal species (rat, mouse, hamster, IARC,
1987b).

The possibility of accidental exposure to CCl4 has been
dramatically demonstrated (Deng et al., 1987) and results in

acute and often severe responses. However it is the presence

of the embryo, susceptible to low exposure rates of
industrial chemicals and environmental contaminants, that
necessitates the assessment of reproductive hazards. Recent
reviews (Iannaccone et al., 1987; Spielmann et al., 1989)
suggest that possible teratogenic, as well as embryotoxic
and embryolethal effects of chemicals on preimplantation
embryos need investigation.

Assessments of reproductive effects of CCl4 in the past
have tended to use inappropriate exposure routes and/or very
high doses resulting in maternal toxicity and conflicting
results. Little if any emphasis has been placed on
determining the effects of exposure during the
preimplantation period. Current evidence suggests that CC14
at high doses is a significant embryolethal threat and an
embryotoxic threat at lower doses. The male reproductive
system appears to be more susceptible to CC14 toxicity than
the female reproductive system.

This study tests the effects of orally ingested
(gavage) CC14 at two dose levels during the preimplantation
period in the mouse, The doses of 1/100 and 1/10 of the LD50
are given in corn oil once a day on days 1 - 5 of pregnancy
to female mice fasted for 2 hours. Corn oil is the
traditional and most commonly used vehicle for delivery of
water insoluble compounds and has been chosen in this study,
although some recent reports suggest that emulphor may be
the vehicle of choice for oral administration of CC14 (Kim

et al., 1990a, 1990b). These authors reported that corn oil

modified the pharmacokinetics of absorption of CCl4
resulting in a moderation of the hepatotoxic action of CCl4,
however Bull et a1. (1988) reported an enhancement in the
hepatotoxic effect of a closely related chemical, chloroform
(CHC13) when orally administered in corn oil as oppposed to
emulphor. It appears that more work is required to determine
which vehicle is most appropriate for oral delivery of VOCs.

Additional experiments are proposed to study the
effects of CC14 on the mouse in vitro fertilization (IVF)
system. Initially the toxicity of emulphor in the IVF system
is assessed. Emulphor is capable of maintaining constant
concentrations of VOCs in solution over long periods of
time, however it has been reported to be too toxic to use as
a vehicle in suspended hepatocyte cultures (O'Hara et al.,
1989). Finally, the effect of CC14 on IVF will be determined
by recording the percent of two cell embryos obtained in the

presence of different concentrations of CC14.

LITERATURE REVIEW

overview of Carbon Tetrachloride Toxicity

Carbon tetrachloride, commonly abbreviated as CC14 is a
colorless, volatile organic compound (VOC) of molecular
weight 153.8 daltons (physiochemical characteristics in
Table 1). The Chemical Abstract series has registered CC14
(registration number 56-23—5) as tetrachloromethane and the
International Union of Pure and Applied Chemistry (IUPAC)
refers to C£l4 as 141,1,1 tetrachloromethane. Common names
for CC14 include carbona, carbon chloride,
tetrachlorocarbon, perchloromethane and methane
tetrachloride. The International Agency for Research on
Cancer (IARC, 1987b) classified CCl4 as a class 23
carcinogen stating that evidence for carcinogenicity in
humans is inadequate although some studies showed an
increase in carcinogenesis of occupationally exposed workers
but the increase was not statistically significant. In
animals however, the evidence of carcinogenicity is
sufficient. Liver neoplasms are commonly seen in rodents
after acute and chronic exposure to CC14 although some
authors suggest that carcinogenesis may be more directly
related to the cirrhosis that is initiated by CC14 (Louria

and Bogden, 1980).

6

Table 1. Physiochemical characteristics of CC14

 

Description:
Boiling-point:
Freezing-point:
Density:
Refractive index:
Solubility:

Volatility:
Stability:

Reactivity:

Conversion factor:

color$ess liquid
76.7 C

'23.o°c

1.585 (d25/4)

1.4607 (n20/D)
miscible with ethanol, diethyl ether,
chloroform, benzene, solvent naptha
and most fixed and volatile oils
vapor pressure is 91.3 mm at 20 C
decomposes to phosgene in presen e of
limited quantity of water at 250 C;
noncombustible
when dry, nonreactive with commonly
used construction metals (iron and
nickel); reacts, sometimes
explosively with aluminum and its
alloys; reduced to chloroform with
zinc and acid; forms telomers with
ethylene and vinyl compunds under
pressure in presence of a peroxide
initiator
1 ppm in air is equivalent to
6.3 mg/m

 

IARC, 1979

7

The genotoxic and mutagenic action of CC14 is
undefined. Diaz Gomez and Castro (1980) found adducts of
14C-CC14 irreversibly bound to liver DNA in vivo. The
IARC monographs (1987a) reported that there is no data on
genotoxicity of CC14 in humans and that animal in vivo and
in vitro data was negative while studies in Saccharomyces
cerevisiae and Aspergillus yielded positive results.

Regnault produced CC14 in 1839 by chlorinating
chloroform (CHCL3) and shortly afterwards Dumas produced
CC14 by the chlorination of methane (CH4). In 1843 Kolbe
reacted chlorine with carbon disulfide (C82) to form CCl4
(IARC, 1979). Currently CC14 is commercially produced by 6
companies in the U.S.A. by three processes 1) as a coproduct
with tetrachloroethylene, usually by the chlorination of
propane or ethane propylene 2) the chlorination of carbon
disulfide 3) the chlorination of methane (IARC, 1979; EPA,
1987). Large scale production of CCl4 began in the U.S.A. in
1907 and by 1914 it was estimated that 4.5 x 106 kg were
produced annually. Reduced demand for CC14 led to a 3.5%
decrease in jproduction. from 1970-1980, however the 1980
production rate was 3.22 x 108 kg. It was estimated that a
further 1% drop in production would occur annually through
1985 giving an estimated production of 3.06 x 108 kg of CCl4
in 1985 (EPA, 1987).

The half life of hydrolytic decomposition of CC14 has
been estimated at 70,000 years, while the evaporative half

life is only 29 minutes (EPA, 1980). Thus, small spills of

8

CC14 rapidly volatize into the atmosphere, while larger
spills have the potential to settle under a water layer due
to the high specific gravity, low water solubility and high
chemical stability of CC14. CC14 is primarily of
anthropogenic origin, however small amounts of CC14 are
believed to be produced in the troposphere by solar-induced
photochemical reactions of chlorinated alkenes, primarily
from tetrachloroethylene (IARC, 1979).

The major use of CCl,; is in the production of
fluorocarbons, other applications include a component of
fire extinguisher solutions, a grain and industrial
fumigant, formulation of petrol additives and as a component
of pesticide mixtures. Before a November 11th 1970 ban by
the United States Food and Drug Administration (U.S.F.D.A.)
CCl4 was used as a domestic solvent and spot remover and is
still used for this purpose in industry, notably the dry
cleaning industry although it has been largely been replaced
by perchloroethylene (IARC, 1979; EPA, 1980). Traditionally
CCl4 had been used as a treatment for hookworm (1920's) , a
component of anesthetics and even in shampoos. Its use in
the treatment of hookworm is one of the few documented
studies of orally ingested CCl4 except in suicide cases
(Louria and Bogden, 1980; EPA, 1980).

Absorption of CCl,; in mammals occurs readily in the
lungs and gastrointestinal tract and at a slower rate
through the skin. A typical average exposure for an adult

male has been estimated at 13 rig/day from inhalation, 9

9

ug/day from fluid intake and 4 ug/day from food intake (EPA,
1987). A, recently' published study in Italy of volatile
halogenated hydrocarbons (Gilli et al., 1990) found that
atmospheric concentrations of CCl4 were correlated with
intensity of industry, commercialization and demographic
density. Disturbingly the concentration of CCl4 indoors was
consistently greater than that outdoors especially in
winter. A Total Exposure Assessment Methodology (TEAM) study
was carried out in New Jersey, North Carolina, and North
Dakota between 1979 and 1988. More than 20 VOC's were
measured in personal and outdoor air, drinking water and in
the expired air of participants. CC14 was identified as one
of the ten most common VOC's. As in the Italian study, the
concentrations of CC14 tended to be greater in indoor air
samples than outdoor air samples and breath concentrations
were consistently higher than outdoor concentrations
(Wallace et al., 1987).

In humans, symptoms of acute exposure include narcosis,
gastric irritation, nausea, vomiting, belching, flatulence,
drowsiness, headache, dizziness, fatigue, visual field
constriction, and moderate to severe damage to the lungs,
kidneys and liver (Bogden and Louria, 1980; EPA, 1987). Many
cases of acute exposure to CC14 have been described (1900's-
1950's) although the validity of many early studies is
doubtful as the hepatic disorders seen from CC14 exposure
are similar to effects of hepatitis B and other viruses that

were not (or could not be) screened for.

10

Some recent outbreaks of CCl4 poisioning have been well
documented. An outbreak of CC14 poisoning was reported in an
isopropyl alcohol packaging plant affecting 14 workers of
which four developed renal failure or hepatitis (Folland et
al., 1976). Most victims experienced some of the following
symptoms: nausea, vomiting, headache, weakness or abdominal
pain. Another outbreak of CCl4 poisioning occurred in a
Taiwanese color printing factory, again poisioning was
attributed to the combined use of isopropyl alcohol and CC14
as solvents (Deng et al., 1987). Recent interest in CCl4
toxicity has stemmed from the identification of CCl4 as one
of many chemicals found in toxic waste dumps and land fill
sites. Liver dysfunction was reported in residents exposed
to water sources containing leachate from a toxic dump site
in Tennessee (Meyer, 1983) . CCl4 was the major contaminant
detected in private wells with concentrations ranging from
61 - 18,700 ug/l. CC14 has also been detected at the
infamous Love Canal dump site at Niagara Falls, New York
(Silkworth et al., 1984).

Animal models have been used to test the toxicity of
complex waste mixtures obtained from these dump sites in
which it is virtually impossible to assign a particular
chemical as responsible for any given effect. Researchers
have discovered that serum battery enzyme assays performed
in vivo or in vitro provide better predictions of
hepatotoxicity than single serum enzyme assays. It is

usually thought that CC14 is the most likely hepatotoxin

11

especially considering the ability of other chemicals to
potentiate the effect of CCl4 (Simmons et al., 1988;
DeMarini et al., 1989). Other groups have investigated the
use of in vitro hepatocyte cultures as models of in vivo
hepatotoxicity in experimental animals. Long and Moore
(1988) compared the inhibition of the endoplasmic reticulum
Ca2+ pump, stimulation of phosphorylase a and inhibition of
glucose - 6 - phosphatase due to CC14 exposure in vivo and
in hepatocyte cultures. These researchers found the sequence
of events to be the same in both systems, with the onset of
every event occurring significantly earlier in vitro. In
vitro cytotoxicity of selected VOC's (including CC14) was
found to be poorly correlated to the in vivo hepatoxicity
(Dahlstrom-King et al., 1990). The researchers stated that
assessment of release of alanine transaminase (ALT) could
not accurately select known hepatotoxic chlorinated
hydrocarbons in in vitro culture systems.

The action of CCl4 as a hepatotoxicant and to a lesser
extent a kidney and lung toxicant has been demonstrated in
humans and others since the 1920’s. Recently some
researchers have used the hepatotoxic action of CC14 to
severely compromise liver function in laboratory animals
with the aim of studying the effects of liver dysfunction on
other physiological systems. For example the effect of liver
dysfunction induced by CC14 on the development of hepatic
encephalopathy (Yamamoto, 1990a) and blood coagulation

variations (Vazquez et al., 1990) have been studied. It is

12

hoped that direct effects of CCl4 are considered in the
assessment of physiological problems induced by liver
dysfunction as these studies could help in the assessment of
effects of CC14 on other systems such as the reproductive
system. Kaminski et al. (1989) reported a direct effect of
CC14 on humoral and cell-mediated immune responses in the
mouse, with T lymphocytes and macrophages exhibiting a
greater sensitivity to CC14 exposure. A dose-dependent
suppression of T cell function was observed at
concentrations of CC14 that had no effect on liver, kidney,
lung or body weight. This is an important report as it
suggests that CCl4 can specifically affect specialized
cellular functions apart from the more common damage seen in
liver, kidney and lung, at concentrations where no overt

signs of toxicity are observable.

Bioactivation and Cytotoxic Mechanism of Carbon
Totrachlcrida

Since the discovery of the hepatotoxic action of CCl4,
research has centered on determining its mechanism of
action. A diverse range of experimental systems such as,
perfused isolated liver, cultured or suspended hepatocytes,
microsomal preparations and in vivo analysis have been used,
yet the exact nature of CC14-induced cytotoxicity is still
unclear. Although we have presently investigated the effects

of CC14 on early reproductive events, the ultimate result

13

of CC14 exposure is hepatocyte destruction. Thus it is
important to understand the mechanism of action.

The metabolism of xenobiotics is generally considered
to be a two step process, occurring primarily in the liver.
The first step involves the addition of an electrophilic
substituent to the xenobiotic as catalyzed by
monooxygenases, reductases or hydrolases termed phase 1
reactions. The second step involves a conjugation reaction
with a variety of possible endogenous substances that
increase the water solubility of the metabolite in the phase
2 reactions (Neal, 1980). Butler (1961) described metabolism
of CC14 to chloroform and postulated the intermediate
formation of a free radical produced by the homolytic
cleavage of a carbon-chlorine bond forming a chlorine ion
and trichloromethyl radical ('CC13). It is now generally
accepted that CC14 is bioactivated by the cytochrome P450
monooxygenase system forming highly reactive free radical
intermediates (Recknagel and Glende, 1973; Recknagel et al.,
1989) . However the identification of the trichloromethyl
radical in vivo, and to some extent in vitro, by the spin
trapping technique is still controversial (Rosen and
Rauckman, 1982). The key, one electron reductive
dehalogenation has been shown to be catalyzed by a specific
isozyme of cytochrome P450 (Noguchi et al., 1982). Ekstrom
et al. (1989) described the participation of a human ethanol
inducible cytochrome P450 in the metabolism of CCl4.

Endoplasmic reticulum is the major source of hepatocyte

14

cytochrome P450 (Wolf et al., 1980). More recently Tomasi et
al. (1987) demonstrated that 'CC13 is generated by
mitochondrial metabolism of CC14 which may explain early
destruction of some mitochondrial function. One of the major
effects of CCl4 on mitochondria is the reversible uncoupling
of oxidative phosphorylation.

Highly reactive radical species such as 'CCl3 and
'OOCC13 have extremely limited range of action due to the
quantity of electrophilic targets at their molecular site of
origin. Despite this, eventually all parts of the
hepatocyte, in particular the plasma membrane, are
pathologically affected and it is these effects that are the
endpoint of CCl4 toxicity. Thus metabolism of CCl4 in the
endoplasmic reticulum must set into motion a set of
secondary' mechanisms which ultimately cause cytotoxicity
(Ungemach, 1987). With regard to the nature of the secondary
mechanism two major hypotheses exist: 1) long lived toxic
products of lipid peroxidation diffuse from the endoplasmic
reticulum to other parts of the cell and, 2) a toxogenic

2+ concentration upon

increase in hepatocyte cytosolic Ca
CC14 metabolism results in cell death. These two hypotheses
are discussed below.
Hypothesis 1. Lipid peroxidation and generation of toxogenic
substances:

Lipid peroxidation is the metabolism of lipids through

pathways involving intermediate formation of lipid

peroxides, hydroperoxides and endoperoxides (McCay et al. ,

15

1984). The formation of 'CC13 from CC14 as catalyzed by P450
occurs at a maximal rate in the absence of oxygen while the
formation of the trichloromethylperoxy radical ('OOCC13) is
exquisitively sensitive to oxygen concentration (Keiczka and
Kappus, 1980) . The 'OOCCl3 has been suggested to be the
reactive metabolite that initiates lipid peroxidation
(Albano et al., 1982). Conner et a1. (1986) suggested that
'OOCCl3 is the starting point of the oxidative pathway that
generates the carbon dioxide anion free radical (CO-‘2) and
the eventual conversion to respiratory C02.

The double allylic hydrogen atom of polyenoic fatty
acids are highly susceptible to abstraction by free
radicals. Hydrogen abstraction by 'CCl3 results in the
formation of a lipid free radical (L') and chloroform
(CHC13) while 'OOCC13 results in L' and trichlorocarboxylic
acid (Cl3COOH) formation (Recknagel et al., 1989). Lipid
radicals (L') react rapidly with oxygen to form lipid peroxy
radicals (LOO') which. abstract. a Ihydrogen from (adjacent
fatty acids to form LOOH and perpetuate L' formation. Also
important in the propagation of L' is the regeneration of
peroxy’ (LOO') and. alkoxy (LO') lipid radicals from. the
interaction of LOOH with either Fe (111) or Fe (11) (Simic
and Taylor, 1987) which can then initiate new chains of
peroxidation. De Groot et al. (1988) utilized an oxystat
system which was capable of maintaining steady state partial
pressure of oxygen at low levels with a constant amount of

respiring biomasss for extended periods of time. The

16

researchers reported that lipid peroxidation induced by CC14
occurred in two stages exhibiting different 02 requirements.
A direct relationship between lipid peroxidation and loss of
cell viability was established while conditions where
production of ’CCl3 were maximal but peroxidation was
minimal caused little loss of cell viability.

CC14 induced lipid. peroxidation has been shown to
inactivate both cytosolic and membrane bound enzymes such as
cytochrome P450, glucose - 6 - phosphatase and UDP —
glucoronyltransferase. The inactivation of these enzymes
has been clearly demonstrated in vitro but usually at high
concentrations of CC14. The extent of in vivo inactivation
at relevant exposure levels is not as clear (Horton and
Fairhurst, 1987). Recently it has been suggested that CCl4-
induced lipid peroxidation also causes the inactivation of
glycerol - 3 - phosphate acyltransferase (GPAT) which is
believed to be involved in the rate limiting step of
glycerolipid synthesis (Thomas and Poznasky, 1990) . These
workers proposed that inactivation of GPAT by lipid
peroxidation could inhibit membrane lipid repair and thus
accelerate membrane disintegration initiated by CC14
bioactivation and promote cytotoxicity.

It is not clear to what extent CCl4-initiated in vivo
lipid peroxidation proceeds. Propagation of lipid
peroxidation can be terminated through a variety of
mechanisms, such as condensation-reactions involving

radical-radical interactions (Simic and Taylor, 1987). These

17

condensation reactions result in cross linking of fatty acyl
side chains of phospholipids and highly abnormally branched
chains of fatty acids (Link et al., 1984). In addition to
physio-chemical termination there are many physiological
mechanisms to cope with oxidative stress. A-Tocopherol
(vitamin E) is a well known free radical scavenger. It
reacts with lipid hydroperoxy radicals at a much faster rate
than do saturated fatty acid bonds. However the
concentration of A-tocopherol is very low in microsomal
membranes and for this to be an effective peroxidation chain
breaker other mechanisms must exist to regenerate the active
form of Artocopherol (Recknagel et al., 1989). It has been
suggested that ascorbic acid (McCay, 1985) and glutathione
may play a role in the regeneration of A-tocopherol. Urisin
and Bindoli ( 1987 ) described a phospholipid hydroperoxide
glutathione peroxidase that directly reduces phospholipid
hydroperoxides to unreactive alcohol derivatives. Miyazawa
et al. (1990) demonstrated the formation and accumulation of
phospholipid hydroperoxides, especially phosphatidylcholine
hydroperoxide in the livers of CCl4-exposed rats, suggesting
that phosphatidylcholine is the most susceptible lipid class
to lipid peroxidation in vivo. These workers demonstrated
that dietary A-tocopherol inhibits phosphatidylcholine
hydroperoxidation suggesting a normal physiological role of
A—tocopherol in coping with oxidative threat.

CCl4-reactive metabolites may also be quenched by

covalent binding to cellular molecules. Diaz Gomez and

18

Castro (1980) found 14CC14 metabolites bound to endoplasmic
reticulum proteins and lipids and liver nuclear DNA but not
RNA. The process of lipid peroxidation is violently
destructive to endoplasmic reticulum. There have been many
reports of minor CC14 exposure protecting aginst further
exposure to larger doses (Lindstrom and Anders, 1977). This
is believed to be due to CCl4-mediated destruction of
cytochrome P450 in the endoplasmic reticulum membrane. The
precise nature of the destruction of cytochrome P450 is
uncertain. One hypothesis is that the propagation of lipid
peroxidation is responsible and another that free radicals
generated by cytochrome P450 reduction of CCl4 directly
bind to the cytochrome P450 molecules.

Lipid peroxidation yields a variety of stable,
diffusable endproducts which have been suggested as the
second messengers that diffuse from the site of CCl4
bioactivation to distal cellular sites resulting in cell
death (Comporti, 1985). These endproducts, particularly the
4- hydroxyalkenals have been linked to most physiological
manifestations of CCl4 toxicity such as loss of glucose-6-
phosphatase activity, inhibition of protein synthesis and
increased permeability of the plasma membrane. However
increasing evidence is being found that 4-hydroxyalkenals
are completely detoxified by the glutathione S-transferase
enzyme system and that the concentration of these products

in vivo does not correspond to the concentrations required

19

for toxicological effects. in ‘vitro (Recknagel et al.,
1989).

A significant increase in arachidonic acid (AA) levels
has been observed in hepatocyte culture following exposure
to low concentrations of CC14 (Chiarpotto et al., 1990). The
increase in AA levels is believed to be due to activation of
phospholipase A2 by CCl4 via a mechanism independent of CCl4
bioactivation. The relevance of this finding to the actual
cause of CC14 induced cytotoxicity is unclear as inhibitors
which prevent activation of phospholipase A2 and increases
in AA do not protect against cell membrane damage induced by
cc14.

Hypothesis 2. Ca2+ hypothesis:

Alternatively, lipid peroxidation may destroy the
ability of the endoplasmic reticulum to sequester Ca2+
leading to an increase in the cytosolic free Ca2+ level. It

2+ acts as the

has been proposed that this increase in Ca
second messenger ultimately responsible for cell death
(Recknagel, 1983; Recknagel et al., 1989). Early in vitro
studies using concentrations of CC14 above lmM showed very
rapid increases in cytosolic Ca2+ due to entrance of Ca2+
from the culture medium into hepatocytes (Brattin and
‘Waller, 1984). It. has been shown that these saturating
concentrations of 1 mM and above elicit hepatotoxicity by
direct solvent effects resulting in increased permeability

of the cell membrane and influx of Ca2+ from the medium

(Berger and Sozeri, 1987) . The relationship between the

20

solvent effects and other pathological effects of CCl4 in
causing toxicity is still under debate. Ca2+-activated
fluorescent labels have given contradictory results in

2+ release from intracellular stores in

analysis of Ca
response to CCl4 challenge (Brattin et al., 1985; Long and
Moore, 1987). While it is accepted that general lipid
destruction in the endoplasmic reticulum results in loss of
Ca2+ into the cytosol in vitro (Long and Moore, 1987), there
is still controversy over the possibility of loss of Ca2+
stores from the mitochondria either due to mitochondrial
P450 bioactivation of CC14 or the uncoupling of oxidative
phosphorylation by CC14 (Recknagel et al., 1989).

Recently it has been shown that Ca2+ levels in rat
liver in vivo rise before significant cell membrane damage
is apparent. Within 30 min of CCl4 challenge it was found
that cytosolic phosphorylase a activity increased in
response to increased cytosolic free Ca2+ (Tsokos-Kuhn,
1989). This confirmed that phosphorylase a activity

2+

increased and endoplasmic reticulum Ca pump activity

decreased in isolated hepatocytes exposed to CCl4 (Long and
Moore, 1986). Further in vitro evidence for the Ca2+
hypothesis was gained by Srivastava et al. (1990) using
hepatic microsomal preparations. These workers used
concentrations of CC14 (0.5 - 2.5mM) that have been measured
in rat plasma following acute dosing and showed a
significant decrease in the ATP dependent uptake of Ca2+. It

was proposed that 'CC13 directly oxidizes the sulfhydryl and

21

2+ pump resulting in a

possibly tryosoyl residues of the Ca
decreased uptake of Ca2+ by endoplasmic reticulum even in
the absence of lipid peroxidation. Parola et al. (1990)
reported that in vivo administration of CC14 to rats results
in a step 2 inhibition of the plasma membrane Ca2+-ATPase.
This enzyme has been shown to be a plasma membrane Ca2+ pump
that only operates by displacing Ca2+ from inside the cell
to the outside. Although the exact CC14 induced mechanism of
pump inhibition is not known, the first step of pump damage
has been demonstrated. to be independent of lipid
peroxidation. Damage to the pump is proposed to exacerbate
the increase in cytosolic free Ca2+ levels as the cells
ability to remove excess Ca2+ to the exterior is diminished.
Controversially other experiments have found that
incubation of hepatocytes with 0.172 mM CCl4 caused a rapid
decrease in both mitochondrial and extramitochondrial stores

of Ca2+ without an associated increase in cytosolic free

Ca2+ levels (Albano et al., 1989). An increase in

2+ 2+

mitochondrial Ca and endoplasmic reticulum Ca was found
after rats were treated with 96 mg/kg CCl4 (Yamamoto,
1990b). This is opposite to other researchers who have found

2+ content in endoplasmic reticulum after

a decrease in Ca
CC14 exposure. However Yamamoto (1990b) reports that this
finding supports the Ca2+ hypothesis as an increase in
mitochondrial and endoplasmic reticulum Ca2+ levels reflects

an increase in cytosolic free Ca2+ levels.

22

The Ca2+ hypothesis suggests that an initial rise in

2+ is due to selective inhibition of the

cytosolic free Ca
Ca2+ pump in the endoplasmic reticulum and results in
activation of Ca2+-dependant enzymes that lead to increased
permeability of the plasma membrane, rapid influx of
extracellular Ca2+ and cell death. In support of this
hypothesis, phospholipase A2 was found to be stimulated in
isolated hepatocytes in parallel with increasing free

2+ concentrations (Glende and Pushpendran,

cytosolic Ca
1986). Phospholipase C has also been shown to be directly
activated by CCl4 in vivo and in ‘vitro and activation
corresponded with a loss of membrane phospholipid content
resulting in membrane dysfunction (Lamb and Schwertz, 1982;
Coleman, 1988) . Furthermore it has been demonstrated that
oxidative damage similar to that caused by CCl4 stimulates
phospholipases as part of the cellular protection system
against oxidative stress (van. Kuijk, 1987). Thus it is
unclear whether phospholipase stimulation is a protective
event or whether CCl4 and the resultant increase in

2+ could overactivate phospholipases resulting

cytosolic Ca
in membrane damage. The rise in cytosolic free Ca2+ levels
could theoretically activate other enzyme systems resulting
in pathological effects. Recent evidence has indicated that
the Ca2+-dependent endonucleases are not stimulated in vitro
or in vivo in response to CC14 exposure (Long et al., 1989),

thus endonuclease damage of DNA resulting in initiation of,

or contribution to the hepatoxicity of CC14 is unlikely.

23

Another set of Ca2+-dependent enzymes, the protein kinase C
system, has been shown to be inhibited in isolated
hepatocyte culture exposed to CCl4 (Poli et al., 1988). The
prospective inhibitory compounds are the 4-hydroxyalkenals
produced by lipid peroxidation and found in concentrations
similar to those required to inhibit protein kinase C
activity in isolated experiments (Poli et al., 1988). other
authors contest any toxicological role for the 4-
hydroxyalkenals. While this report is contrary to the Ca2+
hypothesis the inhibition of protein kinase C could be
involved in CCl4-initiated cell injury due to the impaired
formation of phosphoproteins.

In summary a complex cascade of events links CC14
metabolism, generation of free radicals, lipid peroxidation,
4-hydroxyalkenal formation, macromolecular and free radical
interactions and loss of Ca2+ homeostasis. The relative
importance of each event in the ultimate development of cell
death upon exposure to cytotoxic levels of CCl4 is still
open to conjecture. Future studies utilizing other cell
culture systems affected by CC14 could be assessed for the
endpoint. of CCl4 toxicity, ‘thereby' contributing' to
deciphering the cytotoxic action of CC14. These systems

could include kidney cortical slices (Smith, 1988) and the

in vitro fertilization system used in the present studies.

24

Essential Aspects of Early Mouse Pregnancy

There have been many detailed reviews of mouse
reproduction. Mature female mice exhibit a regular 4-6 day
estrous cycle that is characterized by changing structure
and function of the sex organs dramatically demonstrated by
the continual growth. and regression of the ‘uterine and
vaginal epithelial lining. Females that are maintained in a
regular light cycle tend to ovulate 3-5 hours after the
onset of the dark period during the estrous phase (Champlin
et al., 1973).

After ejaculation sperm begin to reach the ampulla
region of the oviduct within 10 minutes and once
capacitation has been achieved (less than one hour), sperm
penetrating the cumulus mass surrounding the oocytes come in
contact with zona pellucida protein receptors that are
believed to trigger the acrosome reaction (Bleil and
Wassarman, 1983) . Acrosome reacted sperm are capable of
fertilizing the oocytes in the ampulla region of the
oviduct. Some of the events immediately preceding the
acrosome reaction are, penetration of oocyte outer layers
resulting in sperm fusion with the oocyte’s plasma membrane
stimulating ionic changes within oocytes, and initiating
the cortical reaction which blocks polyspermy. Pronuclear
formation is followed by DNA synthesis, metabolic changes,
mitosis and the first embryonic cleavage to form a two cell
embryo (Whittingham, 1979). For the next few days the embryo

divides slowly with little increase in mass occurring.

25

During this time the uterine lining is developing and
preparing for implantation under ovarian hormone control.

Up to the early 8 cell stage there is good
evidence that the blastomeres of the mouse embryo are
equipotent (Rossant and Vijh, 1980). During the next stages
of development the embryo ‘undergoes compaction and
blastulation during which events significant to toxic insult
occur e.g. development of gap junctions for cell to cell
communication (Lo and Gilula, 1979) and expression of
cytochrome P450 monooxygenases (Spielmann and Vogel, 1989).
During these stages the development of two distinct cell
lineages, the trophectoderm and inner cell mass (ICM)
begins.

The embryo enters the uterus on day 4 at the
blastocyst stage and implantation occurs on d4-5.
Implantation is under hormonal control and requires
synchronous development of embryonic and uterine tissues.
The trophectoderm is responsible for invasion of the
maternal uterine tissue and develops into the extraembryonic
tissues including the placenta while the ICM develops into
the fetus proper (Enders et al., 1981).

Between the fifth and tenth day of gestation there
is a dramatic increase in the growth rate of the embryo
during which time the basic body systems are established.
Pairs of somites are formed along the body axis followed by
initiation of the development of the neuronal and

respiratory systems and establishment of the limb buds.

26

During this period of embryogenesis, exposure to toxicants
may result in teratogenesis (Hogan et al., 1985).

The duration of gestation in the mouse is strain
dependent; in the B6D2F1 strain used in this work it is 19
days. At birth, typical of all mammalian species, the mouse
is still relatively underdeveloped and relies completely on

maternal care for survival.

Reproductive Toxicology

Reproductive health hazards are foremost in the minds
of many modern day toxicologists. The adverse reproductive
effects of many’ metals such. as lead, mercury, cadmium,
arsenic and lithium have been described in both humans and
experimental animals. Chemicals including insecticides,
herbicides, fungicides polybrominated biphenyls (PBBs) and
polychlorinated biphenyls (PCBs) have been valuable
agricultural or industrial chemicals due to their chemical
stability, low volatility and nonflammability, the same
characteristics that govern their persistence in the
environment and accumulation in the food chain. These
compounds are also believed to be potential reproductive
hazards. Vinyl halides, widespread in the manufacture of
plastics are believed to cause sexual dysfunction in men and
increase fetal mortality. Organic solvents, the group to
which CCl4 belongs have a potential exposure to an estimated

10 million workers daily and yet only limited studies have

27

been conducted to ascertain their reproductive threat (U.S.
Congress Office of Tech. Assess., 1985).

Reproductive hazard to the male is usually perceived as
a decreased libido or sexual ability due to toxic effects on
hormonal balance or effects on the peripheral or central
nervous systems. Most commonly a reduction in gamete
production, viability or normality can occur from chemical
insult. Fertility hazards in the female are similar to those
in the male although the time periods at which the gametes
are most susceptible to toxic insult differ. Although the
male gametes are more susceptible to mutagens the recovery
of a normal genetic complement occurs very rapidly. Any
mutation of female gametes remains permanently in the
limited population of oocytes (Barlow and Sullivan, 1982;
Christian, 1983a).

The most perplexing reproductive hazard effects are
those on the development of the embryo and fetus. Smith and
Costlow (1985) stated that it is the potential, unknown
presence of the conceptus in the workplace or other
hazardous environment which requires society to provide
effective control of exposure to hazardous chemicals. Karrh
et al. (1981) defined an embryo-fetotoxin as any chemical
which manifests an effect upon the conceptus during any
stage of gestation, from fertilization until birth. This
includes the induction of the death of the conceptus, any
structural malformation or any physiological dysfunction.

Also involved is any manifestation of growth retardation,

28

developmental delay of a physical or psychological nature at
birth or the subsequent postnatal period. This definition
also fits the term teratogen, however Staples (1975)
specifies that a true teratogen must produce adverse effects
on the embryo at concentrations below those necessary to
produce overt signs of toxicity in the mother. Maternal
exposure to chemicals that specifically effect the embryo is
possible during the critical periods of embryogenesis
(Kretchmer, 1978).

The scope of the problem is astonishing. The human
reproductive system is relatively inefficient with only 50%
of all conceptions resulting in normal offspring. A third
of all conceptuses are believed to be lost before pregnancy
is recognized and an approximate 15% of all recognized
pregnancies have been estimated to end in spontaneous
absorption. A further 8% of births exhibit developmental
abnormalities that interfere with survival or quality of
life (Smith and Costlow, 1985). It has also been estimated
that 2-3% of birth defects can be attributed to known drugs
and environmental chemicals (Karrh et al. 1981).
Interestingly, various surveys suggest that less than 1% of
industrial chemicals have been adequately studied for their

potential reproductive hazard (Karrh et al., 1981).

In Vivo and In Vitro Bysteas for Reproductive Toxicity
The goal in the evaluation of hazardous chemicals is

the determination of the toxicological profile using in vivo

29

and in vitro animal studies to allow assessment of the
potential risk to humans. Schardein and Keller (1989) have
discussed the four well established manifestations of
developmental toxicity in laboratory species. These are:

1. Growth. retardation, generally' measured. by fetal body
weight and crown-rump length;

2. Embryolethality, in mammalian species, this is seen as a
decrease in implantation sites or increase in resorption or
stillbirth. In humans embryolethality most commonly results
in miscarriage, spontaneous abortion or stillbirth.

3. Malformation, is sometimes classed as structural
malformation or teratogenesis and includes primarily
functional and metabolic disorders.

4. Functional disorders, this class of developmental
toxicity is not as well defined as the other three and
includes dysfunction of any biological system. More
specifically Vorhees (1986) states that impaired cognitive,
affective, social, arousal, reproductive and sensorimotor
behaviors are the major effects in this class.

It is not within the purview of this literature review
to discuss the complex field of human risk assessment and
the relationship of animal models to human toxicology.
Rather it is to highlight the in vivo and in vitro test
systems for the analysis of reproductive toxicity.

The in vivo tests for reproductive toxicity can be
divided into two main types, single generation and

multigenerational studies. In 1966 the U.S.F.D.A. published

30

guidelines for single generation studies (Christian, 1983b).
These consist of three treatment protocols. The segment 1
studies are usually carried out in rodents and are used to
assess the toxicants effect on mating behavior and gonadal
function, conception rates, maintenance of pregnancy,
parturition, lactation and postnatal development. Males are
dosed for 60 days before mating to cover one cycle of
spermatogenesis and females are dosed for 14 days prior to
mating and throughout gestation until sacrifice. Half of the
females are sacrificed at day 13 of gestation to ascertain
number of corpora lutea, implantation sites and live or
resorbing’ embryos. The ‘remaining females are allowed ‘to
deliver and nurse the offspring with evaluation of gestation
length, litter size, sex ratio, body weight and normality of
pups. Segment 2 studies test the toxicant over the period of
teratogenesis. In rats or mice the females mated with
unexposed males are exposed to the toxicant during days 6-15
of gestation or more uncommonly, days 6-17 (mouse) or days
6-19 (rat). The females are sacrificed on the day prior to
parturition and the fetuses examined for viability, weight,
and congenital abnormalities. Soft tissue, and skeletal
examination of the fetuses are commonly performed. Segment 3
studies expose the mated female (with unexposed males)
during the perinatal and postnatal period. Generally
exposure begins around day 15 of gestation and is continued
for 21 days postpartum. Litter size, pup weight and

normality are evaluated at birth and at selected intervals

31

until weaning. While these three protocols are generally
accepted, some authors suggest that a more segmental
approach is just as valid, with exposure of chemicals
limited to defined periods of the reproductive process
(Barlow and Sullivan, 1982).

Fabro et a1. (1982) described a teratogenic test
protocol in CD-1 mice with toxicant exposure on days 6
through 10 of gestation. These authors developed a probit
model from calculation of adult lethality and teratogenic
concentrations of eight test chemicals, enabling the
estimate of a tDso (teratogenic dose in 50% of the
population). Cummings (1990) suggested the establishment of
a dose response curve for exposure to chemicals from days 1
to 8 of pregnancy. Subsequently the author described three
test protocols to localize toxic effects on the pre and post
implantation period, embryo or uterine physiology and rate
of embryo transport relative to uterine preparation for
implantation. Kline et al. (1981) discussed the problems in
adapting models for reproductive toxicity from pre-
established models for carcinogenesis. These workers
suggested that a multiple exposure model was better suited
to exposure of the conceptus. Contrary to the traditional
view that chemical/toxicant effects are all or none
(resulting in death or normal offspring) (Austin, 1973),
recent studies have demonstrated abnormalities in term
fetuses from maternal preimplantation exposure to genotoxic

chemicals (Iannaccone et al., 1987; Spielmann and Vogel,

32

1989). Takeuchi (1984) exposed pregnant mice on days 2, 3
and 4 of gestation to 10 mg/kg of methylnitrosourea (MNU)
(maternal LD50 is 400 mg/kg). Examination of the fetuses on
day 18 revealed malformations including exencephaly, cleft
palate and abnormal tails. Bossert and Iannaccone (1985)
exposed blastocysts obtained from nonexposed mothers to MNU
and subsequently transferred the blastocysts to nonexposed
surrogate mothers. Examination of transferred blastocysts on
day 12 or 15 revealed a dose dependent decrease in fetal
body length and an increase in abnormal tissue formation.
Vogel et al. (1989) cultured preimplantation embryos
obtained from pregnant mothers exposed to MNU on day 3 of
pregnancy. The embryos demonstrated a significant inhibition
of development in vitro, a reduction in cell numbers and an
increase in structural chromosomal defects suggesting that
within hours of intraperitoneal (i.p.) injection MNU is
capable of reaching the embryo and inducing the
malformations seen at term. When pregnant mice were exposed
to a single i.p. injection of 40 mg/kg MNU on day 2 of
gestation there was 100% embryolethality but no maternal
toxicity. However a single i.p. injection of 20 mg/kg MNU on
day 2 induced developmental abnormalities of vertebrae,
ribs, long bones and kidneys in 40% of the live fetuses
(Spielmann et al., 1989). These experiments with MNU
indicate that chemicals can induce teratogenic malformations
through exposure of pre implantation embryos , at

concentrations that cause no overt maternal toxicity.

33

Other experiments have. demonstrated similar effects
with other chemicals e.g. NiClz (Storeng and Jonsen, 1981),
cyproterone acetate and medroxyprogesterone acetate (Eibs et
al., 1982), and methylmethanesulfonate (Fabro et al., 1984).
However the preimplantation teratogenic nature of these
chemicals is not as clear due to maternal toxicity,
inability to directly associate the chemical effect to
embryo damage or the long half life of chemicals possibly
resulting in initiation of the toxic effect during
postimplantation fetal development.

In vivo and in vitro methods for assessing early
developmental toxicity have recently been described
(Spielmann, 1987; Spielmann and Vogel, 1989). These include
in vitro culture of maternally exposed cleavage stage
embryos and. transplantation intol ‘unexposed surrogate
mothers. The authors suggest that evaluation of in vivo
exposure to chemicals during the preimplantation period
should include determination of malformations, growth
retardation, and fetal survival as well as the traditional
assessment of implantation failure.

Blazak et al. (1985) determined possible indicators of
normal sperm production in Fischer 344 rats. These authors
suggested that a complete analysis of the normal parameters
of male reproduction should include, testicular sperm
production, epididymal sperm number, sperm transit time and
motility for each species. Their findings indicated that the

current practice of determining caudal epididymal sperm

34

number as a ‘useful measure of toxic effects on
spermatogenesis is not accurate based on the wide variation
normally found in this parameter. However, in vitro
assessment of sperm motility in the presence of the test
substance can be a useful measure of toxicity. In addition
lactate dehydrogenase isozymes in plasma and testis can be
used as markers of testicular damage (Reader et al., 1988).
Urinary creatine has also been suggested to be a possible
marker of testicular damage, in studies which showed that
increasing urinary creatine and reduction in relative testis
weight are dose related (Rawcliffe et al., 1989). However
other authors have demonstrated that decreased testis weight
is not a good measure of toxic effects on sperm production
rate (Blazak et al., 1985).

With the growing concern over the number of chemicals
with unknown reproductive effect, increasing costs of whole
animal in vivo assessment and the desire to uncover
mechanisms of reproductive toxicity, more toxicologists are
defining in vitro assay systems to test potential
reproductive toxicants. The use of human cumulus granulosa
cells obtained from in vitro fertilization and embryo
transfer procedures can be used for in vitro toxicant
screening (Hughes et al., 1990). A recent review edited by
Kimmel and. Kochhar (1990) evaluates in ‘vitro assessment
systems for determining toxicant effects on cell-cell gap
junctional communication, cell migration and contractility,

embryonic cell and tissue proliferation, cell adhesion,

35

human placental function, the pharmacokinetics of

teratogenicity and approaches to study embryonic cell death.

Reproductive Effects of CCl4

Chatterjee (1966) administered a single i.p. injection
of 1.5 ml/kg of CCl4 in coconut oil to sexually mature male
rats after they had been fasted for 24h. At sacrifice, four
days later the testes were noticeably retracted into the
abdomen. There was a significant decrease in both testes and
seminal vesicle weight. Histological studies showed atrophic
changes and definite spermatogenic arrest. Furthermore, CC14
administration produced a significant increase in adrenal
weights and noticeable fat accumulation in the liver.
Treatment of male rats with 1.5 ml/kg of CCl4 i.p. and
phentolamine (Chatterjee, 1967a) prevented any effects of
CCl4 on the gonadal, adrenal or hepatic jparameters and
treatment with ascorbic acid (Chatterjee, 1967b) prevented
any gonadal toxicity (preventitive effects on the liver or
adrenal gland were not reported). Adams et al. (1952)
reported a chronic inhalation study of CCl4 in the rat.
Moderate to marked degeneration of the germinal elements of
the testis was found after exposure to 200 or 400 ppm for 7
h/day, 5 days/week. Severe general toxicity also occurred as
9 of 15 rats died during the 200 ppm treatment and 13 of 15
died during the 400 ppm treatment. Lower exposure rates of
400 ppm for 1 h/day, 5 days/wk for 6 weeks elicited no

effects on the testis and limited general toxicity. Adams et

36

al. (1952) also exposed guinea pigs to 400 ppm for 7 h/day
5 days/wk resulting in the death of most animals within 4
weeks. and.‘minor’ nonspecific jpathological changes in 'the
testis.

Male rats were given a single i.p. injection of 1.5
ml/kg of CCl4 in coconut oil every day for either 10, 15, or
20 days (Kalla and Bansal, 1975). These authors found a
decrease in epididymal, seminal vesicle, prostate and testis
weight after all three treatment periods. However, the
decrease in testis weight was greatest for the 10 day
treatment while the decrease in seminal vesicle weight was
more pronounced in the animals treated for 15 and 20 days.
Histological examination of testicular tissue revealed no
abnormalities in the: 10 day’ group, slight reduction in
spermatogenic cell number and an increase in lumen size in
the 15 day group while the 20 day treatment group showed
massive changes in the arrangement and number of germ cells,
luminal diameter integrity of basement membrane and
interstitial material. No documentation of other toxic
effects was presented. Seiler (1987) orally dosed male mice
with 2000 mg/kg of CCl,; and measured the amount of 3H-
thymidine incorporated into testicular DNA. CC14 treatment
resulted in a 50% reduction in incorporation of label into
testicular DNA. The author described this technique as a
novel way to detect testicular carcinogenic and mutagenic
chemicals but no measure of CCl,; toxicity to testicular

cells was mentioned. de Toranzo et al. (1978) measured a 20-

37

36% decrease in rat testicular cytochrome P450 content 3 or
6 h after i.p. administration of 1 ml/kg of CC14. The
authors reported that 14C adducts of microsomal lipids were
found in both the testis and liver. While significant lipid
peroxidation was found in the liver 6 h after the CCl4 dose,
no significant lipid peroxidation was found in the testis
indicating that loss of cytochrome P450 in the testis is due
to direct metabolite attack initiated by CC14 and not by
lipid peroxidation. Spin-trapping of free radicals by A-
phenyl N-tert-butyl nitrose (PBN) yielded a similar
resonance pattern in liver and testicular microsomal
preparations 2 h after i.p. injection of 1 ml of a 12% CC14
solution in the gerbil (Ahmad et al., 1987). The resonance
pattern was characteristic of the 'CC13 adduct indicating
that cytochrome P450 in the testis can metabolize CCl4 to
the highly reactive 'CCl3 adduct.

Alumot et al. (1976) performed a long term exposure
trial with rats fed mash that had been fumigated with CCl4.
They were able to overcome decreasing CCl4 residues in the
feed by using appropriate storage containers and training
the rats to feed before significant loss of CCl,; occurred.
After 6 wk the males and females fed mash containing either
80 ppm CC14 or 200 ppm CCl4 were mated and thereafter, while
treatment continued the animals were mated every 2 months.
No significant difference in litter size or pup weight at
birth or 10 days after birth between control and treated

groups was found. After five pregnancies the reproductive

38

efficiency in all groups began to decrease dramatically,
with the distribution of sterile males not significantly
different between control and treated groups. Higher
concentrations of CC14 were not used in long term
reproductive studies as significant paternal toxicity was
seen after a six week preliminary trial period, during which
no reproductive assessment was made.

The actual effect of exposure to CCl4 on testicular
function is difficult to assess from the above studies as
most of the researchers administered CCl4 by i.p. injection,
a very unlikely exposure route that tends to bypass the
first pass effect of the liver (Lukas et al., 1971). Also
the majority of testicular damage was seen in experiments
where significant toxicity of other organ systems occurred
or the majority of test animals died. Fasting has been shown
to change, the toxic effect of halogenated hydrocarbons
including CC14 (Harris and Anders, 1980) and some of the
above workers fasted animals before exposure to CC14. The
study by Alumot et al. (1976) used levels of CC14 where no
reproductive effects in male or female rats were seen,
however the precise relationship between CCl4 bound to food
molecules and entry into the body was not considered thus it
is possible that the rats were actually exposed to CCl4
levels well below doses where any effect is likely to be
seen. Chatterjee et al. (1966, 1967a, 1967b) and Kalla and
Bansal (1975) proposed that the testicular damage in CCl4

treated males is due to adrenal hypertrophy and disruption

39

of the pituitary-hypothalamic-gonada1 axis but this has yet
to be proven. Other reports (Amad et al., 1987; deToranzo et
al., 1978) suggest that testicular damage is mediated
through cytochrome P450 bioactivation of CC14.

Toxic effects can interfere with female reproduction
either by altering hormone metabolism or directly disrupting
one or more of several crucial periods - the estrous cycle
leading to ovulation, corpora lutea function,
preimplantation embryo development, implantation and
postimplantation embryo development.

Only a few studies have investigated the effects of
CC14 on sex hormone metabolism. Levin et al. (1970) found
that as little as 0.06 ml/kg of CC14 administered orally
decreased the metabolism of estradiol-17B and estrone by 72%
after 24 h in isolated microsomes. Pretreatment with CCl4
before the administration of 3H-estradiol-17B or 3H-estrone
led to an increase in uterine weight and labelled compounds
in the uterus. The authors showed that the increase in
tritiated compounds in the uterus increased with increasing
doses of CC14. Furthermore, pretreatment with CC14 was found
to inhibit the total body metabolism of estrone. All
concentrations of CC14 tested caused marked increases in
plasma transaminase associated with liver damage.
Unfortunately no assessment of CCl4 inhibition of estrogen
metabolism on the normal reproductive states or pregnancy
was presented. Hipkin (1969) reported that a single i.p.

injection of 0.1 m1 CCl4 inhibited the uterotrophic response

40

to administration of 0.5 I.U. human chorionic gonadotropin
(hCG) or pregnant mares serum gonadotropin (PMSG) in rats.
However no effect on the uterotrophic response was seen when
5 1.0. hCG or PMSG was administered after CC14
administration. Conversely to Levin et al. (1970), Hipkin
(1969) did not see any potentiation of the uterotrophic
effect of estrone after CC14 administration. This difference
is difficult to explain as the exact effect of CCl4 on
pituitary secretion of follicle stimulating hormone (FSH)
and luteinizing hormone (LH) and secretion of estrogen, also
the physiological response to the stress caused by CC14
toxicity, is not clear.

A recent report (Larroque and Lange, 1987) using liver
microsomes isolated from male rats treated with the
cytochrome P450 inducer triacetyloleandomycin, demonstrated
that progesterone metabolism was altered by the presence of
20 mM CCl4. The authors did not investigate or discuss the
relationship of CC14 effects on in vivo progesterone
metabolism in male or female rats and normal reproductive
function. The use of a P450 inducer and very high
concentrations of CCl4 were not related to any in vivo
effect.

Inhibition of the estrous cycle, a decrease in uterine
weight and a variable effect on ovarian weight was seen in
fasted, sexually mature rats after i.p. injection of 1.5
ml/kg of CC14 (Chatterjee and Mukherji, 1966; Chatterjee,

1967a, 1967b, 1968). Exposure to CC14 during pro-estrus

41

(determined by vaginal smear) led to persistant vaginal
estrus, while exposure during diestrus resulted in the
maintenance of a diestrus smear. Persisting corpora lutea
and some atretic follicles were seen in rats following CCl4
injection with significant atrophy of ovarian structures
noted in histological examinations. As in the experiments
with male rats, the toxic effects of CC14 in female rats was
alleviated by treatment with phentolamine (Chatterjee,
1967a) and ascorbic acid (Chatterjee, 1967b) with
concomitant restoration of normal adrenal function. In
contrast to this inhibition of the estrous cycle, Khominska
(1974) reported that when normally cycling rats received 3
ml/kg of CCl4 s.c. four times on one day the mean length of
the estrous cycle increased from 4.56 to 5.21 days with the
estrous phase prolonged by greater than one day.

A number of workers have investigated the effects of
CC14 on pregnancy. In a study of preimplantation
development, McLaughlin (1964) injected 160, 80, 32 mg of
CCl4 into the yolk sac of fresh fertile chick eggs.
Respectively, 0, 40, 80 % of eggs hatched and no report of
teratogenesis or birth defects in hatched eggs was given.
Poorly reported studies by Heine et al. (1964, cited in
Barlow' and Sullivan, 1982) and. Neumann (1977, cited in
Barlow and Sullivan, 1982) stated that no malformations
occurred when rabbits were given 50 or 60 mg/kg of CCl4
orally on day 6 of pregnancy. Preimplantation blastocysts

were obtained from rabbits fed 0.6 ml/kg CC14 on day 5 or

42

1.0 ml/kg on days 4 and 5 and examined histologically (Adams
et al., 1961). The blastocysts recovered on day 6.5 of
pregnancy from rabbits treated with the lower dose were
normal while blastocysts recovered from the high dose group
exhibited some cellular degeneration of the embryonic disc
and some trophoblastic cells contained very large nuclei
with prominent nucleoli. No correlation between embryo
toxicity and implantation success was studied.

s.c. injection of 0.8 ml or 0.3 ml CC14 orally was
used in a study to examine the effects of severe liver
damage on normal pregnancy (Wilson, 1954) . Treatment was
given for 2 or 3 days with 2-3 doses per day between days 7
and 11 of pregnancy. Of the 29 rats treated, 59% failed to
produce offspring of which 38% lost their entire litter due
to early resorption. A further 21% of pregnant mothers died
and the remaining mothers, except one, produced normal
litters. Little emphasis can be placed on this study as
exact dosing schedules were not reported and high
concentrations of CCl4 were used, in fact the study was not
designed to determine the effects of CCl4 on pregnancy but
that of severe liver damage.

Roschlau and Rodenkirchen ( 1969) gave 0.1 ml CC14
either s.c. or i.p. to pregnant mice on day 16 or day 18
followed by sacrifice on day 19. The authors reported that
only one of approximately 50 mothers died although the
normal pattern of liver necrosis was apparent in the rest.

There was dramatic fetal mortality in the group injected

43

i.p. on day 16, while fetal liver damage was most evident
in the group injected on day 18. Unfortunately an indirect
cause of fetal death cannot be ruled out as all maternal
groups displayed disturbed placental circulation and
placental necrosis.

Khominska (1974) also investigated the effect of CCl4
induced hepatotoxicity' in. pregnant rats. Preimplantation
losses were found to increase from 5% in controls to 21% in
treated rats and postimplantation losses increased from 7%
to 18% after female rats were injected with 3 m1/kg CC14 in
4 s.c. injections on one day and mated 7 days later.

A report by Gilman (1971), documented exposure of rats
for 8 h/day for 5 days between days 10-15 of pregnancy, to
250 ppm CCl4. There was no significant effect on skeletal
abnormalities, litter size, numbers of resorptions or
stillbirths. However a significant increase in pup deaths
from day 1-4 and 4-21 postpartum was found.

Dowty et al. (1976) reported a study of 11 cord blood-
maternal blood paired samples and found that the
concentration of CC14 in the cord blood was either equal to,
or greater than the concentration in maternal blood. The
authors suggested there may be a selective transfer of CCl4
to the infant. No effect of CC14 on maternal status or on
the infants was reported. Tsirelnikov and Tsirelnikova
(1976) gave 0.15 ml/kg CCl4 orally once on either day 12,
14, 16 or 18 of pregnancy in the rat and reported reduced

glycogen levels and increased division rate of cells of the

44

chorionic epithelium of the placenta. Administration on days
12 or 14 also led to destruction of the chorionic epithelium
of the labyrinthine portion of the placenta. Unfortunately
no data on fetal survival or development was given.

A number of studies have reported the relative
resistance of the fetal liver to CCl4 induced damage in
comparison to the maternal liver (Bhattacharyya, 1965;
Roschlau and Rodenkirchen, 1969; Kyutukchiev and Matrova,
1971), even if fetal mortality is very high. Tsirelnikov and
Dobrovolskaya (1973) reported that exposure of maternal rats
to 3 ml/kg CCl4 did not induce morphological changes in
fetal liver unless exposure was on day 19 of pregnancy,
although exposure on earlier days led to decreased fetal
liver metabolism and delayed glycogen synthesis. Kretchmer
(1978) suggested that cytochrome P450 monooxygenase enzyme
systems may be active in early fetal life and Spielmann and
Vogel (1989) state that the onset of activity of the phase 1
and cytochrome P450 monooxygenase systems and phase 2
conjugation enzyme systems coincides with the fOrmation of
the blastocyst cavity in the preimplantation mouse embryo.
Interestingly Khominska (1974) documented the increased
sensitivity of 4 wk old rats, born from rats exposed to CCl4
before pregnancy, to CCl4 induced hepatotoxicity, presumably
due to delayed induction of cytochrome P450.

Schwetz et al. (1974) evaluated the effects of
subanesthetic concentrations of CCl,; on rat embryonal and

fetal development coupled with careful assessment of

45

maternal toxicity. No significant effect on fetal
resorptions, litter size or conception rates in rats exposed
to 300 or 1000 ppm CC14 on days 6-15 of gestation was
reported, although 1 in 23 litters in the 1000 ppm group was
completely resorbed while none of 43 litters was resorbed in
the control group. At both CC14 concentrations significant
reduction in fetal body weight and crown-rump length
occurred however no gross morphological abnormalities were
observed. A significant occurrance of subcutaneous edema in
fetuses was noted in the 300 ppm group and a significant
increase in sternebral anomalies in fetuses of the 1000 ppm
exposed mothers also occurred, however neither effect
occurred in both groups clouding the overall significance of
this teratogenic effect. Further confounding the assessment
of teratogenicity and embryotoxicity, the occurrence of
overt maternal toxicity was high. The food consumption of
mothers in both dose groups dramatically decreased over the
40 day exposure jperiod and. correspondingly there ‘was a
significant decrease in maternal body weight. Hepatotoxicity
of maternal dosed groups was also seen, as measured by
increasing serum enzyme levels during the dose period and
gross morphological changes in the liver typical of CC14
toxicity.

A more recent study (Clemedson, 1989) investigated the
embryotoxicity of CC14 in the post-implantation rat embryo
culture system and in ovo chick embryos. Embryos recovered

from the rat at 9.5 days of gestation were exposed in vitro

46

to 10, 100, 300, 600 or 1000 mu g/ml of CCl4 in rat serum
with or without the rat S-9 mix liver microsomal activating
system. Without the 8-9 mix in the system no toxic effects
were seen with concentrations of 300 mug/ml or less.
Concentrations of 600 mug/ml and above affected the somite
number, growth and morphology of embryos and was interpreted
as a general toxic effect. Addition of the S-9 mix resulted
in the same generalized toxicity evident at concentrations
of 300 mug/ml and above. The chick embryos were exposed to
CCl4-vapor at concentrations of 25, 35 and 75 ppm, with 25
ppm and above resulting in a reduction of somite number and
a decreased growth rate. The authors concluded that these
experiments indicate that there is no specific malformation
pattern induced by exposure to CCl4 or its activated
metabolites. Another set of experiments utilizing the in ovo
exposure of chick embryos to CC14 described the
establishment of neuronal and astrocyte cell cultures from
exposed chicks (Clemedson et al., 1989b) . Exposure of the
embryos to 25 ppm CCl4 on days 5 - 8 idid not affect
subsequent in vitro development of neuronal cells. However a
reversible effect on the growth rate and respiratory
activity of cultured astrocytes was found.

As in the male reproductive effects of CC14, the
effects on female reproduction are unclear. Many studies
used inappropriate exposure regimens and high doses
resulting in maternal toxicity. The exact effect of CC14 on

estradiol metabolism and corresponding uterotrophic growth

47

is unclear as different authors present opposite effects.
Opposite effects are also seen in the studies of CC14 on
estrous cycles, where: one report suggests CCl4 exposure
stops the cycle in either estrus or diestrus, while another
suggests a small increase in cycle length.

Many studies reported. effects of CC14 on selective
reproductive functions but did not study the effects in
relation to prevention of pregnancy or embryo development.
It appears that the predominant effects of CCl4 on the
developing embryo are an inhibition in developmental rate,
or embryo lethality. However it is not clear whether this
is a direct effect or an indirect effect resulting from
maternal toxicity such as damage to the placenta. Only one
report of significant teratogenesis was found and the result
was unclear as the teratogenic effect was not dose
dependent. Clearly more decisive investigation of the toxic
effect of CCl4 on reproductive function in both male and

females is required.

MATERIALS AND METHODS

Animals

Female BGDZFI mice were obtained from one of two
sources. Six week old females used for in vivo experiments
were obtained from The Jackson Laboratories (Bar Harbor,
ME). All other’ :mice (B6DZF1) were obtained by' mating
C57BL/6 females with DBA/2 males at the Endocrine Research
Center. All animals were housed in a 12 h light : 12 h dark
photoperiod at 27°C. Food and water was available ad libitum

except where mentioned.

In vivo experiments

Mating procedure: On the evening of their arrival, females
from The Jackson Laboratories were placed with fertile
males in a ratio of 1:1 and checked on three subsequent
mornings for the presence of vaginal plugs. Most females
with plugs were obtained on the third morning as expected
from the Whitten effect of induction of estrus (Whitten,
1971). Females were randomly assigned to one of three groups
(control, 1/100 LD50, 1/10 L050) on day 1 of pregnancy (=
day of plug) and housed in groups of 5 until day 15 at which

time they were separated. A minimum of four animals per

48

49

group per replicate were used for the two replicates giving
a total of 31 animals: 10 control, 10 1/100 LD50 and 11 1/10

LDSO.

CCl4 solutions: At room temperature, CCl4 (catalog number
CAS 56-23-5, spectrophotometric grade, Mallinckrodt Int. ,
Paris, KY) was dissolved in corn oil on the first day of
treatment and stored at room temperature in light protected,
sealed bottles. All solutions were stirred using a teflon
coated magnetic bar on each day immediately before use.
Animals received either 0.2ml corn oil day"1 (control),
1/100 LD50 (82.63 mg/kg) or 1/10 LD50 (826.3 mg/kg) in 0.2ml
corn oil. The LD50 of 8263 mg/kg was obtained from the
Materials Safety Data Sheet OH504310 (Occupational Health
Services Inc., New York, New York). All procedures using

C014 were performed in a certified fume hood.

Experimental protocol:

Gavage protocol: Females were gavaged by placing a curved
gavage needle (size 18, Perfektum, New Hyde Park, NY)
attached to a lml glass syringe into the stomach through the
esophagus. Prior to the actual experiments, 10 animals of a
similar size to the experimental groups were gavaged with
0.2ml 2% methylene blue and killed by anesthetic overdose
using tribromoethanol while the needle was in place. The

position of the needle and localization of the dye to the

50

stomach was checked to ensure that no organ damage occurred
as a result of the procedure.

Gavage took place mid-morning on each of day 1 through
5 of pregnancy after a 2 h period of fasting. Within each
group animals were gavaged in random order and the order of

administration to the groups was randomized on each day.

Maternal parameters: Maternal weight (g) was determined on
days 1, 8 and 15 of pregnancy and the 22nd day postpartum.
On that day the females were killed by cervical
dislocation, the kidneys and liver removed, blotted dry,
and weighed. A portion of the liver and the kidneys (one
sliced longitudinally, the other horizontally) were stored

in formalin for histological processing.

Neonatal parameters: On the day of birth, litter size was
determined and each neonate was weighed. Crown-rump (C-R)
length was measured and individuals were sexed and checked
for obvious birth abnormalities e.g. extra/missing digits,
cleft palate, exencephaly and spina bifida. After recording
these parameters, litter size was reduced to 6, where
possible leaving equal numbers of males and females.
Neonatal weight and C-R were measured again on day 8,
15 and 22. In addition, lower incisor eruption and eye
opening was assessed in all pups on day 11 and 15
respectively. On day 22 neonates were weaned and two of

each sex allowed to grow to maturity (6 wk), the others were

51

killed by C02 asphyxiation. At 6 wk the four remaining young
were killed by cervical dislocation and the ovaries or

testes removed, blotted dry and stored in Bouin's fixative.

Statistical analysis: All in vivo parameters were analyzed
by one way ANOVA. When statistical significance between
control and treatments were found pairwise analysis of
treatment and control was performed using Dunnett's t test

(Gill, 1987).

In vitro experiments

Culture Medium: Brinster's medium for oocyte culture (BMOC-
3, Gibco, Grand Island, New York) was used for incubation of
oocytes and sperm in the center well of Falcon organ tissue
culture dishes (Becton-Dickinson and Co . , #3037 ,
Cockeysville, MD). Alternatively, BMOC medium was prepared
in the laboratory and used in the outer well of the tissue
culture dishes. The constituents of BMOC and BMOC-3 are

listed in Table 2 (Brinster, 1971).

52

Table 2. Components of two culture media (BMOC and BMOC-3)
used for IVF.

 

 

Component BMOC BMOC-3

NaCl 119.37 mM 94.90 mM
Na'lactate --- 20.11 mM
Na'pyruvate 1.02 mM 0.51 mM
KCl 4.78 mM 4.78 mM
CaC12'2H20 1.71 mM 1.28 mM
KH2P04 1.20 mM 1.20 mM
MgSO4'7H20 1.19 mM 1.19 mM
NaHCO3 25.07 mM 25.07 mM
Bovine serum albumin (BSA) --- 5 g/L

Glucose 5.55 mM 5.55 mM

 

Superovulation: Six - eight week old B6D2F1 females were
injected s.c. with 8 I.U. pregnant mare serum gonadotropin
(PMSG) followed 45 - 49 h later with 8 I.U. human chorionic
gonadotropin (hCG) s.c.. The PMSG and hCG were obtained from

Sigma Chemical Co. (St. Louis. MO).

Emulphor: Emulphor (EmulphorR EL-620, Rhone-Poulenc,
Princeton, NJ) was dissolved in BMOC-3 to the concentrations
of 0.1%, 0.01%, 0.001% and 1 ml was added to the center well
of duplicate dishes at the same time that 1 ml of BMOC-3 was
added in the center well of duplicate control dishes. 0.5 ml
of BMOC-3 was added to the center well of plates designated
for addition of the epididymis. BMOC medium (3 ml) was added

to the outer well of all dishes. The dishes were then placed

53

in a 5% C02 incubator at 37.50 C approximately 16 h before

IVF .

CC14: The BMOC and BMOC-3 media were added to the culture
dishes 16 h before IVF as in the emulphor experiments
except that the CCl4 was not added at this time. CCl4 was
dissolved in t-butanol (Catalog number B 2138, Sigma
Chemical Co.) to a concentration 100 times the final
concentration in a total volume of 10 pl to be added
directly to the 1 ml BMOC-3 medium at the time stated. Final
concentrations of CC14 in the center well of organ tissue

culture dishes were 0.05, 0.5, 1, 2, 5 or 10 mM.

Gamete recovery and IVF: Twelve hours after females received
hCG, 2 or 3 males of 6 - 10 months of age were sacrificed by
cervical dislocation for each IVF experiment and a single
epididymus was placed directly into the center well of a
organ dish containing 0.5 ml BMOC-3. The epididymus was then
repeatedly punctured (approximately 30 times) with a 25 G
needle before the dish was returned to the incubator for 1 -
1.5 h.

Approximately 45 min after sperm collection the
superovulated females were sacrificed by cervical
dislocation for collection of the cumulus mass containing
the oocytes. One side of each reproductive tract (ovary,
oviduct and uterine horn) was placed in the outer well of

the organ dishes containing 3 ml of BMOC. The cumulus mass

54

was removed from the oviduct under a dissecting microscope,
quickly washed and placed in the center well of a treatment
or control organ dish. The cumulus masses were randomly
placed in either treatment or control across replicates to
avoid the same female donating to the same treatment group.

For the emulphor experiments the cumulus mass was added
to dishes already containing the desired concentrations of
emulphor. For the CCl4 experiments, the organ dishes were
placed in the incubator after cumulus mass addition while
the CCl4 solutions were jprepared. CC14 solutions were
added to the organ dish containing the cumulus mass, with
care to avoid directly adding the 10 ul CCl4 or 10 pl t-
butanol (control) on top of the cumulus mass.

Immediately after the addition of the cumulus mass (for
emulphor experiments) or the CCl4/t-butanol, 50ul of sperm
suspension was added directly to the center well of the
dishes taking care that each replicate was inseminated from
a different, randomly selected male. All treatment and
control dishes were replaced in the incubator for 26 h
before assessment of 2 cell oocytes (which represent

fertilized eggs) was conducted.

Statistical analysis: All statistical analysis of IVF
experiments used Chi square and Bonferronni Chi square

contingency tables(Gill,1987).

RESULTS

In vivo experiments

Maternal Parameters: Replicate treatment data sets were
pooled for tests of statistical significance. A total of 60
female 86D2F1 mice were mated with a pool of fertile male
B602F1 mice. Thirty-five females were designated as pregnant
due to the presence of a plug, of these 31 were assigned to
experimental groups. All 31 treatment females maintained
pregnancy to term and a total of 294 pups were delivered.
Body weight (mean values i SEM) of dams recorded on
days 1, 8 and 15 of pregnancy are listed in Table 3. No
significant difference in maternal body weight across
treatments on the days measured were seen. Dams were
sacrificed on day 22 postpartum (day 1 = day of parturition)
and liver and kidney weights were recorded, body weight was
also recorded to enable calculation of relative organ
weights presented as means 1 SEM in Table 4. Relative and
absolute liver and kidney weights and maternal weights were
not significantly different across treatment groups. Tissue
samples of liver and kidney were prepared and preserved in
formalin for possible histopathological examination at a

later date.

55

56
Table 3. Maternal body' weight1 of B6D2F1 mice during

 

 

pregnancy after oral exposure to CCl4 on days 1 - 5 of
pregnancy.
Day Control 1/100 LD50 1/10 LDso
1 21.3 i 0.19 21.0 i 0.22 21.2 x 0.19
8 22.4 i 0.29 22.0 i 0.35 21.5 i 0.25
15 32.1 i 0.60 32.3 i 0.66 31.4 i 0.35

 

1 Expressed as means 1 SEM in g; N 10-11.

Table 4. Maternal body, liver and kidney weights1 of B6D2F1
mice on day 22 postpartum after oral exposure to CC14 on
days 1 - 5 of pregnancy.

 

 

Control 1/100 LD50 1/10 LD50
Body 30.1 t 0.73 30.3 i 0.86 28.3 _ 1.02
Liver
absolute 2.0 i 0.1 2.1 i 0.1 2.0 i 0.1
relative 6.8 r 0.2 6.9 i 0.2 6.9 i 0.1
Kidney
absolute 0.43 i 0.01 0.43 i 0.02 0.42 i 0.02
relative 1.44 r 0.20 1.42 i 0.02 1.47 i 0.02

 

1 Maternal body weights are mean i SEM in g; absolute liver
and kidney weights are mean 1 SEM in 9; relative weights
are mean percent body weight in g 1 SEM, N = 10 - 11.

57

Litter size and the percent male pups measured on the
day of birth are expressed as means 1 SEM and presented in
Table 5. No significant differences were seen in litter size

or the percent of male pups across treatment groups.

Table 5. Litter size and percent of males in litters exposed
to CCl4 by gavage to the dam on days 1 - 5 of pregnancy.

 

 

Control 1/100 L050 1/10 LD50
Litter size 9.6 i 0.4 9.5 i 0.5 9.4 i 0.2
Percent male 52.9 i 5.1 50.4 i 5.4 46.3 i 4.1

 

Results are mean i SEM, N = 10 - 11.

Neonatal parameters: Pup weight and crown rump length were
measured on days 1, 8, 15 and 22 postpartum and are
presented as means i SEM in Table 6 and 7 respectively. A
significant difference between control and treated groups
was seen on day 15 postpartum. No other significant
differences were seen across treatment groups in pup weight
or crown rump length at any other time.

No malformations were detected in any pup on day 1
postpartum (observations not presented). The normal
development of pups was assessed from observation of incisor
eruption on day 11 and eye opening on day 14. The results

are presented as means i SEM in Table 8.

58

Table 6. Mean pup weight1 of B6D2F1 mice on days 1 - 22
postpartum after exposure to CCl4 by gavage to the dam on

days 1 - 5 of pregnancy.

 

 

 

Day Control 1/100 LD50 1/10 LD50
1 1.29 i 0.02 1.31 i 0.04 1.32 i 0.02

8 4.71 r 0.12 4.89 i 0.08 4.81 i 0.07
15 9.28 i 0.19 9.29 i 0.16 8.77 t 0.10a
22 12.09 i 0.25 12.32 i 0.36 12.55 i 0.17

1 Expressed as the mean SEM of the mean per dam in g, N =

10 -11

a significantly different from control, Dunnett's t test,

p < 0.05

Table 7. Crown-rump length1 of pups on days 1 — 22
postpartum after exposure to CCl4 by gavage to the dam on
days 1-5 of pregnancy.

 

 

Day Control 1/100 LD50 1/10 LD50
1 24.4 i 0.2 24.4 i 0.2 24.2 r 0.1
8 35.6 i 0.4 35.9 i 0.3 35.6 i 0.2
15 46.1 i 0.9 45.9 i 0.3 45.9 i 0.2
22 56.0 t 0.4 56.3 i 0.4 56.3 i 0.1

 

1 Expressed as the mean 3 SEM of the mean per dam in

10 - 11

mm,

N

59

Table 8. Percentage of pups exposed to 1/100 and 1/10 L050
CCl by gavage to the dam on days 1 - 5 of pregnancy showing
incisor eruption on day 11 and eye opening on day 14.

 

 

Control 1/100 L050 1/10 LD50
Incisor eruption 75.0 i 2.2 72.2 i 2.2 78.5 r 2.2
Eye opening 83.3 i 1.4 83.3 i 1.4 84.9 i 2.1

 

Results are mean i SEM of the mean per dam, N = 10 - 11

In vitro experiments

Emulphor: In vitro fertilization trials for emulphor and
CCl4 were pooled for statistical analysis. Emulphor at 0.1%,
0.01% and 0.001% was added to organ tissue culture dishes
the night before IVF trials. Table 9 shows the number of two
cell embryos, total number of ova and the percent
fertilization for all emulphor trials. The fertilization
rate for all emulphor’ concentrations tabulated. was

significantly different from the control.

CCl4: CCl4 was added directly to the center well of organ
culture dishes, containing the cumulus before addition of
sperm, to the concentrations of 0.05 mM, 0.5 mM, 1 mM, 2 mM,

5 mM and 10 mM. The total number of two cell embryos, total

60

number of ova and percent fertilization for all CCl4 trials

are shown in Table 10.

Table 9. Effect of emulphor1 on mouse in vitro fertilization

 

 

 

Dose 2 cell total ova % fertilized
control 85 93 91.4
0.1% 23 100 23.0a
0.01% 32 65 49.2a
0.001% 58 90 64.4b

1 g/100ml

a

significantly different from control, p < 0.001
p < 0.0

b significantly different from control,

Table 10. Effects of CC14 (mM) and t-butanol (v/v) on mouse
in vitro fertilization.

 

 

 

Dose 2 cell total ova % fertilization
control 298 321 92.8

1% t-butanol 215 240 89.6

0.05 179 197 90.9

0.5 169 186 90.9

1.0 49 109 45.0a

2.0 29 94 30.9a

5.0 23 75 21.93
10.0 9 75 12.0a
a

significantly different from t-butanol, p < 0.001

61

The vehicle for delivery of CC14 was t-butanol and the
percent fertilization in 1% t-butanol was not significantly
different from the control. Thus t-butanol was used as the
control for further statistical analysis. CCl4 at
concentrations of 0.05 mM and 0.5 mM had no significant
effect on fertilization while 1 mM, 2 mM, 5 mM and 10 mM
CCl4 caused a significant decrease in fertilization. A
significant linear relationship (p < 0.001) between 1 mM, 2
mM, 5 mM and 10 mM CCl4 and decrease in fertilization was
found.

In the presence of lmM, 2 mM, 5 mM and 10 mM CC14
abnormal ovum forms were observed. The number of abnormal
forms, total number of ovum and percent abnormal forms are
listed in Table 11. All doses of CCl4 resulted in a
significant increase in abnormal forms and a significant
linear relationship (p < 0.001) was found to exist between
dose and percent of abnormal forms.

A pair of typical two cell embryos can be seen in
Figure 1a to be compared to the abnormal forms seen at CCl4
concentrations above 1 mM demonstrated in Figure 1b. The
abnormal forms are characterized by a compact dense cellular
region surrounded by pycnotic structures. These represent

degenerating oocytes.

62

Table 11. Effect of CCl4 (mM) on ovum morphology in mouse in
vitro fertilization.

 

 

 

Dose abnormal total ova % abnormal
1% t-butanol 0 175 0
1.0 9 109 8.0a
2.0 15 94 16.0a
5.0 69 105 65.7b
10.0 53 100 53.0b
g significantly different to t-butanol, p < 0.001

significantly different to t-butanol, p < 0.01

63

a)

b)

 

Figure 1. Appearance of normal two cell embryos (a), and
abnormal forms seen at CC14 concentrations above 1 mM (b).

DISCUSSION

CCl4 is a common industrial chemical and a ubiquitous
environmental contaminant. Disturbingly, levels of CCl4 are
frequently greater in indoor air than outdoor air samples
(Wallace et al., 1987; Gilli et al., 1990). The effects of
acute exposure to lCCl4 are *well documented and include
centrilobular necrosis and hemorrhage of the liver followed
by regeneration, proximal followed by distal tubule damage
in. the kidney, peripheral and. central neurological
disturbances, a host of overt symptoms such as vomiting,
disorientation, nausea and headache (Louria and Bogden,
1980). Interestingly, 50,000 persons infected with hookworm
were reported to have been treated with 3 to 4 ml of CC14
orally during the 1920's with few reports of any toxic side
effects. The most severe cases of CC14 acute toxicity are
often associated with dual exposure to chemicals such as
ethanol, isopropyl alcohol and chlordecone (Folland et al.,
1986; Mehendale, 1989). CCl4 has been designated as a class
2B carcinogen (IARC, 1987b) which states that evidence for
human carcinogenicity is insufficient, although Blair et al.
(1979) reported that. workers exposed to CC14 over long
periods in the dry-cleaning industry showed. higher than
expected numbers of cancer related deaths for each race-sex

group but this was not statistically significant.

64

65

Although a moderate amount of research has been
reported on the reproductive effects of CCl4 in experimental
animals the results are unclear. Many of the reports have
contradictory findings and often overt signs of maternal
toxicity are seen. The routes of exposure are usually not
biologically relevant as most studies exposed animals via
i.p. or s.c. injections. Very little research has been
reported on CC14’s effect during the preimplantation stages
of pregnancy.

This work was designed to investigate the effects of
1/10 LD50 and 1/100 L050 of CC14 administered by oral gavage
in corn oil once a day on days 1 - 5 of gestation (day 1 =
day of plug identification). These dose levels did not cause
any overt signs of maternal toxicity although the average
maternal weight in the high dose groups was consistently
lower than the other groups on day 8 and 15 of gestation and
day 22 postpartum, these differences were not statistically
significant. The average maternal liver and kidney weight of
the high dose group measured on day 22 postpartum was also
less than the other two groups but not statistically
significantly. No overt morphological damage of maternal
liver or kidney was seen on day 22 postpartum however it
should be noted that this examination was 36 days after the
last CC14 exposure.

Hayes et al. (1986) reported that gavage of mice for
14 days with 625 mg/kg of CCl4 in corn oil led to measurable

liver damage. Kaminski et a1. (1990) found that 500 mg/kg

66

delivered orally to mice once a day for seven days resulted
in a specific effect on the immune system. These authors
also reported a decrease in total hepatic microsomal
protein, aniline hydroxylase activity and aryl hydrocarbon
hydroxylase activity following treatment with CC14 for seven
days with doses as low as 5 mg/kg. Additionally Kim et al.
(1990b) reported. that even though. corn oil reduced the
hepatoxicity of CC14, a hepatotoxic effect was still found
when rats were gavaged once with 25 mg/kg of CCl4 in corn
oil.

Although it is not possible to predict the exact
extent of liver toxicity expected to be seen in this study
from the literature, it appears highly likely that some loss
of liver function will occur at both doses of CCl4 used in
this study. Manifestation of liver toxicity depends upon the
route of exposure, administration protocol, administration
vehicle, species and strain of animal and even the weight of
the animal. The important point is that no signs of overt
maternal toxicity were seen in this study that would
confound the assessment of any reproductive effect.

Exposure of pregnant mice to 1/10 LD50 and 1/100 L050
(which is equivalent to 826.3 mg/kg and 82.63 mg/kg or 0.52
ml/kg and 0.05 ml/kg respectively) over the first five days
of gestation. has had no effect on fetal mortality' and
development or postnatal abnormality and development.
Average litter size of all groups was not statistically

significant and no still births were observed indicating

67

that CCl4 had. no effect. on implantation, resorption or
induction of any embryo lethal effects. Average crown-rump
length of pups measured on days 1, 8, 15 and 22 postpartum
was not significantly different across treatments. No
significant difference in average pup weight was seen across
treatments except on day 15 when only the high dose was
significantly different from the control. This difference is
not interpreted as biologically significant as no other
difference in pup weight was seen and the day 15 pup weights
within litters were more dispersed than at other times.
Also, crown-rump length is a more sensitive measure of
neonatal growth and no differences were seen in this
parameter.

Recent reviews (Iannaccone et al., 1987; Spielmann et
al., 1989) have reported that exposure to chemicals during
the preimplantation period can lead to teratogenesis. MNU
has been shown to act on preimplantation embryos resulting
in teratogenesis visualized at birth (Takeuchi, 1984). This
study provides evidence that maternal exposure during the
preimplantation period to CCl4 at 1/10 L050 or 1/100 L050
does not result in teratogenesis in mice.

The genotoxic potential of CC14 is unclear, it acts as
a mutagen in some yeast culture systems but not in bacterial
systems, CC14 metabolites bind mammalian DNA in vivo and in
vitro but no mutagenic effect has been reported. Spielmann
et al. (1989) suggests that teratogenesis induced by

chemicals during the preimplantation period is due to a

68

mutagenic event. The fact that no teratogenic effect of CCl4
was found in this study provides more evidence that CC14 is
not genotoxic in mammalian systems. CCl4 exposure in this
study did not result in delayed development assessed by
observing incisor eruption and eye opening.

In conclusion this study clearly demonstrates that
exposure of maternal mice by oral gavage to 1/ 10 LD50 and
the 1/100 LD50 of CC14 once a day from day 1 through 5 of
gestation has no embryotoxic, embryolethal or teratogenic
effects.

These results agree quite well with the literature in
that CC14 induction of embryotoxic and or embryolethal
effects occur at concentrations where significant overt
maternal toxicity is seen. However this report is
significant in that no other studies were found where the
effects of CCl4 were determined during the preimplantation
period and most previous studies demonstrated high levels of
maternal toxicity.

In the above experiments CCl4 was administered in vivo
approximately 12 hours after the expected time of
fertilization. Thus an additional set of experiments
examined the effect of CCl4 on fertilization utilizing the
well established mouse in vitro fertilization system.

Initially the use of emulphor as a vehicle for delivery
of CC14 in vitro was assessed. Emulphor proved to be highly
toxic at all concentrations tested. A linear relationship

between inhibition of fertilization and emulphor was

69

established. This result agrees with those of O'Hara et al.
(1989) who reported that emulphor was too toxic to be used
as a vehicle for the study of CCl4 and CHC13 in isolated
hepatocytes. It appears that emulphors’ ability to maintain
relatively constant levels of VOC's in solution will not be
useful in in vitro systems due to a high level of toxicity.
Toxicity testing was undertaken using t-butanol as a vehicle
to solubilize the CC14 before addition to the IVF system. A
comparison between t-butanol and no vehicle demonstrated
that t-butanol had no effect on the IVF system at the
concentration used to deliver CCl4. The ability of CC14 to
elicit a toxic effect in vitro has been repeatedly
demonstrated in hepatocyte culture systems at concentrations
ranging from 0.02 mM - 10 mM.

In these experiments CCl4 at 0.05 mM and 0.5
mM was found to have no statistically significant effect on
the percent of fertilization. While the concentrations of 1
mM through 10 mMI had. a markedly significant effect on
fertilization. A linear relationship between dose and
inhibition of fertilization was established with the lowest
effective dose of 1 mM inhibiting fertilization by
approximately 50%. Interestingly the inhibition of
fertilization was not just marked by a reduction in 2 cell
embryos and an increase in one cell ova. An ovum exhibiting
a very dense constricted cellular/nuclear region and a dust
like circle of degraded cell constituents surrounding the

dense body, was observed. The appearance of these forms was

7O

statistically significant at all doses of CC14 from 1 mM to
10 mM and a dose related increase in these forms was
established.

Recently some authors have reported that rapid
halogenated hydrocarbon toxicity in isolated hepatocytes
resulting in morphological alterations is due to a direct
solvent effect (Berger et al., 1987; Berger and Sozeri,
1987). Interestingly, Kaminski. et al. (1989, 1990) have
reported that CC14 suppression of the immune system exhibits
a very flat dose response curve suggesting that
bioactivation of CCl4 is required for this effect. However
direct solvent toxicity appears to be very dose dependent.

The results of this IVF study clearly demonstrate a
dose dependent effect of CCl4 indicating that the effect
seen is a direct solvent effect and not a specific one.
However Dahlstrom-King' et (al. (1990) studied eight
chlorinated hydrocarbons in the isolated rat hepatocyte
system and found that toxicity did not relate to
physiochemical properties (notably the oil/water partition
coefficient) of the chemicals. In fact the authors found
that the in vitro toxicity did not correlate well with the
in vivo toxicity of the eight chemicals. The appearance of
the abnormal ovum in this study corresponds well with
morphological changes in hepatocytes reported to be due to
solvent action (Berger et al., 1987). However it is
interesting to note that at 1 mM CC14 a 50% reduction in

fertilization occurs with only a small highly variable

71

percent of abnormal forms seen, while at 10 mM CCl4 there is
an approximate 80% reduction in fertilization with a more
consistent 58% appearance of abnormal forms. This could
indicate that at the lower effective levels of 1 mM and 2 mM
CCl4 there are other' mechanisms apart from ‘the solvent
effect in operation resulting in the inhibition of
fertilization.

One of these mechanisms could be a disruption of Ca2+
homeostasis which is critical to fertilization and proposed
as a mechanism of CCl4 induced hepatotoxicity. It should be
pointed out that at 0.5 mM CC14 only a two-fold reduction
from 1 mM CCl4 (where a significant 50% reduction is seen)
there is no effect on the fertilization. This could be
interpreted in favor of either direct solvent effects of
CCl,; or some other mechanism. Obviously more research is
required to determine the effect of C014 on the IVF system
and perhaps some insight into the general mechanism of CCl4
cytotoxicity may be gained.

Finally Kim et al. (1990a) reported that a single oral
dose of 25 mg/kg CCl4 in the rat can result in a plasma
concentration of 0.3 mM and Srivastava et al. (1990) have
stated that concentrations of 0.5 - 2.5 mM CC14 can be found
in the blood of intoxicated animals. The results from this
IVF study suggest that a severe decrease in fertilization

rate would occur if the ovum were exposed to levels of CCl4

above 1 mM.

SUMMARY AND CONCLUSIONS

CCl4 is a halogenated hydrocarbon primarily of
anthropogenic origin. The major source of CC14 comes from
the manufacture of fluorocarbons. CC14 is one of the ten
most common VOC's found in dump sites, water supplies, urban
and industrial air samples. The IARC has classified CCl4 as
a class ZB carcinogen. Most reports of CCl4 effects on
reproduction are clouded by high maternal toxicity,
inappropriate exposure routes or conflicting findings.

Administration of 826 mg/kg and 82.6 mg/kg of CC14 in
corn oil by gavage in B6D2F1 mice during the first five days
of pregnancy resulted in no embryotoxic, teratogenic or
embryolethal effects. At concentrations above 1 mM, CC14 had
a significant effect on the mouse in vitro fertilization
system.

In conclusion:

1. Oral administration of CCl4 to BGD2F1 mice had no effect
on maternal liver, kidney or body weight. No effect on
litter size, sex ratio, pup weight, crown-rump length or
normal postpartum development was seen.

2. Emulphor is too toxic to be used as a vehicle for
delivery of volatile/insoluble chemicals in the mouse IVF

system.

72

73

3. CCl4 has a significant effect on in vitro fertilization
resulting in a decrease in the percent fertilization and an
increase in abnormal ovum form.

4. Abnormal ovum forms are most likely due to a direct
solvent effect. which. is seen. in Zhepatocyte cultures at

concentrations of CC14 above 1 mM.

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