Among the mono- and dichloroanilines, 3,5-Dichloroaniline (3,5-DCA) is the most potent nephrotoxicant in vivo and in vitro. However, the role of renal biotransformation in 3,5-DCA induced nephrotoxicity is unknown. The current study was designed to determine the in vitro nephrotoxic potential of 3,5-DCA in isolated renal cortical cells (IRCC) obtained from male Fischer 344 rats, and the role of renal bioactivation and oxidative stress in 3,5-DCA nephrotoxicity. IRCC (~4 million cells/ml) from male rats were exposed to 3,5-DCA (0-1.0 mM) for up to 120 min. In IRCC, 3,5-DCA was cytotoxic at 1.0 mM by 60 min as evidenced by the increased release of lactate dehydrogenase (LDH), but 120 min was required for 3,5-DCA 0.5 mM to increase LDH release. In subsequent studies, IRCC were exposed to a pretreatment (antioxidant or enzyme inhibitor) prior to exposure to 3,5-DCA (1.0 mM) for 90 min. Cytotoxicity induced by 3,5-DCA was attenuated by pretreatment with inhibitors of flavin-containing monooxygenase (FMO; methimazole, N-octylamine), cytochrome P450 (CYP; piperonyl butoxide, metyrapone), or peroxidase (indomethacin, mercaptosuccinate) enzymes. Use of more selective CYP inhibitors suggested that the CYP 2C family contributed to 3,5-DCA bioactivation. Antioxidants (glutathione, N-acetyl-L-cysteine, α-tocopherol, ascorbate, pyruvate) also attenuated 3,5-DCA nephrotoxicity, but oxidized glutathione levels and the oxidized/reduced glutathione ratios were not increased. These results indicate that 3,5-DCA may be activated via several renal enzyme systems to toxic metabolites, and that free radicals, but not oxidative stress, contribute to 3,5-DCA induced nephrotoxicity in vitro.

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; ~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.

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.

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 (~4×106 cells/ml) were incubated with an aminophenol (0.5 or 1.0 mM) or vehicle for 60 min at 37° C with shaking. In some experiments, cells were pretreated with an antioxidant or cytochrome P450 (CYP), flavin monooxygenase (FMO), peroxidase or cyclooxygenase inhibitor prior to 4-A2CP (1.0 mM). 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.

Chlorinated anilines are commonly used in the production of agricultural products. The purpose of this study was to explore the role of renal biotransformation in 3,4,5‐trichloroaniline (3,4,5‐TCA) nephrotoxicity using an in vitro model. Isolated renal cortical cells (IRCC; 3 ml: 4.0 X 106 cells/ml) from male Fischer 344 rats were treated with 0.5 mM 3,4,5‐TCA or DMSO (vehicle control) for 120 minutes. Cytotoxicity was determined using lactate dehydrogenase (LDH) release assays. In some experiments, cells were pretreated with either an antioxidant [ascorbate (2.0 mM), glutathione (1.0 mM), N‐acetyl‐L‐cysteine (2.0 mM) or alpha‐tocopherol (1.0 mM)], a flavin monooxygenase (FMO) inhibitor [methimazole (1.0 mM), or N‐octylamine (1.0)], a cytochrome P450 (CYP) inhibitor [metyrapone (1.0 mM) or piperonyl butoxide (PIBX; 1.0 mM)], a cyclooxygenase inhibitor [indomethacin (1.0 mM)] or a peroxidase inhibitor (mercaptosuccinate, 0.1 mM). 3,4,5‐TCA nephrotoxicity was not attenuated by pretreatment with methimazole or N‐octylamine, suggesting that FMO mediated oxidation doesn’t play a significant bioactivating role in 3,4,5‐TCA nephrotoxicity. Pretreatment with indomethacin or mercaptosuccinate significantly attenuated toxicity. The CYP inhibitor PiBx, but not metyrapone, also significantly attenuated 3,4,5‐TCA toxicity. All antioxidant pretreatments significantly attenuated 3,4,5‐TCA nephrotoxicity. These results suggest that free radicals play a role in 3,4,5‐TCA nephrotoxicity in vitro, and that these species arise from biotransformation of 3,4,5‐TCA by cyclooxygenase and peroxidase activity and/or via CYP isozymes inhibited by PiBx but not metyrapone.