Roles of Necrosis, Apoptosis, and Mitochondrial Dysfunction inS-(1,2-Dichlorovinyl)-L-cysteine Sulfoxide-Induced Cytotoxicity in Primary Cultures of Human Renal Proximal Tubular Cells

Abstract

S-(1,2-Dichlorovinyl)-L-cysteine (DCVC)is the penultimate nephrotoxic metabolite of the environmental contaminant trichloroethylene. Although metabolism of DCVC by the cysteine conjugate ␤-lyase is the most studied bioactivation pathway, DCVC may also be metabolized by the flavin-containing monooxygenase (FMO) to yield DCVC sulfoxide (DCVCS). Renal cellular injury induced by DCVCS was investigated in primary cultures of human proximal tubular (hPT) cells by assessment of time-and concentration-dependent effects on cellular morphology, acute cellular necrosis, apoptosis, mitochondrial function, and cellular glutathione (GSH) status. Confluent hPT cells incubated with as little as 10 M DCVCS for 24 h exhibited morphological changes, although at least 100 M DCVCS was required to produce marked changes. Acute cellular necrosis did not occur until 48 h with at least 200 M DCVCS, indicating that this is a high-dose, late response. The extent of necrosis was similar to that with DCVC. In contrast, apoptosis occurred as early as 1 h with as little as 10 M DCVCS and the extent of apoptosis was much less than that with DCVC. Mitochondrial function was maintained with DCVCS concentrations up to 100 M, consistent with hPT cells only being competent to undergo apoptosis at early time points and relatively low concentrations. Marked depletion (Ͼ50%) of cellular GSH content was only observed with 500 M DCVCS. These results, combined with previous studies showing protection from DCVC-induced necrosis and apoptosis by the FMO inhibitor methimazole, suggest that formation of DCVCS plays a significant role in trichloroethylene-induced renal cellular injury in hPT cells.

Role of mitochondrial dysfunction in cellular responses to S-(1,2-dichlorovinyl)-l-cysteine in primary cultures of human proximal tubular cells

“…Figure 6). As found previously in hPT cells [32][33][34], rPT cells [9,[12][13][14][15][17][18][19][20]26,31], and rabbit proximal tubules [21][22][23][24][25], hPT cells in the current study exhibited decreases in respiration. Modest effects were observed at 10 μM DCVC and none were detected at 1 or 5 μM DCVC.…”

“…Mechanistic studies on the responses of hPT cells to DCVC revealed many of the same effects as observed in rPT cells, including necrosis, apoptosis, repair, proliferation, and mitochondrial dysfunction [32][33][34]. Although it is clear that, like in rPT cells, mitochondria are early and prominent intracellular targets of DCVC in hPT cells, we do not know whether induction of mitochondrial dysfunction is obligatory for subsequent cytotoxicity or whether other mechanisms are important in the various responses of hPT cells to DCVC.…”

“…hPT cells were isolated from fresh human kidneys by a modification of the procedure described by Todd et al [42] as described previously [30,[32][33][34]43]. Cells were identified as being derived from the proximal tubules by their high activities of brush-border membrane enzymes (i.e., alkaline phosphatase and γ-glutamyltransferase) and low activity of the glycolytic enzyme hexokinase, as described previously [9,26,[30][31][32][33][34]43,44].…”

“…Cells were identified as being derived from the proximal tubules by their high activities of brush-border membrane enzymes (i.e., alkaline phosphatase and γ-glutamyltransferase) and low activity of the glycolytic enzyme hexokinase, as described previously [9,26,[30][31][32][33][34]43,44]. Human kidneys were purchased from Life Legacy Foundation (Tucson, AZ; formerly International Bioresearch Solutions) or the International Institute for the Advancement of Medicine (Edison, NJ).…”

“…Only cells that excluded > 90% trypan blue or exhibit < 10% leakage of LDH were used for subsequent primary culture. Basal medium and supplements for hPT primary culture were as described previously [32][33][34]43]. Basal medium was a DMEM:F-12 (1:1) without serum.…”