&EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600 1-79-007
January 1979
Research and Development
Development of
Isolated Mammalian
Embryo
Techniques for
Toxic Substance
Screening
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-79-007
January 1979
DEVELOPMENT OF ISOLATED MAMMALIAN
EMBRYO TECHNIQUES FOR TOXIC
SUBSTANCE SCREENING
by
R. F. Williams, Q. S. Inman, and L. C. Ulberg
Reproductive Physiology Research Laboratory
North Carolina State University
Raleigh, North Carolina
Contract No. 68-02-1769
Project Officer
Neil Chernoff
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U,S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial
products constitute endorsement or recommendation for use.
ii
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FOREWORD
The many benefits of our modern, developing, industrial society
are accompanied by certain hazards. Careful assessment of the relative
risk of exisitng and new man-made environmental hazards is necessary for
the establishment of sound regulatory policy. These regulations serve
to enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Reserach Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pollutants. The Laboratory participates in the development and
revision of air quality criteria documents on pollutants for which national
ambient air quality standards exist or are proposed, provides the data for
registration of new pesticides or proposed suspension of those already in
use, conducts research on hazardous and toxic materials, and is primarily
responsible for providing the health basis for non-ionizing radiation
standards. Direct support to the regulatory function of the Agency is
provided in the form of expert testimony and preparation of affidavits as
well as expert advice to the Administrator to assure the adequacy of health
care and surveillance of persons having suffered imminent and substantial
endangerment of their health.
The large number of compounds finding their way into the environment
has increased the need for reliable short-term testing procedures which will
identify those agents in need of further detailed testing. The study described
in this report is an effort to develop such a technique for teratology screening
using isolated mammalian embryos.
F. G. Hueter, Ph.D.
Director
Health Effects Research Laboratory
ill
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ABSTRACT
A potential screen for assessing teratogenic potential of compounds
in mammals was tested. The technique involves testing isolated mammalian
embryos in culture by direct exposure to agents. Embryos from three
species of mammals were used; the mouse, rabbit and swine. Four dif-
ferent substances were studied; 5-fluorouracil, cadmium, cacodylic acid
and sodium chloride. About 3300 embryos from mice were used in the
development of the technique. In general, the results of the study
indicate that young mammalian embryos are responsive to a variety of
contaminants that may be found in their environment. Different concen-
trantions of the contaminant will elicit a different kind of response in
the embryo. Higher concentrations result in death, but a lower con-
centration may retard the development of the embryo. Still other
concentrations will interfere with the matabolic function of the embryo.
Cadmium appears to be the most toxic of the substances studied. It
causes the highest rate of embryo death. On the other hand, fluorouracil
results in a negative linear relationship between the concentration of
the substance and the incorporation of leucine into protein. It appears
that some substances are effective over a narrow range while others are
effective over a wider range. It is concluded that the procedure as
reported is capable of determining the type of effect that a contaminant
will have on the development of the mammalian embryo.
iv
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TABLE OF CONTENTS
Page
DISCLAIMER ii
FOREWORD iii
ABSTRACT iv
LIST OF TABLES vii
LIST OF FIGURES viii
SCOPE OF WORK 1
Background and purpose of Contract 1
Scope of Work 1
REVIEW OF LITERATURE 3
MATERIALS AND METHODS 16
Animal Strains 16
Culture Media 16
Embryo Recovery and Culture 16
Mouse Embryos 16
Rabbit Embryos 16
Swine Embryos 16
Incubation 17
Experimental 17
Mouse Embryos 17
Rabbit Embryos 18
Swine Embryos 18
Transfer of Cultured Embryos to Pseudopregnant Recipients 19
Photography 19
Statistical Evaluation 19
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RESULTS
Mouse Embryos
Cadmium
5-Fluorourac il
Cacodylic Acid
Sodium Chloride
Embryo Transfer
Rabbit Embryos
Cadmium
5-Fluorouracil
Cacodylic Acid
Sodium Chloride
Swine Embryos
DISCUSSION
SUMMARY
BIBLIOGRAPHY
APPENDICES
Appendix A.
Appendix B.
Brinster's Medium for Ova Culture II
Synthetic Oviduct Fluid
Appendix C. Protocol for Superovulation of Rabbits
20
20
20
20
30
30
30
30
39
39
39
39
39
46
49
50
55
56
58
60
vi
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TABLES
Page
Table 1 Developmental and metabolic response of mouse embryos
cultured in the presence of cadmium 27
Table 2 Developmental response of mouse embryos cultured in the
presence of fluorouracil 28
Table 3 Metabolic response of mouse embryos cultured in the
presence of fluorouracil 29
Table 4 Developmental response of mouse embryos cultured in the
presence of cacodylic acid 35
Table 5 Metabolic response of mouse embryos cultured in the
presence of cacodylic acid 36
Table 6 Developmental response of mouse embryos cultured in the
presence of sodium chloride • 37
Table 7 Metabolic response to mouse embryos cultured in the
presence of sodium chloride 38
Table 8 Developmental and metabolic.response of rabbit embryos
cultured in the presence of cadmium 41
Table 9 Developmental and metabolic response of rabbit embryos
cultured in the presence of fluorouracil 42
Table 10 Developmental and metabolic response of rabbit embryos
cultured in the presence of cacodylic acid 43
Table 11 Developmental and metabolic response of rabbit embryos
cultured in the presence of sodium chloride 44
Table 12 Metabolic response of swine embryos cultured in the
presence of fluorouracil or cadmium 45
vii
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FIGURES
3
Fig. 1 Incorporation by mouse embryos of H-leucine into acid
precipitable protein after 24 hours of incubation
with cadmium
Fig. 2 Developmental response of mouse embryos following 27
hours of culture with fluorouracil
3
Fig. 3 Incorporation by mouse embryos of H-leucine into acid
precipitable protein after 24 hours of incubation
with fluorouracil
Fig. 4 Developmental response of mouse embryos following 27
hours of culture with cacodylic acid
Fig. 5 Incorporation by mouse embryos of H-leucine into acid
precipitable protein after 24 hours of incubation
with cacodylic acid
Fig. 6 Developmental response of mouse embryos following 27
hours of culture with sodium chloride
3
Fig. 7 Incorporation by mouse embryos of H-leucine into acid
precipitable protein after 24 hours of incubation
with sodium chloride
3
Fig. 8 Incorporation by rabbit embryos of H-leucine into acid
precipitable protein after 24 hours of incubation
with cadmium
viii
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SCOPE OF WORK
Background and Purpose of Contract. The determination of teratogenir.
potential of environmental contaminants must be evaluated and become a part of
the data base available to the EPA in support of its legislatively mandated
regulatory function. Terata are malformations resulting from abnormal growth
and development in-utero. Conventional techniques of testing teratological
effects of pollutants involve exposure of embryos, in vivo, followed by subsequent
analysis of effect on the developed fetus. Such conventional techniques are both
time consuming and expensive. There is a need for the development of relatively
rapid testing methodologies which will be predictive of, and substitute for con-
ventional teratology testing. Rapid screening methodology is essential if the
agency is to address itself to the large numbers of environmental chemical con-
taminants.
Scope of Work. The contractor shall conduct experiments to develop rapid
testing methodologies as substitutes for conventional teratology testing methods.
The contractor shall develop teratology screening methods which employ in vitro
exposure of embryos to known teratogens for the purpose of determining the feasi-
bility of using embryonic uptake of radiolabeled protein and nucleic acid
precursors as biochemical indicators of growth and development.
In the development of screening techniques the contractor shall utilize
four compounds for embryo exposures. The compounds shall be subject to approval
by the EPA Project Officer and shall include known teratogens, selected pesti-
cides and/or other synthetic organic environmental contaminants. Testing will be
conducted on pre-implantation embryos of three mammalian species; mice, rabbits
and domestic swine.
The initial testing in development of screening techniques shall be con-
ducted using mice embryos. This testing shall be conducted as follows:
1. Eight-cell stage embryos are to be flushed from the oviduct
and grown in an appropriate culture medium that has been
proved to support normal growth and development. Experimental
embryos will be grown in a medium containing the test sub-
stance for up to three days. Their uptake of radiolabeled pro-
tein-and nucleic acid-precursors will then be determined as the
biochemical indicator of growth and development.
2. Additional exposed embryos will be redeposited into uteri of
recipient animals to determine viability following in vitro
exposure to these chemicals.
3. Studies shall be performed to assess:
a. rate of growth during cell divisions occurring in vitro
of both exposed and unexposed embryos,
b. survival rate to term of exposed and unexposed embryos
redeposited in uncontaminated uteri of recipient animals.
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Upon completion of the above studies involving mice, the contractor
shall apply the testing protocols developed to rabbits and domestic swine. The
testing of rabbits and swine shall be conducted according to the general methods
described above. Upon completion of all testing and studies, the contractor
shall provide a Final Report detailing the work performed and recommending
specific techniques which may be applied to rapid screening of teratological
effects.
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REVIEW OF LITERATURE
The thalidomide disaster of 1962 drew the attention of many people to tho
question of the deleterious effect of drugs and environmental agents upon human
reproduction (Epstein, 1973; Nanda, 1975). Prior to this catastrophe little
consideration was given to the matter of foreign materials affecting the embryo.
For at least the first half of the present century the embryo or fetus was
viewed as being protected behind the shield of the placenta and oblivious to the
environment of the mother.
Today, the attitude has shifted to one of alarm because of the believed de-
fenseless condition of the developing organism. The actual situation lies between
these two extreme views (Wilson, 1975). However, even this actual situation
warrants concern since it has been estimated that 2000 new chemicals are developed
each year, and that 200 will reach measurable quantities in the environment
(Wilson, 1975). Some of these compounds may be carcinogens, mutagens, or tera-
togens, and the populace does need to be protected from such side effects (Durban
and Williams, 1972; Heath et al., 1975). The importance of this is observable in
the area of developmental anomalies. Ten to twenty percent of these errors in
development can be attributed to gene and chromosomal anomalies. The remainder
are believed to occur by interaction of the genotype of a developing organism
with its environment (Gottschewski, 1974).
From the standpoint of reproduction, concern about deleterious effects
must extend beyond developmental anomalies and include embryo-lethability. It
must be noted that these effects are not restricted to just new chemicals, drugs,
food additives, pesticides, etc., but that in actuality all compounds at some
dosage will be harmful (Sullivan, 1974; Nanda, 1975; Wilson, 1975). The embryo-
and fetal-lethality induced by an agent are grouped under the term developmental
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toxicity, while a teratogen is a drug, chemical, virus, or physical agent which
causes a structural or functional alteration (Staples, 1975). Mechanistically
terata are caused by a mutation, a chromosome abberration, an interference with
mitosis, an altered nucleic acid synthesis, a lack of precursors for metabolism,
an altered energy metabolism, enzyme inhibition, osmolar imbalance, or altered
membrane characteristics (Wilson, 1973b).
Adverse effects of these agents can develop at any time during a pregnancy
(Nanda, 1975); however, the period of development may be divided into periods
of differing susceptibility. It is generally believed that from the time of
fertilization until implantation the susceptibility is low, but is present;
death of the embryo is the common manifestation of exposure of the embryo to
a noxious agent. After implantation and until the completion of organogenesis,
the embryo is most vulnerable to assault by some agent. This is the period
during which the teratogenic response of the embryo will occur. Finally, from
the end of organogenesis until parturition the detrimental effect of a compound
will be exhibited as reduced growth or a functional deficiency (Sullivan, 1974;
Nanda, 1975; Wilson, 1975; Wilson, 1973a).
The task of evaluating compounds for deleterious effects has been a difficult
one. Suggestions for a protocol for drug evaluation have ranged from the very
simple to the very complex. A simplistic design is the one given by Sullivan
(1974) based upon the sensitivity of the mother vs the fetus. If the dose re-
quired to induce abnormalities in the fetus approaches that dose which is toxic
to the mother, then the drug or chemical, etc., should be relatively safe. When
anomalies are induced at levels well below those of maternal toxicity the com-
pound is unsafe. Examining known teratogen in this light has revealed that most
would have been identified as safe compounds and would have passed this test.
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Following the thalidomide disaster, the Food and Drug Administration initi-
ated the three generation reproduction tests for pesticides and drugs. These
regulations were eventually extended to cover tests for teratogenicity (Epstein,
1973).
These regulations were designed to test the low dosage, long term effects
of pesticides and food additives, and later extended to other environmental
agents. For the purpose stated, these guidelines are adequate, but for studying
the teratogenicity of these compounds, the procedures will not suffice because
some health problems arise from short term exposure if it occurs at a critical
time. Suggested protocols have been prepared which view the question of short
term effects. The FDA evaluation is divided into three phases: 1) fertility and
general reproduction, 2) teratology study, 3) prenatal and postnatal effects. In
phase I males are treated before mating and females treated before mating, during
pregnancy, and during lactation. The young are examined at 13 days gestation, at
term, and during nursing. During phase II, treatment occurs on days 6 through 15
and the fetuses are examined 1 to 2 days before term. The prenatal and postnatal
phase is composed of treatment during the last third of pregnancy and throughout
lactation. The offspring are evaluated for survival and growth (Wilson, 1975).
The common test species are laboratory animals (rats, mice, hamsters, rabbits,
etc.).
The evaluation of drugs for side effects encounters a number of problems
with these recommendations. Of greatest difficulty is the problem of metabolism.
Many compounds as they exist in the environment do not have adverse effects, but
occasionally from the metabolites found by an organism there are teratogenic,
carcinogenic, or mutagenic effects (Druckrey, 1974). Therefore, it is important
that the species used for testing metabolizes the compound in a manner similar
-------
to that in the human. If this is not the case a harmful compound may pass the
examination or a safe substance may be blocked from distribution. The metabolic
similarity of the test species must be examined from the standpoint of the embryo,
the dam, and the placenta. Not only is the placenta important because of its
ability to metabolize compounds but also because of its transport function. By
regulating the passage of many materials to the embryo it regulates the embryo's
exposure to environmental agents. This variable of exposure must be considered
in screening methodologies as part of dosage. The transport functions within
the mother will alter the level of the agent reaching the embryo. These consider-
ations must be under advisement when a dosage for an experimental animal is
selected; in addition, the route of administration must be as close to that of the
human as possible (a dietary component being investigated should not be adminis-
tered intravenously). Examination of the test animals should be thorough; not
superficial.
If the compound is tested for teratogenicity the fetuses should be examined
both internally and externally (Palmer, 1974; Wilson, 1975; Lister, 1974). Within
such screening studies the number of animals is often small. Ideally the number
should be large. Especially since a small increase in occurrence of a rare
deformity is significant; it may require hundreds or thousands of litters to de-
tect just a dabbling in the rate of such an occurrence (Palmer, 1974; Wilson,
1975; Epstein, 1973).
To alleviate these difficulties Wilson (1975) has proposed the following
changes for improving teratological listing:
1) Use short duration dosage as well as long term.
2) Use one species other than rabbits and rodents which preferably metabolizes
as humans do.
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3) Determine etnbryotoxicity levels for experimental animals and project down-
ward to an acceptable level for man; not up from an estimated maximum dor.e.
4) Evaluation of postnatal function
5) Use primates to screen agents likely to be used during human pregnancy.
In 1969 an advisory panel for the Department of Health, Education and Wel-
fare recommended that:
1) Teratogenic effects should be studied in experimental animals.
2) Test agents should be administered during active organogenesis.
3) Parameters should be a) the incidence of abnormal litters, b) the number of
abnormalities/litters and c) the incidence of specific congenital abnormali-
ties, d) the incidence of fetal mortality, e) maternal weight gain during
pregnancy and f) maternal and fetal organ/body weight ratios.
4) Some animals should be allowed to give birth to determine abnormalities in
the prenatal period.
5) Two species of animals should be used.
6) The animals should be of various nutritional conditions.
7) A variety of routes of administrations should be used.
8) Enough dosages should be used to cover high accidental exposure and low
chronic exposures (Epstein 1973).
These HEW proposals and those of Wilson (1975) would complicate the present
screening procedures and greatly increase the cost. To circumvent this Wilson
(1975) calls for a shortcut method of screening. This may be advised by under-
standing the mechanism of teratogenicity and relating new compounds to these
mechanisms. Also, simple biological systems may provide information about the
deleterious effect of the compound being screened. Similarly, Axelrod (1972) has
called for a system that would be rapid and objective. He suggests that this might
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be achieved through small animal models and/or biochemical test which would
provide a data on changes that occur before anatomical malformations would be
observable. He suggests the use of enzyme action as an end point.
A number of possibilities of rapid and thorough screening systems have been
investigated. These have ranged from the culture of bacteria to the in vitro
growth of post implantation mammalian embryos. Some degree of industrial accep-
tance has been given to a "quick" test for evaluating new compounds for carcino-
genicity. On the belief that many carcinogens are mutagens a test was developed
by Bruce Ames which consists of observing strains of Salmonella (after exposure
to a chemical) for mutations from a histidine dependent strain to one that is not
dependent upon histidine's being present in the culture media. Though a number
of industries have accepted this test the National Cancer Institute has not endorsed
it (Kalato, 1976).
Considerable effort was expended upon the development of a teratogen screen-
ing system utilizing chick embryos. However, acceptance did not occur since it
appeared as though these embryos were too susceptible to chemically induced mal-
formations. Also, little success was achieved with compounds other than those
that were water soluble. Results of this system encouraged some to propose further
evaluations (Gebhardt, 1972), while others saw the results as a menace to human
safety (Goldberg, 1975).
For screening systems the utilization of various types of cell and tissue
culture techniques have been prepared by Moscona (1975). These consisted of study-
ing the effect of agents upon 1) cleavage of the egg, 2) growth of embryonic
myocardial and skeletal muscle cells in culture, 3) innervation of muscle in vitro,
4) reconstruction of tissues from embryonic cell suspensions (e.g. chondrocytes to
cartilage), 5) interactions between epithelial and mesenchymal tissues, and 6) in-
duction of systems by hormones. In vivo, alterations in each of these systems
8
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during embryonic development can result in developmental anomalies. Similarly,
Rajan (1974) has proposed the in vitro culture of human organs as test tissue
until it is possible to grow undifferentiated human embryos j.n vitro.
Organ culture of fetal tail vertebrae has been utilized to show a detrimental
effect of 5-fluorouracil (Yashihara, 1968). This pyrimidine is a patent teratogen,
and in this culture system showed a detrimental effect upon the development of
the vertebrae with a definite relationship between the length of exposure and the
degree of deformity.
The value of organ culture in testing for a teratogen has been demonstrated
by Kochbar (1975) with the culture of mouse limb buds. Detrimental effects of
vitamin A, bramadeoxyuridine, antiglutaneres, thalidomide, B-ameropropicnitribe
have occurred when limb buds have been exposed in vitro. It has been found that
organs cultured in this manner are more sensitive to teratogenic agents than when
in the embryo; however, with further testing such a system may provide a reliable
indicator of teratogenicity.
There has been developed by D.A.T. New (1975) a system for culturing post
implantation embryos of rodents. Rat embryos of 7 to 15 somites can be cultured
relatively successfully to 28 somites, and 22 to 28 somites can be cultured to 40
somites. Possible usage for teratogen screening has been reported by New (1975)
and the following advantages of the system listed: a) low cost and simple culture
media required of rodents b) embryos grown successfully during the period of organo-
genesis c) direct observation of the embryos are allowed d) precise control of test
conditions e) unsuspected causes of malformation revealed and f) freedom from ma-
ternal metabolism and possibly of placental metabolism. The last can also be a
disadvantage along with growth periods of only 1 to 3 days.
In 1966 Turbow reports using the above system to evaluate the teratogenic dye
9
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trypan blue. Low and high concentrations of the dyes were tested. Teratogenic
effects were observed at low concentrations when the embryos were injected within
the yolk but not when the embryos with an intact yolk sac were immersed in the
dye. At high concentrations a deleterious effect occurred with either method.
Modification of New's procedure has been reported by Kochbar (1975) and used
3
to test known teratogens by measuring the incorporation of H-thymidine into DNA
and labelled praline into proteins. Both vitamin A and DON (6-diazo-5-oxanor-
3 °
leucine) caused a reduction in the incorporation of H-thymidine into DNA. The
degree of depression was positively related to the level of teratogen added to the
culture medium.
Vitamin A also reduced the level of protein synthesis. DON was not reported
as being part of the amino acid study. This discussion of methodology has pre-
sented a variety of techniques which have been evaluated as possible tests for
deleterious effects of environmental agents. One area that has not been fully in-
vestigated for contributions to this field is the use of preimplantation mammalian
embryos.
One area of use of preimplantation embryo methodologies has been the attempt
to separate the susceptibility of the embryo to a teratogen from effects upon the
embryo as a result of a maternal response to the teratogen. The procedurie used
was the transfer of embryos prior to implantation from one genetic strain of mice
to another (one strain of high susceptibility and one of low susceptibility). In
two cases in which glucocorticoids were administered during organogenesis to sus-
ceptible strains of mouse embryos (Ajax and Swiss) after they had been transferred
to resistant strains, the number of anomalies was reduced (Vetter, 1971; Takano,
et al., 1972).
Using the same procedure Marsk et al. (1971) found the susceptibility to be
10
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embryonic in nature since the number of anomalies was not reduced when embryos
previously transferred to a resistant strain were treated with cortisone. The
same procedure was used by Marsk et al (1975) to study sodium salicylate. In this
case the embryos of a resistant strain transferred to a susceptible strain in-
creased in number the terata observed.
Embryo transfer has shown that some compounds do have an effect during the
preimplantation period that will cause developmental anomalies. Treatment of mice
with cyclophosphamide prior to transfer decreased the number of embryos surviving
after transfer as compared to controls and a malformation of the maxilla occurred
in transferred embryos which did not occur in treated untransferred controls.
With actinomycin D in treated untransferred controls the number of embryos sur-
viving decreased with increasing dosages of the drug. There was no observable
effect on survival rate and no malformations in treated transferred embryos
(Speilmann et al., 1974).
R. L. Brinster (1975) has discussed from a broad viewpoint the possible roles
which preimplantation embryos could play in teratology. He believes that they
could be used to investigate the mechanism of action of teratogens upon embryonic
cells. Normally the only effect of exposure of preimplantation embryos to tera-
togens is death; the response may result because of the small number of cells
composing the embryo at such stages. In these embryos which die the mechanisms of
cellular alteration may be the same as those which occur during abnormal organo-
genesis. During the organogenesis surviving cells are large enough in number to
form the organ, but in an anomalous form.
Brinster proposed that parameters for a screening system could be either
morphology of the embryo or measurements of metabolic capabilities.
The morphological aspect has been investigated. In 1964, M.F. Hay reported
11
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an altered morphology of rabbit blastocyst after the dam had been treated with
thalidomide. Lutwak-Mann et al. (1969) examined the effects of anti-metabolites,
metabolic inhibitants, anti-mitotic agents, metabolites, and 02 on rabbit blasto-
cysts exposed in vivo and cultured in vitro; cultured and exposed in. vitro, and
exposed in vivo and not cultured.
From the study no general conclusion about a group of embryos could be drawn.
Many compounds had no effect when the dams were treated and the embryos cultured
in vitro, but did have an effect when exposure was in vitro. Only 6-mercaptopurine
riboside had an effect when treatment was given the dam, and no effect on blastocyst
morphology when exposure was in vitro. All other in vivo effects occurred in vitro.
When embryos were exposed in vivo and cultured in vitro the most common response
was no change in morphology during culture. An exception was 2-deoxyglucose which
had a detrimental effect in vivo, but the embryos recovered in vitro.
In their discussion the authors warned that in vivo testing would always be
needed, and that some compounds showed no effect on the blastocyst in vivo or in
vitro but are known to be teratogenic when given during organogenesis.
In other sections of this review it was noted that several authors have called
for a biochemical analysis which could be used to test for deleterious effects of
environmental agents. Brinster (1975) proposed the measurement of metabolic
functions of preimplantation embryos as such a system. Such a proposal can be made
because a large volume of literature exists on the metabolism of the preimplantation
embryo. Nucleic acid synthesis in the mouse has been investigated with the use of
auto-radiography and found to occur at a much earlier stage of development than
in amphibians (Izquierdo, 1965); duration of phases of the cell cycle has been de-
termined by Luthardt and Donahue (1975) using this technique. Qualitative and
quantitative measurements of nucleic acid synthesis have been made using labelled
12
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precursors and techniques of separating various classes of nucleic acids in the
mouse (Monesi and Salfi, 1967; Ellem and Gwatken, 1968; Piko, 1970; Monesi et ,il,
1970; Epstein and Daentl, 1970; Epstein and Daentl, 1971; Daentl and Epstein, 1971;
Epstein et al., 1971; and Murdoch and Wales, 1973) and in the rabbit (Manes, 1969;
and Manes, 1971). Protein synthesis has been monitored in the mouse by determining
the amount of radioactivity incorporated into protein from labelled amino acids
(Monesi and Salfi, 1967; Thomson and Biggers, 1966; Brinster, 1971; and Epstein and
Smith, 1973) and also in the rabbit by the same methodology (Manes and Daniel, 1969).
Proteins synthesized during various cleavage stages of mouse (Van Blerkom and
Brockway, 1975) and rabbit (Van Blerkom and Manes, 1974) have been separated by
polyacrylamide gel electrophoresis and quantitatively evaluated using autoradio-
graphy. The sensitivity of such procedures for detecting variations in metabolism
have been demonstrated with investigations upon altered embryo development. Using
autoradiography, Sanyal and Meyer (1970 and 1972) observed that delayed implantation
3
of rat blastocysts resulted in fewer nuclei incorporating H-thymidine than in con-
trols and that delayed embryos treated with estrogen and progesterone to simulate
the conditions at implantation had an increased level of incorporation as compared
3
to untreated delayed embryos. A similar observation was made with H-uridine
(Jacobson et al., 1970).
Investigating mouse embryos undergoing delayed implantation as a result of
ovarectomy, Weitlauf (1972) found that these blastocysts had a lower level of in-
corporation of labelled amino acids into protein than did controls. It was also
observed that after culturing delayed blastocysts for 8 hours, the incorporation
increased to near that of controls. This observation is an example of the delin-
eation of a maternal inhibitory effect utilizing biochemical techniques on pre-
implantation embryos.
13
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Direct effects of hormonal steroids upon embryos have been investigated in
rabbits and mice. Daniel (1964) observed fragmentation of rabbit embryos cul-
tured in vitro with estrogen and cleavage inhibition with progesterone. It was
later found that the cleavage inhibition was reversible if the embryos were placed
in fresh media or if the level of proteins and amino acids in the culture was in-
creased (Daniel and Levy, 1964). Synthetic steroids of oral contraceptives were
also observed to cause inhibition and fragmentation of preimplantation rabbit em-
bryos (Daniel and Cowar, 1966). The incorporation of **-C labelled amino acids into
mouse embryos was found to be unaffected at the morula stage, slightly increased in
early blastocyst, and significantly increased in expanded blastocysts following
growth in culture media containing estradiol 17-B (Smith and Smith, 1971). An
example of the valuable information about environmentally induced problems that
can be obtained from investigations with preimplantation embryos has been reviewed
by Ulberg and Sheean (1973). Periods of transitory loss of fertility occur in
many species following exposure of the female to high environmental temperatures.
In cattle and sheep a decrease in fertility occurs for each degree rise in body
temperature during the time of the first cleavage. Four and eight cell rabbit em-
bryos transferred from heat stressed females to normal does failed to survive. No
effect was observed when unfertilized ova from control and heat stressed ewes were
transferred to ewes whose reproductive tract contained spermatozoa; this obser-
vation demonstrated that the detrimental effect of increased body temperature
occurs after fertilization. Heat stressing of spermatozoa in vivo reduced embryo
survival rate; exposure of first cleavage rabbit embryos in vitro resulted in de-
creased survival and retarded development. Mouse embryos respond in the same manner
as other species mentioned. In vitro culture of heat stressed embryos with
%-uridine indicated an increased incorporation into RNA above that of control embryos.
14
-------
With this example it is possible to see the identification of an environmental
problem (reduced fertility); the delineation of the level of the problem (the
embryo); and the biochemical evaluation of the effect of the environment (increased
RNA synthesis).
With results such as these, and those on delayed implantation, and hormonal
effects the utilization of these methodologies for possible screening of effects
of environmental agents is certainly warranted.
The purpose of the investigation being reported was to evaluate the use of
preimplantation embryo culture as a screening system for the deleterious effects
of environmental agents. The parameters used for evaluation of the system were the
ability of the embryos to cleave, the rate at which development occurred, the sur-
3
vival of embryos after transfer to dams, and incorporation of H-leucine into
proteins.
15
-------
MATERIALS AND METHODS
Animal Strains. Mice were of an Institute of Cancer Research randombred
stock maintained in the Animal Science Department, North Carolina State University.
Rabbits were New Zealand whites obtained from the Franklin Rabbit Ranch, Wake
Forest, North Carolina. Virgin females were of an 8-1/2 Ib. minimum weight. Pigs
were crossbred gilts and boars from an experiment station research farm. One boar
was a pure bred Yorkshire.
Culture Media. For constituents of the culture media see the appendix.
Mouse and rabbit embryos were grown in Brinster's Medium for Ova Culture II (BMOC );
pig embryos were cultured in synthetic oviduct fluid (SOF).
Embryo Recovery and Culture
Mouse Embryos. Normally ovulating female mice were sacrificed on day 2 (day
0 = day of vaginal plug) by cervical dislocation. The oviducts were excised, and
flushed with BMOC2 utilizing a 30 gauge needle, 2 cc syringe and a Wild binocular
dissecting scope with 12x magnification. Embryos were pooled from all females on
a given day, washed once, separated into groups of 10 eight-cell embryos (at 50x)
and placed in appropriate 10 x 75 mm culture tubes containing 1 ml of BMOC£.
Rabbit Embryos. Superovulated rabbits (see appendix for regime) were sacri-
ficed by exsanguination and embryos recovered from the oviduct by flushing it with
5 ml of BMOC2 into a petri dish approximately 42 hours after mating. Eight-cell
embryos were pooled, washed once, separated into groups of 5, and placed in appro-
priate tissue culture wells containing 1 ml of ZMQCn' Eight.culture wells were
located in a covered petri dish containing approximately 20 ml of water.
Swine Embryo's. Pig embryos were flushed from the uterus and oviduct with SOF
for flushing (see appendix) on either day 3 or 4 (day 0 = 1st day of estrus) under
surgical conditions. Embryos were pooled, washed once, grouped according to cell
16
-------
stage, and divided evenly (according to cell stage) between control and treatment
groups and placed in 10 x 75 mm culture tubes containing 1 ml of either treated
or untreated SOF.
Incubation. The 10 x 75 mm culture tubes and petri dishes containing the
culture wells were placed in an incubator at 37°C with an atmosphere of 5% C0? and
95% air prior to the addition of embryos. Humidity was maintained with an open
container of water.
Experimental
The culture media into which embryos were placed contained either no environ-
mental agent or one of four compounds being investigated. The compounds and their
chemical form were: cadmium (CdCl«), 5-fluorouracil, cacodylic acid (sodium cacody-
late) and NaCl. The concentrations of the materials used were calculated upon the
basis of the moeity of interest, and the appropriate amount was added to the cul-
ture medium. On a given day the embryos were divided between at least one control
culture and several dilutions of one contaminant.
3
Mouse Embryos. Following 24 hrs of culture 5 fiC± of H-leucine (specific
activity 60 Ci/mmole, New England Nuclear) in 50 01 of H20 was added to the control,
each of the dilutions of the contaminant, and at least one tube containing 1 ml of
BMOC2 but no embryos (blank for incorporation measurement). After 3 additional
hours of culture the embryos were removed from an individual culture tube, placed
on a watch glass and classified at 50x as cleaved (ICM-expanded blastocyst with
inner cell mass), blastocyst (small blastocoele cavity), morula, or abnormal (16
or 32 cells, or irregular morphological conformation), not cleaved, degenerated
(blastomeres lyzed and cytoplasm contained within the zona pellucida) or frag-
mented (repeated cytoplasmic division of the cytoplasm, but no nuclear division
of the blastomeres). Cleaving and non-cleaving embryos were separated and were
17
-------
transferred to 1 ml of cold BMOCo in separate 10 x 75 mm culture tubes.
The contents were decanted onto a Millipore filter (.45j/ pore size) and
filtered with a vacuum. The tube was rinsed three times with 1 ml of cold 5%
TCA (trichloroacetic acid) and a total of 25 ml of acid was passed through the
filter. The filters were placed in a scintillation vial, dried, dissolved in a
scintillation cocktail, and radioactivity determined in a Packard Tri-Carb liquid
scintillation spectrometer. By a computer transformation the counts/minute were
converted to disintegrations/minute using data from an automatic external standard.
Rabbit Embryos. Following 24 hours of culture 25 vC± of %-leucine (specific
activity = 53 Ci/m mole, Amersham-Searle) in 50,,/il of H^O was added to the control
and each of the dilutions of the contaminant. After 3 additional hours of culture
the embryos were classified at SOX in their culture wells as cleaved (16 cell or
morula), not cleaved, degeneratred, or fragmented. Embryos were transferred to a
10 x 75 mm culture tube containing 1 ml of BMOC2 + leucine (O.OlAg./liter). A
volume of BMOC2 equivalent to that used to transfer the embryos was placed in a
separate tube and processed as a blank. The solution was frozen at -20°C and
stored at least overnight. Following rapid thawing the embryos were rapidly frozen
(dry ice and acetone or liquid nitrogen) and thawed at 37°C five times; 2 ml of 10%
TCA was added to each tube, and the tubes stored at 4°C overnight. Contents were
decanted onto a Millipore filter (.45// pore dize) and filtered. Two ml of an ice
cold 10% TCA washing were passed through the filter followed by 10 ml of hot 10%
TCA (80-90°C) and 5 ml of ice cold 70% ethanol. The filters were placed in scin-
tillation vials, dried, dissolved in scintillation fluid and counted as were the
mouse embryos.
Swine Embryos. After equal division of the embryos according to cell stage,
5 f-C± of H-leucine (specific activity 60 Ci/m mole, New England Nuclear) in 50 ^1
18
-------
of H^O was added to each culture tube. Following 24 hours of culture the embryos
were removed from their culture tubes, placed on a watch glass, and classified as
to stage of cell division. The embryos were then placed in 1 ml of cold BMOC2
and decanted onto a Millipore filter and treated in the same manner as were the
mouse embryos.
Transfer of Cultured Embryos to Pseudopregnant Recipients. Embryos were
grown in vitro as described previously. At the end of 24 hours the embryos were
classified, and picked up with a finely drawn glass pipette. The uterus (exposed
through mid-ventral laparatomy-rabbits; high lumbar laparatomy-mice) was punctured
with a hypodermic needle (mice - 27 gauge; rabbits - 20 gauge); the pipette was
inserted; and the embryos were expelled into a uterine lumen.
All females were pseudopregnant. Mice were day 2 (day 0 = vaginal plug from
vasectomized male) and rabbits were approximately 66 hours pseudopregnant (induced
by mating and removing embryos at 42 hours, or by LH injections).
Photography. All photographs of embryos were taken as wet mounts with a
camera attached to a binocular microscope.
Statistical Evaluation. Cleavage patterns were analyzed by chi-square analy-
sis; leucine incorporation by control and treated embryos was evaluated by least
squares analysis of variance; and treated embryos were examined for a linear
relationship between the level of contaminates and leucine incorporation by
regression analysis.
19
-------
RESULTS
Mouse Embryos
Over 3300 8-celled embryos were used to investigate the effect of cadmium,
5-fluorouracil, cacodylic acid, and sodium chloride on the development of
preimplantation embryos. The results of these experiments are presented in
Tables 1-7, Figures 1-7, and Photographs 1-8.
Cadmium. A dramatic effect of cadmium in embryo development is presented
in Table 1 and Figure 1. The percentage of embryos cleaving is significantly
reduced after treatment with 1 ppm or 100 ppb Cd (0 and 12%, respectively) as
compared to controls (99%). Cadmium treated embryos were not classified as to
stage of development. At 100 ppb Cd the level of incorporation of %-leucine
was significantly reduced to only 13% of the control value, while at 10 ppb the
incorporation was significantly elevated above controls (2486 DPM vs 1592 DPM).
Embryos characteristic of controls and Cd treatment (1 ppm) are presented in
Photographs 1 and 2, respectively.
5-Fluorouracil. Table 2 and Figures 2 and 3 clearly illustrate the dele-
terious effect of 5-fluorouracil (F) on embryo development. The number of embryos
cleaving is significantly reduced at 100 ppm, 10 ppm and 1 ppm F (76%, 86%, 93%)
vs controls (100%).
The embryos treated with fluorouracil were classified as to stage of develbp-
"\
ment at the end of the culture period. There is a dramatic retardation Of
development at 100 ppm, 10 ppm, and 1 ppm F. Besides growth retardation there is
also an obvious morphological abnormality in these embryos. The frequency of
development of this malformation decreases with increasing dilution of the anti-
metabolite. Embryos treated with fluorouracil are presented in Photographs 3,
4, 5, 6 and 7. Leucine incorporation was significantly reduced by all of the
fluorouracil dilutions (Table 3). There exists a highly significant (P < .001)
linear relationship between the level of fluorouracil treatment and the incorporation
of leucine. For this analysis by linear regression the control data were excluded.
20
-------
PHOTOGRAPHS
1. Untreated (control) embryos - morula stage
2. Cadmium treated embryo - uncleaved degenerating embryo
characteristic of treatment with 1 ppm and 100 ppb Cd
3. & 4. Fluorouracil treated embryos (100 ppm) - embryos
show abnormal cleavage characteristic of fluorouracil
treatment*
5. Fluorouracil treated embryos (10 ppm) - 3 abnormally cleaved
embryos and 1 resembling a normal embryo*
6. Fluorouracil treated embryos (1 ppm) - 3 normal appearing
embryos and one abnormal*
7. Fluorouracil treated embryos (100 ppb) - 2 normal morula*
8. Uncleaved 8-cell embryo representative of treatment with
10 ppt or 1 ppt cacodylic acd,d or 10 ppt NaCl
*Ratio of normal and abnormal is characteristic of the treatment
group.
21
-------
*
1
3
22
-------
••••
r
5
7
-
6
8
23
-------
S3
2500
2000
1500
1000
500
[ | Embryos cleaving
H-lauclM Incorporated by
Embryos fragmenting, degen-
erating, or not cleaving
H-leucme Incorporated by •
bryos fragmenting, degener-
ating, or not cleaving
100
80
60
40
20
CONTROL
1 PPM
100 PPB
10 PPB
Concentration off cadmium in the incubation medium
Fig. 1
Incorporation by mouse embryos of H-leucine into acid pre-
cipitable protein after 24 hours of incubation with cadmium
-------
to
II Emory** cleaving
n agmentlng, degen
eratlng, or not cleaving
^^ Cleaving embryos forming a tnorula
HJ^ Cleaving embryos forming a Mastocyst
(JJJ Cleaving embryos forming an Inner cell mass
Embryos cleaving abni
100 - m
CONTROL 1OO PPM 10 PPM
Concentration of fluorouracil in the incubation medium
I PPM
100 PPB
10 PPB
Fig. 2
Developmental response of mouse embryos following 27 hours of culture with fluorouracil
-------
N> ,
250O
20OO
1500
1000
SOO
t~J Embryo* doovwit
• Embryoo fracmmtl
^m (onoratlnc, or not <
T»-t»ueln« lneorp«nl«l by
•nbmtt fra(iiMiitln|( do—
, or not cluvhic
10O
80
60
40
20
f
K
I ^
s
n
s I
if
CONTROL 100 PPM 10
1 PPM
1OO PPB
10 PPB
Concentration of fluorouracil in th* incubation medium
Fig. 3 Incorporation by mouse embryos of H-teucine into acid precip-
Itable protein after 24 hours of incubation with fluorouracil
-------
Table 1. Developmental and metabolic response of mouse embryos cultured in the presence of cadmium
Embryos cleaving
Treat-
ment
Control
1 ppm
100 ppb
10 ppb
Total No.
of embryos
277
220
219
280
No. of in- No. of
cubations embryos
29 274
3 27
31 273
Percent DPM
of total
99 1592
oc
12C 139C
98 2486C
Embryos fragmenting, degenerating
or not cleaving
No. of in-
cubations
3/Oa
24
21/20a
6/Oa
No. of
embryos
3/Ob
200
192/191b
7/Ob
Percent
of total
1
100C
88°
2
DPM
104
247
number of incubations in which embryos responded in a given manner/number of these incubations used
for incorporation determinations
Number of embryos having a specific response/number of these embryos used for incorporation
determinations
cSignif icantly different from control
.05)
-------
Table 2. Developmental response of mouse embryos cultured in the presence of fluorouracil
o>
Treat- No. of in- Total No.
ment cubations of embryos
Control 13 126
100 ppm 4 38
10 ppm 12 114
1 ppm 11 101
100 ppb 9 85
10 ppb 7 67
Embryos fragmenting,
degenerating, or not
cleaving
0
0%
&
19
17%c
3
3%c
0
0%
0
Embryos
cleaving
126
100%
29
76%°
95
83%c
98
97%c
85
100%
67
100%
Stage of development of
cleaving embryos
ICM
5
4%
0
0%c
0
0%
o%c
2
2%
4
6%
B
73
58%
0
0%c
10
11%C
12
12%c
39
46%
45
67%
M
48
38%
14
48%c
34
36%c
82
84%c
44
52%
17
25%
A
0
0%
15
52%c
51
54%c
4
4%c
0
0%
1
2%
Significantly different from control (P < .05)
-------
Table 3. Metabolic response of mouse embryos cultured in the presence of fluorouracil
Embryos cleaving
Treat-
ment
Control
100 ppm
10 ppm
1 ppm
100 ppb
10 ppb
Total No.
of embryos
126
56
124
101
85
75
No. of in-
cubations
13
6
13
11
9
8
No. of
embryos
126
47
105
98
85
75
Percent
of total
100
84
85
97
100
100
Embryos fragmenting, degenerating
or not cleaving
DPM No. of in- No. of
cubations embryos
2522
293cd 3 9
796cd 5/2a 19/13b
916cd 3/Oa 3/Ob
1263cd
2249cd
Percent DPM
of total
0
16 0
15 429
3
0
0
aNumber of incubations in which embryos responded in a given manner/number of these incubations used for
incorporation determinations.
Number of embryos having a specific response/number of these embryos used for incorporation determinations.
CSignificantly different from control (P < .05).
Significant linear relationship between the concentration of fluorouracil and incorporation of leucine
(P < .001). Does not include control data.
-------
Cacodylic Acid. Cacodylic acid had a much less dramatic effect upon embryo
development and metabolism than did the previous compounds. Concentrations of
10 ppt and 1 ppt were required to inhibit cleavage, and the only treatment which
resulted in a significant alteration in development was 1 ppt. Though 87% of the
embryos failed to cleave when cultured with 10 ppt cacodylate, the remaing 14%
appeared to develop at a normal rate (Table 4, Figure 4). No significant effect
of the treatment procedure as a whole was detected; but when the control was
omitted and the remaining treatments analyzed for a linear relationship, signifi-
cance was achieved (Table 5, Figure 5). Photograph 8 is an embryo characteristic
of the uncleaved ones resulting from treatment with 10 ppt and 1 ppt cacodylate.
Sodium Chloride. Only treatments of 10 ppt and 1 ppm NaCl significantly
prevented cleavage (100% and 14%, respectively) as compared to 7% for control
embryos. Treatments with 1 ppt, 100 or 10 ppm resulted in a significant improve-
ment in the ability of the embryos to cleave. A significant retardation in embryo
development occurred at 1 ppm, 100 ppb and 10 ppb; a significant improvement took
place when the embryos were grown in 10 ppm NaCl (Table 6, Figure 6). Leucine
incorporation corresponded to the developmental results. Treatments of 1 ppt,
10 ppm and 1 ppm significantly increased the incorporation of leucine while a
significant decrease was noted with levels of 10 ppm, 100, 10 and 1 ppb (Table 7,
Figure 7).
Embryo Transfers. Embryos cultured in vitro for 24 hours were transferred to
both horns of 75 pseudopregnant females. Of these females 5 were pregnant on day
12. Because of this low success rate transfers of treated embryos were not made.
Rabbit Embryos
Over 800 rabbit embryos were cultured in various concentrations of cadmium,
5-fluorouracil, cacodylic acid, and sodium chloride. During 24 hours of culture
in BMOC2 the 8-cell embryo cleaved into a morula.
30
-------
100
80
60
EimbryM cleaving
Embryos
Ctoavfnc einbtyo*
a monite
or not
Embryo* cleaving abnormally
CONTROL
Concentration of cacottylk acid in the incubation medium
100 PPB
Fig.4 Developmental response of mouse embryos foNowiny; 27 hours of culture with cacodylic acid
-------
(jO
to
a«
o
II
I?
o e
• a
i!
£ «
o S
750
600
450
300
150
Embryo*
Embryo*
fMMratkig, or not
Incorporated by
mcorporatad by «
bryo* fragnwiTtlnc, d*g*m>r-
atlnf, or not cleaving
100
80
60
40
20
*
I?8
1*
•5 9
I!
if
2
CONTROL
10 PPT
1 PPT
1OO PPM
10 PPM
1 PPM
100PPB
Concentration of cacodylic acid in the Incubation medium
Fig. 5 Incorporation by mouse embryos of •*H-leucine into acid precipitable
protein after 24 hours of incubation with cacodylic acid
-------
OJ
OJ
tu
II
II
Fig. 6 Developmental recpome of mourn* embryo* Mkru^ng 27 hour* of cutture wtth (odhim chtorMe
-------
Embryo* cleaving
3H-huclne Incorporated by
cleaving embryo*
OJ
5400
52SO -
«
600
45°
«»
iso
Embryos li agnienUiia, do- E3 ^H-leuclne Incorporated by em-
generating, or not cleaving ~ bryo* fragmenting,' degenerating,
or not cleaving
CONTROL 10 PPT 1 PPT 1OO PPM 10 PPM
Concentration of •odium chloride in the Incubation medium
- 100
- 40
- 20
Fig. 7 Incorporation by mouse embryos of ^Meucine into acid precipitaMe protein after 24 hours of incubation
with sodium chloride
-------
Table 4. Developmental response of mouse embryos cultured in the presence of cacodylic acid
Ul
Treat- No. of in- Total No.
ment cubations of embryos
Control 23 221
10 ppt 10 96
1 ppt 9 86
100 ppm 24 230
10 ppm 14 140
1 ppm 14 135
100 ppb 14 131
Embryos fragmenting, Embryos
degenerating, or not cleaving
cleaving
3
1%
83
87%
n%c
3
1%
0
0%
2
2%
0
0%
218
99%
13
14%
77
90%c
227
99%
140
100%
133
99%
131
100%
Stage of development of
cleaving embryos
ICM
23
11%
0
0%
0
0%
31
14%
28
20%
14
11%
18
14%
B
108
50%
5
39%
12
16%
107
47%
66
47%
47
35%
62
47%
M
87
40%
8
62%
65
84%C
89
39%
46
33%
72
54%
51
39%
A
0
0%
0
0%
o%c
0
0%
0
0%
0
0%
0
0%
cSignificantly different from control (P < .05)
-------
Table 5. Metabolic response of mouse embryos cultured in the presence of cacodylic acid
UJ
Embryos cleaving
Treat-
ment
Control
10 ppt
1 ppt
100 ppm
10 ppm
1 ppm
100 ppb
Total No.
of embryos
221
96
94
230
140
135
131
No. of in-
cubations
23
3/la
10/9a
24
14
14
14
No. of
embryos
218
13/10b
85/84b
227
140
133
131
Percent
of total
99
14
90
99
100
98
100
Embryos fragmenting, degenerating
or not cleaving
DPM No. of in-
cubations
519 2/Oa
0 9
147 1
390 3/Oa
487
407 2/Oa
671
No. of
embryos
3/Ob
83
9
3/Ob
2/Ob
Percent
of total
1
86
10
1
0
2
0
DPM
136
0
aNumber of incubations in which embryos responded in a given manner/number of these incubations used for
incorporation determinations
Number of embryos having a specific response/number of these embryos used for incorporation determinations
cSignificantly different from control (P < .05)
-------
Table 6. Developmental response of mouse embryos cultured in the presence of sodium chloride
CO
Treat No. of in- Total No.
ment cubations of embryos
Control 14 134
10 ppt 9 83
1 ppt 9 91
100 ppm 10 96
10 ppm 7 62
1 ppm 7 65
100 ppb 9 75
10 ppb 9 87
1 ppb 7 62
Embryos fragmenting, Embryos
degenerating, or not cleaving
cleaving
10
7
83
100C
1
1%C
1
1%C
0
0%C
9
14%°
8
11%
8
9%
0
0%c
124
93%
0
0%c
90
99%C
95
99%C
62
100%c
56
86%c
67
89%
79
91%
62
100%c
Stage of development of
cleaving embryos
ICM
9
7%
0
0%
2
2%
2
2%
4
6%
1
2%c
0
0%c
2
3%c
0
0%c
B
40
32%
0
0%
25
28%
43
45%
29
47%
10
18%c
25
37%c
21
27%c
23
37%6
M
75
61%
0
0%
63
70%
50
53%
29
47%
43
77%c
42
63%c
56
71%c
39
63%c
A
0
0%
0
0%
0
0%
0
0%
0
0%
2
4%c
0
0%c
0
0%c
0
0%c
' L'.-antly different from control (P < .05)
-------
Table 7. Metabolic response to mouse embryos cultured in the presence of sodium chloride
CO
oo
Embryos cleaving
Treat-
ment
Control
10 ppt
1 ppt
100 ppm
10 ppm
1 ppm
100 ppb
10 ppb
1 Ppb
Total No.
of embryos
134
83
91
96
62
65
75
87
62
No. of in-
cubations
13
—
9
10
7
6
8
8
7
No. of
embryos
124
90
95
62
56
67
79
62
Percent
of total
92
0
99
99
100
86
89
91
100
DPM
346
355C
295C
423C
431C
189C
235C
268C
Embryos fragmenting, degenerating
or not cleaving
No . of in-
cubations
3/la
9
I/O3
I/O3
1
1
1
No. of
embryos
10/8b
83
l/0b
l/0b
9
8
8
Percent of
total
8
100
1
1
0
14
11
9
0
DPM
85
126
5265
506
290
aNumber of incubations in which embryos responded in a given manner/number of these incubations used for
incorporation determinations
Number of embryos having a specific response/number of these embryos used for incorporation determinations
cSignificantly different from control (P < .05)
-------
Cadmium. A significant effect on the number of embryos cleaving was caused
by 100 ppm and 10 ppm (0 and 0 vs. 98% for control). Since all cleaving embryos
achieved the morula stage no effect on development was observed. Among the
3
cleaving embryos there was no significant difference in H-leucine incorporated
into protein (Table 8, Figure 8).
Fluorouracil. All embryos treated with fluorouracil, as well as those which
served as controls, were morula at the time of examination. There was no effect
of treatment upon cleavage or stage of development (Table 9). In Table 9 a
disparity can be observed in the two types of blanks processed. Those blanks
taken from control media were much higher than those of treatment media. This
may have been caused by an unknown microbial growth in the culture wells during
the incubation. This disparity in blanks and the response of embryos in 100 ppb
F do not allow for a meaningful interpretation of the data.
Cacodylic Acid and Sodium Chloride. In each of these sets of data, Tables
9 and 10 respectively, the blank is high and the data for the leucine incorporated
is erratic. No interpretation is possible from these data. The data on the
embryos' ability to cleave is acceptable. It can be observed that either 10 ppt
of cacodylic acid or NaCl, and 1 ppt of cacodylic acid inhibits cleavage.
Swine Embryos
From 31 gilts 187 embryos were placed in culture and treated as a control
or treated with a dilution of fluorouracil or cadmium. Few of the embryos
completed one cleavage when cultured in SOF, and the incorporation data do not
reflect valid measurements (Table 12).
39
-------
-p-
o
900
600
300
r~l Embryo* cleaving
B Embryo* fragmenting, de-
generating, or not cleaving
^l-leucme Incorporated by
cleaving embryo*
ni-touchw Incorporated by
•mbryo* fragm*ntlng, dogon-
•ratlng, or not cleaving
100
80
60
40
20
CONTROL
100 PPM
10 PPM
100 PPB
10
Fig. 8
Concentration of cadmium In the incubation medium
Incorporation by rabbit embryos of 3H-teucln« Into acid preclpitabto
protein after 24 hours of Incubation with cadmium
-------
Table 8. Developmental and metabolic response of rabbit embryos cultured in the presence of cadmium
Treat-
ment
Control
100 ppm
10 ppm
1 ppm
100 ppb
10 ppb
1 ppb
Total No.
of embryos
40
25
25
25
25
20
20
1
No. of in-
cubations
8
-
-
5
5
4
4
Embryo clea-\
No. of
embryos
39
—
—
25
25
20
20
ring
Percent DPM
of total
98 757
oc
Oc
100 882
100 767
100 771
100 721
Embryos fragmenting , degen
or not cleaving
No., of in- No. of Percent
cubations embryos of total
l/0a l/0b 2
5 25 100
5 25 100
- — 0
0
0
- — 0
ierating
DPM
74
263
Blank
166
aNumber of incubations in which embryos responded in a given manner/number of these incubations used for
incorporation determinations.
Number of embryos having a specific response/number of these embryos used for incorporation determinations.
Significantly different from control (P < .05)
-------
Table 9. Developmental and metabolic response of rabbit embryos cultured in
the presence of fluorouracil
NJ
Embryo cleaving
Treat-
ment
Control
1 ppt
100 ppm
10 ppm
1 ppm
100 ppb
10 ppb
Blank1
Blank2
Total No.
of embryos
34
15
30
25
20
20
15
No. of in-
cubations
7
3
6
5
4
4
3
11
3
No. of
embryos
34
15
30
25
20
20
15
Percent
of total
100
100
100
100
100
100
100
DPM
24,662
444
59
772
56
0
82,337
69,059
1,779
1
Subtracted from control
"Subtracted from treatments
-------
Table 10. Developmental and metabolic response of rabbit embryos cultured in the presence of cacodylic acid
Treat-
ment
Control
10 ppt
1 ppt
100 ppm
10 ppm
1 ppm
100 ppb
Blank
Total No.
of embryos
53
29
30
25
30
25
25
No . of in-
cubations
11
—
5
5
6
5
5
5
Embryo cl
No. of
embryos
53
—
25
24
30
25
25
eaving
Percent
of total
100
Oc
83C
96
100
100
100
DPM
27,741
—
102,730
27,419
21,512
14,036
61,406
3,832
Embryos fragmenting, degenerating
or not cleaving
No. of in- No. of Percent DPM
cubations embryos of total
o
6 29 100C 9,401
1 5 17C 0
I/O3 l/0b 4
o
o
o
aNumber of incubations in which embryos responded in a given manner/number of these incubations used for
incorporation determinations.
Number of embryos having a specific response/number of these embryos used for incorporation determination.
cSignificantly different from control (P < .05).
-------
Table 11. Developmental and metabolic response of rabbit embryos cultured in the presence of sodium chloride
Treat-
ment
Control
10 ppt
1 ppt
100 ppm
10 ppm
1 ppm
100 ppb
10 ppb
Blank
Total No.
of embryos
34
34
35
35
34
30
25
25
No. of in-
cubations
7
2
7
7
7
6
5
5
16
Embryo clea
No. of
embryos
33
6
30
34
33
30
22
22
ving
Percent
of total
97
18C
86
97
97
100
88
88
DPM
49,596
0
21,963
0
0
19,469
14,375
25,171
37,568
Embry<
No. of in-
cubations
l/0a
6
2
I/O3
l/0a
1
1
js fragment
or not cl
No. of
embryos
l/0b
28
5
l/0b
l/0b
3
3
ing, degener
eaving
Percent
of total
3
82C
14
3
3
0
12
12 11
at ing
DPM
417
0
0
,075
aNumber of incubations in which embryos responded in a given manner/number of these incubations used for
incorporation determinations.
"Number of embryos having a specific response/number of these embryos used for incorporation determinations.
cSignificantly different from control (P < .05)
-------
Table 12. Metabolic response of swine embryos cultured in the presence of fluorouracil or cadmium
Treatment
Control
100 ppm F
10 ppm F
1 ppm F
100 ppb F
Control
10 ppm Cd
1 ppm Cd
No. of incubations
16
3
5
3
5
7
3
3
No. of embryos
71
11
22
14
19
26
13
11
o
DPM of H-leucine incorporated/
5 embryos
632
1206
409
620
1026
361
244
580
-------
DISCUSSION
It is obvious from these observations that a direct relationship exists
between the morphology and function of the mammalian embryo and the concentration
of certain contaminants which may reach the embryo's environment during its early
stages of development. Four different substances are used in the culture medium
to demonstrate this relationship. Two of the substances, cadmium and fluor-
ouracil, used in this investigation are known to be teratogenic. A third substance,
cacodylic acid, is a substance which is of interest because it comes from a family
of compounds known to have teratogenic effects. The fourth substance, sodium
chloride, a substance which is required for normal embryo growth; was used as a
control substance. It is teratogenic but only at very high concentrations (Nishi-
mura and Mujamata, 1969).
In order to avoid being misled by responses which are limited to only one
species these substances were tested in embryos from three different species of
mammals; the mouse, the rabbit and the porcine. Much of the preliminary obser-
vation and development of technique was done on mouse embryos because of their
availability and ease in handling logistic problems. The rabbit was used as
representative of that class of species which have a slightly different repro-
ductive pattern in that ovulation is induced rather than spontaneous. The porcine,
the third species studied, was used as a representative of a non laboratory
species, a species intermediate between the classical laboratory animal and the
primate.
These results support the concept that young mammalian embryos in vitro are
responsive to a variety of contaminants which can be incorporated in their environ-
ment. Different concentrations of the material in the environment influences the
type of response elicited in the embryo. The higher concentration results in the
46
-------
death of the embryo, but a lower concentration of the material may retard embryo
development. Still other concentrations of the material will interfere witli the
function of the embryo as measured by protein synthesis. For example, cadmium
appears to be the most toxic of the substances studied. It causes a high rate
of death in the embryos subjected to it. On the other hand, fluorouracil results
in a negative linear relationship between the concentration of the substance and
the incorporation of leucine into protein. These data suggest that substances
such as cadmium are effective over a very limited range of concentrations. How-
ever, this substance may also provide linear relationships providing sufficient
intermediate concentrations were used in the culture media. To demonstrate the
sensitivity of the embryo to contaminates further, it is apparent that certain sub-
stances in proper concentrations cause a different response among the individual
cells of the embryo. For example, a concentration of 100 ppm of fluorouracil will
alter the morphology of some of the blastomeres within a given embryo while other
blastomeres appear to be normal.
The observations made on embryos from a second species, the rabbit, demon-
strate that this system is effective in more than just the mouse. The response
of rabbit embryos as measured by cleavage rate is similar to that observed in the
mouse embryos when subjected to the same compound. This indicates that the effect
is not limited to one species.
Observations made of swine embryos indicate that a prerequisite for this
system requires a. culture medium which will support embryo growth in vitro. It is
obvious from these observations that the precise environmental condition required
for normal growth in vitro is different for the three different species. This
simply indicates the precise sensitivity of the mammalian embryo to its immediate
environmental surroundings. There was no indication from these observations, given
-------
the proper culture medium for normal growth, that swine embryos would not respond
similarly to mouse and rabbit embryos when subjected to these compounds. Con-
sequently, these observations suggest that the embryo from any mammalian species,
including primate, would respond to pollutants in the manner described in this
study providing a culture medium has been developed which supports normal embryo
growth in vitro for this particular species. Culture media for normal embryo
growth are now available for a number of mammalian species including the primate.
The results of this study support the concept that young mammalian embryos
can be used to detect the presence of harmful substances in the environment as a
preliminary testing system. It would be rapid, inexpensive and efficient. The
observations indicate that such a system is feasible and its continued develop-
ment should be pursued.
48
-------
SUMMARY
A one year contract was awarded to develop and test the concept that young
mammalian embryos could be used to measure the harmful effects of contaminants
that may be teratogens.
Embryos from three species of mammals were used; the mouse, rabbit and swine.
Four different substances were studied; 5-fluorouracil, cadmium, cacodylic acid
and sodium chloride. About 3300 embryos from mice were used in the development
of the technique.
In general, the results of the study indicate that young mammalian embryos
are responsive to a variety of contaminants that may be found in their environ-
ment. Different concentrations of the contaminant will elicit a different kind
of response in the embryo. Higher concentrations result in death, but a lower
concentration may retard the development of the embryo. Still other concentrations
will interfere with the metabolic function of the embryo. Cadmium appears to be
the most toxic of the substances studied. It causes the highest rate of embryo
death. On the other hand, fluorouracil results in a negative linear relationship
between the concentration of the substance and the incorporation of leucine into
protein. It appears that some substances are effective over a narrow range while
others are effective over a wider range.
Observations from these studies suggest that the contaminants act similarly
on mammalian species. However, it is obvious that each species has its own precise
environmental requirements for normal growth in vitro.
It is concluded that the procedure as reported is capable of determining the
type of effect that a contaminant will have on the development of the mammalian
embryo.
49
-------
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54
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Appendices
55
-------
Compound
NaCl
Appendix A
BRINSTER'S MEDIUM FOR OVICULTURE (BMOC2)
mM
Grams/Liter
Na Lac*2
Na Pyr7
KC1
CaCl
MgS04.7H20
94.88
25.00
0.25
4.78
1.71
1.19
1.19
25.00
5.5460
2.2530
0.0280
0.3560
0.1890
0.1620
0.2940
2.1060
1 ml
0.2 ml
1.0000
1.0000
Solids are added to 1 liter of double glass distilled H20 and filtered through
a Millipore filter (pore size, 0.45/j) into a sterile flask. After filtration,
flask openings are covered with sterile gauze, and the flask is placed in a 37°
incubator (atmosphere 5% C02, 95% air) overnight. Sterile serum bottles are filled
with the medium using a sterile Cornwall tubing apparatus under an ultra violet
hood. After bottling, pH is checked. Ideal pH after gassing and bottling is
7.4 + 0.05. BMOC2 is stored under refrigeration for up to 3 months.
Penicillin**
Streptomycin***
f
Bovine serum albumin, fraction V
Glucose*
*4.73 ml of 60% sodium lactate can be used per liter instead of neutralized
lactic acid. (Sigma, DL-Lactic Acid, 60% Grade V, Sodium Salt).
**Add 10 ml sterile double glass distilled H20 to 1 million units penicillin; add
1 ml of this solution to 1 liter of culture medium (Squibb, Buffered Potassium
Penicillin G, 1 million units) 100 units/ml.
56
-------
***Add 4 ml sterile double glass distilled H20 to 1 gm Strep; add 0.2 ml of this
solution to 1 liter of culture medium (Lilly, Streptomycin Sulfate U.S.P.)
50 Mg/ml.
Sources for chemicals
^Fisher for Scientific Matheson, Coleman & Bell
Sigma "Nutritional Biochemicals
3Allied Chemicals 7Calbiochem
^J. T. Baker Chemical
57
-------
Compound
Appendix B
SYNTHETIC OVIDUCT FLUID (SOF)
Gram/liter of stock
solution (0.154 M,
except where noted)
Milliliters of stock
solution for 50 ml of
culture medium
NaCl1
KC11
KH2P041
CaCl2.2H205
MgCl2
N3HC021
Na Lac**2
Na Pyr7
Glucose*
9.001
11.482
20.958
14.702*
14.665
12.938
6.469
11.100 (0.1M)
34.96
2.325
0.386
0.855
0.159
8.140
1.071
0.165
dry 13.5 mg
Bovine serum albumin
(fraction V) +6
Penicillin
Streptomycin
H2°
dry 1.6 gm
100 units
50
0.990
From refrigerated stock solutions and solids, SOF is mixed and filtered through
a Millipore filter (pore size, .45 ) into a sterile flask. Penicillin and
streptomycin are added as in BMOC2- After filtration, flask openings are covered
with sterile gauze. Medium is gassed overnight in a 37°C incubator with an
atmosphere of 5% CO and 95% air. The following morning the medium is put in
Sterile serum bottles using a sterile Cornwall tubing apparatus. A bacteriology
hood-with UV lighting is used for bottling the medium. SOF is refrigerated until
needed.
*CaCl2 = 0.100 M stock
**4. 73 ml of 60% sodium lactate can be used per liter instead of neutralized lactic acid.
58
-------
-1- For a flushing solution of SOF 50 mg BSA was added instead of 1.6 gm
Sources of chemicals
Fisher Scientific Matheson, Coleman & Bell
2Sigma "Nutritional Biochemicals
o 7
JAllied Chemicals Calbiochem
^J. T. Baker Chemical
59
-------
Appendix C
PROTOCOL FOR THE SUPEROVULATION OF RABBITS
1. Inject 0.4 mg FSHP (Armour Baldwin) in methyl cellulose* (1 mg/ml) for
4 days, subcutaneously.
2. On the fourth day of FSH treatment the doe is mated to a fertile male.
3. Breeding is followed by intravenous injection of 2.5 mg of PLH (Armour
Baldwin). Diluent is saline (5 mg LH/ml of saline).
*Methyl cellulose (100 centipoise - Fisher). Add 1 mg. methyl cellulose per ml
distilled H?0 and put in serum bottles with serum stoppers. Autoclave at 15 Ibs.
250°F for 30 min.
60
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-6QO/1-79-Q07
3. RECIPIENT'S ACCESSION NO.
TITLE ANCLSUBTITLE
5. REPORT DATE
Development of Isolated Mammalian Embryo
Techniques for Toxic Substance Screening
6. PERFORMING ORGANIZATION COPE
AUTHOR(S)
R.F. Williams, Q.S. Inman and L.C. Ulberg
8. PERFORMING ORGANIZATION REPORT NO.
PERFORMING ORGANIZATION NAME AND ADDRESS
North Carolina State University
Raleigh, NC
January 1979
10. PROGRAM ELEMENT NO.
1LA629
11. CONTRACT/GRANT NO.
68-02-1769
2. SPONSORING AGENCY NAME AND ADDRESS
US Environmental Protection Agency
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A potential screen for assessing teratogenic potential of compounds in mammals
was tested. The technique involves testing isolated mammalian embryos in culture by
direct exposure to agents. Embryos from three species of mammals were used; the
mouse, rabbit and swine. Four different substances were studied; 5-fluorouracil,
cadmium, cacodylic acid and sodium chloride. About 3300 embryos from mice were
used in the development of the technique. In general, the results of the study
indicate that young mammalian embryos are responsive to a variety of contaminants
that may be found in their environment. Different concentrations of the contaminant
will elicit a different kind of response in the embryo. Higher concentrations
result in death, but a lower concentration may retard the development of the embryo.
Still other concentrations will interfere with the metabolic function of the embryo.
Cadmium appears to be the most toxic of the substances studied. It causes the
highest rate of embryo death. On the other hand, fluorouracil results in a negative
linear relationship between the concentration of the substance and the incorporation
of leucine into protein. It appears that some substances are effective over a
narrow range while others are effective over a wider range. It is concluded that
the procedure as reported is capable of determining the type of effect that a
contaminant will have on the development of the mammalian embryo.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFieRS/OPEN ENDED TERMS C. COSATI h'icld/Group
Embryo! ogy
Organics
Trace Metals
Screening Techniques
06, T
18. DISTRIBUTION STATEMENT
Release To Public
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. Of PAGES
69
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
61
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