.315. i... .. n . flu...th.u.z.. :1.‘ .hnu :- hf.......d-. . y . .4»... x; “Halli; «'32 . .-... . s... .9 1. n!“ ,‘.v LI RABIES iiiiiii ii ii; \\\\\\\\\\\\\\\\\\\i 3129 This is to certify that the dissertation entitled The Effects of Polychlorinated Biphenyls on Dopaminergic PC12 Cells presented by William Gartley Robertson Angus has been accepted towards fulfillment of the requirements for Ph VD degree in .Eharmacolngy- Environmental Toxicology 77 / Ld)7ZL(/( ,2/ / Major professor Date /c/// 7/7// MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University PLACE ll RETURN BOXto roman” Momma: your record. TO AVOID FINES Mumpn or More data duo. DATE DUE DATE DUE DATE DUE MSU IoAn Nflmdivo NONE“ Opponunily lm W1 THE EFFECTS OF POLYCHLORINATED BIPHENYLS ON DOPAMINERGIC PC12 CELLS BY William Gartley Robertson Angus A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1994 ABSTRACT . THE EFFECTS OF POLYCHLORINATED BIPHENYLS ON DOPAMINERGIC PC12 CELLS BY William Gartley Robertson Angus Polychlorinated biphenyls (PCBs) are putative neurotoxicants. The dopaminergic model cell system PC12 cells were used to examine the hypothesis that subchronic exposure of dopaminergic cells to PCBs would result in a decrease in cellular dopamine that resulted from a decrease in the synthesis of dopamine, and manifested itself as a decrease in the evoked release of dopamine. Levels of cellular catechols in cells subchronically exposed to Aroclor 1254 or PCB congeners were determined by high performance liquid chromatography coupled to electrochemical detection (HPLC/ECD), and normalized to DNA content. The release of dopamine from subchronically PCB-exposed PC12 cells in response to 56 mM potassium was determined and normalized to DNA content. Results indicated that subchronic exposure of PC12 cells to Aroclor 1254 or PCB congeners resulted in dose-dependent decreases in cellular dopamine that manifested themselves as a decrease in the release of dopamine in response to depolarization with 56 mM potassium. The data from studies using PCB congeners indicated that the effects of PCBs on amounts of cellular dopamine and dopamine released was specific for each congener. Gas chromatography coupled to electron capture was used to determine the amounts of PCBs in the cells at media concentrations of PCBs at which decreases in cellular dopamine and evoked release of dopamine were observed. The concentrations of PCBs in the cells increased as levels of dopamine decreased, however, there was a lag time between the association of PCBs with the cells and the decrease in dopamine. Possible mechanisms by which PCBs cause decreases in levels of dopamine were examined by measuring 1) the uptake of 3H-tyrosine, 2) the synthesis of dopamine from 38- tyrosine, and 3) the ability of supplemental dihydroxyphenylalanine (DOPA) to be converted to dopamine. Aroclor 1254 did not cause a decrease in the uptake of hr- tyrosine, but did causeaa decrease in the conversion of DOPA to dopamine, suggesting that one way in which Aroclor 1254 decreases cellular dopamine is by inhibition of the conversion of DOPA to dopamine by L-aromatic amino acid decarboxylase. DEDICATION I dedicate this publication to my wife, Tamera for her support and patience. I also dedicate this work to our unborn child and unborn children everywhere that they may not have to experience the consequences of exposure to environmental contaminants. iv ACKNOWLEDGMENTS I would like to acknowledge my deepest appreciation to Dr. Margarita Contreras for her excellent guidance and assistance in helping me obtain this degree. I thank Dr. K.J. Lookingland for his assistance with HPLC. I also thank the other members of my Guidance Committee, Dr. L.J. Fischer, Dr. W.E. Braselton, and Dr. J.P. Giesy for their guidance and assistance. I would further like to acknowledge the assistance of Pat Rumler, Tanya Leinieke, Vivianne Vargas, and Annette Feugaroa with these studies. A special thanks to Dr. Annette Fleckenstein for invaluable consultation and moral support. TABLE OF CONTENTS List of Tables List of Figures Abbreviations Chapter 1 Chapter Chapter Chapter Chapter Introduction Polychlorinated Biphenyls Neurotoxicity of PCBs PC12 Cells Dopamine as a Neurotransmitter Dopamine Synthesis and Metabolism in PC12 Cells Hypothesis and Aims Materials and Methods Effects of Polychlorinated Biphenyls on Cellular Dopamine in PC12 Cells Introduction Results Discussion Effects of Polychlorinated Biphenyls on Release of Dopamine From PC12 Cells Introduction Results Discussion Association of Polychlorinated Biphenyls With PC12 Cells Introduction Results Discussion vi viii ix xii 10 18 22 23 33 36 54 55 58 80 93 94 95 103 114 115 116 128 Chapter 6 Mechanisms by Which Polychlorinated Biphenyls Decrease Cellular Dopamine in PC12 Cells Introduction Results Discussion Chapter 7 Summary Review of Aims Discussion Appendix 1 Structure and Nomenclature of Congeners Used Appendix 2 GC/MS Parameters Appendix 3 DNA Content Versus Cell Number Bibliography vii 134 135 135 144 153 155 161 176 177 178 179 Table Table Table Table Table Table LIST OF TABLES Effects of Aroclor 1254 on DNA and Protein Synthesis Percent of PC12 Cells Expressing Neurites and Number of Neurites per Cell Following Subchronic Exposure to Aroclor 1254 and NGF Effects of Aroclor 1254 and Various PCB Congeners on Levels of Cellular Dopamine in PC12 Cells Aroclor 1254 and PCB Congeners Associated with PC12 Cells Biphenyl Associated with PC12 Cells Structure and Nomenclature of Congeners Used viii 62 71 78 123-124 127 176 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 3.6 3.7 3.8 3.9 LIST OF FIGURES EEE Structure of Polychlorinated Biphenyl 3 Environmental Contamination by PCBs 6 Synthesis of Dopamine 24 Effects of Subchronic Exposure to Aroclor 1254 on Cellular Catechols 59 Subchronic Exposure of Undifferentiated PC12 Cells to Aroclor 1254 60 Media Lactate Dehydrogenase Levels Following Subchronic Exposure to Aroclor 1254 64 Subchronic Exposure of NGF-stimulated Differentiating PC12 Cells to Aroclor 1254 65 Effect of NGF Pretreatment on Aroclor 1254-mediated Decreases in Cellular Dopamine 66 Effect of Aroclor 1254 on NGF-induced Neurite Outgrowth 68 Effect of Aroclor 1254 on NGF-induced Neurite Elongation 69 Subchronic Exposure of PC12 Cells to 11 PCB Congeners 72-76 Cellular Dopamine Content Following Acute Exposure to Aroclor 1254 or 2,2'- Dichlorobiphenyl 79 Dopamine Released by PC12 Cells in Response to 56 mM K 96 ix Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure BEE Effects of Exposure to Aroclor 1254 on Release of Dopamine From PC12 Cells 98 Effects of PCB Congeners on Release of Dopamine From PC12 Cells 100-102 Fractional Release of Dopamine From PC12 Cells 105 Cellular Association of Aroclor 1254 With PC12 Cells Over Time 118 Association of Aroclor 1254 and PCB Congeners With PC12 Cells 119 Relation of Cellular Dopamine to Cellular Aroclor 1254 Concentration in PC12 Cells 120 Effects of Aroclor 1254 and Biphenyl on Cellular Dopamine 126 Uptake of'ifl-tyrosine Into PC12 Cells 137 Uptake of Amino Acids or Choline by PC12 Cells Exposed to Aroclor 1254 138 Uptake of 3H-tyrosine Into PC12 Cells Exposed to PCB Congeners 139 Effects of Subchronic Exposure to Aroclor 1254 on Cellular Catechols 141 Synth sis of SH—DOPA and 3H-Dopamine From H-tyrosine 142 Effects of DOPA Supplementation on DOPA and Dopamine in Aroclor 1254- exposed Cells 143 Effects of DOPA Supplementation on Cellular Dopamine and DOPA in Tetrachlorobiphenyl-exposed Cells 145 Effects of DOPA Supplementation on Cellular Dopamine and DOPA in Hexachlorobiphenyl-exposed Cells 146 Operating Parameters For Gas Chromatograph and Mass Spectrometer 177 BEE Figure A.2 Cellular DNA Content as a Reflection of Cell Number 178 xi ALD DH 'BDNF an, com DA DBH DNA DOPA DOPAC Ca/CAMPK ECD EPI cc GC/MS HPLC LNAA MAO+ MPP MPTP NE NGF OCN OSS PBS PCB PKA PKC PNMT TCDD 5-HT 5-HTP 6-OHDA ABBREVIATIONS Aldehyde dehydrogenase Brain derived neurotrophic factor Tetrahydrobiopterin Catechol-O-methyltransferase Dopamine Dopamine fl hydroxylase Deoxyribonucleic acid L-3,4-dihydroxyphenylalanine Dihydroxyphenylacetic acid Calcium/calmodulin-dependent protein kinase Electrochemical detection Epinephrine Gas chromatography Gas chromatography coupled to mass spectrometry High performance liquid chromatography L-aromatic amino acid decarboxylase Large neutral amino acids Monoamine oxidase N-methyl-4-phenylpyridinium ion 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Norepinephrine Nerve growth factor Octachloronaphthalalene 0ctyl sodium sulfate Phosphate buffered saline Polychlorinated biphenyl Protein kinase A Protein kinase C Phenylethanolamine-N-methyltransferase 2,3,7,8-Tetrachlorodibenzo-p-dioxin Tyrosine hydroxylase Serotonin 5-Hydroxytryptophan 6-Hydroxydopamine xii E J l] . ! 1 E. l J Polychlorinated biphenyls (PCBs) are chlorinated aromatic hydrocarbons consisting of two six-carbon aromatic rings joined at the 1-position with chlorine atoms attached in various combinations at the other 10 positions (Figure 1.1; for reviews see Safe 1984, 1994, 1990; Safe et al., 1987; Silberhorn et al., 1990; Hutzinger et al., 1974). There are 209 possible combinations of chlorine arrangements called congeners. These PCB congeners can be classified into two groups, coplanar and non-coplanar, based on the orientation of the two phenyl rings. PCBs used in industry were marketed as mixtures of these congeners. For the 0.8. and Great Britain, the Monsanto Company was the major producer of PCB mixtures, which were marketed under the trade name Aroclor. The various Aroclor mixtures were identified by a four digit code whereby 12 represented the code for chlorinated biphenyl. The second two digits, such as 54 or 60, indicated the approximate weight of chlorine in the compound as a percent of total weight. Hence, Aroclor 1254 consists of a mixture of chlorinated biphenyls with about 54% of its weight coming from chlorine atoms (Hutzinger et al., 1974). Further, the percentage of chlorine in Aroclor mixtures indicates the degree of congener’ chlorination in the ‘mixture. For instance, Aroclor 1254 contains mostly tetra-, penta-, and hexachlorobiphenyls: whereas Aroclor 1260 contains mostly m. Brtho Ortho Mata 3 - 2 29 39 Run 4’ 1 1’ 4? 5 6 6’ 5’ Meta (Ortho Ortho Meta Figure 1.1 structure of Polychlorinated Biphenyl 4 highly chlorinated congeners -- penta- through octachlorinated biphenyls (Hutzinger et al., 1974). PCBs were used industrially until 1977 for such purposes as hydraulic fluids, plasticizers, heat transfer fluids, wax extenders, lubricants, dielectric fluids in capacitors and transformers, and sealants for farm silos. The properties that made these compounds appropriate for'the above industrial uses, namely resistance to thermal breakdown, high dielectric current, hydrophobicity, and miscibility with organic solvents (Safe, 1984) have also allowed them to become persistent, bioaccumulative environmental contaminants. Due to the widespread use of PCBs and ignorance of their toxic effects on biological systems, disposal of these compounds was unregulated and unsupervised during most of the timespan of their 'USEu Consequently, substantial amounts of these compounds are currently circulating and redistributing throughout the environment (Nisbet and Sarofim, 1972: Safe et al., 1987). A geographic area of particular interest, due to large scale PCB contamination, is the Great Lakes. PCBs in sinks and sources in the lithosphere leach into ground water and runoffs, ending up in large rivers, inland lakes, and eventually the Great Lakes. PCBs in the hydrosphere can. be taken ‘up and bioaccumulated by fauna within the lake biosphere and then biomagnified up the food chain (Figure 1.2). Death and Figure 1.2 Environmental Contamination by PCBs PCBs can enter lakes and rivers through direct contamination, runoff, or atmospheric deposition. Once within the hydrosphere, one route of distribution that PCBs can undergo is to be bioaccumulated and biomagnified up trophic levels to top predatory species, which are in turn consumed by man. Other routes of distribution include removal from active circulation by sedimentation, or recirculation into the atmosphere through evaporation and windborne droplets containing particulates. Figure taken from Safe et al., 1987. N.H ousmwh .. .. 352.com .. ,. .......=eE.pe-. a. mom ,. .. . “152.183: III 8:808; _ 8:88... H l H l 8:81.685: uco 322283003 I .1 III ozoaao .llllinllll so. H- 9-5 HHHH- |\I~ ii II. I .I too-co: # h I -.|il umwzmmozo>x H 5:283. uzwzamozhk 8:38... 7 decay of aquatic organisms and their predators cause new sinks of PCBs to be formed, some of which become quiescent and result in removal of PCBs from active circulation, others of which are again redistributed throughout the ecosystem. Further, evaporation from contaminated bodies of water (the Great Lakes, for example) and from contaminated terrestrial sinks allows for airborne deposition and redistribution. of ‘PCBs into tareas not. previously contaminated. The net result of this airborne distribution pattern is that there is no place on earth where one can escape being exposed to PCBs (Risebrough and deLappe, 1972: Parkinson and Safe, 1987). PCBs can gain access to ‘the ‘body' through dermal absorption, inhalation, or ingestion. Currently, the most common means of exposure to the general public is through ingestion of foods contaminated with PCBs. This is especially true in the Great Lakes region where game fish such as trout and salmon are reported to have bioaccumulated relatively high levels (Swain, 1983). Once in the body, the more lightly-chlorinated congeners (di- through tetrachlorobiphenyls) are readily metabolized by the liver and excreted” However, highly-chlorinated congeners, those with five or more chlorine atoms, tend to be deposited in adipose tissue and other tissues with high lipid content, and can be highly concentrated in human milk. The toxicity caused by PCBs is dependent.upon.age, sex, 8 and species of the animal, as well as the congeners involved (Safe, 1990). PCBs are not acutely toxic, except in very ihigh doses (Safe, 1984). There is increased toxicity with increased duration of exposure to PCBs. Further, there is a latent period between PCB exposure and onset of the symptoms of toxicity (Parkinson and Safe, 1987). Some symptoms appear after a few days or weeks, while other symptoms become apparent only after months of exposure. In addition to neurotoxic effects, which will be discussed in more detail later, some of the toxic effects of PCBs that have been described in humans and animals are listed below (Parkinson and Safe, 1987): 1. A wasting syndrome, entailing progressive weight loss that unrelated to a decrease in food intake. 2. Skin. disorders such. as hyperpigmentation and chloracne. Dermal lesions are regarded as some of the most important and characteristic signs of PCB intoxication. 3. Hyperplasia of the epithelial lining of the extrahepatic bile duct, gall bladder, and urinary tract. 4. Lymphoid involution or atrophy of the thymus and spleen that is accompanied by immunosuppression and bone marrow and hematic dyscrasias. Immunotoxic symptoms of PCBs include decreased resistance to host infections, depressed T-cell response to mitogens, and 9 delayed hypersensitivity. 5. Hepatomegaly and liver damage, which varies with age, sex, and species. Some signs of hepatotoxicity include proliferation.of smooth endoplasmic reticulum, focal necrosis of hepatic parenchyma, and increases in cytoplasmic lipid droplets. 6. Porphyria. ‘7. Endocrine and reproductive dysfunctions such as altered plasma steroid and thyroid hormone levels: menstrual irregularities, increased incidence of spontaneous abortion, hemorrhage and anovulation in females; and testicular atrophy and decreased spermatogenesis in males. 8. Teratogenesis, including cleft palate and kidney malformations. 9. Carcinogenesis, namely hepatocarcinoma. Studies on the structure-activity relationships of PCBs have shown. ‘that coplanar’ congeners ‘with. chlorine substitutions at the para-position, or para- and meta- positions appear to be toxic in the same manner as 2,3,7,8- tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD: Hansen, 1987). Coplanar congeners can interact with the cellular receptor for 2,3,7,8-TCDD, the Ah receptoru 'The Ah receptor has been shown to mediate the immunotoxic effects of PCBs (Parkinson and Safe, 1987). Most of the other known adverse effects brought about by PCB exposure are also believed to be 10 mediated through the Ah receptor. PCB congeners with para-, or para- and.meta-chlorine substitutions are more likely to have both phenyl rings in the same plane and thus are more apt to fit into the 2,3,7,8-TCDD binding site on the Ah receptoru .However, addition of one or more chlorines in the ortho position decreases the likelihood that both phenyl rings will remain in the same plane, thus reducing the ability of the PCB congener to bind to the Ah receptor. Interestingly, coplanar congeners are usually only present in very low levels in PCB mixtures, such as those contaminating the environment. Mono- and diortho- substituted analogues, however, are prevalent in these PCB mixtures. PCBs are differentially metabolized in the human body. Lightly-chlorinated congeners tend to be metabolized more rapidly, usually to mono- or dihydroxylated metabolites that are excreted in the urine and feces (Safe, 1984). Highly- chlorinated congeners are strongly lipophilic and quite resistent to metabolic breakdown. WW Acute exposure of humans to high levels of heat- degraded PCBs has occurred in Japan in 1968 (the Yusho poisoning) and in Taiwan in 1979 (the Yu Cheng poisoning), and chronic exposure to PCBs has been described for workers in industries manufacturing and using PCBs. Children born to Yusho victims demOnstrated retarded growth, abnormal 11 tooth development, hyperpigmentation, small birth size and lower birthweight (Safe, 1987) . Yu-Cheng children have been described as being stunted in growth, delayed in developmental milestones , displayed def icits in developmental testing and demonstrated abnormalities in behavioral assessments (Rogan et al., 1988; Guo et al., 1994). The Yu-Cheng children further displayed continuing cognitive deficits at 4 to 5 years of age (Chen et al., 1992) . Interestingly, children born to Yu-Cheng mothers up to 6 years after exposure had ceased also displayed cognitive deficits similar to those seen in siblings exposed in utero in 1979. A proviso with respect to the data collected on Yusho and Yu-Cheng victims is that the heat- degraded PCBs also contained other halogenated species related to PCBs, such as polychlorinated dibenzofurans. Furthermore, it was suggested that children born to women consuming PCB-tainted Great Lakes fish also had small birth size and low birthweight (Jacobson et al. , 1983, 1984; Fein et al., 1984). The observed effects of PCBs on the offspring of PCB-exposed women demonstrate that PCBs exert deleterious effects on the developing embryo. PCBs can cross the placenta from the maternal to the fetal bloodstream and affect the developing embryo. Milk is another source of infant exposure, as PCBs are concentrated in milk. A number of studies have been conducted to evaluate the 12 effects of PCBs on the developing nervous system. These studies included the use of rats, monkeys, human epidemiological surveys, and cell cultures. Children born to women consuming PCB-contaminated fish to those born to "non-fish eating" women were compared for the purpose of examining cognitive abilities and behavior (Jacobson et al., 1983). Deficits were noted in visual recognition memory as measured by Fagan's test (a measure of infant response to novel visual stimuli) in seven-month old infants exposed in utero to PCBs (Jacobson et al., 1985). There was a dose-dependent relationship between poorer scores in Fagan's test and levels of intrauterine exposure to PCB, as determined by PCB levels in the umbilical cord serum. An effect on visual recognition memory in infants exposed to PCB via lactation was not seen. These data suggest that intrauterine exposure to PCBs may have effects on neurological development. These same children were again tested at 4 years of age for PCB—related deficiencies (Jacobson et al. , 1990) . Intrauterine exposure to PCBs was associated with lower scores in both the verbal memory and numerical (quantitative) memory. Interestingly, PCB exposure via lactation was positively related to memory and verbal scale performance, indicating that there is no adverse effects on verbal and quantitative memory from exposure to PCBs in milk. Thus, it was suggested that intrauterine exposure to PCBs is associated with poor visual 13 recognition memory, and poor verbal and quantitative abilities. Another epidemiological study (Rogan et al., 1986) reported that infants exposed to PCBs in utero demonstrated hypotonicity and hyporeflexia in a dose-dependent relationship. Furthermore, higher transplacental exposure was associated with lower psychomotor scores at both 6 and 12 months of age (Gladen et al., 1988). There was no relationship between these deficits and nursing exposure, again suggesting that PCB-mediated deficits are due primarily to in utero exposure to PCBs. By 2 years of age, these children no longer displayed any deficiencies (Gladen and Rogan, 1991) . These data directly contradict the results published by Jacobson et a1. (1990), leaving the matter unresolved as to the long-lasting effects of PCBs on young children. Taken together, the results from the above studies suggest the possibility that intrauterine, transplacental exposure to PCBs does affect human neurological and behavioral development. Therefore, maternal exposure to PCBs does affect the neurological development of humans. Studies were done with rats to further examine the transfer of PCBs from.mother to offspring. Accumulation of 1l'C-labeled PCB was measured in rat dams and pups given an oral dose of 10 mg/kg Kaneclor 600 (a PCB mixture similar to Aroclor; Takagi et al., 1986). The dams accumulated 44% of 14 the total dose given, while pups accumulated only 0.003%. Newborn and suckling rat pups' concentrations of PCBs had the following distribution -- liver’> heart > skin > muscle > blood > lung > brain. PCB concentrations then decreased upon weaning. Congener accumulation of Aroclor 1254 in rat pups has also been investigated (Shain et al., 1986), and it was determined that the maternal to fetal PCB transfer was not correlated simply with physical and chemical properties of PCBs. Bioaccumulation of congeners in the brain was not correlated with extent of chlorination, suggesting that the number of chlorines did not influence entry into the brain as much as other characteristics of PCB congeners. These investigators suggested that accumulation of congeners is associated with chlorination of the 2 and 4 positions (ie. ortho and para) of the second phenyl ring, but not with chlorination of the 2 and 5, or 2 and.3 positions (ie. ortho and meta). Experiments performed on rats relating ingestion of PCB mixtures to effects on the amounts of brain dopamine (Seegal et al., 1986a, 1991b), suggesting that when a dose of Aroclor 1254 or 1260 equal to 1/10 of the rat LD50 was administered by gastric lavage, the levels of dopamine and dihydroxyphenylacetic acid (DOPAC), a dopamine metabolite, were significantly decreased in the caudate region of the brain, but not in the lateral olfactory lobe. When similar doses of Aroclor 1254 were mixed with chow and fed to rats 15 for 30 days as a chronic exposure, dopamine concentrations _in the striatum and lateral olfactory tract were decreased. No decreases in dopamine were seen in other areas of the brain. These data suggest that more brain regions are affected by chronic doses of PCBs than are affected by a single acute dose. In non-human primates, PCBs also affect behaviors and amounts of dopamine in the brain. After seven months on PCB-containing diets (either 2.5 ppm or 5 ppm Aroclor 1248) infants that were born to a group of rhesus monkeys and survived tended to be hyperactive and did not learn reversal tasks as readily as the control animals, and this altered behavior persisted for two years (Allen et al., 1979). Infants born to these same female monkeys one year after removal from PCB-laced diets showed similar characteristics to those born during the time of the PCB-containing diets, suggesting that PCBs can remain within maternal adipose stores and affect offspring even after direct exposure to PCBs has ceased for a length of time. Work by other investigators (Bowman et al., 1978, 1981: Bowman and Heironimus, 1981) revealed that simian infants born to PCB- exposed mothers had low birthweights and were hyperactive up to 12 months of age; but became hypoactive by 44 months of age. The in' utero PCB-exposed offspring demonstrated deficits in spatial and color discrimination reversal tasks, and this retarded learning was correlated to the level of 16 PCB exposure in utero. Since the effects of PCBs on learning in animals were observed using complex discrimination tasks and schedule—controlled performance in monkeys, these findings indicate that it is the higher, cognitive levels of CNS function that are disturbed by exposure to PCBs. Exposure of adult monkeys to Aroclor 1016 (bi- to tetrachlorobiphenyls) resulted in decreases in dopamine in the caudate, putamen, substantia nigra, and hypothalamus (Seegal et al., 1990) that lasted for up to 44 weeks after exposure to PCBs ended (Seegal et al., 1994). Interestingly, three ortho-substituted congeners (2,4,4'; 2,2',4,4'; and 2,2',5,5') accounted for most of the PCBs that were accumulated in the brains of these animals (Seegal et al., 1990). Hence, the data suggested that ortho- substituted PCBs can gain access to various brain regions and decrease the dopamine content. Again, monkeys were used to determine effects of different PCB mixtures (Aroclors 1016 and 1260) on brain dopamine levels (Seegal et al., 1991a) . With Aroclor 1016, decreases in dopamine were again seen in the caudate, putamen, substantia nigra, and hypothalamus, and the same three congeners were seen to accumulate in these brain regions. Yet, when the mixture Aroclor 1260 (mostly pentachlorobiphenyls and larger) was used, only in the caudate, putamen, and hypothalamus were decreases in dopamine levels observed. However, with 17 Aroclor 1260, the total amount of bioaccumulation of PCBs was much greater than with the 1016. Further, of the congeners making up greater than 5% of total brain PCB residue, most were hexa- or heptachlorinated di-ortho- substituted congeners. These data support the notion that ortho-substituted congeners are 1) more likely to accumulate in the brain (in agreement with the results of Shain et a1. (1991)), and 2) likely to be responsible for changes in brain dopamine levels. Supporting the hypothesis that ortho-substituted congeners are the effectors of decreased brain dopamine are studies on the structure-activity relationships of the neurotoxic effects of PCBs in PC12 cells (Shain et al. , 1991) . Congeners with ortho- or ortho— and para-substitutions were reported to be the most potent in decreasing cellular dopamine. Substitutions in the meta- position reportedly enhanced the neurotoxic effects of ortho- , but not of ortho- and para-substituted congeners. Congeners structurally similar to 2,3,7,8-TCDD, such as 3,3'4,4'-tetrachlorobiphenyl and 3,3',4,4',5'- pentachlorobiphenyl, did not cause a decrease in cellular dopamine, suggesting that the Ah receptor does not mediate the effects of PCBs on brain dopamine content. Furthermore, increasing congener chlorination did not correlate with a decrease in potency. Only when one ring of the biphenyl was completely chlorinated, was there a definite decrease in potency, suggesting that the positions of chlorination 18 rather than the number of chlorines is important in mediating the neurotoxicity of PCBs. Furthermore, the parent PCB congeners, and not their metabolites, were the active mediators in reducing dopamine levels (Shain et al., 1991) . To determine whether the three specific congeners that accumulated in the brains of PCB-treated adult monkeys (2,4,4'; 2,2',4,4'; and 2,2',5,5') were responsible for the decreased amounts of dopamine in specific brain regions, they were applied to rat pheochromocytoma PC12 cells, a model dopaminergic system (Seegal et al., 1990). These congeners caused a decrease in cellular dopamine content. However, application of TCDD-like congeners failed to elicit the. decrease, confirming that the Ah receptor is not involved in this toxicity. Aroclor 1254 also produced a dose-dependent decrease in cellular dopamine in PC12 cells in comparison to vehicle—treated control cells (Seegal et al., 1989). These findings suggested that PCBs may affect theadopamine-containing PC12 cells in the same way that they affect the dopaminergic neurons in the brain. The evidence compiled from human, animal, and cell culture studies suggest that PCB mixtures cross the placenta into the developing embryo, enter neurons or their progenitors, and affect the neurological development of the organism. 19 mm PC12 cells were isolated by Greene and Tischler (1976) from a solid rat pheochromocytoma of a New England Hospital Deaconess white rat. These small (6—14 u) round cells with a doubling time of 48 to 96 hours, contain acetylcholine, and various catecholamines (dopamine and norepinephrine) in separate granular chromaffin-like vesicles. The catecholamines are secreted in a calcium-dependent manner in response to depolarization or nicotinic or muscarinic cholinergic agonists (for reviews, see Guroff, 1985; Fujita et al., 1989). PC12 cells contain the synthetic enzymes for catecholamines: tyrosine hydroxylase (TH), L-aromatic amino acid decarboxylase (LAAAD) , dopamine beta hydroxylase (DBH) , and phenylethanolamine N-methyltransferase (PNMT). Metabolizing enzymes are also present: monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). Dopamine is the primary catecholamine in PC12 cells. Addition of nerve growth factor (NGF) to PC12 cells causes a morphological and physiological transformation of the cells into "sympathetic-like neurons". The changes brought about by NGF provide a basis by which the PC12 cell line may be used as a model system to study neuronal differentiation (Guroff, 1985) and effects of neurotoxins (Shafer and Atchison, 1991). NGF is a neurotrophic protein that can be isolated from male mouse submaxillary glands. The 78 form.of NGF, contains alpha, beta, and.gamma subunits 20 and has a molecular weight of about 140,000 KDa. The 2.58 form, which has biological activity, is a dimer of noncovalently linked beta subunits with a molecular weight of approximately 26,000 KDa. When PC12 cells are exposed to NGF, a number of remarkable phenomena occur. These events can be categorized into immediate early (within seconds), delayed early (within minutes to hours), and late events (hours to days). One of the earliest changes seen in PC12 cells exposed to NGF is a ruffling of the membrane, followed by changes in membrane properties that alter the ability of the cells to adhere to plastic substrates. Other early events that occur with NGF administration include the transcription-dependent induction of the protooncogenes c-fos, c-jun, v-src, and H-ras and N- rasa It is believed.that the induction of the protooncogene proteins play an important role in later events linked to NGF exposure. Together with protooncogene induction come changes in ion fluxes, alterations in second messenger systems such as changes in the activities of cyclic AMP- dependent protein kinase (PK-A) and protein kinase C (PK-C) , activation or increased levels of cytoskeleton-associated proteins such as GAP 43, and cytoskeletal rearrangements. Patterns of protein phosphorylation are also altered. Later changes in the PC12 cells caused by NGF exposure include the outgrowth of neurites. This change is usually apparent at 24 to 48 hours after NGF addition. Upon 21 continued exposure to NGF, the neurites become extensive and branched. By 14 days of NGF exposure, PC12 cells have obtained a fully differentiated sympathetic-like neuronal phenotype, demonstrating similar properties to those of a neuron, such as electrical excitability and release of neurotransmitters in response to electrical stimulation or administration of agonists. Removal of NGF from the medium results in retraction of the neurites, however, readdition of the factor causes their immediate reissuance. NGF binds to two receptor types located on the surface of the PC12 cell, a low- affinity NGF receptor which has a Kd value of approximately 1 x 104 M, which is not believed to mediate the biological response to NGF; and a high- affinity NGF receptor, with a Kd of approximately 1 x 10'11 M, which is considered the biologically relevant form of the receptor. The NGF high-affinity binding protein is the trk protooncogene product, pp140"*, which has tyrosine kinase activity (Kaplan et al., 1991a,b; Ohmichi et al., 1991; Klein et al., 1991). The pp140"* is distinct from the low- affinity receptor defined by Green and Greene (1986) as demonstrated by immunoprecipitation studies, indicating that the pp140"k is not the low-affinity NGF receptor with an associated protein (for review see Ross, 1991). At the present time, there is great literature debate as to the roles of the high and low affinity receptors in differentiation of neuronal cells. 22 E . H ! 't! The neurotransmitter dopamine appears to be highly integrated in many types of behaviors and in cognition. Dopaminergic projections within the CNS are extensive and occur in, among other regions, the neostriatum, limbic regions, and cortex (Cooper et al., 1991; Graybiel et al., 1994). Dopamine is associated with various behaviors, including: sensorimotor control and learning (substantia nigra, striatum, nucleus accumbens; Cousins et al., 1993; Aosaki et al., 1994), hyperactivity and hypoactivity (mesolimbic, substantia nigra, striatal, nucleus accumbens: Richardson and Tizabi, 1994), motivation and reinforcement (nucleus accumbens; Salamone, 1994), behavioral inhibition (prefrontal cortex), grooming (Francis et al., 1994), aggression (Micsek et al., 1994: Francis et al., 1994), lordosis (striatum; Pednekar and Mascarenhas, 1993), food and.water seeking behaviors (caudate and nucleus accumbens; Pal and Thombre, 1993). Dopamine has recently also been implicated in cognition and learning (Graybiel et a1. , 1994; Crossman, 1987: Albin et al., 1989: Malpaini et al., 1994). Many of the deficits observed in children involved in epidemiological studies on prenatal exposure to PCBs include decreased cognitive and motor functions, suggesting that the deficits in their behaviors may well have a dopaminergic component to them. Additionally, the data obtained on decreased brain dopamine content in prenatally exposed mice 23 (Agrawal et al., 1981) support the hypothesis that some of the neurological deficits associated with prenatal PCB exposure have at least partial underlying dopaminergic components” However, this is not to suggest that all of the deficits seen due to an in utero exposure to PCBs can be attributed solely to a decrease in basal ganglionic dopamine content. Effects of PCBs on other transmitter systems such as acetylcholine and neuropeptides warrant investigation. Further, both prenatal and postnatal exposure to PCBs have been reported to affect endocrine the system (Safe, 1994). The endocrine system plays an important role in the development.of the nervous system, and thus some.alterations in endocrine function may also play a role in the cognitive and behavioral deficits associated with prenatal PCB exposure. Investigations into this possibility have not yet been reported. n ' b 's ' PC s Changes in dopamine content and synthesis are at the crux of this thesis, therefore, a discussion of them is in order; Figure 1.3 depicts aigeneric dopaminergic cell. The synthesis of catecholamines involves several reactions. Tyrosine is transported into the cell where the phenyl ring is hydroxylated by TH to form 3,4-dihydroxyphenylalanine (DOPA). The formation of DOPA by TH is the rate-limiting step in the synthesis of catecholamines. DOPA is then decarboxylated by L-aromatic amino acid decarboxylase 24 TH LAAAD f Tribune—b DOPA —» Dopamine DOPAC Figure 1.3 synthesis of dopamine The amino acid precursor, tyrosine, is actively taken up into the cell where it is hydroxylated by the rate limiting enzyme, tyrosine hydroxylase (TH) to form 3,4- dihydroxyphenylalanine (DOPA). DOPA is decarboxylated by the cytosolic L-aromatic amino acid decarboxylase (LAAAD) to form dopamine. Dopamine may then be packaged into dense core vesicles for release in response to a depolarizing stimulus, or acted upon by an aldehyde reductase (ALD DH) and monoamine oxidase (MAO) associated with the mitochondria to form dihydroxyphenylacetic acid (DOPAC). Each enzyme along the biosynthetic pathway requires a cofactor, for TH, this is tetrahydrobiopterin (8H,); for LAAAD, the cofactor is pyridoxal phosphate. 25 (LAAAD) to form dopamine. Dopamine is then taken up into storage vesicles where it may be hydroxylated at the beta position to form norepinephrine by dopamine B hydroxylase (DBH) , which is located primarily in the vesicular membrane. Each of the enzymes involved in catecholamine biosynthesis require cofactors. Tyrosine hydroxylase requires tetrahydrobiopterin (BH,) , while LAAAD requires the presence of pyridoxal phosphate. Ascorbate is the cofactor required for DBH activity. In PC12 cells, dopamine is the primary catecholamine since the level of ascorbate is low in these cells (Fujita et al., 1989). .A number of factors can affect the rate of catecholamine synthesis. Cell density can have an effect on TH and DBH levels in PC12 cells (Badoyannis et al., 1991). TH, TH mRNA, and dopamine levels increase when cells are cultured at a high density. However, when PC12 cells are recultured from high to low density, the dopamine, TH, and TH mRNA levels decrease. In contrast, the DBH mRNA level decreases at high density, thereby decreasing the production of norepinephrine. Data from the work published by Seegal et a1. (1989) indicate that the release and degradation of dopamine is not increased in PC12 cells acutely exposed to PCBs, suggesting that the PCB-mediated decrease in dopamine may not be due to decreased synthesis. The levels of DOPA were not reported to be elevated in PC12 cells treated with Aroclor 1254, 26 suggesting that L-aromatic amino acid decarboxylase is not inhibited by PCB exposure (see Seegal et al., 1989). Inhibition of LAAAD by PCBs would have resulted in DOPA accumulation. The amounts of cellular norepinephrine and dopamine in acutely Aroclor 1254-exposed cells decreased in a parallel fashion, indicating that in the PCB-treated cells, DBH activity was not altered (Seegal et al., 1989). If DBH activity was impaired, dopamine would have increased, due to a slower rate of conversion of dopamine to norepinephrine. Therefore, since the activities of LAAAD and DBH have been reported to be unaffected by exposure to PCB, it appears, based on the reports of Seegal et al. , that PCB-mediated reduction in dopamine may be due to inhibition of TH activity. However, results of studies by Seegal et a1. omit the possibility that the decrease in levels of cellular dopamine may be due to an alteration in the uptake of tyrosine, the amino acid precursor for dopamine. In order to be taken up into dopaminergic cells, tyrosine must cross the blood-brain barrier and the neuron plasma membrane. The transport of tyrosine across the blood-brain barrier is active (Guroff et al., 1961) and thought to be facilitated, in part, by a Nai-independent transporter designated system L (Wiesel et a1. , 1991) . This transporter also transports large neutral amino acids (LNAA) . In addition to system L, another Nat-independent amino acid carrier which prefers aromatic amino acids, 27 designated system T, may also play a role in controlling the uptake of tyrosine into the brain and nerve cells (Stewart et al., 1989). Several studies using synaptosomes isolated from rat brain as a model have suggested that there are multiple transport routes for tyrosine into neurons. Work by Aragon et al. (1981) suggests that the uptake of tyrosine into an osmotically active space was dependent on a Nai gradient, and was stimulated by a membrane potential. Kinetic data suggested two uptake systems with different affinities for tyrosine. Expanding on the findings of Aragon et al. (1981), Diez-Guerra and colleagues (1986, 1989) indicated that L-tyrosine enters the cell through three pathways, each with different Km values, and each with different roles in the regulation of cellular tyrosine levels. The authors suggested that the transporter with the lowest K“I (6 uM) may play a regulatory role in catecholamine biosynthesis (Diez-Guerra et al., 1986). Historically, it has been believed that tyrosine hydroxylase activity is the rate-limiting factor in the synthesis of dopamine and other catecholamines. However, this may not always be the case. In the last twenty or so years, numerous researchers, led by Drs. Richard Wurtman and John Fernstrom, have published data suggesting that availability of aromatic amino acid precursors for various neurotransmitters may be rate limiting for synthesis of neurotransmitters, in some situations. This precursor- 28 dependent limitation to neurotransmitter synthesis has been (well established for serotonin (5-HT) and its precursor tryptophan (Carlsson and Lindqvist, 1978, Fernstrom, 1983). For dopamine and its precursor tyrosine, the relationship is more convoluted. There have been some governing principles put forth by Wurtman (1984) relating the synthesis of neurotransmitters such as serotonin, acetylcholine, and the catecholamines, and the plasma levels of their precursors: "1. The limiting step in the biosynthesis of the transmitter must be catalyzed by a low-affinity enzyme . . . tyrosine hydroxylase -- which at normal substrate concentrations, is unsaturated with the precursor. 2. This enzyme must not be subject to significant end-product feed-back control when the neuron containing it is fired. 3. The amount of the enzyme's substrate (the neurotransmitter precursor) present within the neuron must depend on its concentration in the plasma, either because the nerve terminal is unable to make the precursor ... and obtains it solely by influx from the plasma, or because, even though the neuron can synthesize the precursor ... , it tends to lose it by efflux into the plasma at a rate that varies inversely with the precursor's plasma concentration. 4. .A mechanism must exist which facilitates the precursor's passage from the bloodstream to the brain, and vice versa (i.e., a blood- brain barrier transport system): moreover, the affinity of 29 this mechanism for the circulating precursor must be relatively low. 5. Plasma levels of the precursors must, in fact, change under normal conditions." Indeed, it has been demonstrated that these principles do apply to the catecholamines and their precursor, tyrosine (Lehnert and Wurtman, 1993: Wurtman, 1984: Fernstrom, 1983; Wurtman et al., 1981: and Wurtman and Fernstrom, 1976). Traditionally, TH is believed to be saturated with tyrosine, under normal conditions in the brain, and since TH has a slower rate than LAAAD, is the rate limiting step in catecholamine synthesis. Many studies have demonstrated that tyrosine availability can be rate-limiting for catecholamine synthesis, but only under conditions where the neurons in question are rapidly firing, as in normally highly active, catecholaminergic brain regions, or in catecholaminergic neurons responding to a pharmacological challenge. This condition of rapid firing of the neuron results in phosphorylation of TH, thus decreasing the K. for the cofactor, 8H,, and increasing the Ki for end-product inhibition. This, in effect, causes TH to perform at a maximal rate, thus making tyrosine availability rate limiting (see Fernstrom, 1983: Milner and Wurtman, 1986: Wurtman, 1988: and Lehnert and Wurtman, 1993) . However, recent data have suggested that high activity is not necessarily required for tyrosine availability to have effects on catecholamine synthesis and release. Using 30 microdialysis, in vivo studies have shown that tyrosine availability can affect unstimulated catecholamine release in the brain (Lehnert and Wurtman, 1993: Kreutz et al., 1990; and Acworth et al., 1988). Thus, there are many avenues for polychlorinated biphenyls to disrupt the synthesis of dopamine. For instance, PCBs can alter the uptake of tyrosine through a variety of mechanisms, namely, competitive or non- competitive inhibition of amino acid uptake, or disruption of the uptake protein subSequent to increased fluidization of the membrane. One report (Wurtman et a1. , 1974) suggested that there is not a linear relationship between cellular tyrosine levels and DOPA formation, that a small decrease in cellular tyrosine can lead to a sizable decrease in DOPA synthesized. Hence, a small disruption of tyrosine uptake by PCBs could lead to a serious decrease in levels of cellular dopamine. Another route for PCBs to cause decreased dopamine synthesis is by affecting the activity of TH. TH is a tetrameric enzyme with a molecular weight of approximately 210-220 KDa with individual subunits having a molecular weight of approximately 62,000 KDa (Markey et a1. , 1980) . Iron and BH, are the cofactors needed for TH activity (Wang et al. , 1991) . TH activity can be inhibited by dopamine through a negat ive feedback loop . Phosphorylation of the TH enzyme has been reported to cause a decrease in the Km for BH, and an increase in the Ki for 31 dopamine (Albert et al., 1984: Haycock. and Haycock, 1990), resulting in an increase in TH act;vity. The phosphorylation of TH can be catalyzed by several protein kinases. Agents activating’ PKA, PKC, and calcium/calmodulin-dependent protein kinase (Ca/CAMPK) increase phosphorylation of TH at Serine 43 (Campbell et al.,1986; Haycock, 1990). Ca/CAMPK phosphorylates an additional site as well (Campbell et al., E986), which as been reported to be Serine 19 (Haycock, 199i) . Vulliet et a1. (1989) have also described a proline-iirected kinase capable of phosphorylating TH at Serine 8. Theoretically, PCBs could alter the IgnEor BH, or alter the Ki for dopamine. Alterations in Km are likely to come from alterations in the phosphorylation of the enzyme. Phosphorylation by a calcium/calmodulin-dependent protein kinase enhances the activity of tyrosine hydroxylase without altering the kinetic association constants. However, a CAMP-dependent protein kinase may phosphorylate the enzyme, resulting in decreased Km for BH‘, and increased Ki for dopamine which leaves the enzyme activity dependent on the amount of tyrosine available to be utilized (Lehnert and Wurtman, 1993). It is possible that PCBs may affect the activity of tyrosine hydroxylase by conpeting either with substrate (tyrosine), BH,, or dopamine, thereby disrupting the kinetics and regulation of the enzyme. Finally, PCBs may disrupt dopamine synthesis by altering the activity of 32 LAAAD, which converts DOPA to dopamine. LAAAD is a dimer (with a molecular weight of approximately 100 KDa (Coge et al., 1989). LAAAD converts DOPA to dopamine, and also 5- hydroxytryptophan (5-HTP) to serotonin (5-HT). For activity, LAAAD requires the presence of pyridoxal pyrophosphate, B6, with a K“I of approximately 0.28 uM (Young et al., 1993; Hadjiconstantinou et al., 1993) . The me of LAAAD for DOPA is about 37 nmol/mg protein/20 min, while the K. for DOPA is around 32 DM (Young et al., 1993; Hadj iconstantinou et a1. , 1993) . Neuronal and non-neuronal isoforms of the enzyme have been reported (Morgan et al., 1986, Krieger et al., 1991), although there appears to be only one gene (Albert et al., 1987), indicating possible tissue-specific alternative splicing of the RNA. However, at this time, there appears to be debate in the literature about how many forms of decarboxylase exist (see Melamed et al., 1980 and Sims et al., 1973, reviewed in Ebadi and Simonneaux, 1991) . The activity of LAAAD appears to be regulated acutely by PKA, resulting in a change in er without alterations in K. values (Young et a1. , 1993) . Both D1 and D2 receptors have been reported to chronically regulate striatal LAAAD. Antagonists for the dopamine receptors increase the activity of striatal LAAAD, while agonists decrease the activity (Hadjiconstantinou et al., 1993) . The increase in activity caused by dopamine receptor antagonists appears to be caused by an increase in er and 33 not alterations in K. values. Hence, PCBs could have effects on any of the enzymes involved in dopamine synthesis or on the uptake of tyrosine into cells. Inhibition of tyrosine uptake or dopamine synthesis could result in a decrease in the release of dopamine since it has been shown that newly synthesized catecholamines are preferentially released (Kopin et al., 1968: Besson et al., 1969; Gewirtz and Kopin, 1970). Another deficit in the understanding of the significance of PCBs causing a decrease in the amount of cellular dopamine is information on a functional significance of the decrease. Because cellular levels of dopamine decline, is this enough to manifest an alteration in the release of dopamine? At this time, the effects of polychlorinated biphenyls on the release of dopamine are unknown. H !l . l E' Polychlorinated biphenyls have been reported to be associated with various neurological deficits in animals and humans, some of which may have a dopaminergic component. Acute studies using dopaminergic cell culture systems have suggested that PCBs cause decreases in cellular dopamine, and that ortho substituted PCB congeners play a major role in the deleterious effects of PCBs on dopaminergic cells. The purpose of these studies was to examine the subchronic effects of polychlorinated biphenyls on the dopaminergic function of PC12 cells. Specifically, the goal was to 34 examine the hypothesis that polychlorinated biphenyls cause _decreases in levels of cellular dopamine in PC12 cells that result from altered synthesis of dopamine, and is manifested as a decrease in the release of dopamine in response to a depolarizing stimulus. The scope of the research included several specific aims: . Determine the subchronic effects of Aroclor 1254 and selected PCB congeners on cellular dopamine content, using undifferentiated and differentiating PC12 cells as a model system. . Determine whether the neurotrophic factor, nerve growth factor can protect dopaminergic cells from damage caused by subchronic exposure to Aroclor 1254, using PC12 cells as a model system. . Determine whether subchronic exposure of PC12 cells to Aroclor 1254 and selected PCB congeners results in altered release of dopamine in response to stimulation with 56 mM potassium, and whether the PCB-mediated decreases in cellular dopamine contributed to any alterations observed. . Determine the amounts of Aroclor 1254 or PCB congeners associated with PC12 cells after a subchronic exposure, and whether the amounts of PCBs associated with the cells relate to the effects seen in the cells after subchronic exposure to PCBs. . Examine possible mechanisms to explain the decreases in amounts of cellular dopamine and release of dopamine from 35 PC12 cells following subchronic exposure to Aroclor 1254. Targets for investigation include 1) the effects of Aroclor 1254 on the uptake of tyrosine, 2) the effects of Aroclor 1254 on the rates of conversion of 3H-tyrosine to 3H-DOPA and 3H-dopamine, and 3) the effects of Aroclor 1254 on LAAAD, using supplementation of cell growth medium with DOPA and examining the cellular dopamine content. I 36 KB ODS 37 Materials Nerve growth factor (NGF) was isolated from male mouse submaxillary glands by the procedure of Bocchini and Angeletti (1969). Stock solutions of.Aroclor 1254 (courtesy of Dr. J. Quensen, Michigan State University) were prepared using’ dimethyl sulfoxide (DMSO) as a solvent. Stock solutions of.Aroclor 1254 were diluted into the cell culture medium to achieve the desired concentrations. The final DMSO concentration in the culture medium was at most 0.1% (v/v). Polychlorinated biphenyl congeners [2,2'-dichlorobiphenyl (PCB 04); 3,3'-dichlorobiphenyl (PCB 11); 4,4'- dichlorobiphenyl (PCB 15) ; 2,4,4'-trichlorobiphenyl (PCB 28); 2,2',4,4'-tetrachlorobiphenyl (PCB 47); 2,2',5,5'- tetrachlorobiphenyl(PCBSZ):2,3,4,4',5-pentachlorobiphenyl (PCB 114): 2,3',4,4',5-pentachlorobiphenyl (PCB 118); 3,3',4,4',5-pentachlorobiphenyl (PCB 126); 2,2',3,3',4,4'- hexachlorobiphenyl (PCB 128): and 2,2',4,4',5,5'- hexachlorobiphenyl (PCB 153)] were obtained from AccuStandard, Inc. (New Haven, CT: see Appendix I for PCB congener structures). Stocks of congeners were prepared in DMSO, then diluted into the culture medium to achieve the desired medium concentration. The final DMSO concentration in the medium was at most 0.5% (v/v). The reagents L-3,4- dihydroxyphenylalanine (L-DOPA), 3—hydroxytyramine (dopamine, DA), L-3,4-dihydroxyphenylethanolamine (norepinephrine, NE), and 3,4-dihydroxyphenylacetic acid 38 (DOPAC) were purchased from the Sigma Chemical Co. (St. Louis, MO) and standard solutions were prepared in 0.1 M HCl at a concentration of 10 ng/ml. Hoechst 33258 (bis- benzamide) was also purchased from the Sigma Chemical Co. (St. Louis, MO). Octyl sodium sulfate (038) was obtained from Eastman Kodak Co. (Rochester, NY). (Methyl-3 H)thymidine and (methyl-3H)-choline were obtained from Amersham Corp. (Arlington Heights, IL) or New England Nuclear (Boston, MA), while L-(ring 3,5-3H)-tyrosine, L-“C- leucine and L-“C-arginine were obtained from New England Nuclear. All other chemicals were of reagent or high performance liquid chromatography (HPLC) grade. WWW: PC12 cells were grown in monolayer form in growth medium consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with 6% fetal bovine serum, 6% horse serum, 100 ug/ml of streptomycin, 100 units/ml of penicillin, and 25 mM HEPES at pH 7.4. Cells were incubated at 37°C in a humidified atmosphere containing 6% C02, and subcultured at least once per week. t' PC e ls for etermination of Ce We To examine the effects of PCBs on amounts of dopamine in PC12 cells treated simultaneously with or without NGF, cells were seeded on polylysine-coated 6-well plates in cell culture medium with or without Aroclor 1254, in the presence 39 and absence of NGF. Cells exposed only to 0.1 % (v/v) DMSO (in the presence and absence of NGF were used as controls. DMSO alone did not alter levels of cellular dopamine. After treatment, cells at an approximate density of 2.1 x 10" cells/cm2 were harvested following incubation in 1.0 ml of phosphate buffered saline (PBS) containing 0.05% trypsin and 0.53 mM EDTA for 10 minutes at room temperature. The PBS consisted of 138 mM NaCl, 2.7 mM KCl, 7.3 mM KHZPO“ and 8 mM NaZHPO‘. An additional 2 ml of ice-cold PBS was added and the cells were subsequently collected. Each well was then washed with an additional 2 m1 of ice-cold PBS. After trituration of the cells, 1 ml of each cell suspension was removed for determination of DNA content. The remainder of each sample was centrifuged at 5000 g x min at 4°C. Subsequently, each pellet was resuspended in 0.5 ml of 0.2 M HClO‘ containing 0.263 mM EGTA and incubated on ice for 30 min. The samples were again centrifuged at 5000 g x min and the supernatants were stored at -20°C until assayed for amounts of cellular catechols. To examine the effect of Aroclor 1254 on amounts of dopamine in NGF-pretreated PC12 cells, cells were seeded in polylysine-coated 6-well plates in DMEM with or without NGF and cultured for various periods of time in the absence of PCBs. Every 4 days, fresh DMEM with or without NGF was added. After the appropriate incubation period without PCBs, 25 pg/ml Aroclor 1254 was then added. After 2 days of 40 exposure to Aroclor 1254, the cells were then harvested and .prepared for DNA and HPLC analysis as described above. The effects of various PCB congeners on amounts of catechols in PC12 cells was investigated by seeding cells and harvesting them as described above. After three days of exposure, cells at an approximate density of 2.1 x 104 cells/cm2 were harvested and prepared for DNA determination and HPLC analysis of cell extracts as described above. I! .!. EEHEQH-B! Determination of cellular DNA content was performed according to the method of Labracca and Paigen (1980) . Briefly, the samples were centrifuged at 5000 g x min, the supernatant aspirated, and the pellet resuspended in 650 pl of an assay buffer consisting of 2.0 M NaCl, 0.05 M Nazi-1P0), and 2.0 mM EDTA.at pH 7.4. The samples were sonicated and 50 to 300 pl aliquots were incubated with 1.2 ml of Hoescht 33258 and 0 to 250 pl of assay buffer. The final volume of the incubation was 1.5 ml. Relative fluorescence was measured with a spectrophotofluorometer using excitation and emission wavelengths of 356 and 448 nm, respectively. The DNA content in samples was quantitated from the relative fluorescence values, using calf thymus DNA as a standard. 9‘3. - ‘0’! ° '°°'.Ll_!‘ '2‘ A 0, 21°. ’ b0 .- ‘. Amounts of the catechols DOPA, DOPAC, and dopamine in cell extracts were measured using HPLC coupled with electrochemical detection, based on a modification of the 41 method of Seegal et a1. (1986). Catechols were eluted Ithrough a C-18 reverse phase column at a rate of 0.9 to 1.0 ml/min with a mobile phase consisting of 2.6 mM NaHZPO“ 0.05 to 0.064% 088, 0.1 mM EDTA, 2.5% triethylamine, and 20 to 22.5% methanol at pH 3.3. The catechols were then oxidized on a glassy carbon electrode at a potential of 0.775 V, versus a Ag/AgCl reference electrode, and the resulting signals were quantitated as peak height. The minimal sensitivity of the system for determining amounts of catechols was 25 pg. Peak heights of standards containing 1 ng of catechols were used to quantitate catechols in each sample. Following quantitation of levels of catechols in the cell extracts, values were calculated as pg/ng DNA and reported as percent of control value in the absence of PCBs. WW Induction of neuronal differentiation in PC12 cells was accomplished by incubation of the cells with 100 ng/ml of NGF. To determine the effects of Aroclor 1254 on neurite outgrowth, photographs.of random fields of cells were taken. Photographic images were digitized and analyzed using the BIOQUANT computer system (R a M BIOMETRICS, INC.) to determine percent of cells with neurites, number of neurites per cell, and length of neurites. Neurites were defined as processes issuing from the cell that were at least one cell diameter in length. 42 Week Tot determine the effects of .Aroclor 1254 on DNA synthesis, cells were synchronized by culturing the cells for 72 hours in a low serum-containing medium consisting of DMEM with 5 ng/ml selenous acid, 5 ug/ml insulin, 5 pg/ml transferrin, 0.075% bovine serum albumin, and 2% horse serum. Cells were then reintroduced to the normal serum- containing DMEM, plated onto polylysine-coated 12-well plates, and treated with or without Aroclor 1254. After 3 days, cells were exposed to 0.5 uCi/ml of (methyl- 3H)thymidine for 6 hours. Cells were harvested by the method of Madhukar et a1. (1989). Briefly, the cells were washed with PBS containing 0.5 mM M9012 and 0.9 mM CaClz. The cells were then incubated with 0.5 ml of 10% trichloroacetic acid for 10 minutes on ice, and then washed sequentially with 70% and 95% ethanol. Solubilization of cells was accomplished by incubation in 0.5 ml of 1 N NaOH. The base in the samples was neutralized with 0.5 ml of 1 N HCl, and the radioactivity in each sample was determined by scintillation counting. Results were reported as dpm/ng DNA. To determine the effects of Aroclor 1254 on protein synthesis in PC12 cells, a modification of the method of Madhukar et al. (1989) was used. 114DNA ffi" I ' fitY‘f'l’ I V I' "U’I‘I Z3AA¥5 u. q (IO 150- 125- 28‘ 75-! (I O l I 118DOPA A 1180A O IIBDOPAC O 1180NA VV'Tr I V I ‘IT"' I I' V V‘UU'I N III I O 175 erases i 150. Percent of DMSO-treated Control 125- 100- I 126 DOPA A 1260A O 126 DOPAC o 126 DNA 0 VIII r t I W'I'I" 1 10 ' 100 Media Concentration (uglml) Figure 3.8 Continued 76 175 150- 125- 100- ‘5 III I N (I 21.337454 I 128 DOPA A 1280A O 128 DOPAC O 1280M 175 'TTY' V 1' I Y I'VV‘ 150- 125- Percent of DMSO-treated Control a a s N I! l 2.2.4.455 F I 15300PA A 1530A O 15300PAC O 1530M O V'U'l 1' U rIjUY—rr fit UT'I'VVI' 1 10 100 Media Concentration (uglml) Figure 3.8 Continued 77 a decrease in dopamine. Cellular dopamine decreased with exposure 1x: 2,2',4,4'-tetrachlorobiphenyl; 2,2',5,5'- tetrachlorobiphenyl: 2,3,4,4',5-pentachlorobiphenyl: 2,3',4,4',5-pentachlorobiphenyl: 3,3',4,4',5— pentachlorobiphenyl; 2 , 2 ' , 3 , 3 ' , 4 , 4 '-hexachlorobiphenyl; and 2,2',4,4',5,5'-hexachlorobiphenyl. Only 4,4'- dichlorobiphenyl did not cause a dose-dependent decrease in cellular dopamine. For the coplanar congener 3,3',4,4',5- pentachlorobiphenyl, the dose-dependent decrease in DNA content occurred at media PCB concentrations identical to those at which decreases in amounts of cellular catechols were observed. The media PCB concentration Icfl,values were calculated for the effects of PCB.congeners and Aroclor 1254 on amounts of cellular dopamine (Table 3.3). .Aroclor 1254 had the lowest 1C“, for decreasing cellular dopamine and 2,2 ' ,4,4 ' ,5,5'-hexachlorobiphenyl had the highest. The effects of a 6 hour exposure of quiescent cells to 2,2'- dichlorobiphenyl were examined using the method of Seegal et a1. (1989) to try to explain differences between our results with. the congener, and those of Seegal pertaining to cellular amounts of catechols. The results indicated that cellular dopamine, DOPA and DOPAC were unaffected by exposure of the cells to 2,2'-dichlorobiphenyl until the media concentrations reached 100 pg/ml, where a modest decrease occurred (Figure 3.9). 78 Table 3.3 Effect of Aroclor 1254 and Various PCB Congeners on Levels of Cellular Dopamine in PC12 Cells l , Cellular Dopamine -. ICSO t S.E.M. | F _ _.—._.—.———.—_~—— Aroclor 1254 7 212' 4 d 2L2'1515' 4 31.8 i’ 3.0 2,3',4,4',5 4 38.1 i 2.8 313',4,4',5 4 27.6 1" 1.6 2,2',3,3',4,4' 4 34.4 i . ll . 41-8 a 1- J PC12 cells were exposed to various media concentrations of Aroclor 1254 or selected PCB congeners for 3 days. The determination of cellular dopamine was performed as described in Chapter 2, "Materials and Methods". n.d. not determinable since amounts of cellular dopamine never decreased below 50%. Data represent the mean i S.E.M. ICso values determined from 4 to 7 separate experiments for each congener. 79 200 ..a (II O l 0| 0 l + Aroclor 1254 —e— 22' Percent of DMSO-treated Control 3 O 0 '1" I V UNIV“ U I 'U'U'U U U U'UV'U I I I I 0.1 1 10 100 Media PCB Concentration (uglml) Figure 3.9 Cellular Dopamine Content Following Acute Exposure to Aroclor 1254 or 2,2'-Dich1orobiphenyl PC12 cells were exposed for 6 hours to Aroclor 1254 or 2,2'- dichlorobiphenyl. Cellular dopamine content was determined as described.in.Chapter 2, "Materials and.Methodsfi. .Amounts of cellular dopamine were normalized to protein levels as determined by incorporation of C-leucine as described by Seegal et al., 1989. Values represent the mean.r S.E.M. for 4 separate measurements. 80 DISEHSfiIQN Aroclor 1254: Exposure of cells to Aroclor 1254 for 6 hours has been reported.to be sufficient to attain a maximal decrease of approximately 70% in cellular dopamine at a media concentration of 100 pg/ml of Aroclor 1254 (Seegal et al., 1989). In the present studies, 100 pg/ml of Aroclor 1254 was lethal to the cells by 24 hours, suggesting that this concentration of PCBs elicits a cytotoxic effect. To further determine the cytotoxicity of lower concentrations of Aroclor 1254 at 3 days of exposure in PC12 cells, total media concentrations up to and including 50 pg/ml of the PCB mixture were further examined. The results of studies on the incorporation of (methyl-3H)-thymidine as a measure of DNA synthesis, incorporation of 1l'C-leucine as a measure of protein synthesis, and leakage of LDH into the culture medium as a measure of membrane integrity all suggested that at concentrations of Aroclor 1254 up to and including 25 pg/ml, there was no generalized cytotoxicity at 3 days. At a 50 pg/ml media concentration of Aroclor 1254, where DNA content began to decrease, decreases in sit-thymidine and 1"C- leucine incorporation indicated that the cells were beginning to show signs of cytotoxicity suggesting that at 50 pg/ml or greater, Aroclor 1254-mediated decreases in cellular dopamine may be at least partially attributable to general cytotoxicity. However, at media concentrations of Aroclor 1254 lower than 50 pg/ml, cytotoxicity cannot 81 account for the PCB-induced decrease in the amounts of cellular dopamine and thus may be due to decreases in dopamine synthesis. Furthermore, these data support the use of DNA content measurements as indicators of general cytotoxicity. In undifferentiated, non-NGF-treated cells, the PCB mixture Aroclor 1254 caused dose-dependent decreases in cellular dopamine and its major metabolite, DOPAC. Amounts of cellular DOPA, however, were elevated, compared to control cells. These results suggest that Aroclor 1254 may affect the enzyme converting DOPA to dopamine, LAAAD. An examination of the possible mechanisms for the effects of Aroclor 1254 on cellular dopamine will be addressed in Chapter 6. Amounts of DOPAC in PC12 cells exposed to Aroclor 1254 decreased in a dose-dependent manner, along with amounts of cellular dopamine. Whether the decreased cellular amounts of DOPAC were due to a direct effect of Aroclor 1254 on the enzyme converting dopamine to DOPAC, monoamine oxidase (MAO), or if the decreases in DOPAC solely reflects a diminished amount of dopamine available to be metabolized is not known at the present. However, if MAO were inhibited, and increase in dopamine would be expected. More study into the mechanism for the dose-dependent decreases in cellular DOPAC will be required to elucidate this issue. In undifferentiated, or non-NGF-exposed PC12 cells, the 82 dose-response curves depicting both cellular dopamine and DNA content were shifted to the left with longer exposure, up to 3 days. There was no significant shift in the dose- response curves between 3 and 7 days, however, suggesting that the maximal ability of .Aroclor 1254 to decrease cellular dopamine levels was attained within 3 days. PC12 cells in the process of differentiating responded to Aroclor 1254 in the same manner as that observed with undifferentiated cells. Differentiating PC12 cells, those exposed simultaneously to NGF and Aroclor 1254, also demonstrated shifts to the left in the dopamine and DNA dose-response curves between 1 and 3 days of exposure. The shifts were of the same magnitude as those seen with undifferentiated PC12 cells. Thus the data suggest that concomitant exposure of PC12 cells to NGF and Aroclor 1254 did not result in any increase or decrease in the sensitivity of these differentiating dopaminergic cells to decreases in.cellular dopamine or DNA.content.as a result of exposure to Aroclor 1254, when compared to undifferentiated PC12 cells. Further, the data suggest that differentiating dopaminergic PC12 cells exposed to PCBs beginning in the early stages of differentiation may be as susceptible to PCB-mediated decreases in cellular dopamine as are undifferentiated cells. However, exposure of PC12 cells to NGF’for 7 or 14 days prior to exposure to Aroclor 1254 resulted in partial 83 protection from the decreases in cellular dopamine caused by Aroclor 1254. Attenuation of the effects of other neurotoxic compounds such as 6-hydroxydopamine (6—OHDA) , MPTP, and N-methyl-4-phenylpyridinium ion (MPP‘) by neurotrophic factors have previously been shown. Preincubation with.brain-derived.neurotrophic factor (BDNF) or NGF was shown by Spina et a1. (1992) to offer a degree of protection from the neurotoxic effects of both 6-OHDA and MPPA. In SH-SY5Y human neuroblastoma cells, 24 hours of pretreatment with either BDNF or NGF prior to exposure to 6- OHDA or MPPi resulted in significant protection from these neurotoxicants. In primary nigral dopaminergic cell cultures, 6 days.or'4 days of pretreatment with BDNF offered partial protection from the neurotoxic effects of 6-OHDA or MPP’, respectively. Further, Hyman et a1. (1991) have demonstrated that a 24 hour preincubation of dopaminergic embryonic rat ventral mesencephalic cells with BDNF resulted in partial protection from the neurotoxic effects of MPTP. It is apparent that neurotrophic factors can play an important role in attenuating the ability of toxic compounds to damage dopaminergic cells. In the case of PCBs, the mechanisms by which NGF offers partial protection from the Aroclor 1254-mediated decrease in cellular dopamine are unclear and merit further investigation. There appear to be multiple effects of Aroclor 1254 on PC12 cells. For instance, effects on neurite elongation and 84 effects on cellular dopamine, may or may not be related since the dose-response curves for these effects are different. It is unclear why the simultaneous treatment of PC12 cells with NGF did not afford partial protection from the effects of Aroclor 1254, but pretreatment with NGF did partially'protect.the.cellsa Possibly, NGF induced an.event during the first 7 days of differentiation that allowed the cells to be partially resistent to the dopamine-depleting effects of Aroclor 1254. This event, however, was apparently blocked or altered in the presence of Aroclor 1254, since concurrent administration of NGF and Aroclor 1254 resulted in similar decreases in dopamine as were seen in cells treated only with.Aroclor 1254. This suggests that cells in early differentiation are more susceptible to the effects of PCBs than more differentiated cells are, consequently, embryos in the earliest stages of development may be the most sensitive. Aroclor 1254 did not block the NGF-induced morphological differentiation of PC12 cells. With concomitant exposure to NGF, Aroclor 1254 caused a dose- dependent increase in neurite elongation in PC12 cells without altering the percentage of cells bearing neurites or the number of neurites per cell, suggesting that Aroclor 1254 enhanced NGF-stimulated elongation of neurites. It is possible that the mechanism underlying this enhanced neurite elongation may be related to ion flux and homeostasis, 85 especially that of Ca“, since Ca” is involved in the regulation of neurite elongation (Lankford and Letourneau, 1989). Alterations in Ca” flux and homeostasis have been reported in cerebellar granule cells upon exposure to PCB congeners (Kodavanti et al. , 1993a) . Furthermore, PCB congener-related.alterations.injphosphoinositide signalling and protein kinase C (PKC) translocation have also been reported (Kodavanti et al. , 1993b) . PKC has been implicated in cell growth and also plays a role in neuritogenesis, hence, the effects of PCBs on neurite elongation in PC12 cells may also involve PKC. Other, non-calcium, non-PKC mediated mechanisms may also be involved. Further work will be required to determine the mechanism by which PCBs enhance neurite elongation. While it is clear that concomitant exposure of PC12 cells to NGF and Aroclor 1254 results in enhanced neurite elongation, it is unknown whether these neurites form synapses with other cells, or if synapses formed are operable. Hence, more research will be required to determine the physiological consequences of the PCB- mediated enhanced neurite elongation in PC12 cells. PCB Congeners: Of the 11 PCB congeners examined, those with at least 4 chlorine substitutions generally caused a dose-dependent decrease in cellular dopamine in PC12 cells. Amounts of cellular DOPA were increased by 2 di-ortho tetrachlorobiphenyls (2,2',4,4'-tetrachlorobiphenyl and 2,2',5,5'-tetrachlorobiphenyl), and 1 di-ortho 86 hexachlorobiphenyl(2,2',4,4',5,5'-hexachlorobiphenyl)..All three of these congeners can be found in Aroclor 1254 (Hutzinger' et al., 1974), and two of these congeners (2,2',4,4'-tetrachlorobipheny1 and. :2,2',5,5'- tetrachlorobiphenyl) have been found to be concentrated in the brains of adult monkeys who had ingested PCBs (Seegal et a1. 1990, 1991). These animals demonstrated a decrease in amounts of brain dopamine that was apparent up to 44 weeks following cessation of exposure to PCBs (Seegal et al, 1994). The increase in cellular DOPA suggests that these congeners may, like Aroclor 1254, decrease the activity of LAAAD. In addition to their effects on cellular dopamine and DOPA, the more highly substituted PCB congeners caused a general decrease in the cellular DOPAC. As with Aroclor 1254, the mechanism(s) for this decrease are not known, but may result from a decrease in available substrate, and/or may also be due to direct effects of PCBs on MAO. The PCB congeners with 2 or 3 chlorine substitutions- tested failed to decrease the amount of cellular dopamine in PC12 cells until media levels of PCBs reached a cytotoxic concentration of 100 pg/ml. Amounts of cellular DOPA were unaffected by these more lightly chlorinated congeners, however, amounts of cellular DOPAC were decreased by 2,2'- dichlorobiphenyl; 3,3'-dichlorobiphenyl: and 2,4,4'- trichlorobiphenyl. These 3 congeners decreased DOPAC without a concomitant or prior decrease in the amount of 87 cellular dopamine. These results suggest the possibility that these congeners may affect MAO directly, since the levels of substrate appeared to be unaffected by these PCBs. Hypothetically, if these lightly substituted PCB congeners were directly affecting MAO, the amounts of cellular dopamine would be expected to increase. However, it is possible that inhibition of TH by the end product, dopamine, may have compensated for the expected increase in the amount of cellular dopamine by causing less DOPA.to be formed, ‘The only congener tested that was wholly without effect on levels of catecholamines in PC12 cells was 4,4'- dichlorobiphenyl. The reasons for this are unknown. The PCB congener 2,2'-dichlorobiphenyl has been reported to be the most potent PCB congener for decreasing cellular dopamine in acutely exposed PC12 cells (Shain et al., 1991). In our system, 2,2'-dichlorobiphenyl failed to cause any changes in cellular catechols, except for a decrease in the metabolite DOPAC. The reasons for discrepancy are unknown, but it may be that a difference in the clone of PC12 cells utilized was sufficient to cause such.a.discrepancyu Even when the methods used by Seegal et a1. (1989) were duplicated using our PC12 cells, 2,2'- dichlorobiphenyl failed to affect the amount of cellular dopamine in our PC12 cells. The PCB congener 3,3',4,4',5-pentachlorobiphenyl, has been reported to act through the Ah-receptor to exert its 88 effects (Safe 1984). In PC12 cells, 3,3',4,4',5- pentachlorobiphenyl appeared to be toxic, decreasing in DNA content (used as an indicator of cytotoxicity) beginning at 10 pg/ml media concentration. Indeed, 3,3',4,4',5- pentachlorobiphenyl caused decreases in cellular dopamine, DOPA, and DOPAC that concurred with decreased DNA content, suggesting that with this congener, cytotoxicity may be the cause of the decreased cellular catechols in PC12 cells. For a number of selected congeners (2,2'- dichlorobiphenyl:2,2',4,4'-tetrachlorobiphenyl:2,2',5,5'- tetrachlorobiphenyl: 2,3,4,4',5-pentachlorobiphenyl: 2,3',4,4',S-pentachlorobiphenyl: 2,2',3,3',4,4'- hexachlorobiphenyl: and 2,2',4,4',5,5'-hexachlorobiphenyl) and Aroclor 1254, IC.50 values were determined based on the media concentrations of PCBs at‘which the amount.of cellular dopamine decreased by 50%. 2,2'-dichlorobiphenyl was the exception, since cellular dopamine never fell below 50% at media concentrations up to 50 pg/ml. The congeners 2,2',5,5'-tetrachlorobiphenyl; 2,3',4,4',5- pentachlorobiphenyl: 2 , 2 ' , 3 , 3 ' , 4 , 4 ' -hexachlorobiphenyl: and 2,2',4,4',5,5'-hexachlorobiphenyl each comprise a large percentage of Aroclor 1254 (4.2%, 9.5%, 1.5%, and 6.1%: Hansen, 1987). By examining these congeners, it was hoped that one or some of the components of Aroclor 1254 that caused the decrease in cellular catechols observed in PC12 cells may become more apparent. Aroclor 1254 was the most 89 potent of the PCBs examined for decreasing cellular dopamine in PC12 cells. Surprisingly, for the individual congeners for which ICso values could be calculated, the values were similar to each other, and were about twice the value for Aroclor 1254. However, the PCB congeners may differentially associate with the cells, and thereby have different potencies to reduce cellular dopamine. This possibility will be investigated in Chapter 5 of this dissertation. These data on the ICfl,values for congeners and Aroclor 1254 also raised a question as to whether PCB congeners in a mixture, such as Aroclor 1254, interact with each other to enhance each others' effects. 'The effects of 3 congeners on dopamine levels in PC12 cells has been previously examined (Seegal et al., 1990). The data suggested that a mixture of the congeners was more effective at decreasing cellular dopamine than any one of the component congeners alone. These data indicate that more work will be needed to examine possible interactions of individual PCBs within a mixture of congeners, and how possible congener interactions may affect dopaminergic cells. In the current studies, increasing the ortho substitution from non-ortho to di-ortho appeared to lead to a slightly enhanced ability of a PCB congener to decrease cellular dopamine without a concurrent decrease in DNA content, supporting the findings.of Shain et al., 1991, The congener 2,2'-dichlorobiphenyl appeared to be an exception 90 in our system, even though 2,2'-dichlorobiphenyl has been reported to be the most potent congener for decreasing cellular dopamine in PC12 cells (Shain et al., 1991). Data presented earlier indicated that exposure of the cells to the non-ortho substituted congener 3,3'4,4',5- pentachlorobiphenyl was toxic to the cells and decreased cellular dopamine could be related to decreases in DNA content. However, with exposure to increasing media concentrations of the mono-ortho substituted congener 2 , 3 ' , 4 , 4 ' , 5-pentachlorobiphenyl and the di-ortho substituted congener 2,2',3,3',4,4'-hexachlorobiphenyl, the amounts of cellular dopamine began to decline at concentrations lower than those at which decreases in DNA content were observed. This suggests that these last two congeners may have effects on the biosynthetic pathway for dopamine that is not due to cell death. Furthermore, a comparison of the dose-response data from 3,3',4,4',5-pentachlorobiphenyl: 2,3',4,4',5- pentachlorobiphenyl and 2 , 2 ' , 3 , 3 ' , 4 , 4 '-hexachlorobiphenyl suggest that di-ortho substituted congeners are more efficacious at decreasing cellular dopamine in PC12 cells than mono-ortho substituted congeners: which in turn appear to be more efficacious at decreasing cellular dopamine than non-ortho substituted congeners at non-cytotoxic media concentrations. These findings support the conclusions drawn by Shain et al. (1991) that ortho substituted congeners may be primary effectors in the ability of PCBs to 91 decrease cellular dopamine. However, Shain et al. (1991) suggested that substitution in the meta positions decrease the efficacy of a congener to decrease cellular dopamine, especially if the congener were substituted at the para position. Congeners with ortho, para and minimal meta patterns of substitution were reported to be very effective at decreasing cellular dopamine. Data from our studies, however, suggests that chlorine substitution in the meta-5 positions may be important. in predicting’ the ability' of a congener to decrease cellular dopamine, possibly via decreasing dopamine synthesis. For instance, 3, 3 '-dichlorobiphenyl (which could be called 5,5'-dichlorobiphenyl) failed to decrease amounts of cellular dopamine in PC12 cells: however 2,2 ' ,5,5'- tetrachlorobiphenyl was the most potent congener for decreasing the amounts of cellular dopamine. Furthermore, increasing the ortho substitution of 4,4'-dichlorobiphenyl to 2 , 4 , 4 '-trichlorobipheny1 to 2 , 2 '4 , 4 '-tetrachlorobiphenyl , does not enhance the ability of the congener to cause a decrease in cellular dopamine content. Therefore, these data suggest that, while ortho substitution may be required, it is not in itself sufficient for a PCB congener to cause a decrease in cellular dopamine. Hence, PCBs, either as a mixture (Aroclor 1254) or as individual congeners, generally' appear' tot decrease ‘the amount of cellular dopamine in PC12 cells. Some PCB 92 congeners and Aroclor 1254 also affected cellular DOPA and DOPAC. The mechanism( s) explaining these phenomena are unknown, but may include alterations in tyrosine uptake, TH activity, or LAAAD activity. Furthermore, increasing the ortho substitution of the congener appeared to enhance its ability to decrease cellular dopamine: and the substitution of the meta-5 position may also be an important determinant in predicting the ability of a congener to cause a decrease in cellular dopamine. Concurrent exposure of PC12 cells to PCBs and NGF’ did not alter the PCB-mediated decrease observed in cellular dopamine, however, pretreatment of PC12 cells with.NGF'prior to exposure to PCBs appeared to protect against the cellular dopamine decreases. Furthermore, Aroclor 1254 enhanced the elongation of neurites of cells exposed concomitantly to NGF, indicating that PCBs have a multitude of effects on these cells. 93 94 INTBQQQQIIQH Using a model dopaminergic cell system, the PC12 rat pheochromocytoma cell line, data have been obtained demonstrating that both acute and subchronic exposure of dopaminergic cells to PCBs, either' as mixtures or as individual congeners, can result in a dose-dependent decrease in cellular dopamine content (Seegal et al., 1989, 1990: Angus and Contreras, 1994: Chapter 3). To our knowledge, no studies have yet investigated the relationship between the PCB-mediated decrease in cellular dopamine and possible changes in the amount.of dopamine released from the cells. Thus, cells were exposed to various media concentrations of Aroclor 1254 or six individual PCB congeners [2,2' dichlorobiphenyl (PCB 04): 2,2',5,5' tetrachlorobiphenyl (PCB 52): 2,3',4,4',5 pentachlorobiphenyl (PCB 118): 3,3',4,4',5 pentachlorobiphenyl (PCB 126): 2,2',3,3',4,4' hexachlorobiphenyl (PCB 128): and 2,2',4,4',5,5' hexachlorobiphenyl (PCB 153)] to determine their effects on the amounts of dopamine released spontaneously or in response to stimulation of the cells with 56 mM Ki. Four of these congeners, 2,2',5,5' tetrachlorobiphenyl, 2,2',3,3',4,4' hexachlorobiphenyl, 2,3',4,4',5 pentachlorobiphenyl and 2,2',4,4',5,5' hexachlorobiphenyl, are found in relatively great abundance in Aroclor 1254, comprising 4.2%, 1.5%, 9.5%, and 6.1% of the mixture, 95 respectively (Hansen, 1987). The congener 3,3',4,4',5 pentachlorobiphenyl is a coplanar PCB congener known to interact with the Ah-receptor; thus it acts through a similar mechanism as 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), and is representative of the dioxin-like class of PCBs. The congener 2,2' dichlorobiphenyl was tested because it has been reported to be the most potent congener in decreasing cellular dopamine in PC12 cells (Shain et al., 1991) although my previous results disagree (Chapter 3). 32811.15 PC12 cells displayed both a spontaneous and Ki-evoked release of dopamine. The amount of evoked released dopamine increased over time for cells exposed to media concentrations of 25 pg/ml Aroclor 1254 (Figure 4.1). An exposure time of 5 min was chosen for use in these studies. Exposure of the cells to 0.1 to 0.5% of the vehicle, dimethylsulfoxide (DMSO), had no effects on either the amount of spontaneous or Ki-evoked release of dopamine from PC12 cells, the amount of cellular dopamine, or the DNA content (data not shown). For all of the aforementioned parameters, the intraexperimental variability between naive and DMSO-treated cells was at most 10%. Treatment of PC12 cells for 3 days with various media concentrations of Aroclor 1254 led to a dose-dependent decrease in both spontaneous and Ki-evoked release of 96 120 Dopamine Released (% of maxinal) ctr' 'U 'I‘TVUIj'II Iir" U I rttr'j'. I rtf" 0 1 2 3 4 5 6 7 Time (minutes) Figure 9.1 Dopamine Released by PC12 Cells in Response to 56 mM K PC12 cells were stimulated to release dppamine by exposure to a HEPES buffer containing 56 mM K . The buffer was collected and analyzed for amounts of dopamine as described in Chapter 2, "Materials and Methods". Each point represents the mean f S.E.M. for 3 separate experiments. 97 dopamine (Figure 4.2) . The spontaneous and Ki-evoked release of dopamine from the cells decreased significantly at 10 pg/ml media concentration of Aroclor 1254, which did not cause decreases in DNA content (Figure 4.2) . Further, the dose-response curves depicting the decreased release of dopamine and cellular dopamine caused by Aroclor 1254 appear to coincide (Figure 4.2). Spontaneous release of dopamine began to decline at a media Aroclor 1254 concentration of 10 pg/ml, but then remained relatively steady at an approximate 30% decrease. To examine the specificity of the effects of PCBs on cellular dopamine and amounts of dopamine released, PC12 cells were exposed for 3 days to various media concentrations of six different PCB congeners. The effects of the different PCB congeners on the release of dopamine from PC12 cells were variable, as follows: 2,2' dichlorobiphenyl caused no decrease in cellular dopamine, spontaneous or evoked release of dopamine at media concentrations up to and including 50 pg/ml (Figure 4.3). At media concentrations greater than 50 pg/ml, there were decreases in released dopamine and cellular dopamine, coincident with a decrease in DNA content. 2,2',5,5' tetrachlorobiphenyl demonstrated.the ability to cause similar dose-dependent decreases in the amounts of cellular dopamine and evoked release of dopamine at media concentrations up to and including 100 pg/ml (Figure 4.3). 98 175 _ 1504 . O .b 8 L) 125. 3 § 100. *‘F 8 75- 2 CI ‘5 so- 0 o 5 a 25- El DNA A SpontDAReIeaee \ O CellulerDA e EvekedDAReIeaee N \ 0 .-.., . . . . ...., .7 . . . ....Qh——~ 1 10 100 Media Concentration Aroclor1254 (uglml). Figure 4.2 Effects of Exposure to Aroclor 1254 on Release of Dopamine From PC12 Cells PC12 cells were exposed to various.concentrations of.Aroclor 1254 in the culture medium for 3 days. The release of dopamine, and the cellular dopamine and DNA contents were determined as described in Chapter 2, "Materials and Methods". Each point represents the mean i S.E.M. for 3 to 7 separate experiments. 99 The spontaneous release of dopamine decreased at a media concentration of 10,pg/ml, and remained steady'at.a decrease of about 70%. Only at media concentrations greater than 50 pg/ml was there a decrease in the DNA content (Figure 4.3). 2,2',4,4',5,5' hexachlorobiphenyl decreased cellular dopamine and the amount of dopamine released in response to a stimulation by 56 mM K+ at media concentrations up to and including 100 pg/ml (Figure 4.3). As was seen.with.Aroclor 1254, the spontaneous release of dopamine decreased by about 30% at 10 pg/ml media concentration of this PCB congener. Amounts of DNA began to decline at media PCB concentrations greater than 50 pg/ml. 3,3'4,4',5 pentachlorobiphenyl caused DNA content and cellular dopamine to decline significantly at media concentrations of 10 pg/ml. The amount of evoked released dopamine declined between 10 and 25 pg/ml (Figure 4.3) . Amounts of dopamine spontaneously released were elevated. Visual observation of the cells treated with 3,3'4,4' ,5 pentachlorobiphenyl indicated that this PCB congener caused blebbing and vacuole formation on or in the cells. 2,3' ,4,4 ' ,5 pentachlorobiphenyl did not affect cellular dopamine or the amount of evoked dopamine released until media concentrations exceeded 25 pg/ml (Figure 4.3). The amount of spontaneously released dopamine was unaffected at media PCB concentrations up to and including 50 pg/ml. 100 Figure 4.3 Effects of PCB Congeners on Release of Dopamine From PC12 Cells PC12 cells were exposed to various concentrations of PCB congeners in the culture medium for 3 days. The spontaneous and evoked release of dopamine, the cellular dopamine and DNA contents were determined as described in Chapter 2, "Materials and Methods". Each point represents the mean 1 S.E.M. for 3 to 4 separate experiments. 101 2,2' 0 DNA 0 Spent. DA Release I Cellular DA A Evoked DA Release c ‘I"' r I I III... I U I II 150_ 2,2',5,5' Percent of DMSO-treated Control 0| 0 o II'I' v - attrit' r u I'UUII' 1 10 100 Media Concentration (uglml) Figure 4 . 3 102 175 150- 125- 100‘ 75‘ 50- 25- 3,334,435 i 175 150q 125- 100- 75- 50- 25- o rrrri r U’ f'rIUI' U T I I'UUT' 175 150‘ 125- 100- Percent of DMSO-treated Control o UUUI' V I ITVV'I' r I letfi Media Concentration (uglml) Figure 4.3 Continued 103 2,2',3,3',4,4' hexachlorobiphenyl caused dose-dependent decreases in cellular dopamine and the amount of dopamine released in response to a stimulation by 56 mM K5 (Figure 4.3) at media congener concentrations.of’up to and including 100 pg/ml. Spontaneous release of dopamine was unaffected by this PCB congener. DNA content decreased at media concentrations of greater than 50 pg/ml. The fraction of total dopamine released upon stimulation of PC12 cells with 56 mM K did not change significantly with increasing media concentration of Aroclor 1254 (Figure 4.4). The fraction of total dopamine released in response to K? for each of the individual PCB congeners examined also did not change significantly with increasing media concentrations of PCBs (Figure 4.4). 215911551211 In general, polychlorinated biphenyls caused a significant decrease in the evoked release of dopamine from PC12 cells. 'This decrease in the evoked release of dopamine followed a similar concentration-response relationship as the decrease in cellular dopamine content caused by PCBs. These observations suggest that the decrease in the release of dopamine may be related to the decrease in cellular dopamine. PCBs have been suggested to cause a decrease in dopamine synthesis (Seegal et al., 1989, 1990: Angus and Contreras, 1994). Since it has been reported that newly synthesized catecholamines are preferentially released in 104 Figure 4.4 Fractional Release of Dopamine From PC12 Cells The amount of dopamine, in ng, released by the cells when stimulated with 56 mM K was divided by the total amount of dopamine, in ng, from each sample. Each bar represents the mean i S.E.M. for 4 separate experiments. v . v 925: 25.33 cozmbcoocoo mon— flows. ommwowmpo ommwowmpo ommwowmwo ommmdemwo 105 :: -. .... -. :. -. :— -. J 0 9 -2 to. -9 roam. O I: I. 2 9 m. 2.0.. Smuosmpo Smuemeo Smuopmpo moi ..o G 0 HI m I.” w w. 9 -2 m. a. m. 106 response to a depolarizing stimulus (Kopin et al., 1968: Besson et al., 1969: Gewirtz and Kopin, 1970), a decrease in dopamine synthesis could contribute to the observed decrease in the evoked release of dopamine from PCB-treated cells. The spontaneous release of dopamine from PC12 cells decreased after exposure to Aroclor 1254, 2,2',5,5'- tetrachlorobiphenyl and 2,2'4,4'5,5'-hexachlorobiphenyl at media concentrations of PCBs that did not decrease DNA, but did decrease the amount of cellular dopamine. For these three PCBs, decreased dopamine synthesis could lead to decreased spontaneous release of dopamine. Data from these studies suggest that the meta-5 positions may be important in determining the propensity of a congener to decrease spontaneous release of dopamine, since the 5-positions of both of the congeners decreasing spontaneous release of dopamine are chlorine substituted. By inference, this pattern of substitution may thus be important in predicting a congener's potential to cause a decrease in the amount of cellular dopamine, possibly via decreasing dopamine synthesis, since dopamine may be spontaneously released from cells by diffusion across the membrane from the cytoplasm, prior to being packaged into vesicles or metabolized into dihydroxyphenylacetic acid (DOPAC). Also, a 2,2'-and-5,5' substitution pattern appears to be common in both of the congeners causing a decrease in the spontaneous release of dopamine from PC12 cells. These two congeners, 2,2',5,5'- 107 tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl, make up a significant component of Aroclor 1254 (4.2% and 6.1%, respectively: Hansen, 1987), and thus, may partially account for the action of Aroclor 1254 on spontaneous release of dopamine. In general, increasing the ortho substitution from non- to di-ortho appeared to lead to a slightly enhanced ability of a PCB congener to decrease both the evoked release of dopamine, and cellular dopamine content. The congener 2,2' dichlorobiphenyl was an exception in our system. Upon exposure of the cells to the non-ortho substituted congener, 3,3'4,4',5 pentachlorobiphenyl, amounts of DNA and evoked dopamine were substantially decreased at low media concentrations. This suggested that the decreases in DA release due to this congener were related more to the congener's cytotoxicity than to effects of the congener on the synthetic pathway for dopamine per se. Yet, exposure to increasing media concentrations of the mono-ortho substituted congener 2 , 3 ' , 4 , 4 ' , 5 pentachlorobiphenyl and the di-ortho substituted congener 2,2',3,3',4,4' hexachlorobiphenyl caused a decrease in evoked dopamine release to decline at media concentrations of the congeners that were less than those needed to decrease DNA content. These results suggest that 1) these two ortho substituted congeners (2,3',4,4',S-pentachlorobiphenyl and 2,2',3,3',4,4'-hexachlorobiphenyl) may have effects on the 108 biosynthetic pathway for dopamine that is not due to cell death, and 2) even though the degree of chlorination is the same between 3,3',4,4',5- and 2,3',4,4',5- pentachlorobiphenyl, the degree of ortho substitution contributed to the neurotoxicity and cytotoxicity of each congener. These data further suggest that, while ortho substitution may be required, it is not sufficient for observed effects on the evoked or spontaneous release of dopamine from PC12 cells in the absence of cytotoxicity. This supposition is supported by the observation that 2,2'- dichlorobiphenyl is a di-ortho substituted congener that does not decrease evoked release of dopamine from PC12 cells. These findings are further supported by the conclusions drawn by Shain et al. (1991) that ortho substituted. congeners may’ be primary effectors in the ability of PCBs to decrease amounts of cellular dopamine (see Discussion of Chapter 3). However, Shain et al. (1991) did not investigate the effects of PCB congeners on the release of dopamine from PC12 cells. An interesting observation is that none of the PCB congeners tested, except for 3 , 3 ' , 4 , 4 ' , 5 pentachlorobiphenyl (due to its cytotoxicity), were more potent than Aroclor 1254 in decreasing evoked release of dopamine or cellular dopamine content. This observation might encourage speculation about cooperative, permissive, or synergistic effects of PCB congeners on this dopaminergic system. It 109 has been suggested (Seegal et al., 1990) that a mixture of PCB congeners was more effective at decreasing the amount of cellular dopamine than any one of the component congeners alone. More work will be needed to determine potential interactions of individual congeners within a mixture of PCBS. The exact mechanism(s) defining how polychlorinated biphenyls alter evoked DA release are unclear. Sites of action by PCBs could include 1) the enzymes involved in the synthesis of dopamine, 2) the packaging of dopamine into vesicles for release, and/or 3) the mechanisms involved in the release of dopamine form the cells. Obviously, if the synthesis of dopamine is decreased, either through effects of PCBs on biosynthetic enzymes or by decreasing the uptake of the precursor amino acid tyrosine, the amount of newly synthesized dopamine available for release will be reduced and subsequently, the amount of dopamine released in response to a stimulus will be reduced. The results of the present studies demonstrate that the fraction of dopamine released as a percentage of total available cellular dopamine is not affected by increasing media concentrations of PCBs. These findings suggest that the packaging of the dopamine into vesicles is not affected by PCBs, since an inhibition in the packaging of dopamine would have resulted in a decrease in the fraction of dopamine released. These data further suggest that decreases in evoked release of 110 dopamine may be directly related to decreases in the synthesis of dopamine, rather than to alterations in the mechanisms involved in the release of dopamine. If PCBs were stimulating or inhibiting the process of release of dopamine, the fraction of dopamine released in response to K+ would either increase or decrease compared to control cells. Polychlorinated biphenyls decrease the amounts of cellular dopamine and dopamine released in response to a depolarizing stimulus in PC12 cells. In the brain, dopamine is an important neurotransmitter in the numerous cognitive, behavioral, and emotional functions. Dopamine is also important in the regulation of motor functions, as demonstrated by a decrease in dopamine content in the substantia nigra of persons with Parkinson's disease. Together, this information suggests the possibility that an exposure of neuronal dopaminergic tissue to PCBs in utero may alter the distribution and kinetics of dopamine, potentially leading to cognitive, behavioral, emotional, and/or motor functions of the developing organism. This hypothesis is supported by studies involving the effects of in utero exposure of animals and humans to PCBs (Jacobson, et al, 1990, Bowman et al., 1981, Schantz et al., 1991). A decrease in the release of dopamine from dopaminergic neurons may not be great enough to result in an overt demonstration of cognitive, emotional or behavioral 111 deficits, or a Parkinsonian-like symptomology. However, deficits may occur under conditions of stress, such as learning new tasks or testing. In previous studies (Angus and Contreras, 1994) , 25 pg/ml of Aroclor 1254 caused about a 90% decrease in cellular dopamine content at 3 days. Furthermore, there was an approximate 80% decrease in the amount of dopamine released from cells exposed to the same media concentrations of Aroclor 1254. Decreases of this magnitude in PC12 cells raise the question of the functional significance of an 80-90% reduction in cellular dopamine. Does a decrease of this magnitude translate into a physiological deficit? Insight into this question can be gained through studies using the neurotoxin 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP) in monkeys. Monkeys treated subchronically with low doses of MPTP and displaying no gross motor deficits or overt Parkinsonian symptoms were shown by Taylor et al. (1990a,b) to have persistent deficits in acquisition and performance of tasks requiring complex cognition. Subtle deficits in motor coordination were also noted. Further, HVA (a dopamine metabolite) was reduced by approximately 65% in the cerebra- spinal fluid during the time period when these animals were tested for the complex cognitive tasks. Similarly, monkeys exposed chronically to MPTP that were motor asymptomatic for Parkinsonism displayed deficits in complex cognition tasks (Schneider and Kovelowski, 1990: Schneider, 1990). 112 Alterations in behaviors, such as increased irritability and decreased attentiveness, were also noted. Subtle motor deficits were observed in the MPTP-treated monkeys which were especially apparent during the testing of the complex cognition. Some of these subtle motor deficits included difficulty in reaching for and manipulating food rewards during the testing procedures, which indicated effects on fine motor coordination. Dopamine content in certain brain regions of these motor asymptomatic, MPTP-treated monkeys was significantly decreased. The amount of dopamine in the striatal was decreased 84-98%, while dopamine levels in the putamen were decreased some 80-90%. Therefore, in answer the question of functional significance for a 70-90% decrease in cellular dopamine levels, theoretically, there could be subtle deficits in cognition and motor coordination with dopamine decreases of this magnitude. In fact, the work of Jacobson et a1. (1985, 1990), Rogan and Gladen (1992) and Gladen and Rogan (1991), cited in the introduction suggest that, in humans, there are indeed deficits in cognitive processes associated with PCB exposure. Deficits in cognitive, behavioral, emotional, and motor abilities have been described in animal models in which MPTP has decreased the amount of dopamine in the brain, and such deficits have also been described in humans exposed to PCBs in utero. Exposure of dopaminergic PC12 cells to PCBs 113 resulted in a decrease in cellular dopamine to extents consistent with decreases observed in the brains of animals exposed to the dopaminergic neurotoxin MPTP. PCBs also decreased the evoked release of dopamine from PC12 cells that appears to be directly related to decreased cellular dopamine content. Together, the data suggest that polychlorinated biphenyls are agents capable of altering the function of dopaminergic cells. This altered function may underlie some of the PCB-mediated behavioral and cognitive deficits observed in children exposed in utero to PCBs. 'The current studies suggest that a decrease in the amount of cellular dopamine caused by a subchronic exposure to PCBs can manifest itself as a decrease in the evoked release of dopamine in dopaminergic PC12 cells. Further research needs to be done to examine mechanisms by which PCBs can decrease cellular dopamine and decrease evoked release of dopamine. Possible mechanisms by which PCBs act to cause the putative alterations in levels of cellular dopamine will be:discussed in Chapter 6. 1 3!. , ‘13. 114 CHARTER 5 31;]. 1' 3.2i!f 115 W Polychlorinated biphenyls are putative neurotoxins, especially to dopaminergic cells, either upon acute or subchronic exposure (Seegal et al., 1989, 1990: Shain et al., 1991: Angus and Contreras, 1994). Data from previous chapters from this dissertation have demonstrated that both PCB mixtures and congeners decrease cellular dopamine, which functionally manifests itself as a decrease in the release of dopamine in response to a depolarizing stimulus. Although the evidence indicates that PCBs alter dopamine levels and synthesis, it is unclear at ‘what cellular concentrations of PCBs these effects are seen. Only one study has attempted to quantitate the amount of PCBs that are associated with cells when PCB-mediated decreases in cellular dopamine are observed (Shain et al., 1991) . The weaknesses of that study were that 1) the procedure used in the study analyzed for PCB.content in cell remnants previously extracted for determination of cellular catechols, therefore the amount of PCBs measured may have been underestimated: and 2) the study involved an acute exposure of PC12 cells to Aroclor 1254. Extraction of whole, subchronically PCB-exposed cells may provide a better indication of the cellular concentration of PCBs at a given media concentration that results in decreased cellular dopamine in the subchronically exposed cells. Thus, in the current studies, PC12 cells were subchronically exposed for 116 three days to either DMSO or Aroclor 1254 or PCB congeners. The PCBs were then extracted from the whole cells and amounts of PCBs in the cells were quantitated using gas chromatography coupled to electron capture, as described in Chapter 2. To support a contention that the effects on cellular dopamine seen with exposure of PC12 cells to PCBs were not due just to exposure to an aromatic hydrocarbon, cellular concentrations of and effects of unchlorinated biphenyl were also determined in biphenyl-treated cells. Cellular biphenyl concentrations were determined in subchronically exposed PC12 cells using gas chromatography coupled to mass spectroscopy. Results of these studies suggested that PCBs were more effective at decreasing cellular dopamine than biphenyl: and that this is likely the case because the cellular PCB concentrations were greater than the cellular concentrations of the unchlorinated biphenyl. Further, a study involving PCB congeners suggested that the association of PCB congeners with the cells appears to vary with degree of chlorination and pattern of substitution, however, no discernable structure-activity relationship could be identified. 395913;: A timecourse depicting the amounts of Aroclor 1254 contained in PC12 cells after various periods of exposure, indicated that the amount of Aroclor 1254 associated with 117 the cells increased over time (Figure 5.1). However the maximal concentrations of PCBs were associated with the cells at 1 day of incubation, and the cellular concentrations decreased in the days following to 7 days, which was the last time examined (Figure 5.1). Interestingly at 6 hours of exposure to Aroclor 1254, the amounts of Aroclor 1254 in the cells was similar to the amounts determined in the cells after 3 days of exposure. Studies examining the partition of 25 pg/ml Aroclor 1254 in serum-free culture medium indicated that less than 1% was in the medium proper: however, if 12% serum was added to the medium, greater than 90% of the PCBs were found to be associated with the medium. Hence, serum in the media appeared to be a source for PCB binding in this test system. The data depicting the amounts of PCBs in the cells in relation to the amounts of PCBs in the media indicated that beyond.a«certain:media concentration of PCBs, the amounts of PCBs in the cells increased at.a greater rate than that seen at lower media concentrations of PCBs (Figure 5.2) . In other words, the serum in the medium acted as a buffer for PC12 cells against the accumulation of Aroclor 1254. A.comparison of the effects of.Aroclor 1254 on.cellular dopamine and amount of cellular Aroclor 1254 at a given media concentration of Aroclor 1254 indicated that the cellular concentrations of PCBs increased as amounts of cellular dopamine decreased (Figure 5.3). Media ”9) h is ?' (n .3 30~ 10- Cellular Aroclor1254 N O 3 ¢ . b) O O 250- 200- 150- 100- 50- Ceiiular Aroclor 1254 (uM) <3 o ' 2 I 4 I é I 8 Time (days) Figure 5.1 Cellular Association of Aroclor 1254 with PC12 Cells Over Time PC12 cells were exposed to a media concentration of 35 pg/ml Aroclor 1254 for various amounts of time. Amounts of Aroclor 1254 in each sample (top) and cellular Aroclor 1254 concentrations (bottom) were determined as described in Chapter 2, "Materials and Methods". Each point represents the mean r 5.0. of 2 measurements. 119 5000 .2. -I— Aroclor1254 3 r T :40004 + 2'2 .2 -a— ZTAA' E l I e g 3000- -+- 2.2.55 0 c o o an 2000- O o. a '5 1000- '5 O 0‘ ‘ — i l i 0 so 100 150 200 250 Media PCB Concentration (uM) 1500 -e— 2.3.44.5 —e— 2.3.44.5 + 2,2',3.3'.4,4' 1°°°1 -e- 2.2.4.4255 500d Cellular PCB Concentration (uM) o I i l I 0 50 100 1 50 200 250 Media PCB Concentration (uM) Figure 5.2 Association of Aroclor 1254 and PCB Congeners With PC12 Cells PC12 cells were exposed to various media concentrations of Aroclor 1254 or 7 PCB congeners for 3 days. Cellular concentrations of PCBs were determined as described in Chapterz, "Materials and Methods". Points represent the means i S.E.M. for 3 separate measurements. The molecular weight of Aroclor 1254 used for calculations of molarity was 327 and was taken from Hutzinger et al., 1974. 120 .s O O q (I i 504 Cellular Dopamine (°/. DMSO-treated Control) 8 D O 4 1' V T'V'v v I *1 v vav 10 100 Cellular Aroclor 1254 (ug) A 150 250~ " g ~100 100‘ Cellular Concentration Aroclor 1254 (uglml) I a .5 o 8 .(IOJIUOO Paleafl‘OSWO 'I.) euimedoa “IDIIOO O .1 "1 ' """'1'6 Media Concentration Aroclor1254 (uglml) v ' "£60 Figure 5.3 Relation of Cellular Dopamine to Cellular Aroclor 1254 Concentration in PC12 Cells Data from effects of Aroclor 1254 on cellular dopamine content and from the cellular concentration of Aroclor 1254 was graphed onto one figure to visualize the relationship between the two processes. Top: Cellular dopamine and cellular Aroclor 1254 concentrations have an inverse relationship as media concentrations of Aroclor 1254 increase. Bottom: The decreases in cellular dopamine are directly related to increasing cellular Aroclor 1254 concentrations. 121 concentrations used for the determination of the cellular amounts of Aroclor 1254 were chosen based on the ICfl,values for Aroclor 1254 that caused a decrease in levels of cellular dopamine (see Chapter 3), with one media concentration above the ICso and one media concentration below the chr The percent of total PCB added to the media that was associated with the cells was determined, as well as the cellular concentration of PCBs (Table 5.1). Generally, about 1 to 3% of the total Aroclor 1254 added to the culture medium was associated with the cells. Further, PC12 cells appeared to concentrate the Aroclor 1254 from.the medium. A media concentration of Aroclor 1254 of 25 pg/ml (which was used in the timecourse studies) represented a molar concentration of 76.5 pM. Yet, the cellular concentrations of Aroclor 1254 was around 230 to 280 pM in the timecourse studies. To determine if the effects on cellular dopamine observed with Aroclor 1254 could be duplicated with a non- chlorinated aromatic hydrocarbon, PC12 cells were exposed to various media concentrations of unchlorinated biphenyl. The concentrations of media biphenyl were chosen based on the molar concentrations of PCBs previously examined. This was done to compare the effects of the different compounds over the same range of media concentrations. Subchronic exposure of PC12 cells to polychlorinated biphenyls (Aroclor 1254) for 3 days was significantly more effective AT decreasing 122 Table 5.1 Cellular Concentrations of Aroclor 1254 and PCB in PC12 Cells The amounts of cellular Aroclor 1254 or individual PCB congeners in subchronically treated PC12 cells was determined as described in Chapter 2, "Materials and Methods". Values represent the mean i S.E.M. for 3 determinations. 123 Table 5.1 Aroclor 1254 and PCB Congeners Associated With PC12 C0118 Media % of total PCB Cellular Cellular conc. Conc.in associated conc. (pM) pg/ml with cells (pg/ml) 7"» u = PCB 04 10 (44.8) 1.65 i 0.83 8.54 i 3.64 38.0 i 16.0 25 (112) 1.15 i 0.25 18.34 i 8.15 103.4 1 0.4 so (224) 1.92 i 0.46 304.7 1 1,363.7 : 135.91 609.5 PCB 47 10 (31.3) 2.75 i 0.23 18.0 i 3.74 61.7 t 12.9 47 (161) 2.94 i 0.2 203.62 1 697.7 1 234.4 68.42 60 3.20 i 1.2 997.37 1 435 3,417 r 1,491 (205.6) PCB 52 10 (31.3) 1.53 i 0.31 9.47 i 1.56 32.4 i 5.36 32 2.94 i 0.35 47.65 i 6.42 163.2 1 22.0 (109.6) 50 (171) 3.61 i 0.28 441.13 1 1,511.4 1 74.59 255.6 PCB 114 10 (30.6) 2.75 i 0.31 19.57 i 3.44 60.0 i 10.6 33 2.94 t 0.80 77.92 i 238.8 1 58.4 (101.1) 19.05 50 (153) 2.44 t 0.05 134.85 1 413.3 f 60.5 19.74 124 Table 5.1 Aroclor 1254 and PCB Congeners Associated With PC12 Cells (Continued) Media % of total PCB Cellular Cellular conc. Conc. in associated conc. (pM) with cells (pg/ml) ..... I PCB 118 10 (30.6) 2.84 i 0.93 13.27 i 6.36 29.6 i 4.9 38 2.40 t 0.87 46.91 i 7.11 143.8 1 21.8 (116.5) 50 (153) 1.69 i 0.20 105.72 i 256.9 1 149.8 43.62 PCB 128 10 (27.7) 2.14 i 0.38 12.77 i 2.33 35.4 i 6.43 34 (94.2) 5.01 i 0.18 140.36 1 389.0 1 72.9 26.32 50 (139) 1.98 i 0.16 399.9 1 720.7 1 216.7 248.52 PCB 153 10 (27.7) 2.13 0.20 12.99 i 2.20 36.0 i 6.1 41 1.57 i 0.39 51.79 i 9.43 143.5 1 26.2 (113.6) 60 3.52 i 0.04 355.86 1 986.4 1 121.6 (166.3) 76.69 Aroclor 1254 5 (15.3) 2.47 i 0.06 15.70 i 0.86 48.0 i 2.7 18 (55.1) 2.00 i 0.20 41.54 i 0.89 127.0 1 2.7 35 4.40 i 0.70 232.88 1 712.1 f 89.7 (107.0) 29.34 125 cellular dopamine in PC12 cells than 3 days of subchronic exposure to equimolar media concentrations of biphenyl (Figure 5.4). The amounts of PCBs determined to be associated with subchronically treated cells was on the order of two to three magnitudes greater than biphenyl for exposure to equimolar media concentrations -- the cellular concentrations of biphenyl was in the nM range, as compared to the pH range for PCBs (Tables 5.1 and 5.2). Less than 1% of the total biphenyl added to the culture medium was found in the cells (Table 5.2). The relationship between subchronic exposure of PC12 cells to individual PCB congeners and the cellular concentrations of those congeners at media concentrations which decreased cellular dopamine was also examined. The media concentrations of individual congeners examined included the Icfl,concentration, 10 pg/ml, and either 50 or 60 pg/ml (60 pg/ml was used for congeners whose ICso concentrations were 40 pg/ml or greater). The cellular concentrations of PCB congeners, reported either as pg/ml or as pM, (Table 5.1) changed with differing amount of congener chlorination. IHigher concentrations of lightly substituted congeners such as 2,2'-dichlorobiphenyl, or the tetrachlorobiphenyls were associated with the cells, as compared to penta- or hexachlorobiphenyls (Table 5.1) . However, the cellular concentrations of PCB congeners did not vary significantly among congeners with the same number 126 150 ..s O O l 0| 0 l -l- Aroclor1254 -O— Biphenyl Dopamine (% DMSO-treated Contol) c I V U V VijVI I I V V ‘fTVI I V I I ‘ffi 1 10 100 1000 Media Concentration (uM) Figure 5.4 iEffects of.Aroclor 1254 and.Biphenyl on Cellular Dopamine PC12 cells were exposed to various media concentrations of either Aroclor 1254 or biphenyl for 3 days. The amounts of cellular dopamine were determined as described in Chapter 2, "Materials and Methods". Each point represents the mean i S.E.M. for 3 to 5 separate experiments. 127 Table 5.2 Biphenyl Associated With PC12 Cells Media Conc. of Biphenyl % of Total Biphenyl Assoc. with Cellular Conc. of Biphenyl (119/m1) Cellular Conc. of Biphenyl 50 0.061 t 0.001 773.33 i 74.91 5.02 i 0.49 100 0.177 t 0.021 6,348.22 1 41.20 i 1.41 218.84 500 0.158 t 0.011 56,971.78 1 369.0 1 64.05 9,867.84 PC12 cells were subchronically exposed to various media concentrations of unchlorinated biphenyl. Amounts of cellular biphenyl were determined as described in Chapter 2, "Materials and Methods" . for 3 measurements. Values represent the mean i- S.E.M. 128 of chlorine substitutions, but not the same patterns of substitution (for example 2 , 2 ' , 3 , 3 ' , 4 , 4 ' -hexachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl: Figure 5.2: Table 5.1). Furthermore, there 'was an inverse relationship between amounts of cellular dopamine and cellular concentrations of PCB congeners for each congener examined (data not shown), as previously described for Aroclor 1254 above. E° . A question that needed to be addressed in the research for this dissertation concerned the effects of PCBs on dopaminergic cells -- are the decreases in cellular dopamine after acute or subchronic exposure of PC12 cells to PCBs due specifically to PCBs or can other non-halogenated aromatic hydrocarbons, sudh as biphenyl, cause similar effects on cellular dopamine at similar molar concentrations in the culture medium? Results suggest that, at a given media concentration, Aroclor 1254 was greater than 2 orders of magnitude more potent at causing a decrease in cellular dopamine in PC12 cells than unchlorinated biphenyl. Therefore, these data suggest that a degree of halogenation is necessary for' the aromatic hydrocarbon to cause a decrease on cellular dopamine in PC12 cells. A.determination of the cellular concentrations of PCBs and biphenyl in subchronically exposed PC12 cells indicated that, over the same range of molar concentrations in the 129 media, cellular concentrations of PCBs were 10 to 1000 times greater than cellular concentrations of unchlorinated biphenyl. Thus, the apparent greater potency of Aroclor 1254 to decrease cellular dopamine compared to unchlorinated biphenyl may be due to an increase in the ability of Aroclor 1254 to become associated with the cells as compared to biphenyl. PCBs have been described as being more lipophilic with increasing chlorination, and, since the molecular volume of chlorinated biphenyl is greater than the molecular volume of unchlorinated biphenyl, these factors may provide at least a partial explanation for the higher cellular concentrations of Aroclor 1254 with the cells compared to unchlorinated biphenyl. A timecourse examining how rapidly cellular concentrations of Aroclor 1254 increased in PC12 cells demonstrated that by 6 hours, the cellular concentrations of PCBs were maximal. Even though the total amounts of Aroclor 1254 associated with the cells increased over time, the cellular concentrations of Aroclor 1254 decreased by 7 days. This decrease cellular PCB concentration may be due to a limited availability of Aroclor 1254 in the medium due to its binding to serum proteins, coupled with an exponentially dividing cell population, however more studies will be needed to examine the kinetics of PCB binding and dissociation in serum-containing media to be able to address this issue. PCBs bind to serum proteins, especially albumin 130 (Mohammed et al., 1990: Borlakoglu et al., 1990a,b, 1993: Busbee et al., 1985), thus the amount of PCBs available for interaction with the cells may be limited; an increase in cell number'could therefore, under these:conditions, lead to a decreased cellular concentration of PCBs. In Chapter 3, the data indicated that the dose-response curves for cellular dopamine shifted significantly to the left between 1 and 3 days of exposure to Aroclor 1254 indicating an increased sensitivity of cellular dopamine content to lower media concentrations of Aroclor 1254 (Figure 3.2). The results of the current experiments demonstrate that the cellular concentrations of PCBs are actually the same at 3 days and at 6 hours. These observations suggest that the effects of Aroclor 1254 on cellular dopamine and evoked release of dopamine from PC12 cells are not immediate and require a lag time to manifest themselves. The cellular events that occur during the lag between PCB exposure and decrease in cellular dopamine are unknown, but may involve alterations in second messengers, phosphorylation of enzymes, or transcription or translation of DNA or RNA. Further studies will be needed to determine the sequelae of events occurring between the time of PCB exposure and the manifestation of the decrease in cellular dopamine content in PC12 cells. To examine the relationship between increasing cellular concentrations of Aroclor 1254 and decreasing amounts of 131 cellular dopamine, the amount of cellular dopamine and cellular concentrations of Aroclor 1254 were plotted against the molar concentration of Aroclor 1254 in the culture medium. Results indicated that the cellular Aroclor 1254 concentration was inversely related to the amount of cellular dopamine. Studies examining the cellular concentrations of individual PCB congeners provided interesting results, as the cellular concentrations of PCBs of the different PCB congeners examined varied. The di- and tetrachlorobiphenyls accumulated to the highest concentrations in PC12 cells, while the penta- and hexachlorobiphenyls had lower cellular PCB concentrations. Congeners with the same degree of chlorine substitution, but different patterns of chlorine substitution appeared not to differ greatly with respect to cellular PCB concentrations. It was discussed in Chapters 3 and 4 that PCB congeners differ slightly in their ability to idecrease cellular' dopamine and decrease the evoked release of dopamine from PC12 cells. One factor that may play a role in the ability of an individual PCB congener to decrease dopamine is the ability of the different individual congeners to accumulate in the cells to different extents. The amount of a congener's chlorination appears to relate to how greatly it associates with the cells, for instance, di- and tetrachlorobiphenyls accumulate in cells to higher concentrations than penta- and hexachlorobiphenyls do. 132 However, as was reported by Shain et al. (1986), these data did not distinguish a structure-activity relationship describing the most effective patterns of chlorine substitution for the prediction of how congeners accumulate in cells. Another observation made during the current studies related to the concentrations of PCBs in the cell culture medium versus the cellular concentrations of PCBs. For the PCB mixture Aroclor 1254, 25 pg/ml media concentration translated to a concentration of 76.5 pM. However, in the timecourse experiments, for example, the cellular concentrations of Aroclor 1254 were on the order of 150 to 280 pM. These data strongly suggest that PC12 cells concentrate PCBs out of the culture medium. This notion is supported by studies examining the distribution of PCBs in the body tissues of humans (Luotamo, 1991) and animals (Takagi et al., 1986). Generally, the amounts of PCBs in the blood are a fraction of the levels seen in stationary tissues such as brain or adipose, suggesting that these tissues take up and concentrate PCBs out of the blood. The current studies suggest that the decreases in cellular dopamine associated with PCBs are not caused by unchlorinated aromatic hydrocarbons, at least not over the range of :media concentrations examined. in the current studies. This may be so because of the differences in the molecular volume, which impacts the lipophilicity, between 133 halogenated and non-halogenated aromatic compounds. Furthermore, PC12 cells are able to concentrate PCBs out of the culture medium. Finally, the extent to which cellular dopamine content is reduced is dependent on the cellular concentrations of PCBs, however, there is a lag time between the association of PCBs with the cells and the manifestation of the decrease in cellular dopamine. 134 135 W The toxic effects of PCBs have been studied in numerous organ systems in a variety of species, including man. Recent research has suggested that PCBs have detrimental effects on the nervous system, and particularly the dopaminergic portion of the nervous system. Studies by Seegal et al. (1989) and Angus and Contreras (1994) have demonstrated that PCB mixtures, such as Aroclor 1254, can cause a dose-dependent decrease in cellular dopamine in PC12 rat pheochromocytoma cells, a dopaminergic model system. However, the mechanism( s) by which PCBs cause this decrease is unknown. Previous studies (Chapter 4) have demonstrated that PCBs decrease the spontaneous and evoked release of dopamine from PC12 cells: therefore, the decreases in cellular dopamine caused by exposure to PCBs is not due to an increase in the release of dopamine by the cells. Furthermore, the decreases in cellular dopamine are associated with a decrease in cellular DOPAC. Thus, PCBs do not cause a decrease in cellular dopamine by increasing the metabolism of dopamine. Hence, PCBs probably effect a decrease in cellular dopamine content through a decrease in the synthesis of dopamine. The current studies were undertaken to examine the effects of PCBs on several steps in the biosynthetic pathway for dopamine. The results indicate that PCBs do not affect the uptake of tyrosine, the amino acid precursor for dopamine, but do decrease the 136 conversion of DOPA to dopamine. BE§QLI§ The uptake of 3H-tyrosine proved to be linear with respect to time through 90 min (Figure 6.1). The uptake of L-“C-leucine, L-“C-arginine, and 3H-methyl-choline was also linear with respect to time (data not shown). A timepoint of 40 min was chosen for the subsequent uptake studies since it was in the linear portion of the uptake curves. The results of the experiments examining the uptake of radiolabeled amino acids and choline into PC12 cells demonstrated that media concentrations of PCBs below 50 pg/ml did not affect the uptake of these substances (Figure 6.2). At high 50 pg/ml media concentrations of Aroclor 1254, there was a decrease in the uptake of amino acids concomitant with a decrease in the DNA content in the sample, which was used as an indicator of cytotoxicity. PCB congeners also did not affect the uptake of iH- tyrosine below media concentrations of 50 pg/ml (Figure 6.3). At media concentrations of 50 pg/ml, the uptake of tyrosine decreased with 2,2',5,5'-tetrachlorobiphenyl and 2,2',3,3',4,4'-hexachlorobiphenyl. The congener 2,3',4,4',5-pentachlorobiphenyl did not significantly increase the uptake of 3Iii-tyrosine, while the congener 2,2',4,4',5,5'-hexachlorobiphenyl had no effect on the uptake of 3H-tyrosine at any media concentration examined. Exposure of PC12 cells to Aroclor 1254 resulted in an 137 2000 dpmlng DNA 0 - . - . . , . , . O 20 40 60 80 100 Time (minutes) Figure 6.1 Uptake of 3H-tyrosine Into PC12 Cells PC12 cells were exposed to 3 pCi of 3H-tyrosine for 0, 2.5, 5, 10, 20, 40, or 90 min. The uptake of tyrosine was determined as described in Chapter 2, "Materials and Methods". A representative graph is shown. 138 15C 3 ‘3 O 125‘ l U ...}. . _ . __ ._ u --------- e— - — -..- .- — d - 3 100- l I \f N y \ w ‘ I? 1 \ _ _ § \ o . T t U) 75 5 C3 “5 50- o\° x +14C-Arginine E 25- --e--3H-cnoun. o- —*— 14CvLeucine :3 —.e— ammo... 0 v . w ......1 . . . . vrvv. 0.1 1 10 100 Media Concentration Aroclor 1254 (uglml) Figure 6.2 Uptake of Amino Acids or Choline by PC12 Cells Exposed to Aroclor 1254 - PC12 cells were exposed to various media qpncentrationsuof Aroclor 1 54 for 3 days. e uptake of C-arginine, C- leucine, H-tyrosine, and H-choline were determined as described in Chapter 2, "Materials and Methods". Each.point represents the mean t S.E.M. for 3 to 8 separate experiments. 139 160 120- (L ‘3 .‘3 C o o '0 ‘3 8 9:" C m 2 <3 9. 80- o 32 0 x g a 40' [:1 2.2.5.9 C . . "5 0 2.3.4.4.5 e . 3‘ A 2.2.3.3.“ 0 o 2.2.4.4355 0.1 1 10 100 Media Concentration (uglml) Figure 6.3 Uptake of 3li-t:yrosine Into PC12 Cells Exposed to PCB Congeners PC12 cells were exposed to various media concentrationssof different PCB congeners for 3 days. The uptake of H- tyrosine was determined as described in Chapter 2, "Materials and Methods". Each point represents the mean i S.E.M. for 3 to 4 separate experiments. 140 increase in the cellular DOPA as the amount of cellular dopamine declined (Figure 6.4). This increase in cellular DOPA suggested that LAAAD was being inhibited by exposure to PCBs. However, the effects of PCBs on the activity of tyrosine hydroxylase were also unknown. To examine the effects of Aroclor 1254 on the activity of TH and LAAAD, the conversion of SH-tyrosine to z'H-DOPA and SH-dopamine was measured. PC12 cells treated for 3 days with 25 ug/ml Aroclor 1254 demonstrated an increase in the synthesis of 3IrI-DOPA from Bil-tyrosine after 120 min. Exposure to 25 ug/ml Aroclor 1254 for 3 days also caused a decrease in the synthesis of’lH-dopamine, that was apparent at both 30 and 120 min (Figure 6.5). To further investigate the effects of Aroclor 1254 on LAAAD, various concentrations of DOPA were added to the culture medium of cells treated for 3 days with or without 25 ug/ml Aroclor 1254. The exposure to DOPA occurred for 6 hours following the exposure to 25 ug/ml.Aroclor 1254. With increasing concentrations of DOPA in the culture medium, the cellular DOPA content increased to the same extent in both PCB-treated and DMSO control cells (Figure 6.6). However, even though the content of cellular DOPA was similar in the control and experimental cells, the amount of cellular dopamine in PCB-treated cells was still decreased approximately 75% compared to the amount in vehicle-treated control cells (Figure 6.6). 141 450 400- —O" DOPA 350 .. + Dopamine /. 300- / 250 - / 200- ' P 150- Percent of DMSO-treated Control . . . ... 1 10 100 Media Concentration of Aroclor 1254 (uglml) Figure 6.4 Effects of Subchronic Exposure to Aroclor 1254 on Cellular Catechols PC12 cells were exposed to various media concentrations of Aroclor 1254 for 3 days. Cells were harvested and amounts of cellular DOPA, DOPAC, and dopamine were determined as described in Chapter 2, "Materials and Methods". Each point represents the mean : S.E.M. for 3 separate experiments. 142 0.5-1 -e—masoom -a--mc:|or12254°opA < .. + omsom 250$ D —e— Aroclor1254m . C” C E 90.2- ' 13 u ' ——e 0 l I 0 30 6'0 90 150 Time (min) Figure 6.5 Synthesis of 3fi-DOPA and 3H-Dopamine From 3a- tyrosine P012 cells were exposed to media concentratiogls of 25 ug/ml groclor 1254 for 3 days. The conversion of H-tyrosine to H-DOPA and H-dopamine was determined as described in Chapter 2, "Materials and Methods". Each point represents the mean i S.E.M. for 6 separate measurements. 143 25- + omso om N O l .5 0| 1 + DMSODA —A- Aroclor1254DA A O l T DOPA or Dopamine (pglng DNA) O-mv’ . . ... . ......I . v r.....' 1 10 100 1000 Media Concentration DOPA (uM) Figure 6.6 Effects of DOPA Supplementation on DOPA and Dopamine in Aroclor 1254-exposed Cells Cells were incubated for 6 hours with various media DOPA concentrations following subchronic exposure to Aroclor 1254. Amounts of cellular DOPA and dopamine were determined as described in Chapter 2, "Materials and Methods". Each point represents the mean i S.E.M. for 3 to 5 separate experiments, and are representative of cells treated with or without 25 ug/ml of Aroclor 1254. 144 Various PCB congeners also decreased the activity of LAAAD. Cells were exposed for 3 days to 25 ug/ml of the PCB congeners 2,2',4,4'-tetrachlorobiphenyl; 2,2',5,5'- tetrachlorobiphenyl: 2,2',3,3',4,4'-hexachlorobiphenyl: 2,2',4,4',5,5'-hexachlorobiphenyl: or DMSO, followed by 6 hours of exposure to various concentrations of DOPA in the medium. In both PCB-exposed and vehicle-treated control cells, cellular DOPA content increased to the same extent (Figures 6.7 and 6.8). However, even though cellular DOPA content in the PCB-treated and control cells was comparable, there was still a decrease of about 50% in cellular dopamine in the congener-treated cells compared to the controls. Discussion Subchronic exposure of PC12 cells to Aroclor 1254 did not affect the uptake of 3H-tyrosine into PC12 cells at non- cytotoxic media concentrations of PCBs. The uptake of 5%- tyrosine into PC12 cells decreased at 50 ug/ml of Aroclor 1254 after'3 days of exposure, but the decreases in cellular dopamine content caused by Aroclor 1254 began at 10 ug/ml after 3 days of exposure (see Chapter 3, Figure 3.2); therefore a decrease in the uptake of’lfl-tyrosine into the cells could not account for a decrease in cellular dopamine content caused by Aroclor 1254. These data suggest that exposure of the cells to Aroclor 1254 does not result in a decrease in cellular dopamine by causing a decrease in the uptake of amino acids or other precursors by the cells. 145 20- -IFwnwomnA :2. -4I-22A§m,A is -fik-2zssm,A n gt“ —a—omsoDA I! E —0- 23.410, q, 45,. 2.2.5.5DA / go. / m /// 8. I; 4: _____ —A '5 //// // < 5-1 /// / 8 ./ / 0' ' ' V ‘ ' ' ' ' I ' r ' ‘ ' ' ' '1 10 100 1000 Media DOPA Concentration (uM) Figure 6.7 Effects of DOPA Supplementation on Cellular Dopamine and DOPA in Tetrachlorobiphenyl-exposed Cells PC12 cells were exposed to supplemental DOPA in the media for 6 hours following subchronic exposure to tetrachlorobiphenyl. The cellular DOPA and dopamine contents were determined as described in Chapter 2, "Materials and Methods". Each point represents the mean i S.D. for 3 separate measurements, and are representative of cells treated with or without 25 ug/ml of either 2,2',4,4'- or 2,2',5,5'-tetrachlorobiphenyl. 146 b O .J —e- 22.3.3.4.4' no“ a O l - i. . iii-4.435300» —B— mason, .0- 2233363. - A- - 2.2.4.4255 DA DOPA or Dopamine (pg/n9 DNA) " 3 Q Media DOPA Concentration (uM) Figure 6.8 Effects of DOPA Supplementation on Cellular Dopamine and DOPA in Eexachlorobiphenyl-exposed Cells PC12 cells were exposed to supplemental DOPA in the media for 6 hours following subchronic of exposure to hexachlorobiphenyls. The amounts of cellular DOPA and dopamine were determined as described in Chapter 2, "Materials and Methods". Each point represents the mean t S.D. for 3 measurements, and are representative of cells treated with or without 25 ug/ml of 2,2',3,3',4,4'- or 2,2',4,4',5,5'-hexachlorobiphenyl. 147 Additionally, the PCB congeners 2,2',5,5'- tetrachlorobiphenyl; 2,3',4,4',5-pentachlorobiphenyl: 2,2',3,3',4,4'-hexachlorobiphenyl: (n: 2,2',4,4',5,5'- hexachlorobiphenyl did not significantly decrease or increase the uptake of 3H-tyrosine into PC12 cells at media concentrations below 50 ug/ml. As previously mentioned, these four congeners are present in relatively large percentages in the PCB mixture Aroclor 1254 (Chapter 3). Cellular dopamine content began to decrease around media concentrations of 10 to 25 ug/ml of these congeners (Chapter 3, Figure 3.7): but the uptake of 3H-tyrosine by these congeners was not effected until media concentrations reached 50 ug/ml. Therefore, decreases in the uptake of fiP— tyrosine into the PC12 cells caused by these congeners cannot account for the congeners' abilities to decrease cellular dopamine. Tyrosine and leucine are both taken up, though not exclusively, through a membrane transport system designated system L (Christensen, 1989; Stewart et al., 1989; Diez- Guerra and Gimmenez, 1989; Diez-Guerra et al., 1986), therefore, they act as controls for the effects of PCBs on the uptake of each other. Arginine was chosen to represent a charged amino acid, while choline was used to determine if the uptake of the precursor for the other neurotransmitter produced by PC12 cells (acetylcholine) was affected. However, exposure to Aroclor 1254 did not alter the uptake 148 of 1l'C-leucine, 1l’C-arginine, or 3H-methyl choline into PC12 cells. Thus, PCBs do not appear to have effects on transport systems for amino acids or choline, suggesting that PCBs do not have a generalized effect on plasma membrane proteins. Subchronic exposure to Aroclor 1254 caused a dose- dependent increase in cellular DOPA content, suggesting that LAAAD may be inhibited. Thus, conversion of 3H-tyrosine to 3'H-DOPA and 3H-dopamine was used to examine the effects of subchronic exposure to Aroclor 1254 on the synthetic enzymes for dopamine in PC12 cells. Results indicated that the amounts of newly synthesized DOPA in PCB-treated cells increased significantly over amounts in control cells, while the amounts of newly synthesized dopamine were decreased compared to controls. These data indicate that Aroclor 1254 decreased the activity of LAAAD. The increase in newly synthesized DOPA in the PCB-treated cells likely reflected an accumulation of DOPA due to decreased LAAAD activity. However, these findings do not completely rule out an additional effect of PCBs on TH. A recent report (Seegal and Shain, 1994) suggests that TH activity is inhibited by PCBs in NEIlS neuroblastoma cells. In order to determine the effects of Aroclor 1254 on TH, its kinetic parameters, and cofactors, more work will be required. In order to further investigate the effects of Aroclor 1254 on the activity of LAAAD, cell culture media was A /_f.... 149 supplemented with various concentrations of DOPA for 6 hours after subchronic exposure to Aroclor 1254, and this treatment did not restore cellular dopamine content in the PCB-treated cells to control levels, suggesting that Aroclor 1254 inhibited LAAAD in a manner that cannot be overcome by increasing cellular DOPA content. The effects of selected PCB congeners on the activity of LAAAD were also investigated. Subchronic exposure of PC12 cells to the congeners 2, 2 ' 5, 5 '-tetrachlorobipheny1 and 2,2',4,4',5,5'-hexachlorobiphenyl caused an increase in cellular DOPA content and a decrease in cellular dopamine, similar to the results seen with subchronic exposure of PC12 cells to Aroclor 1254. Yet, subchronic exposure to the congeners 2 , 2' , 4 , 4 ' ~tetrachlorobiphenyl and 2 , 2 ' , 3 , 3' , 4 , 4 ' - tetrachlorobiphenyl did not cause an increase in cellular DOPA content, even though they did cause decreases in cellular dopamine (Chapter 3, Figure 3.7). Hence, the PCB congeners were paired -- 2,2'4,4'- with 2,2'5,5'- tetrachlorobiphenyl, and 2,2',3,3',4,4'- with 2,2 ' ,4,4 ' ,5, 5'-—hexachlorobiphenyl -- and amounts of cellular DOPA and dopamine were determined following a 3 day subchronic exposure to 25 ug/ml of each congener, and 6 hours of DOPA supplementation in the media prior to cell harvest. The expected results were to see 2,2 ' ,5, 5'- tetrachlorobiphenyl and 2 , 2 ' , 4 , 4 ' , 5 , 5 ' -hexachlorobiphenyl decrease LAAAD activity, since they increased cellular DOPA 150 content, but to see no effects on LAAAD activity by the other two congeners, since they do not cause an increase in cellular DOPA. The actual results of these studies indicated that for all four congeners, cellular DOPA content increased to the same extent in PCB-treated and vehicle- treated control cells, reflecting increased concentrations of DOPA in the media. However, cellular dopamine was decreased compared to controls for all four congeners, suggesting that each congener had decreased the activity of LAAAD. These results suggest that a decrease in the activity of LAAAD may be only one mechanism by which PCBs can decrease cellular dopamine. The observations that the congeners 2,2 ' ,4,4'-tetrachlorobiphenyl and 2,2 ' ,3,3 ' ,4,4 '- hexachlorobiphenyl decrease cellular dopamine, decrease activity of LAAAD, but do not increase cellular DOPA content, suggest that these two congeners may also be affecting TH as well as LAAAD. A decrease in TH activity might prevent an apparent accumulation of DOPA in the cells secondary to decreased LAAAD activity. More studies will be required to determine if this indeed is true. L-aromatic amino acid decarboxylase is a dimer with a molecular weight of approximately 100 KDa (Coge et al., 1989). For activity, LAAAD requires the presence of pyridoxal pyrophosphate, Bar with a 1g. of approximately 0.28 pH (Young et al., 1993; Hadjiconstantinou et al., 1993). The V.“ of LAAAD for DOPA is about 37 nmol/mg protein/20 151 min, while the K; for DOPA is around 32 pH (Young et al., 1993; Hadjiconstantinou et al., 1993). Neuronal and non- neuronal isoforms of the enzyme have been reported (Morgan et al., 1986, Krieger et al., 1991), although there appears to be only one gene (Albert et al., 1987). The activity of LAAAD appears to be regulated acutely by PKA, resulting in an increase in Va“, without alterations in K... values (Young et al. , 1993) . Chronically, striatal LAAAD has been reported to be regulated through dopamine receptors, both.cn and DZ. Antagonists for the dopamine receptors increase the activity of striatal LAAAD, while agonists decrease the activity (Hadjiconstantinou et al., 1993; Rossetti et al., 1990). The increase in activity caused by dopamine receptor antagonists appears to be caused by an increase in Vfim, and not alterations in Kfi*values. Thus, the activity of LAAAD appears to be regulated and this regulation can result in the altered synthesis of dopamine. To confirm the effects of PCBs on LAAAD, in vitro studies using the isolated enzyme need to be done. PCBs might alter the cellular level of the cofactor, pyridoxal pyrophosphate, reduce the binding of the cofactor to LAAAD, or alter the activity or amount of the enzyme. PCBs have been suggested to affect second messenger systems (Kodavanti et al., 1993a,b) and could thereby be altering the activity of LAAAD (see Introduction for' a discussion of LAAAD regulation). More work needs to be done to determine the 152 mechanism for the PCB-mediated decrease in LAAAD activity. Polychlorinated biphenyls appear to inhibit the activity of LAAAD in PC12 cells as one mechanism by which they cause a decrease in cellular dopamine. PCBs do not appear to alter the uptake of amino acids or choline, nor do they appear, from the data obtained in these experiments, to overtly alter the activity'of'TH; ‘Work will have to be done on in vitro enzyme preparations in order to better understand the effects of PCBs directly on the enzymes for dopamine synthesis. m1 m1 153 154 Polychlorinated biphenyls are putatively neurotoxic to dopaminergic cells. Studies involving animals and humans (see Chapter 1) have described cognitive and behavioral deficits in individuals exposed to PCBs in utero. In humans, in utero exposure to PCBs has been implicated in impaired visual recognition memory, deficits in verbal and numeric manipulatory abilities, decreased psychomotor skills, and hypotonia and hyporeflexia (Fein et al., 1984, Jacobson et al., 1985, 1984, 1990; Gladen and Rogan, 1991; Rogan and Gladen, 1992; Chen et al., 1992). Prenatal exposure of animals to PCBs is also associated with defects, such as learning disabilities, hyperactivity, decreased muscular strength, and a behavior described as a "spinning syndrome" (Chou et al., 1979; Bowman et al., 1979, 1981: Agrawal et al., 1981; Tilson et al., 1990). These PCB- mediated deficits in learning and behavior may be due, in part, to altered dopaminergic function in the brain, as a decrease in brain dopamine content and number of dopamine receptors has been reported in mice prenatally exposed to PCBs (Agrawal et al., 1981). At the cellular level, direct acute (Seegal et al., 1989) exposure of dopaminergic cells to PCBs also results in decreased cellular dopamine. Thus, the data indicate that a decrease in cellular levels of dopamine may potentially play a role in the cognitive and behavioral deficits observed in humans and animals following prenatal exposure to PCBs. 155 The results from studies by Seegal et al. (1989) indicated that acute, 6 hour exposure of dopaminergic PC12 cells to Aroclor 1254 caused a dose-dependent decrease in cellular dopamine content. However, in.a prenatal exposure situation, the exposure of developing dopaminergic cells to PCBs would be on a subchronic to chronic basis. Therefore, studies were needed to examine the subchronic exposure of dopaminergic cells to PCBs. The current studies were undertaken to examine the hypothesis that.polychlorinated biphenyls decrease cellular dopamine in PC12 cells, which results from a decreased dopamine synthesis and, which manifests itself as a decreased release of dopamine in response to a depolarizing stimulus. in: investigate this hypothesis, five specific aims were addressed. Each is listed, along with a brief summary of the results below. Following a review of the specific aims and the brief summary of their results, a.more comprehensive discussion will bring together aspects of the results of this dissertation as they pertain first to Aroclor 1254, then to individual PCB congeners. ev' ' s Aim 1 Determine the subchronic effects of.Aroclor 1254 and select PCB congeners on cellular dopamine content, using undifferentiated and differentiating PC12 cells as a model system. Subchronic 3-day exposure to Aroclor 1254 decreased cellular dopamine content in a dose-dependent fashion in 156 both undifferentiated and differentiating PC12 cells. Amounts of cellular dopamine decreased at lesser medium concentrations of Aroclor 1254 with longer times of exposure, as indicated by a shift to the left in the dose- response curves for cellular dopamine between 1 and 3 days of exposure. In both undifferentiated and differentiating cells, the maximal effect of Aroclor 1254 on cellular dopamine content was observed at 3 days of exposure. Cellular dopamine content was decreased in a dose- dependent manner upon subchronic exposure to the following PCB congeners: 2,2',4,4'-tetrachlorobiphenyl: 2,2',5,5'- tetrachlorobiphenyl: 2,3,4,4',5-pentachlorobiphenyl: 2,3',4,4',5-pentachlorobiphenyl; 2,2',3,3,'4,4'- hexachlorobiphenyl; and 2,2',4,4',5,5'-hexachlorobiphenyl. The coplanar congener 3,3',4,4',S-pentachlorobiphenyl was toxic to the cells. The lightly substituted congeners 2,2 '- dichlorobiphenyl' 3,3'-dichlorobiphenyl; 4,4'- dichlorobiphenyl; and 2,4,4'-trichlorobiphenyl were ineffectual at decreasing cellular dopamine content in the PC12 cells. Aim 2 Determine whether the neurotrophic factor, NGF can protect dopaminergic cells from damage caused.by subchronic exposure to Aroclor 1254, using PC12 cells as a model system. A preincubation of the cells with NGF for 0 to 3 days prior to subchronic exposure to Aroclor 1254 did not result in any protection from the dopamine-decreasing 157 effects of Aroclor 1254. However, preincubation of the cells with NGF for 7 or 14 days resulted in an attenuation of the decrease in cellular dopamine content caused by subchronic exposure to Aroclor 1254. Aim 3 Determine whether subchronic exposure to PC12 cells to Aroclor 1254 and selected PCB congeners results in altered release of dopamine in response to stimulation.with 56 mM potassium, and whether the PCB-mediated decreases in cellular dopamine contribute to any alterations observed. Subchronic exposure of PC12 cells to Aroclor 1254 resulted in dose-dependent decreases in the evoked release of dopamine. The dose-response curves for evoked dopamine release and cellular dopamine content appeared to coincide, indicating a possible cause and effect relationship. The fraction of evoked dopamine released from the cells was unchanged with increasing PCB concentrations, indicating that Aroclor 1254 did not affect the packaging of dopamine into vesicles or the mechanisms for the release of dopamine. The PCB congener 2,2'-dichlorobiphenyl failed to decrease evoked release of dopamine. Meanwhile, the decreased release of dopamine caused by the congener 3,3',4,4',5- pentachlorobiphenyl was attributable to the congener's toxicity; The congeners 2,2',5,5'-tetrachlorobipheny1: 2,3',4,4',5-pentachlorobiphenyl: 2,2',3,3',4,4'- hexachlorobiphenyl; and 2 , 2 ' , 4 , 4 ' , 5 , 5 ' -hexachlorobiphenyl all caused dose-dependent decreases in the evoked release of 158 dopamine. For all four congeners, the dose-response curves for evoked release of dopamine closely followed the decrease in cellular dopamine content, again indicating a possible cause and effect relationship. The fraction of evoked dopamine released by the cells following subchronic exposure to any one of the six congeners examined did not change with increasing amounts of PCB in the media. These results indicate that the packaging and release mechanisms for dopamine were not affected by these PCB congeners and suggested that the decrease in amount of cellular dopamine was responsible for the decrease in dopamine release. Aim 4 Determine the concentrations of Aroclor 1254 or PCB congeners in the cells following a subchronic exposure, and whether the cellular PCB concentrations are related to the effects observed on cellular dopamine content and evoked release. A timecourse examining the rate of association of Aroclor 1254 with PC12 cells indicated that the PCBs are associated with the cells after 6 hours of exposure. However, effects of Aroclor 1254 on cellular dopamine content are not optimal until 3 days of exposure, thus there is a lag time between maximal association of Aroclor 1254 with the cells and a maximal manifestation of the decrease in cellular dopamine content. PC12 cells also concentrated Aroclor 1254 from the culture medium. An examination of the relationship between cellular Aroclor 1254 concentration and cellular dopamine content indicated an inverse relationship, 159 indicating a direct effect of Aroclor 1254 on cellular dopamine. PCB congeners also demonstrated an inverse relationship between cellular PCB concentration and cellular dopamine content. PCB congeners were concentrated by the cells from the medium, however, they accumulated to different extents within the cells. There appeared to be no discernable structure-activity relationship to describe the accumulation of PCB congeners into the cells. Aim 5 Examine possible mechanisms to explain the decrease in amounts of cellular dopamine and evoked release of dopamine from PC12 cells following subchronic exposure to Aroclor 1254. It was observed that subchronic exposure to Aroclor 1254 resulted in a dose-dependent increase in cellular DOPA content, suggesting that LAAAD was being inhibited. However, to rule out the possibility that the uptake of tyrosine was also being inhibited by subchronic exposure to Aroclor 1254, it was determined that the uptake of 3H-tyrosine was not affected at media Aroclor 1254 concentrations at which decreased cellular dopamine content was observed. An examination of the synthesis of 3H-DOPA and 3H--dopamine from 3H-tyrosine indicated that DOPA synthesis was elevated following subchronic exposure to Aroclor 1254, while synthesis of dopamine was substantially decreased. These results indicated an inhibition of LAAAD. Furthermore, following supplementation of culture medium 160 with DOPA, Aroclor 1254-exposed cells were able to take up DOPA to achieve a cellular content equivalent to control cells, however, they were unable to convert the DOPA to dopamine to the same extent as in control cells, further indicating that one mechanism by which Aroclor 1254 may decrease cellular dopamine content is by inhibition of LAAAD. However, not all PCB congeners increased cellular DOPA content. The congeners 2,2',5,5'-tetrachlorobipheny1 and 2,2',4,4',5,5'-hexachlorobiphenyl did increase cellular DOPA, and were experimentally paired with the congeners 2,2',4,4'-tetrachlorobiphenyl and 2,2',3,3',4,4'- hexachlorobiphenyl, congeners which did not increase cellular DOPA. These congener pairings were made to test the hypothesis that inhibition of LAAAD was a major mechanism causing the decrease in cellular dopamine content. Theoretically, only 2,2',5,5'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl should have caused an inhibited conversion of supplemented DOPA to dopamine, since they increased cellular DOPA levels. Surprisingly, cellular dopamine content in the cells exposed to any one of the 4 congeners were decreased, even though the cellular DOPA contents of the PCB-treated cells matched the cellular DOPA contents of the control cells. These data indicate that inhibition of LAAAD is only one mechanism by which PCBs can decrease cellular dopamine. 1.. 161 E . . Aroclor 1254: Subchronic exposure of undifferentiated or NGF— stimulated differentiating dopaminergic PC12 cells to the PCB mixture Aroclor 1254 resulted in dose-dependent decreases in cellular dopamine (Chapter 3). Yet, if PC12 cells were pretreated with NGF for at least 7 days prior to exposure to Aroclor 1254, the decrease in levels of cellular dopamine was partially attenuated. These data suggest that cells early in the process of differentiation are equally susceptible to the dopamine-decreasing effects of Aroclor 1254 as undifferentiated cells, however cells that receive the insult after differentiation had begun were less susceptible to the effects of Aroclor 1254 on cellular dopamine. The decreases in cellular dopamine content did manifest themselves as a decrease in the evoked release of dopamine (Chapter 4). Further, the fraction of dopamine released from the cells in response to a depolarizing stimulus did not change with increasing media concentrations of Aroclor 1254, suggesting that 1) the mechanisms for the packaging of dopamine into vesicles was not inhibited, otherwise the fraction released would have decreased; 2) the mechanisms for the release of dopamine from PC12 cells were neither stimulated nor inhibited, or else a corresponding increase or decrease in the fraction of dopamine released would have 162 been observed; and 3) the decreased evoked release of dopamine was caused by a decrease in the amount of cellular dopamine available for packaging. A decrease in the levels of cellular dopamine could, by decreasing the evoked release of dopamine, lead to learning and behavioral, as well as physical deficits. As discussed in Chapter 4, a substantial decrease (approximately 85 to 90%) in the brain dopamine content were observed in primates treated subchronically with very low doses of MPTP. These monkeys remained motor asymptomatic until challenged to perform a task requiring fine motor coordination skills. Further, these normal-appearing and behaving animals displayed cognitive and behavioral defects when challenged to perform tasks involving cognition and motor coordination. It is therefore possible that brain dopamine content can be significantly compromised without an overt manifestation of cognitive, behavioral, or motor deficits. However, under conditions involving stress, such as testing, the deficits may reveal themselves. The PCB mixture Aroclor 1254 decreased amounts of cellular dopamine and evoked release of dopamine at media concentrations between 10 and 50 ug/ml. Therefore, in a culture dish containing 2 ml of media, the total amount of Aroclor 1254 present was 20 to 100 ug. However, the culture medium that the cells were grown in contained 12% serum, and PCBs have been reported to bind to serum proteins, 163 especially albumin (Mohammed et al., 1990: Borlakoglu et al., 1990a,b) . Hence, the cellular concentrations of Aroclor 1254 under the experimental conditions used for the current studies was unknown. The concentrations of Aroclor 1254 in cells cultured in a 12% serum-containing medium was determined using GC/MS, and the amount of cellular Aroclor 1254 was normalized to the volume of the cells. The data suggested that 1) only about 2% of total Aroclor 1254 added to the culture medium became associated with the cells; and 2) dopaminergic cells concentrate PCBs from the cell culture medium. A time course examining the rate of association of Aroclor 1254 with PC12 cells indicated that a nearly maximal concentration of PCBs became associated with the cells by six hours; however, data from Chapter 3 demonstrated that the maximal effect of Aroclor 1254 on levels of cellular dopamine lagged behind at three days of exposure. Moreover, results from the current studies indicated that the cellular concentrations of Aroclor 1254 and the amount of cellular dopamine were inversely related (Chapter 5) . Together, the results from Chapter 5 suggest that Aroclor 1254 had a direct effect on cellular dopamine in PC12 cells that resulted, after a lag time, from exposure to Aroclor 1254. The mechanisms and pathways involved in the lag will require further investigation. Aroclor 1254 did not appear to decrease cellular dopamine content by 1) increasing metabolism of dopamine, as 164 a decrease in cellular DOPAC was observed as levels of cellular'dopamine:decreased; or 2) increasing the release of dopamine, since release decreased as amounts of cellular dopamine decreased. Elimination of Aroclor 1254 action on metabolism and release of dopamine oriented the focus of the studies on the synthetic pathway for dopamine. .Aroclor 1254 could therefore potentially affect LAAAD, TH, or the uptake of tyrosine into the PC12 cells. Following subchronic exposure of PC12 cells to Aroclor 1254, the uptake of HP- tyrosine into the cells did. not decrease until media concentrations of Aroclor 1254 reached 50 ug/ml: however levels of cellular dopamine began to decrease at 10 pg/ml. Therefore, a decrease in the uptake of tyrosine could not account for the decrease in the levels of cellular dopamine. Studies examining the synthesis of 3H-DOPA and 3H-dopamine from 3H-tyrosine indicated that Aroclor 1254 increased the synthesis of’3H-D0PA and a decreased the synthesis of 3H- dopamine; hence .Aroclor 1254 appeared. to (decrease the activity of LAAAD to cause decreases in cellular dopamine content. The synthesis of BH-DOPA was not reduced in Aroclor 1254-treated cells, which may indicate that Aroclor 1254 did not inhibit TH activity. However, the lack of an effect of Aroclor 1254 on the activity of TH still needs to be directly confirmed with in vitro studies involving the enzyme. To further examine the effects of Aroclor 1254 on the f.. 165 activity of LAAAD, the culture medium of subchronically exposed cells was supplemented with DOPA. Cellular DOPA content increased with increasing amounts of DOPA in the medium in both Aroclor 1254-treated and control cells. However, cellular dopamine content in the Aroclor 1254- treated cells was still significantly lower than in control cells. This effect of PCBs on LAAAD to cause a decrease in the synthesis of dopamine is probably one mechanism by which PCBs cause a decrease in dopamine levels. Thus, further in vitro studies on isolated TH and LAAAD themselves will be required to confirm the effects of PCBs on the enzymes involved in dopamine synthesis. PCB Congeners: The interactions of Aroclor 1254 and individual PCB congeners with PC12 cells resulted in a variety of effects on amounts of cellular dopamine, DOPAC, and DOPA; and on the release of dopamine. Furthermore, the ability of a PCB congener to become associated with cells differed. A subchronic exposure of PC12 cells to 2,2'- dichlorobiphenyl; 3,3'-dichlorobiphenyl; and 4,4'- dichlorobiphenyl generally failed to have significant effects on cellular dopamine or the evoked release of dopamine at media concentrations of the congeners that did not cause a general cytotoxicity. Very high cellular concentrations of the congener 2,2'-dichlorobiphenyl (1,363.7 pH at 50 ug/ml) were observed, even though this 166 congener failed to elicit significant effects on cellular dopamine content or evoked release of dopamine until cytotoxic media concentrations of 100 ug/ml were reached. These data suggest that the amount of PCB congener associated with the cells is not predictive of the ability of the congener to decrease cellular dopamine. Meanwhile, 2,2',4,4'- and 2,2',5,5'-tetrachlorobiphenyl differed greatly in their abilities to alter cellular dopamine and accumulated in the cells. The congener 2,2',4,4'-tetrachlorobiphenyl had no significant effects on cellular DOPA content, and decreased amounts of cellular dopamine and DOPAC at media concentrations of 50 ug/ml. Yet, the congener 2 , 2 ' , 5 , 5 ' -tetrachlorobiphenyl greatly increased the level of cellular DOPA, while significantly decreasing levels of cellular dopamine and DOPAC at media concentrations as low as 25 ug/ml. While the 2,2',5,5'- tetrachlorobiphenyl , but not the 2 , 2 ' , 4 , 4 ' - tetrachlorobiphenyl did cause an increase in cellular DOPA, both congeners appeared to decrease LAAAD activity, as supplementation of cell culture medium with DOPA failed to restore the synthesis of dopamine to control levels. Interestingly , these two congeners , 2 , 2 ' , 4 , 4 '- tetrachlorobiphenyl and 2 , 2 ' , 5 , 5 ' -tetrachlorobiphenyl , accumulated in the cells to different concentrations, 61.7 + 12.9 and 32.4 i 5.4 at 10 ug/ml media concentrations, respectively; and these differences could not be attributed 167 to any physical or chemical property differences between them. These findings further support the contention made earlier that association of congener with the cells is not necessarily predictive of the ability of the congener to cause a decrease in cellular dopamine; that is to say that a larger cellular concentration of PCBs does not necessarily translate into a greater decrease in cellular dopamine content. The three pentachlorobiphenyls examined, 2,3,4,4',5-; 2, 3', 4 , 4 ' , 5-; and 3 , 3 ' , 4 , 4 ' , 5—pentachlorobiphenyl , presented an interesting situation. The congener tested representative of the dioxin-like, coplanar PCB congeners, 3 , 3 ' , 4 , 4 ' , 5-pentachlorobiphenyl , decreased cellular dopamine, DOPAC, and DOPA together with DNA, suggesting that this congener elicited its effects through general cytotoxicity . The effects of 3 , 3 ' , 4 , 4' , 5- pentachlorobiphenyl on the evoked release of dopamine, and a determination of its cellular concentration was not performed in these studies since its effects were attributed to cytotoxicity. The congeners 2, 3,4,4 ' , 5- and 2, 3 ' ,4,4 ' ,5- pentachlorobiphenyl both decreased levels of cellular dopamine and DOPAC at concentrations that did not cause a decrease in DNA content. These congeners did not alter levels of cellular DOPA. Of the two congeners, the cellular concentration of 2 , 3 , 4 , 4 ' , 5-pentachlorobiphenyl was slightly greater than the cellular concentration of 2 , 3 ' , 4 , 4 ' , 5- 168 pentachlorobiphenyl. Further, a difference in the abilities to decrease levels of cellular catechols was also seen between the hexachlorobiphenyls examined, 2,2',3,3',4,4'- hexachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl, even though no difference was observed in the cellular concentrations of these congeners. The congener 2,2',4,4' ,5,5'-hexachlorobiphenyl increased levels of cellular DOPA while decreasing levels of cellular dopamine and DOPAC. The congener 2,2',3,3',4,4'-hexachlorobiphenyl decreased levels of cellular dopamine and DOPAC, but did not increase the cellular DOPA content. However, as will be discussed later, DOPA supplementation of the culture medium did not return cellular dopamine content to that of controls in cells treated with either congener. Both of these congeners decreased the evoked release of dopamine from the PC12 cells. The association of PCB congeners with the PC12 cells did not appear to follow any structure-activity relationship. Patterns of substitution and extent of chlorination did not relate to the ability of the congeners to accumulate in the cells. For example, the di- and tetrachlorobiphenyls accumulated in the cells to the largest molar concentrations, followed by the hexachlorobiphenyls and then the pentachlorobiphenyls. PCBs have been reported to become more hydrophobic as chlorination increases; hence, it would be expected that the hexachlorobiphenyls would 169 become associated with the cells to the greatest extent. Further, as mentioned above, congeners with the same number of chlorine substitutions did not become accumulate in the cells 'to ‘the same concentrations“ These observations support ‘the findings. of Shain. et (al. (1986) that the association of a congener with PC12 cells is not due to a generalizable pattern of chlorination of the congener, the extent of congener chlorination, or the physical properties of the congener (such as molecular volume): but rather association with cells depends on the structure of the congener itself. Selected congeners were used to determine the effects of individual PCBs on the uptake of tyrosine into the PC12 cells. The congeners 2,2',5,5'-tetrachlorobiphenyl: 2,3',4,4',5-pentachlorobiphenyl; 2,2',3,3',4,4'- hexachlorobiphenyl; and 2,2',4,4',5,5'-hexachlorobiphenyl, all of which constitute large percentages of Aroclor 1254, failed to significantly increase or decrease the uptake of z’H--tyrosine into PC12 cells at non-cytotoxic media concentrations. As with Aroclor 1254, the uptake of tyrosine decreased at 50 ug/ml, while levels of cellular dopamine declined beginning at around 10 ug/ml media PCB congener concentration. These data suggested that individual PCB congeners do not decrease levels of cellular «dopamine by decreasing the uptake of the precursor amino acid for dopamine synthesis, tyrosine. 170 Furthermore, studies involving supplementation of culture media with DOPA indicated that pgth of the tetrachlorobiphenyls (2,2',4,4'- and 2,2',5,5'- tetrachlorobiphenyl) and 99511 of the hexachlorobiphenyls (2,2',3,3',4,4'- and 2,2',4,4',5,5'-hexachlorobiphenyl) examined appeared to inhibit LAAAD, even though only one member of each pair tested (2,2',5,5'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl, respectively) increased levels of cellular DOPA. The two congeners that increased levels of cellular DOPA were expected to demonstrate an inhibition in LAAAD activity, while the other two congeners were not. These results suggested that the inhibition of LAAAD is only one mechanism by which PCB congeners can decrease in cellular dopamine content. It is possible that TH activity may be compromised, in combination with the activity of LAAAD or not, by the congeners that decreased cellular dopamine content and did not increase that of cellular DOPA. Hence, further work will be needed to determine other mechanisms by which PCB congeners can cause decreases in cellular dopamine. Caveats: PC12 cells are commonly used as model dopaminergic cell systems (Guroff, 1985: Fujita et al., 1989). These cells are, however, limited models. As with neural dopaminergic cells, PC12 cells synthesize and store catecholamines (Greene and Rein, 1977a,b). They also release 171 catecholamines upon stimulation by depolarization or following drug stimulation (Schubert et al., 1980), and contain specific 'uptake systems for' the. catecholamines (Greene and Rein, 1977a,b). Most neuronal cells have feedback inhibition of catecholamine synthesis and release (Cooper et al., 1991), which may occur in PC12 cells, although the issue is currently unclear (Courtney et al., 1991; Althaus et al., 1991). Both types of cells appear to have tyrosine hydroxylase as the rate limiting enzyme for catecholamine biosynthesis, however, PC12 cells can synthesize only small quantities of norepinephrine because of a lack of the cofactor for dopamine beta hydroxylase, ascorbate. Supplementation of PC12 cells with ascorbate has been successful in restoring the activity of DBH and increasing NE in the cells (Greene and Rein, 1979). Due to the lack of ascorbate and the pursuant decreased NE content, epinephrine is not easily detected in these cells. IHowever, EPI and its synthetic enzyme phenylethanolamine-N- methyltransferase (PMNT) have been determined to be present in PC12 cells (Byrd et al., 1986). Furthermore, PC12 cells have been used as a model system for the investigation of the regulation and activity of TH (Haycock, 1990). Another difference between PC12 cells and neuronal catecholaminergic cells is that LAAAD cannot be inhibited in the PC12 cells by the decarboxylase inhibitor NSD 1015 (WGR Angus, unpublished results). Furthermore, PC12 cells are an immortalized cell 172 line derived from a pheochromocytoma, a tumor of adrenal cortical origin, whereas neuronal cells are of central nervous system origin. Neuronal catecholaminergic cells require neurotrophic factors, such as NGF or BDNF for survival. PC12 cells, on the other hand do not require these factors for survival, but do require NGF for differentiation. Upon differentiation with NGF, PC12 cells, like neuronal cells, are electrically excitable and can form synapses with other cell types. Moreover, one report (Knudson and Poland, 1980) has suggested that cultured cells and cell lines are not as sensitive to the effects of dioxins and related compounds as are cells in vivo. Therefore, while PC12 cells are used as models for catecholaminergic cells, they do have some distinct differences from true neuronal cells. PCBs have been suggested to affect humans in a variety of ways (see Introduction for a discussion and references). Determination of the direct effects of PCBs on humans is difficult due to confounding factors, such as, for example, co-exposure to other potentially toxic compounds, uncertainty about exact amounts of exposure, routes of distribution of PCBs to various tissues, and motility of humans between regions of greater and lesser contamination. Some of these confounding factors can be limited by the use of laboratory animals. Under laboratory conditions, the availability of PCBs can be monitored, as well as amounts of 173 exposure, limitations to co-exposure to other toxins, and mobility of the animals to areas of differing contamination. However, neither in animal or human studies, can the direct mechanistic effects of PCBs on certain cell types be examined. Cell culture systems are advantageous for these investigations because a large quantity of tissues can be generated.and.the environment.and exposures of the cells can be directly controlled by the investigators. Further, tissue culture systems are easier to manipulate for mechanistic studies and lend themselves to analysis more easily than do animals or humans. However, as discussed above, cells in culture, such as PC12 cells, are not always accurate models for normal cells. Thus, for both cell culture systems and animal models, extrapolating the results back to either an in vivo situation or to humans, respectively, is uncertain at best. Human investigations, animal model systems, and cell culture systems all have distinct advantages and disadvantages, but used in a coordinated investigation into a toxicological problem, can prove invaluable for determining the actions of a toxic compound. In the current studies, PCBs were introduced into cell culture medium containing serum and cells. The availability of the PCBs to the cells was in saturating excess, which is not likely the situation in vivo. As previously mentioned, only about 0.1% of total PCBs administered to pigeons is 174 found to be associated with the serum (Borlakoglu et al., 1993). Only about 1% of total PCBs were found to be associated with brain tissue. While these data support the hypothesis that neuronal cells can concentrate PCBs from blood, they do not specify what regions of the brain contain what concentration of PCBs. Moreover, larger percentages of total PCBs administered were found associated with non- neural tissues such as liver and adipose, indicating that the availability of PCBs to the brain.are low initially, and when the kinetics and dynamics of exchange of PCBs between tissues and blood serum are factored in, one can only speculate about how closely the current studies mimic the events of neural exposure to PCBs in vivo. Nonetheless, the current studies did, for the first time, relate the effects of PCBs on cellular dopamine content, cellular concentrations of PCBs, and the relationship in time between exposure of cells to PCBs and a manifestation of an event. In conclusion, polychlorinated biphenyls can cause decreases in levels of cellular dopamine in dopaminergic PC12 cells. The decreased levels of cellular dopamine can manifest themselves as a decrease in the evoked release of dopamine in response to a depolarizing stimulus. For Aroclor 1254 and PCB congeners that decrease cellular dopamine, the extent of the decrease reflected an increased cellular concentration of Aroclor 1254 or congener. However, the accumulation of the various PCB congeners in 175 the cells does not appear to have any determinable structure-activity relationship. The effects of PCBs on cellular levels of dopamine are direct, but require a lag time before manifestation. Furthermore, one mechanism by which PCBs may cause decreased cellular dopamine is through inhibition of LAAAD, however, this does not appear to be the only mechanism by which PCBs may affect dopaminergic cells. More research will be required to further the understanding of the effects of PCBs on dopaminergic cells. APPENDICES 176 APPENDIX 1 Table A.l. Structure and Nomenclature of Congeners Used IUPAC Name PCB Congener Name 3,3'-Dichlorobiphenyl PCB 11 " 4,4'-Dichlorobiphenyl PCB 15 n 2,4,4'-Trichlorobiphenyl PCB 28 2,2',4,4'-Tetrachlorobiphenyl PCB 47 2,2',5,5'-Tetrachlorobiphenyl PCB 52 2,3,4,4',5-Pentachlorobiphenyl PCB 114 2,3',4,4',5-Pentachlorobiphenyl PCB 118 3,3',4,4',5-Pentachlorobiphenyl PCB 126 2,2',3,3',4,4'-Hexachlorobiphenyl PCB 128 2,2',4,4',5,5'-Hexachlorobiphenyl PCB 153 §IBHQIQB§§ Ci CI CI Cl 04 11 15 CI Cl Cl CI Cl CICI CI Cl CI Cl C' 114 C 118 C' 126 CI