&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

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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

-------
     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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   in vitro.   J. Embryo Exp. Morph. 15:387-395.

Ulberg,  L. C., and Sheean,  L. A. (1973).  Early development of mammalian embryos
   in elevated ambient temperatures.  J. Reprod. Fert. Suppl.  19:155-161.

Van Blerkom, J. and Manes,  C.  (1974).  Development of preimplantation rabbit
   embryos in  vivo and in vitro.   2. Comparison of qualitative aspects of protein
   synthesis.  Dev. Biol. 40:40-51.

Van Blerkom, J. and Brockway, G. 0. (1975).  Qualitative patterns of protein
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   424-425  (Abst.).

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                                         53

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   Produce Congenital Defects.  American Elsevier Publishing Company, Inc. New
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Yashihara, H.  (1968).  The effect of 5-fluorouracil on the development of tail
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                                        54

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Appendices
       55

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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

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***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

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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

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-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

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                                  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

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                                  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
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                                             61

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