2-Amino-5-chlorophenol toxicity in renal cortical slices from Fischer 344 rats: effect of antioxidants and sulfhydryl agents.

2-Amino-5-chlorophenol is nephrotoxic through an unidentified mechanism. This study examined the in vitro toxicity of 2-amino-5-chlorophenol in renal cortical slices from Fischer 344 rats and specifically assessed induction of lipid peroxidation and depletion of renal glutathione. Renal cortical slices exposed to 0, 0.25, 0.5, and 1 mM 2-amino-5-chlorophenol exhibited a concentration- and time-dependent increase in lactate dehydrogenase (LDH) leakage. Pyruvate-directed gluconeogenesis was diminished in a concentration-dependent manner following a 90-min incubation with 0, 0.25, 0.5, and 1 mM 2-amino-5-chlorophenol. Lipid peroxidation was induced within 60 min by 1 mM 2-amino-5-chlorophenol in renal slices relative to control tissue. Total glutathione (GSH) levels were decreased below control values within 30 min of exposure to 0.5 and 1 mM 2-amino-5-chlorophenol. These results indicated that GSH levels were decreased prior to the appearance of increased LDH leakage and diminished membrane integrity. 2-Amino-5-chlorophenol toxicity was increased in renal slices isolated from animals pretreated with buthionine sulfoximine (BSO, 890 mg/kg ip). Pretreatment of renal slices with the phenolic antioxidant N,N'-diphenyl-1, 4-phenylenediamine (DPPD, 50 microM) or the iron chelator deferoxamine did not reduce 2-amino-5-chlorophenol cytotoxicity. These results suggest that 2-amino-5-chlorophenol toxicity was not mediated through an iron-dependent mechanism. 2-Amino-5-chlorophenol cytotoxicity was reduced by a 15-min pre-incubation with 2 mM ascorbate or a 30-min preincubation with the thiol-containing agents GSH (1 mM) or dithiothreitol (1 mM, DTT). Pretreatment with GSH, DTT, or ascorbate reduced LDH leakage and lipid peroxide generation induced by 2-amino-5-chlorophenol. These results suggest that 2-amino-5-chlorophenol cytotoxicity involved free radical generation through an iron-independent mechanism. Toxicity was reduced by the presence of the antioxidant ascorbate or by addition of glutathione.

Cephaloridine in vitro toxicity and accumulation in renal slices from normoglycemic and diabetic rats.

Previous work has shown a reduction in cephaloridine nephrotoxicity in a diabetic rat model. The following studies examined in vitro cephaloridine toxicity in renal slices from normoglycemic and diabetic Fischer 344 rats. Diabetes was induced by acute intraperitoneal injection of 35 mg/kg streptozotocin. Renal cortical slices were isolated from normoglycemic and diabetic animals. Tissues were exposed to 0-5 mM cephaloridine for 15-120 min. Pyruvate-directed gluconeogenesis was diminished in all groups exposed to 2-5 mM cephaloridine for 60-120 min. Leakage of lactate dehydrogenase (LDH) was apparent only in the normoglycemic group in the presence of 4-5 mM cephaloridine for 120 min. LDH leakage was not increased at any cephaloridine concentration in the diabetic tissue. Total glutathione levels were compared in renal cortical slices exposed to cephaloridine for 30-120 min. Baseline values for glutathione were comparable between normoglycemic and diabetic tissue suggesting that the mechanism for reduced toxicity was not due to higher glutathione levels in diabetic tissue. Total glutathione levels were diminished more rapidly in normoglycemic than diabetic tissue by incubation with 5 mM cephaloridine. Comparison of cephaloridine accumulation indicated that diabetic tissue accumulated less cephaloridine than the normoglycemic group when tissues were incubated with 0-2 mM cephaloridine. However, renal slice accumulation was similar between normoglycemic and diabetic groups following in vitro incubation with 4-5 mM cephaloridine. These results suggest that the mechanism for reduced in vitro cephaloridine toxicity in diabetic tissue cannot be limited to differences in accumulation and must include an unidentified cellular component.

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.

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.