Hepatic and renal nonprotein sulfhydryl concentration following toxic doses of hexachloro-1,3-butadiene in the rat: The effect of Aroclor 1254, phenobarbitone, or SKF 525A treatment

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

57, 79-87 (1981)

Hepatic and Renal Nonprotein Sulfhydryl Concentration following Toxic Doses of Hexachloro-1,3-butadiene in the Rat: The Effect of Aroclor 1254, Phenobarbitone, or SKF 525A Treatment1 EDWARD A. LOCKANDJOHN ISHMAEL Biochemical

Mechanisms and Pathology Sections, Imperial Chemical Industries Central Toxicology Laboratory, Alderley Park, Nr Macclesfield. Cheshire SKI0 4TJ. United Kingdom

Received

June 24, 1980: accepted

September

Limited,

10. 1980

Hepatic and Renal Nonprotein Sulthydryl Concentration following Toxic Doses of Hexachloro-1,3-butadiene in the Rat: The Effect of Aroclor 1254, Phenobarbitone, or SKF 525A Treatment. LOCK, E. A., AND ISHMAEL, J. (1981). Toxicol. Appl. Pharmacol. 57, 79-87. A single ip administration of hexachloro-l,3-butadiene (HCBD) to male rats at 300 m&kg produced a marked increase in liver and kidney water content, and organ-to-body weight ratio, over 24 hr. In the same animals the total nonprotein sulfhydryl content (NP-SH) of the liver was decreased, the nadir being a 60% reduction at 6 hr, whereas renal NP-SH remained unchanged. By 24 hr marked necrosis of the straight portion of the proximal renal tubules was seen, whereas the liver showed slightly fatty changes when examined by light microscopy. Increasing doses of HCBD up to 900 mg/kg ip produced a dose-related decrease in liver NP-SH, with no alteration in renal NP-SH. Treatment of rats with Aroclor 1254 prior to HCBD administration appeared to produce a small increase in the nephrotoxicity of HCBD without producing any liver changes, whereas treatment with either phenobarbitone or SKF 525A prior to HCBD administration did not modify the toxicity. It is suggested that in the kidney HCBD may be activated via a different metabolic pathway than in the liver and that this metabolism may be enhanced by Aroclor 1254 treatment.

Hexachloro-1,3-butadiene (HCBD) is a byproduct in the manufacture of perchloroethylene synthesized by the chlorination of hydrocarbons. In the male rat, the LD50 is about 250 mg/kg when given orally and 200 mg/kg when given ip (Gradiski et al ., 1975). The kidney appears to be the major target organ where HCBD produces necrosis of the proximal tubules (Murzakaev, 1967; Gradiski et al., 1975; Kociba et al., 1977; Lock and Ishmael, 1979; Bemdt and Mehendale, 1979). Renal necrosis was also seen

following inhalation exposure (Gage, 1970), dermal application (Duprat and Gradiski, 1978), and following 90-day feeding studies in rats at 150 and 450 ppm in the diet (Harleman and Seinen, 1979). Several halogenated hydrocarbons are known to produce liver and/or kidney necrosis with an associated depletion of liver nonprotein sulfhydryl content (NP-SH), e.g., vinyl chloride (Hefner et al. , 1975); vinylidene chloride (Jaeger et al., 1974); and chloroform (Johnson, 1965; Brown et al., 1974). The degree of organ toxicity and depletion of NP-SH produced by these compounds can be altered by treatment with inducers or inhibitors of xenobiotic mixed-function

* Presented in part at the Eighteenth and Nineteenth Annual Meeting of Society of Toxicology in New Orleans, La., March ll- 15, 1979, and Washington D.C., March 9-13, 1980. 79

0041-008X/81/010079-09$02.00/0 CopyrIght b 1981 by Academic Press, Inc. All rights of reproductmn in any form reserved.

80

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AND

oxidase activities (Jaeger et al., 1976; Ilett et ul., 1976; Kluwe et al., 1978). In this study we have examined (1) the effect of HCBD on liver and kidney NP-SH and (2) the effect of various treatments known to alter mixed-function oxidase activities on HCBD-induced organ necrosis. METHODS Trecltment ofanimals. Male, Alderley Park (Wistarderived) albino rats were maintained in an air-conditioned animal room with a l2-hr light-dark cycle at an ambient temperature of 22 t 1°C and relative humidity of 56 + 7%. Rats weighing between 160 and 200 g were fasted for 16 hr prior to dosing and for the entire experimental period thereafter, except where otherwise stated. Water was available ad lihirum. HexachloroI $butadiene’ (HCBD) was administered ip, at doses ranging from 10 to 900 mg/kg in corn oil at 5 ml/kg. Control animals were fasted for the same period of time as treated animals and dosed ip with corn oil at 5 ml/kg. Drugs used for treatments were injected according to the following schedules: phenobarbitone (PB), 75 mgikgiday, ip, for 3 days in saline at 5 ml/kg. the animals then being fasted on Day 3 prior to dosing, with HCBD; polychlorinated biphenyl, Aroclor 12543 (A-12541, 500 mgikg, ip, at 2.5 ml/kg in corn oil, the animals being left 5 days and then fasted on Day 5 prior to dosing with HCBD; SKF 525A4, 75 mg/kg, SC, at 5 ml/kg in saline 1 h prior to dosing with HCBD. In studies with SKF 525A which continued for 24 hr. rats were given a second booster dose about 6.5 hr after HCBD administration. Control animals for these treatments were given, PB. SKF 525A. or A-1254 followed by corn oil at 5 ml/kg, ip. For NP-SH measurements animals were dosed between 8 AM and IO AM. with three or four corn oil-treated and four to six HCBD-treated animals killed at each time point. With fasted rats we observed no significant diurnal variation in liver or kidney NP-SH. Results are expressed as a percentage of the control carried out at the same time. Erperimenrtrl procedure. At the required time after dosing each animal was killed by decapitation and blood collected from the trunk into beakers containing heparin. The liver was removed. the total wet weight determined, and portions (approximately 1 g) used for Z British Drug Houses, Poole, Dorset. U.K., >9970 purity. ‘$ A gift from Monsanto Company, St. Louis, MO. ’ 2-Dimethylaminoethyl-2-diphenylpropylacetate, hydrochloride salt, a gift from Smith, Kline and French Limited. Welwyn Garden City. Her&, U.K.

ISHMAEL the determination of water content, NP-SH content, and histological assessment. The wet weight of the kidneys was determined after removal of the capsule and extraneous fat. A portion of the left kidney was used for water content determination and the remainder for histological assessment. The right kidney was used for NP-SH determination. Analyrical methods. Water content was determined as weight loss on drying to constant weight at 105°C. Nonprotein sulfhydryl content was determined by the method of Beutler et (II. (1%3) on tissue extracts prepared in buffered ethanol (Johnson, 1966). For the measurement of oxidized glutathione, 3 ml of liver homogenate (15’?G, w/v) in 0.1 M sodium phosphate buffer, pH 7.4, was incubated with glutathione reductase (25 units/g liver protein) and NADPH I I mg) at 37°C for IO min to reduce the oxidized glutathione. The reaction was then stopped by the addition of sulfosalicylic acid (4%. w/v) and after ccntrifugation, 1 ml of supernatant was assayed for NP-SH. A sample of liver homogenate containing NADPH hut no added glutathione reductase was carried thr-ough the procedure and the difference between the two measurements (i.e., with and without added glutathione reductase) was used to estimate the concentration ofoxidized glutathione in the liver homogenate. Plasma urea was determined by the method of Marsh c’t cii. (1965). Alkaline phosphatase (EC 3. I .3. I.) and alanine aminotransferase (EC 2.6.1.2.) were determined using a Boehringer test kit with a Vitraton AKES analyzer on samples of plasma. Histopnthology. A portion of liver and kidney was fixed in 10% formal saline and paraffin sections (5 pm) were prepared and stained with hematoxylin and eosin. Stcltistic~.r. Statistical analysis between control and treated means were determined by Student’s I test. and p values ~0.05 considered signific.ant.

RESULTS The Effkt of HCBD on Lilvr clnci Kidnr> Water Content und Orgun-to-B&?7 Weight Ratio in Normal, A-12.54-, PB-. und SKF 525A-Treated Rots

A single administration of HCBD (300 mg/kg, ip) produced. within 24 hr. a significant increase in liver and kidney:body weight ratios (Table 1). which was associated with an increase in tissue water content (Table 1). The increase in kidney water was significant by 6 hr and appeared to reach a

HEXACHLOROBUTADIENE

81

NEPHROTOXICITY

TABLE

1

THE EFFECT OF HCBD (300 mg/kg, ip) ON LIVER OR KIDNEY ORGAN-TO-BODY WEIGHT RATIOS AND WATER CONTENTS IN RATS TREATED WITH PHENOBARBITONE, AROCLOR 1254, SKF 525A, OR CORN OIL Liver Treatment” Corn oil Corn oil + HCBD PB PB + HCBD A-1254 A-1254 + HCBD SKF 525A SKF 525A + HCBD

Organ/body weight x 100 3.08 4.50 3.97 4.99 5.91 7.28 3.52 4.41

2 f k * ? 2 + +

0.04” 0.07’ 0.06 0.10’ 0.36 0.51c 0.09 0.08’

Kidney Water (g &Ok dry wt) 2.28 2.79 2.29 2.59 2.25 2.55 2.28 2.71

+ 2 k k k k f +

0.02 0.04’ 0.03 0.03’ 0.64 0.08’ 0.03 0.04’

Organ/body weight x 100 0.42 0.55 0.40 0.52 0.42 0.56 0.41 0.53

+ c * 2 5 2 5 2

Water (g HZO/g dry wt)

0.01 0.01’ 0.01 0.01’ 0.02 0.01’ 0.01 0.01’

3.56 4.32 3.51 4.39 3.34 4.40 3.40 4.33

” k + k + f 2 +

0.01 0.06c 0.03 0.15’ 0.04 O.OY 0.03 0.08’

n Rats were treated with PB, A-1254, or SKF 525A (as described under Methods), and then dosed with HCBD in corn oil at 5 ml/kg body wt, or just corn oil alone and then killed 24 hr after this injection. h Values are expressed as means ? SE with at least five observations per group. c Significantly different from animals given similar treatment but no HCBD.

plateau by 16 hr, when there was a 29% increase in kidney water (Fig. 1A). Rats treated with PB, A-1254, or SKF 525A alone showed an increased 1iver:body weight ratio compared to corn oil-treated animals, whereas the ratio of liver water: dry weight did not change (Table 1). In these same animals the kidney:body weight ratio and the ratio of kidney water:dry weight did not change when compared to animals given no treatment (Table 1). Administration of HCBD (300 mg/kg, ip) to rats treated with PB, A-1254, or SKF 525A produced by 24 hr a significant increase in 1iver:body weight ratio and in the ratio of liver water:dry weight compared with animals given the treatment but no HCBD (Table 1). There was also a significant increase in kidney:body weight ratio and in the ratio of kidney water:dry weight compared with animals given the treatment but no HCBD (Table 1). The time course of water accumulation in the kidney following HCBD was not altered by treatment with PB or Al254 (Fig. 1A). With increasing concentrations of HCBD kidney water content increased (Fig. 1B). Treatment with A-1254 but not

PB followed by increasing concentrations of HCBD produced a significant increase in kidney water (Fig. 1B). Hexachloro-I Damage

,3-butadiene-Induced

Kidney

Renal tubular necrosis involving the outer stripe of the outer medulla was present in all animals given 300, 200, or 100 mg/kg HCBD at 24 hr. Glomeruli were unaffected. The extent of necrosis was assessed by histological examination of one kidney section, scored on a I+ to 4+ basis (Table 2). At 50 mg/kg 6 out of 13 animals showed minimal necrosis involving isolated straight proximal tubules, while no damage was seen in the remaining 7 animals. At 20 mg/kg none of the 13 animals examined showed necrosis (Table 2). Histological assessment indicated that A-1254 treatment had slightly enhanced the nephrotoxicity of HCBD (Table 2). Whereas treatment with PB or SKF 525A did not appear consistently to alter the incidence or severity of HCBDinduced renal necrosis (Table 2). The only alteration seen in the morphology of the

82

LOCK AND ISHMAEL

-rl-rrr

alanine aminotransferase (Table 3). Administration of HCBD to rats treated with PB or SKF 525A did not appear to alter this response, whereas HCBD administration to animals given A-1254 produced a significant increase in plasma urea, above that produced by HCBD alone (Table 3) without altering plasma alkaline phosphatase or alanine aminotransferase activity. Treatment of animals with A-1254 followed by increasing concentrations of HCBD produced a dose-related increase in plasma urea above that seen for HCBD administration alone (Fig. 2).

24 Tlmelhrl

TABLE THE

EFFECT

NECROSIS BY 24 hr

I M

I I I 100 150 al0 Dose of HCBD Imqlkg,

I

250

OF VARIOUS

PRODUCED

2 TREATMENTS

BY

HCBD

ON RENAL

I\

1.11

RAT

1 m

FIG. 1. Kidney water content in corn oil-, phenobarbitone-. and Aroclor-1254-treated rats, at various times after HCBD, 300 mgkg, ip, and 24 hr after varying doses of HCBD, ip. Results are mean & SE of the appropriate control, with at least four measurements per time point. Corn oil + HCBD (0). PB + HCBD (A), A-1254 + HCBD (m). The control values for kidney water content expressed as (g H,O/g dry wt) were: corn oil, 3.53 + 0.02 (66); PB, 3.45 i 0.02 (22): A-1254, 3.42 it 0.04 (19).

liver at the light microscopic level was a slight fatty change in some of the rats given 300 mgkg. However, the livers of all A- 1254treated animals showed some vacuolation of the hepatocytes and some small localized areas of focal necrosis, and HCBD did not alter the incidence of this. Treatment of animals with SKF 525A or PB alone produced no specific changes in the liver or kidney. Associated with the renal necrosis, HCBD produced by 24 hr a significant increase in plasma urea and plasma alkaline phosphatase with no change in plasma

PB

A- I:54

3w 200 IO0 50 20 IO 0

1 7 6 I? I2 3 9

7 IO 4 9

4 I

4 I I

6 ?

7 I

” Treatment regimes are given under Methods. ’ The extent of renal necr~s,s was scored by the f~llowmg ~:ntena. 0. absent: I+. minimal mvolvmg isolated straight proxmx~l tubules: ?+: moderate. mvolvingseveral straight proxtmal tubule-:? * extensive, seen as a dlstmct band of damage m the outer stripe uf thr wter medulla with some tubular cysts: 4+. severe. a more dtffuse band of damage mvolving the outer medulla and mner cortex wth many tubular cysts. ’ One ammal died m this group.

HEXACHLOROBUTADIENE

Effect of HCBD on Kidney and Liver Nonprotein Sulfydryl Content

HCBD (300 mg/kg, ip) decreased the concentration of liver NP-SH (mainly reduced glutathione) such that it reached a nadir of about 40% of control by 6 hr (Fig. 3). It then gradually increased approaching control values by 16-24 hr and was about 140% of control by 48 hr (Fig. 3). The observed decrease was not due to the oxidation of glutathione to its disfulfide dimer, since oxidized glutathione accounted for less than 5% of the total glutathione in the livers of control and HCBD treated rats 4 hr after dosing (data not shown). In contrast, the renal NP-SH content gradually increased from about 6 hr, until by 48 hr it was 250% of control (Fig. 4). Increasing concentrations of HCBD up to 900 mg/kg produced by 4 hr a dose-related decrease in TABLE

3

THE EFFECT OF HCBD (300 mgikg, ip) ON PLASMA UREA, PLASMA ALANINE AMINOTRANSFERASE, AND PLASMA ALKALINE PHOSPHATASE IN RATS TREATED WITH PHENOBARBITONE AROCLOR 1254, SKF 525A. OR CORN OIL

Treatment” Corn oil (9) Corn oil + HCBD (18) PB (6) PB + HCBD (4) A-1254 (15) A- I254 + HCBD 17) SKF 525A (6) SKF 525A + HCBD (8)

Alanine aminotransferase (W/ml)

Urea (ItIM) 6.7 k 0.5”

Alkaline phosphatase (NJ/ml)

II 2 I

204 i-

7

1.4’ 0.5 1.2’ 1.5

13 k I 11 t 3 922 9% I

339 i218 2 311 +1932

14’ 14 17’ 8

38.8 k 5.9’ 6.4 2 0.2

921 11 f I

309 f 12’ 235 2 19

23.2 -c 1.5’

10 -’ 1

403 i 21”

24.7 7.4 25.7 8.2

t 2 2 +

” Rats were treated with PB. A-1254, or SKF 525A as described under Methods and then dosed with HCBD in corn oil at 5 ml/kg body wt. or just corn oil alone and then killed 24 hr after this injection. b Values are expressed as means ? SE. with the number of observations in parentheses. r Significantly different from animals given similar treatment but no HCBD.

83

NEPHROTOXICITY

""1

HCBD (mgikgl

FIG. 2. The effect of varying doses of HCBD on plasma urea in corn oil-, phenobarbitone-, and Aroclor1254-treated rats, 24 hr after ip administration. Results are mean i- SE with at least three and usually six animals per time point. Corn oil + HCBD (a), PB + HCBD (A), A-1254 + HCBD (m).

liver NP-SH, the maximum effect occuring at about 750 mg/kg; no effect being seen at 150 mg/kg (Fig. 5). However, no doserelated decrease in renal NP-SH content was seen (Fig. 5). Dissection of the kidney to remove the papilla and outer cortex, to enable measurement of NP-SH on the damaged portion of the kidney, again showed no change l-4 hr after 900 mg/kg HCBD, e.g., control (3 hr) 2.51 2 0.23 pmol/g wet weight compared with HCBD (3 hr) 3.00 & 0.15 pmollg wet weight. The Effect of HCBD on Liver and Kidney Nonprotein Sulfydryl Content in Rats Treated with A-1254, PB, or SKF 525A

Rats treated with PB or Al254 but not SKF 525, alone, showed a marked increase in liver NP-SH concentration compared to corn oil-treated animals (legend to Fig. 3). In these same animals the kidney NP-SH concentration did not change when compared to animals given no treatment (legend to Fig. 4). Treatment of animals with A-1254 or PB, followed by HCBD (300 mglkg, ip), produced by 6 hr only a 25 and 40% deple-

84

LOCK AND ISHMAEL

FIG. 3. Liver nonprotein sulfydryl content in corn oil-. SKF 525A-, phenobarbitone-, and Aroclor-1254treated rats at various times after HCBD, 300 mg/kg, ip. Results are means ? SE expressed as a percentage of the appropriate control carrier out at the same time. with at least four animals per time point. Corn oil + HCBD (0). PB + HCBD (A), A-1254 + HCBD (W), SKF 525A + HCBD (A). The mean ? SE of all the control values for liver NP-SH expressed as pmol/g wet wt were: corn oil. 4.73 it 0.11 (43); PB, 6.17 t 0.10 (22); A-1254, 6.97 k 0.19 (16); SKF 525A. 4.76 t 0.23 (12).

there was a marked swelling of the kidney and an increase in plasma urea and plasma alkaline phosphatase activity, the earliest indication of damage being an increase in renal water content 6 hr after dosing (Fig. 1A). The necrosis produced by HCBD was located primarily in the outer stripe of the outer medulla but after large doses there was some extension into the cortex. The reason for the location of the damage at the cortico-medullary junction may be that compounds are concentrated in this region prior to their excretion or reabsorption and those cells may be exposed therefore to high concentrations of HCBD or its metabolites. Alternatively cells in this region are known to have mixed-function oxidase activity. which can be increased by 3-methylcholanthrene (Wattenberg and Leong, 1962; Zenser et ul.. 1978) or 2,3,7,%tetrachlorodibenzodioxin treatment (Fowler et al.. 1977) and it may be

"1 tion in liver NP-SH, respectively (Table 3). Whereas treatment with SKF 525A did not appear to markedly alter liver NP-SH depletion produced by HCBD (Fig. 3). In the kidney SKF 525A treatment, followed by HCBD produced a marked increase in renal NP-SH at early times, whereas PB and A-1254 treatment did not alter the response of the kidney NP-SH to HCBD (Fig. 4).

?

DISCUSSION This study confirms earlier observations (Murzakaev, 1967; Gradiski et al., 1975; Lock and Ishmael, 1979; Berndt and Mehendale, 1979) that HCBD produces a dosedependent renal tubular necrosis in the rat. In this series of experiments we observed minimal tubular necrosis in some animals following 50 mg/kg HCBD, whereas in a previous study we had not (Lock and Ishmael, 1979). Associated with the renal necrosis,

FIG. 4. Kidney nonprotein sulfydryl content in corn oil-. SKF 525A-. phenobarbitone-. and Aroclor-1254treated rats at various times after HCBD, 300 mgikg, ip. Results are means + SE expressed as a percentage of the apropriate control carried out at the same time with at least four animals per time point. Corn oil + HCBD (0) PB + HCBD (A). A-1254 + HCBD (m), SKF 525A + HCBD (A). The mean 2 SE of all the control values for kidney NP-SH expressed as gmolig wet wt were: corn oil. 2.66 2 0.06(44); PB. 2.56 2 0.09(18): A-1254.2.69 i 0.07(16): SKF 525A, 2.56 ? 0.10 (I?).

HEXACHLOROBUTADIENE

2

Control

: 5

2 c”

I 150

1 3w

I 450

I 600

I 759

1 WXJ

I 150

I 3w

I 450

I 600

I 750

do0

-Liver

HCBDlmqikql

FIG. 5. The effect of varying doses of HCBD on nonprotein sulfydryl content in rat kidney and liver. Results are means ? SE with at least three animals per time point.

that HCBD is metabolically activated in this region. HCBD caused swelling of the liver. The increase in water occurring to a similar extent in noninduced and induced rat livers (Table l), with no obvious morphological alterations at the light microscopic level. The reason for the rapid swelling of the liver is not known. Other workers have reported changes in the liver after HCBD administration. When dosed at 15.6 mg/kg/day for 13 weeks it increased liver: body weight ratios in male rats, the liver having increased basophilic, flocky granulation on histological examination (Harleman and Seinen, 1979). In a 2-year chronic feeding study an increase in the urinary excretion of coproporphyrin was seen at the two highest doses of HCBD, suggesting some alteration in heme synthesis in the liver (Kociba et al., 1977). Little information is available on the metabolic fate of HCBD in mammalian systems. This study shows that HCBD administration depletes liver but not kidney

NEPHROTOXICITY

85

NP-SH (mainly glutathione). It is known that glutathione is used in the conjugation of foreign compounds that are then excreted as mercapturic acids (Boyland and Chasseaud, 1969). In addition and perhaps more importantly, the role of glutathione in the body is to protect vital nucleophilic sites in hepatocytes, renal tubular cells (and presumably other tissues) from electrophilic attack by alkylating metabolites of drugs as shown for acetaminophen (Mitchell, 1973: McMurty et al., 1978) and bromobenzene (Jollow et al., 1974). The differences observed in NP-SH depletion between the liver and kidney produced by HCBD may be due to several factors. 1. The glutathione S-transferase for which HCBD is a substrate in the liver may not be present in the kidney. There are several differences in the composition of the various glutathione S-transferases present in rat liver and kidney (Hales, et al., 1978). 2. The kidney cells in which HCBD is concentrated may not be those which contain the glutathione S-transferase for which HCBD is a substrate. 3. HCBD might be metabolically activated in both the liver and kidney, but the metabolic route in the liver may be mainly via a detoxification pathway using glutathione, whereas in the kidney it may be via a toxification route with little if any glutathione conjugation. HCBD administration to rats treated with A-1254, an agent reported to increase both liver and kidney mixed-function oxidase activities (McCormack et al., 1979) produced a smaller decrease in liver NP-SH than that seen with HCBD alone, and also enhanced the renal toxicity. This small enhancement of nephrotoxicity (about 1.5 fold), was seen as a shift in the HCBD doseresponse curve for kidney water (Fig. 1B) and plasma urea (Fig. 2). In agreement with these findings, histopathology showed a small increase in the severity of kidney damage produced by HCBD in A-1254-

86

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treated rats compared with rats treated with HCBD alone (Table 2). HCBD administration to rats treated with either PB, an agent known to increase the liver but not the kidney mixed-function oxidases (Uehleke and Greim. 1968: Jakobsson er al., 1970; Lake et a/. , 1973); or SKF 525A, an inhibitor of hepatic mixed-function oxidases (Anders and Mannering, 1966) did not appear to alter the rate of liver NP-SH depletion or renal toxicity. Support for the hypothesis that HCBD requires metabolic activation to produce renal necrosis could be obtained by examining (a) whether a metabolite of HCBD becomes covalently bound to kidney and/or liver macromolecules in ~ivo, and (b) whether treatments which alter necrosis, correlate with the binding observations. From this study it is concluded that HCBD produces necrosis of the straight portion of the proximal renal tubule and that this damage can be enhanced by treatment with A-1254. This suggests that HCBD requires metabolic activation to produce renal damage. ACKNOWLEDGMENTS The authors would like to thank Ms. J. M. Bishop, Ms. M. Hamilton, and Mr. B. Conolly for valuable technical assistance, and Mr. C. Gore for the plasma analysis.

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DUPRAT. P., AND GRADISKI. D. (1978). (:utaneous toxicity of hexachlorobutadiene. A~,rtr Ph~nz~rcol. Tmicol. 43, 346-353. FOWLER, B. A., HOOK, G. E. R.. AND Luc~t R. G. W. ( 1977). Tetrachlorodibenzo-p-dioxin induction of renal microsomal enzyme systems: Ultrastructural effects on pars recta (S3) proximal tubule cells of the rat kidney. ./. Pharmac~d. E.rp. 7hr 203, 712-721. GAGE. J. C. ( 1970). The subacute inhalation toxicity of 109 industrial chemicals. &it. J. I/X/. <2lrrl. 17, I - 18. GRADISKI, D., DUPRAT, P., MAGADCIR, J. L., AND FAYLIN, E. (1975). Etude toxicologique experimentale de I’hexachlorobutadiene. E//r. .I. To.ric o/ 8. IXO187. HARLEMAN. J. M., AND SEINEN. W. (1979). Shortterm toxicity and reproduction studies in rat4 with hexachlorobutadiene. Toskol. Appl. Pk~rnu~~~~/. 47, I-14. HALES. B. F.. JAEGER. V.. AND NEIMS. A. 111. (1978). Isoelectric focusing of glutathione S-transferases from rat liver and kidney. Biochvm. ./. 175. 937943.

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HEXACHLOROBUTADIENE

NEPHROTOXICITY

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47, 95- 104.

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