THESIS Michicm State University 3 1293 10063 3944 This is to certify that the thesis entitled HALOGENATED ALIPHATIC HYDROCARBON NEPHROTOXICITY presented by William Michael Kluwe has been accepted towards fulfillment of the requirements for Ph . D . degree in PharmacolOgy & Toxicology / ' . /l¢ajor€rk<;'essor Date 5—1—79 0-7639 «WWWVHbNN» _ #AA OVERDUE FINES ARE 25¢ PER DAY 7 PER ITEM ‘ Return to book drop to remove this checkout from your record. HALOGENATED ALIPHATIC HYDROCARBON NEPHROTOXICITY By William Michael Kluwe 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 ,s ," 1??? ABSTRACT Halogenated Aliphatic Hydrocarbon Nephrotoxicity by William Michael Kluwe Dietary ingestion of polybrominated biphenyls (PBB) and polychlo— rinated biphenyls (PCB) increased renal and hepatic aryl hydrocarbon hydroxylase (AHH) activities in a dietary concentration-dependent manner. Mixed—function oxidase (MFO) activities were also induced in liver and kidney by i.p. administration of 2,3,7,8—tetrachlorodibenzo— p-dioxin (TCDD) and 3—methylcholanthrene (3MC),but sodium phenobarbital (Nan) increased hepatic MFO activities only. Renal and hepatic AHH activities and cytochrome P—450 (P—450) con— centrations in male, Fischer 344 rats were increased by treatments with PBB, PCB and 3M0. Nan increased hepatic AHH activity and P—450 content only. The rates of increase (and decline to normal values) of AHH activities following single oral doses of P33, PCB and 3MB were much greater in the kidney than in the liver. Treatment with 3MC increased the susceptibilities of renal and hepatic AHH to inhibition by u-napthflavone (ANF) in_vitro while Nan increased the susceptibility of hepatic but not renal AHH to inhibition by metyrapone (MET). PBB and PCB increased the susceptibility of renal AHH to inhibition by ANF but did not alter the susceptibility of hepatic AHH to inhibition by ANF or MET. Renal AHH was significantly William Michael Kluwe less susceptible than hepatic AHH to inhibition by SKF 525—A and MET. Low concentrations of ANF stimulated hepatic AHH activity in_gitrg but inhibited renal AHH activity. Renal AHH activities were less suscep- tible than hepatic AHH activities to reduction by i.p. administration of SKF 525—A and piperonyl butoxide (PB). Treatments of mice with PBB and PCB potentiated the nephrotoxicity and hepatotoxicity of carbon tetrachloride (C614) in rats and mice. PBB, PCB and HCB also increased total lipid content of the liver but not the kidney. Treatments of mice with PBB and Nan increased the hepatotoxicity of chloroform (CHCl3). CHCl3 nephrotoxicity was increased by PBB but decreased by PCB, 3MC and TCDD. Preadministration of PB reduced the toxicity of CHCl3 in mice. Administration of SKF 525—A before (120 min) CHClS, and SKF 525-A or PB after (60 min) CHCl3, potentiated CHCl3 toxicity. CHCl3 depleted renal and hepatic glutathione (GSH) in intact mice in a dose—dependent manner. PBB enhanced CHCl3 depletion of renal and hepatic GSH. PCB blocked CHCl3 depletiOn of renal GSE but did not alter CH013 depletion of hepatic GSH. Diethyl maleate reduced renal and hepatic GSH concentrations and increased the susceptibility of mice to CH013 toxicity. Incubation of (140)—CHC13 with renal and hepatic microsomes re- sulted in the covalent binding of radioactivity to microsomal protein (290 pmoles/mg protein/5 min, liver; 15 pmoles/mg protein/5 min, kidney). Hepatic microsomes from PBB and PCB treated mice bound more radioactivity than hepatic microsomes from control mice. Renal William Michael Kluwe microsomes from PCB treated mice bound more radioactivity than renal microsomes from control and PBB treated mice. Radioactivity was covalently bound to renal and hepatic endoplas- mic reticulum (ER), mitochondria (M), cytoplasmic protein (CP) and nuclear RNA and DNA following i.p. administratiOn of (14C)-—CHCl3 to mice. The magnitude of binding to hepatic M and CP was increased by pretreatment with PBB and PCB but binding to renal ER, M, CP, RNA and DNA was decreased. Clearance of radioactivity from venous blood was a first—order process and occurred more rapidly in PBB and PCB pretreated mice than in control mice. The results of this dissertation suggest that the nephrotoxicities of halogenated aliphatic hydrocarbons may depend, in part, on biotrans— The formation to a metabolite which is re3ponsible for the toxicity. site of toxic metabolite formation may be liver, kidney or both. ACKNOWLEDGEMENTS I would like to express my sincere appreciation to Drs. Steven Aust, Theodore Brody, Bernard Schwetz and Robert Roth for constructive criticism of the dissertation research and especially to Dr. Jerry Hook for moral support and intellectual guidance throughout my graduate studies at Michigan State University. I would also like to thank Forest Clark, Cathy Herrmann, Robert McNish, Harriett Sherman and Kenneth Smithson for their excellent technical assistance. Most of all I would like to thank Cathy, who stood fast beside me through some exciting, but often difficult, times. Much of this dissertation is owed to her patience and soothing presence. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii LIST OF TABLES— vi LIST OF FIGURES — viii INTRODUCTION— —-— l A. The Kidney as a Target Organ w 1. Incidence and costs of human nephropathy ---------- l 2. Pathophysiology of toxic nephroPathy 2 3. Aspects of renal structure and function predispo- sing to kidney injury 4 4. Nephrotoxic chemicals 6 B. Metabolic Activation of Toxicants 8 l. Reactive metabolites and tissue injury 8 2. Locations and functions of drug-metabolizing enzyme (DME) systems 9 3. Balance between enzymatic toxification and detoxi- fication— 13 4. Tissue—specific metabolic activation 16 C. Biotransformation Capacity of the Kidney l7 1. Characteristics—— — l7 2. Physiological and toxicological significance of renal biotransformation 20 D. Nephrotoxicity of Halogenated Aliphatic Hydrocarbons--- 23 1. Incidence 23 2. Toxicological manifestations 26 3. Mechanisms of toxicity 28 . Purpose— 31 . Objectives — — 32 MATERIALS AND METHODS- 34 A. Animals and Experimental Diets —-- 34 B. Toxicity Tests 34 1. Serum analyses—- — 34 iii TABLE OF CONTENTS (continued) Page 2. Renal cortical slice accumulation of PAH and TEA-— 35 3. LDS determinations 36 4. Hisgology 36 5. Definitions of toxicity 37 C. Analyses of Components of the Drugdmetabolizing Enzyme Systems 38 1. Tissue preparation 38 2. p—Chloro-N-methylaniline N-demethylase (PCNMA) assay . 38 3. Aryl hydrocarbon hydroxylase (AHH) assay —————————— 39 4. Biphenyl—Z-hydroxylase (BP-Z—OH) and biphenyl—4- hydroxylase (BP-4—OH) assays 40 5. Cytochrome P-450 assays 40 D. Individual Experiments 41 l. Time—dependency and organ-specificity of induction and inhibition of renal and hepatic drug-metaboli- zing enzyme systems 41 2. Effects of stimulation and inhibition of renal and hepatic drug—metabolizing enzyme systems on CHCl3 and C01 toxicity 43 3. Interactions of CHCl3 with renal and hepatic glutathione (GSH)-— — 47 4. Covalent binding of CHCl3 metabolites in mice ————— 49 E. Statistics 52 RESULTS- 54 A. Enzyme Induction and Inhibition 54 1. Effects of length of exposure and dietary concen— tration of PBB and PCB on renal and hepatic AHH activities 54 2. Effects of Nan, 3MC, PCB and TCDD on renal and hepatic enzymes in mice 57 3. Effects of single and multiple administration of PBB, PCB, Nan and 3MC on renal, hepatic and testicular enzyme activities and cytochrome P-450 concentrations in rats 57 4. Inhibition of renal and hepatic AHH activity in Vitro 69 5. Time—dependent effects of single doses of SKF 525—A and PB on renal and hepatic PCNMA and AHH activities in rats and mice 73 iv TABLE OF CONTENTS (continued) Page B. Enzyme Modulation and CCl4 and 011013 Toxicity ---------- 76 1. Effects of dietary PBB and PCB on C014 toxicity in mice 76 2. Effects of PBB, PCB and HCB on CCl4 toxicity in rats 81 3. Effects of PBB and PCB on hepatocellular GPT and GOT activities in rats 91 4. Effects of maternal consumption of PBB on the toxicities of CCl4 and CHCl3 in developing male rats— 101 5. Effects of dietary PBB on CHCl3 toxicity in mice-- 101 6. Effects of Nan, 3MC, PCB and TCDD on CHCl toxi— city in mice- 107 7. Effects of SKF 525—A and PB on CHCl3 toxicity in mice — 114 C. Interactions of CHCl3 with GSH 114 1. Effects of PBB and PCB on CHC13-induced depletion of GSH 114 2. Effects of diethyl maleate on GSH depletion and CHCl3 toxicity in mice 121 D. Covalent Binding of 011013 Metabolites in Mice 126 1. Covalent binding of CHC13 metabolites to renal and hepatic microsomal protein in Vitro 126 2. Clearance of CHC13 and metabolites from blood and covalent binding to total renal and hepatic pro- tein in Vivo 126 3. Covalent binding to subcellular fractions in vivo- 135 DISCUSSION— 139 A. Chemical Modulation of Renal Drug Metabolism 139 B. Renal Drug Metabolism and CCl4 Nephrotoxicity —————————— 156 C. Renal Drug Metabolism and CHCl3 Nephrotoxicity --------- 163 SPECULATION- 177 A. Phosgene and CHCl3 Toxicity- — 177 SUMMARY— --- 180 BIBLIOGRAPHY— 184 Table 10 11 LIST OF TABLES Page Nephrotoxic Halogenated Aliphatic Chemicals ———————————— 24 Dietary concentration-dependent induction of aryl hydrocarbon hydroxylase (AHH) activities by polybro- minated biphenyls (PBBs) and polychlorinated biphenyls (P033) 55 Time-dependent induction of aryl hydrocarbon hydroxy— lase (AHH) activities by polybrominated biphenyls (PBB) and polychlorinated biphenyls (PCB) -------------- 56 Induction of mixed—function oxidases (MFOs) in liver and kidney 58 Measurements of aryl hydrocarbon hydroxylase (AHH) activities, p-chloro-N-methylaniline N—demethylase (PCNMA) activities and cytochrome P—450 concentrations in liver, kidney and testis of naive rats —————————————— 59 Time—dependent effects of polybrominated biphenyls (PBB), polychlorinated biphenyls (PCB), sodium pheno- barbital (Nan), and 3—methylcholanthrene (3MC) on the location of soret maxima of reduced cytochrome P—450 difference spectra 63 a—Napthoflavone (ANF) inhibition of renal aryl hydro— carbon hydroxylase (AHH) activities in Vitro at various times after a single dose of polybrominated biphenyls (PBB) or polychlorinated biphenyls (PCB) ——————————————— 70 Effects of dietary polybrominated biphenyls (PBB) and polychlorinated biphenyls (PCB) on liver weight/body weight (LW/BW) and kidney weight/body weight (KW/BW)——— 77 Effects of polybrominated biphenyls (PBB) on the acute LD50 of C014 80 Effects of aromatic organohalides on liver weight/body weight (LW/BW) and kidney weight/body weight (KW/BW)——- 82 Effects of aromatic organohalides and CCl4 on lipid Content of Liver (L) and kidney (K) ———————————————————— 83 vi LIST OF TABLES (continued) Table Page 12 Effects of aromatic organohalides and CCl4 on 48—hour survival ----------------------------------------------- 85 13 Effects of aromatic organohalides and C014 on 48-hour body weight gain --------------------------------------- 86 14 Effects of dietary polybrominated biphenyls (PBB) and polychlorinated biphenyls (PCB) on hepatic GPT activity 100 15 Effects of dietary polybrominated biphenyls (PBB) on liver weight/body weight (LW/BW) and kidney weight/body weight (KW/BW) ----------------------------------------- 106 16 Percent of administered dose of CHC13 remaining in liver and kidney 3, 6 and 12 hr after i.p. administra— tion --------------------------------------------------- 132 17 Covalent binding of CHCl3 metabolites to subcellular fractions in vivo —————————————————————————————————————— 136 18 Covalent binding of CHCl3 metabolites to RNA and DNA in vivo ———————————————————————————————————————————————— 138 Figure l 2 10 ll 12 13 LIST OF FIGURES Page Metabolic fate of xenobiotic compOunds ————————————————— 14 Time—dependent induction of cytochrome P-450 in liver, kidney and testis —————————————————————————————————————— 60 Time—dependent induction of aryl hydrocarbon hydroxy— lase (AHH) activity in liver, kidney and testis ———————— 64 barbital (Nan) or 3—methylcholanthrene (3MC) —————————— 67 Inhibition of renal and hepatic microsomal aryl hydro— carbon hydroxylase (AHH) activities in Vitro by allyl— isopropylacetamide (AIA), SKF 525— , metyrapone (MET), piperonyl butoxide (PB) and d-napthoflavone (ANF) —————— 71 Time—dependent inhibition of p—chloro—N-methylaniline N—demethyla (PCNMA) and aryl hydrocarbon hydroxylase (AHH) activities by SKF 525—A and piperonyl butoxide (PB) ———————————————————————————— ----------------------- 74 Effects of polybrominated biphenyls (PBB) and poly— chlorinated biphenyls (PCB) and 0014 on SGOT and PAH S/M ———————————————————————————————————————————————————— 78 Effects of aromatic organohalides and CCl4 on renal 002 and PAH S/Ms --------------------------------------- 87 Effects of aromatic organohalides and CCl4 on SGPT activities --------------------------------------------- 89 Sections of livers from rats receiving 0.00 ml/kg CC14— 92 Hepatic tissue from rats receiving 0.03 ml/kg CCl4 ————— 94 Hepatic tissue from rats receiving 0.25 ml/kg CCl4 ————— 96 Hepatic tissue from rats receiving 2.00 ml/kg CCl ————— 98 4 viii LIST OF FIGURES (continued) Figure 14 15 16 17 18 19 20 21 22 23 24 25 26 Page Effects of exposure to polybrominated biphenyls (PBB) during development of body weight at 52 days of age---— 102 Effects of polybrominated biphenyls (PBB) and C014 on SGPT and PAH S/M 104 Effects of dietary polybrominated biphenyls (PBB) and i.p. CHCl3 on SGOT activity 108 Effects of dietary polybrominated biphenyls (PBB) and i.p. CHCl3 on BUN concentration and PAH S/M 110 Effects of chloroform and inducers of mixed—function oxidases (MFOs) and SGPT activity —— 112 Effects of chloroform and inducers of mixed-function oxidases (MFOs) on PAH S/M 115 Effects of CHC13 and piperonyl butoxide (PB) or SKF 525—A (SKF) on SGPT and PAH S M— — 117 Reduction of renal and hepatic glutathione (GSH) con- tent by CHC13- 119 Depletion of renal and hepatic reduced glutathione (GSH) by diethyl maleate 122 Effects of diethyl maleate and CHCl3 on SGPT and PAH S/M— ————— 124 Covalent binding of CHCl3 metabolites to microsomal protein in Vitro — 127 Covalent binding of CHCl3 metabolites to renal and hepatic protein in vivo and residual radioactivity in kidney and liver ~ 129 A) Blood concentrations of radioactivity versus time. B) Log blood concentrations of radioactivity versus time— — — 133 ix INTRODUCTION A. The Kidney as a Target Organ 1. Incidence and costs of human nephropathy It is generally believed among health professionals that renal dysfunction is a major factor in human disease, either as the primary cause of disease, a contributing factor, or as a major symptom. Though accurate estimates of the overall incidence of kidney—related maladies are not currently available, more than 12 million persons in the United States aIOne are known to suffer from chronic debilitating diseases of the kidney and urinary tract (including prostatic enlarge— ment, urolithiasis, chronic urinary tract infection and neuromuscular disorders of bladder control), and renal failure is the probable cause of death in an estimated 80,000 to 100,000 fatalities yearly (DHEW—NIH, 1978). In addition to the human suffering wrought, kidney—related diseases are quite probably a significant contributing factor to rapidly rising health care costs. Though total expenditures for the diagnosis and treatment of kidney—related diseases are not known, the cost of a single federal program providing Medicare benefits to approximately 40,000 victims of end-stage renal disease (32,556 renal dialysis patients and 4,450 recipients of kidney transplants) was greater than 1 billion dollars in 1977 and is expected to rise to 3 billion dollars by 1984 (DHEW-NIH, 1978). These indications of the high incidence and economic costs of human nephropathies suggest that laboratory and clinical investiga— tions leading to the elucidation of the causes and consequences of renal injury as well as to its prevention may be of great benefit to mankind. Although the percentage of clinical nephropathies originating from or exacerbated by occupational and environmental exposure to nephrotoxicants is not known, the great variety of chemicals demon— strated to be nephrotoxic in experimental animals and their wide use in medicine, agriculture and manufacturing industries indicate that the potential for chemically induced nephrotoxicity is great. 2. Pathophysiology of toxic nephropathy Chemically induced toxic nephropathy was defined by Schreiner and Maher (1965) as an ”adverse functional or structural change in the kidney due to the effect of a chemical . . . inhaled, injected, ingested or absorbed, or which yields toxic metabolites with an iden— tifiable effect on the kidney". While the consequences of such toxic insults to the kidney can be many and varied, the most important, perhaps, is renal failure, a condition in which the regulation of body fluid and solute balance is lost. Prolonged renal failure is incompatible with continued survival and artificial means (hemodialysis) must be employed if the victim is to survive. Renal failure in humans is characterized by oliguria (less than 400 m1 urine per day) or anuria, a low urine to plasma urea and creatinine ratio (urine is isotonic), and a high fractional sodium excretion (Levinsky, 1977). Renal resistance is increased with a concomitant decrease in renal blood flow. Such nonspecific signs 3 provide few hints of the etiology of this condition. Renal failure has been produced in experimental animals by administration of a variety of chemicals and mechanical constriction of the renal artery. Two general models of acute renal failure have emerged from studies on experimental animals. In the first, the vasoconstrictor model, in- creased resistance in the pre—glomerular vasculature appears to be responsible for tubular ischemia and resultant cellular hypoxia and the loss of energy—dependent tubular functions (Levinsky, 1977; Stein et_a1,, 1978). In the other, the nephrotoxic model, tubular dysfunction appears to be caused by direct disruptive effects of nephrotoxicants on epithelial tubular cells, primarily those of the proximal tubule (Levinsky, 1977; Stein e£_al,, 1978). It is generally believed that renal failure in humans, as a clinical entity, most likely encompasses aspects of both experimental models and that renal ischemia and tubular necrosis are interdependent phenomena (Levinsky, 1977). Acute renal failure may gradually revert to normal renal function when eXposure to the toxicant is stOpped, but the condition may be permanent if the initial injury is severe. Although morpholOgical and functional characteristics of experimentally induced toxic nephropathy have been described in detail (Biber §£.§l:s 1968; Dach and Kurtzman, 1976; McDowell §£_§13, 1976), the animal studies reported to date have failed to clearly identify critical subcellular lesions and mechanisms of injury. However, it is likely that the initiating event in many cases occurs on the molecular level and that the overall response of the kidney to the initial lesion may be determined by the types of cells affected, 4 the quantitative amount of tissue damage produced and, in the case of immature animals, developmental status of the kidney at the time of exposure to the toxicant. The mature kidney, for example, is com— posed of anatomically segregated groups of cells with highly special— ized functions, and even damage limited to a relatively small, dis— crete population of cells may upset the functiOnal interrelationships between diverse cell groups and produce a generalized loss of func— tional renal capacity. The fetal kidney, in contrast, is both ana— tomically and functionally immature and insult occurring during this period generally manifests as structural anomalies such as renal agenesis, renal cysts or hydronephrosis (Gibson, 1976). The relative amount of tissue damage initially produced may also determine the overall renal respOnse to chemical insult because the normal kidney contains a significant functional reserve capacity that allows for the maintenance of normal kidney function despite the loss of func— tional tissue. The kidney, furthermore, is capable of limited regeneration of damaged tubular epithelium (Foulkes and Hammond, 1975). The effects of these factors on renal reSponse to molecular lesions greatly complicate detection of renal injury and elucidation of the pathophysiology of toxic nephropathies. 3. Aspects of renal structure and function predisposing to kidney injury Several factors contribute to the overall sensitivity of the mammalian kidney to chemical injury. These include a high rate of perfusion, high oxygen demand, active and passive transport of chemicals across tubular epithelium and the serial arrangement of the nephrons. Although human kidneys comprise only 4% of body weight, they receive nearly 25% of resting cardiac output, most in the corti— cal region, enhancing the possibility that renal cells will be exposed to large amounts of blood-borne toxicants. In addition, the kidney has a high rate of oxygen consumption and is very sensitive to cellular anoxia (Berndt, 1976; Venkatachalam g£1§1., 1978). Equally important in determining sensitivity to toxicants is the anatomical and functional segmentation of the nephron, the basic unit of the kidney. In brief, each nephron is composed of a vascular, a glomeru— lar, and a tubular component. The vascular component regulates the initial flow of blood to the nephron but also remains closely asso— ciated with the tubular component with which it exchanges fluids and solutes (Tischer, 1976). Filtration of fluids and solutes from capillary blood into the tubular lumen occurs in the glomerular component and absorption, secretion and excretion of fluid and solute occurs largely in the tubular component. Because of the serial arrangement of the nephron components direct toxicant—induced damage and loss of normal function in one nephron component may indirectly alter function in the other components and produce a generalized loss of functional renal capacity. The tubule appears to be the part of the nephron that is most sensitive to chemically induced injury (Schreiner and Maher, 1965). One reason for this sensitivity may be that renal tubular cells are exposed to much higher concentrations of certain toxicants than are other cells. For example, nearly 99% of the fluid volume filtered at the glomerulus is reabsorbed across tubular epithelium, most in the proximal portion of the tubule. Thus, proximal tubular cells are exposed to large quantities of filtered, reabsorbed toxicants and cells of the pars recta as well as cells of the distal tubule are exposed to high concentrations of filtered, non-reabsorbed toxicants. Further— more, the countercurrent multiplier system, dependent on high medullary tissue oncotic pressure and low medullary blood flow, may lead to the development of prolonged, high concentrations of toxicants in the renal medullary interstitium. Additionally, high intracellular concentrations of toxicants may be produced by the active transport of toxicants from blood or tubular fluid into tubular cells. Even toxi— cants tightly bound to plasma proteins may be accumulated intra- cellularly by active transport mechanisms (Foulkes and Hammond, 1975). Finally, cells of the proximal tubule contain cytochrome P—450 and several mixed—function oxidases (MFOs) that are potentially capable of metabolizing xenobiotic chemicals to reactive, toxic products (Uehleke and Greim, 1968; Fowler §t_a1,, 1977; Kluwe gt_§l,, 1978). These properties, therefore, may be largely reSponsible for the high sensitivity of renal tubular cells to chemical toxicity. 4. Nephrotoxic chemicals A great number of chemicals with diverse structural and physical characteristics have been identified as nephrotoxicants. Many of these compounds are of great importance to medicine, agricul— ture and the manufacturing industries (Schreiner and Maher, 1965; Hook §E_§1., 1978a). In addition, some food additives and food contami— nants, many naturally occurring fungus—derived food contaminants and recognized pollutants of air and water produce renal injury when administered to experimental animals (Suzuki g£_§l,, 1975; Krogh 3; al., 1976; Thacker and Carlton, 1977; Ross 35 gl,, 1978). However, the lack of sensitive, non—invasive diagnostic techniques for detecting functional renal damage and the absence of a centralized system for the reception and assimilation of reports cencerning suspected cases of chemically induced nephropathy may delay or prevent the recognition of the nephrotoxic effects of many such chemicals in man. For these reasons it is difficult to assess the importance of nephrotoxic chemicals in the etiology of human kidney disease. Therapeutic agents recognized as nephrotoxicants include many non—narcotic analgesics, general anesthetics, x—ray contrast materials, and numerous antibiotics (Mazze gg.§1., 1976; Appel and Neu, 1977; Hook e£_a1., 1978a). Except for non—narcotic analgesics, access to nephrotoxic drugs generally is restricted to persons under medical care and the hazard of renal injury can be minimized by judicious drug use and careful menitoring for signs of renal dysfunction. Nephrotoxic agents to which exposure generally occurs in the absence of medical supervision, that is, the non—therapeutic chemicals, include many metals (As, Bi, Cd, Cr, Hg, Pb, Pt, Ur), organic solvents, glycols, monomeric chemicals (e.g., vinyl chloride), chemical flame retardants, pesticides and fungal toxins (Flea and Larson, 1965; Schreiner and Maher, 1965; Berndt and Hayes, 1977; Kociba §£_§1,, 1977; Osterberg e£_§1,, 1977; Hook 35 gl,, 1978a; Ross gE-al,, 1978). Although attempts have been made to identify chemical properties and structures common to nephrotoxicants, such endeavors to date have not been fruitful. 8 B. Metabolic Activation of Toxicants 1. Reactive metabolites and tissue injury In 1947 Miller and Miller reported that the administration of several chemical carcinogens to rats resulted in covalent binding of the carcinogens to hepatic proteins. The initial studies docu- menting the covalent binding of carcinogens to genetic molecules were published ten years later (Wheeler and Skipper, 1957). Subsequent studies have demonstrated that nearly all chemical carcinogens bind covalently to macromolecules; either in their native form (alkylating agents) or after metabolism to electrophilic products (Miller, 1970; Cavalieri EE.§l'a 1978). Current evidence indicates that the non- specific alkylation or arylation of critical informational macromole- cules may be an initiating event in the neOplastic transformation of mammalian cells by carcinogenic chemicals (Miller and Miller, 1974; Miller and Miller, 1977). Enzymatic metabolism within mammalian cells, furthermore, may largely determine the carcinogenic potential of certain classes of chemicals. Qualitative and quantitative differ- ences in the metabolism of foreign compounds in various tissues and species appear to contribute greatly to the tissue—Specificities and species—specificities of chemical carcinogens (Bartsch g; g1., 1977). More recently, the metabolism of xenobiotic chemicals to toxic, reactive intermediates has been implicated in chemically induced mutageneSis, teratogenesis and necrogenesis and in the develop— ent of certain blood dyscrasias and immunological disorders (Brodie, 967; Ames §E_§1,, 1973; Gillette gt El-: 1974). It has been proposed, or example, that strong electrophilic products of xenobiotic 9 metabolism covalently bind to nucleophilic sites on cellular macro— molecules in a nonspecific manner (Gillette, 1974). Sufficient alkylation of essential macromolecules may lead to cell dysfunction and cell death. Relationships between the generation of reactive metabolites and the development of acute tissue injury have been studied most extensively in the rodent (primarily rat and mouse) liver, where positive correlations have been demonstrated between the binding of reactive metabolites to hepatic proteins and lipids and the dis— ruption of membrane structures, loss of hepatocyte functiOn and hepato— cellular necrosis (Gillette, 1977). Few positive correlations between alkylation in non—hepatic organs and acute tissue injury, however, have been reported (Mitchell 3; g1., 1977). Although direct cause and effect relationships between covalent binding and acute hepatic inju— ries have not been unequivocally demonstrated, many investigators have prOposed that covalent binding may be a mechanism of toxicity and that he generation of reactive intermediates is the critical step in the activation of many hepatotoxicants (Gillette, 1974; Mitchell and Iollow, 1975; Thorgeirsson and Wirth, 1977). 2. Locations and functions of drug—metabolizing enzyme (DME) systems Xenobiotic chemicals undergo at least four basic types of etabolism in mammals; oxidation, reduction, hydrolysis and conjuga— ion. The metabolic activation of many toxicants is believed to be adiated primarily by cytochrome P—450—dependent MFOs, a hetero— anous group of membrane-bound enzymes that catalyze the oxidation numerous endogenous and exogenous c0mpounds. MFG—mediated .idations include: aliphatic and aromatic hydroxylations, N and 0 A, ll, "'7‘?“ W—1- 7- Herr-r 10 dealkylatiOns, N oxidations and hydroxylations, S demethylation, sulfoxidation, desulfuration and the deamination of primary and secondary amines (Goldstein g£_§1,, 1974). It is evident, therefore, that MFOs can accommodate a great variety of substrates. Hepatic MFOs have been studied extensively in_yit£g, Homo— genates of liver can be fractionated to obtain endoplasmic reticulum (ER) membrane fragments (microsomes) containing high specific activi— ties of MFOs. By such investigations it has been determined that MFO reactions consume molecular oxygen and reduced pyridine nucleotides (NADPH) and require a specific hemoprotein (or a group of related hemoproteins) that acts as a terminal oxidase in the enzymatic reaction, and an unidentified lipid component (Goldstein §£.al., 1974). Cyto— chrome P—450 (P—450), the hemoprotein, is so—named because the difference Spectrum of the reduced C0:hemoprotein complex displays an absorption maximum at 450 nm. Several forms of P—450 have been iso- ated from rat liver (Grasdalen e£_a1,, 1975; Ryan EE.él-: 1975; Euengerich, 1977; Mailman g£_al,, 1977; Ullrich and Kremers, 1977), 1nd each appears to have different affinities for MFO substrates. his has led to speculation that quantitative and qualitative MFO ctivities within a single tissue, as determined by ig_vitro investi— ations with microsomes, may be regulated in part by the relative ancentrations of the different forms of P—450 present in the tissue Illrich and Kremers, 1977). P-450—dependent, MFG-mediated oxidations, as studied in £33, c0nsume one mole of melecular oxygen and two reducing equiva— nts (2 electrons) for each mole of substrate oxidized. The preducts 11 of the reaction are an oxidized substrate molecule and a mole of H20. In brief, the reaction is thought to occur in the following manner: the substrate binds to oxidized P—450, this complex is reduced by an electron from a reduced flavoprotein molecule (the electron is origi— nally from NADPH), molecular oxygen ”binds" with the reduced P—450— substrate complex and another electron (from NADPH or NADH) is intro— duced, whereupon the complex decomposes and releases oxidized sub— strate (containing an atom of oxygen from molecular oxygen), oxidized P—450 and H20 (from the reduction of an atom of oxygen) (Goldstein g; g1,, 1974). The wide substrate specificity of the P—450—dependent MFO system allows for the oxidative metabolism of a great variety of chemicals, both endogenous and exogenous, by a single enzyme system. P—450—dependent MFOs are generally located in cellular membranes, primarily in the ER but also in the mitochondria and the nuclear envelope (Ghazarian and DeLuca, 1974; Kashnig and Kasper, 1969). Mitochondrial MFO activities are particularly high in the adrenal cortex where extensive steroid hydroxylation occurs (Zam— paglione and Mannering, 1973). Although MFO systems have been identi— ied in many extrahepatic tissues (lung, kidney, adrenal, intestinal pithelium, skin, gonads, placenta) the specific activities of MFOs 'n hepatic ER, in general, are much greater than these in extrahepatic rgans (Litterst 9311;, 1975, 1977; Fry filial” 1978). Many sub— trates for MFOs are lipid soluble, a property which may facilitate iffusion across the plasma membrane and dissolution into the ER embrane prior to binding to P-450. 12 Non—microsomal oxidations and microsomal reductions have been less well—characterized. Their relationships to the metabolic activation of toxicants is not presently clear, though the metabolism of carbon tetrachloride to a reactive species appears to be mediated by microsomal reduction (Uehleke and Werner, 1975; Sipes g£.§1., 1977). Enzymatic hydrolysis, on the other hand, is generally re— stricted to esters and amides (Goldstein e£_§1,, 1974) though the presence of an enzyme in the ER membrane that catalyzes the hydrolysis of epoxides (epoxide hydratase) has been reported recently and appears to be of great toxicological significance (Brooks, 1977; Oesch EE.§l-, 1977). Synthetic reactions include glucuronidation (a microsomal reaction), acetylation (a non—microsomal reaction), mercapturic acid formation (non—microsomal) and sulfotransferase reactions (non- microsomal). DMES were originally described as "detoxification" systems, largely because the pharmacological activities of many therapeutic drugs were reduced by enzymatic degradation and the excretion of lipophilic toxicants was frequently hastened by enzymatic biotrans- formation (Goldstein SE g1., 1974). More specifically, however, DMEs appear to convert lipophilic substrates into more polar products that can be readily excreted in the urine or feces (Goldstein g£.a1., 1974). In certain instances, however, the products of microsomal metabolism, though more polar than the parent compound, may be :hemically unstable species that rapidly react with appropriate, )roximate, cellular molecules in a nonspecific manner (Gillette, 1977; ollow and Smith, 1977). Examples of such reactions include oxidative 13 dehalogenations of chlorinated aliphatic hydrocarbons and epoxidations of polycyclic aromatic hydrocarbons (Docks and Krishna, 1976; Pohl §£_§l., 1977; Cavalieri gg_§l., 1978). The metabolites produced by the above reactions are generally strong electrophiles that attack nucleophilic sites on proximate macromolecules in a nonspecific manner (Miller and Miller, 1970, 1974; Gillette, 1974, 1977). 3. Balance between enzymatic toxification and detoxification The extent of damage produced in a specific tissue by a highly reactive intermediate is probably proportional to the innate sensitivity of the affected tissue to the presence of the reactive species and proportional to the concentration of reactive metabolites in the tissue. Thus, tissues possessing mechanisms to protect against electrophile injury or to repair electrophile damage will probably be less sensitive than tissues without such protective mechanisms. In addition, the molecular lesion produced may be of greater functional consequence in some tissues than in others. Where these factors are equal, however, relative tissue injury may be directly proportional to the amount of reactive metabolite formed within the tissue. Most P—450—dependent MFO activities appear to be first— order reactions under in_yi££g_conditions (and, presumably, under in vivo conditions) (Goldstein 25.§l:’ 1974; Jollow and Smith, 1977). The rate of generation of reactive metabolites, therefore, is depen— ent upon the affinity of the enzyme for the substrate and the amount f substrate available (Gillette, 1977). Availability of substrate generally the parent compound) in biological systems can be directly l4 affected by the presence of competing pathways of metabolism and by the excretion of substrate in an unchanged form. In addition, toxic, reactive metabolites can frequently be enzymatically transformed to more stable, non-toxic products (Brooks, 1977; Jollow and Smith, 1977). These principles are illustrated in Figure 1 and have been reviewed recently by Jollow and Smith (1977). The relative activities of enzymatic metabolism to toxic products and to non—toxic products within a specific tissue may determine, to a large extent, the sensitivity of that particular tissue to chemical injury. That is, the balance between enzymatic toxification and detoxification reac- tions determines the concentration of reactive species present in the tissue at any specific time. Modifications of the relative acti— vities of pathways 1, 2 or 3 in Figure 1 may change the percentage of parent compound metabolized to a toxic product via pathway 3 and, Figure l Excretion (unchanged) ’1‘ E (l) I ' (2) . PARENT x Metabolite A COMPOUND I (non-toxic) l l E (3) l ‘1’ (4) Metabolite B (toxic) ) Metabolite C (non-toxic) hereby, cause an increase or decrease in the net amount of metabo— ite B formed. Similarly, changes in the activities of pathways 3 or 15 4 may alter the concentration of metabolite B, the proximate toxicant, within the tissue. Therefore, if tissue damage occurs as a result of the actions of a toxic, reactive intermediate on sensitive, cellular macromolecules then the extent of injury produced may be dependent on a set of interrelated pharmacokinetic parameters. Reactions depicted by pathway 3 in Figure l are generally catalyzed by P—450—dependent microsomal MFOs and those of pathway 2 by MFOs or non-oxidative cytosolic enzymes (Jollow and Smith, 1977). Pathway 4 enzymes generally catalyze conjugation reactions (UDP— glucuronyl transferase, glutathione—S-transferases, 3'-phosphoadeno— sine—5'—phosphosulfate transferase), but may also catalyze hydrations of electrophilic epoxides (Goldstein EE.E$>, 1974; Brooks, 1977; Jerina and Bend, 1977). Conjugations with glutathione (glutathione- S—transferases) and sulfate (sulfotransferases) are catalyzed by several different forms of cytosolic enzymes. The fact that many reactive metabolites thought to be generated at the ER are conjugated with glutathione and sulfate by cytosolic enzymes suggests that even reactive metabolites may possess limited mobility within the cell. UDP-glucuronyl transferase and epoxide hydratase, though, are micro— somal enzymes and may metabolize reactive intermediates to more stable products prior to relocation from the ER membrane. Recent investigations have demonstrated that complex aroma— ic molecules may undergo oxidative metabolism at multiple sites. ,4—Benzo(a)pyrene, for example, can be oxidized to the 4,5—, 7,8—, r 9,10—epoxides (Conney gt_al,, 1977). Benzo(a)pyrene-7,8—epoxide an then be hydrated to benzo(a)pyrene—7,8—dihydrodiol and this 16 compound may be subsequently oxidized to benzo(a)pyrene—7,8—dihydro— diol-9,10—epoxide, a very potent mutagen and cytotoxicant (Borgen et_al., 1973; Sims et_al., 1974). Sequential metabolism of chemicals may enable relatively stable metabolites (e.g., benzo(a)pyrene—7,8— dihydrodiol) to travel from the site of formation to other tissues where activation to the*proximate toxicant (e.g., benzo(a)pyrene—7,8— dihydrodiol-9,lO—epoxide) may occur. For such chemicals the reaction sequences illustrated in Figure 1 may oversimplify the relationship of metabolism to toxicity. 4. Tissue-specific metabolic activation The toxic effects of reactive intermediates are thought by many investigators to result from their chemical instability (Gillette, 1977; Jollow and Smith, 1977). Since biological membranes resist the passage of polar and ionic species, and since reactive chemical moieties exhibit short half—lives, it is unlikely that a truly reactive intermediate formed in one tissue would travel to another tissue and there produce direct injury. Bartsch gt_al. (1975, 1977), for example, reported a strong correlation between the abilities of various tissues to metabolize N—nitrosamine derivatives to reactive products and the sensitivities of the various tissues to nitrosamine injury. That is, N—nitrosamines appear to be metabolized to proximate toxi— cants and tissues most active in this biotransformation reaction are, accordingly, most susceptible to nitrosamine injury, implying that a cause and effect relationship exists between metabolic activation nd tissue injury. Toxic metabolites with greater chemical stability, owever, may produce injury in tissues distant from the site of l7 Jlite formation, especially if distant tissues are more sensitive groximate tissues to the molecular effects of the toxic metabo- The neurotropic carcinogen 3,3—dimethyl-l—phenyltriazene, for Le, is metabolized in the liver to a weak alkylating agent, 3— l-l—phenyltriazene, but produces brain tumors rather than hepatic s (Preussmann at al., l969a,b). Brain tissue does not appear to ate (demethylate) 3,3-dimethyl-l-phenyltriazene (Preussmann, ,b), but exhibits a relative inability, in comparison to the , to excise 06-methylguanine, a methylated DNA base produced by hyl-l—phenyltriazene in brain and liver DNA that is likely to .base miSpairing during nucleotide synthesis. The estimated 'life of 3-methyl-l—phenyltriazene, the toxic metabolite, in »us medium (pH 7.40, 37°C) is slightly greater than one minute. time appears sufficient to allow for transport from the in_vivo ‘of formation (liver) to the target site (brain) since 3-methyl- , nyltriazene injected i.p. methylated DNA bases in both liver rain (Bartsch §£.§ir: 1977). For relatively stable toxic olites such as 3—methyl—l—pheny1triazene there may be little ation between organ—specific biotransformation and organ- ic injury. That is, damage appears to be dependent largely on sponse of the tissue to the molecular lesion. iotransformation Capacity of the Kidney . Characteristics DMEs, including P—450—dependent MFOs, are present in kidney her extrahepatic organs. Many substrates for hepatic microsomal l8 lation appear to undergo the same types of reactions when incubated 1 renal microsomes, thOugh the specific activities of the enzymes )1ved are generally much lower in the kidney than in the liver :terst EE.E£'9 1975, 1977; Fry et al., 1978). The subcellular LtiOflS and actions of renal DMEs are generally similar to those :ribed for the liver. The renal P—450—dependent MFO system in the rat has been acterized by Jakobsson e£_al, (1970) and Orrenius §t_al, (1973). greement with earlier investigations (Kato, 1966; Ichihara gt_al,, ), they reported that renal MFOs, like hepatic MFOs, required ced pyridine nucleotides as cofactors, mOlecular oxygen, and a rane—bound hemoprotein. The hemoprotein has been variously :red to as cytochrome P—450, cytochrome P—450K, and cytochrome L (henceforth referred to as P—450) because of the absorption Ium at 454 nm in the reduced CO:hemoprotein difference spectrum. IOSt apparent difference between renal and hepatic MFOs is that pecific activities of most substrate oxidations and the concen- ons of P—450 in renal microsomes appear to be only 10—30% of in hepatic microsomes (Jakobsson et_al,, 1970; Orrenius et al., Litterst at al., 1975, 1977; Fry §£_al., 1978). In centrast, >ecific activities of m and w—l hydroxylations of fatty acids :arly equal in renal and hepatic microsomes, prompting specula— hat renal MFOs may primarily oxidize endogenous fatty acids than lipophilic xenobiotic chemicals (Jakobsson gt_al,, 1970; iE.§£-’ 1972; Jakobsson and Cinti, 1973). The current metho— for assessing renal microsomal MFO activities in Vitro is l9 tsically that methodology developed to maximize the specific activi— Les of hepatic microsomal MFOs. Relatively little has been reported )ncerning the optimization of renal MFO activities in_yi££g (e.g., [, temperature, substrates, cofactor concentrations, antioxidant >ncentrations). Thus, renal MFO activities, as currently measured ;Xi££2) may underestimate the biotransformatiOn capacity of the dney. Additional differences between renal and hepatic MFOs in dents (largely mice and rats) include dichotomies in sex—related fferences in enzyme activities, differential responses to stress .g., fasting, altered diet), and a resistance of renal MFOs to the iuctive effects of phenobarbital (Litterst gt_al,, 1975, 1977; labra and Fouts, 1974). Such differences suggest that control of ) activities is organ specific; for example, renal MFO activities : intrarenally controlled. An alternate explanation for the low activities of MFOs in a1 microsomal preparations in yitgg may be that such enzymes are fined to a relatively small population of renal cells. MFOs :ar to be components of the membrane of the smooth ER, an organelle Ld in highest renal concentrations in the S cells of the proximal 3 les. The concentration of S3 cells is greatest in the pars recta katachalam_§£_al., 1978), that portion of the proximal tubule with greatest apparent susceptibility to toxicant damage and the :est activity of active organic ion transport. If P-450—dependent are largely confined to this renal cell type, then homogenates of kidney or kidney cortex, either of which contain greatly diluted 20 ncentrations of the centents of S3 cells, understandably exhibit low tivities toward most substrates for hepatic microsomal oxidation. support of the localization of high concentrations of P—450— pendent MFOs in S3 cells, Fowler EE.§£- (1977) have demonstrated at the anatomical distribution of MFO activities within the kidney assly corresponds to the intrarenal distribution of 83 cells. In iition, Zenser and Davis (1978) reported that renal MFO activities 1 P—450 concentration exhibited a cortical—papillary gradient Lghest in the cortex and outer medulla and lowest in the papilla), )attern similar to the distribution of S3 cells in the kidney. rler.gt_al. (1977) also reported that treatment of rats with i,7,8—tetrachlorodibenzo-p—dioxin greatly increased renal MFO acti— ies and increased the amount of smooth ER in S3 cells, though tomically adjacent 82 cells and distal tubular cells were not ected. These studies suggest that P-450—dependent microsomal MFO ivities in S3 cells may be quantitatively similar to those in etic parenchymal cells. Fractionation of microsomes from whole 1ey homogenates, however, results in a dilution of microsomes from :ells and may be responsible for low specific activities of renal 2. Physiological and toxicological significance of renal biotransformation The urinary excretion of lipophilic agents is known to he need by conjugation reactions that increase the polarity of genous and exogenous compounds and retard their passive reabsorp— (Goldstein gt_§l,, 1974). The activities of many conjugating 21 enzymes, as measured by £2.22EEB techniques, are nearly as high in the kidney as in the liver (Chhabra and Fouts, 1974; Litterst EE 31., 1975; Fry EE.§$:’ 1978), suggesting that renal enzymes may be important in the excretion of some lipophilic substances. For example, renal biotransformation has been demonstrated to increase the urinary excretion of certain chemicals (Quebbeman and Anders, 1973; Acara §E_al., 1977) and to alter the activities of some hormonal agents (Blackwell gt_al,, 1975). In addition, the oxidation of 25-hydroxy— cholecalciferol to l,25—dihydroxycholecalciferol, the form of vitamin D most active in promoting intestinal absorption of calcium and bone mineral mobilizatiOn (Omdahl and DeLuca, 1971; Tanaka and DeLuca, 1971), appears to occur primarily in the kidney and is mediated by a P—450—dependent mitochondrial MFO (Omdahl and DeLuca, 1973; Ghazarian and DeLuca, 1974, 1977). Thus, renal MFOs may be important in main— ‘taining mineral homeostasis. The kidney also appears to be very active in synthesizing and degrading prostaglandins (Blackwell fig 31,, 1975; Zenser et_§l,, 1977), derivatives of arachidonic acid believed to participate in control of renal function. Thus, renal drug metabolism may be important in maintaining renal function. The overwhelming capacity of the liver for xenobiotic meta— olism clearly makes it the dominant organ in the quantitative bio— ransformation of toxicants. For reasons specified earlier, however, he kidney may largely be responsible for the generation of chemically eactive metabolites that produce renal injury. Hill §t_§l. (1975), or example, have reported great strain differences in the nephro— oxicity of CHCl3 in male mice, an apparent genetic phenomenon, 22 though strain differences in the hepatotoxicity of CHCl were slight. 3 Similarly, male mice were markedly more sensitive to the nephrotoxi- city of CHCl than were female mice, though sex differences in the 3 hepatotoxicity of CHCl3 were not apparent (Klaassen and Plaa, 1967). These results may be due to inherent differences in the response of the kidneys in male and female mice and various strains of mice to CHCl3 or to sex-related and strain—related differences in renal bio- transformation of CHC13. Reports indicating that treatment with testosterone, a hormone that alters xenobiotic metabolism, enhanced the susceptibility of female mice to CHCl nephrotoxicity without 3 producing morphological changes in the kidney and that castration reduced the sensitivity of male mice to CHCl nephrotoxicity suggest 3 that the latter possibility, sex-related differences in renal bio— transformation of CHCl3,is the more likely reason for the insensi— tivity of female mice to CHCl nephrotoxicity (Krus g; §l°: 1974). 3 In addition, Ilett g5 El. (1973) reported that more CHCl3 metabolites were bound in kidneys of male mice than in kidneys of females fellow— ing i.p. injection of CHCl3 but no sex difference was observed in binding to liver. Thus, renal metabolism may be important in the activation of CHCl3 to a reactive metabolite. Weekes (1975) has reported that a strong correlation exists between the abilities of renal microsomes from several strains of mice to metabolize dimethyl— nitrosamine to mutagenic products in_yigrg_and the sensitivities of these same strains of mice to dimethylnitrosamine—induced renal tumorigenesis. Thus, renal MFO activities, though low when measured in whole kidney homogenates, may be of toxicological significance. 23 D. Nephrotoxicity of Halogenated Aliphatic Hydrocarbons 1. Incidence The chemical and physical characteristics of nephrotoxic compounds are many and varied, as mentioned previously. One commonly used group of chemicals that has consistently proven to be nephrotoxic across a wide spectrum of mammalian species is the halo— genated aliphatic hydrocarbons. A partial list of widely—used halo— genated aliphatic hydrocarbons reported to be nephrotoxic in experi— mental animals is contained in Table 1. Although significant inci— dences of human intoxications resulting in renal failure have been reported for only 3 of these compounds (CHCl3 and 0014, Von Oettingen, 1964; methoxyflurane, Mazze, 1976), the lack of extensive documenta— tion of human renal damage produced by the other halogenated alipha— tics should not be taken as an indication of their safety. Rather, human exposure to the rest of the compounds in Table 1 may currently be insufficient for the development of human nephropathies or for their detection by insensitive, non—invasive techniques. Of recent concern is the increasing presence of halogenated tliphatic hydrocarbons, principally halomethanes and haloethanes, in :urface waters used for municipal water supplies (Deinzer gE_§l., 978) and the potential consequences to human and environmental ealth of such pollution (Kuzma §£_al,, 1977; Cantor gt_al,, 1978; raybill, 1978). Sources of halogenated aliphatic hydrocarbon con— 1mination of the general environment include agricultural runoff 1d industrial effluents. Additionally, haloalkanes may be produced ‘unintentional chlorination of organic materials during sewage 24 TABLE 1 Nephrotoxic Halogenated Aliphatic Chemicals Chemical allyl chloride carbon tetrachloride chloroform dibromochloropropane 1,1—dichloroacetylene 1,1—dichloroethylene 1,3—dichloropropene ethylene dibromide hexachlorobutadiene methoxyflurane 1,1,2—trichloroethane trichloroethylene tris(2,3—dibromopropyl) phosphate trihalomethanes (most) Reference Ross §£_§l., 1978 Ross g£_§l., 1978 Ross 23 gl., 1978 Torkelson §t_al,, 1961 Reichert g£.al., 1978 Jenkins and Andersen, 1978 A TorkelSOn and Oyen, 1977 Ross §£.§1., 1978 Ross §£_§1., 1978 Mazze, 1976 Klaassen and Plaa, 1966 Klaassen and Plaa, 1966 Osterberg SE g1., 1977 VOn Oettingen, 1964 25 sterilization (Deinzer §E_§l,, 1978). Contamination of surface waters with trace quantities of chlorinated aliphatic hydrocarbons is of concern, in part, because at least three halogenated aliphatic chemi— cals of industrial importance, 0014, CHCl3 and hexachlorobutadiene, have been reported to produce tumors in rodents (CHCl3 and hexachloro— butadiene produced renal tumors, Kociba 25.21:: 1977; Ross §§_31., 1978). Furthermore, a positive correlatiOn has been found between the presence of CHCl3 in drinking water and the incidence of renal tumors in males in certain industrialized regions of the United States (Cantor §£_§1., 1978). Very little, however, is known about the potential risk of leoplastic and nOn—neOplastic renal injury from long‘term exposure to .ow concentratiOns of halogenated aliphatic hydrocarbOns. Many of :he compounds listed in Table l are used in chemical manufacturing rocesses and may be of special toxicological significance to occupa— ional health. Although occupationally associated nephropathies do ot appear to be common, two factors may obscure the relationship etween occupation and kidney disease. First, there is an inability > detect renal damage with common clinical tests until functional aserve capacity has been overwhelmed (and the victim is near death). :cond, there is frequently a lengthy latency period between chemical posure and recognition of nephrotoxicity. Thus, the actual inci— nce of chemical nephropathy may be greater than is currently recog— zed. 26 In a recently published list, the National Science Founda— tion ranked organic chemicals according to their potential for pro— ducing harm to humans and the envirOnment, based on parameters such as potencies as toxicants, yearly production volumes, rates of re— lease into the environment, and biodegradation (Stephenson, 1977). Of the ten compounds perceived most hazardous, six were halogenated aliphatic hydrocarbons known to be nephrotoxic. Thus, it would seem that there should be concern for the possibility of renal injury resulting from industrial and occupational exposure to halogenated aliphatic hydrocarbons even though the incidence of recognized chemical nephropathy is currently low. 2. Toxicological manifestations The nephrotoxic effects of 0014 were recognized as early as the turn of the century (cited in Smetana, 1939) and clinical descrip- tions of CCl4 nephropathy from that time until the present abound in the literature (see Ross §£_gl,, 1978). In brief, human ingestion or inhalatiOn of large quantities of CCl4 (20-400 m1) is generally followed by the rapid onset of nonspecific symptoms such as gastrointestinal disturbances and intense abdominal pain (Smetana, 1939; Schreiner and Maher, 1965). Oliguria or anuria, depending on the severity of the intoxication, deVelops over the ensuing 3—9 day period. Should the patient survive this period, the anuria progresses to oliguria (which may last for as long as 60 days), then to a transient state of iuresis and finally to normal renal function. Renal biopsies from urvivors of 0014 intoxications exhibited tubular swelling and non— pecific degenerative cellular changes that were most severe in the 27 area of the proximal tubules. Microscopic lesions were similar in appearance, but more severe, in renal tissue from CCl4-induced fata— lities examined at the time of autopsy. Frank renal cellular necrosis does not appear to be produced by even fatal amounts of 0014 (Smetana, 1939; Von Oettingen, 1964; Schreiner and Maher, 1965). The path0physiology of CCl4 intoxication in experimental animals (primarily rats, mice and rabbits) appears to be quite similar to that reported in humans except that the time sequence is shorter. That is, CCl4—induced renal dysfunction occurs long after elimination of the parent CCl4 from the body (2—3 days), damage is most severe in the area of the proximal tubules and either the animal dies or renal function slowly returns to normal (Striker EE_fll-’ 1968). The clinical manifestations of intoxication with CHCl3 and other halogenated aliphatic hydrocarbons have not been characterized as well as those of C014. Studies with experimental animals, however, suggest that most halogenated aliphatic compounds specifically damage the proximal tubules of the kidney (see references in Table l). The lesiOn is evident histologically as a swelling of tubular epithelial cells that progresses, with increaSing severity of intoxication, to necrosis and sloughing of tubular cells followed by tubular regenera— tion and a return to normal renal function or by tissue calcification and terminal renal failure. In contrast to the liver, where signs of 'ntoxication are maximal within 24 hr, renal damage appears to be 0st severe within 2—7 days after exposure (see references in Table l). he functional manifestations of halogenated aliphatic hydrocarbon 28 intoxication are basically those expected following damage to the proximal tubule; proteinuria, glucosuria, increased fractional excre— tion of sodium and decreased clearance of p—aminohippuric acid (Sirota, 1949; Von Oettingen, 1964; Striker EE.§132 1968). Although the renal sequelae of intoxication with a great number of halogenated aliphatic compounds appear to be qualitatively similar, it is not known if halogenated aliphatic hydrocarbons produce renal injury by a common mechanism. 3. Mechanisms of toxicity The strength of the carbon—halogen bond was originally believed sufficient to prevent substantial metabolic degradation of halogenated aliphatic hydrocarbons within mammalian cells. Early references to the pharmacokinetics of halogenated anesthetic gases, for example, implied that such compounds were excreted from the body wholly in an unchanged form. Zeller, however, had reported in 1883 (cited in Rubenstein and Kanics, 1964) that serum chloride concen— trations in dogs chronically depleted of chloride were significantly elevated by anesthesia with CHClB, suggesting that extensive dechlo— rination had occurred ig_yiyg, Subsequent investigations have clearly demonstrated that substantial dehalogenation of organic com- pounds can occur in humans and experimental animals and, in fact, indicate that the primary method of excretion of some chlorinated hydrocarbons involves hepatic oxidative dehalogenation (Rubinstein and Kanics, 1964; Leibman, 1965; Brown EE él-, 1974a). The nephro— toxicity of at least one VOlatile anesthetic, methoxyflurane, has been attributed to hepatic defluorination and the resulting increase in 29 rerum concentrations of fluoride ion, a nephrotoxic agent (Mazze, 976). The relationships between metabolism of halogenated aliphatic .ydrocarbons and their nephrotoxicity, however, have not been ,ddressed extensively. In contrast, considerable efforts have been evoted to studying the relationship of metabolism to the hepato— oxicity of halogenated aliphatic chemicals. It appears, in many .ases, that these compounds are metabolically activated to hepatotoxi— ants. That is, the parent compound, which is relatively innocuous o the liver, is metabolized by hepatic enzymes to toxic products that reduce hepatocellular necrosis (Mitchell and Jollow, 1975). Sensi— ivity of the liver to the damaging effects of many halogenated ali— hatic hydrocarbons can be greatly altered by modulation of the enzyme ystems involved in xenobiotic metabolism (Suarez g£_§l,, 1972; larlson, 1975). Further support for the involvement of metabolites in halo— nated aliphatic hydrocarbOn hepatotoxicity is provided by correla- ions between the chemical reactivity of such compounds and their Jilities to produce liver damage. The hepatotoxicities of equimolar nounts of a series of trihalomethanes (CHBr3 > CHCl3 > CHI3), for (ample, is inversely proportional to the carbon—halogen bond energy 2-1 > C—Cl > C—Br), suggesting that metabolic activation occurs st readily on molecules with labile bonds. Accordingly, increased .logenation destabilizes the molecular structures of haloalkanes d increases their hepatotoxicity, but stabilizes the molecular uctures of haloalkenes and decreases their hepatotoxicity (Bonse Henschler, 1976). The reactive, hepatotoxic metabolites 30 generally appear to be strong electrophiles that react with nucleo- philic sites 0n cellular macromolecules in a nonspecific manner and nay thereby produce cellular dysfunction, as discussed previously. Relatively little is known about the role of biotransforma— tiOn in the development of halogenated aliphatic hydrocarbon nephro— toxicity. Ilett g£.gl. (1973) and Reid (1973), however, reported that metabolites of chlorobenzene, bromobenzene and CHCl3 became covalently bound to renal proteins upon i.p. injection of the parent chemicals into mice. Renal binding in intact mice was greatest in the area of the proximal tubules, the site of halobenzene and CHCl3— induced renal damage. It seemed possible, therefore, that renal MFOs, vhose activities are greatest in the proximal tubule, may have trans— formed the parent compounds into reactive, nephrotoxic products in a lanner similar to that postulated to occur in the liver. Renal licrosomal protein, however, exhibited little or no capacity to enerate reactive metabOlites iE_XlE£2 (Ilett_g£_§1., 1973; Reid, 973). The authors speculated that reactive metabolites formed in 1e liver might have travelled to the kidney as plasma protein—bound )mplexes where they were released again as reactive, toxic inter— :diates (Ilett §E_§l,, 1973; Reid, 1973). As discussed previously, wever, quantitative, ig_vitro measurement of renal microsomal zyme activities may underestimate the biotransformation capacities segregated renal cell types (e.g., S3 cells). In addition, the hniques of in Vitro covalent binding used by Ilett g£_gl. (1973) Reid (1973) may be of questionable worth (when renal microsomes used) since Sipe5_gg_§l. (1977) using the same technique, was 31 nable to demonstrate a dependency of covalent binding of CHCl3 to ‘enal microsomal protein on the presence of molceular oxygen and ’-450 and inhibition of covalent binding by C0. h Purpose The primary purpose of this investigation was to elucidate the. 1echanisms by which halogenated aliphatic hydrocarbons produce acute renal injury. For this reason the roles of hepatic and renal biotrans- Formation and covalent binding in the development of halogenated ali— >hatic hydrocarbon nephrotoxicity were evaluated. This required, to L certain extent, characterization of the biochemical and physiolo— ;ica1 responses of the liver and kidney to toxic halogenated aliphatic ompounds and determination of the capacities of kidney and liver for enobiotic metabolism, their relative responses to enzyme induction nd inhibition, and the manner in which such responses affected the ephrotoxicities and hepatotoxicities of selected chlorinated ali— Iatic compounds. Attempts were also made to relate pharmacokinetics—— »sorpti0n, distribution, excretion, and metabolism--to organ—specific xicities in intact animals and to determine the role of changes in armacokinetic behavior in the potentiation and inhibition of halo— cane toxicity. In addition, the roles of reactive and electrophilic :ermediates in CHCl3 nephrotoxicity were investigated. Insights into the mechanisms of halogenated aliphatic hydrocarbon irotoxicity in rodents may aid in the development of experimental :ls with which to study human response to such toxicants. Appro— te experimental models are vital for extrapolation of toxicity 32 data from experimental animals to man. Such knowledge may also aid in predicting the toxicological consequences of chronic exposure to low concentrations of halogenated aliphatic chemicals, the potential for synergistic effects between organohalides, and the potential toxicity of newly—synthesized halogenated aliphatic compounds. It is hoped that the results of this investigation will aid in understanding the roles of hepatic and renal biotransformation, reactive metabolites, and covalent binding of reactive metabolites in the development of halogenated aliphatic hydrocarbon nephrotoxicity. Additionally, it is hOped that these results can be applied to the assessment of such parameters in human response to nephrotoxicants. _' 1. Objectives The specific objectives of this investigation are listed below. 1. To determine the time-dependency and dietary concentration— lependency of induction of drug—metabolizing enzyme systems by .ietary ingestion of polybrominated biphenyls and polychlorinated iphenyls. 2. To determine the effects of treatment with sodium pheno— arbital, 3—methylcholanthrene, polychlorinated biphenyls and 2,3,7,8— etrachlorodibenzo—p—dioxin on renal and hepatic drug—metabolizing izyme systems. These compounds were used to modify chlorinated Liphatic hydrocarbon toxicities. 3. To examine the types of cytochrome P-450 induced in the ,dney by polybrominated biphenyls, polychlorinated biphenyls, 3— thylcholanthrene and sodium phenobarbital. 33 4. To determine the effects of treatment with SKF 525—A and iperonyl butoxide on renal drug—metabolizing enzyme systems. These ompounds were used to modify chloroform toxicity. 5. To evaluate the effects of several inducers of drug— etabolizing enzyme systems (polybrominated biphenyls, polychlorinated iphenyls, hexachlorobenzene) on carbon tetrachloride toxicity. 6. To determine the effects of inhibitors of drug—metabolizing nzyme systems (SKF 525—A, piperonyl butoxide) on chlorform toxicity. 7. To elucidate the relationship between glutathione concen— rations in target tissues and chloroform toxicity. 8. To examine the dependency of organ—specific chloroform .oxicity on covalent binding of chloroform metabolites to kidney and .iver. MATERIALS AND METHODS A. Animals and Experimental Diets ICR mice and Sprague-Dawley rats were purchased from Spartan Research Animals (Haslett, MI) and Fischer 344 rats from Harlan Industries (Indianapolis, IN). All animals were maintained in sani- tary, ventilated animal rooms under controlled humidity, temperature and light-cycle for the duration of the experiments. Unless indi— cated otherwise, all animals were young adults at the time of use. Experimental diets were prepared by mixing polybrominated biphenyls (PBB, Firemaster BP-6, Velsicol Chemical Co., St. Louis, MI) or polychlorinated biphenyls (PCB, Aroclor 1254, Monsanto Chemical Co., St. Louis, MO) in acetone slowly and evenly into finely—ground rodent pellets (Wayne Lab Blox, Chicago, IL). Analyses showed that the actual concentrations of PBB and PCB in diets prepared in this manner were within 8% of calculated concentrations. Control diet was formulated by mixing an equivalent amount of acetone into the ground diet. 3- .Tgxicity Tests l. Sgrum analyseg Freshly—drawn whole blood was allowed to clot for 90-120 min .t room temperature then carefully centrifuged and the serum fraction ithdrawn. Glutamic oxaloacetic transaminase (GOT) and glutamic 34 35 pyruvic transaminase (GPT) activities were determined in aliquots of serum (S) using Sigma reagent kits (Sigma Chemical Co., St. Louis, 0) and quantified as Sigma—Frankel units per ml serum. Urea nitro— en concentrations (BUN) were determined in aliquots of serum using Sigma reagent kit. 2. Renal cortical slice accumulation of PAH and TEA The abilities of renal cortical Slices to accumulate the rganic anion para—aminohippurate (PAH) and the organic cation tetra— thylammonium (TEA) were determined in the following manner. The nimals (rats or mice) were weighed, killed by cervical dislocation r by decapitation and the kidneys removed, decapsulated, weighed nd placed in ice—cold saline (0.9% NaCl). Thin slices (approxi— ately 0.5 mm) of renal cortex were cut with a razor blade and 100-150 g of tissue (wet weight) incubated in 2.0 ml of a phosphate—buffered edium (Cross and Taggart, 1950) containing lxlO—3M acetate, lxlO_4M 6M PAH (Sigma) EA (Eastman Organic Chemicals, Rochester, NY), 5.8x10- 1d trace quantities of (3H)-PAH and (14C)—TEA (New England Nuclear, )ston, MA). The slices were allowed to incubate for 90 min (to aach equilibrium) at 25°C under an atmosphere of 100% 02. They are then removed, blotted, weighed and homogenized in 1.5 m1 of 10% ‘ichloroacetic acid (TCA), brought to a final volume of 5.0 ml with stilled water and mixed thoroughly. A 1.0 ml aliquot of medium was xed with 1.5 ml of 10% TCA and 2.5 m1 of distilled water (total lume of 5.0 ml). The TCA solutions were then centrifuged to parate the precipitated protein and 1.0 ml aliquots of the Super— tant fraction added to 10.0 ml of Aqueous Counting Scintillant (ACS, 36 Amersham Corp., Arlington Heights, IL) and radioactivity determined by liquid scintillation spectrometry. Disintegrations per min (dpm) were determined using (14C)—toluene and (3H)—H20 (New England Nuclear) as internal standards. The slice-to—medium ratios,or S/M, of PAH and TEA were calculated as dpm/g tissue (wet weight) divided y dpm/ml incubation medium. . 3. £250 determinations LD50 values for chloroform (CHCl3) and carbon tetrachloride CC14) were determined in male mice by injecting solutions of 011013 r CCl4 in corn oil (total injection volume of 5 ml/kg) into the peri— .oneal cavity and determining the cumulative number of deaths every 24 :r thereafter. At least 10 animals were used at each dose. All urviving animals were observed for an additional 18 days after njection of CHCl3 and C014. LD50 values, 95% canfidence intervals 1 nd potency ratios were calculated by the method of Litchfield and ilcoxin (1949). 4. Histology Samples of liver and kidney were fixed in buffered formalin 3.7% formaldehyde in 0.3 M sodium phosphate buffer, pH 7.2), Ibedded in paraffin blocks, sectioned at 5 microns, mounted on glass .ides and stained with hematoxylin and eosin under the direction of '. V.L. Sanger, Michigan State University, Department of Pathology, ,st Lansing, Michigan. Black and white photographs were taken at X magnification using Ektapan film (Eastman-Kodak 00., Rochester, ). Histopathological examination was performed by Dr. V.L. Sanger. 37 For specific experiments, samples of liver and kidney were so stained with periodic acid-Schiff (PAS), with and without prior astase digestion, and evaluated for epithelial changes by Dr. J. rnstein, William Beaumont Hospital, Department of Anatomic thology, Royal Oak, MI, 48072. Formalin—fixed samples of liver were so stained with Gomori's trichrome, Wilders reticulin, Perl's rrocyanide for iron, and rubeanic acid for capper. Sections were luated for cellular necrosis and vacuolization, nuclear enlargement lyploidy), inflammatory cell infiltration, and fibrosis or septa— n on an arbitrary scale of O—3+ (negative, mild, moderate, severe) Dr. J. Bernstein. Regenerative activity was estimated by counting otic figures per 40 random high—power fields. 5. Definitions of toxicigy Elevations of SGPT and SGOT activities were used as indica— rs of CCl and CHCl3 hepatotoxicity. The hepatotoxic effects of 4 .4 were also defined histologically, in selected eXperiments, as Iatocellular necrosis. Decreases in PAH and TEA S/Ms were used as indicators of 4 and CHCl3 nephrotoxicity. Intoxication with CHCl3 also in— ased BUN concentrations and total kidney weight. Kidneys from 4-intoxicated rats were examined by light microsc0py1nn:histolo— a1 evidence of cellular injury was not detected. Losses of body weight and death were also used as indicators toxicity though the mechanism of such effects is unknown. C. Analyses o l. Tissu Sampl several times a K01 buffer, pH grinder (0.10—0 were punctured Tris-KCl buffer were then centr and unbroken ce. 14,000 x g for J the 14,000 x g g the rest of the 60 min at 2°C. resuspended in I (Dr 20 EM Tris-1 stored for later again at 100,00C resuSpended in I for 2-3 days unt um PCNMA 2. fraction 0f reua Impfer and Brugg mixtures Were as 4 x , 38 . Analyses of Components of the Drug—metabolizing Enzyme Systems 1. Tissue preparation Samples of liver and kidney were weighed, minced, washed everal times and homogenized in 3 volumes of ice—cold 20 mM Tris—1.15% Cl buffer, pH 7.40, using a motor—driven Potter—Elvehjem tissue rinder (0.10—0.15 mm clearance). The tunica albuginea of the testes ere punctured and the extruded contents homogenized in 3 volumes of ris—KCl buffer as described for livers and kidneys. The homogenates ere then centrifuged at 600 x g for 5 min at 2°C to separate nuclei nd unbroken cells, and the supernatant fractions centrifuged at 4,000 x g for 25 min at 2°C to separate mitochondria. An aliquot of e 14,000 x g supernatant fraction was removed and refrigerated and he rest of the supernate decanted and centrifuged at 100,000 x g for 3 min at 2°C. The resulting pellets (microsomal fraction) were asuspended in the original volume of 20 mM Tris—1.15% KCl buffer )r 20 mM Tris-1.15% KCl—l mM EDTA, pH 7.40, if the pellet was to be :ored for later determination of cytochrome P—450) and centrifuged ;ain at 100,000 x g for 30 min at 2°C. The final pellet was then ,suspended in Tris—KCl buffer for enzyme assays or stored at —70°C r 2—3 days until assayed for cytochrome P—450 content. 2. prhloro—N—methylaniline N—demethylase (PCNMA) assay PCNMA activity was determined in the 14,000 x g supernatant action of renal, hepatic and testicular homogenates as described by fer and Bruggeman (1966). Final concentrations in the incubation xtures were as follows: 0.4 mM NADP, 15 mM MgC12, 10 mM nicotinamide, nM semicarbazide, 8 mM glucose—6—phosphate, l U/ml glucose—G—phOSphate J___‘ e+ dehydrogenase E CA)- ”mi“ at E. (1951), 3-10mg/m1 (tee 3. fl AHH a fraction or the hepatic and tes (1968) and Oesc mixtures were a Tris, 5.8 mM g1 genase and 1.0 T were 0.25-0.65 '1 (testis) when t3 0.3 mg/ml (liVPJ x g pellet fracr fluorometricallj 1 cm light path} relative fluores tion of quinine fluorescence uni length, 460 mm). The in C°rP-, Philadelp Westchester, PA) Nude)“ NJ), met 39 ehydrogenase and 3 mM p—chloro—N-methylaniline (Calbiochem, La Jolla, LA). Protein concentrations, as determined by the method of Lowry £__l, (1951), were 2.0—2.5 mg/ml (liver), 4—5 mg/ml (kidney) and ;-10 mg/ml (testis). 3. Aryl hydrocarbon hydrogylase (AHH) assay AHH activity was determined in the 14,000 x g supernatant ’raction or the resuspended 100,000 x g pellet fraction of renal, repatic and testicular homogenates as described by Nebert and Gelboin T1968) and Oesch (1976). Final concentrations in the incubation Lixtures were as follows: 0.4 mM NADP, 0.25 mM NAD, 3 mM MgC12, 60 mM ris, 5.8 mM glucose-6-phosphate, l U/ml glucose-6-phosphate dehydro- enase and 1.0 mM 3,4-benzo(a)pyrene (Sigma). Protein concentrations ‘ere 0.25—0.65 mg/ml (liver), 1-4 mg/ml (kidney) and 3—4 mg/ml testis) when the 14,000 x g supernatant fraction was used, and 0.1— .3 mg/ml (liver) and 1—2 mg/ml (kidney and testis) when the 100,000 g pellet fraction was used. Product formation was estimated Luorometrically using an Aminoo Spectrofluorimeter (quartz cuvettes, cm light path) and activity (formation of product) eXpressed as alative fluorescence units per mg protein per min. A 10 uM solu— .on of quinine sulfate in 0.05 M H2804 produced 2X103 relative uorescence units (excitation wavelength, 365 nm; emission wave— ngth, 460 nm). The inhibitory effects of SKF 525—A (Smith, Kline and French fp., Philadelphia, PA), piperonyl butoxide (PB, Chem Service, :tchester, PA), allyl—isoPropylacetamide (AIA, Hoffman-LaRoche, Iley, NJ), metyrapone (MET, Aldrich Chemical Co., Milwaukee, WI) and a-napthOfl‘ .12 m were < except that the mixture was 1X] added to “him 1x10'3u. Stock distilled water sorbitan mono-0 acetone. The V incubation miXt comprised 17. (V. tions (no addit: conducted. 4. Bipher (BF-4- BP—2-C x g supernatant to the method of incubation mixtu m1 glucose~6~pho and 10.0 mM biph. 0.204 mg/ml (l: 5. Cytochr Cytochr (100,000 x g pell homogenates were Photometer (B eckm 4O u—napthoflavone (ANF, Aldrich) on renal and hepatic AHH activities yit£9_were determined using the procedure outlined above for AHH ept that the concentration of 3,4—benzo(a)pyrene in the incubation ture was lxlOfiAM. Stock solutions of the various inhibitors were ed to achieve concentrations of 0 M, lxlO—6M, 1x10_5M, lxlO_%fi or 0_3M. Stock solutions of SKF 525—A and MET were formulated in tilled water, stock solutions of AIA and PB in 1% poloxyethylene bitan mono-oleate (Tween 80, Sigma) and stock solutions of ANF in tone. The various stock solutions of inhibitors Were added to the ubation mixtures in such a manner that the stock solution vehicle prised 1% (v/v) of the total incubation mixture. Control incuba— ns (no additions of inhibitors or inhibitor vehicles) were also ducted. 4. Biphenyl-Z—hydroxylase (BP—Z—OH) and biphenyl—4—hydroxylase (BP-4-OH) assays BP—Z—OH and BP—4—OH activities were determined in the 14,000 supernatant fraction of renal and hepatic homogenates according :he method of Creaven_e£.al. (1965). Final concentrations in the bation mixtures were as follows: 0.3 mM NADP, 2.5 mM MgC12, 3.8 1ucose—6—phosphate, 0.5 U/ml g1ucose—6—phosphate dehydrogenase 10.0 mM biphenyl (Eastman Organic). Protein c0ncentrations were 3.4 mg/ml (liver) and 0.8-1.2 mg/ml (kidney). 5. Cytochrome P—450 assays Cytochrome P—450 concentrations in the microsomal pellets 000 x g pellet fraction) of hepatic, renal and testicular enates were determined spectrally using a dual—beam spectro— Deter (Beckman UV5260). The microsomal pellets were suspended in 100 mM sodium : —1 of 91 111111019 CI p-450 from the baselines 0f t} difference Spec mm 1. Tine- —h— inhib syste a. taining 25, 100 days. Another g or 200 ppm of P( with an equivale livers and kidne x g supernatant b. taining 0 or 200 injection of TCDI 20:1) 0 or 16 “g, Sacrifice s o e]: (Sigma), 50 lug/kg 41 DO mM sodium phosphate buffer, pH 7.20, and an extinction coefficient 91 mmole_lcm—l used to calculate the concentrations of cytochrome 450 from the differences in soret maxima near 450 nm and the 490 nm selines of the sodium dithionite—reduced CO:hemoprotein—complex fference Spectra (Omura and Sato, 1964). Individual Experiments 1. Time—dependency and organ—specificity of induction and inhibition of renal and hepatic drug—metabolizing enzyme systems a. Effects of length of exposure and dietary concentrations of polybrominated biphenyls (PBB) and polychlorinated biphenyls (PCB)on renal and hepatic AHH activities: ICR, male mice (15—20 g) were maintained on diets con— ning 25, 100, or 200 ppm of PBB or 25, 200 or 400 ppm of PCB for 14 8. Another group of mice received diets containing 100 ppm of PBB 200 ppm of PCB for 21 days. COntrol mice received diet formulated 1 an equivalent amount of acetone. The mice were sacrificed, the ars and kidneys removed and AHH activities determined in the 14,000 supernatant fraction of renal and hepatic homogenates. b. Effects of sodium phenobarbital (Nan), 3—methylcholan— threne (3MC), PCB and 2,3,7,8—tetrachlorodibenzo-p— dioxin (TCDD) on enzyme activities in kidney and liver in mice ICR, male mice (15—20 g) were maintained on diets con— ing 0 or 200 ppm of PCB for 28 days or were given a single i.p. ction of TCDD (Dow Chemical Co., Midland, MI) in corn oil:acetone, , 0 or 16 ug/kg (total injection volume of 5 m1/kg), 72 hr before ifice. A separate set of mice received i.p. injections of Nan na), 50 mg/kg in distilled water, Once daily for 4 consecutive days, or 3MC (Easma: Secutive days- 5ml/k8 and th‘ The mice were 1 of AH, PCNMA: supernatant fra concentrations 1 in distilled wal sure studies ma] dose of PBB (90 or a single i.p. 24, 72, or 216 h of corn oil. F0 ceived successiw hr before sacrif: 72 and 96 hr beft volumes of corn c were killed at 8 sacrifice and AHH 42 or 3MC (Eastman Organic), 25 mg/kg in corn oil, once daily for 3 con— secutive days. The total injection volumes for Nan and 3MC were 5 ml/kg and the final doses were administered 24 hr before sacrifice. The mice were killed, livers and kidneys removed, and the activities of AHH, PCNMA, BP-2—OH and BP—4—OH determined in the 14,000 x g supernatant fraction of renal and hepatic homogenates. c. Effects of single and multiple doses of PBB, PCB, Nan2 and 3MC on renal, hepatic and testicular enzymes and gytochrome P—450 in rats PBB, PCB and 3MC Were dissolved in corn oil to achieve .oncentrations of 18, 18, and 8 mg/ml, respectively. Nan was dissolved n distilled water at a cancentration of 33.5 mg/ml. For single expo- ure studies male, Fischer 344 rats (125-150 g) received a single oral ose of PBB (90 mg/kg), PCB (90 mg/kg) or 3MC (40 mg/kg) by gavage, r a single i.p. injection of Nan (75 mg/kg) and were sacrificed 9, 4, 72, or 216 hr later. Control rats received an appropriate volume 3 corn oil. For multiple-dose studies male, Fischer 344 rats re- :ived successive oral doses of PBB or PCB (90 mg/kg) 24, 48, and 72 ' before sacrifice, or i.p. injections of Nan (75 mg/kg) 24, 48, and 96 hr before sacrifice. Control rats received appropriate lumes of corn oil. Administrations were timed so that all animals re killed at 8 a.m. Kidneys,livers and testes were removed upon orifice and AHH and PCNMA activities and cytochrome P—450 concentra— Jns determined. In selected cases, the sensitivities of renal and >atic AHH to inhibition by MET and ANF were evaluated in_vitro. (125—150 g) rer Pharmaceuticals 75 mg/kg in wat were killed 1, PCNMA determine and hepatic hon 2. Effec drug- a. taining 100 ppm received diet f mice were then 0.005, 0.025, 0 Volume of 51111, Blood was collar and BUN deter-mil abilities of re Ilined. ! taining 0, 20’ C a Single i.p_ ir total inJ'eCtion mined in all thr 43 d. Time—dependent effects of single doses of SKF 525-A and piperonyl butoxide (PB) on renal and hepagig enzyme activities in mice and rats ICR, male mice (15—20 g) and Fischer 344, male rats 25-150 g) received single i.p. injections of PB (80% pure, ICN armaceuticals, Plainview, NY), 600 mg/kg in corn oil, or SKF 525—A, mg/kg in water (total injection volumes of 5 ml/kg). The animals :e killed 1, 2, 4, or 12 hr later and the activities of AHH and [MA determined in the 14,000 x g supernatant fractions of renal . hepatic homogenates. 2. Effects of stimulation and inhibition of renal and hepatic drug—metabolizing enzyme systems on CHCl3 and C012 toxicity a. Effects of PBB and PCB on CC14 toxicity in mice ICR, male mice (15—20 g) were maintained on diets con— ning 100 ppm of PBB or 200 ppm of PCB for 28 days. 'COntrOl mice eived diet formulated with an equivalent amount of acetone. The a were then challenged with a single i.p. injection of CC14, 0.000, )5, 0.025, 0.125, or 0.625 ml/kg in corn oil, total injection ime 0f 5 ml/kg, and sacrificed 48 hr later by decapitation. >d was collected, the serum fractions separated, and SGOT, SGPT BUN determined. Livers and kidneys were removed, weighed and the ities of renal cortical slices to accumulate PAH and TEA deter— d. Another group of mice was maintained on diets con— ing 0, 20, or 100 ppm of PBB for 20 days and then challenged with igle i.p. injection of one of several doses of CCl4 in corn oil, L injection volume of 5 ml/kg. LD values for C014 were deter— 50 1 in all three groups. on diets conta third group of corn oil, by g; doses). A four with an equival every 72 hr for 21) with a sing or 2.00 ml/kg, hr later by a b ties of SGOT, 8 directly from t trations and fo Elkhart, IN). abilities of re] mined. Thin 31: incubated in a I saturated With ( receptacle and t an Oxygen-SeHsit Yellow Springs, of 02 CODSumed p from Samples of The residlles Wer Pressed as mg li- 44 b. Effects of PBB, PCB and hex achlorobenzene (HCB) on CClg toxicity in rats Sprague—Dawley, male rats (125—150 g) were maintained a diets containing 100 ppm of PBB or 200 ppm of PCB for 20 days. A aird group of rats received HCB (98% purity, Aldrich), 30 mg/kg in arm oil, by gavage every 72 hr for a period of 20 days (total of 7 )ses). A fourth group (controls) was maintained on diet formulated .th an equivalent amount of acetone and received corn oil by gavage 'ery 72 hr for a 20 day period. All rats were then challenged (day ) with a single i.p. injection of CCl4 in corn oil, 0.00, 0.03, 0.25 2.00 ml/kg, total injection volume of 5 ml/kg, and were killed 48 later by a blow to the head. Blood was collected and the activi— es of SGOT, SGPT, and BUN determined. Urine samples were collected rectly from the bladder and analyzed for protein and glucose concen— 1tions and for pH with reagent sticks (Hema—Combistix, Miles Labs., chart, IN). Kidneys and livers were removed, weighed and the Llities of renal cortical slices to accumulate PAH and TEA deter— Led. Thin slices (approximately 0.5 mm thick) of renal cortex were .ubated in a phosphate—buffered medium (Cross and Taggart, 1950) urated with 02 at 30°C in an air—tight, magnetically—stirred eptacle and the rate of oxygen consumption (002) determined using oxygen—sensitive electrode (Yellow Springs Instrument Co., low Springs, OH). Renal Q02 values were expressed as microliters )2 consumed per g tissue per minute. Total lipids were extracted n samples of liver and kidney by the method of Folch E£.§l- (1957). residues were dried to a constant weight and lipid content eX‘ ;sed as mg lipid (dry weight) per g of tissue (wet weight). C. on diets conta Control rats r acetone. The samples of liv: 20 mil Tris-1.1. of Tris-KCl bu: earlier. The 1 2°C to separate centrifuged at The resulting s 100,000 x g for measured in the kits. Protein 3011‘?) fraction; GOT and GPT act: of 100,000 x g 5 and per 100 g of d. lr-+ Ira-1 I diets containing were normalized from the dams at randomized , Sepa 45 c. Effects of PBB and PCB on hepatocellular GPT and GOT activities Sprague—Dawley, male rats (125-150 g) were maintained diets containing 100 ppm of PBB or 200 ppm of PCB for 20 days. ntrol rats received diet formulated with an equivalent amount of etone. The rats were killed on day 21 by a blow to the head and mples of liver removed, weighed, washed several times in ice-cold mM Tris—1.15% KCl buffer (pH 7.40) and homogenized in 3 volumes Tris-KCl buffer in a Potter—Elvehjem tissue grinder, as described rlier. The homogenates were then centrifuged at 600 x g for 5 min at 3 to separate nuclei and unbroken cells and the supernatant fraction 1trifuged at 14,000 x g for 25 min at 2°C to separate mitochondria. : resulting supernatant fraction was decanted and centrifuged at 1,000 x g for 60 min at 2°C and the activities of GOT and GPT _sured in the 100,000 x g supernatant fraction using Sigma reagent 5. Protein concentrations in the 100,000 x g supernatant (cyto— ic) fractions were determined by the method of Lowry_e£_al. (1951). and GPT activities were expressed as Sigma—Frankel units per m1 00,000 x g supernate, per mg of 100,000 x g supernatant protein, per 100 g of total body weight. d. Effects of maternal consumption of PBB on the toxici— ties of CHClq and CCl, in developing male rats J H- Timed—pregnant, Sprague—Dawley rats were placed on 3 containing 0 or 100 ppm of PBB on day 8 of gestation. Litters normalized to 10 pups each at birth. The pups ware separated the dams at 26 days of age (the approximate time of weaning), omized, separated by sex and placed on diets containing either 0 ppm of PBB (al and half of th 100 ppm of PBB PBB diet). 0n Body weights w challenged wit? 0.00, 0.03, 0.2 The rats were I 0f SGOT and SC} kidneys and liv renal cortical taining 0’ l, 2‘ with a Single i 50'0 1ll/kg in c< were sacrificed livers and kidne and the abilitie were detQUHined. f. [HIM I—l 3110 or Napb as d. m' ice were Challei 0.25 or 0.75 1111/} F'— 46 pm of PBB (all of the male pups from dams consuming 0 ppm PBB diet nd half of the male pups from dams consuming 100 ppm PBB diet) or )0 ppm of PBB (half of the male pups from dams consuming 100 ppm SB diet). Only male pups Were used for the remainder of this study. idy weights were determined at 52 days of age and the rats then iallenged with a single i.p. injection of CHCl3 or CCl4 in corn oil, 00, 0.03, 0.25, or 2.00 m1/kg, total injection volume of 5 mllkg. e rats were killed 48 hr later, blood collected and the activities SGOT and SGPT and the concentrations of BUN determined. The ineys and livers were removed and weighed and the abilities of 1al cortical slices to accumulate PAH and TEA determined. e. Effects of dietarnyBB on CHClO toxicity in mice J ICR, male mice (15-20 g) were maintained on diets con- .ning 0, l, 25, or 100 ppm of PBB for 14 days and then challenged .h a single i.p. injection of CHCl 0.0, 0.5, 2.5, 5.0, 25.0, or 33 O ul/kg in corn oil, total injection volume of 5 ml/kg. The mice e sacrificed 24 hr later by decapitation, blood collected and hrs and kidneys removed and weighed. SGPT, SGOT and BUN values the abilities of renal cortical slices to accumulate PAH and TEA a determined. f. Effects of Nan, 3MC, PCB and TCDD on CHCl, toxicity in mice J ICR, male mice (15—20 g) were treated with PCB, TCDD, or Nan as described previously (Methods, Section le). The were challenged with a single i.p. injection of CHCl 0.00, 0.05, 3’ or 0.75 ml/kg in corn oil, total injection volume of 5 ml/kg, 24 1____ a i hr after the f TCDD, or after animals were 5 and BUN and th and TEA determ PB, 600 mg/kg, min after a Si; in corn oil, 11 killed 24 hr at SCOT and BUN ar PAH and TEA det 3~ Inter a. taining 100 ppm lated with an e. then challenged 0'00, MS. 0.25 and sacrificed 2 kidneys were hon for reduch 110n- 1959). Selected 47 after the final dose of Nan or 3MC, 72 hr after a single dose of DD, or after 28 days on diet containing 200 ppm of PCB. The imals were sacrificed 24 hr later, blood collected, and SGPT, SGOT d BUN and the abilities of renal cortical slices to accumulate PAH d TEA determined. g. Effects of SKF 525—A and PB on CHCl3 toxicity in mice ICR, male mice (15—20 g) received i.p. injections of , 600 mg/kg, or SKF 525—A, 75 mg/kg, either 120 min before or 60 1 after a single i.p. injection of CHCl3, 0.00, 0.25, or 0.75 ml/kg corn oil, in a total injection volume of 5 m1/kg. The mice were Lled 24 hr after CHCl3 administration, blood collected and SGPT, )T and BUN and the abilities of renal cortical slices to accumulate 1 and TEA determined. 3. Interactions of CHClO with renal and hepatic glutathione (GSH) ’ a. Effects of PBB and PCB on CHClo—induced GSH depletion in mice J ICR, male mice (15—20 g) were maintained on diets con— ing 100 ppm of PBB or 200 ppm of PCB or on a control diet formu— d with an equivalent amount of acetone for 20 days. The mice were challenged with a single i.p. injection of CHCl3 in corn oil, , 0.05, 0.25, or 0.75 ml/kg, total injection volume of 5 ml/kg, sacrificed 2 hr later by cervical dislocation. Livers and eye were homogenized in 20 volumes of ice—cold 6% TCA and analyzed reduced non—protein thiol content using Ellman's reagent (Ellman, ). Selected samples (from naive and treated animals) were also analyzed speci Cohn and Lyle reduced non-pr kidney homogen the kidneys an an analytical b. tion of diethyl oil, total inje by cervical dis cortical, medul liver and the v volumes of ice- separate TCA~de non‘Protein thi. Ellman's reagen- assay Specific ; that at least 9( natant fraction liver and the V5 described Previo C. [Olm r—I injection of die 48 ralyzed specifically for reduced GSH content using the method of >hn and Lyle (1966) and it was determined that at least 90% of the iduced non—protein thiols in the supernatant fractions of liver and dney homogenates were reduced GSH. The concentrations of GSH in e kidneys and livers were calculated using reduced GSH (Sigma) as analytical standard and expressed as ug GSH/g tissue (wet weight). b. Depletion of renal and hepatic GSH by diethyl maleate ICR, male mice (15—20 g) received a single i.p. injec— >n of diethyl maleate (80% pure, Aldrich), O or 600 mg/kg in corn _, total injection volume of 5 ml/kg, and were sacrificed 2 hr later cervical dislocation. Kidneys were removed, cut lengthwise and 'tical, medullary and papillary sections dissected. Samples of 'er and the various sections of the kidney were homogenized in 20 umes of ice—cold 6% TCA. The homogenates were centrifuged to arate TCA—denatured protein and the concentrations of reduced, —protein thiols in the supernatant fractions determined using an's reagent. Analyses of random samples using a fluorimetric y specific for reduced GSH (Cohn and Lyle, 1966) demonstrated at least 90% of the reduced, non—protein thiol in the super— nt fraction was reduced GSH. The concentrations of GSH in the r and the various sections of the kidney were calculated as ribed previously. c. Effects of diethyl maleate on GSH concentrations and CHClg toxicity in mice J ICR, male mice (15—20 g) were given a single i.p. tion of diethyl maleate, 0 or 600 mg/kg, followed in 90 min by a single i.p. 1' The mice were 1 collected. SGT slices to accun 4. £__l a. taining 100 ppm an equivalent 3 killed by cervi genized in 3 we and renal and h (Methods, Secti with (140)-CHC1 in tightly-stop Ed. (1973). as f01lows: 0. glucose-6_ph08p. 0.020 mM CHC13 W—fi _.._-....._ -. _ ~—-—.~.~.-.--~—-.4-—=—r—-= .__-_ _ . . ~ ~ -- —. 49 single i.p. injection of CHC13, 0.000, 0.033, 0.100 or 0.300 ml/kg. 3 mice were killed 24 hr after CHCl3 administration and blood was Llected. SGPT, SGOT and BUN and the abilities of renal cortical Lces to accumulate PAH and TEA.were determined. 4. Agovalent binding of CHCl.3 metabolites in mice J__ a. Covalent binding of CHClO metabolites to renal and hepatic microsomal_protein in Vitro ICR, male mice (15-20 g) were maintained on diets con— .ning 100 ppm of PBB or 200 ppm of PCB or on a diet formulated with equivalent amount of acetone for 20 days. The mice were then led by cervical dislocation and kidneys and livers removed, homo- ized in 3 volumes of ice-cold 20 mM Tris-1.15% KCl buffer (pH 7.40) renal and hepatic microsomes isolated as described previously thods, Section C). Renal and hepatic microsomes were incubated 1 (;4C)—CHC1 l 3 :ightly—stoppered 25 ml Erlenmeyer flasks as described by Ilett (New England Nuclear) and a regenerating system EL. (1973). Final concentrations in the incubation mixtures were Follows: 0.12 mM NADH, 0.20 mM NADPH, 2.0 mM nicotinamide, 2.0 mM .ose-6—phosphate, 1 U/ml glucose-6—phosphate dehydrogenase and 0 mM CHCl3 (1 uCi/ml), all dissolved in 20 mM Tris-1.15% KCl er, pH 7.40. Protein concnentrations, as determined by the method oer.§£_§lx (1951), were 1.0 mg/ml (liver) and 2.0 mg/ml (kidney). r; 14.9 mCi/mmole, radiochemical purity, 99%. The material, as nased from.NeW England Nuclear, was dissolved in dimethylforma— to achieve a concentration of 20 mM and stored at -70°C. Radio— rity in the stock solution was determined each time it was used )rrect for loss due to volatilization during handling. The reactions 1 102 TCA. The ( extracted sever as described b: radioactivity . protein samples determination c internal stand; b. taining 100 pp}; equivalent amoi single i.p. inj- 110 uCi/kg. Se from the retro- 15, 30, 45’ 60, W3. Blood 5 ment COW DOWIIE scintillation g C . Benized in 3 we homogenates Wer resulting Pelle 50 he reactions were stopped after 5 min by the addition of ice—cold 0% TCA. The denatured protein was separated by centrifugation and xtracted several times with hot (50°C) chloroformrmethanol (2:1), 5 described by Ilett gt 3;, (1973), to remove non-covalently bound adioactivity. (The final extracts contained no radioactivity.) The rotein samples were then dissolved in 1 N NaOH and aliquots used for etermination of protein and radioactivity using (14C)—toluene as an iternal standard. b. Clearance of CHQlD and metabolites from blood and covalent binding Eb total renal and hepaticgprotein ICR, male mice (15—20 g) were maintained on diets con— tining 100 ppm of PBB, 200 ppm of PCB or on diet formulated with an [uivalent amount of acetone for 14 days and then challenged with a .ngle i.p. injection of (14C)-CHC1 1.75 mmoles/kg (0.10 ml/kg), 3, .0 uCi/kg. Samples of whole venous blood (10 pl) were withdrawn ’om the retro-orbital sinus into heparinized glass capillary tubes , 30, 45, 60, 90, 120 and 180 min after administration of (14C)— Cl Blood samples were dissolved in Soluene—350 (Packard Instru- 3. nt Co., Downer's Grove, IL) and radioactivity determined by liquid intillation spectrometry using (14C)—toluene as an internal standard. 2 mice were sacrificed by cervical dislocation 3, 6 or 12 hr after 313 administration. Livers and kidneys were removed and homo— [ized in 3 volumes of ice—cold distilled water. Aliquots of the logenates were mixed with an equal volume of ice—cold 10% TCA, the :tures centrifuged to separate the denatured proteins, and the ulting pellets extracted 5 times with hot (50°C) chloroform: methafl01’ 2:1 quots of resuS‘: quots of whole was determined C. 14 ( C)-CHCl (l. 3 excised, minced 0.005 M Hepes ( Sigma) buffer, volumes of 0.3 tissue grinder for 10 min at 2 saved, the pell sucrose—0.005 M fuged again at were extracted (1972). RNA co at 260 nm, pH 1 mined by the me' X 8 Centrifugat; t he suPernatant SuSPended in 0 f before. This p I 51 hanol, 2:1 (the final extracts contained no radioactivity). Ali— ts of resuspended protein were then dissolved in l N NaOH and ali— ts of whole homogenates dissolved in Soluene-350. Radioactivity determined by liquid scintillation spectrometry. c. Covalent binding of CHCl3 metabolites to subcellular fractions in vivo Mice were sacrificed 3 hr after an i.p. injection of C)—CHC13 (1.75 mmoles/kg, llO uCi/kg). Kidneys and livers were ised, minced, washed several times with ice—cold 0.3 M sucrose- 05 M Hepes (N—Z—hydroxyethylpiperazine—N'-2-ethane sulfonic acid, ma) buffer, pH 7.40. The tissues were then homogenized in 5 umes of 0.3 M sucrose—0.005 M Hepes buffer with a Potter—Elvehjem sue grinder (0.10—0.15 mm clearance) and centrifuged at 1,000 x g 10 min at 2°C. The supernatant fractions were decanted and ed, the pellets rehomogenized in the original volume of 0.3 M rose-0.005 M Hepes—0.2% Triton X-100 buffer, pH 7.40, and centri— sd again at 1,000 x g for 10 min at 2°C. Nucleotides (RNA and DNA) a extracted from this pellet by the method of Goodman and Potter ’2). RNA concentrations were determined spectrophotometrically 160 nm, pH 1.0 (Pomerai §£_al., 1974) and DNA c0ncentrations deter— :d by the method of Ceriotti (1958). The supernatant fractions saved from the initial 1,000 centrifugation were centrifuged at 8,000 x g for 10 min at 2°C, supernatant fractions decanted and saved, and the pellets re- ended in 0.3 M sucrose—0.005 M Hepes buffer and centrifuged as re. This process was repeated again (total of 3 centrifugations at 8,000 x g) homogenized 1n centrifugation the supernatan 60 min at 2°C. sucrose-0.005 1 min at 2°C, an- reticulum frac fractions were protein separa1 times with hot was then dissol aliquots of mic Folch it 11: (1 stream of N2, 8 to dry under N2 Radioactivity i tide solutions Using (14C)~tol Statis - w variance (COmPl 52 : 8,000 x g) and the final pellet, termed the mitochondrial fraction, rmogenized in 2 ml of distilled water. The supernatant fractions from the initial 8,000 x g :ntrifugation were centrifuged at 14,000 x g for 25 min at 2°C and .e supernatant fractions decanted and centrifuged at 100,000 x g for min at 2°C. The resulting pellets were resuspended in 0.3 M crose—0.005 M Hepes buffer, centrifuged again at 100,000 x g for 60 n at 2°C, and the final pellet, termed the microsomal or endoplasmic ticulum fraction, homogenized in 2 ml of distilled water. One ml aliquots of the microsomal and mitochondrial actions were mixed with 1 ml of ice—cold 10% TCA, the denatured )tein separated by centrifugation, and the pellets extracted 5 1es with hot (50°C)chloroformzmethanol, 2:1. The protein pellet ; then dissolved in l N NaOH. Lipids were extracted from 1 ml .quots of microsomal and mitochondrial fractions by the method of .ch at a1. (1957), the extracts allowed to dry under a gentle eam of N2, and the residues dissolved in 1.5 m1 CHCl3 and allowed dry under N2, twice, to remove non-bound, residual radioactivity. ioactivity in the lipid residues, protein solutions and nucleo— e solutions was determined by liquid scintillation spectrometry 1g (14C)—toluene as an internal standard. Statistics Unless stated otherwise, all data were analyzed by analysis of Lance (completely randomized design or blocked design) and treat— : means were compared using the Least Significant Difference or the Student-Ne of significanr The lines 14C—activity 1‘ 011013 was conf curves (contro variance and t as the criteri 53 the Student—Newman-Keuls tests (Sokal and Rohlf, 1969). The criterion of significance in all cases was p<0.05. The linearity of the curves relating the log concentrations of l4C—activity in the blood to the time after administration of (14C)- CHCl3 was confirmed by regression analysis. Data from the three curves (control, PBB and PCB) were then analyzed by analysis of co— variance and the slopes compared using Student's Eftest with p<0.05 as the criteriOn of significance (Sokal and Rohlf, 1969). A. Enzyme In 1. Effe PBB , The 1 vities was pro} diet when ICR, of PBB or 25, I and hepatic AH} containing 25 F Cient to Produc aPPeared to be activities than than renal AHH creasing the le to 21 days did 1 activities of r« Table 3). The slightly greate] the same diEts j PBB and PCB may (data not Shown) RESULTS A. Enzyme Induction and Inhibition 1. Effects of length of exposure and dietary c0ncentration of PBB and PCB on renal and hepatic AHH activities The magnitude of increase in renal and hepatic AHH acti— vities was proportional to the concentration of PBB and PCB in the diet when ICR, male mice were fed diets containing 25, 100 or 200 ppm of PBB or 25, 200 or 400 ppm of PCB for 14 days (Table 2). Both renal and hepatic AHH activities Were increased in mice consuming diets :ontaining 25 ppm of PBB for 14 days, though 25 ppm of PCB was insuffi- :ient to produce an increase in AHH activities. In general, PBB rppeared to be a more potent inducer of both renal and hepatic AHH Lctivities than was PCB, and hepatic AHH appeared to be more sensitive .han renal AHH to the inductive effects of dietary PBB and PCB. In— reasing the length of dietary exposure to PBB and PCB from 14 days 0 21 days did not change the magnitudes of the increases in specific ctivities of renal and hepatic AHH (activities per mg of protein, able 3). The liver weight—to—body weight ratio, however, was lightly greater in mice fed PBB and PCB for 21 days than in mice fed 1e same diets for 14 days, suggesting that continued ingestion of SB and PCB may increase total hepatic biotransformation capacity lata not shown). 54 Dietary Co; Hydroxyl. c . l Significan d . Slgnifican p<0.05, 55 TABLE 2 Dietary COncentration—Dependent Induction of Aryl Hydrocarbon Hydroxylase (AHH) Activities by Polybrominated Biphenyls (PBBs) and Polychlorinated Biphenyls (PCBs) . . . a AHH Act1V1t1es Treatment ppm Liver Kidney b b PBB 25 216il9b c 150il4b c PBB lOO 438i37 ’0 d 310il3 ’0 PBB 200 942i41 ’ ’ 466i26 ’ ’ PCB 25 139il4b c 85i 6 c PCB 200 339i47b’c d 292i12b’c d PCB 400 598i56 ’ ’ 478:13 ’ ’ Cl . Relative fluorescence i l S.E., N=6 animals. 0.08i0.01 (kidney). units/mg protein/min, mean Z of control Control values: 12.10i2.10 (liver), bSignificantly greater than control, p<0.05. cSignificantly greater than 25 ppm, p<0.05. dSignificantly greater than 100 ppm (PBB) or 200 ppm (PCB), p<0.05. Time-Dep (AHH) A PBB PBB PCB PCB aRele Z 01 Valr bNuml 56 TABLE 3 Time—Dependent Induction of Aryl Hydrocarbon Hydroxylase (AHH) Activities by Polybrominated Biphenyls (PBB) and Polychlorinated Biphenyls (PCB) . b AHH Activitiesa Treatment Time . . Liver Kidney PBB, 100 ppm 14 d 438i37 310i13 PBB, 100 ppm 21 d 413:26 302i21 PCB, 200 ppm 14 d 339i47 292i12 PCB, 200 ppm 21 d 267il7 300:15 a . . . . Relative fluorescence units/mg protein/min, mean Z of control i l S.E., N=6 animals. Control values: 12.10i2.10 (liver), 0.08i0.01 (kidney). 29Number of days (d) on designated diet. are are; The single i.p. it and hepatic ME treatment with renal and hepa MFO activities 3. am NiPh tifi Cytor activities in 1, in Table 5, En were greater in testis. Hepat PBB and PCB but and testicnlar } PCB’ Nan or 3m hepatic and rena however, was gre treatment with p creased p-450 co P450 COntent Wa: The im ( the soret maXinu ence speCtrum of 57 2. Effects of Nan, 3MC, PCB and TCDD on renal and hepatic enzymes in mice The effects of multiple i.p. injections of Nan and 3MC, a ingle i.p. injection of TCDD and dietary consumption of PCB 0n renal 1d hepatic MFO activities are summarized in Table 4. In general, :eatment with 3MC, TCDD and PCB increased the activities of both anal and hepatic MFOs while treatment with Nan increased hepatic ‘0 activities, only. 3. Effects of single and multiple administrations of PBB, PCB, Nan and 3MC on renal, hepatic and testicular enzyme activi- ties and cytochrome P—450 concentrations in rats Cytochrome P—450 (P—450) concentrations and AHH and PCNMA tivities in kidney, liver and testis from naive rats are summarized Table 5. Enzyme activities and P—450 concentrations, in general, :e greater in liver than in kidney, and greater in kidney than in stis. Hepatic P—450 centent was increased by single oral doses of i and PCB but not by single doses of Nan or 3MC (Figure 2). Renal testicular P-450 contents were not affected by single doses of PBB, , Nan or 3MC. Multiple doses of PBB, PCB and 3MC increased both atic and renal P-450 centents. The magnitude of this effect, ever, was greater in the liver than it was in the kidney after atment with PBB and PCB (Figure 2). Multiple doses of Nan in— 1sed P-450 content in the liver but not in the kidney. Testicular i0 c0ntent was not increased by any of the inducing chemicals used. The induced P—450, in many cases, exhibited soret maxima soret maximum is the absorbance peak near 450 nm in the differ— spectrum of the dithionite—reduced CO:hemoprotein complex. See Induction 0 Organ Liver Kidney Liver Kidney Liver Kidney Liver Kidney M PCNMA. AHH, BP- X 8 suPernatanl treated With N; Percentage of c *Significant 11 c N=4 animals (c ACtivities in LiVer: , PCMIA, 00] 0.lli0,01 min; BP-4. of Proteir Kidney; , PCNMA, 0.c 0.01:0,01 per min; E mg 0f PrOt AbbrEViations: 2,3,7 biPhe (PCNM hydro 58 TABLE 4 Induction of Mixed-Function Oxidases (MFOs) in Liver and Kidney Enzyme Activities Or a Inducing g n Chemical AHH PCNMA BP—Z—OH BP—4—OH mean Z control i l S.E. Liver Nan: 125:22 201i14* 137i42 331i56* Kidney Nan 93:14 106r11 ioorzo 100i30 Liver 3MC: 346i53* 87:21 n.d. n.d. Kidney 3MC 286i21* 113:19 n.d. n.d. Liver TCDD: 945il86* n.d. iisrio 141:20 Kidney TCDD 5560i1200* n.d. 200:100 200:50 Liver PCB: 356i49* 217i36* n.d. n.d. PCB lei36* l65i18* n. . n.d. Kidney PCNMA, AHH, BP—Z—OH and BP-4—0H activities Were measured in the 14,000 x g supernatant fraction of homogenates of livers and kidneys of mice treated with Nan, 3MC, TCDD or PCB. Activities are presented as a percentage of control i l S.E. n.d. = Not determined. *Significant increase (p<0.05). aN=6 animals. bN=5 animals 0N=4 animals (data from Hook_e§flal., 1978b)- Activities in control mice were as follows: Liver: AHH, 11.20il.50 fluorescence units per mg of protein per min; PCNMA, 0.139i0.012 O.D. units per mg of protein per hr; BP—2-OH, 0.11i0.01 nmol of 2—hydroxybiphenyl produced per mg of protein per min; BP-4—OH, 2.08i0.28 nmol of 4-hydroxybiphenyl produced per mg of protein per min. Cidney: AHH, 0.05i0.01 fluorescence units per mg of protein per min; PCNMA, 0.080i0.009 O.D. units per mg of protein per hr; BP—Z-OH, 0.01i0.01 nmole of 2—hydroxybiphenyl produced per mg of protein per min; BP—4-OH, 0.02i0.01 nmol of 4-hydroxybiphenyl produced per mg of protein per min. bbreviations: sodium phenobarbital (Nan), 3—methylcholanthrene (3MC), 2,3,7,8—tetrachlorodibenzo—p—dioxin (TCDD), polychlorinated biphenyls (PCB), p—chloro—N—methylaniline N-demethylase (PCNMA), aryl hydrocarbon hydroxylase (AHH), biphenyl—2- hydroxylase (BP-2-OH), biphenyl-4-hydrOXy1ase (BP-4-OH) Parameter CYtochmme P- 59 TABLE 5 Measurements of Aryl Hydrocarbon Hydroxlyase (AHH) Activities, p—Chloro-N—methylaniline N—demethylase (PCNMA) Activities and Cytochrome P—450 Concentrations in Liver, Kidney and Testis of Naive Rats Parameter Organ: Liver Kidney Testis Iytochrome P—450a 0.57li0.06l 0.110i0.019 0.059i0.016 LHHb 33lil6 6.20 i0.66 1.83 i0.37 ’CNMAO 0.457i0.023 0.229i0.012 0.105i0.011 AHH and PCNMA activities were measured in the 14,000 x g Supernatant fractions of hepatic, renal and testicular homogenates. Values are represented as the mean i l S.E. of 6 animals. a . nmoles per mg microsomal protein. 19 . . . . Relative fluorescence unlts per mg protein per min. cnmoles product (p—chloro—N-methylaniline) formed per mg protein per min. 2»me ‘ wn ¥N%—¥ I mine. In)... 0 . $203. " W. 8“ “W «1 IUL * >5 0 . 00! 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I . 8.. mg: 0 £02 - com mwOO u F532 .2.- wi—h a v. .— 05 NR VN o q q a — OO— — I_| n CON i can 259 4 F :23. l r o3 * $2.. 0 O DU.— # 00m N muswflm “moo - :52 2 m2: is e av... 05 llu' l 1081NOD % OSV-d BWOIIHDOIK.) .1 I— . l 52: an Vac § .3. § § «J M o 3 w W a 1 9 25m: 0 5205. NA m $5: N I. a .0 Us; 309.522 :1 a o. a. 2“ Ill" 3 m w W a .1 v m x m 2me d m. 520:. I FT 52. O O on“. D A I... O 3 m. 0 W a 1 .7 C. o x .nov m l. as hepatic soret hr after a si 3MC) (Table 6). MSG were si shifts appear hr for 3MC) . Mult hepatic and re 3 rdativaly tion of hepati and Peaked at deses of PBB, were not incre Chemicals , —7—‘_ '__ ‘ ‘ fi—‘m’: _ 62 Methods, Section C5) of shorter wavelengths than P-450 from naive rats. After multiple doses of PBB, PCB or 3MC, hepatic soret maxima were shifted from 450 nm to 448 nm, and renal soret maxima were shifted from 454 nm to 453 or 452 nm (Table 6). Treatment with Nan did not produce shifts in soret maxima of renal or hepatic P—450. Though a single oral dose of PBB, PCB and 3MC produced shifts in hepatic soret maxima within 9 hr, maximum shifts appeared to occur 72 hr after a single dose and were still apparent at 216 hr (except for 3MC)(Table 6). Effects of PBB, PCB and 3MC on soret maxima of renal P-450 were similar to those on hepatic P-450 except that maximum shifts appeared to occur at 9—24 hr and had disappeared by 216 hr (72 hr for 3MC). Multiple doses of PBB, PCB and 3MC greatly increased both hepatic and renal AHH activities while multiple doses of Nan produced a relatively modest increase in hepatic AHH only (Figure 3). Induc- tion of hepatic AHH activity, in general, was first evident at 24 hr and peaked at 72 hr after single doses of PBB, PCB and 3MC. Renal H activities, however, were greatly increased at 9 hr, maximally increased at 24 hr and had fallen considerably by 72 hr after single oses of PBB, PCB and 3MC (Figure 3). Testicular AHH activities ere not increased by single or multiple doses of the inducing hemicals. PCNMA activity in liver was increased (approximately 2X) by ultiple doses of PBB, PCB and Nan (data not shown). Renal and esticular PCNMA activities in rats, however, were not significantly ncreased by PBB, PCB, Nan or 3MC (data not shown). Treatment Control PBB PCB Nan 3MC L-‘t—‘l—‘r—IH Control PBB PCB 63 TABLE 6 Time-Dependent Effects of Polybrominated Biphenyls (PBB), Polychlorinated Biphenyls (PCB), Sodium Phenobarbital (Nan), and 3eMethylcholanthrene (3MC) on the Location of Soret Maxima of Reduced Cytochrome P+450 Difference Spectra ph— a f or ' Treatment Organ W velength (nm) 0 the s et maX1mum Time (hr): 9 24 72 216 Multi-dose Control Liver 450 450 450 450 450 PBB , Liver 449 449 449 449 448 PCB Liver 449 449 448 449 448 Nan Liver 450 450 450 450 450 3MC Liver 449 449 449 450 448 Control Kidney 454 454 454 454 454 PBB Kidney 453 453 453 454 452 PCB Kidney 453 453 453 454 453 Nan Kidney 454 454 454 454 454 3MC Kidney 454 453 454 454 452 Rats were sacrificed 9, 24, 72 or 216 hr after a single dose of PBB, PCB, Nan or 3MC, or 24 hr after the final dose of a multiple-dose regimen. Cytochrome P—450 was determined in the microsomal pellets and the soret maximum in the vicinity of 450 nm recorded to the earest wavelength. Values are represented as the means of 4 nimals rounded off to the nearest integer. 64 Figure 3. Time-dependent induction of aryl hydrocarbon hydroxylase (AHH) activity in liver, kidney and testis. Symbols represent the means i 1 S.E. (N=4 rats) of the activities of AHH in animals sacrificed 9, 24, 72 or 216 hr after a single dose of polybromi— nated biphenyls (PBB), polychlorinated biphenyls (PCB), sodium phenobarbital (Nan) or 3-methylcholanthrene (3MC). Bars represent the means i 1 S.E. (N=4 rats) of the activities of AHH in animals sacrificed 24 hr after the final dose of a multiple—dose regimen of PBB, PCB, Nan or 3MC. T, testis; K, kidney; L, liver. Open symbols or bars, significantly greater than in control rats; closed symbols or bars, not significantly greater than in control rats; p<0.05. *Significant difference between percent increases in liver and kidney, p<0.05. S CONTROL AHH ACTlVIYY s s S CONT’ROI. AHH AC TIVIYY § § § :3 3 if 924 72 X CONTROL AHH ACTIVITY 1 CONTROL AHH ACTIVITY 65 1300 FEB 1600- O O LIVER l n KIDNEY uoo- A rssns —l 1200- * 2 E 0 U 1000- u * E son a C < 600 i * < 400 200 .d—H__n_ 924 72 m r K L TIME hr MULTl-DOSE "0° Nan 1600- O LIVER I KIDNEY uoo- A rEsns _, o a: E 1200- 0 U R 1000- >- : 300- E < I 600- E 400- 200- ® 924 72 216 T K L TIME 5, MULTl-DOSE Figure 1600 800 400 * PCB ‘ O O LIVER I I: KIDNEY - A rEsns r K I. TIME I" MUlTl-DOSE 3MC O O LIVER I D KIDNEY A TESTIS 9 24 72 216 r K L YIME hr MUlTl-DOSE The AHH activitie hepatic homog using MET and P-450-depende Gelboin, 1975 hepatic AHH a hepatic AHH a testicular AH values) while nearly 307., a (testicular d appeared to i Slight extent concomitantly inhibitory ef. oPPosite effe, COmPaI‘iSOn to ANF but hepat: 0f MET (Figure of PBB and PC] to the inhibiI rats, however: to that of mi t0 the inhib it 66 The inhibitory effects of ANF and MET (lxlO_4M) in_yi££g_on AHH activities in the 14,000 x g supernatant fractions of renal and hepatic homogenates are illustrated in Figure 4. Inhibition studies using MET and ANF have previously been used to classify the type of P—450—dependent AHH activity induced by various agents (Wiebel and Gelboin, 1975; Goujon g£_al., 1972). MET preferentially inhibits hepatic AHH activity induced by Nan while ANF preferentially inhibits hepatic AHH activity induced by 3MC. ANF reduced hepatic, renal and testicular AHH activities to approximately 30% of normal (uninhibited values) while MET reduced hepatic and testicular AHH activities to nearly 30%, and renal AHH activities to nearly 60% of normal values (testicular data not shown). Treatment with multiple doses of Nan appeared to increase the susceptibility of hepatic AHH (and, to a slight extent, renal AHH) to the inhibitory effects of MET while concomitantly reducing the susceptibility of hepatic AHH to the inhibitory effects of ANF (Figure 4). Treatment with 3MC had the opposite effect; renal and hepatic AHH became more susceptible (in comparison to AHH from control animals) to the inhibitory effects of ANF but hepatic AHH became less susceptible to the inhibitory effects of MET (Figure 4). Hepatic AHH from rats receiving multiple doses of PBB and PCB did not exhibit net alterations in susceptibilities to the inhibitory effects of ANF and MET. Renal AHH from these same rats, however, responded to ANF and MET in a manner quite similar to that of renal AHH from rats treated with 3MC. That is, renal XHH from PBB and PCB—treated rats exhibited increased sensitivity :0 the inhibitory effects of ANF (Figure 4)- Inhibition 0f 67 Figure 4. d—Napthoflavone (ANF) and metyrapone (MET) inhibition of aryl hydrocarbon hydroxylase (AHH) activities ig_vitro after multiple doses of polybrominated biphenyls (PBB), polychlorinated biphenyls (PCB), sodium phenobarbital (Nan) or 3-methylcholan— threne (3MC). Bars represent the means i l S.E. (N=4 rats) of the activities of AHH, in the presence of ANF (lxlO_4M) or MET (lxlO‘4M), from rats sacrificed 24 hours after the final dose of a multiple-dose regimen of PBB, PCB, Nan or 3MC. The data are expressed as percentages of normal (no inhibitor present) values. *Significantly different from control (C), p<0.05. ( X of normal value ) AHH ACTIVITY 2% ES 23 68 ( % of normal value) AHH ACTIVITY * * LIVER . . n, + + H— an + '5 >1: IT * - a [*1 c PBB PCB Nan 3MC c PBB PCB Nan 3MC KIDNEY H— +' + ;- +I H- + * * [3 c PBB PCB Nan 3MC c PBB PCB Nan 3MC ANF(IXIO'4M) METUXIOAM) Figure 4 69 testicular AHH by ANF and MET was unaltered by the administration of multiple doses of PBB, PCB, Nan and 3MC (data not shown). Table 7 illustrates the time—dependencies of PBB and PCB— induced alterations in susceptibility of renal AHH to the inhibitory effects of ANF. Of the times evaluated (9, 72 and 216 hr after a single dose of PBB or PCB), increased susceptibility to the inhibitory effects of ANF appeared to be maximal at 9 hr. Although still more susceptible to ANF than renal AHH from control animals, renal AHH from PBB and PCB-treated rats was less susceptible to the inhibitory effects of ANF at 216 hr (72 hr for PCB-treated rats) than at 9 hr (Table 7). Thus, the time courses of the PBB and PCB—induced in— creases in the susceptibility of renal AHH to the inhibitory effects of ANF paralleled the time courses of the PBB and PCB-induced in- creases in renal AHH activity (Figure 3). 4. Inhibition of renal and hepatic AHH activity in vitro The effects of several inhibitors of MFO activities on renal and hepatic microsomal AHH activities in_yi££9_are illustrated in Figure 5. Tween 80, the detergent used for dissolution of AIA and PB, produced significant inhibition of microsomal AHH activity when present in the medium at a concentration of 0.01% (v/v). Hepatic AHH activity appeared more sensitive to inhibition by Tween 80 (activity reduced to 45% of control values) than did renal AHH activity (reduced to 65% of control values) (Figure 5). Renal and hepatic microsomal AHH activities, however, were not further reduced by the presence of AIA in the incubation mixtures (Figure 5). PB, on the other hand, decreased the activities of renal and hepatic AHH, and renal and 70 TABLE 7 a—Napthoflavone (ANF) Inhibition of Renal Aryl Hydrocarbon Hydroxylase (AHH) Activities In Vitro at Various Times After a Single Dose of Polybrominated Biphenyls (PBB) or Polychlorinated Biphenyls (PCB) AHH Activity (% of normal values) i 1 S.E. Treatment Time (hr): 9 72 216 Control 33:1 30:4 33:3 PBB grid 12:1“ zorza’b PCB 6ila 20i1a’b 17r3a’b Rats were sacrificed 9, 72 or 216 hr after a single dose of PBB or PCB. AHH activity in the 14,000 x g supernatant fraction of renal homogenates was determined in the pre— sence of ANF (lxlO‘4M). Values represent the means i l S.E. of 4 animals. The data are expressed as percentages of normal (no inhibitor present) values. aSignificantly different from control, p<0.05. Significantly different from 9 hr, same treatment, p<0.05. 71 Figure 5. Inhibition of renal and hepatic microsomal aryl hydro— carbon hydroxylase (AHH) activities ig_vitro by allyl—isopropyl- acetamide (AIA), SKF 525-A, metyrapone (MET), piperonyl butoxide (PB) and a—napthoflavone (ANF). AHH activities were determined in the presence of AIA, SKF 525-A, MET, PB or ANF; 0, 1x10‘6, 1x10" , lino-4, or 1x10-3M, and with no inhibitors or vehicles present (control). Activities are expressed as percentages of control, mean i 1 S.E. Circles represent renal microsomal activities and squares represent hepatic microsomal activities. Open symbols, significantly different from control (no inhibitor). *Significant difference between percent inhibition in renal and hepatic AHH even though both were significantly inhibited. N=5 independent experiments, p<0.05. SKF 525-A 300 _ _ :5 100 ANF g . 3 250 _ "'5 so 33 z~ 200 - IE 2 I 150 _ I I < 100 100 0 so so _ ' CI 1_ l 1 l 1‘. l l -6 -5 -4 -3 -6 -5 -4 -3 Log Inhibitor Concentration (M) Figure 5 atic AHH appeared to be equally susceptible to inhibition by in— asing concentrations of PB in the incubation mixture. The presence SKF 525—A and MET in the incubation mixtures produced concentration— endent decreases in the activity of hepatic AHH but increases, at concentrations, in the activity of renal AHH (Figure 5). Addition ANF to the incubation mixtures produced large increases in hepatic . . . . -5 act1v1ty that were max1mal at a concentration of 1x10 M and lined as the concentration of ANF was increased. In contrast, ANF concentrations of lxlO_5M and greater inhibited renal AHH acti— y (Figure 5). 5. Time—dependent effects of single doses of SKF 525—A and PB on renal and hepatic PCNMA and AHH activities in rats and mice Administration of a single i.p. injection of SKF 525—A, 75 'kg, to Fischer 344 rats produced a transient decrease in hepatic MA activity, an effect that was maximal 2 hr after dosing, and a longed reduction in hepatic AHH activity (Figure 6). Renal PCNMA ivity was not reduced by a single treatment with SKF 525—A; renal activity was reduced by SKF 525—A, but not to the extent that atic AHH activity was reduced. Administration of PB, 600 mg/kg, rats produced only a slight reduction in hepatic PCNMA activity a prolonged reduction in hepatic AHH activity (Figure 6). Renal activity was also reduced by treatment with PE, though not to extent that hepatic AHH activity was reduced. not affect the activity of renal PCNMA. Qualitatively similar llts were obtained when these experiments were c0nducted on ICR, : mice (W.M. Kluwe and J.B. Hook, unpublished observations). 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Enzyme Modulation and COIL and CHClQ Toxicity ‘1’ J 1. ‘Effects of dietary PBB and PCB on 0014 toxicity in mice Maintenance of ICR, male mice on diets containing 100 ppm of PBB or 200 ppm of PCB for 28 days markedly increased liver weight- to—body weight ratios (Table 8) without significantly affecting total body weight. The effect of PBB on liver weight was greater than that of PCB, even though PCB was present in the diet at a greater concen- tration than was PBB. Kidney weight—to-body weight ratios were not affected by ingestion of PBB or PCB (Table 8). Ingestion of PBB and, to a lesser extent PCB, appeared to enhance the susceptibility of mice to the toxic effects of C014. The CCl4-induced rise in SGOT activity in PBB—treated mice was greater than that in control mice 24 hr after administration of 0.125 mllkg CC14, and greater in PCB-treated mice than in control mice after administra- tion of 0.625 m1/kg 0014 (Figure 7). The CCl4-induced increases in SGPT activities were affected by PBB and PCB in manners similar to those of SGOT (data not shown). PBB appeared to increase the suscep- tibility of mice to 0014 nephrotoxicity. Although administration of up to 0.625 m1/kg CCl to control and to PCB-treated mice failed to pro- 4 duce a decrease in the abilities of renal cortical slices to accumulate PAH and TEA (PAH and TEA S/Ms), as little as 0.125 ml/kg CCl4 produced significant decreases in PAH and TEA S/Ms in PBB—treated mice (Figure 7, TEA data not shown). The LD50 values for CCl4 in mice were inversely related to the concentrations of PBB in the diet (Table 9). Ingestion of 20 ppm of PBB in the diet for 20 days increased the susceptibility of mice 77 TABLE 8 Effects of Dietary Polybrominated Biphenyls (PBB) and Polychlorinaed Biphenyls (PCB) on Liver Weight/Body Weight (LW/BW) and Kidney Weight/Body Weight (KW/3W) Diet LW/BW i l S.E. (X 100) KW/BW i l S.E. (x 100) C 5.93i0.09a 1.51:0.03 PCB 8.29:0.20a 1.42i0.04 PBB 13.62i0.35 1.42i0.04 Mice were maintained on control diet (C) or the same diet formulated to contain 100 ppm of PBB or 200 ppm of PCB for 28 days. aSignificantly greater than control (C), p<0.05. 78 Figure 7. Effects of polybrominated biphenyls (PBB) and polychlori— nated biphenyls (PCB) and 0014 on SGOT and PAH S/M. Mice were main— tained on control diet (C) or the same diet formulated to contain 100 ppm of PBB or 200 ppm of PCB for 28 days prior to a single i.p. injection of one of several doses of CC14. Mice were killed 48 hr later and SGOT and PAH S/Ms determined. *Significant difference in comparison to mice receiving the same dose of €014 but ingesting control diet (C), p<0.05. SGOT (U lml) % DECREASE IN PAH S/M 600 _ 500 400 300 200 100 2O 4O 60 80 0.0 ml / kg 039;! on § .9... PCB-.- N=6 0.005 ml/ kg ‘p"’ 60 000 000 'O’ 090 090 .... 909 00 . 0.! 00! '00 000 '00 9 9. '90 9". 99 on '09 on '09 .90 ’OO' 00. '00 090 '09 ’Q ’00 .00 'OO' OOQ '00 001 9.9.0. 09 . . .. \\ 9090? / I . / (/ /////// C PCB PBB PRE-TREATMENT Figure 7 79 0.625ml/ kg * OJZSml/kg 1 * l 0.025ml/kg ‘ 0 w 53 \‘ '9’9’ :2 $32: __ .. - a P : as B c BB PRE-TREA‘I’MENT CCl4 0.025 ml/kg 0.125 ml/kg 0.625ml/ kg * § . ID \ a \\ “‘ C PCB PBB C PCB PBB 80 TABLE 9 Effects of Polybrominated Biphenyls (PBB) on the Acute LD50 of CCl 4 Dietary PBB(ppm) 96-hour LD50 (ml/kg) Potency Ratio 0 1.84 (1.55-2.18) 20 1.00 (0.82—1.22)a 1.84 (1.46-2.32)“ 100 0.28 (0.25—0.31)a’b 6.57 (5.48—7.88)a’b Mice were fed diets formulated to contain 0, 20, or 100 ppm polybro- minated biphenyls (PBB) for 20 days prior to a single injection of CC14, in corn oil, in a total volume of 5 ml/kg. Each dose of CCl4 was administered to 10 animals. Deaths occurring within 96 hr after 001 administration were recorded. The 95% confidence limits are in parentheses following LD50 and potency ratio values. Potency ratio is defined as the LD50 in mice ingesting 0 ppm PBB divided by the LDSO in mice ingesting 20 or 100 ppm PBB. CZ... . . . Significant decrease/increase in comparison to 0 ppm PBB. Significant decrease/increase in comparison to 20 ppm PBB. 81 to the lethal effect of CCl by nearly a factor of 2 while ingestion 4 of lOO.ppm of PBB in the diet for 20 days increased susceptibility to the lethal effect of 0014 by a factor of more than 6. The maximum cumulative number of deaths produced by CCl4 in both control mice and PBB—treated mice occurred 96 hr after administration of CClA. 'Mice surviving the initial 4 days after CCl4 administration were observed for an additional 2 weeks during which time no deaths occurred. In addition, the dose—response (cumulative death) curves (plotted on log vs. probit scales) were parallel, suggesting that the mechanism of 0014—induced death was the same in the control and the'PBB-treated mice. 2. Effects of PBB, PCB and HCB on CCl, toxicity_in rats G'- Ingestion of the aromatic organohalides, HCB, PBB and PCB, by male, Sprague—Dawley rats over a 20 day period produced hepatomegaly; liver weight-to—body weight ratios were increased (Table 10) but total body weights were not affected by the aromatic organohalides (data not shown). Kidney weight-to—body weight ratios, in contrast, were unaffected by aromatic organohalide treatment. The increase in relative liver size was accompanied by an increase in hepatic lipid content, though renal lipid content was not affected (Table 11). Treatment of rats with single injections of 0014 (0.25 or 2.00 ml/kg) also increased hepatic lipid content, an effect that was not additive to that of PBB, PCB or HCB on hepatic lipid content (Table 11). Renal lipid content was not increased by treatment with 0014 nor by any combination of C01 and aromatic organohalides. 4 82 TABLE 10 Effects of Aromatic Organohalides on Liver Weight/Body Weight (LW/BW) and Kidney Weight/Body Weight (KW/3W) Treatmenta LW/BW i 1 S.E. (x 100) KW/BW i 1 S.E. (x 100) Control 4.54i0.08b 0.745:0.011 HCB 6.08i0.06b c 0.776i0.0l3 PBB 7.06:0.15b’ 0.753:0.013 PCB 5.77i0.06 0.766i0.053 a . Sprague-Dawley, male rats were treated over a 20 day period with hexachlorobenzene (HCB), polybrominated biphenyls (PBB) or polychlorinated biphenyls (PCB). Control rats received the appropriate vehicles. Significantly greater than control, p<0.05. cSignificantly greater than HCB or PCB, p<0.05. N=6 except HCB, 2.00 ml/kg CCl4, where N=4. W. _~.V___ _ 83 .mHmEHcm muz .mo.ove .Amaoanm>v «Hoe wx\aa oo.o poo pamaumonuoum mamm moo wafl>fimomu mamafidm ou domfiummaoo aw omwonoaw unmoflmwowflmo .q .mo.ovm H00 wo udsoam mEMm .Houudoo ou domflummaoo oH ommouoaw udmofiMHome Q a .wa\aa oo.N-oo.ov H00 mo mmmov Hmum>mm mo moo fiufls wmwooaaozo omsu wow AHouudoov moHoH£o> mumwum loaded man no “momv mahaosmfln mommafluoanohaom .Ammmv mahdoomfln mommaflaounhaom .Amomv maouaonouoasomxoo suHB weapon mop om m Ho>o woumonumum one? wumu mama .mmasmmlodwmnmm o -- -- No.“ Hem.wm «a.mhwa.em oo.~ e oe.a “we.ae eo.m HHH.mm em.e “wa.oa mo.mamm.mm mN.o e em.a Mme.ne Ho.a Ham.ae HN.H HmA.wm mm.ano~.mm AQHUHsm>V 00.0 M -- -- nm.omflam.mw pea.mhmm.qe oo.N a ee.oanwm.we eo.m How.ae om.H HmH.om pea.mhmo.me mm.o a nmm.aanoe.me om.onnoa.oe Ha.m Hoe.em mm.mham.me mo.o g nno.m hom.ee nma.m Hem.am ANowe Hom.mm oo.NHHH.mm Aeneasm>v 00.0 a Ame\aav «Hoe l1. mum mmm mom Honucoo amwuo .m.m H H Aaanm\mev namuaoo vegan "enemaummuumum AMV aocwam was Ago nm>aq wo udmudoo wHQHA Go «HUD wdm m®@HHM£ocmmHO UHumEOH¢ mo muommwm Ha m4m¢H 84 Ingestion of PBB, PCB and HCB increased the susceptibility of rats to the lethal effects of i.p.-administered CCl4 (Table 12). Accordingly, losses in body weight induced by C01 occurred following 4 lower doses of CCl in PBB, PCB and HCB-treated rats than in control 4 rats (Table 13). Treatment with HCB, PBB and PCB, alone, did not signifi- cantly affect the abilities of renal cortical slices to accumulate PAH and TEA (Figure 8, TEA data not shown). Rats treated with HCB, PBB and PCB, however, exhibited significant decreases in PAH and TEA S/Ms after receiving as little as 0.25 ml/kg CC14, though a decrease was not apparent in control rats until the dose of CCl4 was increased to 2.00 ml/kg (Figure 8). The combined effects of CCl4 and the aromatic organohalides on renal cortical slice respiration paralleled their effects on PAH and TEA S/Ms, though in an inverse manner. Increases in renal cortical slice 00 were evident in renal tissue from control 2 rats only after administration of 2.00 ml/kg 001 while Q02 from PBB 4 and PCB—treated rats was increased after as little as 0.25 ml/kg CCl 4 (Figure 8). Treatment with HCB, PBB and PCB similarly increased the susceptibility of the liver to the toxic effects of C014. Increases in SGPT activities were detected in PCB—treated rats after as little as 0.03 ml/kg CCl4 (Figure 9). Furthermore, increased SGPT activities were detected in HCB, PBB and PCB-treated rats, but not in control rats, following administration of 0.25 ml/kg CC14. Control rats exhi- bited increased SGPT activities after receiving 2.00 ml/kg CCl4 (Figure 9). 85 TABLE 12 Effects of Aromatic organohalides and CCl4 on 48-hour Survival Number gf Survivors/Number Treated Pretreatment CCl4 (ml/kg): 0.25 2.00 Control 6/6 6/6 HCB 6/6 4/8 PBB 6/6 0/6 PCB 6/9 0/6 a48-hour survival refers to the ability of HCB, PBB and PCB— pretreated rats or control rats to survive for 48 hr after a single injection of CC14. Sprague-Dawley, male rats were pretreated over a 20 day period with hexachlorobenzene (HCB), polybrominated biphenyls (PBB), polychlorinated biphenyls (PCB) or the appropriate vehicles and then challenged with one of several doses of CCl4 (0.00-2.00 ml/kg). 86 TABLE 13 Effects of Aromatic Organohalides and C014 on 48-hour Body Weight Gain b Percent of Original Body Weighta i 1 S.E. Pretreatment : Control HCB PBB PCB CCl4 (ml/kg) 0.00 (vehicle) lO7i2 108:1 llliZ llli2 0.03 106il 104ild 102i10’d 100:20’d 0.25 100:1d 94t10’d 86ilc’d 84i28’d 2.00 87i2d 88i2d ___ ___ aDetermined as body weight at time of sacrificee-body weight 48 hr prior to sacrifice (time of C014 administration) x 100. SpraguewDawley, male rats were pretreated over a 20 day period with hexachlorobenzene (HCB), polybrominated biphenyls (PBB), polychlorinated biphenyls (PCB) or the apprOpriate vehicles and then challenged with one of several doses of 0014 (0.00-2.00 ml/kg)- C3 . . . . . . Significantly lower than in control animals receiVing the same amount of CC14, p<0.05. Significantly lower than in animals receiving the same pretreatment but 0.00 ml/kg CCl4 (vehicle), p<0.05. N=6 except HCB, 2.00 ml/kg 0014, where N=4. 87 Figure 8. Effects of aromatic organohalides and 001 on renal Q0 and PAH S/Ms. Rats treated subacutely (20 days)with hexachloro— benzene (HCB), polychlorinated biphenyls (PCB), polybrominated biphenyls (PBB) or vehicle were sacrificed 48 hr after i.p. injec— tion of 0014 (0.00, 0.03, 0.25 or 2.00 ml/kg) and renal 002 and PAH S/Ms determined. Q02 values are depicted as pl 02 consumed/g tissue/min. Control values for PAH S/M and Q02 are illustrated on the right portion of the figure. N=6 animals except HCB, 2.00 ml/kg, where N=4 animals. TSignificant difference in comparison to animals receiving the same arOmatic organohalide but 0.00 ml/kg 0014, p<0.05. *Significant difference in receiving the same dose of 001 (vehicle), p<0.05. comparison to animals 4 but no aromatic organohalide % Decrease in PAH S/M N N s 3 5‘ o 5 002( pl /gram/min) O 88 CONTROL PRETREATMENT PAH S/M Elven-nae 13.10t0.63 HCB I3.42t 0.89 ++ ** .......... .......... ....... ........... 'g E PBB 13.9mm '5? I.“ 8 % PCB lO.65t0.65 O. "E .3 00000 E 003 0.25 2.00 + 1 + h- * PRETREATM ENT CONTROL 002 Cl VEHICLE I46 2 6 _ w 1’ HCB 150: 4 \ 2 _ § 3 g PBB 153 t 8 § g m PCB 145 t 6 § 5 § 22 003 0.25 2 00 CCI4 (ml/kg) Figure 8 89 .mo. vm H00 mo omow mEmm woo wdfl>flwomu mamaflsm ou oomwummmoo .m0.0va .qauo wx\HE 00.0 map mvHHmnooowHo oflumEoum 080m 0 SH oodeCMMHw unmoHMHsmHm+ .mamaflsm quz whoa? .wx\aa .emcflanmume mmflufl>fluom Hmum was Amx\ae oo.m no .mN.o noumm y: we wouamenomm ohms oHoHEm> Ho Amomv mammoamflo .Amomv mommaonouoaaomxom sues hamusomnzm .m mudmflm .AoHoH£o>v mpflfimaonmmuo oHumEoum os u:@ 0 CH mommamwwflp osmoHMHomex CSu wow>floomu mamaflam on :omflnmmfio 00.N .mom How umooxo mamafldm 0nz .mo.o oo.ov ease no coauomnan .a.fl wwuwdflhoanokaom .Ammmv wa>do£mflp woumdflBOthaog .mwflufl>fluom Hmwm so «H00 pom mmwflawoosmwno oflumfioum mo muoommm wmumouu muom 90 CON Peudxa 03d PUD 88d m muswflm 3.. x .3 4.3 mg mod 86 I moi mmaw fflflflu mo: 74 30.5; D CV 00 ON— 00— CON OVN (IN/fl) .lclEJS 91 Sections of livers from control, HCB, PBB and PCB—treated rats injected with 0.00 (vehicle), 0.03, 0.25 or 2.00 m1/kg 0014 are illustrated in Figures 10, ll, 12 and 13, respectively. Normal liver histology was exhibited by tissue from control rats receiving no 0014 (Figure 10). Tissue from PBB, PCB and HCB—treated rats re— ceiving no 0014 exhibited dilation of the hepatic vein, hepatocellular swelling and early degenerative changes (e.g., vacuolation) at the periphery of the lobules (Figure 10). With increasing doses of 0014 centrilobular necrosis (swelling and degeneration of the cells closest to the central vein) encompassed an increasing percentage of the liver in samples from control rats (Figures 11—13). Livers from PBB, PCB and HCB—treated rats exhibited similar progressions of centrilobular necrosis but the extent of necrosis in aromatic organohalide—treated rats appeared to be greater than that in control rats after doses of 0.03, and 0.25 ml/kg 0014 (Figures 11 and 12). 3. Effects of PBB and PCB on hepatocellular GPT and GOT acti— vities in rats The effects of dietary PBB and PCB, potent inducers of microsomal enzyme activities, on the activities of GPT and GOT within the liver were investigated to determine if PBB and PCB induced hepatic GPT and GOT activities and to determine if such effects might be responsible for potentiation of 0014—induced elevation of SGPT and SGOT activities. As shown in Table 14, the activities of hepato— cellular GPT, per m1 of 100,000 x g supernatant fraction or per mg of 100,000 x g supernatant protein, were not greater in PBB and PCB rats than in control rats. Relative liver weights, however, were signifi— cantly increased by ingestion of PBB and PCB (Table 11). If the 92 Figure 10. Sections of livers from rats receiving 0.00 ml/kg 0014. Hematoxylin and eosin stain, X40. A) No aromatic organohalide pretreatment. Parts of several lobules can be seen. Central veins are small, sinusoids are visible and hepatic cords present an orderly arrangement. Interlobular spaces are scarcely visible and all liver cells throughout the lobule present an identical appearance. B) Pretreated with hexachlorobenzene. Central veins are slightly dilated and hepatic cells at the periphery of the lobule are vacuolated. Swelling of cells has obliterated sinusoids at the periphery of the lobules. Hepatic cords remain regular in arrangement. 0) Pretreated with polybrominated biphenyls. Appearance of the liver tissue is very similar to that described in 4B. D) Pretreated with polychlorinated biphenyls. The central vein is dilated, sinusoids are nearly obliterated by cellular swelling and light vacuolation of hepatic cells can be seen at the periphery of the lobule. 93 .V .uO ¢afifl .rcuh . .n. 9.9% - Figure 10 94 Figure 11. Hepatic tissue from rats receiving 0.03 ml/kg 0014. Hematoxylin and eosin stain, X40. A) No aromatic organohalide pretreatment. Hepatic cells are swollen; sinusoids are obli— terated except those adjacent to the central vein and cells show vacuolation throughout the lobule. B) Pretreated with hexa— chlorobenzene. The central vein is symmetrical in Outline, sinu- soids are slightly visible and cellular vacuolation can be seen at the periphery of the lobule. 0) Pretreated with polybrominated biphenyls. The central vein is dilated, adjacent sinusoids are open but peripheral hepatic cells are vacuolated. polychlorinated biphenyls. The central vein is markedly dilated and irregular in shape. Sinusoids are Completely obliterated because of swelling of hepatic cells, many cells are necrotic and others are vacuolated. D) Pretreated with Figure 11 96 Figure 12. Hepatic tissue from rats receiving 0.25 mllkg 0014. Hematoxylin and eosin stain, X40. A) No aromatic organohalide pretreatment. The larger dose of 0014 caused a remarkable vacuolation of hepatic cells extending from the periphery to midway of the lobule. B) Pretreatment with hexachlorobenzene. Swollen hepatic cells throughout the lobule obliterate the sinusoids although the central vein remains readily visible. There is remarkable vacuolation of a few hepatic cells at the periphery of the lobule. C) Pretreatment with polybrominated biphenyls. The central vein is markedly dilated, hepatic cells are swollen and necrotic or vacuolated. Many nuclei are pyknotic. D) Pretreatment with polychlorinated biphenyls. Hepatic tissue is in a state of degeneration. Much necrosis has occurred, living cells are swollen, sinusoids are irregular, central veins are obliterated and hepatic architecture is deranged. - HCB '44“ ".""'TI',¢ nu . , ., . .. . _ r *r "iii—twig; ”Tia-fie?“ it??? was .,. «--~=s-.:-;' - meg- r. 3 ., , »-' .i. ~. A > -,‘ 1" ._,. . 4‘ - 1 :. '. ' - ~ 'I"€,s_ .M‘azw‘fi. 73*! V " 1 . A . ‘ ,‘-I ‘~ ‘.'vx’f‘.u$%?“i'?f>f 3 *‘ I N g}. "~ Z. , v: ' " ‘ V l ‘7‘ '7'.) H. ‘ ‘- V i347. ;I%f;.~t f‘afi‘. ‘ " . ,§ .”~,‘A r , 971*? 8:31;. - ~f‘a- ~- . “fit, “:4 _ .. , .~ :‘W'x ,, ~ - :IQ“ L. I _. .> ‘a 8 '-.fi{ 3 \ k-. “A. .‘ 5 . i" it. . ‘ .‘0 f. z . v‘ .v .__» \ » ... : C t - $0 » - b ":9 ‘/ J04 )- a 0‘, ’k. é. , ‘2‘ lfi “0%égffw‘~ Figure 12 98 Figure 13. Hepatic tissue from rats receiving 2.00 ml/kg 0014. Hematoxylin and eosin stain, X40. A) No aromatic organohalide pretreatment. Liver cells are vacuolated and in the region of the central vein, cells have undergone necrosis with subsequent disruption of hepatic cords and sinusoids. The cells at the periphery of the lobule are less severely affected. B) Pretreat- ment with hexachlorobenzene. Some vacuolation of cells can be seen. Necrosis of cells extends from the central vein nearly to the periphery of the lobule. Sinusoids and liver cords are visible only near the periphery of the lobules. 99 . 0.. ...Ip ”Av; Maxi.» . .00 Figure 13 100 TABLE 14 Effects of Dietary Polybrominated Biphenyls (PBB) and Polychlorinated Biphenyls (PCB) on Hepatic GPT Activity Mean GPT activity i l S.E. (% of control) Treatment 3a b 30 U/ml x 10 U/mg U/lOOg x 10 Control 4.72:0.32 442i36 .86i6 PBB 4.17:0.12 (87) 361i15 (82) 118i34 (138)d PCB 5.34i0.48 (113) 423i42 (95) 123i11 (1440CZ GPT activities were measured in the 100,000 x g superna— tant fraction of hepatic homogenates from rats maintained for 20 days on diets containing 100 ppm of PBB or 200 ppm of PCB or diet formulated with an equivalent amount of acetone (control). Activities were expressed per ml of 100,000 x g supernatant fraction (a), per mg of 100,000 x g supernatant protein (b), and per 100 g of body weight (0). Significantly greater than control, p<0.05. N=5 rats. 101 increases in liver weight are taken into account, then GPT activities, per 100 g body weight, were increased by treatment with PBB and PCB (Table 14). Quantitatively similar effects were produced by PBB and PCB on hepatic GOT activities (data not shown). 4. Effects of maternal consumption of PBB on the toxicities of 001? and CHClO in deve10ping male rats Sprague—Dawley, male rats were exposed to PBB preweaning by the inclusion of 100 ppm of PBB in the dam's diet from day 8 of gesta— tion, and to PBB postweaning by the inclusion of 100 ppm of PBB in the diet consumed by the pups postweaning. At 52 days of age rats exposed to PBB continuously (100-100) had lower body weights than rats exposed to PBB during preweaning only (100-0), and rats exPosed to PBB pre— weaning had lower body weights that control rats (0—0) (Figure 14). Ingestion of PBB greatly increased the susceptibility of rats to the hepatotoxic and nephrotoxic effects of 0014. SGPT activities were elevated by as low a dose of 0014 as 0.03 ml/kg in rats exposed to PBB preweaning or continuously, but were increased in control rats only by a much larger dose of 0014 (2.00 ml/kg) (Figure 15). Simi— larly, PAH and TEA S/Ms were reduced in control rats by administration of 2.00 m1/kg 001 but not lower doses, while as low a dose as 0.03 4, ml/kg 001 reduced PAH and TEA S/Ms in rats exposed to PBB preweaning 4 or continuously (Figure 15, TEA data not shown). 5. Effects of dietary PBB on CHClO toxicity in mice k J Ingestion of PBB by ICR, male mice produced dietary concen- tration—dependent increases in liver weight—to—body weight ratios (Table 15). Since body weights were not significantly affected by 102 Figure 14. Effects of exposure to polybrominated biphenyls (PBB) during development on body weight at 52 days of age. Control diet (0-0); 100 ppm PBB fed to dam, pups weaned onto control diet (100-0); 100 ppm PBB fed to dams, pups weaned onto 100 ppm PBB diet (100-100), N=l6. *Significantly less than 0—0, p<0.05. +Significantly less than 100—0, p<0.05. ) m (9 T IGH E w Y D 0 B 103 F41 L. 60 1 L 0 0 1 0.. 4° °-§° ‘2 B P 0 O- 4 1 re 11 g i F 104 Figure 15. Effects of polybrominated biphenyls (PBB) and 0014 on SGPT and PAH S/M. Rats Were sacrificed 48 hr after a single i.p. injection of 0014 (0.00, 0.03, 0.25 or 2.00 ml/kg) and SGPT and PAH S/Ms determined. Control diet (O—O); 100 ppm PBB fed to dam, pups weaned onto control diet (100—0); 100 ppm PBB fed to dams, pups weaned onto 100 ppm PBB diet (100—100). N=4 rats except 100-0, 0.25 ml/kg 0014, where N=2 rats. *Significantly different from 0—0 receiving the same amount of 0014, p<0.05. +Significantly different from 0—0 receiving 0.00 ml/kg 0014, p<0.05. SGPT (u/ml) PAH S/M 105 40- 200 - [:o-o * * § 4: ‘60 ' § 100-0 \ l ‘20 _ $100400 § \ k 80 - § § \ \\ 0.00 0.03 0.25 2.00 CCL4 (ml/kg) 100-0 'IOO-IOO D @ PBB DIET: H" l * * l 1. * +- . \\ 0.03 0.25 2.00 CCL4 (ml/kg) Figure 15 106 TABLE 15 Effects of Dietary Polybrominated Biphenyls (PBB) on Liver Weight/Body Weight (LW/BW) and Kidney Weight/Body Weight (KW/BW) PBB Dieta LW/BW i 1 S.E. (x 100) KW/BW a: 1 S.E. (x 100) Control 5.97:0.23 1.54:0.05 1 ppm 6.10:0.l9b 1.49:0.04 25 ppm 6.91:0.31b 1.47:0.03 100 ppm 9.68i0.38 l.58i0.04 aAnimals were maintained on diets containing 0 (control), 1, 25 or 100 ppm of PBB for 14 days. Each value represents the mean i 1 S.E. of 6 animals. bSignificantly greater than control, p<0.05. 107 dietary PBB the increases in liver weight-to—body weight ratios are indicative of PBB—induced increases in liver size. Kidney weight-to— body weight ratios, in contrast, were not affected by consumption of PBB (Table 15). The hepatotoxicity and nephrotoxicity of CHCl3 were increased by ingestion of PBB. SGOT activities, already slightly elevated in mice ingesting 100 ppm of PBB for 14 days, were further increased by 25 ul/kg CHCl in mice ingesting 100 ppm PBB diet but not 3 in mice receiving lower dietary concentrations of PBB (Figure 16). PBB produced similar effects on CHClB-induced elevations of SGPT activities (data not shown). CHClB-induced increases in BUN and decreases in PAH and TEA S/Ms were also potentiated by PBB in a dietary concentration— dependent manner (Figure 17, TEA data not shown). Increases in BUN and decreases in PAH and TEA S/Ms were evident in control mice only after a dose of 50 ul/kg CHCl3. Increases in BUN, however, were produced by 25 ul/kg of CHCl3 in mice ingesting 25 and 100 ppm of PBB, and decreases in PAH and TEA S/Ms were produced by 25 pllkg of CHCl3 in mice ingesting l and 25 ppm of PBB and by 2.5 ul/kg of CHCl in mice ingesting 100 ppm of PBB (Figure 17). 3 6. Effects of Nan, 3M0, PCB and TCDD on CHClq toxicity in mice J Treatment with Nan appeared to increase the susceptibility of mice to the hepatotoxic effects of CHCl 0.25 ml/kg of CH013 in— 33 creased SGPT activity in Nan—treated mice but not in control mice (Figure 18). Though 3MC and PCB treatment had no marked effects on hepatic response to 0H013, treatment with TCDD may have protected the liver from the toxic effects of CHCl 0.75 ml/kg of 0H013 increased 3; SGPT activity in both control and TCDD-treated mice, but the magnitude 108 Figure 16. Effects of dietary polybrominated biphenyls (PBB) and i.p. CH013 on SGOT activity. Animals were maintained on diets containing 0 (control), 1, 25 or 100 ppm of PBB for 14 days prior to a single administration of 0H013. SGOT was determined 24 hr after CH013 administration. Each value represents the mean i l S.E. of 6 animals. *Significant increase in comparison to 0. 1400 1200 1000 g 800 .3’.’ 'E D 5 600 0 U, 400 200 109 CONTROL DIET SGOT C 242 1 ppm 285 25 ppm 173 100 ppm 385 9(- I l I l l 0.5 2.5 5.0 25.0 50.0 Figure 16 110 Figure 17. Effects of dietary polybrominated biphenyls (PBB) and i.p. 0H013 on BUN concentration and PAH S/M. Animals were main— tained on diets containing 0 (control), 1, 25 or 100 ppm of PBB for 14 days prior to a single administration of CH013. BUN and PAH S/Ms were determined 24 hr after 0H013 administration. Each value represents the mean i 1 S.E. of 6 animals. % Decrease in PAH S/M 100 20 l0 0 .b- O O O 80 lll D CONTROL IE'I’ BUN (5.5) D C 21 l 0--O 1 PPM 22 3 0—0 25 ppm 22 2 I—. I 100 ppm 21 2 n—o 1:1 I o /0 V . e 0“!” I I 1 I 1 0.5 2.5 5.0 25.0 50.0 CHCI3 Lll/ kg .\§_. CONTROL 21151 EAfl S/M 55.5.2 I c 7.81 0.37 0—0 0 1 ppm 7.54 0.39 0—0 a 25 ppm 8.11 0.49 ._. . 100 ppm 8.36 0.40 0—0 D 1 1 1 1 n 0.5 ' 2.5 5.0 25.0 50.0 CHCI3 All/kg Figure 17 112 Figure 18. Effects of chloroform and inducers of mixed—function oxidases (MFOs) on SGPT activity. Control mice or mice pretreated with phenobarbital (PB), 3—methy1cholanthrene (3MC), 2,3,7,8— tetrachlorodibenzo—p—dioxin (TCDD) or polychlorinated biphenyls (PCB) were challenged with single i.p. injections of CH013. Mice were sacrificed 24 hr later and SGPT determined. Each bar repre— sents the mean i 1 S.E. of 6 animals. *Significant difference in comparison to control mice receiving the same dose of CH013 (p<0.05). +Significant increase in comparison to mice receiving the same pretreatment and 0.00 ml/kg of 0H013 (vehicle) (p<0.05). $091 (U/m1) SGPT (U/ml ) 200 160 I20 80 40 200 160 I20 - 80 40 113 k mm mm T T T CONTROL 45:3 - @913 4315 V _ g 3MC 50:10T 005 0.75 CHCI3 (ml/kg) 1. . EREIREAIMENI W CONTROL 43:3 " @ TCDD 49:7 I PCB 45:4 *1. 0.25 7 0.75 c1-1c13 (ml/kg) Figure 18 114 of the increase was significantly greater in control mice than in TCDD— treated mice (Figure 18). Treatment with 3MC, TCDD, and PCB prevented or reduced the magnitude of the 0H013-induced decrease in PAH and TEA S/Ms (Figure 19, TEA data not shown). Nan, however, did not alter the sensitivity of mice to the nephrotoxic effects of CH013. 7. Effects of SKF 525-A and PB on CH013toxicity in mice Mice treated with PB, 600 mg/kg, 2 hr before 0H013 admini— stration exhibited no increase in SGPT activity following administration of 0.25 ml/kg CH013, though this dose of CH013 increased SGPT activity in control mice (Figure 20). Similarly, 0.75 ml/kg CHCl produced 3 less of an increase in SGPT activity in PB-pretreated mice than in control mice. Administration of PB 1 hr after CHCl3 potentiated the CHClB-induced elevation of SGPT activity, as did administration of SKF 525-A, 75 mg/kg, either 2 hr before or 1 hr after 0H01 Treatments 3. with SKF 525—A and PB had qualitatively similar effects on renal response to 0H013; pretreatment with PB partially reduced the magni— tudes of 0H013-induced decreases in PAH and TEA S/Ms (TEA data not shown), but administration of PB after CH013, or SKF 525—A either before or after CHCl lacked this effect (Figure 20). 3, 0. Interactions of 0H013_with GSH 1. Effects of PBB and PCB on CH013-induced depletion of GSH Intraperitoneal injection of CH013 reduced the concentrations of hepatic and renal reduced, non-protein thiols (mostly reduced GSH; see Methods, Section D3) in a dose-dependent manner (Figure 21). Control mice exhibited a significant reduction of renal and hepatic I 115 Figure 19. Effects of chloroform and inducers of mixed-function oxidases (MFOs) on PAH S/M. Control mice or mice pretreated with phenobarbital (PB), 3—methylcholanthrene (3MC), 2,3,7,8—tetra— chlorodibenzo—p-dioxin (TCDD) or polychlorinated biphenyls (PCB) for varying periods of time were challenged with single i.p. injec— tions of CH013. Mice were sacrificed 24 hr later and PAH S/M was determined. Each bar represents mean i l S.E. of 6 animals. *Significant difference in comparison to control mice receiving the same dose of CH013 (p<0.05). iSignificant decrease in comparison to mice receiving the same pretreatment and 0.00 ml/kg of CH013 (vehicle) (p<0.05). % DECREASE IN PAH S/M % DECREASE IN PAH S/M 40 80 20 40 60 80 I] 1 ERL-JREAIMENIEALLSZM § 3MC 0.25 CONTROL 9.153- 0.68 9.52! 0.62 9.48:0.89 0.75 c1-1c13(--I lkg) 0.05 BREIREAIMENIPAHJ‘ZZM CONTROL 0.25 9.07 2 0.49 9.08 3 0.41 9.14:0.67 01% (ml/kg) Figure 19 117 Figure 20. Effects of 0H013 and piperonyl butoxide (PB) or SKF 525—A (SKF) on SGPT and PAH S/M. Mice received a single i.p. injection of PB or SKF 525—A 120 min before (pre) or 60 min after (post) a single i.p. injection of 0H013. Bars represent the mean i l S.E. of 6 animals. *Significantly different than in control mice (0) re- ceiving the same amount of CH013, p<0.05. 118 .3 .3 I; l SGPT (U/ml) 8 100 * PRETREA‘I'MENT: c c P8 P8 SKF SKF c P8 SKF pre post pre post pre post c1103 (ml/kg): 0.00 025 0.75 14 12 10 z \ W a: 8 a 6 4 2 PRETREATMENT= c c P8 P8 SKF SKF c PB SKF pre post pre post pre post CHCI3(ml/kg): 0.00 0.25 0.75 Figure 20 119 Figure 21. Reduction of renal and hepatic reduced glutathione (GSH) content by 0H013: Effects of pretreatment with polybrominated biphenyls (PBB) and polychlorinated biphenyls (PCB). Mice were fed control diet or a similar diet formulated to contain 200 ppm of PCB or 100 ppm of PBB for 20 days prior to a single i.p. injection of CHCl3. Bars represent the mean i l S.E. of 6 animals. *Signifi- cantly different than in control mice receiving the same amount of 0H013, p<0.05. TSignificantly different than in mice receiving the same diet but 0.00 ml/kg 0H013, p<0.05. 120 conmor PCB \\\V P88 Hem-11c GSH ( pg Igm) 1200 0.00 0.05 0.25 1.00 CHCI3 (ml/kg) CONTROL RENAL GSH (pg lam) 600 0.00 0.05 0.25 1.00 CHCI3 (ml /kg) Figure 21 121 reduced GSH content following administration of 0.25 ml/kg CH01 As 3. little as 0.05 ml/kg, however, produced decreases in renal and hepatic GSH contents in mice treated with PBB. Mice treated with PCB, in contrast to control mice, failed to exhibit decreases in renal GSH content following administration of 0.25 ml/kg and 1.00 ml/kg of CH013 (Figure 21). Treatment with PCB did not alter the susceptibility of liver to CHCl3—induced depletion of GSH except at the highest dose of CH013 employed, 0.75 m1/kg, where depletion of hepatic GSH was slightly greater in PCB—treated mice than in control mice. 2. Effects of diethyl maleate on GSH depletion and CH01 O l O O 3 tOXicity in mice The concentrations of reduced non—protein thiols in liver and kidney (primarily reduced GSH) are shown in Figure 22. The concen— tration of GSH in renal cortex was approximately half of that in liver. Medullary GSH content was slightly less than that in cortex and papillary GSH content was approximately half that of cortex. Treat— ment with diethyl maleate, 600 mg/kg, decreased renal and hepatic GSH content. Maximum depletion occurred 2 hr after administration of diethyl maleate. As shown in Figure 22, the concentrations of GSH in both kidney and liver were reduced to approximately 15% of control values by diethyl maleate. Treatment of mice with diethyl maleate, 600 mg/kg, 90 min prior to CH01 administration increased the suscep— 3 tibility of mice to the toxic effects of CH01 As illustrated in 3. Figure 23, 0.033 and 0.100 ml/kg CH01 increased SGPT activity in 3 diethyl maleate-treated mice but not in control mice. 0.100 ml/kg CH013 also decreased PAH and TEA S/Ms (TEA data not shown) in diethyl 122 .moHE Hosusoo :H swam sozoa handeHmwstmm .mHmSHsm m we mumm .wx\mfi 000 .mummHmE Hzaumflw mo COHHMHumHoHEmm nmumm o .oumonS H%£umflm kn Ammwv odomauMuuaw woospoh oflummon pom Hma .m0.0vn .m.m H H Emma mam uuommhmmu HE ONH pooflmwuomm one: wows oh mo comumfimom .NN ousmflm 123 00¢ 000 00m— A ED\ 03 000— Inna 0:53: 000m 00¢N T F l l 71% % Z NN mmsmam Seas. 5532 03.5.00 * m. 332.2 _ .555 005.200 00a 00¢ 000 000— 009 AEQ on v .30 .225. 124 Figure 23. Effects of diethyl maleate and CH013 on SGPT and PAH S/M. Mice received a single i.p. injection of diethyl maleate, 600 mg/kg, 90 min prior to 0H013 and were sacrificed 24 hr later. Bars represent the mean i l S.E. of 6 animals. *Significantly different than in control mice receiving the same amount of CH013, p<0.05. 125 *I Am §m § 0. .m . m mm 8 $0... . o *l\\\\\\\\\\\\\\ m g II M .. .. T§m . m m mm m 1 \\\\\\\\\\\\\\\\\\\\\\§m m mm .11. o. fim \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\m f t1. vi. _ _ ”a“. 2 0 8 6r 4. 2. W m m m. m m W .. .. W... :8}: tom we 2% :s. w 126 maleate-treated mice though control mice were not affected at this dose (Figure 23). Although 0.300 ml/kg CH01 increased SGPT activity 3 and decreased PAH and TEA S/Ms in both control and diethyl maleate— treated mice, the magnitudes of the CH01 effects were greater in 3 diethyl maleate-treated mice than in control mice (Figure 23). The effects of treatments with CHCl3 and diethyl maleate on SGOT activity were similar to their effects on SGPT activities (data not shown). D. Covalent Binding of CH01.2 Metabolites in Mice 1. Covalent binding of CHClQ metabolites to renal and hepatic microsomal protein in vifro Incubation of (140)—0H01 with renal and hepatic microsomes 3 iE_vitro resulted in the covalent binding of radioactivity to micro— somal protein (Figure 24). The amount of radioactivity covalently bound per mg of protein per 5 min was nearly 15 times greater when hepatic microsomes were used than when renal microsomes were used. Hepatic microsomes from PBB and PCB—treated mice, furthermore, bound more radioactivity than hepatic microsomes from control mice. Covalent binding to renal microsomes from PCB-treated mice was quantitatively greater than covalent binding to renal microsomes from control mice (Figure 24). Covalent binding to renal microsomes from PBB—treated mice, however, was indistinguishable from that to renal microsomes from control mice. 2. Clearance of CH01Q and metabolites from blood and covalent binding to total Penal and hepatic protein in vivo Administration of (140)-0H01 to intact mice (1.75 mmoles/kg) 3 resulted in the covalent binding of radioactivity to renal and hepatic proteins (Figure 25). The amount of radioactivity bound, per mg of 127 Figure 24. Covalent binding of CHCl3 metabolites to microsomal protein ip_vitro. ( C)—CH013 was incubated with renal or hepatic microsomal protein for 5 min and covalent binding of radioactivity to protein determined. Data are represented as the mean i l S.E. of 6 independent experiments. *Significantly different than control, p<0.05. DPM BOUND / mg PROTEIN 128 8000 . LIVER * ‘ 6000 p * 4000L 2000 _ ' 3 i c PBB— PCB * 400 _ KIDNEY E E 300 2 .- O. l” E a 200 L 2 :3 2 100 j E _ O c P88 Figure 24 50 40 30 20 IO .. 600 400 200 N|3102ld Bw/aNnoa sa1owd NlilOlld Bw/ aunoa sa1owd 129 .m0.0va .HosuSOU aosm pneumMMHv hausmoflmwomflme .mao>fia was whospfix 0:“ 0H poofiEuouow >0H>Hu emu mm pouawmonmmu mum puma Isfiououn pom hufl>fluomOHpms Hmopfimms mam .0.H smumm a: NH so 0 .m wooflMfisowm mnt ooH paw o>H> mw smouonm UHummm: mom HmnoH on moo .mHmEflnm 0 mo .m.m H H meme omompmu mason Amx\mmaoas ms.av mammo-Aoeav mo moaummumflmaamm E .mmbflfi mom mmomflx :H kufl>fluomOHmmH Hmowmmms Haommmms maomo mo mmepaflm 08848800 .mN mmsmflm 130 nmoles Bound I mg ProIein O. O. Q o “m°'°5 [mg Tissue e m N ,_ ——fi | 1 I 6 ¢ g) 96 S! I I . I 8 g , U n- ’ I —eo- ae -o I I E —-6 l rO- * ‘°" 2 ~ ~ 9 ‘ ~ :4 *1 ~ I I I I l O 0 O O 8 8 .8. a 9 uIeIOId Bw/punog ludp enssu BuI / uIdp nmoles Bound I mg Protein nmoIes/mg Tissue 0. o O «s 01 .9 3 1 r ' I I I ¢ * O'$- ‘9 $ I I .3 J- ’ I u E X I I I me —o.’$——-— .4, \ \ 5 96 —OED;—_ "2 Z . ‘ '- d ‘ -. ~ I L I l 50 00 300 00 100 - ugegmd Bu: / punog uIdp enssu Btu/map I2 6 TIME hours Figure 25 TIME hours .-§\ \§_ ‘\\- ~~.\ 131 hj protein, appeared maximal in control mice 3 hr after CH01 . . 3 administra‘ tion and was quantitatively greater in the kidney than in th e liver, The patterns of total residual radioactivity in the liver and k idney 3, 6 and 12 hr after CH013 administration closely resembled the patterns of covalent binding to renal and hepatic proteins (Figure 25), Mice pretreated with PBB and PCB bound more radioactivity to hepatic proteins and less radioactivity to renal proteins than did control mice (Figure 25). Residual radioactivity in the liver and kidney was affected by PBB and PCB in a similar manner. The percentages of the total administered dose of (14)— CHCl3 remaining in the liver and kidney and covalently bound to renal and hepatic protein 3, 6 and 12 hr after CH013 administration are Summarized in Table 16. Pretreatment with PBB and PCB led to in— creases (by a factor of approximately 2—4X) in the percentage of the administered dose covalently bound to hepatic protein and the per- centage of the dose remaining in the liver, but decreases (by a factor of nearly 2) in the percentage of the administered dose covalently bound to renal protein and the percentage of the dose re— maining in the kidney 3, 6 and 12 hr after 011013 administration (Table 16). The concentrations of total radioactivity (CH013 and metabo— lites) in blood from control, PBB and PCB—treated mice appeared maximal 30 min after i.p. injection of (140)—CH013, 1.75 mmoles/kg (Figure 26A). Maximum blood concentrations were lower in PBB and PCB- treated mice than in control mice and remained lower at each C°1leCtion time thereafter. 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By analysis of co-variance it was determined that the slopes of the lines relating log blood concen- trations of radioactivity to time after CH01 administration from PBB 3 and PCB—treated mice were significantly different from that'of control mice. That is, mice pretreated with PBB and PCB removed CH013 from the blood more rapidly than did control mice. 3. Covalent binding to subcellular fractions in vivo Subcellular fractionation of samples of kidney and liver removed 3 hr after i.p. injection of (140)—CH013 (1.75 nmoles/kg) to control, PBB and PCB-treated mice revealed that covalent binding of radioactivity had occurred to proteins and lipids in mitochondria (mitochondrial fraction), endoplasmic reticulum (microsomal fraction) and to cytosolic proteins (100,000 x g supernatant protein)(Table 17). The magnitude of covalent binding in control mice, per mg of protein or lipid, was nearly equal in renal and hepatic mitochondria and endo— plasmic reticulum. In contrast, the magnitude of covalent binding to hepatic cytosolic protein was much greater than that to renal cytosolic protein. Mice treated with PBB and PCB bound more radioactivity (per mg of protein or lipid) than did control mice to hepatic mitochondria and cytosolic protein but not to hepatic endoplasmic reticulum. 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