muwflgnxlLumummmgmw ' LIBRARY. This is to certify that the thesis entitled PHYSIOLOGICAL AND BIOCHEMICAL SEQUELAE TO PERINATAL EXPOSURE TO POLYBROMINATED BIPHENYLS presented by Kevin Michael McCormack has been accepted towards fulfillment of the requirements for Ph.D. degree in Phar_rnagQ|,ogy & . Toxicology %%34< 7 / Major professor Date H «at/a 77 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. PHYSIOLOGICAL AND BIOCHEMICAL SEQUELAE TO PERINATAL EXPOSURE TO POLYBROMINATED BIPHENYLS BY Kevin Michael McCormack A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology & Toxicology 1979 ABSTRACT Physiological and Biochemical Sequelae to Perinatal Exposure to Polybrominated Biphenyls by Kevin Michael McCormack Polybrominated biphenyls (PBBS) were directly added to the food supply in Michigan. Of particular concern are possible effects of PBBs on mothers and progeny since high concentrations of PBBs have been detected in human milk and PBBs have been reported to undergo transplacental movement. The purpose of this investigation was to determine if physiological and biochemical alterations are produced by prenatal and/or postnatal exposure to PBBs. Dietary exposure of Sprague-Dawley rats to 100 ppm PBBs (Fire- master BP-6) from day 8 through day 20 of gestation did not alter litter size, resorption rate, fetal body weight or length or incidence of gross, soft tissue or skeletal anomalies. Food deprivation in combination with this treatment increased fetal resorption rate and decreased fetal body weight. Treatment of rat pups with 150 or 500 mg/kg PBBs on day l postpartum had no effect on growth, develOpment or mortality. However, after 1 wk postpartum growth and develOpment were retarded and mortality increased in pups born to and suckled by dams fed 100 ppm PBBs from the eighth day of pregnancy. Consistent with these effects, concentrations of PBBs in tissues from animals eXposed Kevin Michael McCormack to PBBs transplacentally and via mother's milk were higher than concen— trations in tissues from rats neonatally treated with a single injec- tion of PBBS. Kidneys from rats exposed to PBBs did not have prominent, if any, macroscopic or microscopic morphological changes. The paucity of renal structural alterations was correlated with a lack of effect of P333 on renal function. Similarly, perinatal eXposure to PBBs did not affect lung or heart weights and had little effect on pulmonary or cardiac function. The liver weight-to-body weight ratio was increased following perinatal exposure to 10 or 100 ppm PBBs and after neonatal treatment with 150 or 500 mg/kg PBBs. Liver enlargement was dose dependent and directly related to hepatic concentrations of PBBs. Increased liver weight may have been due, at least in part, to elevated protein content as hepatic microsomal protein was increased by PBBs. MicroscoPic mor— phologic alterations were also produced by PBBs. Liver from rats perinatally exposed to 100 ppm PBBs had vacuolation, hepatocyte swelling, necrosis, absent or pycnotic nuclei and myelin bodies. Liver from rats treated with P333 also had a decreased concentration of vitamin A and an increased concentration of coprOporphyrin and uroporphyrin. Activity of microsomal enzymes in liver and extrahepatic organs was altered by P333 in a manner that was dependent on dose, age and time following administration. These enzymatic changes produced by PBBs were correlated with modifications in the toxicity and/or duration of action of certain subsequently administered therapeutic agents and environmental chemicals. Thus, even animals that exhibited no visible Kevin Michael McCormack expression of PBBs toxicity were more susceptible to drug interactions after exposure to PBBs. Activity of progesterone hydroxylases was increased in hepatic microsomes prepared from rats perinatally exposed to 100 ppm PBBs. Accelerated metabolism of progesterone in_yi££g_was reflected in_!iyg by a reduced duration of anesthesia following a pharmacologic dose of progesterone. Similarly, responses to exogenously administered estradiol-l78 and testosterone were diminished by perinatal eXposure to PBBs. Radioactivity in serum and target tissues following admini- stration of labeled steroid hormones was also reduced by PBBs. Repro— ductive capacity may be diminished by PBBs as a consequence of en— hanced steroid metabolism. Effects of P338 not only persisted for long periods of time in directly exposed rats but were produced in their descendants. PBBs were transferred from one generation to the next via transplacental movement and excretion into milk. Transferred PBBs produced altera- tions in liver morphology and ability to metabolize xenobiotic and endogenous compounds. Therefore, the health hazard associated with exposure to PBBs may not be limited to a single generation. ACKNOWLEDGEMENTS I wish to express my sincere appreciation to graduate committee members: Drs. Theodore M. Brody, Jerry B. Hook, Steven D. Aust, Michael D. Bailie, Gregory D. Fink and Gilbert H. Mayor for their advice and assistance in the preparation of this thesis. I would expecially like to thank Dr. Jerry B. Hook for his guidance, encourage— ment, and constructive criticism during my graduate education. I would like to thank Drs. James E. Gibson, Stuart Z. Cagen, and John G. Dent for their intellectual contributions during the initial phase of my research career. The excellent technical assi— stance of Lisa F. Lepper and clerical assistance of Diane K. Hummel are gratefully acknowledged. For their encouragement, I would like to dedicate this thesis to Thomas J., Ruth M. and Amy J. McCormack. These studies were supported in part by the National Institute of Environmental Health Sciences, Grant No. ESOOS60. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii LIST OF TABLES Vi LIST OF FIGURES viii INTRODUCTION 1 A. Characteristics of Polybrominated Biphenyls 2 B. Environmental Contamination by Polybrominated Biphenyls 4 C. Biological Effects of Polybrominated Biphenyls --------- 8 D. Biological Effects of Agents that Increase Microsomal Enzyme Activity 13 E. Objectives— 28 MATERIALS AND METHODS 29 A. Animals 29 B. Teratology 29 C. Postnatal Development 30 D. Vaginal Cycle Length 31 E. Histopathology 31 F. Milk Collection 31 G. Quantitation of PBBs 31 H. Liver 33 l. Copr0porphyrin and uroporphyrin concentrations in liver and urine 33 2. Vitamin A concentration in liver and serum -------- 34 I. Preparation of Postmitochondrial Supernatants and Microsomes 34 J. Enzyme Assays 35 . Kidney 39 1. Function in vitrc 39 2. Function in viva 41 iii TABLE OF CONTENTS (continued) Page L. Heart 42 1. Function in vitro 42 2. Arterial pressure 43 M. Lung 44 1. Function in vitro 44 2. Isolated perfused lung 45 Response to Xenobiotics 47 0. Response to Pharmacological Doses of Steroid Hormones-- 48 P. Persistence of Effects 49 1. Single injection 49 2. Perinatal exposure 49 3. Multiple generations 50 Q. Statistics- 50 RESULTS 51 A. Survival, Growth and Development 51 B. Liver 56 C. Kidney 83 D. Heart 99 E. Lung 107 F. Testis and Ovary 107 G. Response to Xenobiotics 119 H. Response to Exogenously Administered Steroid Hormones-- 128 I. Persistence of Effects 143 DISCUSSION 178 A. Survival, Growth and DevelOpment 178 B. Liver 182 C. Kidney 192 D. Heart 194 E. Lung 194 F Extrahepatic Microsomal Enzyme Stimulation 195 G. Response to Xenobiotics 198 iv TABLE OF CONTENTS (continued) Page H. Response to Exogenously Administered Steroid Hormones-- 200 I. Persistence of Effects 204 SUMMARY AND CONCLUSIONS 209 BIBLIOGRAPHY 215 Table 10 ll 12 13 14 LIST OF TABLES Resorption rate, fetal size and whole carcass concen- tration of PBBs among offspring of pregnant rats treated with PBBs and food deprived Postnatal development of rats following neonatal or perinatal treatment with PBBs Tissue concentrations of PBBs following neonatal or perinatal and continuous treatment with PBBs Effect of perinatal eXposure to PBBs on the concentra- tion of vitamin A in serum and liver of rats Concentration of coproporphyrin and uroporphyrin in liver from female rats exposed to PBBs Concentration of c0proporphyrin and urOporphyrin in urine from female rats eXposed to PBBs Packed cell volume (hematocrit) in female rats exposed to PBBs Effect of PBBs on blood urea nitrogen (BUN) Effect of PBBs on glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) Serum calcium concentration and bone mineral mass in female rats exposed to PBBs Effect of perinatal exposure to PBBs on the concentra- tion of vitamin A in kidney of rats Effect of PBBs on the accumulation of PAH and NMN-—---- Effect of PBBs on ammoniagenesis and g1uconeogenesis--— Effect of PBBs on the accumulation of (3H)—d,l-meta— raminol vi Page 52 55 59 72 73 74 75 91 92 95 96 97 98 106 LIST OF TABLES (continued) Table 15 16 17 18 19 20 21 22 23 24 25 Page Effect of P335 on left atrial susceptibility to ouabain-induced arrhythmia, twitch tension and inotro- pic response to ouabain or calcium 108 Effect of PBBs on mean systolic blood pressure—------—- 109 Effect of perinatal treatment with polybrominated bi- phenyls on the clearance of angiotensin I (AI) and 5— hydroxytryptamine (5-HT) by isolated perfused rat lungs 112 Concentration of testosterone and progesterone in serum from rats exposed to PBBs 120 Vaginal (estrus) cycle length and age of vaginal opening and testicular descent in rats exposed to PBBs— 121 Median time to death after administration of bromoben- zene (2820 mg/kg) in 49 day old male rats neonatally treated with polybrominated biphenyls 126 Median time to death after administration of digitoxin in female rats pretreated with PBBs 7 127 Tissue concentrations of PBBs following a single injec- tion 148 Residual effect of PBBs-exposure on blood urea nitrogen (BUN) and activity of serum glutamic pyruvic transami- nase (SGPT) 159 Tissue concentrations of PBBs following perinatal exposure to PBBs 162 Concentration of P333 in liver following perinatal exposure to PBBs 176 vii Figure 10 11 12 LIST OF FIGURES Effect of neonatal or perinatal treatment with PBBs on body wt gain Effect of neonatal or perinatal treatment with PBBs on survival rate-to-weaning Concentration of PBBs in milk from lactating rats fed diet containing 50 ppm PBBs from the 8th day of preg- nancy Effect of neonatal or perinatal treatment with PBBs on liver wt to body wt and kidney wt to body wt ratios---- Hepatic tissue from control rat Hepatic tissue from rat 28 days of age that had been exposed to 100 ppm PBBs from 8th day of gestation _____ Hepatic tissue from rat fed diet containing 100 ppm PBBs for 90 days Hepatic microsomal protein and enzyme activities in rats at various times after treatment with 0 or 150 mg/kg PBBs on day 7 postpartum Hepatic enzyme activities in rats at various times afte treatment with 0 or 150 mg/kg PBBs on day 7 postpartum- Effect of neonatal or perinatal treatment with PBBs on activity of arylhydrocarbon hydroxylase and epoxide hydratase in liver Hepatic progesterone hydroxylase activities in rats treated with 0 or 100 ppm PBBs from day 8 of gestation until they were killed at day 28 postpartum Hepatic monoamine oxidase activity in rats treated with 0 or 100 ppm PBBs from day 8 of gestation until they were killed at day 28 postpartum viii Page 53 57 6O 62 65 67 69 77 r 81 84 86 LIST OF FIGURES (continued) Figure 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Renal tissue from rat fed diet containing 100 ppm PBBs for 90 days Effect of PBBs on fractional sodium excretion------——-— Renal postmitochondrial supernatant protein and enzyme activities in rats at various times after treatment with 0 or 150 mg/kg PBBs on day 7 postpartum Effect of neonatal or perinatal treatment with PBBs on activity of arylhydrocarbon hydroxylase and epoxide hydratase in kidney Effect of P333 on the heart weight-to-body weight ratio Effect of PBBs on the lung weight-to-body weight ratio- Effect of perinatal treatment with PBBs on the activity of angiotensin-converting enzyme and monoamine oxidase in lung— Effect of PBBs on activity of arylhydrocarbon hydroxy- lase and epoxide hydratase in lung ‘ Effect of PBBs on the testis weight— or ovary weight- to-body weight ratios Effect of neonatal or perinatal treatment with PBBs on the duration of anesthesia produced by pentobarbital-—- Effect of PBBs on the duration of anesthesia produced by diethyl ether Effect of perinatal exposure to 0, 10 or 100 ppm PBBs on the duration of anesthesia produced by progesterone- Effect of perinatal exposure to 0 or 100 ppm PBBs on the concentration of equivalents of progesterone-14C in brains of rats treated with progesterone—14C Effect of perinatal exposure to 0 or 100 ppm PBBs on the concentration of equivalents of progesterone-14C in serum of rats treated with progesterone-14C Effect of perinatal eXposure to 0, 10 or 100 ppm PBBs on the increase in seminal vesicle wt-to-body wt ratio produced by testosterone ix Page 88 93 100 102 104 110 113 115 117 122 124 129 131 133 135 LIST OF FIGURES Figure 28 29 30 31 32 33 34 35 36 37 38 39 40 Effect of perinatal exposure to 0, 10 or 100 ppm PBBs on the concentration of equivalents of testosterone- H in testis of rats treated with testosterone-3H ------ Effect of perinatal exposure to O, 10 or 100 ppm PBBs on the concentration of equivalents of testosterone-3H in serum of rats treated with testosterone-3H Effect of perinatal exposure to 0, 10 or 100 ppm PBBs on the increase in uterus wt-to-body wt ratio produced Page 137 139 141 by estradiol-17B Effect of perinatal exposure to 0, 10 or 100 ppm PBBs on the concentration of equivalents of estradiol—17B- 6,7-3H in uterus of rats treated with estradiol-l7B- 6,7-3H— 144 Effect of perinatal exposure to 0, 10 or 100 ppm PBBs on the concentration of equivalents of estradiol-l78- 6,7-3H in serum of rats treated with estradiol-l78- 6,7- H Disappearance of PBBs from the peritoneal cavity of rats after a single intraperitoneal injection --------- Residual effect of perinatal exposure to PBBs on liver 146 150 wt—to—body wt and kidney wt-to—body wt ratios Residual effect of perinatal exposure to PBBs on acti- vity of arylhydrocarbon hydroxylase and epoxide hydra- tase in liver— Residual effect of perinatal exposure to PBBs on acti- vity of arylhydrocarbon hydroxylase and epoxide hydra- 152 155 157 tase in kidney Residual effect of perinatal exposure to PBBs on dura- tion of anesthesia produced by pentobarbital Effect of exposure to PBBs on body weight and the liver wt-to-body wt ratio in subsequent generations --------- Effect of exposure to PBBs on percent survival-to- 160 164 166 weaning in subsequent generations Effect of exposure to PBBs on activity of arylhydrocar- bon hydroxylase and epoxide hydratase in liver in 168 subsequent generations LIST OF FIGURES (continued) Figure 41 42 43 Page Effect of exposure to PBBs on activity of arylhydrocar- bon hydroxylase and epoxide hydratase in kidney in subsequent generations 170 Effect of exposure to PBBs on duration of anesthesia produced by progesterone in subsequent generations ----- 172 Effect of exposure to PBBs on duration of anesthesia produced by pentobarbital in subsequent generations——-- 174 xi INTRODUCTION Accidental addition of polybrominated biphenyls (PBBs) to dairy cow feed in Michigan has resulted in contamination of the food supply (Dunckel, 1975). As PBBs are quite lipid soluble, they accumulate in biological tissues, especially those with a high fat content. Most PBBs are biologically stable, efficiently absorbed from the gastro- intestinal tract, and very slowly eliminated from the body. Extrapo- lation of data obtained in animals suggests that PBBs will persist in tissues throughout a human lifetime (Matthews gt 31., 1977). Biological effects of PBBs in man and animals have not been fully characterized. Hepatic and renal histopathological changes have been produced by PBBs; however, data presently available are inadequate to assess changes in organ function. Although PBBs have been shown to increase activity of microsomal mixed function oxidases (MFO) in liver, the effect of P333 on MFO activity in extrahepatic tissues has not been characterized (Dent g£_al,, 1976a,b). These microsomal enzymes catalyze metabolism of a variety of xenobiotic and endogenous compounds including certain carcinogens, therapeutic agents, vitamins and hormones (Conney, 1967). Thus, some effects of PBBs may be mediated through alterations in MFO activity. The potential hazard to man of such metabolic alterations following PBBs has not been con- clusively established. 2 Of particular concern are possible effects of PBBs on mothers and progeny since high concentrations of P333 have been detected in human milk and P333 have been reported to undergo transplacental movement (Dunckel, 1975; Rickert §£_a1,, 1978). Major organ systems are mor- phologically and functionally immature in very young animals and de- ficient in their capacity to maintain homeostasis when challenged with stressful stimuli. Therefore, mammals including man, may be particu- larly vulnerable to damage produced by PBBs during organogenesis and early postnatal life. A. Characteristics of Polybrominated Biphenyls Polybrominated biphenyls (PBBs) are composed of two six-membered aromatic carbon rings, connected by a carbon-carbon bond, which may be brominated at any position, except the bridge carbons. Firemaster FF- 1 is the commercial name for the product that contained PBBs (Fire- master BP—6) and calcium polysilicate and was added to the food supply. Firemaster BP—6 has been shown by electron capture gas chroma- tography to consist of a mixture of P333. Analysis of the Firemaster mixture using gas chromatography—mass spectrometry demonstrated 12 peaks whose mass spectrum corresponded to compounds with molecular weights indicative of 2 penta-, 4 hexa-, 4-hepta—, and 2 octabromobi— phenyl congeners (Anderson e£_§1,, 1974; Sundstrom gt 31., 1976). Nuclear magnetic resonance was used to identify the structures of congeners in Firemaster BP—6. The two main congeners, 2,4,5,2',4',5'- hexabromobiphenyl and 2,3,4,5,2',4',5'-heptabromobiphenyl, account for 54-68 and 27 percent of the Firemaster BP-6 mixture by weight, re- spectively (Sundstrom gt 31., 1976; Jacobs gt_al,, 1976; Moore §£_al,, 3 1978a). Other congeners identified include: 2,4,5,4',5'-penta-, 2,4,5,3',4'-penta-, 2,3,4,2',4',5'-hexa-, 2,4,5,3',4',5'-hexa-, 2,3,4,5,2',3',4',5'-octa-, and 2,3,4,5,6,2',3',4',5'-nonabromobiphenyl (Moore and Aust, 1978). Firemaster may contain 30 or more components. Trace quantities of hexa-, penta-, and tetrabromonapthalene have been found in this mixture as well as several polar contaminants which have been partially characterized (O'Keefe, 1976; Hass gt 31., 1978). Firemaster is a gray powder that is nearly insoluble in water (11 ppb) but soluble in most organic solvents. Firemaster has a compara— tively low vapor pressure, begins to melt at 72°C and decomposes in the range 300-400°C (Sundstrom gt a1., 1976). The major congener, 2,4,5,2',4',5'-hexabromobiphenyl, melts at 159-160°C and 2,3,4,5,2',4', 5'-heptabromobipheny1 melts at 165-166°C (Moore £5 31., 1978a, 1979). The stability of PBBs to oxidation and hydrolysis in the environment has not been determined. However, part of the Firemaster mixture is unstable to alkaline hydrolysis as reflected by the fact that treat- ment with 2% potassium hydroxide in ethanol resulted in degradation of the major hexa-isomer (Pomerantz gt 31., 1978). PBBs are more photo- reactive than polychlorinated biphenyls (PCBs) and should be protected from light in the laboratory. When dissolved in methanol and exposed to ultraviolet light (3000 A), 2,4,5,2',4',5'-hexabromobiphenyl was 7 times more photolytically reactive than 2,4,5,2',4',5'—hexachloro- biphenyl (Ruzo and Zabik, 1975). 2,4,5,2',4',5'-Hexabromobipheny1 was found to undergo photolytic reductive debromination in methanol to penta— and tetrabromobiphenyl as well as (approximately 1%) dimethoxy- tetrabromobiphenyl (Ruzo and Zabik, 1975). PBBs may be more photo- reactive than PCBs because of enhanced intersystem crossing to a 4 triplet state due to virbonic coupling with the bromines and low carbon-bromine bond energy (71 Kcal/mole) (Kerst, 1974; Ruzo and Zabik, 1975; Matthews £5 31., 1978). The rates and extent of photo— lytic reactivity of PBBs in the environment have not yet been deter- mined. B. Environmental Contamination by Polybrominated Biphenyls Contamination of the environment by PBBs was discovered in the spring of 1974, at least 8 months after 500-1000 pounds of Firemaster had been inadvertently substituted for magnesium oxide in dairy cattle feed (Dunckel, 1975). According to formula, animals were being fed diet with a concentrate containing 4,000 to 5,200 ppm PBBs but some concentrate contained as much as 13,500 ppm PBBs while other livestock and poultry feed mixed in contaminated mills had smaller amounts. Two other probable routes of indirect contamination are recycling of contaminated products and feed swapping between individual farms and feed mills. Milk from Michigan farms had concentrations of PBBs in excess of 600 ppm which if consumed at 500 to 1000 ml per day during the period between contamination and identification of PBBs would have resulted in human intakes of approximately 150 mg PBBs per kg body weight (Fries and Marrow, 1975). Additional human exposures could have come from consumption of poultry, beef or eggs which had concentrations as high as 4,600 (fat), 2,700 (fat) and 4,000 ppm PBBs, respectively. PBBs are usually quantified by the major hexabromobiphenyl peak (2,4,5,2',4',5'-hexa) despite the possibility that other congeners are more toxic. Guidelines for permissible concentrations of PBBs were 5 first established in May, 1974 by the U.S. Food and Drug Administra- tion and included milk and meat, 1.0 ppm; eggs, 0.1 ppm; and feed, 0.3 ppm. These guidelines were revised in November, 1974 to milk and meat, 0.3 ppm; and eggs and furnished feeds, 0.05 ppm, and have been reduced several times since then. All products containing PBBs at or exceeding those guideline concentrations were confiscated. To date more than 30,000 cattle, 5,000 swine and sheep, 1.5 million chickens, 2,600 pounds of butter, 34,000 pounds of dry milk products, 1500 cases of canned evaporated milk, 18,000 pounds of cheese, 5 million eggs, and 865 tons of feed have been destroyed. It has been estimated that between the onset of contamination in the fall of 1973 and the esta- blishment of quarantine of contaminated livestock in the spring of 1974, over 10,000 Michigan residents were exposed to PBBs through consumption of contaminated milk and meat (Dunckel, 1975; Kay, 1977). Extrapolation of data obtained in 1976 suggested that approximately 90% of the residents in Michigan had detectable body burdens of PBBs (Brilliant gt a1., 1978). Although once considered to be solely a Michigan problem, PBBs have recently been found in New York, New Jersey, Pennsylvania, Iowa, Indiana, Wisconsin, Alabama, Mississippi and Texas (Report, 1976; Carter, 1976; Michigan Dept. Agriculture, 1977; Anonymous, 1977). Products from Michigan contaminated with PBBs may have been trans- ported to other states for consumption. It has been estimated that over 600,000 pounds of P333 were directly added to the environment at sites of PBBs manufacture (Neufeld gt a1., 1977). Industrial pollu- tion included emission to the air from vents of the hydrogen bromide recovery system, losses in waste waters resulting from quenching and 6 washing PBBs as they were recovered from the reaction mixture, and losses occurring with landfills resulting from drying, handling and transportation (Neufeld gt 31., 1977). The general population may also be exposed to PBBs from the use of Firemaster as a flame retar- dant. Between 1971 and 1974 almost 12 million pounds of PBBs were marketed as fire retardants used in thermal plastics such as type— writer, television and business machine casings (Kerst, 1974). Most of the products containing PBBs are or will be eventually buried in refuse dumps. PBBs have little tendency to migrate from the thermal plastics into which they are incorporated, however, small quantities of PBBs may leak into streams or the water table, be consumed by scavengers, or enter the atmosphere upon flameless combustion of products containing PBBs (e.g., in refuse dumps or office fires). PBBs entering the atmosphere may then be deposited within a few days onto land or into water and eventually enter food chains. Polybrominated biphenyls are quite lipid soluble and stable and thus, would be anticipated to accumulate in biological tissues espe- cially those with a high fat content. Fish accumulate PBBs as do a variety of mammals including rodents, farm animals and man (Zitko, 1977; Fries and Marrow, 1975; Matthews gt a1., 1977; Willet and Irving, 1975; Kimbrough g£_§1,, 1978). Investigations with Firemaster in rats and farm animals indicate that PBBs are efficiently absorbed from the gastrointestinal tract and very slowly eliminated from the body (Gutenmann and Lisk, 1975; Willett and Irving, 1976; Matthews_gt 31,, 1976). Studies with 2,4,5,2',4',5'-hexabromobiphenyl suggest that under conditions of adequate feed and good health, the 7 concentration of this congener in rat adipose tissue would not be expected to decline appreciably during the animal's lifetime (Matthews gt 31., 1977). Lactating females may eliminate PBBs more rapidly than males or nonlactating females, since lipophilic compounds such as PBBs are eliminated in the lipid portion of milk. Cows consuming PBBs had milk concentrations of PBBs that plateaued at approximately 4 times the dietary concentration (Fries, 1978; Fries gt_al,, 1978). Transfer of PBB congeners from diet to milk is dependent on degree of bromina— tion (Fries and Marrow, 1975). Hexabromobiphenyls have been trans— ferred 5 to 6 times more efficiently than 2,3,4,5,2',4',5'-heptabromo- biphenyl, in cows, suggesting that there is a greater resistance to movement of more brominated congeners across membranes (Fries and Marrow, 1975; Fries, 1978). Concentrations of PBBs (2,4,5,2',4',5'- hexabromobiphenyl) were lower in body fat than milk fat while cows were being fed PBBs (Fries, 1978; Fries gt_§l,, 1978). Following termination of PBB exposure in cows, the relationship between concen- tration of PBBs in milk and body fat approached a 0.4 to 1.0 ratio (Fries, 1978; Fries 35 31., 1978). Of women selected at random from Michigan's lower peninsula, 96% had PBBs in their milk (Eyster, 1976). Human breast milk contains relatively high concentrations of PBBs, which ranged from 0.21 to 92.66 ppm in milk from mothers on quarantined farms in 1974 and 1975 (Cordle gt 31., 19778 Brilliant gt 31., 1978). The concentration of PBBs in human milk ranged from 70 to 131 times the concentration detected in paired blood (plasma) with an average ratio of 100 to 1 (Cordle £5 31., 1978). The concentration of PBBs in blood (plasma) of 8 individuals on quarantined farms ranged from 0.002 to 2.26 ppm in 1974 (Cordle gt 31., 1978). Firemaster handlers employed at the Michigan Chemical Corporation had plasma concentrations of PBBs that ranged from 0.006 to 0.085 ppm (Michigan Chemical Corporation, 1975). The concentration of PBBs in human fat ranged from 61 to 370 times the concentration detected in plasma with ratios of fat to plasma around 175 (Cordle gt_§1,, 1978). These findings suggest that the lipo— soluble PBBs are biomagnified within individuals and are probably biomagnified from species to species while ascending in the food chain. C. Biological Effects of Polybrominated Biphenyls Although at present no human health effects have been unequivo- cally attributed to PBBs, dietary exposure to PBBs has resulted in a variety of toxic manifestations in animals (Kimbrough g£_al,, 1978). Cows fed high concentrations of PBBs lost body weight as a result of aversion to food containing PBBs as well as developing diarrhea (Jackson and Halbert, 1974; Mercer gt él-9 1976). Body weight gain was reduced in rats and monkeys fed lower doses of PBBs (Sleight and Sanger, 1976; Garthoff g£_alf, 1977; Allen gt a1., 1978). However, this effect resulted, at least in part, from decreased food efficiency since weight differences were not attributable to differences in food consumption alone. General weakness, slow wound healing, thymus atrophy, abnormal hoof growth, hematomas and abscesses in peritoneal and thoracic cavities and dermal lesions including subcutaneous hemorrhage, hyperkeratosis with accumulation of keratin in hair follicles of the epidermis and squamous metaplasia with keratin cysts 9 in eyelid tarsal glands have been observed in cows following PBBs (Jackson and Halbert, 1974; Mercer 35 a1., 1976; Moorhead 35 a1,, 1978). Dermal lesions similar to those in cattle have been reported in rhesus monkeys fed PBBs (Lambrecht £5 31., 1978; Allen gt a1,, 1978). Hyperkeratosis, keratotic hair follicles and atrophy and squamous metaplasia of the sebaceous glands were observed on micro- scopic examination of monkey skin (Allen gt a1., 1978). Modifications in erythroid and lymphoid tissues have been pro- duced by dietary exposure to PBBs. Rhesus monkeys fed PBBs had reduced packed cell volume which was associated with bone marrow hypoactivity (Lambrecht gt a1., 1978; Allen gt_§1,, 1978). Leukopenia and immunosuppression have been produced in monkeys, rats, mice and guinea pigs by PBBs (Kimbrough g£_§1,, 1978; Allen gt a1., 1978; Lambrecht g£_§1,, 1978; Fraker and Aust, 1979). Rhesus monkeys fed PBBs had decreased immunoglobulins and altered T-cell function (Lam- brecht g£_§1,, 1978; Allen gt 31., 1978). Exposure to PBBs reduced the i§_yiyg immunoglobulin response and had an adverse effect on B- cells and helper T-cells in mice (Fraker and Aust, 1979). Endocrine alterations also may be caused by PBBs. Rhesus monkeys had lengthened menstrual cycles after PBBs and this effect was corre- lated with flattened and lengthened serum progesterone peaks (Lam- brecht et_§1., 1978; Allen §£_§1,, 1978). In addition to decreased concentrations of serum progesterone, rhesus monkeys fed PBBs had excessive postconceptional bleeding (Allen gt al., 1978). Rhesus monkeys fed PBBs had hypoactive seminiferous tubules (Allen gt_al,, 1978). Bulls fed PBBs had testicular atrophy and reduced spermato- genesis (Jackson and Halbert, 1974; Kimbrough gt 31., 1978). Prenatal 10 administration of PBBs to rats caused a delay in vaginal opening (Harris §£_a1,, 1978). Milk production was decreased in lactating cows which also may have been more susceptible to stress following PBBs (Jackson and Halbert, 1974). Pregnant cows, that did not abort, went overdue 2-4 weeks and their udders did not develop (Jackson and Halbert, 1974; Mercer gt al., 1976). Although PBBs undergo transpla— cental movement and may be lethal to embryos and/or fetuses, their teratogenic potential is low, at least in rodents (Corbett g£_al,, 1975; Preache et a1., 1976; Kimbrough gt_§l., 1978; Rickert gt_§l,, 1978). Kidneys from cows fed diet containing PBBs have been reported to be pale tan-to-gray and almost twice normal size (Jackson and Halbert, 1974; Willett and Irving, 1976). Microscopically, bovine kidneys had collecting ducts that were extremely dilated, convoluted tubules with epithelial degenerative changes such as cloudy swelling, hydropic degeneration and separation from the basement membrane (Moorhead gt 31,, 1977,1978). Kidney enlargement and renal lesions including petechial hemorrhage and hyaline degenerative cyt0p1asmic changes have been observed in rats following treatment with octabromobiphenyl (Aftmosis §£_§1,, 1972; Norris gt 31., 1974). Suggestive of an altera— tion in renal function were findings of polyuria, low urine specific gravity and moderately elevated urinary protein concentration in both cows and rats following PBBs (Sleight and Sanger, 1976; Mercer g£_§l,, 1976). Total protein, albumin and cholesterol were reduced in serum from monkeys after consumption of diet containing PBBs (Allen gt_al., 1978). Cows with low tissue concentrations of PBBs had significantly decreased concentrations of serum calcium, glucose and cholesterol and ll urinary potassium and increased excretion of calcium and protein (Willett and Irving, 1976; Mercer 35 31., 1976,1978). Changes in serum and urinary calcium concentrations may result from renal lesions and/or effects on several hormones including parathyroid hormone and adrenal g1ucocorticoids. Alterations in renal function and/or calcium homeostasis may be responsible, at least in part, for depressed heart and respiratory rates produced in cows fed PBBs (Jackson and Halbert, 1974; Mercer gt a1., 1976). Heart enlargement has been produced in rhesus monkeys fed PBBs (Allen gt al., 1978). Dietary treatment of white leghorn cockerals has resulted in a variety of cardiovascular effects including hydro- pericardium and alterations in cardiac output, heart rate and arterial pressure (diastolic, systolic and mean) (Heineman and Ringer, 1976). Ingestion of PBBs by cows also resulted in hepatic degenerative changes. Abscesses, foci of fatty degeneration and glycogen depletion were seen in liver and serum glutamic oxaloacetic transaminase (SGOT) was increased following PBBs (Jackson and Halbert, 1974; Moorhead gt El}, 1978; Durst 35 El-’ 1978). Livers from rhesus monkeys fed PBBs had hyperplasia of bile duct epithelium and enlarged hepatocytes, the cytoplasm of which contained markedly proliferated smooth endoplasmic reticulum (Allen gt a1., 1978). ExPosure of rhesus monkeys to PBBs also resulted in increased serum glutamic pyruvic transaminase (SGPT) activity (Allen gt al., 1978). Livers from rats consuming PBBs were enlarged and had lesions consisting of swelling, centrilobular cyto— plasmic myelin bodies and vacuolation (Norris £5 31., 1974; Sleight and Sanger, 1976; Dent 95 31., 1976b). Correlated with hepatocyte hypertrophy were increased liver lipid (total lipid, cholesterol, 12 phospholipid and neutral lipid) and massive proliferation of the smooth endoplasmic reticulum (Sleight and Sanger, 1976; Garthoff 35 a1,, 1977). Elimination of certain drugs via liver was stimulated in rats by PBBs in an age-dependent manner (Cagen and Gibson, 1977; Cagen and Gibson, 1978). Microsomal mixed function oxidase (MFO) activity has been in- creased in liver by PBBs (Farber and Baker, 1974; Dent gt 31., 1976a,b). PBBs belong to a class of compounds termed "mixed inducers"; that is, PBBs exhibited stimulating characteristics of both phenobar— bital and 3-methy1cholanthrene (3MC), two agents which induce distinct types of hepatic microsomal enzymes (Sladek and Mannering, 1969a,b). Although mixed stimulation following PBBs may result from combined effects of individual mixture congeners, which are either phenobarbi— tal or 3MC-like stimulators, one or more congeners, such as 2,4,5,3', 4',5'-hexabromobipheny1 could produce both types of stimulation (Dannan gt §l°’ 1978). Following a single i.p. injection of Fire- master BP-6, the pattern of hepatic microsomal enzyme stimulation in adult rats initially resembled phenobarbital, however, later the effect more closely resembled that of 3MC (Dent gt 31., 1976a). Consequently, animals pretreated with a single i.p. injection of PBBs displayed a time-dependent change in susceptibility to bromobenzene (which is metabolized by hepatic MFOs to toxic epoxides) toxicity that coincided with the pattern and time course of stimulation of hepatic MFO activities (Dent gt_a1,, 1977; Roes et 31., 1977; Zampaglione gt 31., 1973). These findings suggest that interactions with endogenous and xenobiotic compounds may be dependent upon time following exposure to PBBs. 13 D. Biological Effects of Agents that Increase Microsomal Enzyme Activity Numerous drugs and environmental contaminants stimulate or inhibit microsomal enzyme function in animals, and this is reflected 12.2122. by modified metabolism and action of therapeutic agents, carcinogens, and a variety of endogenous compounds, such as steroid hormones, vitamins, fatty acids, thyroxin, and bilirubin (Conney, 1967). Since PBBs are environmental contaminants that increase microsomal enzyme activity, they may have pharmacological and toxicological actions resembling those of other agents that stimulate mixed function oxi- dases. Phenobarbital, administered to rats for several days, increased the activity of hepatic microsomal enzymes that metabolize certain drugs. The anticoagulants bishydroxycoumarin and warfarin are examples of therapeutic agents which are metabolized by MFOs (Cucinell gt a1., 1965; Ikeda 22 a1., 1966). Administration of phenobarbital to man reduced the concentration of bishydroxycoumarin and warfarin in plasma and reduced their pharmacological action (Cucinell gt 31., 1965; MacDonald g£_§1,, 1969). Treatment of rats with phenobarbital for several days increased the activity of hepatic microsomal enzymes that hydroxylate androgens, estrogens, progestational steroids, and adrenocortical steroids (Kuntzman g£_§1,, 1964; Conney and Klutch, 1963; Conney £5 31., 1976; Levin g£_§1,, 1968). Accelerated hydroxylation of steroid hormones by hepatic microsomal enzymes of rats treated with phenobarbital was reflected in ziyg_by an altered metabolism and modified physiological action of steroids. Increased progesterone hydroxylase activity l4 induced by phenobarbital was associated with a decrease in the anes- thetic action of large doses of progesterone and a decreased concen- tration of progesterone and its metabolites in the brain and total body of rats (Conney gg‘al., 1966). Prolonged administration of phenobarbital also decreased the anesthetic action of deoxycortico— sterone, androsterone, and A4-androstene-3,l7-dione and accelerated their metabolism by hepatic microsomes. Pretreatment of immature rats with phenobarbital for several days prior to administration of testo- sterone or testosterone propionate inhibited the growth-stimulating effect of these androgens on the seminal vesicles (Levin et al., 1969). Estrone and 178-estradiol metabolism was also increased by phenobarbital (Kuntzman 32 a1., 1964; Welch gt 31., 1971). Stimulated catabolism of these estrogens as well as commonly used oral contra- ceptives was associated with a reduced ability of these compounds to increase uterine wet weight (Levin gt 31., 1968). Patients treated chronically with phenobarbital for anticonvul- sant therapy have an increased incidence of rickets, osteomalacic bone changes, hypocalcemia and elevated serum concentrations of alkaline phosphatase (Kruse, 1968; Dent gt 31., 1970). These symptoms are similar to those resulting from vitamin D deficiency and respond rapidly to vitamin D supplementation (Dent 35 al., 1970). Further suggesting that increased MFO activity is correlated with accelerated catabolism of vitamin D are findings that rats pretreated with phenobarbital were protected from hypercalcemia and renal calcinosis produced by calciferol and that patients chronically administered phenobarbital rapidly converted injected vitamin D -3H to more polar 3 15 metabolites, some of which are biologically inactive (Richens and Rowe, 1970; Hahn 25 a1., 1972). Calcium homeostasis may also be disrupted by other mechanisms following phenobarbital. In_3132, phenobarbital had an inhibitory effect on vitamin D-25—hydroxylase kinetics and stimulated bile excre— tion resulting in reduced serum concentrations of 25-0H-vitamin D (Delvin 35 31., 1977). Inhibition of the energy-dependent calcium transport system in the intestinal mucosa and inhibition of calcium- binding protein synthesis are two additional postulated effects of phenobarbital on calcium homeostasis that may contribute to osteo- malacia (Harrison and Harrison, 1976). Metabolism of vitamin K may also be altered by treatment with phenobarbital. Hemorrhagic episodes occur in some babies born to mothers taking phenobarbital for epilepsy (Mountain e£_§1,, 1970). The coagulation defect mimics that found in vitamin K deficiency and has been prevented by administration of vitamin K, suggesting that phenobarbital stimulates metabolism of vitamin K. Administration of phenobarbital to animals enhanced the enzymatic g1ucuronidation of bilirubin by liver microsomes, stimulated bile flow and accelerated metabolism of bilirubin in yiyg_(Roberts §t_§1,, 1967). Investigations in man indicate that chronic treatment with phenobarbital results in a decreased serum bilirubin concentration in patients with intrahepatic cholestasis and in jaundiced infants (Thompson and Williams, 1967; Crigler and Gold, 1969; Arias gt_al,, 1969). 16 The mechanism by which phenobarbital and a host of other chemi- cals stimulate the synthesis of MFOs is different from the mechanism whereby polycyclic aromatic hydrocarbons produce their inductive effects (Sladek and Mannering, 1969a,b). Early evidence for this difference was the observation that agents such as phenobarbital induced the increased metabolism of endogenous compounds as well as a much larger number of drugs and other xenobiotics than did polycyclic aromatic hydrocarbons such as 3-methy1cholanthrene (3MC) or benzo(a)- pyrene (BP). Administration of phenobarbital resulted in parallel increases in rates of metabolism of certain drugs and hepatic cyto- chrome P450 concentrations and withdrawal of phenobarbital resulted in parallel decreases to basal values suggesting that cytochrome P450 is the rate-limiting component of the microsomal drug metabolizing system (LaDu e£_§1,, 1971). The observation that polycyclic aromatic hydro- carbons increased hepatic cytochrome P450 concentrations with a differ— ential effect on two hepatic microsomal enzymes suggested that P450 was either not rate—limiting or polycyclic aromatic hydrocarbons cause the synthesis of a unique P450 hemoprotein (Sladek and Manner- ing, 1969a,b). A number of observations led to the latter conclusion and the 3MC- or BP-sensitive hemoprotein was termed cytochrome P —450 l or cytochrome P448 (Sladek and Mannering, 1969a,b). Induction of cytochrome P1—450 by polycyclic aromatic hydrocar— bons such as BP, which is found in tobacco smoke and polluted atmos- phere, parallels the induction of an enzyme system in the microsomal fraction of liver and several extrahepatic tissues including kidney, lung, intestine, skin, placenta and mammary gland (Wattenberg §£_al,, 17 1962; Welch §£_§1,, 1968; Nebert and Gelboin, 1969). Various chemi- cals including nicotine, zoxazolamine, phenacetin and polycyclic aromatic hydrocarbons are metabolized into alkene and arene oxides in reactions catalyzed by this NADPH-dependent enzyme complex, activity of which is reflected by activity of the enzyme BP hydroxylase (aryl- hydrocarbon hydroxylase - AHH)(Beckett and Triggs, 1967; Welch g£_§1,, 1969; Pantuck gt 31., 1972). This process, and particularly subse- quent metabolism by the microsomal enzyme epoxide hydratase (EH) and/or conjugation with glutathione by a family of cytosolic trans- ferases can result in detoxification (Jerina and Daly, 1974). How— ever, certain intermediary metabolites (epoxides) produced by AHH are more electrophilic than the parent compound and react readily with critical cellular nucleophiles including DNA, RNA and protein to produce toxic responses (Miller, 1970; Daly g£_§l., 1972; Oesch, 1973; Jerina and Daly, 1974). Although EH often results in detoxification of epoxides such as BP 4,5-oxide, it is also capable of catalyzing the transformation of certain arene oxides, such as BP 7,8—oxide, to precursors, such as BP-7,8-dihydrodiol, of the ultimate carcinogenic and mutagenic forms of parent compounds, such as the isomeric BP 7,8- dihydrodiol-9,10-epoxides of BP (Sims gt 31,, 1974; Wood g£_§l., 1976). Thus, EH plays a dual role in the metabolic activation and inactivation of certain polycyclic aromatic hydrocarbons into muta— genic and carcinogenic metabolites. The toxicity and/or carcinogeni- city of a compound depends on the region in which the molecule is oxidized, which is dependent on the positional Specificities of differ- ent forms of cytochrome P-450 and on MFO activity (Wiebel §t_§1,, 1975). 18 Since metabolites which are ultimate carcinogens or toxicants are highly reactive and unstable, they may not be transported from liver to extrahepatic tissues. Thus, if metabolic activation to an ultimate carcinogen or toxicant is an essential intermediate step in tissue specific toxicity it may occur in target tissues. Tissue differences in metabolism, whether quantitative or qualitative, may be responsible, at least in part, for site specific deleterious effects. The steady— state concentration of an epoxide metabolite within cells of an organ depends, at least in part, on its rates of synthesis and further metabolism. Consequently, the rates of epoxide forming and detoxi- fying enzyme activities in various tissues or cells and the sensi- tivity of cells or tissues to such toxic metabolite(s) may be impor- tant determinants of tissue-specific toxicity, including the initial step in chemical mutagenesis or carcinogenesis. Different metabolites of the potential carcinogens 7,12-dimethylbenz(a)anthracene (7,12- DMBA) and N2-fluorenylacetamide (2-FAA) were produced by microsomes from mammary than from liver and treatment with 3MC produced a shift in 7,12-DMBA metabolism from side chain to ring hydroxylation in liver (Tamulski_ggflal., 1973; Malejka-Giganti £5 31., 1977). However, no such alteration occured in mammary tissue. These findings are suggestive of tissue specific qualitative differences in metabolism. Tissue differences in metabolism may also vary with age. Several studies have revealed that immature animals lack, or possess low acti- vities of many hepatic microsomal enzymes (Pants and Devereux, 1972). Perinatal development of enzymes involved in epoxide metabolism in extrahepatic tissues has not yet been fully characterized despite the 19 potential importance of interactions between chemical carcinogens, (e.g., BP) and extrahepatic tissues. Polychlorinated biphenyls (PCBs) are widespread commercial pollutants that persist in the food chain and have been detected in tissues and milk from many species including man (Risebrough gt_§1,, 1968; Price and Welch, 1972; Hamano 25.31., 1974). Since PBBs are structurally analogous to PCBs and similar in chemical and biological stability they may share many biological and toxicological prOperties (Jacobs 95 a1., 1976; Lee 95 a1., 1977; Rickert gt a1., 1978). Like PBBs, PCBs have increased the liver weight-to—body weight ratio, produced histological changes in liver including fatty infiltration, formation of myelin bodies and proliferation of smooth endoplasmic reticulum and stimulated hepatic microsomal enzymes sensitive to both phenobarbital and BP (Nishizumi, 1970; Kimbrough gt a1., 1972; Norback and Allen, 1972; Alvares £5 31., 1973). On a weight basis, PBBs (Firemaster BP-6 or hexabromobiphenyl) may be three times more potent than PCBs (Aroclor 1254 - 54% chlorine) at increasing hepatic MFO activity in male rats (Farber and Baker, 1974; Garthoff £3 31., 1977). Hepatic microsomal enzyme activity has been increased for at least a m0nth following a single i.p. injection of PCBs (Parkki gt al., 1977). Induction patterns of MFOs in liver exhibit time—dependent profiles after PCBs (Bickers g£_al,, 1974; Parkki 35 a1., 1977). Microsomal enzymes have also been stimulated in extrahepatic tissues such as kidney, lung, skin and placenta following exposure to PCBs (Vainio, 1974; Bickers g£_§l,, 1974). Administration of PCBs to pregnant rats has also resulted in detectable fetal concentrations of PCBs and 20 stimulation of fetal hepatic microsomal enzymes (Hamano et_§l,, 1974; Takagi g£_al,, 1976; Alvares and Kappas, 1975). Rats treated with PCBs had a marked reduction in pentobarbital sleeping time, an indirect measure of pentobarbital metabolism iE.X£!2 (Villeneuve §£_§1,, 1972). The insecticides dieldrin and DDT were more toxic when administered with PCBs (Lichenstein gt_al,, 1969). Pretreatment with PCBs also potentiated the acute toxicity of carbon tetrachloride (Grant gt 31., 1971; Carlson, 1975). In addition to modifying biological responses to certain xenobiotics metabolized by the MFO system, PCBs may alter the metabolism of endogenous compounds such as fat-soluble vitamins, fatty acids and steroid hormones. Vitamin A functions in maintaining reproduction, growth and development (Mason, 1939; Thompson, 1969). Decreased growth rate is one of the earliest and most sensitive indices of vitamin A deficiency (Corey and Hayes, 1972). Insufficient vitamin A can also result in xerophthalmia, night blindness, disturbances in the central nervous system (CNS), respiratory difficulties, skin lesions including hyper- pigmentosis and hyperkeratosis and renal lesions including epithelial vacuolization, hyperkeratization and distention of tubules as well as albuminuria, polyuria and decreased urine osmolality which is poten- tiated in rats by cold stress (Herrin and Nicholes, 1930; Herrin, 1939; WOlbach, 1954; Odagiri and Koyamagi, 1961; Arvy, 1968; Webb 25 a1,, 1968,1970). A variety of clinical symptoms exhibited by patients with Yusho disease were similar to those symptoms noted in vitamin A deficiency. Yusho disease was diagnosed in approximately 1400 Japanese in 1968 and caused by consumption of rice oil contaminated with Kanechlor 400, a 21 commercial mixture of PCBs mainly composed of tetrachlorobiphenyls but also containing numerous chlorinated trace impurities including dibenzo- furans (Kuratsune et 31,, 1972; Nagayama e£_a1., 1975). These indi— viduals had an increased incidence of CNS disturbances, such as short- term memory loss, behavioral changes, and numbness, and respiratory problems such as bronchitis, dyspnea and cough (Kuratsune g£_§1., 1972; Umeda, 1972). More prominent symptoms suggestive of vitamin A deficiency were hyperpigmentation of the skin, mucus membranes and nails, acne-like skin eruptions with follicular accentuation and transient visual disturbances (Kuratsune g£_§1,, 1972; Umeda, 1972; Kimbrough, 1974). Dermal application of PCBs to rabbit ear resulted in hyperplasia and hyperkeratosis of the epidermal and follicular epithelium (Vos and Beems, 1971). Dietary treatment with PCBs reduced the concentration of vitamin A in liver of rats and pregnant rabbits (Cecil g£_al,, 1973; Innami e£_al,, 1976; Villeneuve £5 31., 1971). Body weight gain was retarded in these animals and P083 were reported to cause renal lesions in rats including hydropic degeneration of the convoluted tubules and tubular dilation which are similar to micro- scopic changes observed in kidneys of vitamin A deficient rats (Vos and Beems, 1971; Bruckner 25 31., 1973, 1974). Calcium homeostasis may be altered following PCBs secondary to modification of vitamin D metabolism. Vitamin D mediated calcium metabolism has been reported to be altered in chickens after treatment with P083 (Wong £5 31., 1974). Yusho patients also had symptoms suggestive of an alteration in calcium homeostasis such as an in- creased incidence of dental disorders including exfoliation and 22 fracture of teeth, abnormal eruption of decidious and permanent teeth and abnormal formation and growth of erupted teeth and facial bones (Kuratsune g£_§1., 1972; Umeda, 1972). Vitamin E acts as an antioxidant possibly to prevent oxidation of essential cellular constituents (Wasserman and Taylor, 1972). Chickens fed PCBs had an increased incidence of exudative diathesis which could be reduced by dietary supplementation with vitamin E or selenium (Combs e£_§1,, 1975). Thus, the metabolism of this fat soluble vitamin may also be altered following exposure to PCBs. The porphyrin nucleus is an integral component of hemoglobin and cytochromes. Cytochromes P450 and P448 were increased in liver and hemoglobin, hematocrit and spleen size were reduced following PCBs suggesting that porphyrin metabolism is altered by PCBs (Alvares §£_ .al., 1972; V03 and Beems, 1971; Bruckner gt_§1,, 1974). Consistent with these findings, PCBs have been reported to increase fluorescence of tissues including liver, kidney and bone marrow (Vos gt El-’ 1970, 1971). Excretion of a porphyrin fraction in feces and hepatic iron content were also increased and P033 produced hepatic porphyria (Vos g£_§1,, 1970, 1971; V03 and Notenboom—Ram, 1972; Kimbrough g£_a1,, 1972). Hepatic porphyria in rats following PCBs resembles human porphyria cutanea tarda, a condition resulting from an acquired defect in hepatic porphyrin metabolism, symptoms of which include uropor- phyrinuria, photosensitivity and mechanical fragility of skin (Kim- brough, 1974). Porphyria produced by PCBs is characterized by slow onset, increased excretion of uroporphyrins (URO), coprOporphyrin (COPRO), delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) in urine, accumulation of 8- and 7-carboxyporphyrins in liver and 23 induction of hepatic drug metabolizing enzymes and ALA synthetase, the rate-limiting enzyme in heme synthesis (Goldstein £5 31., 1974; Granick, 1966). Porphyria produced by many hepatOporphyrinogenic chemicals is due primarily to increased activity of ALA synthetase followed by overproduction of porphyrins. However, PCBs may act differently because induction of ALA synthetase occurs rapidly whereas porphyria has a delayed onset (Goldstein et 31-: 1974). Many chemicals that produce hepatic porphyria also induce AHH and other microsomal monoxoygenases as well as cytochrome P448 and the cytosolic enzyme glutathione S—epoxide transferase. Coordinate induction of AHH and cytochrome P448 and possibly these other enzymes by xenobiotics may be a result of binding to a receptor (Poland and Glover, 1974). An induction receptor theory was initially based on observations regarding effects of 2,3,7,8-tetrachlorodibenzo—p-dioxin (TCDD), the most potent inducer of AHH and cytochrome P448 known, and 3MC on induction of AHH (Poland and Glover, 1974). TCDD and 3MC produced parallel log dose-response curves for induction of hepatic AHH in the rat, both agents produced the same maximal response and concomitant administration of maximally inducing doses of both com- pounds resulted in no additive effect. Induction of AHH by TCDD has been correlated with the binding of 3H-TCDD to a high-affinity low capacity stereospecific cytosol receptor protein (i.e., TCDD-receptor) (Poland 35 31., 1976). Structurally related polycyclic aromatic hydrocarbons such as 3MC and halogenated aromatic compounds such as PCB congeners chlorinated symmetrically in both the meta and para positions, that also induce AHH, compete for the binding of 3H-TCDD to 24 the receptor (Poland and Glover, 1974, 1977; Poland gt_al,, 1976; Goldstein §£_§1,, 1977). In addition to being porphyrinogenic, many compounds that bind to the TCDD receptor are acnogenic and immuno- suppressant (Poland et 31., 1976). Phenobarbital, pregnenolone-l6o-carbonitrile and other PCB con- geners, which induce cytochrome P450 and are less efficient inducers of AHH, do not compete with TCDD for binding to a receptor (Poland 33 .31., 1976; Poland and Glover, 1977). By analogy, there may be a similar recognition site for phenobarbital and phenobarbital—like compounds which controls the induction of cytochrome P—450 and its associated enzymes (Poland e£_al., 1976). As previously noted, phenobarbital has produced a variety of biological effects including accelerated steroid metabolism. Metabolism of steroid hormones by hepatic microsomal enzymes was increased i§_yi££g_by pretreatment with PCBs (Nowicki and Norman, 1972). Pretreatment of rodents with PCBs accelerated the catabolism of exogenously administered testosterone, estradiol and progesterone (Orberg and Lundberg, 1974; Orberg and Ingvast, 1977; Orberg and Kihlstrom, 1973; Derr, 1978). These findings suggest that increased microsomal enzyme activity following PCBs is correlated with enhanced metabolism of steroid hormones. Physiological functions controlled by steroid hormones such as reproduction would be expected to be affected least by lowly chlorinated PCBs because of their relative low effi- ciency at increasing mixed function oxidase activity (Bickers g£_a1,, 1972; Orberg, 1976). However, certain PCBs with low chlorine content have estrogenic activity which is reflected as an increase in uterine 25 glycogen content and inhibition of binding of tritiated estradiol to rat uterus cytosol in yitrg_(Nelson, 1974; Bitman and Cecil, 1970; Ecobichon and MacKenzie, 1974). Following administration to rat neonates, an estrogenic mixture of PCBs produced precocious puberty as well as persistent vaginal estrus and anovulation by six months of age (Gellert, 1978). When estrogenic PCB congeners are components of complex PCB mixtures which contain other PCBs and in some cases toxic contaminants such as dioxins and dibenzofurans, direct effects may be obscured by factors including indirect actions resulting from enhanced steroid metabolism. A variety of effects observed after administration of mixtures of PCBs suggest that these compounds may modify endogenous steroid meta— bolism to the extent that physiological sequelae are noted. Adrenal weights and plasma corticosterone concentrations have been increased in mice, rats and rhesus monkeys by PCBs (Wasserman et 31., 1973; Sanders e£_§1,, 1974; Barsotti and Allen, 1975). Urinary androgen in boars and seminal vesicle weight and spermatogenesis in mice have been reduced by pretreatment with PCBs (Platonow gt 31., 1972; Sanders g£_ a1,, 1974; sanders and Kirkpatrick, 1975). Rhesus monkeys consuming diet containing PCBs had lengthened menstrual cycles (Allen and Barsotti, 1976). Women with Yusho disease also had menstrual cycle irregularities, dysmenorrhea and altered serum concentrations of ketosteroids (Kuratsune gt a1., 1972). Various mixtures of PCBs caused uterine atrophy, reductions of plasma progesterone concentra- tion, ovarian stromal changes, and lengthened estrus cycle in rodents 26 (V08 and Beems, 1971; Johnsson_§§”§1., 1976; Orberg and Kihlstrom, 1973; Kimbrough et 31., 1978). Endocrine changes in mammalian females, especially primates, exposed to PCBs suggest that the uterine hormonal environment may be suboptimum for blastocyst implantation and maintenance of pregnancy (Smith and Biggers, 1968; McCormack and Greenwald, 1974; Orberg, 1978). Ova implantation frequency has been reduced in mice treated with PCBs (Orberg and Kihlstrom, 1973; Kihlstrom_g£.a1., 1975; Orberg, 1978). Dose and mixture-dependent reductions in conception rate occurred in monkeys maintained on diet containing PCBs (Allen, 1975; Allen and Barsotti, 1976). Exposure to PCBs reduced the number of rats producing litters after mating (Linder §£_al,, 1974; Johnsson gt a1,, 1976; Keplinger er a1., 1972). Implantation rate reduction in mice may not occur if only one mate is treated with PCBs (Kihlstrom gt a1,, 1975). Fetal tissues from numerous species including monkey, rat and human contain PCBs, indicating that PCBs readily cross the placenta and accumulate in the fetus (Allen and Barsotti, 1976; Takagi e; 31., 1976; Kuratsune £5 31., 1972; Umeda 35 31., 1978). Although the teratogenic potential of PCBs is low, resorption and abortion fre- quencies are increased in rabbits, mink and rhesus monkeys fed PCBs (Villeneuve £2 31., 1971; Ringer £5 31., 1972; Allen and Barsotti, 1976). Of those women with Yusho who gave birth, 2 of 13 had still- born infants (Kuratsune gt al., 1972). Infants born to Yusho patients exhibited skin, liver and growth disorders including low birth weights (Kuratsune §£_a1,, 1972; Umeda £5 31-: 1978). Infant monkeys also had decreased birth weights and focal areas of hyperpigmentation of the 27 skin (Allen and Barsotti, 1976; Allen 3£_31,, 1978). Rabbits trans- placentally exposed to PCBs had reduced thymus size and white cell count, which are associated with a reduced capacity to produce anti- bodies and general immunosuppression (Vos and Bemms, 1971; Miller, 1963). A factor in addition to immunosuppression and vitamin A defi- ciency which could contribute to reduced birth weights and body weight gain following PCBs may be altered energy metabolism. Inhibition of oxidative phosphorylation and respiration have been associated with addition of PCBs 1B.y1££g_to either rat liver mitochondria or heavy beef heart mitochondria (Chesney and Allen, 1974; Pardini, 1971). Elevated concentrations of PCBs in tissues from stillborn babies born to Yusho mothers have been detected even 10 years after the rice- oil incident (Umeda 33 31., 1978). Extrapolation of data obtained in rats indicated that less than 20% of administered hexachlorobiphenyl would ever be excreted (Matthews and Anderson, 1975). Persistence of PCBs in biological tissue is further exemplified by a report that one year after initial diagnosis in 159 patients, 40% showed no remission of symptoms while 10% appeared more severely affected (Kuratsune 33 31,, 1972). Even 10 years later, symptoms of Yusho were observed and PCBs, especially highly chlorinated congeners, detected in tissues of patients diagnosed as having Yusho disease (Umeda 33 31,, 1978). Body burdens of PCBs may be reduced in lactating females as PCBs are mobilized with body fat during lactation and excreted with milk lipids (Hamano 31 313, 1974; Takagi_3£131,, 1976). Signs of PCBs intoxication quickly became more severe in monkeys and rats suckled by mothers maintained on diet containing PCBs (Allen and Barsotti, 1976; Takagi 35 31., 1976). Survival-to-weaning has been reduced and 28 liver weight-to-body weight ratio increased in F rat pups following 2 dietary exposure of both F and F generations to PCBs (Linder 33_31., 0 1 1974). Thus, PCBs, like PBBs, may be passed from one generation to the next via transplacental movement and especially through milk and may produce effects in each generation. E. Objectives The purpose of this investigation was to determine if functional and metabolic alterations are produced by prenatal and/or postnatal treatment with PBBs. Identification of physiological and biochemical sequelae to perinatal exposure to PBBs will assist assessment of the potential health hazard posed by PBBs to developing mammals. Three specific objectives of this research were identified: 1) to determine effects of perinatal exposure to PBBs on survival, growth and develop— ment, organ function, morphology and microsomal enzyme activity; 2) to determine if enzymatic alterations produced by PBBs result in modifi- cations in metabolism of xenobiotic and endogenous compounds; 3) to determine the persistence of effects of PBBs and whether or not effects of PBBs are correlated with tissue concentrations of PBBs. MATERIALS AND METHODS A. Animals Sprague-Dawley rats were either purchased directly or descendants of rats from Spartan Research Animals, Inc., Haslett, Michigan. Adults were female unless otherwise specified. Timed—pregnant rats were obtained between days 1 and 5 of pregnancy or impregnated by males of the same age and treatment from our breeding stock. At birth all litters were normalized to 10 pups; 5 males and 5 females. Pups were weaned at 28 days of age. Rats were not used until after at least 2 days of acclimatization. Animals were maintained in clear polyprOpylene cages at 22°C with a 12 hr light cycle (0700-1900 hr) and were allowed free access to food (wayne Lab Blox, Anderson Mills, Maumee, Ohio) and water. Rats in dietary studies received ground Lab Blox; all others received pelleted chow. Ground diet was prepared by dissolving appropriate quantities of PBBs (Firemaster BP-6, Velsicol Chemical Co., St. Louis, Michigan) in acetone and mixing this solution (20 ml) with 2 kg of ground Lab Blox for approximately 10 min. Controls were fed ground diet with which only acetone had been mixed and allowed to evaporate. B. Teratology Pregnant rats were placed on diet containing 0 or 100 ppm PBBs (approximately 7.0 mg/kg/day) on day 8 of gestation and deprived of 29 30 food for 0 or 48 hr beginning the 10th day of gestation. 0n the 20th day of gestation, fetuses were removed by caesarean section and the number and position of live, dead and resorbed fetuses was recorded. Fetuses were dried on absorbant paper, weighed, measured for crown- rump length with a vernier caliper, sex determined and examined for external anomalies. Each litter was divided into 3 subgroups for further examination and 1 subgroup for determination of whole carcass concentration of PBBs. One was fixed in Bouin's solution for 2 weeks, after which fetuses were sectioned by hand into 2-3 mm sections and examined for soft-tissue anomalies by the method of Wilson (1965). A second subgroup was fixed in 95% ethanol, skinned and eviscerated. Fetal cartilage and ossified skeletons were subsequently stained with alcian blue 8G8 and alizarin red 8, respectively, by the method of Inouye (1976). Stained skeletons were examined for cartilaginous and skeletal anomalies. Organ weight-to-body weight ratios were deter- mined in the third-subgroup following decapitation. C. Postnatal Development Pups were randomly cross-fostered within treatment groups and litters were normalized to 5 males and 5 females on day 1 postpartum. Control values were obtained by pooling data from animals treated with peanut oil alone or suckled by dams maintained on diet containing 0 ppm PBBs. Rats were weighed at 7, 14, 28 (whole litter) 56 and 84 (individual female) days of age. Daily observations were made to determine the age of pinna detachment, incisor eruption, fur develop- ment, Opening of external auditory duct, eye opening, testes descent and vaginal opening. 31 D. Vaginal Cycle Lenth Vaginal cycle length was monitored in 10-14 week old rats that had been exposed to 0 or 100 ppm PBBs from day 8 of gestation. Daily vaginal smears were obtained for at least 14 consecutive days. The interval in days between 2 successive peaks in the frequency of cornified cells was taken as the length of each individual vaginal (estrus) cycle. E. Histopathology Animals used for histologic examination were killed by cervical dislocation and pieces of tissue were immediately cut and fixed in 10% buffered formalin. After fixation, tissues were embedded in paraffin, sectioned at 5 uM and stained with hematoxylin and eosin. F. Milk Collection To obtain milk, animals were anesthetized with ether and treated i.m. with 0.05 USP units (approximately 9 ng/kg) synthetic oxytocin (Haven-Lockhart Laboratories, Shawnee, Kansas). Mammary areas were washed and milk collected using an in line pulsating vacuum pump. G. _Quantitation of PBBs Concentrations of PBBs in peritoneal cavity and tissues following treatment of 7 day old rats with 150 mg/kg PBBs and in milk and tissues from rats dietarily exposed to 50 ppm PBBs were determined by the method of Fehringer (1975). Petroleum (pet) ether (5 ml) was added to tubes containing milk (1 m1), peritoneal swabs (one 2 inch square gauze sponge) shaved fat (1 g) or other tissues (200 mg homo— genized in 0.8 ml water). Samples were mixed with 20 m1 acetonitrile 32 saturated with pet ether. Phases were separated and 100 mg NaCl was added to the acetonitrile phase. The acetonitrile phase was extracted 3 times with 10 m1 portions of pet ether. Pet ether fractions were pooled and reduced in volume to 2 ml which was placed on a Florisil (60-100 mesh) (Fisher, Pittsburgh, Pa.) column (100 mm x 5 mm). PBBs were eluted with 6% diethyl ether in pet ether. After evaporation of pet ether and diethyl ether residues were reconstituted in pet ether (20-100 ul) for injection onto a gas chromatograph. Quantitation was by gas-liquid chromatography with electron capture detection on a Varian model 2100 gas chromatograph (Varian, Palo Alto, Calif.) using a 1.7 M column packed with 1% OV-l. Carrier gas (N2) flow was 30 ml/min. Column temperature was held at 230° and only area of the major peak (2,4,5, 2',4',5'-hexabromobiphenyl) was calculated. Concentrations of P333 in tissues from all other rats used in this investigation were determined using the general AOAC procedure for chlorinated hydrocarbon pesticides (1970). Samples were weighed and ground with sufficient anhydrous sodium sulfate to render them completely dry and pulverized. Powdered tissues were extracted 5 times with 15 ml portions of hexane. Pooled extracts were reduced in volume to 1-2 mls which was placed on a Florisil (60-100 mesh) (Fisher, Pittsburg, Pa) column (500 mm x 22 mm). PBBs were eluted with 200 m1 hexane which was evaporated to dryness and then brought up to desired volume with hexane. Quantitation was by gas-liquid chroma- tography as described except that column temperature was held at 245°C and heights of both major peaks (2,4,5,2',4',5'-hexa- and 2,3,4,5, 2',4',5'-heptabromobiphenyl) were measured. Concentration of PBBs was 33 expressed as ug PBBs (usually as 2,4,5,2',4',5'-hexabromobiphenyl) per g (wet wt tissue) or ml (milk). H. Liver l. Copr0porphyrin and ur0porphyrin concentrations in liver and urine Copr0porphyrin (COPRO) and urOporphyrin (URO) were extracted from liver and urine by the method of Schwartz 3£_31. (1951) and quantified fluorimetrically. Urine was collected for 24 hr in alu- minum foil—covered flasks containing 300 mg sodium carbonate and l'ml toluene. After centrifugation to remove sediment, urine was trans- ferred to separatory funnels containing 75 m1 ethyl acetate (EA), 10 ml water and 5 ml acetate buffer (glacial acetic acid, saturated sodium acetate, water; 1:4:3, v/v, pH 4.8) (EA—A buffer). Liver was homogenized (Potter—Elvehjem homogenizer with a Teflon pestle) in 9 m1 EArA buffer and treated as urine. Samples were extracted for 2 min and phases allowed to separate for 30 min. Aqueous phase was drained into a centrifuge tube containing 0.5 g aluminum oxide. The organic phase was washed twice with 10 ml 1% sodium acetate. Sodium acetate washings and aqueous phase were pooled for URO quantitation; COPRO was extracted from organic phase with four 5 ml 1.5 N HCl washings. Fluorescence was measured using an excitation wavelength of 405 nm and emission wavelength of 603 nm. URO was extracted from aluminum oxide with 20 ml 50% saturated sodium acetate and 40 ml water. URO was subsequently extracted from washings and quantified fluorimetrically as described for COPRO. URO values were multiplied by 0.75. URO and COPRO concentrations were expressed as ug URO or COPRO per 24 hr (urine) or per g wet liver wt. 34 2. Vitamin A concentration in liver and serum Concentration of vitamin A in liver and serum was quantified fluorimetrically by the method of Hansen and Warwick (1968, 1978). Serum (100 ml), water (1 ml) and absolute ethanol (3 ml) were added to a screwcap test tube and shaken for l min. Liver (100 mg) was homo— genized (Potter-Elvehjem homogenizer with a Teflon pestle) in water (1 m1) and absolute ethanol (3 ml) and treated as serum. Petroleum ether (5_m1) was added to each sample. Following extraction for 5 min and centrifugation at 600 g for 2 min, vitamin A was quantified in organic phase using an excitation wavelength of 369 nm and emission wavelength of 483 nm. Concentration was expressed as pg vitamin A per ml (serum) or g wet liver wt. Serum glutamic pyruvic transaminase (SGPT) was assayed spectrophotometrically by the method of Reitman and Frankel (1957). Serum (200 pl), NADH (200 pl of 1 mg/ml stock in 0.01 N NaOH), analine (500 pl of stock 0.2 M, pH 7.5), lactic dehydrogenase (800 units) and distilled water (1.8 ml) were added to a cuvette. o-Ketoglutarate (200 pl of stock 0.1 M, pH 7.5) was mixed into the cuvette to initiate the reaction. Decrease in 0.D. at 340 nm was measured. Activity was expressed as units per m1 serum. I. Preparation of Postmitochondrial Supernatants and Microsomes Animals were killed and organs were excised, weighed and then coarsely chopped into ice-cold 1.15% KCl, pH 7.4, (liver) or 66 mM Tris, pH 7.4 (extrahepatic tissues). Postmitochondrial supernatants (PMS) were prepared by homogenization (Potter-Elvehjem homogenizer with a Teflon pestle) in 3 volumes of medium into which they were 35 excised followed by centrifugation at 10,000 g for 20 min. Microsomes were prepared by centrifuging PMS at 105,000 g for 60 min. The micro- somal pellet was resuspended in 0.25 M sucrose containing 5.4 nm EDTA and 20 mM Tris-HCl, pH 7.4, to a final concentration of 1-3 mg wet weight tissue per ml as described by Dent E£_El° (1976). All assays were performed on the day of supernatant or microsomal preparation. Protein was measured by the method of Lowry 33 31, (1951), using bovine serum albumin as a standard. J. Enzyme Assays In all assays, each incubation mixture contained 0.5 to 2.0 mg protein per milliliter. Reaction mixtures were incubated for 10 (hepatic) or 30 (extrahepatic) minutes at 37°C. Radioactivity was determined as described in Renal Function. Spectrophotometric measure- ments were made using a Beckman dual beam spectrophotometer (Beckman Instruments, Fullerton, Calif.). Fluorescence was quantified after spectrOphotofluorimeter (American Instrument Corp., Model SPF 128, Silver Springs, Maryland) standardization with 0.1 ug/ml quinine sulphate (QS) in 0.05 M H2804 using an excitation wavelength of 365 nm and emission wavelength of 460 nm. Arylhydrocarbon hydroxylase (AHH) was assayed fluorimetrically by the method of Nebert and Gelboin (1968) as modified by Oesch (1976). BenzoCa)pyrene (BP) (Sigma Chemical Co., St. Louis, Mo.) and cofactors were added in 66 mM Tris, pH 7.4, to beakers such that the final volume was 0.95 ml. The incubation mixture contained approximately 0.1 umole BP, 5.8 umol glucose-6-phosphate (G-6-P), 0.95 units G-6—P dehydrogenase (G-6-PD), 3.0 umol MgClz, 0.25 umol B-nicotinamide 36 adenine dinucleotide, reduced form (NADH) (from yeast), 0.4 nmol NAD phosphate (NADP) (from yeast) and 0.27 umol NADPH, reduced form (NADPH) (Type I). The reaction was stepped by addition of 1 ml ice- cold acetone. Acetone was added to blanks prior to incubation. After addition of 6 ml petroleum (pet) ether, samples were extracted with 3 ml 1.0 M NaOH and the pet ether aspirated. Fluorescence was measured in the NaOH layer using an excitation wavelength of 396 nm and an emission wavelength of 522 nm. Activitiy was expressed as fluorescent units (relative to Q3 standard) per mg protein per min. Biphenyl—2-hydroxy1ase (BP-2-0H) and biphenyl-4-hydroxylase (BP- 4-0H) were assayed fluorimetrically by the method of Creaven 35 31, (1965). Biphenyl (Mallinckrodt Chemical, St. Louis, Mo.) and co— factors were added in 50 mM Tris, pH 8.5, to beakers such that the final volume was 2.0 ml. The incubation mixture contained approxi- mately 20.0 umol biphenyl, 7.5 nmol G-6-P, 0.5 units G—6-PD, 5.0 nmol MgClz, 0.6 nmol NADP and 0.27 nmol NADPH. Reactions were stopped by addition of 0.5 ml ice-cold 4.0 M HCl. Blanks were treated with HCl prior to incubation. After addition of 5 ml n-heptane containing 1% v/v isoamyl alcohol, samples were extracted with 3 ml 0.1 M NaOH and the heptane layer aspirated. To the aqueous layer, 0.5 ml 0.25 M succinic acid was added. Fluorescence was measured 20 min later using excitation wavelength of 302 nm and emission wavelength of 410 nm (BP- 4-0H) and again using excitation wavelength of 312 nm and emission wavelength of 422 nm (BP-2-OH). Sample fluorescence was compared to fluorescence of 2-0HIH’and 4-0H BP standards. Activity was expressed as nmol of 2- or 4—hydroxybiphenyl produced per mg protein per min. 37 Ethoxyresorufin-O—deethylase (EROD) was assayed fluorimetrically by the method of Burke and Mayer (1974). Ethoxyresorufin (ER) (gift from R.T. Mayer, College Station, Texas) and cofactors were added in 66 mM Tris, pH 7.4, to beakers to give a final volume of 1.0 ml. The incubation mixture contained approximately 2.5 nmol ER, 0.8 units G-6- PD, 10.0 nmol G—6-P, 0.4 nmol NADP and 0.4 nmol NADPH. The reaction was stopped by addition of 1 m1 ice—cold acetone. Acetone was added to blanks prior to incubation. The reaction mixture was diluted with 5 ml distilled water and fluorescence was measured at excitation and emission wavelengths of 560 nm and 580rmh respectively. Sample fluorescence was compared to fluorescence of resorufin standards. Activity was expressed as nmol resorufin formed per mg protein per min. Hexobarbital hydroxylase (Hex-0H) was assayed by the method of Kupfer and Rosenfeld (1973). (2-140)Hexobarbital (New England Nu- clear, Boston, Mass.) and cofactors were added in 66 mM Tris, pH 7.4, to beakers to give a final volume of 2 ml. The incubation mixture contained approximately 1.2 nmol (2-14C)hexobarbital, 0.8 units G-6- PD, 10.0 umol G-6-P, 10.0 umol MgClz, 0.4 pmol NADP and 0.25 nmol NADPH. Reactions were stopped by addition of 3 m1 ice-cold 1 M citrate buffer, pH 5.5. Blanks were treated with citrate buffer prior to incubation. After addition of 10 m1 l-chlorobutane, samples were extracted with 10 m1 ethyl acetate and 1-chlorobutane layer aspirated. Radioactivity was determined in 200 pl of ethyl acetate layer and in 10 m1 substrate. Activity was expressed as nmol 3-hydroxyhexobarbital per mg protein per min. 38 Epoxide hydratase (EH) was assayed by the method of Oesch 33H31. (1971). To screwcap test tubes containing 50 ml 0.5 M Tris, pH 9.0, and sufficient distilled water to give a final volume of 200 pl was added 10 ul of 43.6 mM (7-3H)styrene oxide (Amersham Corp., Arlington Heights, 111.) in acetonitrile. The reaction was stopped by addition of 3 m1 pet ether. Pet ether was added to blanks prior to incubation. Samples were shaken for 5 min, centrifuged at 600 g for 2 min and placed at -20°C. When the aqueous layer was frozen, pet ether was aspirated. An additional 3 m1 pet ether was placed in test tubes and this process was repeated. To the aqueous layer 1 m1 ethylacetate was added and samples were extracted for 5 min, centrifuged at 600 g for 2 min. Radioactivity was determined in 200 pl of ethylacetate layer and in 10 ul substrate. Activity was expressed as nmol styrene glycol formed per mg protein per minute. Glutathione-S-transferase (GSH-transferase) was assayed spectro- photometrically by the method of Goldstein and Combes (1966). Reduced GSH (Sigma Chemical Co., St. Louis, Mo.) and 227 nmol sulfobromo- phthalein (BSP) were added in 0.1 M Na pyrophosphate, pH 8.2, to beakers such that the final volume was 4.4 m1. Conjugating activity was measured by recording 5 min change in optical density (0.D.) at 330 mu from the time BSP was added. Activity was expressed as nmol BSP conjugate (BSP-GSH) produced per mg protein per min. Progesterone hydroxylases (16o and 6BPH) were assayed using high pressure liquid chromatography (HPLC). Progesterone (Sigma) and cofactors were added in 66 mM Tris, pH 7.4 to beakers to give a final volume of 1.05 ml. The incubation mixture contained approximately 1.1 39 nmol progesterone, 5.8 nmol G-6-P, 0.95 units G-6-PD, 3.0 pmol MgClZ, 0.25 nmol NADH, 0.4 pmol NADP and 0.3 nmol NADPH. Following 60 min incubation, reactions were stopped by addition of 20 m1 chloroform/ methanol (2:1 v/v). Chloroform/methanol was added to the blanks prior to incubation. Progesterone and metabolites in chloroform were partitioned from methanol with 5 ml saline. Following filtration through 0.45 uM Teflon Millipore filters and evaporation of chloroform phase to dryness, progesterone and metabolites were redissolved in acetoni- trile/water (1:1, v/v). Quantitation was by HPLC on a Waters 6000A HPLC with a Waters 450 variable wavelength detector (Waters Inst. Co., Milford, Mass.) using a wavelength of 240 nm and a 300 nm x 3.9 nm column packed with reverse phase Bondapak C Solvent (acetoni- 18' trile/water - 49:51, v/v) flow was 1.3 ml/min. Column pressure was held at 750 psi. Standard curves were prepared using progesterone, 68- and 16o-hydroxyprogesterone (Steraloids, Inc., Wilton, N.H.). Activity was expressed as pmol 16a and 68 HP per mg protein per min. K. Kidney 1. Function in vitro Organic ion transport capacity was estimated 1E_y1££g 3 days following an i.p. injection of either peanut oil (10 mllkg) or 150 mg/kg PBBs in peanut oil to 11 day old pups and after 30 or 90 days exposure of adults to diet containing 0 or 100 ppm PBBs. Following cervical dislocation, kidneys were quickly removed, weighed and placed in ice-cold normal saline. Thin (approximately 0.5 mm) renal cortical slices were prepared freehand. Transport capacity was quantified as 40 the ability of renal cortical slices to accumulate a representative anion, p-aminohippuric acid (PAH), and cation, Ndmethylnicotinamide (NMN) by the method of Cross and Taggart (1950). Slices were placed in 3.0 ml Ringer's solution, pH 7.4, containing 10 mM sodium acetate 5M PAH and 6.0x10"6 and 7.4x10- M NMN (New England Nuclear, Boston, Mass.). Following incubation for 90 min at 25°C under 100% oxygen, slices were removed from the medium, blotted on gauze and weighed. Concentrations of PAH and NMN were determined using l4C—labeled compounds (Ecker 3£M31., 1975). Tissue and medium (0.1 ml) were solubilized in 1.0 m1 Soluene-lOO (Packard, Downers Grove, 111.). After 24 hr 10 ml Dimilume-30 (Packard) scintillation cocktail was added to each sample. Following 48 hr storage in dark at room tem- perature, radioactivity was determined using a Packard model 3380 liquid scintillation spectrometer. Results were.expressed as slice- toamedium ratio, calculated by dividing disintegrations per minute per ml of medium (Ecker 33 31., 1975). Ability of renal slices to produce ammonia and glucose was determined by the method of Roobol and Alleyne (1974). Slices were incubated in 5 m1 Krebs-bicarbonate medium, pH 7.4, containing 2 mM glutamine as substrate. Slices and medium were placed in 15 m1 screwcap test tubes, flushed with 02-C02 (95:5 v/v), capped and incu— bated for 60 min at 37°C. Following incubation, slices were removed from the medium, blotted on gauze and weighed. Immediately after removal of slices, 0.5 ml 10% perchloric acid was added to the incu- bation medium, the suspension centrifuged at approximately 600 g, and the supernatant was assayed for ammonium by the method of Kaplan (1965) and glucose using Glucostat reagents (Worthington Biochemical 41 Corp.). Net production of ammonia and glucose was expressed as micromoles per milligram of wet tissue weight per hour. Vitamin A was quantified in kidney as described for liver. 2. Function in vivo To determine renal function 13 2139, animals exposed to 100 ppm PBBs for 30 or 90 days were anesthetized with 50 mg/kg sodium pentobarbital, i.p., and body temperature was maintained at 37il°C using heat lamps. A PE50 cannula was inserted into the bladder and urine was collected under mineral oil in preweighed vials. The left femoral vein was cannulated for infusion. Both femoral arteries were cannulated to monitor blood pressure using a Statham transducer and a Beckman type RS dynograph (Beckman, Schuler Park, Ill.) and to obtain blood samples. The infusion solution contained 1.0% inulin and 0.6% PAH in normal saline. (3H)Inulin (0.5 uCi/ml) and (14C)PAH (0.5 uCi/ml) were added to the solution (saline), which was infused at 0.019 ml/min using a Harvard infusion pump (Harvard Apparatus, Millis, Mass.). A minimum of 90 min elapsed from the beginning of infusion to initiation of urine collection. Four 30 min urine samples were taken. Blood (0.4 ml) was sampled at the middle of each urine collection. Following the initial clearance period, animals were infused with 1:4 rat plasma/saline solution (4% body weight) and three additional 30 min collections were made. Radioactivity (14C-PAH and 3H-inulin) in urine and plasma was determined as previously described. Sodium concentrations were determined by flame photometry (Instrumentation Labs, Model 143, Boston, Mass.). Blood urea nitrogen (BUN) was deter- mined by the method of Kaplan (1965) and expressed asrmgurea nitrogen per lOOImlof whole blood. 42 L. Heart 1. Function in vitro Sympathetic neuronal transport capacity was estimated 1E_31££g_in hearts from animals 14, 28, 56, 84 or 98 days of age that had been continuously exposed to 0 or 100 ppm PBBs from day 8 of gestation. Following cervical dislocation, hearts were quickly excised, weighed and placed in ice-cold preoxygenated Krebs-Henseleit solution. Thin ventricular slices were prepared with a Stadie-Riggs microtome (A.H. Thomas Co., Philadephia, Pa.). Transport capacity was quantified as the ability of ventricular slices to accumulate a nonmetabolized catacholamine, d,1—metaraminol (MET), by the method of Stickney (1976). Slices were placed in 10 ml modified Krebs-Henseleit solu- tion, pH 7.4, of the following composition (mM): NaHCO 27.2; NaCl, 3’ 118.0; KCl, 4.8; KH2P04, 1.0; MgSOA, 1.2; CaClZ, 2.5; anhydrous dextrose, 11.1. The solution also contained 0.75 mg/ml ascorbic acid and 1x10"7 M MET (approximately 20,000 DPM/ml) (New England Nuclear, Boston, Mass.). Following incubation for 60 min at 37°C under 95% 02-5% C02, slices were removed from the medium, washed with ice-cold saline, blotted on gauze and weighed. Concentration of MET was determined using 3H-MET (Stickney, 1976). Slices were solubilized in 0.5 m1 tetraethylammonium hydroxide. Double-distilled water (1.5 ml) was added to each sample and mixed thoroughly. Of this resultant suspension, 0.1 ml was mixed with 10.0 ml Bray's solution (88% p- dioxane, 10% methanol and 2% ethylene glycol). Sample radioactivity was determined using a Packard model 3380 liquid scintillation spec- trometer. Results were expressed as slice-to—medium ratio, calculated 43 by dividing disintegrations per min per g heart tissue by disinte- grations per min per ml medium (Stickney, 1976). Inotropic response to calcium and ouabain was quantified in hearts from animals 70 days of age that had been exposed to 0 or 100 ppm PBBs from day 8 of gestation. Following cervical dislocation, hearts were quickly excised, weighed and placed in ice-cold preoxy- genated Krebs-Henseleit solution modified as previously described. Left atria were separated from ventricles, trimmed, placed in an atria holder and lowered into a 90 ml bath of modified Krebs-Henseleit solution which was maintained at 30°C and bubbled with 95% 02-5% C02 (Stickney, 1978). Atria were attached to a Grass FT-OBC force trans- ducer (Grass Med. Instruments, Quincy, Mass.). A resting tension of approximately 1 g was applied to each atrium. Following determination of the threshold voltage for electrical stimulation, a voltage of 1.1 times threshold was employed to drive the atrium at a frequency of 1 Hz. Atria were allowed to equilibrate for 60 min during which the bathing solution was changed 4 times (15, 30, 45 and 55 min). Follow- ing equilibration, twitch tension was recorded and Ca2+ (1.0x10-2M) or ouabain (1.5x10-6M) was added to the bath. Contractile height was recorded and results were expressed as percentage increase over control (contractile height prior to addition of Ca2+ or ouabain). 2. Arterial pressure After cannulation of the left femoral artery in pentobarbi- tal (50 mg/kg, i.p.) anesthetized female rats exposed to 100 ppm PBBs for 90 days, systolic arterial blood pressure was monitored via a Statham transducer (Statham, Hato Rey, Puerto Rico) attached to a 44 Beckman-Type RS Dynograph (Beckman Inst., Schiller Park, 111.). Systolic arterial pressure was also measured indirectly in unanes- thetized female rats using tail plethysmography (Narco-Bio Systems, Inc., Houston, Texas). M. Lung 1. Function in vitro Activity of angiotensin converting enzyme (ACE) was quanti- fied by the method of Cushman and Cheung (1971) as modified by Wallace 31_31, (1978). Lungs were excised, weighed and homogenized using a polytron homogenizer (Brinkman Instruments, Westbury, N.Y.) in 4 volumes of ice-cold 100 mM potassium phosphate—300 mM sodium chloride buffer, pH 7.0. Homogenate was centrifuged at 600 g for 10 min and supernatant assayed for ACE activity. Hippuryl-L-histidyl-L—leucine (HHL) was added in 100 mM potassium phosphate-300 mM sodium chloride buffer, pH 8.3, to give a final concentration of HHL of 5.0 mM and final volume of 0.25 ml. Following 30 min incubation at 37°C, the reaction was stopped by addition of 0.25 ml 1.0 N HCl. After addition of 1.5 ml ethyl acetate and centrifugation for 10 min at 2,000 g, 1.0 m1 acetate phase was evaporated at 40°C in 3.0 ml 1.0 M NaCl and the optical density determined at 228 nm using a Beckman dual beam spec— trophotometer (Beckman Instruments, Fullerton, Calif.). A molar extinction coefficient of 9.8 mM-1 cm-1 was employed to convert optical density to molar units. Activity was expressed as nmol hippuric acid produced per mg protein per min. Activity of monoamine oxidase (MAO) was quantified by the method of Roth 35 31. (1979). MAO activity was determined in 45 supernatants prepared as described for ACE activity. (14C)-5—HT was added in 50 mM potassium phosphate-150 mM sodium chloride buffer, pH 7.0, to give a final volume of 2.2 ml. The reaction mixture contained 0.2 umole l4C-5-HT. Following 15 min incubation at 37°C, the reaction was stOpped by addition of 0.2 ml Ba(0H)2 followed by 0.2 ml 0.2 M ZnSO4. After centrifugation for 10 min at 2,000 g the resulting supernatant was analyzed for 5-HT and S-HIAA by the method of Roth 33 ‘31. (1977). Samples were added to columns (0.5 x 1.0 cm) of Bio-Rex 70 (sodium form; pH 6.0) cation exchange resin (Bio-Rad Labs, Rich— mond, Calif.) to separate parent (unreacted) 5-HT from its metabolite 5-hydroxyindoleacetic acid (5-HIAA). 5-HIAA was eluted from the column with 2.5 ml distilled water, 5-HT was eluted from the column with 3.0 ml 0.2 N HCl. Radioactivity in each fraction was quantified after addition of 10 ml ACS (Amersham/Searle Corp., Arlington Heights, Ill.) using a Packard model 3380 liquid scintillation spectrometer. Activity was expressed as nmol S-HIAA produced per mg protein per min. 2. Isolated perfused lung Lungs were perfused by the method of Wallace 33 31. (1979). Rats were anesthetized with sodium pentobarbital, 50 mg/kg, i.p. and administered sodium heparin, 2,000 U/kg, i.v. A PE90 cannula was inserted into the pulmonary artery via the right ventricle. A PE160 cannula was inserted into the trachea and the lungs excised and sus- pended in a perfusion apparatus maintained at 37i1°C with a heat lamp. Lungs were ventilated by negative pressure ventilation at 30 strokes per min with a 95% 02-5% C02 gas mixture and perfused with Krebs- bicarbonate medium, pH 7.4, containing 4% bovine serum albumin in a single pass (nonrecirculating) system. Inflow pressure was continuously 46 monitored using a Grass model 7 polygraph (Grass Med. Instruments, Quincy, Mass.) and a P23AC Statham pressure transducer (Statham, Hato Rey, Puerto Rico). Following 5-10 min equilibration, angiotensin I (Al) was added to the perfusate at a concentration of 1 ng AI/ml. Samples (0.5 m1) of effluent medium were taken at the middle of a 4 min collection period (approximately 2 min following onset of perfusion with AI). After a brief equilibration with drug-free perfusion medium, lungs were perfused with medium containing 14C-5-hydroxytryptamine (5-HT) (Amersham/Searle Corp., Arlington Heights, 111.) at a concentration of 0.1 uM. Samples (0.5 m1) of effluent medium were taken at the middle of a 4 min collection period. When the experiment was terminated lungs were removed from perfusion chamber, blotted on gauze and weighed. Concentration of Al was quantified by radioimmunoassay using a specific antibody to pure Aspl-IleS-AI (CIBA Pharmaceuticl Co., Summit, N.J.) according to the method of Haber 32 31. (1969). Follow- ing incubation of sample at 4°C for 18 hr with antibody and Aspl- [1251]-Ile5-AI, free and antibody—bound AI were separated by activated charcoal (10% suspension) binding of free AI. After centrifugation, radioactivity in resultant fractions was quantified using a Searle model 1185 gamma counter. Concentrations of 5-HT and 5-HIAA were quantified as described for MAO. 47 N. Response to Xenobiotics Duration of anesthesia following 40 mg/kg sodium pentobarbital in water, i.p., was determined in female rats 84 days of age that had been neonatally treated with 150 or 500 mg/kg PBBs or exposed to 100 ppm PBBs from day 8 of gestation. Sleeping time was recorded as the interval from time of injection until the righting reflex was re- gained. Duration of anesthesia following 90 sec in dessicator saturated with diethyl ether was determined in female rats 90 days of age that had been exposed to 100 ppm PBBs from day 8 of gestation. Sleeping time was recorded as the interval from the time animals (pair control and PBBs treated) were removed from the dessicator until the righting reflex was regained. Median time to death following bromobenzene administration was determined in male rats that had been neonatally treated with 150 or 500 mg/kg PBBs. At 49 days of age, 2820 mg/kg bromobenzene in DMSO (approximately a 24 hr LD85 dose which was established in preliminary experiments) was administered to the animals, i.p., and the time to death was measured. Median time to death following digitoxin was determined in female rats that had been exposed to 100 ppm PBBs from day 8 of gestation. At 90 days of age, 10 mg/kg digitoxin in DMSO (approximately a 24 hr LD85 dose which was established in preliminary experiments) was administered to the animals, i.p., and the time to death was measured. 48 0. Response to Pharmacological Doses of Steroid Hormones Rats were exposed to 0, 10 or 100 ppm PBBs from day 8 of gesta- tion until 28 days postpartum when experiments were conducted in offspring, unless otherwise specified. Methods employed in these experiments were similar to those used by others to determine effects of phenobarbital on steroid hormone metabolism (Conney'3£_31., 1966; Levin g£_31,, 1968, 1969). Radioactively labeled steroids were pur- chased from New England Nuclear (Boston, Mass.) and unlabeled steroids from Sigma Chemical Co., (St. Louis, Mo.). Sample radioactivity was determined as described in Renal Function. Male rats were treated with an i.p. injection of 150 mg/kg (140)progesterone ([4-14C], 0.4 uCi/mg) (approximately a LD dose in 30 controls) in dimethylsulfoxide (DMSO). Duration of anesthesia was recorded as time of injection until righting reflex was regained. Following decapitation and blood collection, brains were excised, weighed and homogenzed (Potter-Elvehjem homogenizer with a Teflon pestle) in 3 volumes 66 mM Tris, pH 7.4, at waking or 3 hr after progesterone administration. Aliquots (2.5 m1) of whole brain homo- genate were extracted initially with n-hexane (Hex) and subsequently with water saturated ethyl acetate (EA). Homogenates were extracted twice, 4 volumes and then 2 volumes which were pooled, with each solvent. Following evapoaration to dryness, radioactivity was determined in extracts (1.5 m1). Radioactivity was also quantified in whole brain homogenates (500 pl) and serum (50 ul). 49 Male rats were treated with a s.c. injection of 0 (corn oil) or 20 mg/kg (3H)testosterone ([7-3H(N)], 5 pCi/mg) on day 25 postpartum. At least 6 animals in each greatment group were decapitated 24 hr later, blood was collected and testes excised and weighed. Radio- activity was determined in serum (50 pl) and whole testis. Remaining animals were weighed 72 hr later, sacrificed by cervical dislocation, and seminal vesicles were excised and weighed. Female rats were treated with an i.p. injection of 0 (DMSO), 1 or 3 pg/kg (3H)estradiol-l78 ([6,7-3H], 156 pCi/pg). Body weights were recorded, animals decapitated, blood collected and uteri excised and weighed 4 hr later. Radioactivity was quantified in serum (50 p1) and whole uterus. P. Persistance of Effects 1. Single ipjection Nursing dams and pups (10 per litter) were purchased 3 days postpartum and were acclimatized for 4 days prior to experimental treatment. Following normalization of the litter to 5 males and 5 females on day 7, pups of each litter were treated with a single i.p. injection of 0 (peanut oil, 10 ml/kg) or 150 mg/kg PBBs in peanut oil. Pups were sacrificed by decapitation l, 2, 3, 7, 14, 28 or 63 days following treatment at which times enzyme assays and quantification of tissue PBBs were performed. 2. Perinatal exposure Time pregnant rats were obtained on day 2 of gestation and were acclimatized for 6 days prior to experimental treatment. On day 8 of gestation, the experimental diet containing 0 or 100 ppm PBBs was 50 substituted for the Lab Blox. Litters were normalized to 10 pups (5 male, 5 female) each at birth. At 28 days postpartum all animals were weaned onto Lab Blox containing no PBBs. Enzyme assays, pentobarbital sleeping time, tissue PBBs quantification and histologic examinations were performed at 28 days of age and during the residual phase; 150 and/or 328 days of age. 3. Multip1e generations Timed pregnant rats (F0) were obtained on day 2 of gestation and were acclimatized for 6 days prior to experimental treatment. On day 8 of gestation, the experimental diet containing 0, 10 or 100 ppm PBBs was substituted for the Lab Blox. Litters were normalized to 10 pups (5 male, 5 female) at each generation. At 28 days postpartum all progeny (F1) of dams (F0) dietarily eXposed to PBBs were weaned onto Lab Blox containing no PBBs. After an additional 10-12 wk for matura- tion, littermates (F1) were bred to produce the next generation (F2). Similarly, F littermates were bred at 14-16 wk of age to produce F 2 3' Enzyme assays, pentobarbital sleeping times, progesterone sleeping times and tissue PBBs quantification were performed and survival determined in F1’ F2 and/or F3 at weaning. Q. Statistics Data were analyzed statistically by analysis of variance, either randomized complete block or completely random design. Treatment differences were detected by the least significant difference test (Steel and Torrie, 1960). The 0.05 level of probability was used as the criterion of significance. RESULTS A. Survivial, Growth and Development Dietary exposure of pregnant rats to 100 ppm PBBs with maternal food deprivation for 48 hr produced a decrease in number of live fetuses and fetal body weight and an increase in resorption rate and whole fetal carcass concentration of PBBs (Table 1). Dietary treat- ment with 100 ppm PBBs alone increased whole fetal carcass concentra- tion of PBBs when compared to controls but not 100 ppm PBBs with food deprivation. Separate treatment with 100 ppm PBBs or food deprivation had no effect on number of live fetuses, resorption rate or fetal size. No statistically significant treatment differences in fetal liver or kidney weight-to-body weight ratios or incidence of gross, soft-tissue or skeletal anomalies were detected. Body weight gain of pups suckled by dams fed 100 ppm PBBs and weaned onto 100 ppm PBBs was decreased by 14 days of age (83% of control value) and remained decreased through 84 days of age (82% of control value) (Figure 1). Growth rate was not affected by neonatal treatment with a single i.p. injection of 150 or 500 mg/kg PBBs. Fur development, external auditory direct opening and eye opening were delayed in pups suckled by dams fed 100 ppm PBBs (Table 2). Postnatal physical development, assessed using these development markers as well as earlier occurring pinna detachment and incisor eruption, was not affected by neonatal treatment with 150 or 500 mg/kg PBBs (Table 2). Survival rate was reduced in pups exposed to 100 ppm PBBs by 14 days 51 52 .mo.ove .msamb Houusoo wm>wumomaoo can Eoum ucoHoMMHv hauomoHMchng .AHH mom OH whom HchfiuMumomv mm>fiuemm poem weeds: coaumummw mo om ham Hausa coaumumow mo w hem scum mmmm Bee ooa Ho 0 :ufiz vmumouu mums mumm .muwuuaa w mom .z.m.m n memos mum mosam>o ea.oam.m mma.owwm.m H.onw.m QN.¢HN.¢H easoa we OOH em.ono.q wa.oamo.q H.0Hm.m w.HHm.q HHQH o ooa o.o mm.onqo.q H.0Hm.m H.mnn.m HHNH me o o.o HN.OHmm.q H.0Ho.q o.mnm.o HHMH o o Aw\w:v mmmm va unwfioz Aaov numama ANV monouom moon momsuom Aunv AEQQV mmmoumo maosz econ Hmumm mesa cacao Hmumm no ponuomom mo .02 m>wq «0 .oz soaum>wummn mmmm ovm>fiuemn voom pom mmmm sues woummug comm unmowmum mo wcauemmmo wsoa< mmmm mo coaumuudoocoo mmmoumo maonz can swam Hmumm .mumm coaueuommm H mqm Houusoo mnu Scum moamummmwv unmowmaawam haamofiumwumum m mmumofiwow xmaumum< .mamaaom a mom .z.m.m H momma mum mosam> .vmaawx mum3.%m£u Hana: sowumummw «o w mum aoum mmmm see ooa no 0 :uaB no AH >mev suuwn Hmumm mom ecu so Has unease ca mmmm wx\wa com no omH .o nuHB mmummuu mum3 mum» mamamm .ofimw u3 econ so mmmm :uHB unmaumwuu Houmoauma no Hmumsooa mo mummmm .H ouswwm H ouowam 54 Am>m3m0< 3 on on A , 3 , k L“. on s mm. mm .Oo;.aa H O M .1 on. m M «can can 8. E com .I. .4. .9: 9.558... % 8."an 3a.. 9.3:. on. m :l 5528 D 8» 55 .mo.ovm .oon> Houuooo one 80pm uddhdmmgp maucmoamacwflme .moHHHx mums menu Hausa coaumumow mo w mom Scum mmmm Ema ooa no G :ufle no AH hmwv nuufln umumm mom ago so HHo assume ca mmmm wx\we oom no mmmm wx\wa oma .Aaouucoov HHo assume sues wounds“ mums mameHs< .mumuuHH q ummma on you .z.m.m a when ca means can mosam>o em.OHm.mH ~.0Hq.se N.onm.sa N.ons.qa maeamao use em.ons.me «.0Hw.ae N.OHG.HH ~.oae.ee massage some see .uxm e~.oam.oa ~.ope.m H.oae.m H.onm.m unmaaoem>me use m.ono.w m.oae.m ~.oae.w N.OHH.w aoeuaapm nomaoaH ~.osa.m N.0Ho.m ~.oak.m H.oak.m unmanomume muses meme age ooe mama wx\wa com mmmm wx\wa one Hopuaoo umumemumm osmaummuw ommmm Sofia ucmaummua Haumcflumm so Hmumcomz wofizoaaom mumm mo unmaaoam>mn Houmoumom N mamdfi 56 of age (88% of control value) and remained lower than control through 28 days of age (87% of control value) (Figure 2). Pup mortality was not affected by neonatal treatment with 150 or 500 mg/kg PBBs. Liver, kidney, ovary and fat all contained PBBs following neo- natal or perinatal treatment with PBBs (Table 3). Tissue concentra- tions of PBBs were dose-dependent as neonatal treatment with 150 mg/kg PBBs resulted in lower concentrations of PBBs than 500 mg/kg PBBs. Perinatal and continuous exposure to 100 ppm PBBs resulted in higher tissue concentrations of PBBs than neonatal treatment with a single injection of 500 mg/kg PBBs. 0f the tissues examined, kidney had the lowest and fat the highest concentration of PBBs. More PBBs were found in ovary than in either kidney or liver. Concentration of PBBs in milk was highest at parturition and decreased with time for 14 days (Figure 3). At parturition, the concentration of PBBs in milk was approximately 3-4 times higher than the concentration of PBBs in the maternal diet. By 14 days post— partum, concentration of PBBs in milk and maternal diet were similar. B- £112]: The liver weight-to-body weight ratio was increased in a dose- dependent manner by neonatal treatment with 150 or 500 mg/kg PBBs when determined at 28 days of age (120% and 145% of control value, re- spectively) (Figure 4). At 56 days of age, liver was enlarged after 500 mg/kg PBBs, however, not after 150 mg/kg PBBs (115% and 105% of control value, respectively). The liver weight-to-body weight ratio was increased to a greater extent by 100 ppm PBBs than 150 or 500 mg/kg PBBs (approximately 165% of control value by 100 ppm PBBs at 57 .mo.ovm .ooam> Houuooo wnu Bone muconMMHm unmUfiMchHm kHHmoHumHumum m moumoficsfl xmfiuoum< .mameflsm c How .z.m.m H memos mum mmoam> .moHHHx mums hone Hausa coaumummm mo w how Boum mmmm Bea OOH Ho o sowz no AH hmmv nuufin umumm mum use so HHo unease ca mmmm wx\we oom so omH .o :uHB woumouu muo3 mums mamamm .waficm03I0uloumu Hm>H>H=m so mmmm Sofie unmaummuu Hmumafluma so Hmumaooc mo uoommm .N madman 58 33 U) 223 533‘; “wan. 2:5: 89§e mmam , an __.__{444 an 3 ____{444i ——F g a s a a llNBOHEdI 'IVMAHI'IS AGE [days] Figure 2 Tissue Concentrations of PBBs Following Neonata1 or Perinatal 59 TABLE 3 and Continuous Treatment with P333 TISSUE Treatment Liver Kidney Ovary Fat 28b 150 mg/kg PBBs 2.6:0.4 1.2io.1 -—-- 45.0:13.4 100 ppm PBBS 27.2i6.9 16.5i4.2 ---- 282.2i17.3 56b Control 0.0 0.0 0.0 0.0 500 mg/kg PBBS 5.5i0.3 --- .lill.6 52.3i 6.2 100 ppm PBBs 44.7i5.5 20.6i1.8 1 .3i 4.4 449.6:25.8 aValues are means in pg PBBs/g wet wt tissue i S.E.M. for at least 3 animals. Animals were treated with peanut oil (control), 150 mg/kg PBBs or 500 mg/kg P333 in peanut oil on the day after birth (day l) or with 0 or 100 ppm PBBs from day 8 of gestation until they were killed. bAge of animals in days. 60 Figure 3. Concentration of PBBs in milk from lactating rats fed diet containing 50 ppm PBBs from the 8th day of pregnancy. Values are pg PBBs/ml milk i S.E.M. for at least 4 animals. CONCENTRATION OF PBBs IN MILK Wm” § E 9‘ 0| 0 61 O 3 5 7 DAYS AFTER PA RTURITION Figure 3 14 62 Figure 4. Effect of neonatal or perinatal treatment with PBBs on liver wt to body wt and kidney wt to body wt ratios. Female rats were treated with 0, 150 or 500 mg/kg PBBs in peanut oil on the day after birth (day 1) or with 0 or 100 ppm PBBs from day 8 of gestation until they were killed. Values are means i S.E.M. for 4 animals. Asterisk indicates a statistically significant difference from the control value, p<0.05. 63 D CONTROL E 150 mg/kg PBBs 500 mg/kg PBBs 1°” @ 100 ppm Pass *- 9" o s LIVER wt/Boov wr. (x 100) P P P O O 0 HOME; Wt/Boov WT.(x100) . P :e .-‘ m on o N .0 A 9 M 28 56 AGE (days) Figure 4 va ha pl Ce St f0. W8? Clt ‘-’a< 1h 89* ce] bod app hepe b LreE 64 both 28 and 56 days of age). Liver enlargement produced by PBBs was accompanied by an increase in hepatic microsomal protein. Treatment of 7-day old pups with 150 mg/kg PBBs increased microsomal protein in liver after 7, 14 and 28 days (approximately 130% of control values) (Figure 8). Compared to control liver (Figure 5), liver from animals peri- natally exposed to 100 ppm PBBs had histOpathological alterations (Figure 6). At 28 days the most prominent structural change was vacuolation which gave the cytoplasm a foamy appearance. Some cells had large intracytoplasmic vacuoles that displaced most of the cyto- plasm and pressed the nucleus to the periphery. Occasionally vacuola- tion and necrosis progressed centrolobularly but most was midzonal. Cellular swelling caused the sinusoids to become less apparent. Similar hepatic degenerative changes were observed in adult rats following 90 days exposure to 100 ppm PBBs (Figure 7). Hepatic cells were uniformly enlarged so that sinusoids appeared only as small, clear spaces with an occasional Kupfer cell visible. Many cells were vacuolated. Focal necrosis was found occasionally, however, as in liver following perinatal exposure to PBBs midzonal necrosis was most apparent. Hepatocytes were disrupted, nuclei were absent in some cells and pycnotic in others. Many hepatocytes contained large myelin bodies. Some of these bodies were uniform in appearance, while others appeared to have a lighter central mass surrounded by a wide, darker band. In contrast to effects produced by dietary exposure to PBBs, hepatic degenerative changes were not observed following neonatal treatment with a single injection of 150 or 500 mg/kg PBBs. 65 Figure 5. Hepatic tissue from control rat. stain; x100. Hematoxlyin and eosin Figure 6. 67 Hepatic tissue from rat 28 days of age that had been exposed to 100 ppm PBBs from 8th day of gestation. There is vacuolation. Hematoxylin and eosin stain; x40. ‘ _-—.-EH ‘1‘—— “- 'H’w-J-fl‘q-Jr‘fi—t m "-4.." , v, f Figure 6 69 Figure 7. Hepatic tissue from rat fed diet containing 100 ppm PBBs for 90 days. There are inclusion bodies. Hematoxylin and eosin stain; x400. 7O r f" 3 Figure 7 71 Vitamin A concentration (US/8 wet wt tissue) in liver was reduced at 28 days of age in a dose-dependent manner by perinatal exposure to 10 or 100 ppm PBBs (60% and 43% of control value, respectively) (Table 4). Vitamin A per liver was also reduced at 28 days of age by 10 or 100 ppm P333 (76% and 71% of control value, respectively). Similarly, vitamin A concentration in liver was reduced at 56 and 100 days of age by perinatal and continuous exposure to 100 ppm PBBs (50% and 60% of control values, respectively) (Table 4). Treatment with PBBs had no effect on vitamin A concentration in serum when determined at 28, 56 or 100 days of age (Table 4). Concentration of COPRO and URO in liver decreased with age in control and PBBs treated rats (Table 5). At 28 days of age, hepatic COPRO concentration was increased by perinatal exposure to 100 ppm PBBs (approximately 350% of control value). Hepatic URO concentration was increased in a dose dependent manner at 28 days of age by peri- natal exposure to 10 or 100 ppm PBBs (approximately 150% and 350% of control value, respectively). At 112 days of age, hepatic COPRO concentration was increased by 10 or 100 ppm PBBs (approximately 150% and 350% of control value, respectively). Hepatic URO concentration was only increased by 100 ppm PBBs at 112 days of age (approximately 150% of control value). Concentration of COPRO in urine was increased at 28 and 112 days of age by perinatal and continuous exposure to 100 ppm PBBs (approxi- mately 150% and 200% of control values, respectively) (Table 6). Urinary COPRO was not increased by 10 ppm PBBs at 112 days of age and URO concentration in urine was not affected by PBBs. Perinatal expo— sure to PBBs had no effect on packed cell volume (Table 7). 72 TABLE 4 Effect of Perinatal EXposure to PBBs on the Concentration of Vitamin A in Serum and Liver of Rats Vitamin A Concentration PBBs Age (ppm) (days) Serum (pg/m1) Liver (pg/g) O 28 0.32i0.04 66.1i 5.3b 10 28 0.34i0.03 40.0i 4.7b 100 28 0.31i0.04 28.4i10.l O 56 O.34i0.05 94.5:12.6b 100 56 0.36i0.03 47.2: 9.7 O 100 0.37i0.04 187.0i22.4b 100 100 0.34i0.06 112.6i18.2 aValues are means i S.E.M. for at least 4 animals. Rats were treated with 0, 10 or 100 ppm PBBs from day 8 of gestation until 28 days postpartum at which time vitamin A was quantified. bSignificantly different from the control value, p<0.05. 73 TABLE 5 Concentration of COproporphyrin and Uroporphyrin in Liver from Female Rats EXposed to PBBS Treatment C0pr0porphyrin Uroporphyrin Age: 28 Days Control 0.084i0.012 0.056i0.028 10 ppm PBBs 0.109i0.024b 0.094:o.017 100 ppm PBBs 0.282i0.009 0.206i0.039 b Age: 112 Days Control 0.039i0.005b 0.017i0.004 10 ppm PBBs 0.066i0.015b 0.015i0.005b 100 ppm PBBs 0.147i0.029 0.027i0.004 aValues represent pg/gm wet liver, expressed as means i S.E.M. for at least 3 animals. Animals were exposed to 0, 10 or 100 ppm PBBs from day 8 of gestation until measurements were made. bSignificantly different from control value, p<0.05. 74 TABLE 6 Concentration of Copr0porphyrin and Uroporphyrin in Urine from Female Rats Exposed to PBBs Treatment COproporphyrin Uroporphyrin Age: 28 Days Control 3.15i0.46b 0.12i0.01 100 ppm PBBs 4.67:0.24 0.11i0.01 Age 112 Days Control 1.73:0.41 0.35i0.08 10 ppm PBBs 2.29i0.63b 0.41:0.07 100 ppm PBBs 3.62:0.55 0.28:0.05 aValues represent pg/gm wet liver, expressed as means i S.E.M. for at least 3 animals. Animals were exposed to 0, 10 or 100 ppm PBBs from day 8 of gestation until measurements were made. bSignificantly different from control value, p<0.05. 75 TABLE 7 Packed Cell Volume (Hematocrit) in Female Rats Exposed to PBBs Treatment Packed Cell Volume (%) Control 43.0i0.7 100 ppm PBBs 42.3:0.4 aValues are means i S.E.M. for 10 animals. Animals were exposed to 0 or 100 ppm PBBs from day 8 of gestation until 16 wks postpartum, when measure- ments were made. 76 Activity of hepatic microsomal and cytosolic enzymes increased with age in control and PBBs treated rats (Figures 8 and 9). Admi- nistration of 150 mg/kg P335 to 7-day old rats increased the relative activity of all hepatic enzymes investigated. EROD and AHH activities were increased above controls by 2 days following administration of PBBs with maximal activities occurring after 7 to 14 days (approxi- mately 2000% and 900% of control values, respectively) (Figure 8). Hepatic EH activity was higher in PBBs treated pups 7 days following PBBs and was maximally stimulated by 14 days (approximately 650% of control value) (Figure 8). BP-4-0H, Hex-0H and GSH-transferase activities were all increased above control values by 7 days after administration of PBBs and reached maximal activity 28 days following treatment (approximately 450%, 400% and 140% of control values, re- spectively) (Figure 9). BP-2-0H was stimulated 1 day following treatment of 7 day old rats with PBBs and reached a maximum by 14 days (approximately 500% of control value) (Figure 9). Hepatic AHH and EH were stimulated at both 28 and 56 days of age by perinatal and continuous exposure to 100 ppm PBBs and by neonatal treatment with 150 or 500 mg/kg PBBs (Figure 10). Of these treat- ments, hepatic AHH was increased to the greatest extent in animals exposed to 100 ppm PBBs (approximately 1500% of control value at both 28 and 56 days of age). Stimulation of AHH in liver following 500 mg/kg PBBs was greater than after 150 mg/kg PBBs approximately 550% and 200% of control value, respectively, at 28 days of age). Activity of hepatic AHH following 500 or 150 mg/kg PBBs was greater at 56 than 28 days of age (approximately 1250% and 350% of control value, 77 .mo.ove .Houuaoo wswmsomm mouwm mmefiu m30HHm> um mums ca mmHuH>Huom oexuco mom dfimuoue anacmouofia aflumeom Ionuoo Eoum ucwuomwfie mausmoHWHawfim mmsHm> mmumofiesfl xmwuoum< .mumu m ummma no How .z.m.m + name mes musomouemu usfioa :omm .eouumaumoa n mom so mmmm wx\we omH no 0 sues ucmaummuu .w muowfim 78 zo.h<¢hw.z.20< cub“: ”33 am 3 k . \.. a aunt l .5528 um5>xomo>z 2853:9552 w shaman F: um: [mound Bus / BONBOSBHOIHH BMIV'ISU OO— 0 some. I AOEPZOO In... mwz NEXOQN N. '1: "Q G! M N I- 0 mm lugsaord 6w / 103M!) BNSHAIS 80mm u 3 of 20FIhmm0 IO- -2_u=¢8m¢>xcxhw II. .r a \\ so Ot\\ .. \\ a. .6. \ z \\ o \\\ o \ ... o o \ 4 _ L O— 0— On 0v ugm/ugsamd 6m lmanuosau 99'0“! U lHOIBM 308$”. 13M tub/6w 79 .mo.ova .Houucoo wafimcommounoo aouw odoHoMMHm maucmofimwowfim mosam> woumoamoH xmwuoum< .mumu m ummma um mom .z.m.m H once one mucomowamu usfiom comm .asuummumom m mow so mmmm wx\wa oma no 0 nuwe usoeumouu umumm mmeflu msoaum> um mums ca mmwufi>wuom waknao caumeom .m muswfim 80 ZO.h<¢hm.z.2n—< zwhm< w>zm:...o In) Com" 4015.200- I. mgmummgm: 18 m mhflwfim u uI/ugoxmd 6m 0'5 :45 N' /'|AN3HdI8 ANONOAH'V 39PM u vi a.— 0 Va mm . d0 on WW 9 of Md Me 0.0 um «N v.0 295522.23 $t< 223 mu 3 s n N p 8 Tllllldullllll in 1.1 ......... . ..... /. / . \I _ O and; l 40:55.00 I ' wm<4>x0co>1 INIJ>ZmEa 33888 one I JOChZOU I. I wm<4>XO¢O>I 44....ngOXw: unu/umoul 6w ”ANSI-idle AXOHOAH-z snow u ugm luusrmd Bu: NVLIBUVBOXSH AXOUGAH-E soIom U 81 Figure 10. Effect of neonatal or perinatal treatment with PBBs on activity of arylhydrocarbon hydroxylase and epoxide hydratase in liver. Female rats were treated with 0, 150 or 500 mg/kg PBBs in peanut oil on the day after birth (day l) or with 0 or 100 ppm PBBs from day 8 of gestation until they were killed. Values are means i S.E.M. for 4 animals. Asterisk indicates a statistically significant difference from the control value, p<0.05. ACTIVITY ACTIVITY 82 DCONTROL E150 mg/kg PBBs 500 mg/kg PBBs m 100 ppm PBBs Arylhyd rocarbon 500 Hyd roxylase n s 400 300 200 I00 Epoxide Hydratase 10.0 8.0 6.0 4.0 2.0 0.0 AGE (days) Figure 10 83 respectively, at 56 days of age). Of these treatments hepatic EH activity was also highest following 100 ppm PBBs (approximately 400% of control value at both 28 and 56 days of age). Stimulation of EH in liver following 500 mg/kg PBBs was greater than after 150 mg/kg PBBs (approximately 250% and 150% of control values, respectively, at both 28 and 56 days of age). Metabolism of progesterone, in reactions catalyzed by microsomal enzymes was accelerated 1pfiy1££3_following PBBs. At 28 days of age, 16oPH and 6BPH activities were increased in liver from animals peri- natally exposed to 100 ppm PBBs (approximately 450% and 700% of control value, respectively) (Figure 11). Although microsomal enzyme activities were increased following perinatal exposure to 100 ppm PBBs, activity of the mitochondrial enzyme MAO was decreased in liver by PBBs (approximately 80% of control value)(Figure 12). C. Kidney The kidney weight-to-body weight ratio was not affected by neo- natal or perinatal treatment with PBBs (Figure 4). PBBs also had no consistent effect on renal PMS protein although protein was increased 28 days after treatment of 7-day old pups with 150 mg/kg (135% of control value) (Figure 15). Histopathological changes were not observed in kidney after neonatal treatment with 150 or 500 mg/kg PBBs or perinatal treatment with 100 ppm PBBs when examined at 28 or 56 days of age. However, degenerative histological alterations were noted in adult rats treated with 100 ppm PBBs for 90 days (Figure 13). Renal changes included progressive absolescence of glomeruli. Glomerular tufts were shrunken. "wr 84 Figure 11. Hepatic progesterone hydroxylase activities in rats treated with 0 or 100 ppm PBBs from day 8 of gestation until they were killed at day 28 postpartum. Values are means i S.E.M. for at least 3 animals. Asterisk indicates a statistically significant difference from the control value, p<0.05. 500 400 ACTIVITY (0 O O 85 CONTROL @ 100 ppm PBBs 6B Figure 11 86 Figure 12. Hepatic monoamine oxidase activity in rats treated with 0 or 100 ppm PBBs from day 8 of gestation until they were killed at day 28 postpartum. Asterisk indicates a statistically signifi- cant difference from the control value, p<0.05. 87 Monoamine Oxidase 3.00 2.75 2.50 ACTIVITY 2.25 2.00 .919. ’— CONTROL 100 ppm PBBs Figure 12 88 Figure 13. Renal tissue from rat fed diet containing 100 ppm PBBs for 90 days. There are shrunken and fibrotic glomeruli. Since similar changes were observed in renal tissue from controls but less fre- quently, these alterations may be artifacts. Hematoxylin and eosin stain; x100. Figure 13 90 Bowman's membrane, while not thickened, was diminished in proportion to the shrinking tuft. A single focus of lymphocytes was seen in one kidney. Similar renal histopathological changes were seen in control animals but less frequently. Therefore, these alterations may be artifacts. Although glomerular changes may have been observed microscopi- cally, BUN was not affected by 30 or 90 days exposure to 100 ppm PBBs (Table 8). Similarly, the clearance of inulin (glomerular filtration rate) and the clearance of PAH (effective renal plasma flow) were unaffected by treatment with 100 ppm PBBs (Table 9). Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were not different from controls before or after volume expansion (Table 9). Filtration fraction (GFR/ERPF) was unaffected by PBBs. Urine flow rates (pl/min) were not different between control and PBBs treated animals. Fractional sodium excretion in control and PBBs exposed animals was not significantly different before or following volume expansion (Figure 14). Perinatal and continuous exposure to 100 ppm PBBs had no effect on serum calcium concentration or bone mineral mass at 112 days of age (Table 10). Perinatal exposure to 100 ppm PBBs had no effect on the concentration of vitamin A in kidney (Table 11). Treatment of immature rats with 150 mg/kg PBBs or adults with 100 ppm PBBs for 30 or 90 days had no effect on the 13hy1££3_accumulation of PAH and NMN by thin renal cortical slices (Table 12). Exposure to 100 ppm PBBs did not affect the ability of renal slices to produce ammonia or glucose (Table 13). 91 TABLE 8 Effect of PBBs on Blood Urea Nitrogen (BUN)a Treatment Duration BUN (mg urea nitrogen/100 m1/blood) Control 30 days 17.28i2.75 100 ppm PBBs 30 days 22.19i1.92 Control 90 days 19.06i1.86 100 ppm PBBs 90 days 18.59:1.52 aValues are means i S.E.M. for 4 animals. Adult female rats were fed 0 or 100 ppm PBBs for 30 or 90 days. 92 TABLE 9 Effect of PBBs on Glomerular Filtration Rate (GFR) and Effective Renal Plasma Flow (ERPF) b GFR (m1 / min) ERPF (m1 / min) Period Control 100 ppm PBBs Control 100 ppm PBBs I 2.46i0.l6 2.34i0.21 10.87i0.63 8.77i0.97 II 2.86i0.51 3.14:0.43 10.95il.94 10.67i0.78 III 2.77:0.59 2.51:0.17 9.61i1.54 9.05i1.73 IV 2.40i0.24 1.95:0.27 10.39i1.77 10.66il.95 aValues are means i S.E.M. for 4 animals. Adult female rats were maintained on diet containing 0 or 100 ppm PBBs for 90 days. Rat plasma-saline (4% of body wt) was infused after period I. 93 .mamaflom q How .z.m.m H momma mum mmoam> .mmma mGOHumGHEHmumm Hausaufimmm mmuzu mam Auswflms meon Nev deHmmlmammHa umu qua mums momsmaxo madao> meme mameflcm .cowumcwauoumm Houuooo Houm< .mhmv om now mmmm 8mm ooH so 0 wsflcflmuooo uofiw mom muse mumm .cOHumnoxm sowmom Hmoo«uomum so mmmm mo mommmm .qH ouowflm 94 «a ppsmem Ame: ewes... ON m... Oi m6 name can o2 I name Enno I I md QF 0w QN m.N 0....” md (%) NOIIEHOXE WI'IIOOS 'IVNOIiOVUd 95 TABLE 10 Serum Calcium Concentration and Bone Mineral Mass in Female Rats Exposed to PBBs Treatme t Serum Calcium Bone Mineral n Concentration (pg/m1) Mass (g/cmz) Control 88i6 0.220i0.029 100 ppm PBBs 86i10 0.209i0.028 aValues are means i S.E.M. for 5 (serum calcium concentra- tion) or 10 (bone mineral mass) animals. Animals were exposed to 0 or 100 ppm PBBs from day 8 of gestation until 16 wks postpartum, when measurements were made. 96 TABLE 11 Effect of Perinatal Exposure to PBBs on the Concentration of Vitamin A in Kidney of Rats PBBs Vitamin A Concentration (ppm) (pg/g) 0 4.66:0.83 100 4.93:0.67 aValues are means i S.E.M. for at least 4 animals. Rats were treated with 0 or 100 ppm PBBs from day 8 of gestation until 28 days postpartum at which time vitamin A was quantified. 97 TABLE 12 Effect of PBBs on the Accumulation of PAH and NMNa S/M Ratio Treatment PAH NMN 30 Daysb Control 10.17il.46 5.71:0.40 100 ppm PBBs 9.84:0.87 5.59:0.22 90 Daysb Control 10.49i0.43 5.79:0.10 100 ppm PBBs 10.16i0.76 5.20i0.l7 Acutec Control 8.29:0.81 5.10:0.31 150 mg/kg PBBs 8 56i0.33 5.63:0.17 aValues are means i S.E.M. for at least 3 animals. bAdult female rats were fed 0 or 100 ppm PBBs for 30 or 90 days. cTwo week old rats were treated with PBBs 72 hrs prior to sacrifice. 98 TABLE 13 Effect of PBBs on Ammoniagenesis and Gluconeogenesisa Net Production (pmole/mg wet tissue wt/hr) Treatment Ammonia Glucose Control 3.39:0.09 0.03i0.01 100 ppm PBBs 3.45i0.09 0.03:0.01 aValues are means i S.E.M. for 4 animals. Adult female rats were fed 0 or 100 ppm PBBs for 90 days. 99 Activity of renal microsomal enzymes increased with age in control and PBBs treated rats (Figure 15). Administration of 150 mg/kg PBBs to 7 day old pups increased the activity of both AHH and EROD in kidney (Figure 15). Following 150 mg/kg PBBs, renal AHH and EROD activities were highest after 63 days (450% and 1800% of control values, respectively). Renal EH activity was not increased by 150 mg/kg PBBs (Figure 15). BP—2—0H, BP-4-0H and Hex-0H activities were not detectable in kidney. Renal EH activity also was not affected by neonatal treatment with 150 or 500 mg/kg PBBs or perinatal exposure to 100 ppm PBBs (Figure 16). However, renal AHH was stimulated at both 28 and 56 days of age by all treatments with PBBs (Figure 16). Activity of AHH in kidney was increased to the greatest extent following 100 ppm PBBs and was greater at 56 than 28 days of age (approximately 1900% and 700% of control values, respectively). Renal AHH activity was higher follow- ing 500 than 150 mg/kg PBBs (approximately 200% and 150% of control value, respectively, at 28 days of age). Activity of AHH in kidney following 500 or 150 mg/kg PBBs was greater at 56 than 28 days of age (approximately 650% and 150% of control values, respectively, at 56 days of age). D- Bears. The heart weight-to-body weight ratio was not affected by peri- natal and continuous exposure to 100 ppm PBBs (Figure 17). Exposure to 100 ppm PBBs also had no effect on the 1p_y1££3_accumulation of (3H)—d,l~metaraminol by ventricular slices when determined at 14, 28, 56, 84 and 98 days of age (Table 14). Left atria from 70 day old rats 100 .mo.ove .odam> Homoeoo mam Bouw mucouommww .mumu m ummma um sow .z.m.m H some was mucomoue .ssuumaumon m hem so mmmm wx\we omH go o :uHB mooEumouu noumm moaflu msowum> um mums :H moHuH>Huom seesaw mam cHouone ucmumouoaom Hmfiumconooufieumoa Hmsom .mH ouswwm ucmofiwwowfim haamofiumflumum m moumommcfi xmfluoum< low uefiom Loom 101 ZO_._.<¢Pm_2_EO< Gwhm( m>1 wD.X2w me ooomum 9 mound Bu: 2 some 9 o O O q N O O unw/umuud Btu 103119 JNIHAIS "10w u 0‘0 ionu SAIIVHII no an I.” 0:11.. IIIII o IIIIIIIIIIIIIII OIIII \\ III°\\ / *0 20_h<¢hm.z_20< wau< m>Ihwwo l0|ZED¢Qmw¢>XOIhw h map 0 *0 c name I JOKPZOO I I 2.2.0»: min— 00.0 a 0.0 On On ON 00 ugm/ugsamd 5w NIJI'IUOSJII 53|°W u 'lM anssu 13M ws/Sw 102 Figure 16. Effect of neonatal or perinatal treatment with PBBs on activity of arylhydrocarbon hydroxylase and epoxide hydratase in kidney. Female rats were treated with 0, 150 or 500 mg/kg PBBs in peanut oil on the day after birth (day 1) or with 0 or 100 ppm PBBs from day 8 of gestation until they were killed. Values are means i S.E.M. for 4 animals. Asterisk indicates a statistically signi— ficant difference from the control value, p<0.05. ACTIVITY ACTIVITY 12.0 10.0 8.0 6.0 4.0 2 .0 0A 0.3 0.2 0.1 103 Arylhydrocarbon Hydroxylase D CONTROL E 150 mg/kg PBBs 500 mg/kg PBBs m 100 ppm PBBs *- r‘1 a Epoxide Hyd ratase p L§ 28 AGE(days) Figure 16 104 um How .z.m.m H momma mum mmsHm> so O cu Oomoexo mums mumm .mHmBflfim M ummwfl .oHumu nemea moonuoouucmeB snow; was so wmmm mo nommmm .OoHHHx mums mono HHuao sOHuMumow mo w map aouw mmmm Ema OOH .AH opomam 105 3am“: w0< AH ooomem mu Ono Owd ONO IOOIXI 1M A008 / 1M .lHVBI-I 106 TABLE 14 Effect of PBBs on the Accumulation of (3H)-d,1-Metaraminola Age (days) Treatment S/M Ratio 14 Control 1.79i0.04 100 ppm PBBs l.94i0.10 28 Control 2.68i0.24 100 ppm PBBs 2.83i0.13 56 Control 3.04:0.08 100 ppm PBBs 2.95:0.10 84 Control 3.44i0.26 100 ppm PBBs 3.40:0.24 98 Control 4.01:0.47 100 ppm PBBs 3.93:0.45 aValues are means i S.E.M. for 4 animals. Animals were exposed to 0 or 100 ppm PBBs from day 8 of gestation until experiments were performed. 107 perinatally and continuously exposed to 100 ppm PBBs did not differ from controls in susceptibility to ouabain-induced arrhythmias, control twitch tension or maximum inotroPic response to ouabain (Table 15). However, the maximum inotropic response to calcium was greater in atria from PBBs exposed rats (approximately 200% of control value) (Table 15). Systolic arterial pressure did not differ between controls and adult rats dietarily exposed to 100 ppm for 90 days (Table 16). E. Lung The lung weight-to—body weight ratio was not affected by peri- natal and continuous exposure to 100 ppm PBBs (Figure 18). When determined at 28 days of age, the clearance of AI and 5HT by isolated perfused lungs from rats perinatally exposed to 100 ppm PBBs was reduced (85% and 89% of control values, respectively) (Table 17). Metabolism of 5HT by isolated perfused lungs was also reduced by PBBs (85% of control value) (Table 17). However, perinatal exposure to PBBs had no effect on MAO, ACE or EH activities in pulmonary homo- genates (Figures 19 and 20). Pulmonary AHH activity was increased above controls at 28 and 56 days of age by perinatal and continuous exposure to 100 ppm PBBs (approximately 300% and 550% of control values, respectively) (Figure 20). F. Testis and Ovapy The testis weight- or ovary weight-to-body weight ratios were not affected at 100 days of age by perinatal and continuous exposure to PBBs (Figure 21). Perinatal and continuous exposure to 100 ppm PBBs did not produce' histopathological changes in testis, ovary or uterus 108 TABLE 15 Effect of P333 on Left Atrial Susceptibility to Quabain-Induced Arrhythmia, Twitch Tension and Inotropic Response to Quabain or Calcium Control 100 ppm PBBs Quabain concentration for onset 1x10_5M 5x10-4M of arrhythmia Control twitch tension 1.16i0.25 g 1.11i0.21 g Inotropic response to ouabain (% 3.56i1.70 5.02i2.74 change from control) quabain, 1.5x10'6M Inotropic response to calcium (% 12.14i2.71 24.66i4.08b change from control) calcium, 1.0x10'2M aValues represent means i S.E.M. for 5 animals. Animals were exposed to 0 or 100 ppm PBBs from day 8 of gestation until 70 days postpartum when experiments were performed. bSignificantly different from control value, p<0.50. 109 TABLE 16 Effect of PBBs on Mean Systolic Blood Pressurea * Treatment Unanesthetized Anesthetized Control 122.0i3.0 97.5i4.3 100 ppm PBBs 121.5i2.4 94.5:5.3 _L_‘ aValues are means i S.E.M. for 4 animals. Adult female rats were maintained on diet containing 0 or 100 ppm PBBs for 90 days. Units are mmHg. 110 um new .z.m.m no O on Ommomxo ouoa mumm H wcmwa Guam mwflHm> .mHmeHom m unmoH .OoHHHx oum3 mono HHua: sowumuwow mo w ham Baum mmmm Ema OOH .OHumu uanoB meonIoulustoe wooH use so mmmm mo uoomwm .wH ouome 111 we opomam _m>o3m0< ommo top cos a 30528 D and 00;. cm. P OO.N IOOI-XILM MICE/1M 9Nfl'l 112 TABLE 17 Effect of Perinatal Treatment with Polybrominated Biphenyls on the Clearance of Angiotensin I (AI) and 5-Hydroxytryptamine (5-HT) by Isolated Perfused Rat Lungs AI 5-HT . b . b . . c A Removal % Removal % Metabolized Control 53.16i2.64 79.72i2.09 29.69i1.33 PBBsd 45.3o:2.17° 70.84:2.22° 25.3o:1.33° aValues represent mean i S.E.Mv of 9 determinations in 28 day old rats. Calculated from the transpulmonary difference in perfused AI (1 ng/ml) or 5-HT (0.1 pM/L). cCalculated from the fraction of radiolabel appearing as metabolite in the effluent medium. dDietary PBBs (100 ppm) were administered to the dam from day 8 of gestation to 28 days postnatally. eSignificantly different from control (p<.05). 113 Figure 19. Effect of perinatal treatment with PBBs on the activity of angiotensin-converting enzyme and monoamine oxidase in lung. Rats were exposed to 0 or 100 ppm PBBs from day 8 of gestation until they were killed at day 28 postpartum. Values are means i S.E.M. for at least 4 animals. 25.0 20.0 ACTIVITY a O 10.0 1.25 ACTIVITY '0 O 0 .75 114 Angiotensin-Converting Enzyme Monoamine Oxidase a PBBs Figure 19 115 Figure 20. Effect of PBBs on activity of arylhydrocarbon hydroxy- lase and epoxide hydratase in lung. Rats were exposed to 0 or 100 ppm PBBs from day 8 of gestation until they were killed. Values are means i S.E.M. for at least 3 animals. Asterisk indicates a statistically significant difference from control value, p<0.05. ACTIVITY ACTIVITY 2.0 1.5 1.0 0.5 0.4 0.3 0.2 0.1 Arylhyd rocarbon Hydroxylase it h g n EPOXIdO Hyd ratase _|_ I__ 116 28 AGEldaysl D CONTROL m 100 ppm P883 Figure 20 117 Figure 21. Effect of PBBs on the testis weight- or ovary weight-to- body weight ratios. Rats were exposed to O or 100 ppm PBBs from day 8 of gestation until they were killed. Values are means i S.E.M. for at least 4 animals. Ovary wt./Body wt. [x100] Testis wt./Body wt.lx100l 118 1.2 0.4 0.06 0.04 Control Figure 21 119 when examined at 56 days of age. At 100 days of age, testosterone and progesterone concentrations were not affected by PBBs (Table 18). Perinatal exposure to 100 ppm PBBs also had no effect on age of testes descent, vaginal perforation or estrus cycle length (Table 19). G. Response to Xenobiotics Duration of anesthesia following pentobarbital was decreased at 84 days of age by neonatal treatment with 150 or 500 mg/kg PBBs or perinatal exposure to 100 ppm PBBs (Figure 22). Sleeping time was shortest after pretreatment with 100 ppm P333 (10% of control value). Sleeping time was reduced more by 500 than 150 mg/kg PBBs (25% and 75% of control values, respectively). Duration of anesthesia following diethyl ether was not affected by PBBs. At 90 days of age, sleeping time after diethyl ether for control rats and animals perinatally and continuously exposed to 100 ppm PBBs did not differ (Figure 23). Bromobenzene lethality was enhanced by pretreatment with PBBs. At 49 days of age, the median time to death (LTSO) of animals given approximately an LD85 of bromobenzene was reduced by neonatal treat— ment with 150 or 500 mg/kg P335 (74% and 62% of control value, re- spectively) (Table 20). Digitoxin lethality was decreased by pretreatment with PBBs. At 90 days of age, the LT of animals given approximately an LD f 50 85 ° digitoxin was increased by perinatal and continuous exposure to 100 ppm PBBs (130% of control value) (Table 21). 120 TABLE 18 Concentration of Testosterone and Progegterone in Serum from Rats Exposed to PBBs Treatment Testosterone Progesterone Control 3.77:1.54 366.28i11.10 100 ppm PBBs 4.17i0.37 323.82: 8.31 aValues represent ng/ml, expressed as means i S.E.M. for at least 5 animals. Animals were exposed to 0 or 100 ppm PBBs from day 8 of gestation until 100 days postpartum when serum was collected from males (testosterone) and females (progesterone). 121 TABLE 19 Vaginal (Estrus) Cycle Length and Age of Vaginal Opening and Testicular Descent in Rats Exposed to PBBsa Treatment Parameter Control 100 ppm PBBs Testicular Descent 24.6i0.4 25.0i0.7 Vaginal Opening 37.4il.7 39.1i2.1 Vaginal Cycle Length 4.6i0.3 4.8:0.4 aValues are means i S.E.M., in days, for at least 4 litters (testicular descent and vaginal Open- ing) or 10 animals (vaginal cycle length). Animals were exposed to 0 or 100 ppm PBBs from day 8 of gestation through examination periods. 122 .modvnH .mmcommmu Houucoo osu aoum oucmMQMMHw ucmoHMchHm maamowumfluMum m mmumowvcfl xmfiuoum< .mamefldm m How .z.m.m H mcmoa mum monam> .xoammu wcfluswfiu cocwmwmn mamawam kuc: :OHu00mcw mo mafia mm wwvuouou mm3 mfimozummcm mo aowumuso .m.H wmumumflafiawm mm3 kufinumnoucmm wx\wa oq mafia noflnk um mwm mo numb cw Hausa coaumumow mo w zmw Eoum mmmm 8am ooa Ho 0 sufls no sumac smuwm how so HHo uscmmc ca mmmm wx\ma oom no omH .o :oH3 woumouu oum3 moon mamawm .Hmuwnumnoucmm kn vocab loudmammaummcm mo cofiumusw mnu so mmmm nuw3 ucoaummuu HmumcHuoa no Hmumaooc mo uoowmm .NN muswflm 123 § 3:; alt l|ll|ll|||||||lllllllllllllllllllllllllllll 100 ppm Oppm O O O O O O O IO 0 IO 0 ID ‘00 CM CM 1' '- [ugw] vusaHlsauv :Io Mouvano PBBS Figure 22 124 H mammE mm pmwnoomn songs as mamamm .mamancm q now .z.m.m onm mosam> .vmcnmwmn mm3 xmammn wanunwnn man anus: nonwonmmmw aonm Hm>oamn mo mafia mmB mnmonummam mo conumnso .momm.om now nmsuo ahnumwp on womomxw onus mamanam Mann own mo mxmw om anus: conumummw mo w >mw Bonn mmmm 8am 00H no G on vmmoaxm mnm3 moon .nmsum ahzumnp kn vmoswona mammsumocm mo conumnsw mzu co mmmm mo nommwm .mm onswnm 125 mm .tnwan mmmacfiaoow .obcoo [93$] VISEHLSHNV :IO NOILVHnCI 126 TABLE 20 Median Time to Death After Administration of Bromobenzene (2820 mg/kg) in 49 Day Old Male Rats Neonatally Treated with Polybrominated Biphenyls Pretreatmentb LT50 (95% confidence limit) Control 7.2 (6.1-8.5) 150 mg/kg PBBs 5.3 (4.7-5.9)c 500 mg/kg PBBs 4.5 (3.8-5.5)0 aValues are in hr for 8 (500 mg/kg P333) or 10 (centrol and 150 mg/kg PBBs) animals. bAnimals were treated with peanut oil, 150 mg/kg PBBs or 500 mg/kg PBBs in peanut oil on the day after birth (day l). cSignificantly different from control, p<0.05. 127 TABLE 21 Median Time to Death After Administration of Digitoxin in Female Rats Pretreated with PBBs PBBs (ppm)b LTSO (95% confidence limit) 0 326.1 (287.4-366.8)b 100 424.5 (389.7-472.4) aValues are means in min for 9 animals. Female rats were treated with 0 or 100 ppm PBBs from day 8 of gestation until 90 days postpartum at which time 10 mg/kg digitoxin in dimethyl sulfox— ide was administered i.p. bSignificantly different from the control value, p<0.50. 128 H. Response to Exogenously Administered Steroid Hormones Duration of anesthesia following 150 mg/kg progesterone was reduced by perinatal exposure to 10 or 100 ppm PBBs (27% and 11% of control values, respectively) (Figure 24). Radioactivity was de— creased in whole brain homogenates, HEX and EA extracts 3 hr after (14C)progesterone by 100 ppm PBBs (22%, 13% and 24% of control values, respectively) (Figure 25). The ratio of EA to HEX extractable radio- activity was increased after 3 hr by 100 ppm PBBs (183% of control value). Serum radioactivity was also reduced 3 hr following pro— gesterone by 100 ppm PBBs (56% of control value) (Figure 26). At waking, radioactivity in brain was similar regardless of pretreatment. When determined at waking, EA to HEX extractable radioactivity in brain and radioactivity in serum also were not affected by PBBs. Although PBBs did not alter seminal vesicle weight-to-body weight ratio (SVW/BW), the increase in SVW/BW 72 hr following 20 mg/kg testosterone was diminished by perinatal exposure to 100 ppm PBBs (79% of control) (Figure 27). Radioactivity in testis 24 hr after (3H)- testosterone was reduced by pretreatment with 10 or 100 ppm PBBs (79% and 70% of control value, respectively) (Figure 28). Radioactivity in serum 24 hr after (3H)testosterone was also reduced by exposure to 10 or 100 ppm P333 (81% and 64% of control values, respectively) (Figure 29). Although PBBs did not modify uterus weight—to-body weight ratio (UW/BW), the increase in UW/BW 4 hr following 1 or 3 ug/kg estradiol- 178 was reduced by perinatal exposure to 100 ppm PBBs (86% and 78% of control values, respectively) (Figure 30). Radioactivity in uterus 4 129 Figure 24. Effect of perinatal exposure to 0, 10 or 100 ppm PBBs on the duration of anesthesia produced by progesterone. Rats were treated with 150 mg/kg progesterone i.p. at 28 days of age. Duration of anesthesia was recorded as time of injection until animal regained righting reflex. Values are means i S.E.M. for at least 6 animals. Asterisk indicates a statistically significant difference from the control response, p<0.05. 0) O O N O O DURATION OF ANESTHESIA lminl 8 0| 0 O h 0 0 ‘3 130 E] CONTROL .10ppm PBBs & 100 ppm PBBs Figure 24 131 Figure 25. Effect of perinatal exposure to O or 100 ppm PBBs on the concentration of equivalents of progesterone- C in brains of rats treated with progesterone-14C. Rats were treated with 150 mg/kg progesterone-14C i.p. at 28 days of age. Whole brains were excised 3 hr later. Radioactivity was determined in brain homo- genates and n-hexane and ethyl acetate extracts of homogenates. Values are means i S.E.M. for at least 3 animals. Asterisk indicates a statistically significant difference from control value, p<0.05. 4O 0) O EQUIVALENTS OF PROGESTERONElkg/Ql .5 N O O 132 D CONTROL 8 100 ppm P883 ’0’? * o’o’c ’:’:‘ , , , 0 o c * ’9’. {>9 {pQ ”an! ”fiaf 5.01 5'." 5.0.. TOTAL HEXANE mm ACETATE EXTRACTABLE exrucmue Figure 25 133 Figure 26. Effect Of perinatal exposure to O or 100 ppm PBBs on the concentration of equivalents Of progesterone-14C in serum Of rats treated with progesterone-l C. Rats were treated with 150 mg/kg progesterone-140 i.p. at 28 days of age. Serum was collected 3 hr later. Values are means i S.E.M. for at least 3 animals. Asterisk indicates a statistically significant difference from control value, p<0.05. -» __.. _____‘ EQUIVALENTS OF PROGESTERONElhg/mll 8 8 8 ‘6 8 5‘ 8 8 —I. O 134 El CONTROL Q 100 ppm PBBs Figure 26 135 Figure 27. Effect of perinatal exposure to O, 10 or 100 ppm PBBs on the increase in seminal vesicle wt-to-body wt ratio produced by testosterone. Rats were treated with O or 20 mg/kg testosterone s.c. at 25 days of age. Seminal vesicles were excised and weighed 72 hr later. Values are means i S.E.M. for at least 6 animals. Asterisk indicates a statistically significant difference from the control response, p<0.05. SEMINAL VESICLE Wt/BOOY WT. (x100) 136 coNTROL 10 ppm P333 @100 ppm P333 TESTOSTERONE (mg/ kg) Figure 27 137 Figure 28. Effect of perinatal exposure to O, 10 or 100 ppm PBBs on the concentration of equivalents Of testosterone-3H in testis of rats treated with testosterone-3H. Rats were treated with 20 mg/kg testosterone-3H s.c. at 25 days of age. Testes were collected 24 hr later. Values are means i S.E.M. for at least 6 animals. Asterisk indicates a statistically significant difference from control value, p<0.05. '6: E ‘\~ m E. ”I u': z 3.00 o a: m '— 8 5 m 2.00 l- u. 0 a) '2 m 1.00 a‘ 2 2 O l.l.l 0.00 138 Mo? CONTROL E 100 ppm PBBs Figure 28 139 Figure 29. Effect Of perinatal eXposure to 0, 10 or 100 ppm PBBs on the concentration Of equivalents of testosterone- H in serum Of rats treated with testosterone—3H. Rats were treated with 20 mg/kg testosterone-3H s.c. at day 25 postpartum. Serum was collected 24 hr later. Values are means i S.E.M. for at least 6 animals. Asterisk indicates a statistically significant difference from the control value, p<0.05. co b O P O o 1 .00 EQUIVALENTS 0F TESTOSTERONE-Mlpg/mn 0.00 140 * .CONTROL Figure 29 .10ppm PBBs EIOOppm PBBs 141 .mo.ovm .mwaommmn Honucoo 0:0 aonm moomnmmmnv ucmonmncwnm kaamonuwnumum m mmumowvcn xmnnmum< .wamafiam m nom .z.m.m n mumps mnm mosam> .nmuma n: q wmnwnm3 was wmmnoxm mnms anon: .Epunmmumoa mm mac um .m.n mud IHOmenumm wx\w1 o.m no o.H zun3 pmummnu mnms mumm .mmHIHOmenumm hp pmuavonm Ofiumn OB hvonlou I03 mpnmu: an ammonoan man so mmmm and OOH no 0H .0 On onsmomxm Hmumcnnmm mo nommwm .om mnswnm 142 m om mnswnm .06. . o 2 .o... o o . o 2 .o... .2 o. .2 o. .2 o. .2 o. .0... .ooo. 3.3.3 A 2-3.353 V“ 009‘ v0. 09 v9. 00 v0. 00 v6. 90 v9. .9 v0; 00 VOA 00 99. .ooo. mmma E3 8. a 8mm 53.9% .6528 D 0000. 0 09.0.0 00m0.0 0000.0 (00L X) 'JM AGOB/IM $08310 143 hr after 1 0r 3 ug/kg (3H)estradiol-l78 was reduced by exposure to 100 ppm PBBs (64% and 32% Of control values, respectively) (Figure 31). Radioactivity in uterus following 3 ug/kg (3H)estradiol-l78 was also reduced by 10 ppm PBBs (57% of control value) (Figure 31). Radioacti— vity in serum following 1 or 3 ug/kg (3H)estradiol was reduced by 100 ppm PBBs (62% and 21% Of control values, respectively) (Figure 32). Radioactivity in serum following 1 ug/kg (3H)estradiol-17B also was reduced by 10 ppm PBBs (76% of control value) (Figure 32). I. Persistence Of Effects Microsomal enzyme activity was increased in liver and kidney even 63 days following a single i.p. injection of 150 mg/kg PBBs to 7- day Old rats (Figures 8, 9, 15). Enzyme stimulation at 63 days following treatment was indicative Of persistence Of PBBs in tissues. Detectable concentrations of P338 were found in various tissues at different times after administration of 150 mg/kg PBBs (Table 22). NO sex dependent differences in tissue concentrations Of PBBs were Observed, so data from males and females were combined. Concentration of PBBs in brain was low at all times and remained fairly constant until 28 days following treatment. By day 63, con- centration of PBBs in brain had decreased considerably. Sufficient fat was Obtained for analysis on days 28 and 63. Concentration of P338 in fat increased markedly between days 28 and 63. Concentrations of PBBs in heart and liver were lower than fat and higher than brain. Similar to brain, PBBs in heart and liver decreased markedly between days 28 and 63. Muscle, skin and lung contained similar concentra- tions of P339 through 63 days with concentrations in lung diminishing more rapidly than those in muscle and skin. 144 Figure 31. Effect Of perinatal exposure to 0, 10 or 100 ppm PBBs on the concentration of equivalents Of estradiol-17B-6,7- H in uterus- Of rats treated with estradiol-17B-6,7-3H. Rats were treated with 1.0 or 3.0 ug/kg estradiol-17B-6,7-3H i.p. at day 28 postpartum. Uteri were collected 4 hr later. Values are means i S.E.M. for 5 animals. Asterisk indicates a statistically significant difference from the control value, p<0.05. !° 9° P 9' o o o o EQUIVALENTS 0F ESTRADIOL-17B-6, 7-‘I-Itpg/m9] '3 .0 o 145 CONTROL 10 ppm PBBS m 100 ppm PBBS * * * Q... 5x. {'1’} In 99% .203 {93 292’: 5393 ESTRADIOL- 178 UJQ/ kg 1 Figure 31 146 Figure 32. Effect of perinatal exposure to 0, 10 or 100 ppm PBBS 0n the concentration of equivalents Of estradiol-17B-6,7-3H in serum Of rats treated with estradiol-l7B-6,7-3H. Rats were treated with 1.0 or 3.0 ug/kg estradiol-17B-6,7-3H i.p. at day 28 postpartum. Serum was collected 4 hr later. Values are means i S.E.M. for 5 animals. Asterisk indicates a statistically significant difference from the control value, p<0.05. O) O O 500 400 § 200 100 EQUIVALENTS 0F ESTRAOIOL-17p-6,7-’H [pg/ml] .0 147 U CONTROL 10 ppm PBBS Q 100 ppm PBBS ESTRADIOL-17J3 [Ag/ kgl . Figure 32 .apunmmumom 5 map so ..Q.H .mmmm wx\wa omH :uHB commonu mnos mamanq< .mHmanam 0 now .z.m.m n opmmnn us 003 w\mmmm w: an memos mnm mosam>o 148 m.onm.m o.mno.oa q.an.m H.Hno.q N.HHH.~ H.onm.o o.oanm.oo H.0HH.o mo o.anm.n c.HnH.mH .N.onm.a m.onm.s H.HHN.HH s.cnm.a «.0 Hw.a H.0Hm.o mm m.HHo.m m.mno.cm N.NHO.HH o.HnH.o m.mnm.qm o.onq.m III s.cno.~ «a w.~nm.ma m.mnw.o~ w.anm.oa III 0.0 no.mo q.ono.m III ~.onw.o n o.HnN.mH N.mno.om m.onm.n III o.NHHH.wn o.onm.~ III H.0Ho.o m N.Hnm.ma N.Hnm.ma m.onm.oa m.ono.m m.mano.mw H.Hnm.m III N.ono.m N H.Nnm.ma o.mnm.mH H.Hno.w III q.~anm.oaa m.onq.m III m.onm.H H mung anxm sauna: no>n4 manummuoH unnmm 0mm anmnm conuomncH mammHH noum< whoa GEOfiuoomaH mawafim m wnH3OHHom mmmm mo maonumnuaooaou osmmfie NN mqmufi>mo Hmocounnmm man aonm mmmm mo mosmnmmeammna .mm enswnn 151 Va mm spawns 20_h<¢hm.z=¢n( umbn< m> Honucoo 050 Sony mononOMMHp ucmo Inmncwnm maamonumnumum m mmumonwsn xmnnouw< .mamancm 8 now .z.m.m n mamas one mosam> .xmammn wsnugwnn vmcnmwwn mamansm anus: GOHOOOmsn mo menu mm povnoomn mm3 mnmonummsm mo sonumnsn .Q.H Hounnnmnoucma wx\w8 oq :un3 wmummnu oneB mumn mama own «0 m%mw wmm was mm u< .owm mo mmmw mm on umnp Honucoo ouco vmamma ones mamansm HH< .BSunmaumom mm new nwsonfiu sonumumow mo w new 8onm mmmm Ema OOH no 0 wm>noomn gonna wasp kn vmmnsa can Ou anon ones mumm .HmOnnnwnouaoa up pooswonm mnmmnumosm mo sonumnsv so wmmm ou enamomxo Hmumcnnom mo uoommo Hmsvnmom .mm onswnm 161 mm onsmnm ESE m0< 32 E8 8. a 35:00 D 0N 0m 00.. 00' VI‘SBHlSENV :IO NOIlVHflG [WWI 162 TABLE 24 Tissue Concentrations of PBBS Following Perinatal Exposure 100 ppm PBBs to PBBS Tissue Treatment Liver Kidney Ovary Fat 28b Control 0.0 0.0 0.0 0.0 27.2i6.9 16.5i4.2 117.4il6.9 282.2i17.3 b Control 100 ppm PBBs 0.1 0.0 0.0 0.3 4.2il.4 5.4il.7 57.1i 8.2 98.1i25.l aValues are means in ug/g wet wt tissue i S.E.M. for at least 3 animals. Rats born to and nursed by dams which re- ceived 0 or 100 ppm PBBs from day 8 of gestation through day 28 postpartum at which time all pups were weaned onto diet free of PBBs. Age of animals in days. 163 Body weight at weaning (28 days postpartum) was lower in animals exposed to 100 ppm PBBs from day 8 of gestation (Fl-100) than controls (80% Of control value) (Figure 38). Survival-to-weaning of Fl-lOO was also less than controls (87% of control value) (Figure 39). The liver weight-to-body weight ratio was increased at weaning in Fl-100 rats, their progeny (F2-100) and F -10 (exposed to 10 ppm PBBs 1 from day 8 Of gestation) (165%, 128% and 126% Of control values, respectively) (Figure 39). Activity of AHH was increased at this age in liver from Fl-lOO, F2 1 2 600%, 400% and 130% Of control values, respectively) (Figure 40). -100, F -10 and F ~10 (approximately l400%, 2-100 and Fl-lO (approximately 250%, 125% and 125% of control values, respectively) Activity Of EH was increased in liver from F -100, F 1 (Figure 40). Renal AHH activity was increased in F -100 and Fl-10 (approxi- l mately 700% and 200% of control values, respectively) (Figure 42). Activity Of EH in kidney was not affected by PBBs (Figure 41). Duration of anesthesia following 150 mg/kg progesterone was reduced in Fl-lOO, F2 1-10 and Fz-lO (10%, 16%, 25% and 50% Of control values, respectively) (Figure 42). Pentobarbital sleeping -100, F time was reduced in F -100, F ~10 and F l 2 1 2 75% Of control values, respectively) (Figure 43). Progeny Of F -100, F -10 (4%, 16%, 8% and 2-100 (F3-1OO) did not exhibit a difference from controls in any response quantified in this investigation. Dose and generation related effects were correlated with hepatic concentrations of PBBs (Table 25). The concentration Of PBBs in liver was highest in pups born to and suckled by dams fed diet containing 164 Figure 38. Effect Of exposure to PBBS on body weight and the liver wt-tO-body wt ratio in subsequent generations. Rats (F0) were fed 0, 10 or 100 ppm from day 8 Of pregnancy until 28 days postpartum at which time all pups (Fl) were weaned onto control diet, allowed to mature sexually, and bred with littermates to produce F2. F2 generation from 100 ppm PBBs was bred with littermates to produce F3. Values are means i S.E.M. from at least 7 litters. Asterisk indicates a statistically significant difference from the control value, p<0.05. BODY WT. [9] LIVER w1'./ BODY WT. th003 80 70 60 50 8.50 7.50 6.50 5.50 4.50 165 0 F.-10 r; -_ E-IOO E—IOO Ig-IOO PBBstpml Figure 38 166 Figure 39. Effect of exposure to PBBS on percent survival-to~weaning in subsequent generations. Rats (F0) were fed 0, 10 or 100 ppm from day 8 of pregnancy until 28 days postpartum at which time all pups (F1) were weaned onto control diet, allowed to mature sexually, and bred with littermates to produce F2. F2 generation from 100 ppm PBBs was bred with littermates to produce F3. Values are means i S.E.M. from at least 7 litters. Asterisk indicates a statistically signi- ficant difference from the control value, p<0.05. SURVIVAL TO WEANING [percent] 100 95 90 85 80 75. 167 *- ' ' ' ’9‘ o o I o 4 VII 0 o , 920‘ I o I o o > o I .0... O E-lO Ig-Io E-IOO E—IOO E-IOO PBBS [ppml Figure 39 \‘ § \ § \ \ h R R h 168 Figure 40. Effect of exposure to PBBS on activity of arylhydrocarbon hydroxylase and epoxide hydratase in liver in subsquent generations. Rats (F0) were fed 0, 10 or 100 ppm from day 8 of pregnancy until 28 days postpartum at which time all pups (F1) were weaned onto control diet, allowed to mature sexually, and bred with littermates to pro- duce F2. F2 generation from 100 ppm PBBS was bred with littermates to produce F3. Values are means i S.E.M. from at least 7 litters. Asterisk indicates a statistically significant difference from the control value, p<0.05. 400 ACTIVITY 8 0) o 8 5 o 10.0 8.0 6.0 4.0 ACTIVITY 2.0 0.0 . 169 Ar)Ilhydrocarbon Hydroxylase «*- Epoxide Hydratase * *- I § \ = § 0 5"“) 5'10 5400 6-100 E-IOO PBBs( ppm) Figure 40 170 Figure 41. Effect Of exposure to PBBs on activity of arylhydrocarbon hydroxylase and epoxide hydratase in kidney in subsequent generations. Rats (F0) were fed 0, 10 or 100 ppm from day 8 of pregnancy until 28 days postpartum at which time all pups (F1) were weaned onto control diet, allowed to mature sexually, and bred with littermates to pro- duce F2. F2 generation from 100 ppm PBBS was bred with littermates to produce F3. Values are means i S.E.M. from at least 7 litters. Asterisk indicates a statistically significant difference from the control value, p<0.05. 171 Arylhyd rocarbon Hydroxylase ACTIVITY Epoxide Hyd ratase 0.5 a 0.3 ACTIVITY 0.2 0.] O E—IO I=,-IO E-IOO F,-IOO E-IOO PBBs(ppm) Figure 41 172 Figure 42. Effect of exposure to PBBS On duration Of anesthesia pro- duced by progesterone in subsequent generations. Rats (F0) were fed 0, 10 or 100 ppm from day 8 of pregnancy until 28 days postpartum at which time all pups (F1) were weaned onto control diet, allowed to mature sexually and bred with littermates to produce F2. F2 generation from 100 ppm PBBs was bred with littermates to produce F3. At 28 days Of age, female rats were treated with 150 mg/kg pro- gesterone, i.p. Duration of anesthesia was recorded as the time Of injection until animals regained righting reflex. Values are means i S.E.M. for at least 4 animals. Asterisk indicates a statistically significant difference from the control value, p<0.05. DURATION OF ANESTH ESlAlm in] 01 O O b O O (I) O O N O O .L O O .0 173 i- #- 'O 0 9.9.4 510 E— -10 F-100 F-100 E-1OO Pssslppml Figure 42 174 Figure 43. Effect of exposure to PBBS on duration of anesthesia pro- duced by pentobarbital in subsequent generations. Rats (F0) were fed 0, 10 or 100 ppm from day 8 of pregnancy until 28 days postpartum at which time all pups (F1) were weaned onto control diet, allowed tO mature sexually and bred with littermates to produce F2. F2 genera- tion from 100 ppm PBBS was bred with littermates to produce F3. Duration Of anesthesia was recorded as the time of injection until animals regained righting reflex. Values are means i S.E.M. for at least 4 animals. Asterisk indicates a statistically significant difference from the control value, p<0.05. a. __=___________________________. 175 h E-IOO E-IOO \\. £- '8' 400 PBBSIpme 510 F. new F. Figure 43 176 TABLE 25 Concentration of PBBS in Liver Following Perinatal Exposure to PBBS Treatment Concentration of PBBS (pg/g wet tissue) Control 0.0 F1—100 33.6i7.9 F2-100 4.1iO.6 F -100