Bone marrow and renal injury associated with haloalkene cysteine conjugates in calves

 Almost 40 years ago, it was reported that cattle-feed which had been extracted with hot trichloroethylene and then fed to calves produced renal injury and a fatal aplastic anaemia. The toxic factor was subsequently identified as S-(1,2-dichlorovinyl)-L-cysteine (DCVC). These original findings have been confirmed, a single intravenous dose of DCVC at 4 mg/kg, or 0.4 mg/kg intravenously per day administered for 10 days to calves produced aplastic anaemia, and renal injury after a single dose of 4 mg/kg. The toxicity to calves of a number of other haloalkene cysteine conjugates has been examined to ascertain whether, like DCVC, they produce bone marrow and renal injury. Intravenous administration of the N-acetyl cysteine conjugate of DCVC produced renal but not bone marrow injury at a molar equivalent dose to DCVC, indicating that the calf can deacetylate the mercapturic acid and further that sufficient chemical had reached the kidney to be a substrate for the enzyme cysteine conjugate β-lyase. However, intravenous administration of the α-methyl analogue of DCVC, which cannot undergo metabolism via the enzyme cysteine conjugate β-lyase, was without toxicity at doses about five-fold higher than DCVC. These latter findings provide strong evidence that metabolism of DCVC via the enzyme β-lyase is necessary for bone marrow and renal injury to occur. The cysteine conjugates of perchloro ethylene and hexachloro-1,3-butadiene(HCBD) when given intravenously to calves at molar equivalent doses to DCVC, or above, did not produce either bone marrow or renal injury. In contrast, intravenous administration of the cysteine conjugate of tetrafluoroethylene (TFEC) produced severe renal tubular injury in calves without affecting the bone marrow. In vitro studies with these haloalkene cysteine conjugates showed, like DCVC, that they were good substrates for calf renal cysteine conjugate β-lyase and toxic to renal cells as judged by their ability to reduce organic anion and cation transport by slices of calf renal cortex and inhibit the renal enzyme glutathione reductase. Calves were also dosed either orally or intravenously with HCBD to assess its toxicity. HCBD at higher molar equivalent doses than DCVC produced mid-zonal necrosis in the liver, renal tubular necrosis but no bone marrow injury in calves. The key findings emerging from these studies are (1) that none of the other cysteine conjugates, at molar equivalent doses to DCVC and above, produce bone marrow injury in calves, (2) TFEC produced only renal injury, suggesting that sufficient of the other conjugates had not reached the kidney for metabolism by β-lyase to produce cytotoxicity and (3) that HCBD itself is more toxic than its cysteine or mercapturic acid conjugate, suggesting that pharmacokinetics and disposition are important factors in determining the toxicity of these conjugates to calves. Further studies are needed to understand the basis for the selective toxicity of DCVC to the bone marrow of calves. Received: 16 October 1995/Accepted: 9 January 1996     

The effect of haloalkene cysteine conjugates on rat renal glutathione reductase and lipoyl dehydrogenase activities

An early event in the nephrotoxicity of haloalkene cysteine conjugates is their metabolism by cysteine conjugate beta-lyase to generate a reactive "thiol moiety" which binds to protein. This reactive metabolite(s) has been reported to cause mitochondrial dysfunction. We have examined the effect of three haloalkene cysteine conjugates on the activity of rat renal cortical cytosolic glutathione reductase and mitochondrial lipoyl dehydrogenase, two enzymes which have been reported to be inhibited by S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in the liver. N-Acetyl-S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-L- cysteine (N-acetyl PCBC) produced a time- and concentration-dependent inhibition of glutathione reductase and kinetic studies showed that the inhibition was noncompetitive with a Ki of 215 microM. The enzyme activity from male rat kidney was more sensitive to N-acetyl PCBC than that from female rat kidney. Aminooxyacetic acid, an inhibitor of cysteine conjugate beta-lyase, and bis-p-nitrophenyl phosphate, an amidase inhibitor, blocked the effect of N-acetyl PCBC on glutathione reductase, indicating that metabolism by the cytosol is required to produce enzyme inhibition. S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine (TFEC) and DCVC are also noncompetitive inhibitors of glutathione reductase but are less active than N-acetyl PCBC with Ki's of 2.6 and 6.2 mM for DCVC and TFEC, respectively, DCVC produced a time- and concentration-dependent inhibition of lipoyl dehydrogenase and kinetic studies showed that the inhibition was noncompetitive with a Ki of 762 microM. TFEC and PCBC also inhibit lipoyl dehydrogenase. Aminooxyacetic acid blocked the effect of DCVC, TFEC, and PCBC on lipoyl dehydrogenase, indicating that metabolism by the mitochondrial fraction is required to produce enzyme inhibition. Glutathione reductase activity in the renal cortex of male rats treated with 200 mg/kg hexachloro-1,3-butadiene (HCBD) was inhibited as early as 1 hour after dosing, before signs of marked morphological damage. The activity of lipoyl dehydrogenase was also reduced but was only statistically significant 8 hr after dosing when there was marked renal dysfunction. These findings indicate that the reactive thiol moiety formed by cysteine conjugate beta-lyase cleavage of PCBC can inhibit both glutathione reductase and lipoyl dehydrogenase activities in vivo following HCBD administration. We suggest that such inhibition is a general phenomenon, occurring with diverse and as yet unidentified renal proteins. The critical nature of mitochondrial function and the generation of reactive metabolites within this compartment make this organelle a prime target.     

Deacetylation and further metabolism of the mercapturic acid of hexachloro-1,3-butadiene by rat kidney cytosol in vitro

Hexachloro-1,3-butadiene (HCBD) is more nephrotoxic to female than male rats. Metabolism of HCBD involves conjugation with glutathione followed by formation of the cysteine conjugate S-(pentachloro-1,3-butadienyl) cysteine (PCBD-CYS) and then the mercapturic acid N-acetyl-S-pentachloro-1,3-butadienyl-cysteine (PCBD-NAC). PCBD-NAC is also more nephrotoxic to female rats than male rats. The deacetylation of [¹⁴C]-PCBD-NAC to PCBD-CYS and the binding of radiolabelled metabolites to protein has been studied using renal cytosol preparations from male and female rats in vitro, since a sex-related difference in these reactions could explain the difference in nephrotoxicity found in vivo. PCBD-NAC was rapidly metabolised by renal cytosol. The rate of metabolism was similar with either male or female renal cytosol, and the major metabolite identified was PCBD-CYS. N-Acetylation of PCBD-CYS to PCBD-NAC was not detected in the presence of either male or female renal cytosol. Covalent binding of radioactivity from [¹⁴C]-PCBD-NAC to cytosolic protein could be detected after 5 min incubation, and although the extent of binding was similar for both male and female cytosol at early time periods, after 60 min incubation more binding was found in the presence of male cytosol. Covalent binding was largely prevented by aminooxyacetic acid, an inhibitor of cysteine conjugate -lyase, suggesting a role for this enzyme in the activation of HCBD. These results indicate that the sex differences in the nephrotoxicity of HCBD and PCBD-NAC in the rat are not attributable to differences in the rate of deacetylation of PCBD-NAC to give the proximate nephrotoxin PCBD-CYS.This work was supported by a fellowship from the European Science Foundation granted to I. S. P.     

In vivo nephrotoxic action of an isomeric mixture of S-(1-phenyl-2-hydroxyethyl)glutathione and S-(2-phenyl-2-hydroxyethyl)glutathione in Fischer-344 rats.

An isomeric mixture of S-[(1 and 2)-phenyl-2-hydroxyethyl]glutathione (PHEG), a glutathione conjugate of styrene, is moderately nephrotoxic. Its in vivo nephrotoxicity was characterized by significant elevations in the urinary excretion of glucose, gamma-glutamyl transpeptidase, glutamate dehydrogenase, N-acetyl-beta-D-glucosaminidase and lactic dehydrogenase 24 h after an i.v. administration of PHEG (0.5 mmol/kg) in male Fischer-344 rats. The histologic alterations consisted of moderate tubular damage with proximal tubule vacuolization and accumulation of tubular cast material, indicating an early sign of tubular necrosis. The data suggest that nephrotoxic injury induced by PHEG lies preferentially at the tubular region of the rat kidney involving several subcellular targets. The nephrotoxicity of PHEG was blocked by acivicin, a specific inhibitor of gamma-glutamyl transpeptidase, by phenylalanylglycine, an inhibitor of cysteinylglycine dipeptidase, as well as by probenecid, a competitive inhibitor of renal organic anion transport system. On the other hand, pretreatment with aminooxyacetic acid, a specific inhibitor of renal cysteine conjugate beta-lyase, failed to inhibit the nephrotoxicity of this glutathione conjugate. Similarly, prior administration of alpha-ketobutyrate, an inducer of renal cysteine conjugate beta-lyase, failed to potentiate its nephrotoxicity, suggesting an insignificant role of beta-lyase in such toxicity. A modest decline in renal cellular GSH due to PHEG but without any concomitant oxidation of GSH to GSSG and without any increase in lipid peroxidation indicates that oxidative stress may not be an important mechanism of its nephrotoxicity. Therefore, the following steps at least, are involved in the development of its nephrotoxicity: (1) renal tubular accumulation of PHEG via a probenecid-sensitive transport process; and (2) its renal metabolism via gamma-glutamyl transpeptidase and cysteinylglycine dipeptidase to the corresponding cysteine-S-conjugate.     

Biotransformation, excretion and nephrotoxicity of haloalkene-derived cysteine S-conjugates

The formation of cysteine S-conjugates is thought to play an important role in the nephrotoxicity of haloalkenes such as trichloroethene, tetrachloroethene and hexachlorobutadiene. Glutathione S-conjugates formed from these haloalkenes in the liver are processed to the corresponding cysteine S-conjugates, which may be N-acetylated to mercapturic acids and may be accumulated in the kidney. Haloalkene-derived cysteine S-conjugates are also substrates for cysteine conjugate β-lyases and reactive intermediates are formed in this reaction. The equilibrium between cysteine S-conjugate and mercapturic acid thus influences the extent of β-lyase dependent bioactivation and subsequently the nephrotoxicity of S-conjugates. In this study,␣we compared the rates of N-acetylation in vitro and the biotransformation, excretion and nephro‐ toxicity of S-(1,2-dichlorovinyl)-l-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)-l-cysteine (2,2-DCVC), S-(1,2,2-trichlorovinyl)-l-cysteine (TCVC) and S-(1,2,3,4,4-pentachlorobutadienyl)-l-cysteine (PCBC) in rats after i.v. injection (40 μmoles/kg). Marked differences in the extent of enzymatic N-acetylation were observed; N-acetylation was most efficient with 2,2-DCVC and least efficient with 1,2-DCVC. In urine, within 48 h, most of the given 2,2-DCVC (77% of the recovered dose) and 1,2-DCVC (92%) were recovered as the corresponding mercapturic acids. In contrast, a higher percentage of cysteine S-conjugate and less of the mercapturic acid were recovered in urine after administration of PCBC and TCVC (50 and 23% of dose as mercapturic acid), respectively. Histopathological examination of the kidneys and urine clinical chemistry showed marked differences in the extent of renal damage. Necroses of the proximal tubules were found after TCVC, PCBC and 1,2-DCVC administration in male, but not in female rats. These differences in nephrotoxicity do not correlate with the balance of acetylation/deacetylation. The higher toxicity observed in male rats may indicate the involvement of other parameters such as uptake mechanisms. Received: 30 April 1997 / Accepted: 26 August 1997     

Mechanism of S-(1,2-dichlorovinyl)glutathione-induced nephrotoxicity

S-(1,2-Dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-DL-cysteine are potent nephrotoxins. Agents that inhibit gamma-glutamyl transpeptidase, cysteine conjugate beta-lyase, and renal organic anion transport systems, namely L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), aminooxyacetic acid, and probenecid, respectively, protected against S-conjugate-induced nephrotoxicity. Furthermore, S-(1,2-dichlorovinyl)-DL-alpha-methylcysteine, which cannot be cleaved by cysteine conjugate beta-lyase, was not nephrotoxic. These results strongly support a role for renal gamma-glutamyl transpeptidase, cysteine conjugate beta-lyase, and organic anion transport systems in S-(1,2-dichlorovinyl)glutathione- and S-(1,2-dichlorovinyl)cysteine-induced nephrotoxicity.     

The role of glutathione and cysteine conjugates in the nephrotoxicity of o-xylene in rats

Moderate nephrotoxicity was induced in male and female rats exposed to o-xylene for 4 h at atmospheric concentrations of ∼3000 ppm. The xylene in␣vivo nephrotoxicity resulted in low enzyme leakage from the kidney into the urine. This low leakage was confirmed in 24-h urine by an increase in γ-glutamyltranspeptidase (γGT), N-acetyl-β-d-glucosaminidase (NAG) and alkaline phosphatase (ALP) activities. Compared to the control, both the 24-h urine output and the glucose excretion increased in male and female rats. These increases were probably a result of damage to the renal proximal tubules. The role of the metabolic pathway of glutathione in the emergence of the renal damage observed with o-xylene was investigated in rats. Recent studies indicate that the metabolic pathway of glutathione may be a bioactivation pathway, which is responsible for nephrotoxic effects with several drugs or chemicals. The renal toxicity of three synthesized o-xylene thio-conjugates was investigated in several groups␣of female rats. Administration of S-(o-methylbenzyl)glutathione (i.p., 1 mmol/kg), S-(o-methylbenzyl)cysteine (per os, 1 mmol/kg) or N-acetyl-S-(o-methylbenzyl)cysteine (i.p., 0.75 mmol/kg) to female rats did not induce renal toxicity, as monitored by urinary biochemical parameters (γGT, NAG, ALP, glucose). The data obtained suggest that the glutathione pathway would appear to be only detoxication, and probably does not contribute to the renal toxicity of o-xylene in female rats. Thus, either another metabolic pathway or other intermediate metabolites are probably involved in the nephrotoxic action of o-xylene. Received: 10 February 1998 / Accepted: 9 June 1998     

The effect of probenecid on acute N-(3,5-dichlorophenyl)succinimide-induced nephrotoxicity in the Fischer 344 rat

N-(3,5-Dichlorophenyl)succinimide (NDPS), an experimental agricultural fungicide, has been shown to produce selective nephrotoxicity in rats. Previous studies have shown that a metabolite(s) of extrarenal origin contributes to acute NDPS-induced nephrotoxicity. The purpose of this study was to determine if the organic acid transport inhibitor probenecid could modify the renal toxicity produced by NDPS administration. Male Fischer 344 rats were administered a single intraperitoneal (i.p.) injection of probenecid (60, 90 and 120 mg/kg) or 0.9% saline (1.0 ml/kg) followed 30 min later by NDPS (0.4 or 1.0 mmol/kg, i.p.) or sesame oil (2.5 ml/kg, i.p.) Renal function was monitored at 24 h and 48 h. Probenecid (60 mg/kg) did not markedly alter NDPS-induced renal effects on either post-treatment day. However, pretreatment with probenecid (90 or 120 mg/kg) blocked or attenuated the diuresis, increased proteinuria, decreased tetraethylammonium (TEA), uptake, elevation in blood urea nitrogen (BUN) concentration and increased kidney weight produced by NDPS (0.4 mmol/kg) administration. Only increased kidney weight and BUN concentration, and decreased lactate-stimulated p-aminohippurate (PAH) uptake were altered by probenecid (120 mg/kg) pretreatment when NDPS (1.0 mmol/kg) was given. NDPS-induced changes in renal morphology were not prevented by pretreatment with any probenecid dose. These results suggest that at least one nephrotoxic metabolite of NDPS is an organic acid. However, this acidic metabolite might not be the major nephrotoxic metabolite or a precursor to the major nephrotoxic metabolite(s). The identity of these metabolites remains to be determined.     

N-(3,5-dichlorophenyl)succinimide nephrotoxicity: evidence against the formation of nephrotoxic glutathione or cysteine conjugates.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity via one or more metabolites. Previous studies suggested that glutathione is important for mediating NDPS-induced nephropathy. The purpose of this study was to examine the possibility that a glutathione or cysteine conjugate of NDPS or an NDPS metabolite might be the penultimate or ultimate nephrotoxic species. In one set of experiments, male Fischer 344 rats were administered intraperitoneally (i.p.) NDPS (0.4 or 1.0 mmol/kg) 1 h after pretreatment with the gamma glutamyltranspeptidase inhibitor AT-125 (acivicin) (10 mg/kg, i.p.) and renal function was monitored at 24 and 48 h. In general, AT-125 pretreatment had few effects on NDPS-induced nephropathy. In a second set of experiments, rats were treated i.p. or orally (p.o.) with a putative glutathione (S-(2-(N-(3,5-dichlorophenyl)succinimidyl)glutathione (NDPSG), a cysteine (S-(2-(N-(3,5-dichlorophenyl)succinimidyl)cysteine (NDPSC) (as the methyl ester) or N-acetylcysteine (S-(2-(N-(3,5-dichlorophenyl)succinimidyl)-N-acetylcysteine (NDPSN) conjugate of NDPS (0.2, 0.4 or 1.0 mmol/kg) or vehicle and renal function was monitored at 24 and 48 h. An intramolecular cyclization product of NDPSC, 5-carbomethoxy-2-(N-(3,5-dichlorophenyl)carbamoylmethyl)-1,4-th iazane-3-one (NDCTO) was also examined for nephrotoxic potential. None of the compounds produced toxicologically important changes in renal function or morphology. The in vitro ability of the conjugates to alter organic ion accumulation by cortical slices was also examined. All of the conjugates tested caused a reduction in p-aminohippurate (PAH) accumulation at a conjugate bath concentration of 10(-4) M, but none of the conjugates reduced tetraethylammonium (TEA) uptake. In a third experiment, the ability of the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid (AOAA) (0.5 mmol/kg, i.p.) to alter the nephrotoxicity induced by two NDPS metabolites, N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) or N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA) (0.2 mmol/kg, i.p.), was examined. AOAA pretreatment had no effect on NDHS- or NDHSA-induced nephrotoxicity. These results do not support a role for a glutathione or cysteine conjugate of NDPS or and NDPS metabolite as being the penultimate or ultimate nephrotoxic species.     

The metabolism and nephrotoxicity of tetrafluoroethylene in the rat

Exposure of rats to 6000 ppm tetrafluoroethylene for 6 hr produced marked damage to the proximal tubule of the kidney with no effect on the liver. The toxicity was characterized by very high concentrations of urinary glucose and by marked increases in the concentrations of several urinary enzymes. The no observed effect level for a 6-hr exposure was 2000 ppm. Tetrafluoroethylene was metabolized to S-(1,1,2,2-tetrafluoroethyl)glutathione by rat liver fractions in vitro; the reaction was catalyzed by both microsomal and cytosolic glutathione S-transferases. The rate with microsomes was four times that with cytosol fractions. Evidence for this metabolic pathway in vivo has been obtained by the identification of the cysteinylglycine and cysteine conjugates of tetrafluoroethylene in rat bile. Cytochrome P-450 oxidation, a common metabolic route for haloalkenes, does not appear to occur in the metabolism of tetrafluoroethylene. When administered po to rats, the synthetic cysteine conjugate of tetrafluoroethylene causes renal damage identical to that caused by tetrafluoroethylene itself. The conjugate was metabolized by renal slices in vitro giving pyruvate, ammonia, and a reactive species which caused marked inhibition of organic ion transport into slices. Purified renal beta-lyase also cleaved this conjugate giving stoichiometric amounts of pyruvate and ammonia. The nephrotoxicity of tetrafluoroethylene is believed to derive from the hepatic glutathione conjugate of this compound. Following excretion and degradation of this conjugate in bile, the cysteine conjugate is reabsorbed and further metabolized in the kidney by the enzyme beta-lyase to a cytotoxic species.     

The Role of Glutathione INp-Aminophenol-Induced Nephrotoxicity in the Mouse

ABSTRACT

p-Aminophenol (PAP) produces nephrotoxicity in rats through a mechanism presumably involving oxidation and conjugation with glutathione (GSH). Recently it was found that PAP also causes nephrotoxicity in mice as evidenced by elevated blood urea nitrogen (BUN) and serum creatinine levels. The objective of this study was to further investigate the mechanism and elucidate the role of GSH in PAP-induced nephrotoxicity in the mouse. Male C57BL/6 mice injected ip with various doses of PAP were sacrificed at 12 hr for measurement of BUN and serum creatinine levels and determination of the extent of renal cortical nonprotein sulfhydryl (NPSH) and GSH depletion. PAP depleted renal cortical NPSH content in a dose- and time-dependent manner. Depletion of NPSH in mouse kidney did not occur at PAP doses below 600 mg/kg. Buthionine sulfoximine, an inhibitor of GSH synthesis, decreased nephrotoxicity. Ascorbate, a reducing agent, prevented PAP-induced nephrotoxicity and attenuated renal cortical NPSH depletion. However, acivicin and aminooxyacetic acid, inhibitors of y-glutamyltranspeptidase and β-lyase, respectively, did not prevent toxicity in the mouse. Piperonyl butoxide, an inhibitor of cytochrome P-450 enzymes, enhanced nephrotoxicity and renal cysteine depletion but not GSH depletion. The results suggest that PAP-induced nephrotoxicity in the mouse may involve oxidation and formation of a GSH conjugate.     

Biosynthesis and biotransformation of glutathione S-conjugates to toxic metabolites

The material presented in this review deals with the hypothesis that the nephrotoxicity of certain halogenated alkanes and alkenes is associated with hepatic biosynthesis of glutathione S-conjugates, which are further metabolized to the corresponding cysteine S-conjugates. Some glutathione or cysteine S-conjugates may be direct-acting nephrotoxins, but most cysteine S-conjugates require bioactivation by renal, pyridoxal phosphate-dependent enzymes, such as cysteine conjugate beta-lyase (beta-lyase). The biosynthesis of glutathione S-conjugates is catalyzed by both the cytosolic and the microsomal glutathione S-transferases, although the latter enzyme is a better catalyst for the reaction of haloalkenes with glutathione. When glutathione S-conjugate formation yields sulfur mustards, as occurs with vicinal-dihaloethanes, the S-conjugates are direct-acting toxins. In contrast, the S-conjugates formed from fluoro- and chloroalkenes yield S-alkyl- or S-vinyl glutathione conjugates, respectively, which are metabolized to the corresponding cysteine S-conjugates by gamma-glutamyltransferase and dipeptidases; inhibition of these enzymes blocks the toxicity of the glutathione S-conjugates. The cysteine S-conjugates must be metabolized by beta-lyase for the expression of toxicity; the beta-lyase inhibitor aminooxyacetic acid blocks the toxicity of cysteine S-conjugates, and the corresponding alpha-methyl cysteine S-conjugates, which cannot be metabolized by beta-lyase, are not toxic. Moreover, probenecid, an inhibitor of renal anion transport system, blocks the toxicity of cysteine S-conjugates, which cannot be metabolized by beta-lyase, are not toxic. Moreover, probenecid, an inhibitor of renal anion transport system, blocks the toxicity of cysteine S-conjugates. Homocysteine S-conjugates are also potent cyto- and nephrotoxins. The high renal content of gamma-glutamyltransferase and the renal anion transport system are probably determinants of kidney tissue as a target site. Biochemical studies indicate that renal mitochondrial dysfunction is produced by the cysteine S-conjugates. Finally, some of the glutathione and cysteine conjugates are mutagenic in the Ames test, and reactive intermediates formed by the action of beta-lyase may contribute to the nephrocarcinogenicity of certain chloroalkenes.     

Effect of the organic acid transport inhibitor probenecid on renal cortical uptake and proximal tubular toxicity of hexachloro-1,3-butadiene and its conjugates

Hexachloro-1,3-butadiene (HCBD), its glutathione conjugate (HCBD-GSH), cysteine conjugate (HCBD-CYS), and mercapturic acid derivative (HCBD-NAC) all produce acute necrosis of the pars recta of the proximal renal tubule in the rat. Previous studies have shown that radiolabel from administered HCBD appears to concentrate in the pars recta region. Renal uptake of radioactivity from HCBD-NAC was studied in rats by giving a single ip injection of the chemical and measuring its concentration in plasma and renal cortex 4 hr later. Cortex/plasma ratios (C/P) of HCBD-NAC were 4.35 +/- 0.21 (8 animals) at a dose of 64 mumol/kg and 10.4 +/- 0.55 (5) at a dose of 16 mumol/kg. These ratios were greater than that of inulin [C/P inulin = 1.5 +/- 0.2 (4)]. Thus cortical HCBD-NAC content was significantly greater than can be accounted for by glomerular filtration alone. Prior administration of probenecid (500 mumol/kg), a competitive inhibitor of organic acid transport, to animals receiving 16 or 64 mumol/kg of HCBD-NAC reduced the C/P to 1.03 +/- 0.09 (5) and 0.81 +/- 0.05 (8), respectively. Administration of probenecid in increasing doses (100, 200, 300, and 400 mumol/kg) to animals receiving 64 mumol/kg HCBD-NAC resulted in decreases of the C/P (2.59, 2.29, 1.35, and 0.84, respectively), suggesting a competitive inhibition of cortical HCBD-NAC uptake. The extent of covalently bound radioactivity from 64 mumol/kg HCBD-NAC was significantly greater in the renal cortex (1.11 +/- 0.2 nmol eq/mg protein) than in the liver (0.19 +/- 0.01 nmol eq/mg protein). Prior administration of probenecid (500 mumol/kg) reduced the renal cortical concentration of HCBD-NAC to 0.25 +/- 0.02 nmol eq/mg protein. Increasing doses of probenecid resulted in a progressive decrease in renal cortical covalent binding. When treatment with probenecid led to renal cortical concentrations of less than 120 nmol eq HCBD-NAC/g and an amount of covalently bound material less than 0.4 nmol eq/mg protein the animals were completely protected against the nephrotoxicity, as assessed by plasma urea and histopathological examination 24 hr after dosing. Prior administration of probenecid (500 mumol/kg) also protected rats against the nephrotoxicity produced by HCBD (192 mumol/kg), HCBD-GSH (47 mumol/kg), and HCBD-CYS (36 mumol/kg). It is suggested that the renal cortical accumulation and selective proximal tubular toxicity of HCBD and its conjugates is related to a carrier-mediated transport system.     

The role of glutathione in p-aminophenol-induced nephrotoxicity in the mouse.

p-Aminophenol (PAP) produces nephrotoxicity in rats through a mechanism presumably involving oxidation and conjugation with glutathione (GSH). Recently it was found that PAP also causes nephrotoxicity in mice as evidenced by elevated blood urea nitrogen (BUN) and serum creatinine levels. The objective of this study was to further investigate the mechanism and elucidate the role of GSH in PAP-induced nephrotoxicity in the mouse. Male C57BL/6 mice injected i.p. with various doses of PAP were sacrificed at 12 hr for measurement of BUN and serum creatinine levels and determination of the extent of renal cortical nonprotein sulfhydryl (NPSH) and GSH depletion. PAP depleted renal cortical NPSH content in a dose- and time-dependent manner. Depletion of NPSH in mouse kidney did not occur at PAP doses below 600 mg/kg. Buthionine sulfoximine, an inhibitor of GSH synthesis, decreased nephrotoxicity. Ascorbate, a reducing agent, prevented PAP-induced nephrotoxicity and attenuated renal cortical NPSH depletion. However, acivicin and aminooxyacetic acid, inhibitors of gamma-glutamyltranspeptidase and beta-lyase, respectively, did not prevent toxicity in the mouse. Piperonyl butoxide, an inhibitor of cytochrome P-450 enzymes, enhanced nephrotoxicity and renal cysteine depletion but not GSH depletion. The results suggest that PAP-induced nephrotoxicity in the mouse may involve oxidation and formation of a GSH conjugate.     

Gender differences in the potentiation of N-(3,5-dichlorophenyl)succinimide metabolite nephrotoxicity by phenobarbital

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces acute nephrotoxicity characterized as polyuric renal failure with proximal tubular necrosis. Phenobarbital pretreatment potentiates NDPS and N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS, a nephrotoxic metabolite of NDPS) nephrotoxicity in male rats. The purpose of this study was to determine the ability of phenobarbital pretreatment to potentiate (1) NDHS nephrotoxicity in female rats and (2) N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA, a nephrotoxic metabolite of NDHS) nephrotoxicity in male and female rats. Age-matched male and female Fischer 344 rats (4/group) were pretreated intraperitoneally (ip) with phenobarbital (75 mg/d, 3 d). At 24 h after the last injection of phenobarbital, an ip injection of NDHS (0.025 mmol/kg), 2-NDHSA (0.025 mmol/kg, females; 0.05 mmol/kg, males), or vehicle was given and renal function was monitored at 24 and 48 h post NDPS metabolite or vehicle. Additional groups received the NDPS metabolite or vehicle only and were also monitored for 48 h. In a separate experiment, male Fischer 344 rats were pretreated with piperonyl butoxide (PIBX, 1360 mg/kg) or the PIBX vehicle. 2-NDHSA (0.1 mmol/kg) or vehicle was administered (ip) 30 min after PIBX, and renal function was monitored for 24 h. Phenobarbital markedly potentiated 2-NDHSA nephrotoxicity in male rats as evidenced by increased kidney weight, increased blood urea nitrogen (BUN) concentration, and decreased tetraethylammonium (TEA) accumulation by renal cortical slices. PIBX had no effect on 2-NDHSA nephrotoxicity. Phenobarbital pretreatment did not markedly enhance the nephrotoxic potential of NDHS or 2-NDHSA in female rats. These results indicate that phenobarbital exhibits differential potentiation of NDPS metabolite nephrotoxicity in male and female rats and that the potentiation of 2-NDHSA nephrotoxicity observed in males is not due to cytochrome P-450-mediated oxidative biotransformation.     

The Acute Effects of S-(1,2-Dichlorovinyl)-L-cysteine and Related Chemicals on Renal Function andUltrastructure in the Pentobarbital-Anesthetized Dog: Structure-Activity Relationships, Biotransformation, and Unique Site-Specific Nephrotoxicity

The Acute Effects of S-(l,2-Dichlorovinyl}-L-cysteine and RelatedChemicals on Renal Function and Ultrastructure in the Pentobarbital-AnesthetizedDog: Structure-Activity Relationships, Biotransformation, andUnique Site-Specific Nephrotoxicity. KOECHEL, D. A., KREJCI,M. E., AND RIDGEWELL, R. E. (1991). Fundam Appl. Toxicol. 17,17-33. S-(l,2-Dichlorovinyl)-L-cysteine (L-DCVC), a substratefor the renal cysteine conjugate ß-lyase, and otherrelated chemicals were administered intravenously to pentobarbital-anesthetizeddogs. Six pertinent findings emerged regarding their nephrotoxicity.(1) L-DCVC was acutely nephrotoxic in the dog. (2) The earliestindicator of L-DCVC-induced renal damage was an increase inthe urinary excretion rate of protein. (3) Contrary to resultsfrom other species, L-DCVC induced renal ultrastructural lesionsonly in the S¹, and S² cells of the proximal tubule. (4) Thetoxicity of L-DCVC (23.15 µmol/kg, iv) to S¹ and S² cellsresulted from a direct tubular insult and not from overlappingepisodes of hypoxia or ischemia. (5) L-DCVC could be detectedin plasma only during the first 30 min after its injection.In addition, no L-DCVC and only small amounts of N-acetyl-L-DCVCand S-(1,2-dichlorovinyl)mercaptoacetic acid (DCV-MAA) (1.5%and <1% of the administered dose, respectively) were detectablein urine during the 6 hr following L-DCVC administration. (6)DCV-MAA and chloroacetic acid as well as other compounds thatare not substrates for the renal cysteine conjugate ß3-lyase(i.e., S-allyl-L-cysteine, vinthionine, and S-(l,2-dichlorovinyl)-D,L-a-methylcysteine)were not acutely nephrotoxic. These findings provide indirectevidence for the involvement of ß-lyase in the toxificationof L-DCVC in the dog. 1991 Society of Toxicology.     

Protection from 1-cyano-3,4-epithiobutane nephrotoxicity by aminooxyacetic acid and effect on xenobiotic-metabolizing enzymes in male Fischer 344 rats.

1-Cyano-3,4-epithiobutane (CEB), a naturally occurring nitrile derived from cruciferous plants, causes nephrotoxicity and increased renal glutathione (GSH) concentration in male F-344 rats. This CEB-induced nephrotoxicity is dependent on GSH conjugation and bioactivation. The objectives of the present study were to investigate the effect of CEB on several xenobiotic-metabolizing enzymes and to evaluate the effect of modulators of GSH transport and metabolism on CEB-induced nephrotoxicity and GSH concentration. Animals received 125 mg kg-1 CEB alone or following pretreatment with one of three selective inhibitors of GSH metabolism: acivicin, probenecid or aminooxyacetic acid. There were no significant alterations in epoxide hydrolase (EH), P-450, ethoxyresorufin O-deethylase (EROD) or pentoxyresorufin O-depentylase (PROD) enzyme activity, but renal glutamyl cysteine synthetase (GCS) activity was decreased at 12 and 24 h, as was renal glutathione S-transferase 4 h after CEB administration. Renal ECOD activity was also diminished at 24 h and at 12 and 24 h in liver. Aminooxyacetic acid (AOAA) abrogated the nephrotoxicity, the renal GSH-enhancing effect, and decreased GCS of CEB alone. These findings provide further evidence for the importance of GSH conjugation as a significant pathway in CEB metabolism and the role of a reactive thiol in nephrotoxicity and altered renal GSH.     

Intranephron distribution of cysteine S -conjugate β-lyase activity and its implication for hexachloro-1,3-butadiene-induced nephrotoxicity in rats

The intranephron distribution of two major cysteine S-conjugate β-lyases was determined in order to clarify the role of these enzymes in promoting the nephrotoxicity associated with certain halogenated xenobiotics. Various nephron segments [i.e., glomerulus, early, middle, and terminal portions of the proximal tubule (S₁, S₂, and S₃ respectively), the thick ascending limb, the distal tubule, and the collecting tubule] were isolated by microdissection from collagenase-treated rat kidneys. Each segment was dissected in Hanks' solution, solubilized with Triton X-100, and applied to a micropolyacrylamide gel constructed with a continuous gradient. The gels were subjected to electrophoresis and then incubated in the dark in a solution containing S-(1,2 dichlorovinyl)-l-cysteine (DCVC), sodium α-keto-γ-methiolbutyrate, phenazine methosulfate, and nitroblue tetrazolium. The position of cysteine S-conjugate β-lyase- and l-amino acid oxidase activities in the gels was revealed by the presence of blue formazen dye bands. The relative intensities of the bands were determined by optical scanning with a microdensitometer. Three bands were detected: band I (M_r ˜ 330 000) corresponds to a recently described high M_r cysteine S-conjugate β-lyase whereas band III (M_r ˜ 90 000) corresponds to a lower M_r cysteine S-conjugate β-lyase (identical to cytosolic glutamine transaminase K). Band II (M_r ˜ 240 000) corresponds to l-amino acid oxidase (a unique activity of the B isoform of rat kidney l-hydroxy acid oxidase). β-Lyase activity with DCVC as substrate was detected in the S₁, S₂, and S₃ segments of the nephron but not in other regions of the kidney. The activity was in the order: S₂ = S₃ > S₁. In another series of experiments, rats were killed 24 h after treatment with hexachloro- 1,3-butadiene (HCBD). In whole kidney homogenates the relative intensity of band III (per 22.2 μg tissue wet weight) after a 30 min incubation was induced significantly (by 50%), but the relative intensities of the other two bands were unchanged. On the other hand, in proximal tubules isolated from HCBD-treated rats the relative intensities (per 5 mm of nephron) of peak I of S₂, peak II of S₃, and peak III of S₃ were significantly reduced by 28, 33, and 72%, respectively. These findings suggest that the low M_rβ-lyase is induced by HCBD and that impaired cell function in the segments (especially S₃) results in proteins leaking out of the target cells. To examine the relationship between the nephrotoxic effect of HCBD and cysteine S-conjugate β-lyase activity, the intracellular ATP:protein ratio was quantitated in each nephron segment and in whole kidney homogenates. In HCBD-treated rats the ATP:protein ratio of the S₁, S₂, and S₃ segments was unchanged, decreased by ˜50%, and increased by ˜30%, respectively. In the kidney homogenates of HCBD-treated rats the ATP content was decreased by 32%. However, the loss of ATP was significantly less when the rats were pretreated with aminooxyacetate (a general inhibitor of pyridoxal 5′-phosphate-dependent enzymes, including β-lyase) 1 h before HCBD administration. The results strongly suggest that HCBD is converted to toxic metabolites within the kidney and that this process leads to metabolic derangement and reduction of ATP in susceptible kidney cells. Received: 16 April 1996 / Accepted: 16 July 1996     

Nephrotoxicity of mercapturic acids of three structurally related 2,2-difluoroethylenes in the rat. Indications for different bioactivation mechanisms

The biotransformation and the hepato- and nephrotoxicity of the mercapturic acids (N-acetyl-1-cysteine S-conjugates) of three structurally related 2,2-difluoroethylenes were investigated in vivo in the rat. All mercapturic acids appeared to cause nephrotoxicity, without any measureable effect on the liver. The mercapturic acid of tetrafluoroethylene (TFE-NAC) appeared to be the most potent nephrotoxin, causing toxicity upon an i.p. dose of 50 mumol/kg. The mercapturic acids of 1,1-dichloro-2,2-difluoroethylene (DCDFE-NAC) and 1,1-dibromo-2,2-difluoroethylene (DBDFE-NAC) were nephrotoxic at slightly higher doses, i.e. at 75 and 100 mumol/kg, respectively. In the urine of TFE-NAC-treated rats significant amounts of difluoroacetic acid (DFAA) could be detected. With increasing doses, the relative amount of DFAA in urine increased progressively (5-18% of dose). In urine of rats treated with DCDFE-NAC and DBDFE-NAC, however, the corresponding dihaloacetic acids, dichloroacetic acid and dibromoacetic acid, could not be detected. Formation of DFAA and pyruvate could also be observed during in vitro metabolism of the cysteine conjugate of tetrafluoroethylene (TFE-CYS) by rat renal cytosol. Inhibition by aminooxyacetic acid (AOA) pointed to a beta-lyase dependency for the DFAA-formation. Next to DFAA and pyruvate, also formation of hydrogen sulfide and thiosulfate could be detected. These results suggest that TFE-CYS is bioactivated to a significant extent to difluorothionacyl fluoride, which most likely is subsequently hydrolysed to difluorothio(no)acetic acid and difluoroacetic acid. According to formation of pyruvate, the cysteine conjugates derived from DCDFE-NAC and DBDFE-NAC also were efficiently metabolized by rat renal beta-lyase. However, the formation of corresponding dihaloacetic acids, dichloroacetic acid and dibromoacetic acid, could not be detected in vitro at all. Only very small amounts of hydrogen sulfide and thiosulfate were detected. These results suggest that bioactivation of the latter two conjugates to a dichloro- or dibromothionoacyl fluoride represents only a minor route. Because of better leaving group abilities of chloride and bromide compared to fluoride, rearrangement of the initially formed ethanethiol to a thiirane might be favoured. Based on the present in vivo and in vitro data, it is concluded that the nephrotoxicity of the structurally related mercapturic acids of 2,2-difluoroethylenes is dependent on halogen substitution and presumably the result of at least two different mechanisms of bioactivation.     

Nephrotoxicity and hepatotoxicity of 1,1-dichloro-2,2-difluoroethylene in the rat. Indications for differential mechanisms of bioactivation

1,1-Dichloro-2,2-difluoroethylene (DCDFE) produced marked nephrotoxicity in rats upon an i.p. dose of 150 mumole/kg. At doses higher than 375 mumole/kg, DCDFE also produced hepatotoxicity. Aminooxyacetic acid, an inhibitor of cysteine conjugate beta-lyase, appeared to be slightly nephrotoxic in Wistar rats. Nevertheless it exerted an inhibitory effect on the nephrotoxicity of DCDFE. The N-acetylcysteine conjugate of DCDFE was identified as a major urinary metabolite of DCDFE. When administered as such, this conjugate appeared to be a potent nephrotoxin, without any effect on the liver, indicating that glutathione conjugation of DCDFE is most likely a bioactivation step for nephrotoxicity. The appearance of traces of chlorodifluoroacetic acid in urine of rats treated with higher doses of DCDFE indicates the existence of an oxidative pathway of metabolism of DCDFE, probably involving epoxidation by hepatic mixed-function oxidases. It is speculated that the latter route might account for the hepatotoxicity at higher doses of DCDFE. The nephro- and hepatotoxicity of DCDFE, therefore, most likely are the result of two different mechanisms of bioactivation.