Nephrotoxic Potential of the Three Dichlorobenzene Isomers in Isolated Kidney Cells from Male Fischer 344 Rats

Abstract

Dichlorobenzenes (DCBs) are commonly used as chemical intermediates in the manufacture of a wide range of agricultural, industrial and pharmaceutical products. Previous studies have shown that 1,4-DCB can induce nephrotoxicity in male rats characterized as α2u-globulin nephropathy. Initially, 1,4-DCB binds to α2u-globulin at extra-renal sites. The 1,4-DCB-protein complex then accumulates in kidney and causes protein droplet formation, ultimately leading to cell death and/or eventually tumor formation. The purpose of this study was to explore the direct cytotoxic effects of the three DCB isomers in isolated kidney cells (IKCs) from male Fischer 344 rats. IKCs (~4 million/ml, 3 ml) were incubated with a DCB (0.25-1.0 mM) or dimethyl sulfoxide (DMSO, vehicle) for 30 or 60 min with shaking at 37oC under a 95% oxygen/5% carbon dioxide atmosphere. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release. In separate experiments, IKCs were pretreated with an antioxidant (ascorbate, 1.0 mM; N-acetyl-L-cysteine, 2.0 mM; glutathione, 1.0 mM) prior to the addition of 1,4-DCB (0.5 mM) and followed by a 60 min co-incubation. 1,4-DCB increased LDH release at all times and concentrations tested. 1,3-DCB increased LDH release at 0.5 mM or greater at both time points, while 1,2-DCB only increased LDH release at 0.5 mM or greater at 60 min. Thus, the order of decreasing nephrotoxic potential was 1,4-DCB > 1,3- DCB > 1,2-DCB. Pretreatment with any of the antioxidants tested attenuated 1,4-DCB nephrotoxicity. These results indicate that DCBs can induce nephrotoxicity in vitro without the presence of extra-renal α2u-globulin, 1,4-DCB is the most potent nephrotoxicant among the three DCB isomers in IKCs, and free radicals play a role in DCB nephrotoxicity.