= = — = = _ — — —— — — — — — = = llIlllllllllllllllllllllllllllll 293 01020 5866 flaw": Hi"? This is to certify that the thesis entitled REPRODUCTIVE TOXICITY OF 3,3',4,4'- TEIRACHIoROBIPHENYL IN MICE presented by Jaime Rodriguez has been accepted towards fulfillment of the requirements for M. 8. degree in Zoology mdw/Wzfl/ Major professor Date 11/09/94 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACED! RETURN aoxmmmumummm TOAVOID FlNESntunonorbdmdutodm. ’ DATE DUE DATE DUE DATE DUE l MSU lsAnNflnnllvo NONE“ OW Institution m1 REPRODUCTIVE TOXICITY OF 3,3',4,4'-TETRACHLOROBIPHENYL IN MICE By Jaime Rodriguez A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1 994 ABSTRACT REPRODUCTIVE TOXICOLOGY OF 3,3',4,4'-TETRACHLOROBIPHENYL IN MICE By Jaime Rodriguez There are a possible 209 polychlorinated biphenyls (PCBs) with physicochemical properties ranging from inflammability to lipophilicity. These properties have made them prime candidates for industrial use. However, as a result of their use, improper disposal practices, and accidents, PCBs have found their way into the environment. Ironically, the same properties have made them an environmental pollutant. 3,3',4,4'-Tetrachlorobiphenyl (TCB) is one of the most toxic PCBs. In this study, female C57BU6J mice were gavaged with either 7, 14, or 21 mg/kg of TCB during days 1-5, 6-10 or 11-15 of gestation (day 1 = vaginal plug presence). No maternal or pup parameter studied was significant. In vitro fertilization trials revealed a significant effect of TCB on in vitro fertilization, the number of degenerative ova and the number of abnormal 2-cell embryos. To Spark of Revolutions ACKNOWLEDGMENTS I wish to thank my committee members Drs. Lynwood G. Clemens, Cheryl L. Sisk and W. Richard Dukelow. A special thanks to Dr. Sanjiva D. Kholkute without whose help these past two years could not have been possible. This work was supported by NIH grants HDO7534 and ESO4911. Thanks are also extended to the Office of Urban Affairs through which I received the Minority Competitive Doctoral Fellowship. TABLE OF CONTENTS LIST OF TABLES ......................................................................................... vii LIST OF FIGURES ...................................................................................... viii INTRODUCTION .......................................................................................... 1 LITERATURE REVIEW ................................................................................ 3 Mouse Reproduction and Development ............................................ 3 Polychlorinated biphenyls ................................................................. 4 3,3',4,4'-Tetrachlorobiphenyl ............................................................. 6 Metabolism ........................................................................................ 6 Carcinogenicity .................................................................................. 9 Reproductive Toxicology ................................................................... 11 MATERIALS AND METHODS ...................................................................... 13 In Vivo Fertilization Trials .................................................................. 13 Animals ................................................................................... 13 Chemical ................................................................................. 13 Dosage ................................................................................... 13 Administration ......................................................................... 15 Mating ..................................................................................... 16 Data Collection ....................................................................... 16 Statistical Analysis .................................................................. 17 In Vitro Fertilization Trials .................................................................. 17 Animals ................................................................................... 17 Chemical ................................................................................. 17 Culture Medium ....................................................................... 18 Superovulation ........................................................................ 18 Gamete Recovery and NF ..................................................... 18 Statistical Analysis .................................................................. 20 RESULTS ..................................................................................................... 21 In Vivo Fertilization Trials .................................................................. 21 Maternal parameters ............................................................... 21 Pup parameters ...................................................................... 29 In Vitro Fertilization Trials .................................................................. 29 Fertilization rates for TCB ....................................................... 29 DISCUSSION ............................................................................................... 36 SUMMARY AND CONCLUSIONS ................................................................ 4O BIBLIOGRAPHY ........................................................................................... 41 APPENDIX ................................................................................................... 48 Appendix A ........................................................................................ 48 vi Table LIST OF TABLES Page Identification and physical properities of 3,3',4,4'-tetrachloro- biphenyl (TCB) .................................................................................... 14 Components of the two culture media used in the TCB in vitro fertilization trials .................................................................................. 19 Maternal body weight of CS7BU6J mice administered TCB by gavage on days 6 to 10 of pregnancy ................................................. 22 Liver and kidney weights of female C57BU6J mice administered TCB by gavage .............................................................. 26 Litter size and total number of male and female mice delivered byCS7BL/6J females exposed to TCB by gavage ............................... 30 Crown-rump length of B602F1 mice for days 1 to 22 after dam was exposed to TCB by oral gavage .................................................. 34 Effect of TCB on in vitro fertilization in 8602F1 mice ......................... 35 Effect of TCB on oocyte degeneration and abnormal embryos ........... 35 vii LIST OF FIGURES Figure Page 1. Proposed metabolism of TCB ............................................................... 7 2. Mean body weight pre- and postpartum of C57BU6J mice administered 7 mglkg TCB by gavage ................................................ 23 3. Mean body weight pre- and postpartum of CS7BU6J mice administered 14 mglkg TCB by gavage .............................................. 24 4. Mean body weight pre- and postpartum of C57BU6J mice administered 21 mglkg TCB by gavage .............................................. 25 5. Mean body weight on day 8 prepartum of C57BU6J mice administered TCB by gavage ............................................................. 27 6. Mean body weight on day 15 prepartum of CS7BU6J mice administered TCB by gavage .............................................................. 28 7. Mean pup weight of B602F1 mice for days 1 to 22 after dam was exposed to 7 mglkg TCB by gavage ............................................ 31 8. Mean pup weight of 8602F1 mice for days 1 to 22 after dam was exposed to 14 mglkg TCB by gavage .......................................... 32 9. Mean pup weight of B6DZF1 mice for days 1 to 22 after darn was exposed to 21 mglkg TCB by gavage .......................................... 33 viii Introduction Polychlorinated biphenyls (PCBs) were first produced in the United States in the late 1920s with peak production in the mid 19705. Since that time they have been banned for use in the United States. Polybrominated biphenyls (PBBs) were first produced in the early 19705 and their use is regulated by the United States government. These halogenated aromatic chemical mixtures are highly stable, practically insoluble in water, chemically inert and emit toxic fumes when heated to decomposition. Before 1972, it was common to use PCBs in transformer cooling systems, heat transfer and hydraulic fluids, lubricants, plasticizers, sealants and copy paper. Since 1974, PCBs have been confined to closed systems such as electrical capacitors and transformers, vacuum pumps and gas transmission turbines. On the other hand PBBs were used as flame retardant additives in synthetic fibers and molded plastics, polyesters, polystyrenes and polyoletins, though presently PBBs are not used in consumer products. Because of their persistent nature, both PCBs and PBBs, are long lasting environmental contaminants. PCBs have been detected in mammalian and non-mammalian species in both the industrialized and non- industrialized areas of the world such as the Arctic and Antarctic. As environmental contaminants, PBBs are similarly widespread. PCBs and P883 are retained in body fat and are excreted in feces, eggs and milk. 1 2 Exposure to these chemicals does not only occur through environmental contamination. Accidental exposure to PCBs in Japan and Taiwan and to PBBs in Michigan have occurred and it was this that brought these chemicals into the limelight. Subsequent studies have consistently shown these chemicals to have negative effects on those exposed. The causative agents of the accidental human exposure in Japan and Taiwan had been considered to be the co-contaminants of PCBs such as polychlorinated dibenzofurans that were formed after heating the P085 in rice oil. However, recent studies have demonstrated the presence of extremely toxic PCB congeners in commercial mixtures and tissue samples. Thus, a re—evaluation of the current status of PCBs and their congeners is needed. One of the most toxic PCB congeners is 3,3',4,4'-tetrachlorobiphenyl, TCB. A review of the existing literature on TCB establishes this compound's teratogenicity, and embryo toxic effects, but there is no consensus on whether the parent compound or a metabolite is the active compound. In vivo and in vitro methods for testing toxicity differ, which can lead to conflicting results. Furthermore, TCB studies also show toxicity to vary with the method of administration--injection, ingestion, or inhalation. Reproduction is considered as one of the most viable means of testing a compound's toxicity. Although previous studies have demonstrated TCB's toxic effects, a more thorough analysis is needed. The present work examines the toxic effects of TCB throughout the entire murine gestation period by oral ingestion (gavage) for five consecutive days beginning on either day one for the period of implantation, day five for the period of organogenesis or day 11 for the period of embryogenesis. A vaginal plug presence the day after the female mouse is placed with the male is day 1. LITERATURE REVIEW Mouse Reproduction and Development The mouse estrous cycle is four to five days long. To maintain a regular cycling, a day-night cycle should be kept constant. In natural matings, insemination, ovulation, and fertilization occur during the dark period, usually between 10:00 p. m. and 2:00 am. Thus, twenty-five percent of natural matings in any one night are usually successful. The presence of a vaginal plug, coagulated ejaculum, the next morning permits the recognition of a successfully mated female and such a presence is denoted as Day 1 of pregnancy. A gestational period of 19 to 21 days follows. Females experience a postpartum estrus (Theiler, 1989). The ovulated ova are in the metaphase stage of the second meiotic division until fertilization by spermatozoa has occurred. Fertilization normally occurs in the ampulla of the oviduct. By 24 hours, the zygote(s) or embryo(s) will have moved into the first and second loop of the oviduct and will have completed the first cleavage division, yielding two blastomeres of about equal size. Three days later, the presence of 16 cell embryos (morulae) is evident. Blastomeres are of unequal size and dividing mitotically. Compaction of the embryo is readily apparent after the 8 cell stage. By the third or fourth day, embryos will have moved into the uterus and the blastocoele forms. The embryo is now called a blastocyst. During 3 4 this time, the distance between the uterine blastocysts increases and the corpora lutea become profoundly vascularized. Most blastocytes will have arrived in the uterus by the fourth day clearly spaced and free within the lumen. The zona pellucida will have disappeared and the blastocyst clearly separated into an inner cell mass and trophoblast tissue. At 4 1l2 to 5 days, some embryos will have begun implantation by invading the epithelial lining. Implantation is hormonally influenced, but once established, embryo growth rate increases as a result of the glycogen rich decidua. Development between days 5 and 10 is referred as the period of organogenesis. It is marked by rapid development of the basic body systems. Paired somites appear along the anterior-posterior axis after day 7 leading to a total of 65 pairs with the chronological and linear of appearance of somites being an indicator of embryonic age. Tissues derived from the inner cell mass will develop into the rudimentary excretory, circulatory, digestive and nervous systems. They will also develop into the limb and tail buds. By day 10, the sense organ primordia are noticeable, the primordial germ cells are migrating toward the gonads, and the heart is removing metabolic waste while delivering nutrients to the rest of the embryo. The remaining days of development are a period of embryogenesis. During this time organ systems are finalized and chondrification occurs (Rugh, 1967). Polychlorinated Biphenyls Polychlorinated biphenyls (PCBs) are a class of halogenated aromatic compounds. They were first synthesized in 1881 by Schimdt & Schultz. In the United States, they were first produced in 1929 by the 5 Monsanto Company (Tanabe, 1988). Theoretically, there are 209 possible PCBs. Commercially, they are sold under the trade name of Aroclor and each mixture is distinguished by the four numbers (Ballschmiter et al., 1989, Hutzinger et al., 1974). The first two digits, 12, denote polychlorinated biphenyls and the last two digits denote the percent of chlorine in the mixture. PCB production reached its peak in the early 19705. Gross estimates from 1970 indicate that 20% of sales were in service while the remaining 80% was believe to have been discharged into the environment (Nisbet & Sarofim, 1972). This could have occurred through a variety of means such as improper waste disposal (Shcimdt et al., 1971) and leaching from dumps (Lidgett & Vodden, 1970). Environmental contamination by PCBs was first detected while screening environmental samples for DDT and related compounds (Jensen, 1966). Since then, PCBs have been detected in almost every component of the various global environments. They have been detected in the North Pacific and Indian Oceans (Tanabe & Tatsukawa, 1980) and the Great Lakes in the United States (Mackay et al., 1983). PCBs have been detected in mammalian and non-mammalian species (Jensen et al., 1977, Bennington et al., 1975). Accidental exposure of humans to PCBs has occurred in Japan (Higuchi, 1976) and Taiwan (Chen et al., 1981 ). In both cases, the poisoning was a result of cooking rice in PCB contaminated bran oil. It was believed that the symptoms derived from the poisoning were caused by co- contaminants of PCBs such as polychlorinated dibenzofurans (Masuda & Yoshimura, 1984). Recent studies however, have since detected the presence of 3',4'-tetrachlorobiphenyl, an extremely toxic PCB congener, as a 6 component of the PCB mixture (Kannan et al., 1987, Tanabe et al., 1987, Yoshimura & Yamamoto, 1974). 3,3',4,4'-Tetrachlorobiphenyl (TCB) TCB attains coplanarity due to its non—ortho chlorine substitution in the biphenyl rings and because it is an approximate isostereomer of 2,3,7,8— tetrachlorodibenzo-p-dioxin (T4CDD), it elicits similar toxic and biological responses of dioxins and furans (Safe, 1984). Detection of TCB in PCB mixtures was virtually impossible until recently when a simple and sensitive analytical method was developed to determine the amount of TCB in PCB residue(Tanabe et al., 1987). Studies on TCB indicate that exposure to TCB can result in teratogenicity, carcinogenity and reproductive toxicity (Cornwall et al., 1984; Sargent et al., 1991; Agrawal et al., 1981; Wehler et al., 1990, Lucier et al., 1977). Metabolism PCB mixtures will vary with respect to chlorine content and their relative distribution of individual congeners. PCBs are metabolized directly or via arene oxide intermediates into phenolic metabolites, that can be hydroxylated or conjugated to form catechols and phenolic conjugates (Safe, 1994). Unlike the other coplanar and mono-ortho coplanar, TCB is readily metabolized. Yoshimura and Yamamoto (1973)reported that rats given TCB at an oral dose of 25 mglkg every third day produced three fecal metabolites in addition to the unchanged TCB. No metabolite was detected in the urine. A study of 2,5,2',5'-tetracholorbiphenyl by Van Miller et al. (1975) had different 12:93.th5:oelcmmen loo. .0 _U Com? ._m Ho mSEEmoC .mo... .6 EEBnSoE ommoooi .359“. 8 results. Tritiated TCB was administered to rats. The rats were sacrificed and their tissues examined for the presence of TCB and its metabolites. The results indicated that 66% of TCB and its metabolites was present in the feces and that 10% was present in urine within three days. Yoshimura and his co-workers (Yoshimura et al., 1987) took an extensive look at the metabolic fate of TCB (Figure 1). The PCB congeners are categorized according to their ability to induce liver enzymes. TCB is categorized as an 3-methylcholamthrene (MC-type) and as such is acutely more toxic than phenobarbital (PB-type) inducers (Yoshimura et al., 1979). However, it had been reported that the 5-hydroxy-metabolite of 2,4,3'4'-TCB was more toxic that its parent compound, TCB (Yamamoto and Yoshimura, 1973). The 3,4- epoxy- and 4-hydroxy-metabolite of 2,5,2',5'-TCB have been reported to have similar metabolic activations (Stadnicki and Allen, 1979). Similarly, the metabolites of TCB in a chick embryotoxicity study were more toxic than the parent compound (Klasson-Wehler et al., 1990). These studies suggesting then that the metabolites may contribute to the toxic effects of TCB. The results of Yoshimura et al. (1987) do not corroborate what has been previously reported. Instead, they found the parent compound to be more toxic than both monohydroxy-metabolites. Furthermore, their results suggest that metabolic activation by hydroxylation occur only in PB-type congeners of PCB, but not in MC-type. PCB metabolites can have other biological activities. They can act as uncouplers of mitochondrial activity (Ebner et al., 1987), inhibit P450 dependent enzyme activity (Schmoldt et al., 1977), and can bind prealbumin (Rickenbacher et al., 1986). It has also been reported that 3,3',4',5— tetrachloro-4-biphenylol, a TCB metabolite, binds with high affinity to 9 transthyretin ('l'l'R), a thyroxin transport protein (Brouwer et al., 1990; Brouwer and Van den Berg, 1986). Thus, hydroxylated TCB metabolites are biologically active, but the significance these activities has not been determined. Carcinogenicty Studies with a variety of PCBs have reported that after exposure to the chemical, laboratory rodents will develop an increased incidence of liver lesions, including neoplastic nodules and hepatocellular carcinomas (Kimbrough et al., 1975; Shaeffer et al., 1984). Studies have been carried out using a variety of protocols and both long and short term assays to determine the incidence of tumors and preneoplastic lesions such as nodules and papillomas. After exposure, PCBs promoted hepatocellular carcinomas and neoplastic nodules in the rat (Graham et al., 1988). Similar effects were observed in the mouse skin and lung (Anderson et al., 1991; Poland et al., 1982) When PCBs are given after an initiator, an agent that causes one or more initial basic changes that lead to early pathogenesis, there is convincing evidence that they have a tumor promoting effect (Pereira et al., 1982). Rats fed 3'-methyI-4-dimethylaminoazobenzene for 2 months follow by Kanechlor 400 for 6 months developed a high incidence of hepatocellular carcinomas (Kimura et al., 1976). This assumption was based on the fact that the administration of the initiator by itself, before or concurrently, did not result in tumors. Some PCBs promote effects more in the liver of female than in male rats (Deml & Oesterle, 1982). 10 PCB mixtures will sometimes contain planar and non-planar congeners. Planar congeners bind to Ah receptors, induce cytochrome P450c and cause effects in the liver and immune cells (Safe at al., 1985; Parkison et al., 1981). Osterele & Deml (1981) found the planar congers to elicit growth of preneoplastic foci. The non-planar congeners are less toxic. They are weak promoters, but do cause hepatic enlargement (Preston et al., 1985). Sargent et al.(1991) studied the effects of planar and non-planar congeners in liver and lymphocytes. Rats were exposed to TCB or 2,5,2',5'- tetrachlorobiphenyl by diet for one year. The results of their study demonstrated that the interaction of both TCBs cause a greater incidence toxicity and mutagenicity in peripheral lymphocytes and hepatocytes than with each TCB alone. Studies suggest that when studying the carcinogenic effect of PCBs, the time of administration is important. PCBs are inducers of hepatic microsomal enzymes and these enzymes can metabolize carcinogens to less potent metabolites. Thus, administration of a hepatocarcinogen such as diethylnitrosamine can inhibit carcinogenesis (Berry et al., 1979; Makiura et aL,1974) Because of their lipophilic nature and persistence in the body, Kimbrough (1979) suggested that prolonged administration is not necessary. For example, a single dose of 1 g/kg over a short period caused a high incidence of hepatocellular carcinomas in rats (Kimbrough et al., 1981). This is in contrast to classic tumor promotors such as phenobarbital that has to be administered over an extended period (Pitot et al., 1982). 11 Reproductive Toxicology The effects of PCBs on reproduction were first studied in the mink (Ringer et al., 1972). But since then the reproductive toxicity of many commercial and PCB congeners have been assessed. Golub et al. (1991) reported the relationship between dose and the lowest-observable-adverse— effect levels, LOAELs, for various reproductive and developmental end points. The results demonstrate increased malformations at 66.0 mglkg, reduced litter size at 10 mglkg and decreased reproductive organ weights, lower birth weights, and decreased postnatal weight gain at 1.0 mglkg. Some studies, however, fail to demonstrate teratogenicity after mice and rats have been exposed to PCBs during embryogenesis (Villeneuve et al., 1971; Shiota, 1976). Ronnback (1991)found no observable effect of 1.5 mglkg and 15.0 mglkg TCB administered as a single dose to pregnant mice on day 13 of gestation. Lucier et al. (1977) had reported earlier that no observable effect appears in animals administered less than 16 mglkg/day. Transplacental movement of PCBs has been detected in a number of species such as primates (Allen et al., 1974) and chicken eggs (Platonow and Reinhart, 1973). The treated female rhesus monkeys were fed 250 mglkg in their diet for two months. Of the five, 2 conceived and aborted, 2 never conceived and the fifth conceived and delivered. The infant, however, weighed less than the average rhesus infant. Studies have evaluated the effects of TCB on reproduction, but none have studied its effects throughout the entire mouse gestation period. This study was undertaken to evaluate the effects of TCB on the following stages of mouse development: implantation, organogenesis and embryogenesis. 12 Although accidental or environmental exposure to levels of TCB tested in this experiment are highly unlikely, it is believed that the dose levels are made valid because the lipophilic nature of PCBs allows for bioaccumulation and biomagnification in a variety of species, including mammals. Since PCBs have been detected in water sources administration of TCB by oral gavage is similar to this natural method of exposure. Other studies have not examined the entire mouse gestation period or have selected to test TCB with a protocol that does not mimic natural exposure in the environment. Furthermore, this study also examines the effects of TCB on the mouse in vitro fertilization system. Because of PCBs' lipophilic nature and because PCBs have been detected in human follicular fluid (Trapp et al., 1984), TCB could have detrimental effects on the oocyte. By studying the effects of TCB in vitro, information can also be obtained on its ability to affect fertilization and preimplantion. In vitro examination of TCB allows for further testing to determine the mechanisms of toxicity as well as it decreases the cost of whole animal in vivo testing. Therefore, this study is an attempt to determine as well as clarify the reproductive effects of TCB both in vivo and in vitro. MATERIALS AND METHODS IN VIVO FERTILIZATION TRIAL Animals Male DBA/2J and female CS7BU6J mice were used. These mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and aged 2-3 months and 7-9 weeks, respectively. The B6D2F1 hybrids were produced by mating DBAI2J males with C57BU6J females at the Endocrine Research Center of Michigan State University. All animals were housed under a 12 hour light/dark photoperiod and maintained in an air conditioned room at 24 :l: 2°C. Feed (Mouse Chow® #5015, Purina Mills, Inc.) and water were available ad Iibitum except where mentioned. Chemicals 3,3',4,4'-tetrachlorobipheny|, 99% pure by gas chromatography and flame ionization detector, GC/FID, was purchased in neat form, catalog number C-077N, from AccuStandard, Inc. (New Haven, CT). The chemical formula for 3,3',4,4'-tetrachlorobiphenyl is C5-H3-Cl2-C5-H3-Cl2. Lot numbers used were #70075, #10162 , and #011893. The physical properties of TCB are listed on Table 1. Dosage Each dose administered was based on the lowest lethal published dose (TDLO) of 7 mglkg of body weight (b.w.) in mice according to the 13 14 Table 1. Identification and physical properties of 3,3',4,4'-tetrachlorobiphenyl (TCB). CAS Number: 32598—13-3 RTECS Number: DV8650000 Component: 3,3',4,4'-tetrachloro-1 ,1 '-biphenyl Chemical family: halogen compound, aromatic Description: colorless crystals with a mild aromatic odor Trade names/Synonyms Molecular formula: Molecular weight: Melting point: Solubility in water: Solvent solubility: Reactivity: Decompostion: 3,3',4,4'-PCB, PCB, tetrachlorobiphenyl, biphenyl, TCB, 4-CB, OHSS7117 06-H3-CL2-C6-H3-CL2 291.99 351-354 F (177-179 C) practically insoluble soluble in acetone, ethanol, methylene chloride, oils and organic solvents stable under normal temperatures and pressures; exothermic reaction with liquid chlorine; fire and explosive hazard with oxidizers; attacks plastics, rubber and coaflngs Thermal decompostion products may include toxic fumes of phosgene, toxic and corrosive fumes of chlorides, oxides of carbon (Occupational Health Services, Inc., 1992) 15 M _Da_ngerog§ Progeirties of Industrial Chemicals (1992). Four doses of 3,3',4,4'-tetrachlorobiphenyl were tested. To each 25 mg vial of 3,3'4,4'- tetrachlorobiphenyl, 1 ml of ethyl alcohol, 100%, was added to dissolve the chemical. The volume of liquid administered to each animal was 0.1 ml and doses were reconstituted in 100% pure extra virgin sesame oil (Loriva® SupremeTM Foods, Inc., Hauppauge, NY) and dissolved 3,3'4,4'- tetrachlorobiphenyl to contain the proper concentrations of the test chemical. Control doses consisted of sesame oil and ethyl alcohol.. Doses were prepared in glass bottles that had been wrapped in aluminum foil and autoclaved. Prior to each dosing, the control and treatment solutions were mixed using a Deluxe Mixer (McGaw Park, IL) to assure a homogenous mixture. Doses per treatment were as follows: Low dose = 7 mglkg (TDLO) Medium dose = 14 mglkg High dose = 21 mglkg Administration Each pregnant mouse received five consecutive oral administrations of either the control or treatment solutions beginning on their assigned day: Day 1, Day 6 or Day 11. Gavage was accomplished using an 18 gauge, 3.8 cm curved gavage needle (Perfectum, New Hyde Park, NY) attached to a graded 1 ml glass syringe. The gavage needle was inserted into the esophagus and the test solutions expelled into the stomach. Food was withheld for a period of 2-3 hours before each gavage, and each gavage was administered between 1250 and 1350 hours. 16 Mating On their day of arrival at the laboratory, female mice were grouped four to a Plexiglass cage 7" x 11 1/ " x 5" (Allentown Caging, Allentown, NJ) and allowed a full week of recuperation. Males were housed individually on their day of arrival. Following this adaptation period female mice were put into a male's cage at a 1:1 or 2:1 ratio late in the afternoon. Females were observed for the presence of a vaginal plug the following morning. The presence of a vaginal plug denoted Day 1 of pregnancy. If no plug was observed the female was removed from the male's cage and they were placed together again late in the afternoon. Mated females were caged together and randomly divided into groups on the first day of treatment. A minimum of 15 females were used per group, three replicate groups of 5 per treatment. Data Collection Maternal: Dosed female C57BU6J mice were weighed on days 1, 8, 15 of pregnancy and on days 1, 8, 15 and 22 following parturition. The gestation length and litter size were noted for each female. In addition, the total weight of the litter was recorded along with the number of males and females delivered. On day 22 postpartum, each female was sacrificed by cervical dislocation. The liver and kidneys were examined for abnormalities, excised and weights recorded. The spleen was also observed, excised and its length measured and recorded. The liver and kidneys, one cut in a sagittal plane and the other in a frontal plane, were preserved in a 10 % buffered formalin solution and catalogued for future studies. 17 Pups: On Day 1, each pup was visually inspected for gross abnormalities. Also on Day 1, each litter was reduced to 6 pups, whenever possible on a 1:1 sex ratio. Pup weight and crown-rump length were recorded on days 1, 8, 15 and 22. The day of lower incisor eruption, upper incisor eruption and eye opening was recorded. On day 22, each litter was weaned, reduced to 2:2 and allowed to develop until day 43. On day 43, the pups were weighed and sacrificed. The ovaries and testes were excised, their weights recorded, preserved in Bouin's fixative solution and catalogued for future studies. Statistical Analysis Analysis of variance (ANOVA) for split-plot design was used to test for differences of treatment means with significance at the 5% level. Day 8 data was not analyzed because one group had been treated, one was under treatment and one had not been treated (Gill, personal communication). Sex ratio data was analyzed by a binomial probability test. IN VITRO FERTILIZATION TRIAL Animals Female and male 86D2F1 mice were bred at the Endocrine Research Center by mating C57BU6J females with DBA/2J males. The parental strains were obtained from The Jackson Laboratory (Bar Harbor, ME). Chemical 3,3',4,4'-Tetrachlorbiphenyl was purchased from Accustandard, Inc. (New Haven, CT) in neat form and dissolved in ethyl alcohol. The solution 18 was then suspended in culture medium to obtain the desired doses with a maximal alcohol content of 0.01% (VN). Control dishes were loaded with 1 ml of medium containing 0.01, 0.1, 1.0, or 10.0 pg of the desired compound or control medium. Culture Medium Brinster's medium for oocyte culture, BMOC-3 with 0.4% BSA (GIBCO, Grand Island, NY) was used for the in vitro fertilization trials. A similar medium (BMOC-without BSA) was used in the outer well of Falcon organ tissue culture dishes (Becton-Dickson and Co., No. 3037, Cockeysville, MD). BMOC-3 (1.0 ml) and BMOC (3.0 ml) were placed in the inner and outer wells of each culture dish respectively. The culture dishes, when loaded, were equilibrated overnight in a humidified incubator at 5% 002 + 95% air at 37°C. The components of the two culture media are listed on Table 2. Superovulation Female mice were superovulated by injecting (ip) 10 IU pregnant mares serum gonadotropin and 10 IU human chorionic gonadotropin (hCG, Sigma Chemical Co., St. Louis, MO) 46-48 hours later. Gamete Recovery and IVF Twelve to 15 hours following hCG administration, adult male (3 to 5 months old) mice were sacrificed by cervical dislocation. The cauda epididymides were excised and placed in the inner well of an organ tissue culture dish containing 1.0 ml of BMOC-3. They were then repeatedly 19 Table 2. Components of the two culture media used in the TCB in vitro fertilization trials. Component BMOCa BMOC-3a NaCI 1 19.37mM 94.90mM Na-lactate --- 20.1 1 mM Na-pyruvate 1 .02mM 0.51 mM KCI 4.78mM 4.78mM CaClz-ZHQO 1.71 mM 1.28mM KH2PO4 1 .20mM 1 .20mM MgSO4-7H20 1.19mM 1.19mM NaHCO3 25.07mM 25.07mM Bovine serum albumin (BSA) --- 5 grn/L Glucose 5.55mM 5.55mM aBrinster’s medium for oocyte culture (Brinster, 1971 ). 20 punctured with a 25 gauge needle to release sperm. The sperm suspension thus obtained was incubated for 1.5 hours. Thirty minutes after incubation, motility was assessed and samples showing >60% motility were used for insemination. Approximately 45 minutes after sperm collection, 5 to 7 superovulated mice were killed by cervical dislocation for oocyte recovery. The ovaries and oviducts with part of the uterus were excised and kept in the inner well of the culture dishes containing BMOC-3. The dilated ampullae of the oviducts were carefully pulled apart with fine forceps to release the cumulus masses containing the oocytes. The oocytes were washed once in BMOC-3 and transferred to the control or the treatment organ culture dishes. The cumulus masses were randomly placed in culture dishes. Fifty pl of sperm suspension was added directly to the inner well containing the cumulus mass. All the dishes were replaced in the incubator for 20-24 hours. At the end of the incubation period, each culture dish was scored for the percentage of oocytes fertilized. Oocytes were considered fertilized by the presence of one cell with two pronuclei, one cell with two polar bodies, or 2-cell embryos. Oocytes were considered unfertilized by the presence of a single cell or if degenerative. The abnormal 2 cell embryos were considered as fertilized. The number of degenerative oocytes and abnormal embryos was also recorded. Statistical Analysis Results of the NF trials were analyzed by Chi Square and Bonferroni Chi Square contingency tables. The differences between the groups were also analyzed by individual Chi Square tests. RESULTS In Vivo Fertilization Trials Data from replicate treatment sets was pooled for tests of statistical significance. A total of 300 female C57BU6J mice were mated with a pool of fertile DBA/2J male mice. Of these, 180 were confirmed mated by the presence of a vaginal plug and were allocated randomly to one of the four groups. Maternal Parameters: Since Day 8 data was not analyzed, the selected graphic representations are shown as examples of dose response curves. The body weight of the pregnant dams was recorded on days 1, 8 and 15 of pregnancy and was recorded on days 1, 8, 15 and 22 postpartum. Weight means : SEM for dams treated on days 6 to 10 are listed on Table 3. No effect of dose over treatment period was indicated by analysis of variance for split-plot design. In addition, there was not an interactive effect between the time period of dose administration and the dose administered. There was no effect of gestation period on weight gain on the three period tested. Figures 2, 3 and 4 show the mean body weight for dams treated with 7, 14 and 21 mglkg TCB, respectively. The liver and kidneys of sacrificed dams were excised, weighed and preserved. The absolute and relative weights for these organs are listed on Table 4. No dose, time or dose/time interaction effect was found for maternal liver or kidney weights. Figures 5 and 6 present the mean body weight of mice administered TCB by oral gavage on day 8 and 15 of gestation. 21 22 Table 3. Maternal body weight of CS7BU6J mice administered TCB by gavage on days 6 to 10 of pregnancy. Prepartum Day1 Day8 Day15 Postpartum Day1 Day8 Day15 Day 22 Control 7mg/kg 14mglkg 21 mglkg 19.14i0.2 18.61 10.2 18.47103 18.31 :02 20.63104 1996:02 20.34i0.4 1975:02 30.102504 29.59102 2941:03 28.64i0.2 25.39503 25.58104 24.64;l:0.4 25.48i0.3 2846:03 27.77104 28.16104 2831:04 30.46i0.4 30.01103 30.20i0.4 30.85105 25.69103 25.85_t0.4 26.09i0.4 2622:04 *Mean 1: SEM in grams. 23 " -Jl.':-i1 ’u (a: v, '. :1 ‘ I: 3 it”s-.23. . . .. _.—:-1.“. dogma .3 mo... 9:9: h noLSnEEua 62E 12.3th Co E32332. ES -03 £225 >25 :35. .N 2:2”. 8-2505 oto>8D 918. E- mm on mm (6) W5I9M 24 mp 50 m 50 F ED 9. "s‘ .c. 5 . III . I . ’ :u --g 3-2.30] 23 >8 D 3 >3 I dogma >3 mo... 3 9.3:. S. necouflsEua 3:: 33¢th Cc E3893.— ucu -Eq £925 >62. cue—2 .m 2:9“. mm on mm (6) whim 25 mm 50 mp >8. m >8. 00:: m >8. P >8. _' . “- .« .. a. . .. I .. _. ..._ -.uo-.'oo'-.-8 B 9-8 >8 D 9. >8 I dogma >5 nub Co 9:9: PN caBSEEuo 523an he E33988 ace .0... £925 >62. :85. 6 239". ram I5) NB 26 Table 4. Liver and kidney weights of female CS7BU6J mice administered TCB by gavagef DAYS 1-5 Liver Kidney Absolute Relative Absolute Relative Control 19510.67 7.4910.01 03110.19 11810.03 7 mglkg 19310.06 74710.01 0.301025 11410.04 14 mglkg 18210.08 69610.01 03010.31 11410.03 21 mglkg 18110.08 69710.01 03110.35 12010.03 DAYS 6-10 Liver Kidney Absolute Relative Absolute Relative Control 18910.06 7.351001 0.321023 12610.03 7 mglkg 19110.54 7.421001 0.311023 12110.04 14 mglkg 18810.07 72110.01 0.321022 12210.03 21 mglkg 18910.06 72310.01 0.321024 12210.03 DAYS 11-15 Liver Kidney Absolute Relative Absolute Relative Control 18310.09 69410.01 0.311028 11710.03 7 mglkg 18310.04 6.8210.01 0.3010.16 11310.03 14 mglkg 18510.07 68510.01 0.301022 11310.03 21 mglkg 18610.06 71110.01 0.311023 11710.03 1‘Absolute liver and kidney weights are mean 1 SEM in grams. Relative liver and kidney weights are mean percent body weight in grams 1 SEM. 27 once 9:oE Pm 9:oE 3 9:9: > m7: >a0 Ile| 07$ 25 IT m-.. >mo IT dogma >n 00h no..2a_:_Eua 8:: 3:398 3 83:32: m >8 :0 £225 >25 :85. .m 2:9“. .2500 md F -1 mp .T mdp I. WON FN m.—N lfil WBIOM 28 9:aE FN T omen 9:9: 3 9.38 N. .9200 I I m . RN .. mm m7: 95 Illelll I mdw 07m >mo IIDIII m4 >50 lull . .- m8 mdm Fm .383 >n no... 3:39:25; 03:. afimhmo 3 E3533 mp >23 :6 £925 >95 :85. .m 959“. (III “15PM 29 Table 5 lists the mean 1 SEM of dam litter size as well as the pooled number of males and females for treatment groups. No significant difference was seen in litter size or sex ratio. Pup parameters: Pup weight and crown-rump length were recorded on days 1, 8, 15 and 22 postpartum. Figures 7, 8 and 9 reflect the mean pup weight for treatment groups. No dose, time period, or dose/time period interaction effect was noted in either of these parameters. The crown-rump length mean totals are listed on Table 6. No significant difference was noted for day of eye opening or lower and upper incisor eruption. No anomalies were observed in any of the pups. Each of the other parameters studied showed no significant difference between the treatment groups. In Vitro Fertilization Table 7 shows the effect of TCB on IVF in 86D2F1 mice. Analysis of variance revealed a significant effect of TCB on IVF. The IVF rate dropped as the TCB dose increased in the culture media. TCB at the levels of 1.0 uglml and 10 uglml was significantly lower than the control. The IVF rate of the 10.0 uglml group was also significantly lower than the 1.0 uglml group rate. The effect of TCB on oocyte degeneration and abnormal embryos is shown on Table 8. The rate of both oocyte degeneration and number of abnormal embryos is significantly higher than the control as revealed by chi square analysis. Furthermore, two treatment groups, 1.0 ug/ml and 10.0 It glml, had a significantly higher rate of degeneration and abnormal embryos from than the control group. The data in Tables 7 and 8 were included in a recent publication (Kholkute et al., 1994). Table 5. Litter size and total number of male and female mice delivered by C57BU6J females exposed to TCB by gavage" Control Males Females 7 mglkg Males Females 14 mglkg Males Females 21 mglkg Males Femals Day 1-5 8.110.36 65 56 7.710.21 61 55 7.410.40 56 55 7.91027 60 56 Day 6-10 8.010.17 65 57 8.31021 63 61 8.110.24 60 61 7,510.55 51 61 Day11-15 8.010.32 60 52 8.01022 67 54 8,310.25 60 64 8.010.26 58 62 7FLitter size expressed in mean 1 SEM. 31 w >8 P >8. NN >8. mp >mo 8.: >8. 2.... >8D .3 >8. .883 >: no... 9:9: > 3 E398 an! En: Leta NN 2 w «>8 .8 oo..: Enema 5 2.225 9:. :85. .> 232.... or NP (bl NBIOM 32 NN >mo mp >8. m >8. F >8. m.-:>8l 8... >88 m-_>8l .oagau >2 no... 9.3:: 3. 8 63693 83 Eu: 3:: mu 2 P n>eu .8 3.8 Eamon .0 £225 9:. :85. .m 2:9... 0— NF (5) whim 33 NN >mo mp >8. m >mn. P >mo 2-: >8- 88 >8D m-.>8l .888 >9 mph 9:9: FN 2 88:8 83 E8 85: NN 9 F «>8 .2 ou_E {Noon Co 2.225 :2. :85. .m 959“. OF NF (6) NBIOM 34 Table 6. Crown-rump length of B6D2F1 mice for days 1 to 22 after dam was exposed to TCB by oral gavage.* Dam treated Days 1-5 Postpartum Day 1 Day 8 Day 15 Day 22 Control 28.6102 38.0108 48.6105 54.8103 7 mglkg 27.6101 38.8102 48.1103 54.0106 14 mglkg 27.5101 37.3103 48.9103 54.8105 21 mglkg 28.8108 37.0103 48.4105 54.9106 Dam treated Days 6-10 Postpartum Day 1 Day 8 Day 15 Day 22 Control 27.1103 39.0104 48.5103 56.9106 7 mglkg 27.2103 38.6102 48.4103 56210.1 14 mglkg 27.6103 38.8101 48.0103 56.5104 21 mglkg 27.7103 38.9105 48.6109 56.9103 Dam treated Days 11-15 Postpartum Day 1 Day 8 Day 15 Day 22 Control 28.4104 39.3106 44.2109 56.3104 7 mglkg 28.3104 39.3105 44.4106 56.3106 14mglkg 27.4102 40.3105 45.7107 56.4108 21 mglkg 27.4102 40.2104 45.1103 57210.8 :Mean 1 SEM in grams. 35 Table 7. Effect of TCB on In Vitro Fertilization in 86D2F1 mice. Group Total Ova %Fertilized (mean 1sem) Control 1 1 1 83.81001 1 0.01 uglml 116 77.610.012 0.1 ug/ml 132 72.710.008 1.0 ug/ml 109 688100053 10.0 uglml 173 53.710.0048.b ANOVA on % fertilized in trial for each group revealed a significant effect of TCB, p<0.001. aSignificantly different from control, p<0.01. bSignificantly different from 1 uglml, p<0.01. Table 8. Effect of TCB on oocyte degeneration and abnormal embryos. Group Total Ova No. degenerative Total 2-cell Abnormal 2-cell ova (%) embryos embryos (%) Control 111 3 (2.7) 86 2 (2.3) 0.01 uglml 116 4 (3.4) 84 3 (3.6) 0.1 uglml 132 6 (4.5) 86 4 (4.6) 1.0 11ng 109 9 (8.2) 61 6 (9.8)a 10.0 uglml 173 19 (11)a 69 14 (20.3)a Overall chi square significant, p<0.001. alndividual chi square significantly different from control, p<0.05. DISCUSSION 3,3',4,4'-Tetrachlorobiphenyl was tested at double, triple and the lowest lethal published dose for mice (Lucier et al.,1978). The results of this study demonstrate that female C57BU6J mice administered a single dose of TCB for five consecutive days at 7, 14, and 21 mglkg of body weight by gavage will not experience a significantly different gain in weight for any of the periods of gestation tested. The results of this study did not demonstrate a dose, period or dose/time interaction effect. That is, the doses tested did not affect any of the parameters measured. The time of gestation that the doses were administered was also not a significant factor. Marks et al. (1989) reported that CD-1 mice exposed to TCB by gavage at doses of 16, 32 and 64 mglkg of body weight will experience decreased weight gain between days 6 to 10 as the dose increases, but that mice exposed to doses below 16 mglkg between days 6 to 17 of gestation will not experience a significantly different pattern of weight gain. The data of the present study, are consistent with this study, not having been significantly lower than the control at 7 and 14 mglkg, however, the present study did not demonstrate a significant difference in weight gain at the 21 mglkg dose level. Thus, the effect of TCB could possibly be strain dependent. A similar dose dependent pattern of weight gain was reported by d'Argy et al., (1987). On average, C57BU6 mice administered a single intraperitoneal dose of 6 mglkg or 16 mglkg of TCB on day 12 were found to weigh less on 36 37 day 18 as the dose increased. Data from both studies, however, were not significant when compared to the controls. This study, however, is different in the route of exposure, having chosen to test TCB by i.p. versus oral gavage. In the cotton top marmoset monkey, weightJoss was significant when exposed to a bi-weekly oral dose of TCB of 0.1 mglkg, 1.0 mglkg and 3 mglkg of body weight (van den Berg et al., 1988). Mo Nulty et al., (1980) described a similar pattern of weight loss when rhesus monkeys were exposed to food containing dissolved TCB at 3 ppm. Thus, the effect of TCB on weight gain could be species dependent. In rat studies, Chen et al., (1992) found no difference in weight gain when they were exposed to a single intraperitoneal injection of 150 umollkg of body weight of TCB. Previously however, Leece et al., (1985) had reported an ED50 of 3.3 umol/kg and an ED25 of 2.0 umollkg body weight to induce weight loss when exposed to TCB by a single intraperitoneal injection. The data in this study support the hypothesis that exposure to TCB will cause a decrease in the rate of weight gain. The data in the present study, however, differ from reports in relation to liver and kidney weights. No significant difference in weight for either organ was found between the control and treatment groups in the present study. Mean liver weights were significantly different when marmosets were exposed to 3 mglkg body weight of TCB, but the authors also found no significant difference in mean kidney weight in the same species (van de Berg et al., 1988). Buchmann et al., (1991) found a significant difference in mean liver weight of TCB treated rats compared to the controls at dose levels of 150 umollkg and 15 umol/kg of body weight after a nine week period. 38 Results from this study also indicate that litter size and gestation length were not affected by exposure to TCB. In rats however, gestational length increased when treated with 3 mglkg body weight of TCB. Of the 98% of rats that gave birth between days 20 and 22 of gestation, 80 % gave birth on day 21, the other 18% were divided equally between days 20 and 22. For treated rats, 96% gave birth on days 21, 22 or 23. Of these, 46% gave birth on day 22 or 23 (White et al., 1983). The results of the present study agree with what was reported by Ronnback (1991) who found no significant difference in litter size after exposing C57/Bl dams to a single intraperitoneal injection of TCB on the 13th day of gestation for any of the doses administered ranging from 1.5 mglkg to 15.0 mglkg body weight. The results of the present study also demonstrate that TCB adversely affects in vitro fertilization in the mouse. Treatment reduced the IVF rate significantly at the 0.1, 1.0 and 10.0 ug/ml dose levels. Furthermore it increased the number of degenerative ova and increased the number of abnormal 2-cell embryos. While some studies exist on the reproductive toxicity of TCB on mammalian and non-mammalian species, no other information on the in vitro fertilization toxicity of TCB was located in the literature. Therefore, an evaluation of TCB's effects on the oocyte, spermatozoa, fertilization and preimplantion needed to be conducted. Many environmental toxins have been detected in human follicular fluid and because of TCBs lipophilic nature, it could have serious effects on the fertilization ability of the oocytes (Trapp et al., 1984). 39 But did the TCB actually reach the fetus? This question was thought to be answered through the examination of gross abnormalities and certain growth parameters. Though liver, kidney, ovary and testis samples were preserved, they were not assayed for levels of TCB. By conducting an assay on these tissue samples, one could come to know if TCB actually reached the fetus. A chromosomal analysis on some of the pups could also help determine the toxicity of TCB in mice. Although no anomalies were noted, TCB could have acted mutagenicaly. Hormone levels were also not assayed during the treatment period. By conducting assay of hormone levels during pregnancy, one could possibly start shedding some light on the mechanism of action. Other possibilities of testing TCB's reproductive toxicity are to administer the toxicant before mating or after delivery. By administering TCB before mating, information on it's effects on sexual receptivity and the estrous cycle could be obtained. Because TCB has been detected in the mammary gland and milk, administering the dose after delivery could ensure that the pups are exposed to TCB. Thus, results from this study are in no way comprehensive in any manner. Further studies that examine the mechanisms behind TCB's toxicity are warranted. Summary and Conclusions Polychlorinated biphenyls (PCBs) are persistent environmental contaminants. Tests on animals have revealed these aromatic hydrocarbon compounds to be teratogenic, carcinogenic, and mutagenic. The reproductive effects of PCBs vary depending on the route of administration, amount of administration, time of administration and most importantly, the PCB administered. Oral administration of 7, 14 and 21 mglkg b.wt. of TCB to C57BU6J mice for five consecutive through day 15 of pregnancy resulted in no observable reproductive effects. However, TCB did affect the in vitro fertilization at concentrations of 0.1 jig/ml, 1.0 uglml, and 10.0 jig/ml. The following conclusions are drawn: 1. Oral administration of TCB to pregnant C57BU6J mice had no effect on maternal liver or kidney weight. TCB did not affect litter size or sex ratio. 2. Maternal weight gain was not affected by TCB during any period of gestation tested. 3. Pup weight and crown-rump length were not affected by TCB. lncisor eruption and eye opening were also not affected. 4. After 43 days of development, pup gonadal weight was not affected by TCB. 5. TCB affected B6D2F1 mouse in vitro fertilization. TCB increased the number of degenerative ova and increased the number of abnormal 2-cell embryos. 40 Bibliography Bibliography Allen, J. R., Carstens, L. A. & Barsotti, D. A. (1974) Residual effect of short- term, low level exposure of nonhuman primates to polycholorinated biphenyls. Toxicol Appl. Pharmacol., 30, pp. 440-451. Anderson, L. M., Beebe, L. E., Fox, S. D., lssaq, H. J. 8. Kovatch, R. M. (1991) Promotion of mouse lung tumors by bioaccumulated polychlorinated aromatic hydrocarbons. Exp. Lung. Res, 17, p. 455. Agrawal, A.K., Tilson, H.A. & Bondy, SC. (1981) 3,4,3',4'- Tetrachlorobiphenyl given to mice prenatally produces long-term decreases in striatal dopamine and receptor binding sites in the caudate nucleus. Toxicology Letters, 7, pp. 417-24. Ballschimiter, K., Rappe, C. & Buser, H. R. (1989) Chemical properties, analytical and environmental levels of PCBs, PCTs, PCNs and PCBs. Halogenated Biphenyls, Terphenyls, Naphthalenes, Dibenzidioxins and Related Products. Kimbrough, R. D. and Jenson. A_. A.. Eds. Elsevier-North Holland Amsterdam, p. 47. Bennington, S. L., Conners, P. G., Conners, C. W. & Resebrough, R. W. (1975) Patterns of chlorinated hydrocarbon concentration in New Zealand sub-Antarctic and coastal marine birds. Environ. Pollut., 8, pp. 135-147. Berry, D. L., Slaga, T. J., DiGiovanni, J. & Juchau, M. R. (1979) Studies with chlorinated dibenzo-p-dioxins in a two-stage system of mouse skin tumorigenesis: potent anticarcinogenic effects. Ann. N. Y. Acag. Sci, 320, pp.405-414. Brinster, R. L. (1971) In vitro culture of the embryo. In Sherman Al, ed. Pathways to conception. Springfield, IL: Charles C. Thomas; pp. 245-277. 41 42 Brouwer, A., Klasson-Wehler, E., Bokdam, M., Morse, D. C. & Traag, W. A. (1990) Competitive inhibition of thyroxin binding to transthyretin by monohydroxy metabolites of 3,4,3',4'-tetrachlorobiphenyl. Chemosphere, 20, p.1257. Brouwer, A. & Van den Berg, K J. (1986) Binding of a metabolite of 3,4,3',4'-tetrachlorobiphenyl to transthyretin reduces serum vitamin A transport by inhibiting the formation of the protein complex carrying both retinol and thyroxin. Toxicol. Appl. Pharmacol., 85, p. 301. Buchmann, A., Ziegler, S., Wolf, A., Robertson, L. W., Durham, S. K. & Schwarz, M. (1991) Effects of polychlorinated biphenyls in rat liver: Correlation between primary subcellular effects and promoting activity. Toxicology and Applied Pharmacology. 111, pp. 454-468. Chen, L.-C., Berberian, I., Koch, 8., Mercier, M., Azais-Braesco, V., Glauert, H. P., Chow, C. K. & Rovertson, L. W. (1992) Polychlorinated and polybrominated biphenyl congeners and retinoid levels in rat tissues: Structure-activity relationships. Toxicology and Applied Pharmacology, 114, pp. 47-55. Chen, P. H., Chang, K. T. & Lu, Y. D. (1981) Polychlorinated biphenyls and polychlorinated dibenzofurans in the toxic rice-bran oil that caused PCB poisoning in Taichung. Bull. Environ. Conta_m. Tox_icol.. 26, pp. 489-495. d'Argy, R., Dencker, L., Klasson-Wehler, E., Bergman, A., Darnerud, P. O. & Brandt, l. (1987) 3,3',4,4'-Tetrachlorobiphenyl in Pregnant Mice: Embryotoxicity, Teratogenicity, and Toxic Effevts on the Cultured Embryonic Thymus. Pharmacology & Toxicology, '61, pp. 53-57. Cornwall, G. A., Carter, M. W. & Bradshaw, W. S. (1984) The relationship between prenatal lethality or fetal weight and intrauterine position in rats exposed to diethystillbestrol, Zeranol, 3,4,3',4'-tetrachlorobiphenyl, or Cadmium. Teratology, 30, pp. 341 -349. Deml, E. & Oesterle, D. (1982) Sex-dependent promoting effect of polychlorinated biphenyls on enzyme-altered islands induced by diethylnitrosamine in rat liver. g‘flcinogenesis. 3, pp. 1 449-1 453. Ebner, K. V. 8. Braselton, W. E. (1987) Structural and chemical requirements of hydroxylated polychlorinated biphenyls (PCBOH) for inhibition of energy-dependent swelling of rat liver mitochondria. Chem-Biol. Interact, 63, p. 139. 43 Gill, J. L. Personal communication. Golub, M. 8, Donald, J. M. 8 Reyes, J. A. (1991) Health Perspectives, 94, pp. 245-253. Graham, M. J., Lucier, G. W., Linko, P., Maronpot, R. R. & Goldstein, J. A. (1988) Increases in cytochrome P-450 mediated 17beta-estradiol 2- hydroxylase activity in rat liver microsomes after both acute administration and subchronic administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin in a two- stage hepatocarcinogenesis model. Carcinogenesis, 9, p. 1935. nguchi, K., (Ed.) (1976) PCB poisoning and pollution. Academic Press, New York. Huztzinger, 0., Safe, S. 8. Zitko, V. The chemistm of PCBs. CRC Press, Boca Raton, Florida. 1974. Jensen, S. (1966) Report of a new chemical hazard. New Sci., 32, p. 612. Jensen 8., Kihlstrom, J. E., Olson, M., Lundberg, C. & Orberg, J. (1977) Effects of PCBs and DOT on mink (Mustela vison) during the reproductive season. Ambio, 6, p. 239. Kannan, N., Tanabe, S., Wakimoto, T. 8 Tatsukawa, R. (1987) Coplanar Polychlorinated biphenyls in Aroclor and Kanechlor mixtures. J. Assoc. Off. Anal. Chem., 70, pp. 451-4. Kimura, N. T., Kanematsu, T. & Baba, T. (1976) Polychlorinated biphenyl (s) as a promoter in experimental hepatocarcinogenesis in rats. Z. Krebsforsch. Klin. Onkol., 87, pp. 257-266. Kimbrough, R. D. (1979) The carcinogenic and other chronic effect of persistent halogenated organic compounds. Ann. N. Y. Acad. Sci., 320, pp. 415-418. Kimbrough, R. 0., Grace, D. F., Korver, M. P. & Burse, V. W. (1981) Induction of liver tumors in female Sherman strain rats by ploybrominated biphenyls. J. Natl. Canc. Inst, 66, pp. 535-542. Kimbrough, R. D., Squire, R. A., Linder, R. E., Strandberg, J. L., Montali, R. J. & Burse, V. W. Induction of liver tumors in Sherman strain female rats by PCB Aroclor 1260. Natl. Cancer Inst., 55, p. 1453. 44 Kholkute, S. D., Rodriguez, J. 8 Dukelow, W. R. (1994) Effects of polychlorinated biphenyls (PCBs) on in vitro fertilization in the mouse. Reproductive Toxicology. 8, pp. 69-73. Klasson-Wehler, E. , Brunstorm, B., Rannug, U. 8 Bergman, A. (1990) 3,3',4,4'-Tetrachlorobiphenyl: Metabolism by the chick embryo in ovo and toxicity of hydroxylated metabolites. Chem-M Interactions, 73, pp.121- 132. Leece, B., Denomme, M. A., Towner, R., Li, S. M. A. 8 Safe, S. (1985) Polychlorinated biphenyls: Correlation between in vivo and in vitro quantitative structure-activity relationships (QSARs). Journal of Tofixicology and Environmental Health. 16, pp. 379-388. Lewis, R. J., Sr., Sax's Dangerous Prpperties of Industrial Materials. Eighth Ed., 3, Van Nostrand Reinhold, New York. 1992. Lidgett, R. A. 8 Vodden, H. A. (1970) PCB—the environmental problem. In PCB Conference, Wenner-GRen Center, September 20, 1970. Stockholm: National Swedish Environmental Protection Board. pp. 88-96. Lucier, G. W., Davis, G. J. 8 McLachlan, J. A. (1978) Transplacental toxicology of the polychlorinated and polybrominated biphenyls. The 17th Hanford Biology Symposium Monograph. Eds.: D. Mahlum 8 M. Sikov. (fl Ridge Technical Information Centre, pp. 188-203. Mackay, D., Paterson, S., Eisenreich, S. J. 8 Simmons, M. S. (Eds) (1983) Physical behavior of PC_Bs in t_he Great Lakes. Ann Arbor Science, Michigan. Makiura, S., Aoe, H., Sugihara, 8., Hirao, K., Arai, M. 8 Ito, N. (1974) Inhibitory effect of polychlorinated biphenyl on liver tumorigenesis in rats treated with 3'-methylaminoazobenzene, N-2-fluorenyI-acetamide and diethylnitrosamine. J._N_atl. Ca_ncer Inst. 53, pp. 1253-1257. Marks, T. A., Kimmel, G. L., 8 Staples, R. E. (1989) Influence of symmetrical polychlorinated biphenyl isomers on embryo and fetal development in mice. Fundamental and Applied Toxicology. 13, pp. 681-693. Masuda, Y. 8 Yoshimura, H. (1984) Polychlorinated biphenyls and dibenzofurans in patients with Yusho and their toxicological significance: A review. Americgl. lndust. Med., 5, pp. 31-44. Material Safety Data Sheet. Occupational Health Services, Inc., New York. 1992. 45 McNuIty, W. P., Becker, G. M. 8 Cory, H. T. (1980) Chronic Toxicity of 3,4,3',4'-and 2,5,2',5'-tetrachlorobiphenyls in rhesus macaques. Toxicology and flplied Pharmacology,, 56, pp. 182-190. Nisbet, I. C. T. 8 Sarofim, A. F. Rates and routes of transport of PCBs in the environment. (1974) Environ. Health Perspect, 1, pp. 21-28. Osterele, D. 8 Deml, E. (1981) Promotion effect of varios PCBs and DOT on enzyme altered islands in rat liver. Nauyn-Schmiedberg's Arch. Pharmacol. Suppl. R, 16, pp. 316. Parkinson, A., Robertson, L., Safe, L. 8 Safe, S. (1981) Polychlorinated biphenyls as inducers of hepatic microsomal enzymes: structure-activity rules. gem-Biol. Interfl, 30, p. 271. Pereira, M. A., Herren, S. L., Britt, A. L. 8 Khoury, M. M. Promotion by polychlorinated biphenyls of enzyme altered foci in rat liver. Cancers Letters, 15, pp. 185-190. Pitot, H. C., Goldsworthy, T., Moran, S., Sirica, A. E. 8 Weeks, J. Properties of incomplete carcinogens and promoters in hepatocarcinogenesis. Carcinpgenesis. 7, pp. 85-98. Platonow, N. S. 8 Reinhart, B. S. (1973) The effect of polychlorinated biphenyls (Aroclor 1254) on chicken egg production, fertility, and hatchability. Can. J. Comp. Med, pp. 341-346. Poland, A., Palen, D. 8 Glover, E. (1982) Tumor promotion by TCDD in skin of HRS/J hairless mice. Nature, 300, p.271. Preston, 8. D., Miller, E. C. 8 Miller. J. A. (1985) The activities of 2,2',5,5'- tetrachlorobiphenyl in turmor induction and promotion assays. Carcinogenesis, 6, pp. 451 -453. Rickenbacher, U., McKinney, J. D., Oatley, S. J. 8 Blake, C. C. F. (1986) Structrurally specific binding of halogenated biphenyls to thyroxine transport protein. J. Med. Chem, 29, p.641. Ringer, R.K., Aulerich, R.J. 8 Zabik, M. (1972) Effects of dietary polychlorinated biphenyls on growth and reproduction mink. Proceedings of the 164th National Meetings of the Americal Chemical §ocieg., 12, pp. 149- 154. Ronnback, C. (1991) Effects of 3,3',4,4'-tetrachlorobiphenyl (TCB) on ovaries of foetal mice. Pharmacolgy 8 Toxicology, 69, pp. 340-345. 46 Rugh, R. The Mouse: Its reproduction and development. Burgess Publishing Company, Minneapolis, Minn. 1967. Safe, 8., Bandiera, S., Sawyer, T., Rovertson, L. W., Safe, L., Parkinson, A., Thomas, P. E., Ryan, D. E., Reik, L. M., Levin, W., Denomme, M. A. 8 Fujita, T. (1985) PCBs: structure-function relationships and mechanisms of action. finvirpn. Hialth PersmLct, 60, pp. 47-56. Safe, S. H. (1994) Polychlorinated biphenyls (PCBs): environmental impact, biochemical and implications for risk assessment. Critigl Reviews in Toxicology, 24 (2), pp.87-149. Sargent, L., Dragan, Y. P., Erickson, E., Laufer, C. J. 8 Pitot, H. C. (1991) Study of the separate and combined effects of the non-planar 2,5,2',5'- and the planar 3,4,3',4'-tetrachlorobiphenyl in liver and lymphocytes in vivo. Carcingenesis, 12 (5), pp. 793-800. Schaeffer, E., Greim, H. 8 Goessner, W. (1984) Pathology of chronic polychlorinated biphenyl (PCB) feeding in rats. Toxicol. Appl. Pharmaacol., 75,p.278. Schmidt, T. T., Risebrough, R. W. 8 Grass, F. (1971) Input of polychlorinated biphenyls into California coastal waters from urban sewage outfalls. Bull Environ. Contamin. Toxicol., 6, pp. 235- Schmoldt, A., Herzberg, W. 8 Benthe, H. F. (1977) On the ingibition of microsomal drug metabolism by polychlorinated biphenyls (PCBs) and related phenolic compounds. Chem-Biol. Interact, 16, p. 191. Shiota, K. (1976) Embryotoxic effects of polychlorinated biphenyls (Kanechlors 300 and 500) in rats. Okaiimas Fol. Anat. Jpn, 53, pp. 930-104. Tanabe, S. (1988) PCB problems in the future: Foresight from current knowledge. Environmental Pollption. 50, pp. 5-28. Tanabe, S., Kannan, N., Subramanian, An., Watanabe, S. 8 Tatsukawa, R. (1987a) Highly toxic coplanar PCBs: Occurrence, source, persistency and toxic implicatiions to wildlife and humans. Environmental Pollution, 47, pp. 147-63 Tanabe, S. 8 Tatsukawa, R. (1980) Chlorinated hydrocarbons in the North Pacific and lnidian Oceans. J. Oceanogr. Soc. Japan, 36, pp. 217-26. Theiler, K. The house mogse: Atlas of embgyonic development. Springer- Verlag, New York. 1989. 47 Trapp, M., Bauklaoh, V., 8 Bohnet, H-G. 8 Heeschen, W. (1984) Pollutants in human follicular fluid. Fertil. Steril., 42, pp. 146-148. Vanden Berg, K. J., Zurcher, C., Brouwer, A. 8 Van Bekkum, D. W. (1988) Chronic toxicity of 3,4,3',4'-tetrachlorobiphenyl in the marmoset monkey (Cal/ithn'x Jacchus). Toxicolgy, 48, pp. 209-224. Van Miller, J. P., Hsa, l. C. 8 Allen, J. R. (1975) Distribution and metabolism of 3H-2,5,2',5'-tetrachlorobiphenyl in rats. Proc. Soc. Exp. Biol. Med., 148, pp. 682-687. Villeneuve, D. C., Grant, D. L., Khera, K., Clegg, D. J., Baer, H. 8 Phillips, E. J. The fetotoxicity of a polychorinated biphenyl mixture (Aroclor 1254) in the rabbit and in the rat., Environ. thsiol.. 1, pp. 67-71. White, R. D., Allen, S. D. 8 Bradshaw, W. S. (1983) Delay in the onset of parturition in the rat following prenatal administration of developmental toxicants. Toxicology Letters, 18, pp. 185-192. Yamamoto, H. 8 Yoshimura, H. (1973) Metabolic studies on polychlorinated biphenyl. lll. Chemical and Pharmaceutical Bulletin, 21, pp. 2237-2242. Yoshimura, H. 8 Yamamoto, H. (1974) Metabolic studies on polychlorinated biphenyls. IV. Biotransformation of 3,4,3',4'-tetrachlorobinphenyl, one of the major components of Kanechlor-400. Fukuoka Acta Medica, 65, pp. 5-11. Yoshimura, H., Yonemoto, Y., Yamada, H., Koga, N., Oguri, K. 8 Saeki, S. (1987) Metabolism in vivo of 3,4,3',4'-tetrachlorobiphenyl and toxicological assessment of the metabolites in rats. Xenobiotica, 17, pp. 897-910. Yoshimura, H., Yoshigara, S., Ozawa, N. 8 Miki, M. (1979) Possible correlation between induction modes of hepatic enzymes by PCBs and their toxicity in rats. Ann. N. Y. Acad. Sci., 320, p. 179. Appendix Citizenship: Education: Professional Positions: APPENDIX A CURRICULUM VITA Jaime Rodriguez USA University of Texas at Arlington 1985-1989 Arlington, Texas 76019 Maior: Psychology Honors: Honor Roll University of Texas - Pan American 1989-1992 Edinburg, Texas 78539 Maior: Psychology Minor: Biology Degree: Bachelor of Science 1992 Honors: Psi Chi (National Honor Society in Psychology) National Dean's List Michigan State University 1992-Present East Lansing, Michigan 48824 Maior: Zoology Honors: Minority Competitive Doctoral Fellowship, 1992-1996 Research lntem, Summer 1991 (Full-time) Department of Psychology Michigan State University East Lansing, Michigan 48824 Assisted in a study conducted on the neuromechanics of ferret reproduction, to include behavioral testing, record keeping, histology, daily care of the ferrets, a computerized atlas of the ferret brain and the application of different protocols. Supervisor: Cheryl Sisk, Ph.D. 48 49 Appendix A (cont'd) Teaching Assistant, 1991-1992 (Half-time) Department of Psychology and Anthropology University of Texas - Pan American Edinburg, Texas 78539 Duties as assigned; grade and keep record of all student's statistics homework and offer tutorial service to those needing help. Supervisor: Valerie James-Aldridge, Ph.D. Graduate Assistant, 1992-Present (Half-time) Department of Zoology Michigan State University East Lansing, Michigan 48824 Duties: Research Assistantship, Summer 1993 BS. 111-Cell and Molecules T.A., Fall 1993 ZOL 234-Comparative Anatomy T.A., Spring 1994 ISB 204-Applications of Environmental and Organismal Biology T.A., Fall 1994 Memberships in Professional Associations: Society for the Study of Reproduction, Trainee Membership Midwest Teratological Society, Member Sigma Xi, Associate Membership Professional Activities: ' Administrative Actively recruited minority students to apply for summer research programs at Michigan State University. Papers Presented: 1993 26th Annual Meeting of the Society for the Study of Reproduction Abstract: The effects of perchlorinated terphenyls (PCT) and bromobiphenyls (BP) on in vitro fertilization in the mouse. 1993 First Annual Research Day Department of Zoology Abstract: In vitro and in vivo reproductive effects of 3,3', 4,4'- tertrachlorobiphenyl (4-CB) in the mouse. 50 Appendix A (cont'd) 1992 National Conference on Undergraduate Research Abstract: Behavioral and neuroendocrine effects of testosterone implants in the preoptic area and medial basal hypothalamus. 1991 Committee on Institutional Cooperation Conference Presentation: Behavioral and neuroendocrine effects of testosterone implants in the preoptic area and medial basal hypothalamus. Papers Published 1. Rodriguez, J. Behavioral and neuroendocrine effects of testosterone implants in the preoptic area and medial basal hypothalamus. Conference Proceedings of the 6th Annual National Conference on Undergraduate Research (1992). (Abstract) Rodriguez, J. In Vivo and in vitro reproductive effects of 3,3'4,4'- tetrachlorobiphenyl (4-CB) in the mouse. 1st Annual Research Day Department of Zoology Program (1993). (Abstract) Rodriguez, J., Kholkute, SD, and Dukelow, W.R. The effects of perchlorinated terphenyls (PCT) and bromobiphenyls (BP) on in vitro fertilization in the mouse. Conference Proceedings of the 26th Annual Meeting of the Society for the Study of Reproduction (1993). (Abstract) Kholkute, S.D., Rodriguez, J., and Dukelow, W.R. The effects of polychlorinated biphenyls (PCB) on in vitro fertilization in the mouse. Conference Proceedings of the 26th Annual Meeting of the Society for the Study of Reproduction (1993). (Abstract) Kholkute, S.D., Rodriguez, J., and Dukelow, W.R. (1993) Effects of a pesticide mixture and two herbicide mixtures on in vitro fertilization in the mouse. In Vitro Toxicology. 6: 291-298. Kholkute, S.D., Rodriguez, J., and Dukelow, W.R. (1994) The effects of polybrominated biphenyls and perchlorinated biphenyls on in vitro fertilization in the mouse. Archives of Environmental Contamination and Toxicology. 26: 280-211. 10. 51 Appendix A (cont'd) Kholkute S.D., Rodriguez, J., and Dukelow, W.R. (1994) Effects of polychlorinated biphenyls (PCBs) on in vitro fertilization in the mouse. Reproductive Toxicology. 8: 69-73. Kholkute, S.D., Rodriguez J., Rawlins, R., and Dukelow, W.R. (1994) Glass wool column filtration: Effects on motility, viability, and fertilization ability of the mouse epididymal spermatozoa. Laboratory Animal Science. 44: 537-539. Kholkute, S.D., Rodriguez, J., and Dukelow W.R. Reproductive Toxicity of Aroclor-1254: Effects on oocyte, spermatozoa, in vitro fertlization and embryo development in the mouse. (Reproductive Toxicology, 1994). (In Press) Kholkute, S.D., Rodriguez, J., and Dukelow, W.R. In vitro fertilization and acrosome reaction of the mouse epididymal sperm following exposure to progesterone and 17 a-hydroxyprogestorone. (submitted International Journal of Andrology, 1994). MICHIGAN sran UNIV. LIBRARIES llHIWIIIIIHIIIHIWIIIIIIWINIll”lllllllllllllllllllll 31293®10205866