Nephrotoxic Potential of Putative 3,5-Dichloroaniline (3,5-DCA) Metabolites and Biotransformation of 3,5-DCA in Isolated Kidney Cells from Fischer 344 Rats.

The current study was designed to explore the in vitro nephrotoxic potential of four 3,5-dichloroaniline (3,5-DCA) metabolites (3,5-dichloroacetanilide, 3,5-DCAA; 3,5-dichlorophenylhydroxylamine, 3,5-DCPHA; 2-amino-4,6-dichlorophenol, 2-A-4,6-DCP; 3,5-dichloronitrobenzene, 3,5-DCNB) and to determine the renal metabolism of 3,5-DCA in vitro. In cytotoxicity testing, isolated kidney cells (IKC) from male Fischer 344 rats (~4 million/mL, 3 mL) were exposed to a metabolite (0-1.5 mM; up to 90 min) or vehicle. Of these metabolites, 3,5-DCPHA was the most potent nephrotoxicant, with 3,5-DCNB intermediate in nephrotoxic potential. 2-A-4,6-DCP and 3,5-DCAA were not cytotoxic. In separate experiments, 3,5-DCNB cytotoxicity was reduced by pretreating IKC with antioxidants and cytochrome P450, flavin monooxygenase and peroxidase inhibitors, while 3,5-DCPHA cytotoxicity was attenuated by two nucleophilic antioxidants (glutathione and N-acetyl-L-cysteine). Incubation of IKC with 3,5-DCA (0.5-1.0 mM, 90 min) produced only 3,5-DCAA and 3,5-DCNB as detectable metabolites. These data suggest that 3,5-DCNB and 3,5-DCPHA are potential nephrotoxic metabolites and may contribute to 3,5-DCA induced nephrotoxicity in vivo. In addition, the kidney can bioactivate 3,5-DCNB to toxic metabolites, and 3,5-DCPHA appears to generate reactive metabolites to contribute to 3,5-DCA nephrotoxicity. In vitro, N-oxidation of 3,5-DCA appears to be the primary mechanism of bioactivation of 3,5-DCA to nephrotoxic metabolites.

Role of Free Radicals and Biotransformation in Trichloronitrobenzene-Induced Nephrotoxicity In Vitro.

This study determined the comparative nephrotoxic potential of four trichloronitrobenzenes (TCNBs) (2,3,4-; 2,4,5-; 2,4,6-; and 3,4,5-TCNB) and explored the effects of antioxidants and biotransformation inhibitors on TCNB-induced cytotoxicity in isolated renal cortical cells (IRCC) from male Fischer 344 rats. IRCC were incubated with a TCNB up to 1.0 mM for 15-120 min. Pretreatment with an antioxidant or cytochrome P450 (CYP), flavin monooxygenase (FMO), or peroxidase inhibitor was used in some experiments. Among the four TCNBs, the order of decreasing nephrotoxic potential was approximately 3,4,5- > 2,4,6- > 2,3,4- > 2,4,5-TCNB. The four TCNBs exhibited a similar profile of attenuation of cytotoxicity in response to antioxidant pretreatments. 2,3,4- and 3,4,5-TCNB cytotoxicity was attenuated by most of the biotransformation inhibitors tested, 2,4,5-TCNB cytotoxicity was only inhibited by isoniazid (CYP 2E1 inhibitor), and 2,4,6-TCNB-induced cytotoxicity was inhibited by one CYP inhibitor, one FMO inhibitor, and one peroxidase inhibitor. All of the CYP specific inhibitors tested offered some attenuation of 3,4,5-TCNB cytotoxicity. These results indicate that 3,4,5-TCNB is the most potent nephrotoxicant, free radicals play a role in the TCNB cytotoxicity, and the role of biotransformation in TCNB nephrotoxicity in vitro is variable and dependent on the position of the chloro groups.

4-Amino-2-chlorophenol: Comparative in vitro nephrotoxicity and mechanisms of bioactivation.

Chlorinated anilines are nephrotoxicants both in vivo and in vitro. The mechanism of chloroaniline nephrotoxicity may occur via more than one mechanism, but aminochlorophenol metabolites appear to contribute to the adverse in vivo effects. The purpose of this study was to compare the nephrotoxic potential of 4-aminophenol (4-AP), 4-amino-2-chlorophenol (4-A2CP), 4-amino-3-chlorophenol (4-A3CP) and 4-amino-2,6-dichlorophenol (4-A2,6DCP) using isolated renal cortical cells (IRCC) from male Fischer 344 rats as the model and to explore renal bioactivation mechanisms for 4-A2CP. For these studies, IRCC (∼4×10(6)cells/ml) were incubated with an aminophenol (0.5 or 1.0mM) or vehicle for 60min at 37°C with shaking. In some experiments, cells were pretreated with an antioxidant or cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), peroxidase or cyclooxygenase inhibitor prior to 4-A2CP (1.0mM). Lactate dehydrogenase (LDH) release served as a measure of cytotoxicity. The order of decreasing nephrotoxic potential in IRCC was 4-A2,6-DCP>4-A2CP>4-AP>4-A3CP. The cytotoxicity induced by 4-A2CP was reduced by pretreatment with the peroxidase inhibitor mercaptosuccinic acid, and some antioxidants (ascorbate, glutathione, N-acetyl-l-cysteine) but not by others (α-tocopherol, DPPD). In addition, pretreatment with the iron chelator deferoxamine, several CYP inhibitors (except for the general CYP inhibitor piperonyl butoxide), FMO inhibitors or indomethacin (a cyclooxygenase inhibitor) failed to attenuate 4-A2CP cytotoxicity. These results demonstrate that the number and ring position of chloro groups can influence the nephrotoxic potential of 4-aminochlorophenols. In addition, 4-A2CP may be bioactivated by cyclooxygenase and peroxidases, and free radicals appear to play a role in 4-A2CP cytotoxicity.

3,4,5-Trichloroaniline nephrotoxicity in vitro: potential role of free radicals and renal biotransformation.

Chloroanilines are widely used in the manufacture of drugs, pesticides and industrial intermediates. Among the trichloroanilines, 3,4,5-trichloroaniline (TCA) is the most potent nephrotoxicant in vivo. The purpose of this study was to examine the nephrotoxic potential of TCA in vitro and to determine if renal biotransformation and/or free radicals contributed to TCA cytotoxicity using isolated renal cortical cells (IRCC) from male Fischer 344 rats as the animal model. IRCC (~4 million cells/mL; 3 mL) were incubated with TCA (0, 0.1, 0.25, 0.5 or 1.0 mM) for 60-120 min. In some experiments, IRCC were pretreated with an antioxidant or a cytochrome P450 (CYP), flavin monooxygenase (FMO), cyclooxygenase or peroxidase inhibitor prior to incubation with dimethyl sulfoxide (control) or TCA (0.5 mM) for 120 min. At 60 min, TCA did not induce cytotoxicity, but induced cytotoxicity as early as 90 min with 0.5 mM or higher TCA and at 120 min with 0.1 mM or higher TCA, as evidenced by increased lactate dehydrogenase (LDH) release. Pretreatment with the CYP inhibitor piperonyl butoxide, the cyclooxygenase inhibitor indomethacin or the peroxidase inhibitor mercaptosuccinate attenuated TCA cytotoxicity, while pretreatment with FMO inhibitors or the CYP inhibitor metyrapone had no effect on TCA nephrotoxicity. Pretreatment with an antioxidant (α-tocopherol, glutathione, ascorbate or N-acetyl-L-cysteine) also reduced or completely blocked TCA cytotoxicity. These results indicate that TCA is directly nephrotoxic to IRCC in a time and concentration dependent manner. Bioactivation of TCA to toxic metabolites by CYP, cyclooxygenase and/or peroxidase contributes to the mechanism of TCA nephrotoxicity. Lastly, free radicals play a role in TCA cytotoxicity, although the exact nature of the origin of these radicals remains to be determined.

Role of leukotrienes in N-(3,5-dichlorophenyl)succinimide (NDPS) and NDPS metabolite nephrotoxicity in male Fischer 344 rats.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) can induce marked nephrotoxicity in rats following a single intraperitoneal (ip) administration of 0.4mmol/kg or greater. Although NDPS induces direct renal proximal tubular toxicity, a role for renal vascular effects may also be present. The purpose of this study was to examine the possible role of vasoconstrictor leukotrienes in NDPS and NDPS metabolite nephrotoxicity. Male Fischer 344 rats (4 rats/group) were administered diethylcarbamazine (DEC; 250 or 500mg/kg, ip), an inhibitor of LTA(4) synthesis, 1h before NDPS (0.4mmol/kg, ip), N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS, 0.1, 0.2, or 0.4mmol/kg, ip), or N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA, 0.1mmol/kg, ip) or vehicle. In a separate set of experiments, the LTD(4) receptor antagonist LY171883 (100mg/kg, po) was administered 0.5h before and again 6h after NDHS (0.1mmol/kg, ip) or 2-NDHSA (0.1mmol/kg, ip) or vehicle. Renal function was monitored for 48h post-NDPS or NDPS metabolite. DEC markedly reduced the nephrotoxicity induced by NDPS and its metabolites, while LY171883 treatments provided only partial attenuation of NDHS and 2-NDHSA nephrotoxicity. These results suggest that leukotrienes contribute to the mechanisms of NDPS nephrotoxicity.

Nephrotoxicity induced by N-(3,5-dichlorophenyl)-3-hydroxysuccinamic acid in male and female Fischer 344 rats.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is a more potent nephrotoxicant in female rats than in males. Similarly, nephrotoxicant NDPS metabolites studied to date in male and female rats have also demonstrated gender differences, being twice as potent as nephrotoxicants in females as in males. The purpose of this study was to examine the nephrotoxic potential of N-(3,5-dichlorophenyl)-3-hydroxysuccinimide (3-NDHSA) in male and female Fisher 344 rats to determine if gender differences in nephrotoxic potential also exist for this metabolite. Rats (four per group) were administered a single intraperitoneal (i.p.) injection of 3-NDHSA (0.1, 0.2 or 0.4 mmol kg(-1)) or vehicle, and renal function was monitored at 24 and 48 h. 3-NDHSA 0.1 mmol kg(-1) did not induce nephrotoxicity in male or female rats. In male rats, 3-NDHSA 0.2 mmol kg(-1) induced mild nephrotoxicity seen as diuresis and transient, mild proteinuria. However, 3-NDHSA 0.4 mmol kg(-1) induced marked nephrotoxicity. In female rats, 3-NDHSA 0.2 mmol kg(-1) induced mild nephrotoxicity, as evidenced by transient diuresis and proteinuria. As in males, 3-NDHSA 0.4 mmol kg(-1) induced marked nephrotoxicity. These results indicate that, unlike NDPS and other nephrotoxic NDPS metabolites, 3-NDHSA does not exhibit gender differences in nephrotoxic potential. In addition, in comparison with NDPS and other nephrotoxic NDPS metabolites, 3-NDHSA is a less potent nephrotoxicant that NDHS or 2-NDHSA and similar to NDPS in nephrotoxic potential in male rats.

Mechanistic aspects of 4-amino-2,6-dichlorophenol-induced in vitro nephrotoxicity.

4-Amino-2,6-dichlorophenol (ADCP) is a potent acute nephrotoxicant in vivo inducing prominent renal corticomedullary necrosis. In vitro, ADCP exposure increases lactate dehydrogenase (LDH) release from rat renal cortical slices at 0.05 mM or greater. The purpose of this study was to examine the ability of antioxidants, cytochrome P450 (CYP) and flavin adenine dinucleotide monooxygenase (FMO) activity modulators, indomethacin, glutathione and inhibitors of glutathione conjugate metabolism to attenuate ADCP cytotoxicity in vitro. Renal cortical slices prepared from untreated male Fischer 344 rats (N=4/group) were preincubated at 37 degrees C under a 100% oxygen atmosphere with an inhibitor or vehicle for 5-30 min. ADCP (0.05-0.5mM) or vehicle was added and incubations continued for 120 min. At the end of the incubation period, LDH release was measured as an index of nephrotoxicity. ADCP cytotoxicity was partially attenuated by ascorbate (1.0 or 2.0mM), but not by N,N'-diphenyl-p-phenylenediamine (DPPD), alpha-tocopherol or deferoxamine. Inhibitors of CYP (metyrapone, piperonyl butoxide and isoniazid) and FMO activity modulators (methimazole, N-octylamine) had no effect on ADCP cytotoxicity. Indomethacin or glutathione 1.0mM completely and partially blocked ADCP 0.1 and 0.5mM cytotoxicity, respectively. N-acetylcysteine, AOAA (an inhibitor of cysteine conjugate beta-lyase) and probenecid (an organic anion transport inhibitor), but not AT-125 (an inhibitor of gamma-glutamyl transferase), partially attenuated ADCP 0.1mM cytotoxicity. Overall, these results suggest that reactive metabolites may be produced from ADCP primarily via a co-oxidation-mediated mechanism. The difference in the ability of ascorbate and glutathione to attenuate ADCP-induced cytotoxicity in vitro in kidney cells could indicate that alkylation via the reactive benzoquinoneimine metabolite might be responsible for cytotoxicity rather than a free radical-mediated mechanism.

In vitro nephrotoxicity induced by propanil.

Propanil is a postemergence herbicide used primarily in rice and wheat production in the United States. The reported toxicities for propanil exposure include methemoglobinemia, immunotoxicity, and nephrotoxicity. A major metabolite of propanil, 3,4-dichloroaniline (3,4-DCA), has been shown to be a nephrotoxicant in vivo and in vitro, but the nephrotoxic potential of propanil has not been examined in detail. The purpose of this study was to determine the nephrotoxic potential of propanil using an in vitro kidney model, determine whether in vitro propanil nephrotoxicity is due to metabolites arising from propanil hydrolysis, and examine mechanistic aspects of propanil nephrotoxicity in vitro. Propanil, 3,4-DCA, propionic acid (0.1-5.0 mM), or vehicle was incubated for 15-120 min with isolated renal cortical cells (IRCC; approximately 4 million cells/mL) obtained from untreated male Fischer 344 rats. Cytotoxicity was determined by measuring lactate dehydrogenase release from IRCC. In 120-min incubations, propanil induced cytotoxicity at concentrations >0.5 mM. At 1.0 mM, propanil induced cytotoxicity following 60- or 120-min exposure. Cytotoxicity was observed with 3,4-DCA (2.0 mM) at 60 and 120 min, while propionic acid (5.0 mM) induced cytotoxicity at 60 min. In IRCC pretreated with an antioxidant, cytochrome P450(CYP) inhibitor, flavin adenine dinucleotide monooxygenase activity modulator, or cyclooxygenase inhibitor before propanil exposure (1.0 mM; 120 min), only piperonyl butoxide (0.1 mM), a CYP inhibitor, pretreatment decreased propanil cytotoxicity. These results demonstrate that propanil is an in vitro nephrotoxicant in IRCC. Propanil nephrotoxicity is not primarily due to metabolites resulting from hydrolysis of propanil, but a metabolite resulting from propanil oxidation may contribute to propanil cytotoxicity.

Nephrotoxicity induced by the R- and S-enantiomers of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and their sulfate conjugates in male Fischer 344 rats.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity characterized as polyuric renal failure and mediated via metabolites arising from oxidation of the succinimide ring. Recent findings have suggested that the stereochemical nature of NDPS metabolites may be an important factor in NDPS metabolite-induced nephrotoxicity. The purpose of the present study was to determine the role of stereochemistry in the in vivo nephrotoxicity induced by R-(+)- and S-(-)-N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (R- and S-NDHS) and the in vitro nephrotoxicity induced by their enantiomeric sulfate conjugates, R-(-)- and S-(+)-N-(3,5-dichlorophenyl)-2-hydroxysuccinimide-O-sulfate (R- and S-NSC). Male Fischer 344 rats (four rats/group) were administered intraperitoneally (i.p.) an enantiomer of NDHS (0.05, 0.1 or 0.2 mmol/kg) or vehicle, and renal function monitored for 48 h. R-NDHS (0.1 or 0.2 mmol/kg) had little effect on renal function. In contrast, S-NDHS (0.1 mmol/kg) induced marked nephrotoxicity. The nephrotoxic potential of R- and S-NSC (0.5, 0.75 or 1.0mM) was determined using freshly isolated rat renal cortical cells (IRCC, 3-4 x 10(6)cells/ml). Cytotoxicity was determined by measuring the release of lactate dehydrogenase (LDH) at the end of a 1h incubation period. The LDH release observed in these studies was similar between R- and S-NSC. These results indicate that stereochemistry is an important factor for NDPS metabolite nephrotoxicity and that the role of stereochemistry, at least for NSC, occurs at extra-renal sites.

Effect of three n-acetylamino acids on N-(3,5-dichlorophenyl)succinimide (NDPS) and ndps metabolite nephrotoxicity in Fischer 344 rats.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity in mammals characterized as polyuric renal failure and proximal tubular necrosis. Recent studies have suggested that NDPS-induced nephrotoxicity may be mediated by metabolites arising from the nephrotoxic NDPS metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and/or N-(3,5-dichlorophenyl)-2-succinamic acid (2-NDHSA). The purpose of this study was to examine the effects of N-acetylcysteine (NAC), a nucleophilic agent, and two nonnucleophilic N-acetylamino acids, N-acetylserine (NAS) and N-acetylalanine (NAA), on NDPS and NDPS metabolite-induced nephrotoxicity. Male Fischer 344 rats (4-8/group) were administered intraperitoneally (ip) an N-acetylamino acid (1 mmol/kg) 2 h before an ip injection of NDPS (0.4 mmol/kg), NDHS (0.1 mmol/kg), 2-NDHSA (0.1 mmol/kg), or vehicle. Renal function was then monitored at 24 and 48 h. NAC pretreatment markedly attenuated NDPS-, NDHS-, and 2-NDHSA-mediated nephrotoxicity. The nonnucleophilic N-acetylamino acids (NAS, NAA) only partly reduced NDPS and NDHS nephrotoxicity, and they had little effect on 2-NDHSA nephrotoxicity. These results suggest that reactive NDPS metabolites may be formed from NDHS and 2-NDHSA and that nucleophilic substrates (e.g., NAC) may offer protection from NDPS-induced nephrotoxicity. However, mechanisms other than chemical neutralization of reactive NDPS metabolites may also be contributing to the attenuation of NDPS nephrotoxicity, since nonnucleophilic N-acetylamino acids (e.g., NAA) also provided some protection against NDPS and NDHS nephrotoxicity.

In vitro nephrotoxicity induced by chloronitrobenzenes in renal cortical slices from Fischer 344 rats.

Chloronitrobenzenes are important chemical intermediates in the manufacture of industrial, agricultural and pharmaceutical agents. Toxicity induced by the various chloronitrobenzene isomers in vivo includes hematotoxicity, immunotoxicity, hepatotoxicity and nephrotoxicity. The purpose of the study was to determine the direct nephrotoxic effects of nitrobenzene and ten chlorinated nitrobenzene derivatives using renal cortical slices as the in vitro model. Renal cortical slices were prepared from kidneys of untreated, male Fischer 344 rats and incubated with nitrobenzene (1.0-5.0 mM), a chloronitrobenzene (0.5-5.0 mM) or vehicle for 2 h. At the end of the 2 h incubation, tissue gluconeogenesis capacity (pyruvate-stimulated gluconeogenesis) and lactate dehydrogenase (LDH) release were determined as measures of cellular function and cytotoxicity. Based on decreased pyruvate-stimulated gluconeogenesis and increased LDH release, the order of decreasing nephrotoxic potential was trichloronitrobenzenes>dichloronitrobenzenes>monochloronitrobenzenes>nitrobenzene. Among the mono- and dichloronitrobenzenes, 1-chloro-3-nitrobenzene and 3,4-dichloronitrobenzene were the most potent nephrotoxicants, while the two trichloronitrobenzenes tested exhibited similar nephrotoxic potentials. These results demonstrate that chloronitrobenzenes are directly nephrotoxic in vitro and that increasing the number of chloro groups increases the nephrotoxic potential of the resulting chloronitrobenzene derivative.