In Vitro Inhibition of Mammalian Glutathione Transferases by Selected Nitrobenzenes

Abstract

Five structurally related nitrobenzenes (1,2-dinitrobenzene, 1,3-dinitrobenzene, 1,4-dinitrobenzene, 1,3,5-trinitrobenzene, and picric acid) and Meisenheimer complex [1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate] were evaluated as possible inhibitors of affinity purified mammalian glutathione transferases (GSTs) isolated from human liver or human term placenta and rat fiver. The results suggest that the degree of GST inhibition depends upon both the chemical in question and the enzyme source. Among the nitrobenzenes tested, 1,3,5-trinitrobenzene (TNB) was found to be the most potent inhibitor of GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) from all the sources, whereas 1,3-dinitrobenzene (1,3-DNB) was the least effective. TNB-caused inhibition of GST activity toward CDNB appeared to be isozyme specific in that compared to the enzyme from human term placenta (GSTP1–1), the degree of inhibition of the mixture of GST isozymes present in the fivers of adult rats and humans was low. The enzyme assays conducted with 3,4-dichloro-1-nitrobenzene (DCNB), ethacrynic acid (EA), and 4-nitropyridinei N-oxide also suggested the isozymespecific inhibition of rat fiver GST activity by TNB. The nature of TNB-caused inhibition of GSTP1–1 was competitive with respect to CDNB and yielded a Ki value of 12.5 θ M. With EA, a specific substrate for GSTP1–1, an IC50 value of ∼ 16 θ M was estimated for the GSTP1–1 inhibition by TNB. The Meisenheimer complex, the product of nonenzymatic GSH conjugation with TNB by different GSTs, was found to be the most potent inhibitor of mammalian GSTs, and IC50 values ranged between 1 and 4 θ M when the enzyme activity was assayedusing CDNB. The nature of GSTP1–1 inhibition was noncompetitive with respect to CDNB, with a Ki value of 1 θ M for Meisenheimer complex. Although a precise mechanism was not identified, it is postulated that GSH depletion and/or GST inhibition may contribute, at least partly, to the target organ toxicity caused by exposures of animals to different nitrobenzenes reported in the literature.