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Polybrominated biphenyl-induced potentiation of chloroform toxicity
TOXICOLOGY
AND
APPLIED
Polybrominated
Biphenyl-Induced Toxicity’ W. M.
Department
45. 861-869 (1978)
PHARMACOLOGY
of Pharmacolog.v, Received
KLUWE* Michigan
November
AND
Potentiation
J. B.
State University, I, 1977; accepted
of Chloroform
HOOKY East Lansing,
February
Michigan
48824
8,197s
Polybrominated Biphenyl-Induced Potentiation of Chloroform Toxicity. KLUWE, W. M.. AND J. B. (1978). Toxicol. Appl. Pharmacol. 45, 861-869. ICR male mice were fed diets containing 0, 1,25. or 100 ppm of polybrominated biphenyls (PBB) for 14 days prior to challenge with a single injection of chloroform (CHCl,). Dietary PBB potentiated the CHCl,-induced depression of p-aminohippurate accumulation by slices of renal cortex and the CHCl,-induced rise in blood urea nitrogen concentration in a PBB-intake-related manner. The kidney weight-tobody weight ratio was elevated in PBB mice but not in mice consuming control (0 ppm) diet following administration of 50 pi/kg of CHCI,. Mice ingesting food containing 100 ppm of PBB also exhibited a greater increase in serum glutamic-oxaloacetic transaminase activity than did control mice following CHCl, administration. The 24-hr LDSO of CHCl, in control mice was 1.28 ml/kg, while that in mice consuming 100 ppm of PBB was 0.62 ml/kg. HOOK,
Interest has been kindled recently in the toxicological potential of polybrominated biphenyls (PBB). The commerical fire retardant, Firemaster BP6 (Michigan Chemical Co., St. Louis, Mich.), primarily hexabromobiphenyl, was inadvertently mixed into domestic animal feed in south central Michigan (Mercer et al., 1976), resulting in exposure of commerical livestock to high concentrations of dietary PBB. Secondary exposure of the human population occurred through consumption of meat and dairy products derived from contaminated livestock (Chen, 1977). Though a high intake of PBB has been shown to possibly be teratogenic in rodents (Corbett et al., 1975) and to cause toxicosis in dairy cattle (Moorhead et al., 1977), long-term exposure of adult rats to PBB failed to produce evidence of functional nephrotoxicity though histological signs of glomerular damage were evident (McCormack et al., 1977b). Dent and co-workers have shown that PBB is a potent inducer of hepatic (Dent et al., 1976) and extrahepatic (McCormack et al., 1977a) mixed function oxygenase (MFO) systems. Thus, it is possible that doses of PBB that produce no observable change in renal function could “sensitize” the kidney to challenge by a compound metabolized by the kidney to a toxicant. The effect of PBB-induced stimulation of MFO systems on the hepatic and renal toxicity of chloroform (CHCI,), an agent thought to be metabolized by MFO systems to an ultimate toxicant, was examined to determine if PBB exposure could enhance the toxic potential of chemicals biotransformed to toxicants. i Supported in part by USPHS Grants ES00560 and AM 109 13. * Supported in part by USPHS Training Grant GM01 761. 3 Author to whom reprint requests should be addressed. 861
0041-008X/78/0453-0861 $02.00/O Copyright @ 1978 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
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HOOK
METHODS Male Swiss ICR mice, purchased from Spartan Farms (Haslett, Mich.) were used exclusively in these experiments. Control and treated animals were housed in separate but identical rooms with controlled light cycles, heat, and humidity and adequate ventilation. PBB was dissolved in acetone and mixed slowly and evenly into a finely ground standard rodent diet (Wayne Lablox). Diets containing 0, 1, 25, or 100 ppm of PBB were fed ad libitum for 14 days prior to sacrifice. No differences in food consumption were noted. Futzctiotzal studies. Reagent-grade CHCl, was diluted with corn oil and administered ip in a total volume of 5 ml/kg at doses of 0, 0.5, 2.5, 5.0, 25, and 50 pi/kg 24 hr before sacrifice. Control mice received 5 ml/kg of corn oil. At the time of sacrifice, the animals were weighed and decapitated, and blood was collected in small disposable glass tubes. Kidneys and livers were quickly removed, cleaned of extraneous tissue, and weighed. Thin renal cortical slices were cut free-hand and incubated for 90 min in an oxygenated, phosphate-buffered medium (Cross and Taggart, 1950) containing p-amino.hippurate (PAH), 5 x 1O-5 M. for estimation of organic ion transport capacity. After incubation the slices were removed, blotted, weighed, and homogenized in 10% trichloroacetic acid. Aliquots of medium were treated similarly. The protein precipitate was pelleted by centrifugation, and the supernatant was assayed for PAH by methods described previously (Kluwe and Hook, 1977). with the exception that acidified samples were placed in boiling water for 1 hr to hydrolyze any acetylated product to PAH. Ability of renal cortical tissue to accumulate PAH against a concentration gradient can be expressed as a slice to medium ratio (S/M) where S = milligrams of PAH per gram of tissue and h4 = milligrams of PAH per milliliter of medium. The percentage decrease in PAH S/M was determined using the following formula (Watrous and Plaa, 1972a):
Percentage decrease =
Control PAH S/M - Treated PAH S/M Control PAH S/M - 1
The blood samples were allowed to clot for 60 min on ice and then were gently centrifuged. and the serum was withdrawn and refrigerated. Blood urea nitrogen (BUN) concentrations were determined with Sigma reagents (Sigma Chemical Co., St. Louis, MO.), and serum glutamic-oxaloacetic transaminase (SGOT) activities were determined by the method of Reitman and Frankel(l957). LethaliQv studies. Mice ingesting diets supplemented with 0 or 100 ppm of PBB for 14 days were administered various doses of chloroform (0.20-1.80 ml/kg) ip in corn oil in a total volume of 5 ml/kg. The number of deaths occurring within 24.48, 96, and 168 hr were recorded. Slafistics. Data were represented as the mean 5 SE of six animals and analyzed by blocked analysis of variance. Differences between means were compared using the least significant difference (LSD) test (Steel and Torrie, 1960) with p < 0.05 as the criterion of significance. The LD50 values were determined by the method of Litchfield and Wilcoxon ( 1949).
PBB AND
CHC&
863
TOXICITY
RESULTS Neither liver weight to body weight ratios (LW/BW) nor kidney weight to body weight ratios (KW/BW) were affected by CHCI, administration in animals consuming control diet (Table 1). Liver weights were greatly enhanced by dietary PBB. Animals consuming the diet containing 25 ppm of PBB exhibited LW/BW 119% that of control, while those consuming a lOO-ppm of PBB diet showed LW/BW 179% that of control (Table 1, column 1). KW/BW was not affected by dietary PBB. However, in animals consuming PBB, CHCl, administration resulted in an increased KW/BW (Table 1). The increase was apparent only at the higher doses of CHCl,. TABLE EFFECT OF DIETARY LIVER WEIGHT/BODY
POLYBROMINATED WEIGHT (LW/BW)
1
BIPHENYLS (PBB) AND SUBCUTANEOUS AND KIDNEY WEIGHT/BODY WEIGHT RATIOS”
CHCI, ON (KW/BW)
CHCl, Wkg) Diet Control 1 PPm 25 ppm 100 ppm
Control 1 pw 25 ppm 100ppm
0 5.97 6.10 6.91 9.68
0.50
& 0.23 6.40 & 0.04 + 0.19 6.35 k 0.37 k 0.31b 7.36 + O.lgb t 0.38* 10.02 k 0.35 b
1.54 + 0.05 1.49 f 0.04 1.47 & 0.03 1.58 & 0.04
1.73 & 0.03 1.50 & 0.06 1.44 k 0.07 1.39 & 0.04
2.50
5.0
LW/BW 5.93 + 0.30 6.17 IO.10 7.48 & 0.21b 9.74 k 0.30'
x 100 6.47 + 6.11 + 6.76 i 9.64 k
KW/BW
x 100
1.64 k 0.08 1.54 & 0.04 1.55 + 0.03 1.37 + 0.04
0.40 0.13 0.12 0.25b
1.68 & 0.05 1.47 5 0.04 1.46 f 0.05 1.42 k 0.06
“Animals were maintained on the above diets for 14 days prior CHCI,. Twenty-four hours after CHCI,, the animals were decapitated were weighed. Each value represents the mean (&SE) of six determinations. h Significantly greater than control,p < 0.05.
25.0 6.09 k 0.19 5.61 + 0.15 7.35 k 0.12b 10.14 f0.34b
1.63 k 0.08 1.46 & 0.08 1.76 ? 0.03 1.70 k 0.19
50.0 6.39 6.01 6.46 9.54
+ 0.13 5 0.17 & 0.12 + 0.23h
1.70 & 0.10 1.93 + 0.07h 2.12 & 0.04h 2.21 & O.Ogh
to a single administration ot and exsanguinated. and organs
Serum glutamic-oxaloacetic transaminase (SCOT) activities were elevated slightly by PBB ingestion at a dietary concentration of 100 ppm (Fig. 1). Lower dietary levels were not associated with signs of hepatotoxicity. Administration of CHCI, to mice consuming 0, 1, or 25 ppm of PBB did not result in liver damage as evidence by SGOT activities which were not significantly different from those of non-CHCI,-treated control animals. Mice receiving 100 ppm of PBB. however, responded to increasing doses of CHCl, with drastic elevations of SGOT activity (Fig. 1). In mice receiving the control diet, serum blood urea nitrogen (BUN) was increased only slightly by the highest dose of CHCI, used (Fig. 2). As dietary intake of PBB increased, however, elevation of BUN secondary to CHCI, administration (25 and 50 ,&kg) rose rapidly. The capability of renal cortical slices from control and PBB-treated animals receiving various doses of CHCl, to actively accumulate the organic anion p-aminohippurate
864
KLUWE
AND
HOOK
*
1400
ClET CONTROL SCOT151.) c 242- 40 .-. 1PPrn 285 63 Q-0 25Pm7 173 21 ,-. ppm
27
100
#l-D
335
I
/
I
I
I
CHC13
1
25.0 50.0
2.5 5.0
0.5
+
Ilyn
d/kg
FIG. 1. Effect of dietary PBB and subcutaneous CHCI, on SCOT activity. Animals were maintained on 0-, 1., 25, or 100.ppm PBB-supplemented diets for 14 days prior to a single administration of CHCI,. The mice were decapitated 24 hr after CHCI, administration. blood was collected and allowed to clot for 1-2 hr, and glutamic-oxaloacetic transaminase (GOT) activity was determined in the serum fraction. An asterisk(*) denotes a signilicant increase in comparison to control (0 ppm of PBB), p < 0.05.
(PAH) against a concentration gradient is represented in Fig. 3. Animals on control diet exhibited a significant depression (approximately 30% decrease) in PAH S/M after receiving 25 @/kg of CHCl, (Fig. 3). Smaller doses of CHCI, had no effect in control mice. Dietary PBB, however, appeared to cause a PBB-intake-related increase in loo
-
0 c ippm 25ppm lOOppIn
EO-
s
P -5 i
.-. O-0 l -w n--o
0
.
/
60! 40
-
20
-
’ /“. “/.
.
:: A
e-----op. 1 0.5
I I 2.5 5.0 CHC$
1 1 25.0 50.0
1111 kg
FIG. 2. Effect of dietary PBB and subcutaneous CHCI, on BUN concentration (mg/lOO ml). Animals were maintained on 0-. l-. 2S-. or lOO-ppm PBB-supplemented diets for 14 days prior to a single administration of CHCI,. The mice were decapitated 24 hr after CHCI, administration, blood was collected and allowed to clot for 1-2 hr. and blood urea nitrogen (BUN) concentration was determined in the serum fraction.
PBB
AND
I 0.5
I
CHCl,
865
TOXICITY
I I 2.5 5.0 CHCI, til/kg
I I 25.0 50.0
FIG. 3. Effect of dietary PBB and subcutaneous CHCI, on PAH S/M. Animals were maintained on O-. I-, 25-, or 100.ppm PBB-supplemented diets for 14 days prior to a single administration of CHCI,. The mice were sacrificed 24 hr after CHCI, administration. and thin renal cortical slices were incubated in a phosphate-buffered medium containing p-aminohippurate (PAH). Accumulation of PAH against a concentration gradient was expressed as a PAH slice-to-medium ratio (S/M) where S = milligrams of PAH per gram of tissue and M = milligrams of PAH per milliliter of medium.
susceptibility to CHCl,-induced depression of PAH uptake. Significant decreases in PAW S/M were evident following administration of 0.5 PC/kg of CHCl, (100 ppm of PBB) and 5.0 pllkg of CHCl, (1 and 25 ppm of PBB). At a dose of 25 pllkg of CHCI, the decrease in PAH S/M was approximately 30. 45, 55, and 60% for dietary PBB concentrations of 0. 1, 25, and 100 ppm of PBB, respectively. Potentiation of CHCl, lethality by dietary PBB is shown in Table 2. Maximal lethality was achieved 96 hr after CHCl, administration: mice surviving this initial 4day period survived for at least an additional 2 weeks. Potentiation was perhaps most clearly evident after 0.5 ml/kg of CHCl,, a dose at which all control animals survived for at least 96 hr after CHCl, administration while all of the PBB-treated animals expired (data not shown). The 24-. 48., and 96-hr LD50 values were all reduced in PBB-treated mice (Table 2). The minimum lethal dose (the lowest dose of which at least TABLE EFFECT
LD50 24 hr 48 hr Y6 hr
OF
DIETARY
2
POLYBROMINATED ACUTE CHCI, LD50 Control0
1.28 (0.8-2.7) 1.00 (0.8-2.7) 1.00 (0.8-1.8)
BIPHEN~LS
PBB” 0.62 0.50 0.39
(0.5-1.2) (0.32-0.8) (0.32-0.5)
(PBB)
ON
ControllPBB” 2.08 2.00 2.56
U LD50 was determined by the method of Litchiield and Wilcoxon (1949). Numbers in parentheses represent the lowest dose at which any deaths occurred and the highest dose at which any animals survived. h Ratio of LD50 in control animals to LD50 in PBB-treated animals.
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KLUWE
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HOOK
one animal expired within 96 hr after CHCI, administration) in control mice was less than the maximum lethal dose (the lowest dose at which all exposed animals expired within 96 hr after CHCI, administration) in PBB-treated animals (Table 2).
DISCUSSION The short-chain chlorinated hydrocarbon solvents are thought to produce toxicosis, at least in part, through the actions of a toxic metabolic intermediate (Ilett et al., 1973). While the biotransformation of chemicals to hepatotoxins has been studied extensively (Mitchell et al., 1976), little attention has been given to the biotransformation of chemicals to nephrotoxins. Although CHCI, and Ccl, are decidely toxic to human kidneys (Von Oettingen, 1964), their hepatic effects are much more prominent than their renal effects in the commonly used laboratory rat. Thus, the male ICR mouse was chosen as a model to investigate CHCl, toxicity because of its sensitivity to both the renal and hepatic effects of CHCI,. CHCl,, like Ccl,, is metabolized to a toxic intermediate by MFOs. Sensitivity to CHCl, in mice is strain-dependent. C57BL/6J mice are relatively resistant to CHCI, nephrotoxicity, while DBA/U mice are more sensitive (Hill et al., 1975). The outbred strain used in these experiments (ICR) falls between the two extremes. CC],-induced hepatotoxicity is potentiated by induction of hepatic MFO systems with chemicals such as polychlorinated biphenyls (PCB) (Carlson. 1975a), phenobaribtal (Suarez et al., 1972), 3,4-benzpyrene (Pitchumoni et al., 1972), and other chemicals such as aliphatic alcohols (Traiger and Plaa, 1972; Hasamuro et al., 1974). Other MFO system inducers such as 3-methylcholanthrene (Suarez et al., 1972) or inhibitors such as methylmercury hydroxide (Carlson, 1975b) or dibenamine (Maling et al., 1974) decrease the hepatotoxicity of Ccl,. It appears, then, that multiple pathways exist for hepatic biotransformation of Ccl,, metabolites of some being more toxic than metabolites of others. Like Ccl,, CHCl, appears to be a substrate for several biotransformation pathways. End products of CHCl, metabolism include carbon dioxide (Butler. 1961), carbon monoxide (Ahmed et al., 1977), and perhaps a small amount of methylene chloride (Paul and Rubinstein, 1963). Studies based on in vitro tissue incubations have documented the ability of both renal and hepatic homogenates to metabolize CHCI, (Butler, 1961). Administration of phenobarbital or 3-methylcholanthrene increased the hepatotoxicity of CHCl, in rats (Lavigne and Marchand, 1974) while McMurty et al. (1976) have suggested recently that the nephrotoxicity of certain drugs could be reduced by the administration of chemicals which depressed hepatic and renal MFO activity. It seemed likely, then, that chemicals which stimulated MFO systems would enhance the nephrotoxicity of CHCl, while chemicals which inhibited MFO systems would reduce CHCl,-induced nephrotoxicity. Since PBB is a potent inducer of renal and hepatic MFOs it appeared that mice consuming PBB metabolized a greater proportion of the administered dose of CHCl, to the ultimate toxicant. Results from BUN and SCOT determinations and KW/BW all suggested that PBB enhanced biotransformation of CHCl, to a toxic metabolite in both liver and kidney. Inhibition of renal slice uptake of PAH has been shown to parallel CHCl, nephrotoxicity in rodents (Watrous and Plaa, 1972a). PBB ingestion enhanced CHCl, nephrotoxicity as evidenced by potentiation of the CHCl,-induced decrease in PAH S/M. Furthermore,
PBB
AND
CHCl,
TOXICITY
867
the degree of enhancement of the CHCl,-induced decrease in renal transport capacity was directly proportional to the dietary intake of PBB. While renal tissue contains the enzymatic apparatus to metabolize CHCI, (Butler 1961), results from the experiments reported here cannot rule out the liver as the site of formation of the nephrotoxic metabolite. However, covalent binding of a CHCI, metabolite to hepatic and renal microsomal protein appears to parallel CHCl, toxicity (Brown et a/., 1974). The chemical reactivity of a CHCI, metabolite which could alkylate membrane proteins and lipids would be expected to be too great to allow transport, intact, via the hepatic vein and through the arterial system to the kidney. PBB administration also increased the lethality of CHCI, as evidenced by a reduction in the acute LD50. More striking, perhaps, are the data showing that 96 hr after administration of 0.5 ml/kg of CHCI,, all of the PBB animals died while all of the control animals survived. The cause of death in the LD50 experiments does not appear to be renal in origin. The nephrotoxic effects of CHCI, on PAH transport, most likely mediated by tubular necrosis, appear to be maximal 24 hr after administration of CHCl, and nearly absent by 96 hr (Watrous and Plaa. 1972b). In addition, a prominent sex difference exists in susceptibility of ICR mice to the nephrotoxic effects of CHCI,, but no sex difference exists for acute LD50 (Klaassen and Plaa, 1967). It had been previously suggested that death was due to severe narcosis (Klaassen and Plaa, 1966). but in these experiments all animals regained consciousness within a few hours after CHCI,. Death ensued long after the anesthetic effects of CHCl, had ended. Since the LD50 was reduced in PBB-treated mice at a time when liver and kidney capacity for biotransformation was increased it appears that generation of a lethal metabolite resulted from biotransformation of CHCI,. In summary, dietary intake of PBB for 14 days greatly enhanced the nephrotoxicity and hepatotoxicity of CHCl, in male mice. Since PBB is a potent inducer of hepatic and renal MFO systems, and CHCI, is believed to exert its toxic effects through a biotransformation product, these experiments suggest that dietary consumption of PBB greatly increased CHCI, toxicity by stimulating the biotransformation pathway leading to formation of a toxic CHCl, metabolite. PBB is only one of an ever-increasing group of halogenated chemicals used extensively by industry and agriculture which have found their way into the environment. Several of these chemicals, like PBB, are inducers of MFOs, are highly stable in the environment, and display long tissue half-lifes, once ingested, due to their highly lipophilic nature. Investigations into the toxicity of these chemicals has generally concentrated on direct toxic effects (tissue hyperplasia, necrosis, nervous disorders). Outside of the laboratory, though, humans and animals are exposed to a number of potential toxicants. Some, like CHCl, and Ccl,, are metabolized to toxicants while others, like PBB, can be indirectly toxic by increasing the rate at which chemicals like CHCl, and Ccl, are metabolized to toxicants. Thus, the principal hazard of exposure of humans and livestock to low concentrations of PBB may be increased susceptibility to damage from other toxicants. CHCl,, for example, is a common contaminant of surface waters and is ingested daily in what are normally subtoxic quantities. To victims of PBB exposure, however, the intake of CHCl, that is needed to produce damage may be greatly reduced, as shown for the mouse. Thus, the toxicity testing of chemicals which induce microsomal enzyme activities and may become contaminants of the environment should include an evaluation of their effects on the biotransformation of other common chemicals to toxicants.
868
KLUWE
AND
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ACKNOWLEDGMENTS The expert technical gratefully acknowledged.
assistance of Ms. Cathy Herrmann
and Ms. Harriett
Sherman
are
REFERENCES AHMED, A. E., KUBIC, V. L., AND ANDERS, M. W. (1977). Metabolism of haloforms to carbon monoxide. I. In vitro studies. Drug Metab. Dispos. 5, 198-204. BROWN, B. R., JR., SIPES, I. G., AND SAGALYN, A. M. (1974). Mechanism of acute hepatic toxicity: Chloroform, halothane and glutathione. Anesthesiologq’ 41, 554-56 1. BUTLER, T. C. (196 1). Reduction of carbon tetrachloride in vivo and reduction of carbon tetrachloride and chloroform in vitro by tissues and tissue constituents. J. Phannacol. E,rp. Ther. 134.31 I-319. CARLSON, G. P. (1975a). Potentiation of carbon tetrachloride hepatotoxicity in rats by pretreatment with polychlorinated biphenyls. Toxicology 5, 69-77. CARLSON, G. P. (1975b). Protection against carbon tetrachloride induced hepatotoxicity with methylmercury hydroxide. Toxicology 4, 83-89. CHEN, E. (1977). Michigan: If something odd happens.. . . Atlantic Monthly 240, 2-20. CORBETT, T. H., BEAUDOIN. A. R., CORNELL, R. G., ANVER, M. R., SCHUMACHER. R., ENDRES, J.. AND SZWABOWSKA, M. (1975). Toxicity of polybrominated biphenyls (Firemaster BP61 in rodents. Environ. Res. 10, 390-396. CROSS, R. J., AND TAGGART, J. V. (1950). Renal tubular transport: Accumulation of p-atninohippurate by rabbit kidney slices. Amer. J. Phqdol. 161, 181-190. DENT, J. G., NETTER, K. J., AND GIBSON, J. E. (1976). The induction of hepatic microsomal metabolism in rats following acute administration of a mixture of polybrominated biphenyls. Toxicol. Appl. Pharrnacol. 38.237-249. HASUMURA, Y., TESCHKE, R., AND LIEBER, C. (1974). Increased carbon tetrachloride hepatotoxicity, and its mechanism, after chronic ethanol consumption. Gastroenterologbl 66, 415-422. HILL, R. N., CLEMENS, T. L., LIU, D. K., AND VESELL. E. S. (1975). Genetic control of chloroform toxicity in mice. Science 190, 159-160. ILETT, K. F., REID, W. D., SIPES, I. G., AND KRISHNA, G. (1973). Chloroform toxicity in mice: Correlation of renal and hepatic necrosis with covalent binding of metabolites to tissue macromolecules. Exp. Mol. Pathol. 19, 215-229. KLAASSEN. C. D., AND PLAA, G. L. (1966). Relative effects of various chlorinated hydrocarbons on liver and kidney function in mice. Toxicol. Appl. Pharmacol. 9, 139- 15 1. KLAASSEN, C. D., AND PLAA, G. T. (1967). Susceptibility of male and female mice to the nephrotoxic and hepatotoxic properties of chlorinated hydrocarbons. Proc. Sot. Exp. Biol. Med. 124, 1163-I 166. KLUWE, W. M., AND HOOK, J. B. (1977). Analysis of gentamicin uptake by rat renal cortical slices. Toxicol. Appl. Pharmacol., in press. LAVIGNE, J. G.. AND MARCHAND, C. (1974). The role of metabolism in chloroform hepatoxicity. Toxicol. Appl. Pharmacol. 29,3 12-326. LITCHFIELD, J. T., AND WILCOXON, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96,99-l 13. MALING, H. M., EICHELBAUM, F. M., SAUL, W., SIPES. I. G., BROWN, E. A. B., AND GILLETTE, J. R. (1974). Nature of the protection against carbon tetrachloride-induced hepatotoxicity produced by treatment with dibenamine. Biochem. Pharmacol. 23, 1479-149 1. MCCORMACK, K. M., CAGEN, S. Z., RICKERT, D. E., GIBSON, J. E., AND DENT, J. G. (1977a). Stimulation of hepatic and renal mixed-function oxygenases in developing rats by polybrominated biphenyls. Drug. Merab. Dispos., in press. MCCORMACK, K. M., KLUWE, W. M., RICKEIT, D. E., SANGER, V. L., AND HOOK, J. B. (1977b). Renal and hepatic microsomal enzyme stimulation and renal function following
PBB
AND
CHCI,
TOXICITY
864,
three month dietary exposure to polybrominated biphenyls. Toxicol. Appl. Pharmacol.. in press. MCMURTY. R. J.. SNODGRASS, W. R., AND MITCHELL, J. R. (1976). Metabolic activation of acetaminophen. cephaloridine, and other chemically stable compounds to nephrotoxic metabolites. Clin. Res. 24,407A. MERCER, H. D., TESKE, R. H., CONDON. R. J., FURR. A., MEERDINK, G., BUCK, W.. AND FRIES. G. (1976). Herd health status of animals exposed to polybrominated biphenyls. J. Tosicol. Ewirou. Health 2. 335-349. MITCHELL, J., NELSON, S. D.. AND THORGEIRSSON. S. S. (1976). Metabolic activation. Biochemical basis for many drug-induced liver injuries. In Progress in Liwr Disease (H. Popper and F. Schaffner, eds.), Vol. 15, Chap. 16. Grune and Stratton. New York. MOORHEAD, P. D.. WILLETT. L. B., BRUMM. C. J.. AND MERCER, H. D. (1977). Pathology of experimentally induced polybrominated biphenyl toxicosis in pregnant heifers. J. Amer. l,ef. Med.Assoc. 170.307-313. PITCHUMONI, C. S., STENGER, R. J., ROSENTHAL, W. S., AND JOHNSON. E. A. (1972). Effects of 3,4-benzpyrene pretreatment on the hepatotoxicity of carbon tetrachloride in rats. J. Pharmacol. Exp. Ther. 181,221-233. PAUL, B. P.. AND RUBINSTEIN, D. (1963). Metabolism of carbon tetrachloride and chloroform by the rat. J. Pharrnacol. Exp. Ther. 141, 141-148. REITMAN. S., AND FRANKEL, S. (1957). A calorimetric method for the determination of serum glutamic-oxaloacetic and glutamic-pyruvic transaminases. Amer. J. Clin. Pufhol. 28, 56-7 1. STEEL, R. G. D.. AND TORRIE, J. H. (1960). Principles and Procedures of Statistics, p. 67. McGraw-Hill, New York. SUAREZ. K. A.. CARLSON, G. P., FULLER, G. C., AND FAUSTO. N. (1972). Differential acute effects of phenobarbital and 3-methylcholanthrene pretreatment on Ccl,-induced hepatotoxicity in rats. Tosirol. Appi. Pharmacol. 23, 17 1-I 77. TRAIGER, G. J., AND PLAA, G. L. (1972). Relationship of alcohol metabolism to the potentiation of Ccl, hepatotoxicity induced by aliphatic alcohols. J. Pharmacol. Exp. Ther. 183,48 l-488. VON OETTINGEN, W. F. (1964). Halogenated Hydrocarbons of Industrial and Toxicological Importance, p. 77. Elsevier, Amsterdam. WATROUS, W. M.. AND PLAA, G. L. (1972a). Effect of halogenated hydrocarbons on organic ion accumulation by renal cortical slices of rats and mice. Toxicol. Appl. Pharnzacol 22. 52% 543. WATROUS,
W. M.,
649.
G. L. (1972b). The nephrotoxicity of single and multiple doses of hydrocarbon solvents in male mice. Toxicol. Appl. Pharrnacol. 23. 640-
AND PLAA,
aliphatic chlorinated
AND
APPLIED
Polybrominated
Biphenyl-Induced Toxicity’ W. M.
Department
45. 861-869 (1978)
PHARMACOLOGY
of Pharmacolog.v, Received
KLUWE* Michigan
November
AND
Potentiation
J. B.
State University, I, 1977; accepted
of Chloroform
HOOKY East Lansing,
February
Michigan
48824
8,197s
Polybrominated Biphenyl-Induced Potentiation of Chloroform Toxicity. KLUWE, W. M.. AND J. B. (1978). Toxicol. Appl. Pharmacol. 45, 861-869. ICR male mice were fed diets containing 0, 1,25. or 100 ppm of polybrominated biphenyls (PBB) for 14 days prior to challenge with a single injection of chloroform (CHCl,). Dietary PBB potentiated the CHCl,-induced depression of p-aminohippurate accumulation by slices of renal cortex and the CHCl,-induced rise in blood urea nitrogen concentration in a PBB-intake-related manner. The kidney weight-tobody weight ratio was elevated in PBB mice but not in mice consuming control (0 ppm) diet following administration of 50 pi/kg of CHCI,. Mice ingesting food containing 100 ppm of PBB also exhibited a greater increase in serum glutamic-oxaloacetic transaminase activity than did control mice following CHCl, administration. The 24-hr LDSO of CHCl, in control mice was 1.28 ml/kg, while that in mice consuming 100 ppm of PBB was 0.62 ml/kg. HOOK,
Interest has been kindled recently in the toxicological potential of polybrominated biphenyls (PBB). The commerical fire retardant, Firemaster BP6 (Michigan Chemical Co., St. Louis, Mich.), primarily hexabromobiphenyl, was inadvertently mixed into domestic animal feed in south central Michigan (Mercer et al., 1976), resulting in exposure of commerical livestock to high concentrations of dietary PBB. Secondary exposure of the human population occurred through consumption of meat and dairy products derived from contaminated livestock (Chen, 1977). Though a high intake of PBB has been shown to possibly be teratogenic in rodents (Corbett et al., 1975) and to cause toxicosis in dairy cattle (Moorhead et al., 1977), long-term exposure of adult rats to PBB failed to produce evidence of functional nephrotoxicity though histological signs of glomerular damage were evident (McCormack et al., 1977b). Dent and co-workers have shown that PBB is a potent inducer of hepatic (Dent et al., 1976) and extrahepatic (McCormack et al., 1977a) mixed function oxygenase (MFO) systems. Thus, it is possible that doses of PBB that produce no observable change in renal function could “sensitize” the kidney to challenge by a compound metabolized by the kidney to a toxicant. The effect of PBB-induced stimulation of MFO systems on the hepatic and renal toxicity of chloroform (CHCI,), an agent thought to be metabolized by MFO systems to an ultimate toxicant, was examined to determine if PBB exposure could enhance the toxic potential of chemicals biotransformed to toxicants. i Supported in part by USPHS Grants ES00560 and AM 109 13. * Supported in part by USPHS Training Grant GM01 761. 3 Author to whom reprint requests should be addressed. 861
0041-008X/78/0453-0861 $02.00/O Copyright @ 1978 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
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METHODS Male Swiss ICR mice, purchased from Spartan Farms (Haslett, Mich.) were used exclusively in these experiments. Control and treated animals were housed in separate but identical rooms with controlled light cycles, heat, and humidity and adequate ventilation. PBB was dissolved in acetone and mixed slowly and evenly into a finely ground standard rodent diet (Wayne Lablox). Diets containing 0, 1, 25, or 100 ppm of PBB were fed ad libitum for 14 days prior to sacrifice. No differences in food consumption were noted. Futzctiotzal studies. Reagent-grade CHCl, was diluted with corn oil and administered ip in a total volume of 5 ml/kg at doses of 0, 0.5, 2.5, 5.0, 25, and 50 pi/kg 24 hr before sacrifice. Control mice received 5 ml/kg of corn oil. At the time of sacrifice, the animals were weighed and decapitated, and blood was collected in small disposable glass tubes. Kidneys and livers were quickly removed, cleaned of extraneous tissue, and weighed. Thin renal cortical slices were cut free-hand and incubated for 90 min in an oxygenated, phosphate-buffered medium (Cross and Taggart, 1950) containing p-amino.hippurate (PAH), 5 x 1O-5 M. for estimation of organic ion transport capacity. After incubation the slices were removed, blotted, weighed, and homogenized in 10% trichloroacetic acid. Aliquots of medium were treated similarly. The protein precipitate was pelleted by centrifugation, and the supernatant was assayed for PAH by methods described previously (Kluwe and Hook, 1977). with the exception that acidified samples were placed in boiling water for 1 hr to hydrolyze any acetylated product to PAH. Ability of renal cortical tissue to accumulate PAH against a concentration gradient can be expressed as a slice to medium ratio (S/M) where S = milligrams of PAH per gram of tissue and h4 = milligrams of PAH per milliliter of medium. The percentage decrease in PAH S/M was determined using the following formula (Watrous and Plaa, 1972a):
Percentage decrease =
Control PAH S/M - Treated PAH S/M Control PAH S/M - 1
The blood samples were allowed to clot for 60 min on ice and then were gently centrifuged. and the serum was withdrawn and refrigerated. Blood urea nitrogen (BUN) concentrations were determined with Sigma reagents (Sigma Chemical Co., St. Louis, MO.), and serum glutamic-oxaloacetic transaminase (SGOT) activities were determined by the method of Reitman and Frankel(l957). LethaliQv studies. Mice ingesting diets supplemented with 0 or 100 ppm of PBB for 14 days were administered various doses of chloroform (0.20-1.80 ml/kg) ip in corn oil in a total volume of 5 ml/kg. The number of deaths occurring within 24.48, 96, and 168 hr were recorded. Slafistics. Data were represented as the mean 5 SE of six animals and analyzed by blocked analysis of variance. Differences between means were compared using the least significant difference (LSD) test (Steel and Torrie, 1960) with p < 0.05 as the criterion of significance. The LD50 values were determined by the method of Litchfield and Wilcoxon ( 1949).
PBB AND
CHC&
863
TOXICITY
RESULTS Neither liver weight to body weight ratios (LW/BW) nor kidney weight to body weight ratios (KW/BW) were affected by CHCI, administration in animals consuming control diet (Table 1). Liver weights were greatly enhanced by dietary PBB. Animals consuming the diet containing 25 ppm of PBB exhibited LW/BW 119% that of control, while those consuming a lOO-ppm of PBB diet showed LW/BW 179% that of control (Table 1, column 1). KW/BW was not affected by dietary PBB. However, in animals consuming PBB, CHCl, administration resulted in an increased KW/BW (Table 1). The increase was apparent only at the higher doses of CHCl,. TABLE EFFECT OF DIETARY LIVER WEIGHT/BODY
POLYBROMINATED WEIGHT (LW/BW)
1
BIPHENYLS (PBB) AND SUBCUTANEOUS AND KIDNEY WEIGHT/BODY WEIGHT RATIOS”
CHCI, ON (KW/BW)
CHCl, Wkg) Diet Control 1 PPm 25 ppm 100 ppm
Control 1 pw 25 ppm 100ppm
0 5.97 6.10 6.91 9.68
0.50
& 0.23 6.40 & 0.04 + 0.19 6.35 k 0.37 k 0.31b 7.36 + O.lgb t 0.38* 10.02 k 0.35 b
1.54 + 0.05 1.49 f 0.04 1.47 & 0.03 1.58 & 0.04
1.73 & 0.03 1.50 & 0.06 1.44 k 0.07 1.39 & 0.04
2.50
5.0
LW/BW 5.93 + 0.30 6.17 IO.10 7.48 & 0.21b 9.74 k 0.30'
x 100 6.47 + 6.11 + 6.76 i 9.64 k
KW/BW
x 100
1.64 k 0.08 1.54 & 0.04 1.55 + 0.03 1.37 + 0.04
0.40 0.13 0.12 0.25b
1.68 & 0.05 1.47 5 0.04 1.46 f 0.05 1.42 k 0.06
“Animals were maintained on the above diets for 14 days prior CHCI,. Twenty-four hours after CHCI,, the animals were decapitated were weighed. Each value represents the mean (&SE) of six determinations. h Significantly greater than control,p < 0.05.
25.0 6.09 k 0.19 5.61 + 0.15 7.35 k 0.12b 10.14 f0.34b
1.63 k 0.08 1.46 & 0.08 1.76 ? 0.03 1.70 k 0.19
50.0 6.39 6.01 6.46 9.54
+ 0.13 5 0.17 & 0.12 + 0.23h
1.70 & 0.10 1.93 + 0.07h 2.12 & 0.04h 2.21 & O.Ogh
to a single administration ot and exsanguinated. and organs
Serum glutamic-oxaloacetic transaminase (SCOT) activities were elevated slightly by PBB ingestion at a dietary concentration of 100 ppm (Fig. 1). Lower dietary levels were not associated with signs of hepatotoxicity. Administration of CHCI, to mice consuming 0, 1, or 25 ppm of PBB did not result in liver damage as evidence by SGOT activities which were not significantly different from those of non-CHCI,-treated control animals. Mice receiving 100 ppm of PBB. however, responded to increasing doses of CHCl, with drastic elevations of SGOT activity (Fig. 1). In mice receiving the control diet, serum blood urea nitrogen (BUN) was increased only slightly by the highest dose of CHCI, used (Fig. 2). As dietary intake of PBB increased, however, elevation of BUN secondary to CHCI, administration (25 and 50 ,&kg) rose rapidly. The capability of renal cortical slices from control and PBB-treated animals receiving various doses of CHCl, to actively accumulate the organic anion p-aminohippurate
864
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*
1400
ClET CONTROL SCOT151.) c 242- 40 .-. 1PPrn 285 63 Q-0 25Pm7 173 21 ,-. ppm
27
100
#l-D
335
I
/
I
I
I
CHC13
1
25.0 50.0
2.5 5.0
0.5
+
Ilyn
d/kg
FIG. 1. Effect of dietary PBB and subcutaneous CHCI, on SCOT activity. Animals were maintained on 0-, 1., 25, or 100.ppm PBB-supplemented diets for 14 days prior to a single administration of CHCI,. The mice were decapitated 24 hr after CHCI, administration. blood was collected and allowed to clot for 1-2 hr, and glutamic-oxaloacetic transaminase (GOT) activity was determined in the serum fraction. An asterisk(*) denotes a signilicant increase in comparison to control (0 ppm of PBB), p < 0.05.
(PAH) against a concentration gradient is represented in Fig. 3. Animals on control diet exhibited a significant depression (approximately 30% decrease) in PAH S/M after receiving 25 @/kg of CHCl, (Fig. 3). Smaller doses of CHCI, had no effect in control mice. Dietary PBB, however, appeared to cause a PBB-intake-related increase in loo
-
0 c ippm 25ppm lOOppIn
EO-
s
P -5 i
.-. O-0 l -w n--o
0
.
/
60! 40
-
20
-
’ /“. “/.
.
:: A
e-----op. 1 0.5
I I 2.5 5.0 CHC$
1 1 25.0 50.0
1111 kg
FIG. 2. Effect of dietary PBB and subcutaneous CHCI, on BUN concentration (mg/lOO ml). Animals were maintained on 0-. l-. 2S-. or lOO-ppm PBB-supplemented diets for 14 days prior to a single administration of CHCI,. The mice were decapitated 24 hr after CHCI, administration, blood was collected and allowed to clot for 1-2 hr. and blood urea nitrogen (BUN) concentration was determined in the serum fraction.
PBB
AND
I 0.5
I
CHCl,
865
TOXICITY
I I 2.5 5.0 CHCI, til/kg
I I 25.0 50.0
FIG. 3. Effect of dietary PBB and subcutaneous CHCI, on PAH S/M. Animals were maintained on O-. I-, 25-, or 100.ppm PBB-supplemented diets for 14 days prior to a single administration of CHCI,. The mice were sacrificed 24 hr after CHCI, administration. and thin renal cortical slices were incubated in a phosphate-buffered medium containing p-aminohippurate (PAH). Accumulation of PAH against a concentration gradient was expressed as a PAH slice-to-medium ratio (S/M) where S = milligrams of PAH per gram of tissue and M = milligrams of PAH per milliliter of medium.
susceptibility to CHCl,-induced depression of PAH uptake. Significant decreases in PAW S/M were evident following administration of 0.5 PC/kg of CHCl, (100 ppm of PBB) and 5.0 pllkg of CHCl, (1 and 25 ppm of PBB). At a dose of 25 pllkg of CHCI, the decrease in PAH S/M was approximately 30. 45, 55, and 60% for dietary PBB concentrations of 0. 1, 25, and 100 ppm of PBB, respectively. Potentiation of CHCl, lethality by dietary PBB is shown in Table 2. Maximal lethality was achieved 96 hr after CHCl, administration: mice surviving this initial 4day period survived for at least an additional 2 weeks. Potentiation was perhaps most clearly evident after 0.5 ml/kg of CHCl,, a dose at which all control animals survived for at least 96 hr after CHCl, administration while all of the PBB-treated animals expired (data not shown). The 24-. 48., and 96-hr LD50 values were all reduced in PBB-treated mice (Table 2). The minimum lethal dose (the lowest dose of which at least TABLE EFFECT
LD50 24 hr 48 hr Y6 hr
OF
DIETARY
2
POLYBROMINATED ACUTE CHCI, LD50 Control0
1.28 (0.8-2.7) 1.00 (0.8-2.7) 1.00 (0.8-1.8)
BIPHEN~LS
PBB” 0.62 0.50 0.39
(0.5-1.2) (0.32-0.8) (0.32-0.5)
(PBB)
ON
ControllPBB” 2.08 2.00 2.56
U LD50 was determined by the method of Litchiield and Wilcoxon (1949). Numbers in parentheses represent the lowest dose at which any deaths occurred and the highest dose at which any animals survived. h Ratio of LD50 in control animals to LD50 in PBB-treated animals.
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one animal expired within 96 hr after CHCI, administration) in control mice was less than the maximum lethal dose (the lowest dose at which all exposed animals expired within 96 hr after CHCI, administration) in PBB-treated animals (Table 2).
DISCUSSION The short-chain chlorinated hydrocarbon solvents are thought to produce toxicosis, at least in part, through the actions of a toxic metabolic intermediate (Ilett et al., 1973). While the biotransformation of chemicals to hepatotoxins has been studied extensively (Mitchell et al., 1976), little attention has been given to the biotransformation of chemicals to nephrotoxins. Although CHCI, and Ccl, are decidely toxic to human kidneys (Von Oettingen, 1964), their hepatic effects are much more prominent than their renal effects in the commonly used laboratory rat. Thus, the male ICR mouse was chosen as a model to investigate CHCl, toxicity because of its sensitivity to both the renal and hepatic effects of CHCI,. CHCl,, like Ccl,, is metabolized to a toxic intermediate by MFOs. Sensitivity to CHCl, in mice is strain-dependent. C57BL/6J mice are relatively resistant to CHCI, nephrotoxicity, while DBA/U mice are more sensitive (Hill et al., 1975). The outbred strain used in these experiments (ICR) falls between the two extremes. CC],-induced hepatotoxicity is potentiated by induction of hepatic MFO systems with chemicals such as polychlorinated biphenyls (PCB) (Carlson. 1975a), phenobaribtal (Suarez et al., 1972), 3,4-benzpyrene (Pitchumoni et al., 1972), and other chemicals such as aliphatic alcohols (Traiger and Plaa, 1972; Hasamuro et al., 1974). Other MFO system inducers such as 3-methylcholanthrene (Suarez et al., 1972) or inhibitors such as methylmercury hydroxide (Carlson, 1975b) or dibenamine (Maling et al., 1974) decrease the hepatotoxicity of Ccl,. It appears, then, that multiple pathways exist for hepatic biotransformation of Ccl,, metabolites of some being more toxic than metabolites of others. Like Ccl,, CHCl, appears to be a substrate for several biotransformation pathways. End products of CHCl, metabolism include carbon dioxide (Butler. 1961), carbon monoxide (Ahmed et al., 1977), and perhaps a small amount of methylene chloride (Paul and Rubinstein, 1963). Studies based on in vitro tissue incubations have documented the ability of both renal and hepatic homogenates to metabolize CHCI, (Butler, 1961). Administration of phenobarbital or 3-methylcholanthrene increased the hepatotoxicity of CHCl, in rats (Lavigne and Marchand, 1974) while McMurty et al. (1976) have suggested recently that the nephrotoxicity of certain drugs could be reduced by the administration of chemicals which depressed hepatic and renal MFO activity. It seemed likely, then, that chemicals which stimulated MFO systems would enhance the nephrotoxicity of CHCl, while chemicals which inhibited MFO systems would reduce CHCl,-induced nephrotoxicity. Since PBB is a potent inducer of renal and hepatic MFOs it appeared that mice consuming PBB metabolized a greater proportion of the administered dose of CHCl, to the ultimate toxicant. Results from BUN and SCOT determinations and KW/BW all suggested that PBB enhanced biotransformation of CHCl, to a toxic metabolite in both liver and kidney. Inhibition of renal slice uptake of PAH has been shown to parallel CHCl, nephrotoxicity in rodents (Watrous and Plaa, 1972a). PBB ingestion enhanced CHCl, nephrotoxicity as evidenced by potentiation of the CHCl,-induced decrease in PAH S/M. Furthermore,
PBB
AND
CHCl,
TOXICITY
867
the degree of enhancement of the CHCl,-induced decrease in renal transport capacity was directly proportional to the dietary intake of PBB. While renal tissue contains the enzymatic apparatus to metabolize CHCI, (Butler 1961), results from the experiments reported here cannot rule out the liver as the site of formation of the nephrotoxic metabolite. However, covalent binding of a CHCI, metabolite to hepatic and renal microsomal protein appears to parallel CHCl, toxicity (Brown et a/., 1974). The chemical reactivity of a CHCI, metabolite which could alkylate membrane proteins and lipids would be expected to be too great to allow transport, intact, via the hepatic vein and through the arterial system to the kidney. PBB administration also increased the lethality of CHCI, as evidenced by a reduction in the acute LD50. More striking, perhaps, are the data showing that 96 hr after administration of 0.5 ml/kg of CHCI,, all of the PBB animals died while all of the control animals survived. The cause of death in the LD50 experiments does not appear to be renal in origin. The nephrotoxic effects of CHCI, on PAH transport, most likely mediated by tubular necrosis, appear to be maximal 24 hr after administration of CHCl, and nearly absent by 96 hr (Watrous and Plaa. 1972b). In addition, a prominent sex difference exists in susceptibility of ICR mice to the nephrotoxic effects of CHCI,, but no sex difference exists for acute LD50 (Klaassen and Plaa, 1967). It had been previously suggested that death was due to severe narcosis (Klaassen and Plaa, 1966). but in these experiments all animals regained consciousness within a few hours after CHCI,. Death ensued long after the anesthetic effects of CHCl, had ended. Since the LD50 was reduced in PBB-treated mice at a time when liver and kidney capacity for biotransformation was increased it appears that generation of a lethal metabolite resulted from biotransformation of CHCI,. In summary, dietary intake of PBB for 14 days greatly enhanced the nephrotoxicity and hepatotoxicity of CHCl, in male mice. Since PBB is a potent inducer of hepatic and renal MFO systems, and CHCI, is believed to exert its toxic effects through a biotransformation product, these experiments suggest that dietary consumption of PBB greatly increased CHCI, toxicity by stimulating the biotransformation pathway leading to formation of a toxic CHCl, metabolite. PBB is only one of an ever-increasing group of halogenated chemicals used extensively by industry and agriculture which have found their way into the environment. Several of these chemicals, like PBB, are inducers of MFOs, are highly stable in the environment, and display long tissue half-lifes, once ingested, due to their highly lipophilic nature. Investigations into the toxicity of these chemicals has generally concentrated on direct toxic effects (tissue hyperplasia, necrosis, nervous disorders). Outside of the laboratory, though, humans and animals are exposed to a number of potential toxicants. Some, like CHCl, and Ccl,, are metabolized to toxicants while others, like PBB, can be indirectly toxic by increasing the rate at which chemicals like CHCl, and Ccl, are metabolized to toxicants. Thus, the principal hazard of exposure of humans and livestock to low concentrations of PBB may be increased susceptibility to damage from other toxicants. CHCl,, for example, is a common contaminant of surface waters and is ingested daily in what are normally subtoxic quantities. To victims of PBB exposure, however, the intake of CHCl, that is needed to produce damage may be greatly reduced, as shown for the mouse. Thus, the toxicity testing of chemicals which induce microsomal enzyme activities and may become contaminants of the environment should include an evaluation of their effects on the biotransformation of other common chemicals to toxicants.
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ACKNOWLEDGMENTS The expert technical gratefully acknowledged.
assistance of Ms. Cathy Herrmann
and Ms. Harriett
Sherman
are
REFERENCES AHMED, A. E., KUBIC, V. L., AND ANDERS, M. W. (1977). Metabolism of haloforms to carbon monoxide. I. In vitro studies. Drug Metab. Dispos. 5, 198-204. BROWN, B. R., JR., SIPES, I. G., AND SAGALYN, A. M. (1974). Mechanism of acute hepatic toxicity: Chloroform, halothane and glutathione. Anesthesiologq’ 41, 554-56 1. BUTLER, T. C. (196 1). Reduction of carbon tetrachloride in vivo and reduction of carbon tetrachloride and chloroform in vitro by tissues and tissue constituents. J. Phannacol. E,rp. Ther. 134.31 I-319. CARLSON, G. P. (1975a). Potentiation of carbon tetrachloride hepatotoxicity in rats by pretreatment with polychlorinated biphenyls. Toxicology 5, 69-77. CARLSON, G. P. (1975b). Protection against carbon tetrachloride induced hepatotoxicity with methylmercury hydroxide. Toxicology 4, 83-89. CHEN, E. (1977). Michigan: If something odd happens.. . . Atlantic Monthly 240, 2-20. CORBETT, T. H., BEAUDOIN. A. R., CORNELL, R. G., ANVER, M. R., SCHUMACHER. R., ENDRES, J.. AND SZWABOWSKA, M. (1975). Toxicity of polybrominated biphenyls (Firemaster BP61 in rodents. Environ. Res. 10, 390-396. CROSS, R. J., AND TAGGART, J. V. (1950). Renal tubular transport: Accumulation of p-atninohippurate by rabbit kidney slices. Amer. J. Phqdol. 161, 181-190. DENT, J. G., NETTER, K. J., AND GIBSON, J. E. (1976). The induction of hepatic microsomal metabolism in rats following acute administration of a mixture of polybrominated biphenyls. Toxicol. Appl. Pharrnacol. 38.237-249. HASUMURA, Y., TESCHKE, R., AND LIEBER, C. (1974). Increased carbon tetrachloride hepatotoxicity, and its mechanism, after chronic ethanol consumption. Gastroenterologbl 66, 415-422. HILL, R. N., CLEMENS, T. L., LIU, D. K., AND VESELL. E. S. (1975). Genetic control of chloroform toxicity in mice. Science 190, 159-160. ILETT, K. F., REID, W. D., SIPES, I. G., AND KRISHNA, G. (1973). Chloroform toxicity in mice: Correlation of renal and hepatic necrosis with covalent binding of metabolites to tissue macromolecules. Exp. Mol. Pathol. 19, 215-229. KLAASSEN. C. D., AND PLAA, G. L. (1966). Relative effects of various chlorinated hydrocarbons on liver and kidney function in mice. Toxicol. Appl. Pharmacol. 9, 139- 15 1. KLAASSEN, C. D., AND PLAA, G. T. (1967). Susceptibility of male and female mice to the nephrotoxic and hepatotoxic properties of chlorinated hydrocarbons. Proc. Sot. Exp. Biol. Med. 124, 1163-I 166. KLUWE, W. M., AND HOOK, J. B. (1977). Analysis of gentamicin uptake by rat renal cortical slices. Toxicol. Appl. Pharmacol., in press. LAVIGNE, J. G.. AND MARCHAND, C. (1974). The role of metabolism in chloroform hepatoxicity. Toxicol. Appl. Pharmacol. 29,3 12-326. LITCHFIELD, J. T., AND WILCOXON, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96,99-l 13. MALING, H. M., EICHELBAUM, F. M., SAUL, W., SIPES. I. G., BROWN, E. A. B., AND GILLETTE, J. R. (1974). Nature of the protection against carbon tetrachloride-induced hepatotoxicity produced by treatment with dibenamine. Biochem. Pharmacol. 23, 1479-149 1. MCCORMACK, K. M., CAGEN, S. Z., RICKERT, D. E., GIBSON, J. E., AND DENT, J. G. (1977a). Stimulation of hepatic and renal mixed-function oxygenases in developing rats by polybrominated biphenyls. Drug. Merab. Dispos., in press. MCCORMACK, K. M., KLUWE, W. M., RICKEIT, D. E., SANGER, V. L., AND HOOK, J. B. (1977b). Renal and hepatic microsomal enzyme stimulation and renal function following
PBB
AND
CHCI,
TOXICITY
864,
three month dietary exposure to polybrominated biphenyls. Toxicol. Appl. Pharmacol.. in press. MCMURTY. R. J.. SNODGRASS, W. R., AND MITCHELL, J. R. (1976). Metabolic activation of acetaminophen. cephaloridine, and other chemically stable compounds to nephrotoxic metabolites. Clin. Res. 24,407A. MERCER, H. D., TESKE, R. H., CONDON. R. J., FURR. A., MEERDINK, G., BUCK, W.. AND FRIES. G. (1976). Herd health status of animals exposed to polybrominated biphenyls. J. Tosicol. Ewirou. Health 2. 335-349. MITCHELL, J., NELSON, S. D.. AND THORGEIRSSON. S. S. (1976). Metabolic activation. Biochemical basis for many drug-induced liver injuries. In Progress in Liwr Disease (H. Popper and F. Schaffner, eds.), Vol. 15, Chap. 16. Grune and Stratton. New York. MOORHEAD, P. D.. WILLETT. L. B., BRUMM. C. J.. AND MERCER, H. D. (1977). Pathology of experimentally induced polybrominated biphenyl toxicosis in pregnant heifers. J. Amer. l,ef. Med.Assoc. 170.307-313. PITCHUMONI, C. S., STENGER, R. J., ROSENTHAL, W. S., AND JOHNSON. E. A. (1972). Effects of 3,4-benzpyrene pretreatment on the hepatotoxicity of carbon tetrachloride in rats. J. Pharmacol. Exp. Ther. 181,221-233. PAUL, B. P.. AND RUBINSTEIN, D. (1963). Metabolism of carbon tetrachloride and chloroform by the rat. J. Pharrnacol. Exp. Ther. 141, 141-148. REITMAN. S., AND FRANKEL, S. (1957). A calorimetric method for the determination of serum glutamic-oxaloacetic and glutamic-pyruvic transaminases. Amer. J. Clin. Pufhol. 28, 56-7 1. STEEL, R. G. D.. AND TORRIE, J. H. (1960). Principles and Procedures of Statistics, p. 67. McGraw-Hill, New York. SUAREZ. K. A.. CARLSON, G. P., FULLER, G. C., AND FAUSTO. N. (1972). Differential acute effects of phenobarbital and 3-methylcholanthrene pretreatment on Ccl,-induced hepatotoxicity in rats. Tosirol. Appi. Pharmacol. 23, 17 1-I 77. TRAIGER, G. J., AND PLAA, G. L. (1972). Relationship of alcohol metabolism to the potentiation of Ccl, hepatotoxicity induced by aliphatic alcohols. J. Pharmacol. Exp. Ther. 183,48 l-488. VON OETTINGEN, W. F. (1964). Halogenated Hydrocarbons of Industrial and Toxicological Importance, p. 77. Elsevier, Amsterdam. WATROUS, W. M.. AND PLAA, G. L. (1972a). Effect of halogenated hydrocarbons on organic ion accumulation by renal cortical slices of rats and mice. Toxicol. Appl. Pharnzacol 22. 52% 543. WATROUS,
W. M.,
649.
G. L. (1972b). The nephrotoxicity of single and multiple doses of hydrocarbon solvents in male mice. Toxicol. Appl. Pharrnacol. 23. 640-
AND PLAA,
aliphatic chlorinated