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.

Role of stereochemistry in N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) nephrotoxicity.

The nephrotoxicity induced by the agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is mediated through oxidative metabolites of NDPS. Oxidation of the succinimide ring in NDPS yields the nephrotoxic metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and its hydrolysis product N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA). The oxidation of NDPS on the succinimide ring also introduces an asymmetric carbon atom into these NDPS metabolites, so that R- and S- enantiomers of NDHS and 2-NDHSA are possible. The purpose of this study was to begin to explore the importance of the stereochemical orientation at the asymmetric carbon atom for the nephrotoxicity induced by NDPS metabolites. Male Fischer 344 rats were administered a single intraperitoneal (ip) injection of R-(+)- or S-(-)-2-NDHSA (0.05, 0.1 or 2.0 mmol/kg) or vehicle, and renal function was monitored for 48 h. R-2-NDHSA (0.1 mmol/kg) administration had little effect on renal function. R-2-NDHSA (0.2 mmol/kg) treatment induced mild diuresis on day 1, increased proteinuria, and a small increase in blood urea nitrogen (BUN) concentration, but no change in kidney weight or glucosuria. S-2-NDHSA (0.1 mmol/kg) induced marked nephrotoxicity as evidenced by diuresis on both post-treatment days, increased proteinuria, glucosuria, and increased kidney weight and BUN concentration. No evidence of hepatotoxicity was obtained in any treated group. Thus, the S-isomer of 2-NDHSA is a more potent nephrotoxicant than the R-isomer, and stereochemistry may play a role in NDPS metabolite-induced nephrotoxicity.

Sodium sulfate potentiates N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) nephrotoxicity in the Fischer 344 rat.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity as its major toxicity in rats. Previous studies have shown that NDPS induces nephrotoxicity following oxidation of the succinimide ring to form N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and the hydrolysis product of NDHS, N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA). Our recent work found that sodium sulfate potentiated NDPS nephrotoxicity, suggesting that sulfate conjugation of NDPS metabolites might be a bioactivation step mediating NDPS nephrotoxicity. The purpose of this study was to determine if sodium sulfate also potentiated the nephrotoxicity of the two nephrotoxic metabolites of NDPS and further to see if sodium sulfate potentiated NDHS and 2-NDHSA nephrotoxicity to the same degree. Male Fischer 344 rats (4-16 rats/group) received an intraperitoneal (ip) injection of sodium sulfate (10 mg/kg) 20 min before a non-nephrotoxic dose (0.05 mmol/kg, ip) of NDHS or 2-NDHSA, or vehicle (12.5% dimethyl sulfoxide in sesame oil). Renal function was then monitored over 48 h. Sodium sulfate pretreatment potentiated the renal effects of a non-nephrotoxic dose of NDHS and 2-NDHSA to induce nephrotoxicity. Nephrotoxicity was characterized by diuresis, increased proteinuria, elevated blood urea nitrogen (BUN) concentration, increased kidney weight and proximal tubular necrosis. Differences in the potentiation of NDHS and 2-NDHSA nephrotoxicity by sodium sulfate were also observed as NDHS nephrotoxicity was potentiated to a lesser degree than 2-NDHSA-induced nephrotoxicity. These results support the likelihood that one or more sulfate conjugate(s) of NDPS metabolites contribute to NDPS nephrotoxicity.

Nephrotoxic potential of N-(3,5-dichloro-4-fluorophenyl)succinimide in Fischer 344 rats: comparison with N-(3,4,5-trichlorophenyl)succinimide.

Numerous structure-nephrotoxicity relationship studies from our laboratory have demonstrated that N-(3,5-dichlorophenyl)succinimide (NDPS) is one of the most potent nephrotoxicants among the N-arylsuccinimides. The purpose of this study was to extend our previous structure-nephrotoxicity relationship studies by examining the effect of addition of a fluoro verses a chloro group at the 4-phenyl position in NDPS. Male Fischer 344 rats (four rats/group) received a single intraperitoneal (i.p.) injection of N-(3,5-dichloro-4-fluorophenyl)succinimide (NDCFPS) or N-(3,4,5-trichlorophenyl)succinimide (NTCPS)(0.4 or 0.8 mmol/kg) or vehicle, and renal function monitored at 24 and 48 h. NDCFPS did not induce significant nephrotoxicity at either dose tested. In contrast, NTCPS (0.4 or 0.8 mmol/kg) induced marked nephrotoxicity characterized by diuresis, increased proteinuria, glucosuria, elevated kidney weight and increased blood urea nitrogen (BUN) concentration. NTCPS also induced marked proximal tubular necrosis at both doses tested. Neither NDCFPS nor NTCPS induced hepatotoxicity at either dose tested. The results of these experiments indicate that addition of a fluoro group at the 4-position on the phenyl ring of NDPS produces a nonnephrotoxicant NDPS derivative (NDCFPS), while addition of a chloro group at this site produces an NDPS derivative with similar nephrotoxic potential to NDPS. The mechanism for this differential effect between 4-halophenyl substitution is unclear, but may result from increased hydrolysis of the succinimide ring and/or increased clearance of N-arylsuccinimide metabolites when a fluoro group is added to the 4-position of the phenyl ring.

2-Aminophenol and 4-aminophenol toxicity in renal slices from Sprague-Dawley and Fischer 344 rats.

This study examined differences in toxicity between 2- and 4-aminophenol using a renal cortical slice model. Renal cortical slice toxicity for 2- and 4-aminophenol was also monitored in tissue from Sprague-Dawley and Fischer 344 (F344) rats in order to determine potential strain differences for aminophenol toxicity. Renal cortical slices from Sprague-Dawley and F344 rats (age 50-65 d) were isolated and incubated for 15-120 min with 0-1 mM 2- or 4-aminophenol at 37 degrees C under an oxygen atmosphere. Elevations in lactate dehydrogenase (LDH) leakage from renal cortical slices occurred at lower concentrations of 4-aminophenol than of 2-aminophenol from both strains of rats. Total glutathione levels were more markedly decreased by 4-aminophenol than by 2-aminophenol in renal slices from both strains. LDH release was elevated by 1 mM 2-aminophenol in renal slices from F344 rats, but values were comparable between control and treated in the renal slices from Sprague-Dawley rats. 4-Aminophenol was slightly more toxic to renal slices from F344 than from Sprague-Dawley rats. LDH release was increased, relative to controls, by 0.1 mM in the F344 rats group compared to 0.25 mM in the Sprague-Dawley group. Strain differences were not apparent when comparisons were made of total glutathione levels or rate-limiting substrates of gluconeogenesis. These results indicated that strain differences in toxicity were detected between Sprague-Dawley and F344 rat strains. Based on LDH release, renal cortical slices obtained from age-matched F344 rats were slightly more susceptible than Sprague-Dawley rats to toxicity by 2- and 4-aminophenol.

Gender differences in acute N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) nephrotoxicity in Fischer 344 rats.

N-(3,5-Dichlorophenyl)succinimide (NDPS) is an agricultural fungicide that induces nephrotoxicity as its major toxicity. NDPS is also a more potent nephrotoxicant in female than in male rats. The purpose of this study was to examine the nephrotoxic potential of the two NDPS metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) in age-matched male and female Fischer 344 rats to determine if gender differences exist for the nephrotoxicity induced by the two NDPS metabolites. Rats (4 per group) were administered a single intraperitoneal (ip) injection of NDHS or 2-NDHSA (0.025 or 0.05 mmol/kg) or vehicle, and renal function was monitored for 48 h. Neither compound induced significant nephrotoxicity in male rats at the doses tested. However, in female rats both metabolites induced marked nephrotoxicity at the 0.05 mmol/kg dose level, and treatment with 0.025 mmol/kg 2-NDHSA induced some changes in renal function (transient diuresis, transient proteinuria, decreased organic ion accumulation). Little effect on renal function was induced in females by treatment with 0.025 mmol/kg NDHS. At toxic levels in female rats, the renal lesions were located primarily in the S2 and S3 segments of the proximal tubule. These results indicate that, like the parent compound, gender differences exist in the nephrotoxic potential of NDHS and 2-NDHSA. The results also suggest that in females, as in males, NDPS nephrotoxicity is mediated via NDHS and/or 2-NDHSA. However, it is not clear if the ultimate nephrotoxicant species following NDPS exposure is different in males and females or if the same ultimate nephrotoxicant species is produced in both species but handled differently by male and female kidneys. Thus, further studies are needed to determine the exact nature of the ultimate nephrotoxicant species and the mechanisms of the observed gender differences.

Effects of sodium sulfate on acute N-(3,5-dichlorophenyl)succinimide (NDPS) nephrotoxicity in the Fischer 344 rat.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces acute polyuric renal failure in rats. Results of previous studies have suggested that NDPS may induce nephrotoxicity via conjugates of NDPS metabolites. Thus, the purpose of this study was to examine if administered sodium sulfate could alter NDPS nephrotoxicity. Male Fischer 344 rats (four rats per group) were administered a single intraperitoneal (i.p.) injection of sodium sulfate (0.035, 0.07, 0.35 or 3.5 mmol/kg) or sodium chloride (7.0 mmol/kg) 20 min before NDPS (0.2, 0.4 or 0.8 mmol/kg) or NDPS vehicle (sesame oil, 2.5 ml/kg) and renal function monitored at 24 and 48 h. High dose sodium sulfate (3.5 mmol/kg) markedly attenuated NDPS nephrotoxicity, while sodium chloride had no effect on NDPS-induced renal effects. NDPS nephrotoxicity was also attenuated by a pretreatment dose of 0.35 mmol/kg sodium sulfate, while 0.07 mmol/kg sodium sulfate pretreatment potentiated NDPS 0.2 mmol/kg to produce nephrotoxicity without markedly attenuating NDPS 0.4 mmol/kg to induce renal effects. A dose of 0.035 mmol/kg sodium sulfate did not potentiate NDPS 0.2 mmol/kg to induce nephrotoxicity. These results suggest that sulfate conjugates of NDPS metabolites might contribute to NDPS nephrotoxicity.

4-Amino-2,6-dichlorophenol nephrotoxicity in the Fischer 344 rat: protection by ascorbic acid, AT-125, and aminooxyacetic acid.

A halogenated derivative of 4-aminophenol, 4-amino-2, 6-dichlorophenol (ADCP), is a potent nephrotoxicant and a weak hepatotoxicant in Fischer 344 rats. Although the mechanism of ADCP nephrotoxicity is unknown, ADCP could undergo oxidation to a reactive intermediate, such as a 4-amino-2,6-dichlorophenoxy radical or 2,6-dichloro-1,4-benzoquinoneimine, which can generate additional free radicals and/or covalently bind to cellular proteins. The toxic process might also be mediated by glutathione (GSH) conjugates of ADCP, as suggested for the mechanism of 4-aminophenol nephrotoxicity. In this study, the effects of modulators of oxidation and GSH conjugation-related metabolism or transport on ADCP-induced nephrotoxicity were examined. In one set of experiments, male Fischer 344 rats (four/group) were intraperitoneally (ip) administered ADCP (0.38 mmol/kg) only or coadministered an antioxidant, ascorbic acid (1.14 mmol/kg, ip) with ADCP. Administration of ascorbic acid markedly reduced both functional nephrotoxicity and morphological changes induced by ADCP. Administration of a gamma-glutamyltransferase (GGT) inhibitor, l-(alphaS, 5S)-alpha-amino-3-chloro-4,5-dihydroxy-5-isoxazoleacetic acid (10 mg/kg, ip), or a cysteine conjugate beta-lyase inhibitor, aminooxyacetic acid (0.5 mmol/kg, ip), 1 hr before ADCP (0.38 mmol/kg) challenge partially protected rats against ADCP nephrotoxicity. In contrast, administration of an organic anion transport inhibitor, probenecid (140 mg/kg, ip), 30 min before ADCP had little effect on ADCP nephrotoxicity. The GSH depletor, buthionine sulfoximine (890 mg/kg, ip), was given 2 hr prior to ADCP and only minimal protection was noted. In addition, the nonprotein sulfhydryl (NPSH) contents in renal cortex and liver were determined at 2 hr following the administration of ADCP only or ascorbic acid/ADCP. Ascorbic acid afforded complete prevention of the depletion of NPSH in the kidney and liver caused by ADCP administration and also prevented the elevation of renal glutathione disulfide content induced by ADCP. The results indicate that oxidation of ADCP appears to be essential to ADCP nephrotoxicity and that GSH or GSH-derived conjugates of ADCP may be partly responsible for the nephrotoxic effects of ADCP via a GGT-mediated mechanism.

Characterization of methemoglobin formation induced by 3,5-dichloroaniline, 4-amino-2,6-dichlorophenol and 3,5-dichlorophenylhydroxylamine.

3,5-Dichloroaniline is an intermediate in the production of certain fungicides. This study characterized the capacity of 3,5-dichloroaniline and two putative metabolites to induce methemoglobin formation. In vivo intraperitoneal (i.p.) administration of 0.8 mmol/kg 3,5-dichloroaniline resulted in elevated (P < 0.05) methemoglobin levels at 2 and 4 h after injection and returned to control values within 8 h. In vitro methemoglobin generation was monitored in washed erythrocytes incubated for 60 min at 37 degrees C with 4 and 8 mM 3,5-dichloroaniline. Methemoglobin generation in vitro was higher (P < 0.05) than control values in erythrocytes incubated for 30 min with 0.2-0.6 mM 4-amino-2,6-dichlorophenol or 5-100 microM 3,5-dichlorophenylhydroxylamine. The in vitro methemoglobin generating capacity in decreasing order was: 3,5-dichlorophenylhydroxylamine > 4-amino-2,6-dichlorophenol > > 3,5-dichloroaniline. The results of the in vitro studies further indicated that none of the compounds tested induced lipid peroxidation. Erythrocytes incubated with 5-100 microM 3,5-dichlorophenylhydroxylamine in vitro were associated with depletion of glutathione. These results indicated that: (a) 3,5-dichloroaniline and its metabolites can induce methemoglobin formation; (b) the N-hydroxy metabolite was the most potent inducer of hemoglobin oxidation and (c) glutathione depletion was associated with methemoglobin formation by 3,5-dichlorophenylhydroxylamine.

3,4-Dicholoroaniline acute toxicity in male Fischer 344 rats.

The aromatic amine, 3,4-dichloroaniline (DCA) is an important intermediate in the chemical production of agricultural chemicals. A previous study had shown that nephrotoxicity was apparent 48 h after injection of 3,4-DCA. The purpose of this study was to examine the potential for 3,4-DCA to be toxic to the kidney, liver and urinary bladder 24 h after acute administration. Male Fischer 344 (F344) rats were injected (intraperitoneal (i.p.)) with 0.4, 0.8 or 1.0 mmol/kg 3,4-DCA hydrochloride (HCl) salt (2.5 ml/kg, 25% ethanol). Nephrotoxicity was apparent within 24 h in the 0.8 and 1.0 mmol/kg 3,4-DCA treated group and was characterized by elevated (P < 0.05) blood urea nitrogen (BUN) and kidney weight. Renal cortical slice accumulation ofp-aminohippurate (PAH) was also decreased in the 0.8 and 1.0 mmol/kg 3,4-DCA treated group relative to pair fed controls (PFC). Cellular changes were noted in the liver and bladder 24 h after 3,4-DCA administration. Plasma alanine transaminase (ALT) activity was elevated (P < 0.05) above PFC values 24 h after treatment with 0.8 or 1.0 mmol/kg indicating liver damage was apparent within 24 h. Morphological damage was apparent along the centrilobular region. Hematuria was observed in the 0.8 and 1.0 mmol/kg 3,4-DCA treated groups. Infiltration of erythrocytes and polymorphonuclear leukocytes was apparent within the urinary bladder upon examination by light microscopy. These results indicated that 3,4-DCA was toxic within 24 h and that the target tissues were the kidney, liver and urinary bladder. In vitro studies were conducted to compare the toxicity of two forms of 3,4-DCA, the free base and hydrochloride salt to determine whether chemical form contributes to renal cortical slice toxicity. Lactate dehydrogenase (LDH) release was elevated above control by 120 min exposure to 2 mM 3,4-DCA free base or hydrochloride salt. Pyruvate directed gluconeogenesis in renal slices was decreased relative to control by 0.5 mM 3,4-DCA free base and hydrochloride salt. The results from the in vitro studies indicates that the chemical form did not modify in vitro renal cortical slice toxicity.

Nephrotoxicity of N-(3-bromophenyl)-2-hydroxysuccinimide: role of halogen groups in the nephrotoxic potential of N-(halophenyl) succinimides.

Among N-(halophenyl)succinimides. N-(3,5-dichlorophenyl)succinimide (NDPS) is a potent nephrotoxicant as well as an agricultural fungicide. Although two chloride groups on the phenyl ring are essential to induce optimal nephrotoxicity, the role of halogen groups in NDPS nephrotoxicity is not clear. In this study, N-(3-bromophenyl)-2-hydroxysuccinimide (NBPHS) was prepared as a monohalophenyl derivative of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), an oxidative and nephrotoxicant metabolite of NDPS. The nephrotoxic potential of NBPHS was evaluated in vivo and in vitro to determine the role of halogen groups in N-(halophenyl)succinimide nephrotoxicity. Male Fischer 344 rats (four/group) were administered a single intraperitoneal (i.p.) injection of NBPHS (0.1, 0.4 or 0.8 mmol/kg) or vehicle (25% dimethyl sulfoxide in sesame oil) and renal function monitored for 48 h. Administration of NBPHS (0.8 mmol/kg) induced nephrotoxicity, while very mild changes or no changes in renal function were observed following administration of 0.4 mmol/kg or 0.1 mmol/kg of NBPHS, respectively. Nephrotoxicity induced by NBPHS (0.8 mmol/kg) was characterized by diuresis, transiently increased proteinuria, glucosuria and hematuria elevated kidney weight and reduced tetraethylammonium (TEA) uptake by renal cortical slices, and was not as marked as nephrotoxicity induced by NDHS (0.1 mmol/kg) or NDPS (0.4 mmol/kg). In the in vitro studies the effects of NBPHS on organic ion accumulation, pyruvate-stimulated gluconeogenesis, and lactate dehydrogenase (LDH) release were measured using renal cortical slices. NBPHS decreased p-aminohippurate (PAH) and TEA accumulation at NBPHS bath concentrations of 0.05 mM and 0.5 mM and 0.5 mM or greater, respectively. Renal gluconeogenesis was inhibited by NBPHS at 1 mM bath concentration, while LDH leakage was not increased at NBPHS bath concentrations up to 1 mM. The results demonstrate that NBPHS is a mild nephrotoxicant in vivo and in vitro, but does not have cytotoxic effects to renal tissues at the concentrations tested. From these results, it appears that halogen groups are essential to the nephrotoxic potential of N-(halophenyl)-2-hydroxysuccinimides or N-(halophenyl)succinimides and play an important role in the mechanism of NDPS nephrotoxicity following NDHS formation.

Nephrotoxicity of 4-amino-2-chlorophenol and 2-amino-4-chlorophenol in the Fischer 344 rat.

Aminophenols and halogenated anilines induce nephrotoxicity and mild hepatotoxicity in rats. In this study, the in vivo and in vitro nephrotoxic potential of 4-amino-2-chlorophenol and 2-amino-4-chlorophenol, monochlorinated aminophenols and potential metabolites of 3-chloroaniline, was evaluated. Hepatotoxicity of both compounds was also examined in vivo. Male Fischer 344 rats (four/group) were administered 4-amino-2-chlorophenol hydrochloride (0.4, 0.8 or 1.0 mmol/kg), 2-amino-4-chlorophenol hydrochloride (0.4, 0.8 or 1.2 mmol/kg) or vehicle intraperitoneally (i.p.) and renal and hepatic function monitored for 48 h. Administration of 4-amino-2-chlorophenol (0.8 mmol/kg) induced nephrotoxicity, while only minor changes in kidney function were observed following administration of 0.4 mmol/kg of 4-amino-2-chlorophenol or 0.8 mmol/kg of 2-amino-4-chlorophenol. Increasing the dose of 4-amino-2-chlorophenol to 1.0 mmol/kg or 2-amino-4-chlorophenol to 1.2 mmol/kg resulted in lethality. Nephrotoxicity induced by 4-amino-2-chlorophenol was characterized by diuresis, increased proteinuria, glucosuria, hematuria, elevated blood urea nitrogen (BUN) concentration and kidney weight, and marked proximal tubular damage, while 2-amino-4-chlorophenol induced milder effects on renal function and transient oliguria instead of diuresis. No hepatotoxicity was observed with either compound at any dose tested. In the in vitro studies, the direct effects of 4-amino-2-chlorophenol or 2-amino-4-chlorophenol on organic ion accumulation, pyruvate-stimulated gluconeogenesis and lactate dehydrogenase (LDH) leakage were determined using renal cortical slices. 4-Amino-2-chlorophenol and 2-amino-4-chlorophenol were almost equally effective in inhibiting organic anion or cation uptake and gluconeogenesis or increasing LDH leakage, although small differences in the minimum effective concentrations were present (minimum effective concentration, 0.01-0.5 mM range). These results demonstrate that 4-amino-2-chlorophenol is a more potent nephrotoxicant than 2-amino-4-chlorophenol in vivo. The results also indicate that the addition of a chloride group to aminophenols enhances renal toxicity.

Nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol in Fischer 344 rats: comparisons with 2- and 4-chloroaniline and 2- and 4-aminophenol.

Nephrotoxicity occurs following intraperitoneal (i.p.) administration of 2-chloroaniline or 4-chloroaniline hydrochloride to Fischer 344 rats, but the nephrotoxicant chemical species and mechanism of nephrotoxicity are unknown. The purpose of this study was to evaluate the in vivo and in vitro nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol, metabolites of 4-chloroaniline and 2-chloroaniline. A comparison was also made between the nephrotoxic potential of the aminochlorophenols and the corresponding aminophenols to examine the effect of adding a chloride group on the nephrotoxic potential of the animophenols. Male Fischer 344 rats (4/group) were given an i.p. injection of a chloroaniline or aminochlorophenol hydrochloride (1.5 mmol/kg), and aminophenol (1.0 or 1.5 mmol/kg), or vehicle, and renal function monitored at 24 and 48 h. Both aminochlorophenols induced smaller and fewer renal effects that the parent chloroanilenes in vivo. Also, 4-aminophenol was markedly more potent as a nephrotoxicant that 4-amino-3-chlorophenol, while 2-aminophenol and 2-amino-5-chlorophenol induced only mild change in renal function. In vitro, the phenolic compounds reduce p-aminohippurate accumulation by renal cortical slices at bath concentrations of 0.01 mM, while a bath concentration of 0.50 mM or greater was required for the chloroanilines. However, all compounds reduced tetraethylammonium accumulation at bath concentrations of 0.1-0.5 mM or greater. These results indicate that extrarenally-produced aminochlorophenol metabolites do not contribute to the mechanism of chloroaniline nephrotoxicity. Also, the reduced nephrotoxic potential of 4-amino-3-chlorophenol compared to 4-aminophenol could result from an altered ability of the aminochlorophenol to redox cycle or form conjugates.

Comparison of cephaloridine renal accumulation and urinary excretion between normoglycemic and diabetic animals.

The renal toxicity of cephaloridine is reduced in a streptozotocin diabetic rat model. This study tested the hypothesis that renal cortical cephaloridine accumulation was diminished in diabetic rats. The following studies also investigated whether renal excretion was enhanced in diabetic rats. Male Fischer 344 rats were randomly divided into normoglycemic or diabetic groups. Diabetes was induced by injection (intraperitoneal, i.p.) of 35 mg/kg streptozotocin. Normoglycemic and diabetic rats were injected (i.p.) with 1500 mg/kg cephaloridine. Peak plasma cephaloridine levels were similar in both groups. Renal cortical accumulation was diminished (P < 0.05) in the diabetic group 1 and 4 h after cephaloridine injection. Urinary cephaloridine excretion was enhanced (P < 0.05) in the diabetic group relative to the normoglycemic animals during the first 4 h after cephaloridine injection. Comparisons between normoglycemic and diabetic groups indicated renal cortical cephaloridine accumulation was lower in the diabetic group. These findings would support the hypothesis that reduced cephaloridine toxicity in diabetic animals was due to reduced renal cortical accumulation of the toxin. These data also demonstrate that cephaloridine excretion was enhanced in the diabetic group and may contribute to the diminished renal accumulation.

In Vitro toxicity of 2- and 4-chloroaniline: comparisons with 4-amino-3-chlorophenol, 2-amino-5-chlorophenol and aminophenols.

Chloroanilines have been associated with renal and hepatic toxicity. This study (a) examined the in vitro hepatic and renal toxicity of 2-chloroaniline and 4-chloroaniline, (b) further examined whether aromatic ring hydroxylation would increase toxicity of the parent compound and (c) compared toxicity between respective aminochlorophenol and aminophenol. Renal and hepatic slices were exposed to varying concentrations of 2-chloroaniline, 4-chloroaniline. 4-amino-3-chlorophenol, 2-amino-5-chlorophenol, 2-aminophenol or 4-aminophenol. Toxicity was monitored by measurement of pyruvate-directed gluconeogenesis and leakage of lactate dehydrogenase (LDH). Hepatic tissue was less susceptible to toxicity than kidney tissue for all compounds since LDH leakage was elevated only in renal tissue. Gluconeogenesis was reduced in renal cortical slices exposed to 0.1 mum aminochlorophenols or 4-aminophenol, whereas a concentration of 0.5 mum was necessary for the chloroanilines and 2-aminophenol. LDH release was increased in renal slices by aminochlorophenols and aminophenols but not by the chloroanilines. The nephrotoxic potential in renal cortical slices was 4-aminophenol > 2-amino-5-chlorophenol > 4-amino-3-chlorophenol > 2-aminophenol > 2-chloroaniline = 4-chloroaniline. These results suggest that aromatic ring hydroxylation increased in vitro toxicity of the chloroanilines. Comparison of aminophenols with aminochlorophenols indicated that addition of a halogen can have variable effects on toxicity.

Contractile responses and structure of small bronchi isolated from rats after 20 months' exposure to ozone.

Short-term exposure to high concentrations of ozone has been shown to increase airway responsiveness in normal humans and in all laboratory animal species studied to date. While our knowledge concerning the pulmonary effects of single exposures to ozone has increased rapidly over recent years, the effects of repeated exposures are less understood. The goal of the present study was to determine whether airway responsiveness is increased after near-lifetime exposure to ozone. Airway segments representing approximately eighth generation airways were isolated from Fischer 344 rats of both genders that had been exposed for 6 hr per day, 5 days per week for 20 months to 0, 0.12, 0.5, or 1.0 parts per million (ppm) ozone. Circumferential tension development was measured in isolated airways in response to bethanechol, acetylcholine, and electrical field stimulation. Responsiveness of the airways to the contractile stimuli was described by the effective dose or frequency that elicited half-maximum contraction (ED50) and the maximum response. Since ozone exposure is associated with remodeling of peripheral airways, smooth muscle area was determined and tension responses were normalized to the area measurements. Before normalization of tension data to smooth muscle area, neither the ED50 nor maximum response of small bronchi to the contractile stimuli was altered after chronic ozone exposure. Smooth muscle area was greater in airways isolated from animals that had been exposed to 0.5 ppm ozone. After accounting for smooth muscle area, maximum responses of the small bronchi isolated from male rats were significantly reduced after 0.12 and 0.5 ppm ozone. Although not significant statistically, a similar trend was observed in airways isolated from female rats. These results suggest that the increase in airway responsiveness associated with acute ozone exposure does not persist during near-lifetime exposure. Although the mechanism responsible for the adaptation to the effects of O3 on airway responsiveness is unknown, the results indicate that smooth muscle cell function was compromised by the chronic exposure. The mechanism(s) responsible for mediating this effect and the relevance of these results to humans remains to be determined.

3,5-Dichloroaniline toxicity in Fischer 344 rats pretreated with inhibitors and inducers of cytochrome P450.

3,5-Dichloroaniline (3,5-DCA), a derivative needed in the manufacture of dyes, pesticides and industrial compounds has been reported to induce renal damage. This study investigated whether pretreatment with inducers or inhibitors of P450 altered 3,5-DCA toxicity. P450 levels were induced in male Fischer 344 rats (4-12/group) by pretreatment (i.p.) with phenobarbital (PB, 75 mg/kg/day for 3 days), beta-naphthoflavone (BNF, 100 mg/kg/day for 4 days) or pyridine (PYR, 100 mg/kg/day for 4 days). P450 activity was inhibited by pretreatment with piperonyl butoxide (PiBx) 30 min prior to injection of 3,5-DCA. Upon completion of a designated pretreatment regimen, 0.4 or 0.8 mmol/kg 3,5-DCA was injected into F344 rats. Pair-fed controls were injected with 25% ethanol solution or physiological saline (2.5 ml/kg). The renal changes monitored at 24 and 48 h following treatment with 0.8 mmol/kg 3,5-DCA were characterized by increased blood urea nitrogen (BUN) level and decreased renal cortical slice accumulation of p-aminohippurate (PAH). Plasma alanine transaminase activity (ALT/GPT) was increased 24 h after injection of 0.8 mmol/kg 3,5-DCA while liver wt. was unchanged. PB or PYR pretreatment did not alter the renal or hepatic effects of 3,5-DCA while BNF pretreatment slightly reduced toxicity. In contrast, PiBx pretreatment increased the renal and hepatic changes associated with 3,5-DCA. The results with PiBx suggest that either the parent compound possesses some direct cytotoxicity or that a toxic metabolite was generated through a biotransformation pathway not inhibited by PiBx.

Role of chloride groups in the nephrotoxic potential of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide, an oxidative metabolite of N-(3,5-dichlorophenyl)succinimide.

Although the addition of chloride groups to the phenyl ring of N-phenylsuccinimide (NPS) is known to enhance the nephrotoxic potential of NPS, the mechanism of this enhancement is unknown. One chlorinated NPS derivative, N-(3,5-dichlorophenyl)succinimide (NDPS), is a potent nephrotoxicant which induces marked proximal tubular necrosis at i.p. doses of 0.4 mmol/kg or greater. The purpose of this study was to compare the nephrotoxic potential of 2-hydroxy-N-phenylsuccinimide (HNPS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), an oxidative and nephrotoxicant metabolite of NDPS, to determine the importance of the chloride groups for the nephrotoxic potential of NDHS. Male Fischer 344 rats (4/group) were administered a single i.p. injection of HNPS (1.0 or 1.5 mmol/kg), NDHS (0.1 mmol/kg) or vehicle (25% dimethyl sulfoxide in sesame oil), and renal function measured at 24 and 48 h. HNPS was a nonnephrotoxicant at both doses tested, while NDHS induced marked nephrotoxicity characterized by diuresis, increased proteinuria, glucosuria, elevated blood urea nitrogen (BUN) concentration and kidney weight, decreased organic ion accumulation by renal cortical slices and proximal tubular necrosis. In vitro, HNPS reduced p-aminohippurate (PAH) and tetraethylammonium (TEA) accumulation beginning at HNPS bath concentrations of 0.05 and 0.5 mM, respectively. The results of this study indicate that although HNPS has direct effects on renal function in vitro, HNPS is not a nephrotoxicant in vivo at doses up to 15 times the minimal nephrotoxicant dose of NDHS. Therefore, the chloro groups present on NDHS play an essential role in the nephrotoxic potential of NDHS and contribute to aspects of the nephrotoxic mechanism of NDPS beyond NDPS oxidation to form NDHS.

Cephaloridine nephrotoxicity in diabetic rats: modulation by insulin treatment.

Previous studies have indicated that cephaloridine nephrotoxicity was reduced in diabetic rats. This study determined whether the reduction in toxicity was due to streptozotocin or the diabetic state. Male Fischer-344 rats were injected intraperitoneally with 35 mg/kg streptozotocin to induce diabetes. Insulin (5 U/day, subcutaneously) was begun within 72 h and continued for 10 days. Toxicity was quantitated 48 h after injection of cephaloridine (1500 mg/kg, i.p.) in normoglycemic (NC), diabetic (DC) and diabetic animals treated with insulin (DIC). Cephaloridine produced diuresis, glucosuria, proteinuria, elevated kidney weight and decreased renal cortical slice accumulation of organic ions in the NC group. Cephaloridine toxicity was reduced in the DC group since kidney weight, BUN level and renal cortical slice accumulation of organic anions were similar between treated and control animals. Cephaloridine treatment of the DIC group was associated with increased BUN levels, proteinuria and diminished renal cortical slice accumulation of organic cations. These results indicated that the diabetic state, and not streptozotocin, reduced cephaloridine nephrotoxicity.

Release of prostaglandin E2 and leukotriene C4/D4 from airway segments isolated from rats after exposure to ozone for 20 months.

Metabolites of arachidonic acid have been implicated as mediators of some of the pulmonary effects observed after acute exposure to ozone. Accordingly, recent studies have focused on the effects of acute ozone exposure on the arachidonic acid cascade, however, whether eicosanoid metabolism is altered after chronic exposure to ozone is unknown. To begin to address this issue, we examined the effects of near-lifetime exposure to ozone on release of prostaglandin E2 (PGE2) and leukotriene C4/D4 from airway segments isolated from exposed Fischer-344 rats. Airway segments representing approximately eighth to tenth generation airways were isolated from rats of both genders that had been exposed for 6 h per day, 5 days per week for 20 months to filtered air or 0.12, 0.5 or 1.0 parts per million (ppm) ozone. Basal and stimulated release of eicosanoids were measured in the medium surrounding airway segments using enzymoimmunoassay. Basal release of PGE2 was detected in the medium surrounding airway segments and this release was unaffected by ozone exposure. Incubation of the segments with the calcium ionophore, A23187, increased the release of the prostaglandin; the A23187-induced release of PGE2 was significantly enhanced in airway segments isolated from rats in the 1.0 ppm exposure group. Basal release of leukotriene C4/D4 was not detected in the medium surrounding airway segments regardless of ozone exposure. Measurable amounts of the leukotriene were released during incubation with A23187, however, ozone was without affect on these levels. The results suggest that the cyclooxygenase pathway of the arachidonic acid cascade appears to be affected by ozone exposure. Which of the processes of prostaglandin production and release are affected by chronic ozone exposure remains to be determined.