Renal cysteine conjugate C-S lyase mediated toxicity of halogenated alkenes in primary cultures of human and rat proximal tubular cells

Abstract

Proximal tubular cells from human (HPT) and rat (RPT) kidneys were isolated, grown to confluence and incubated with S-(1,2-dichlorovinyl)-L-cysteine (DCVC), S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) and S-(2-chloro-1,1-difluorethyl)-L-cysteine (CDFEC), the cysteine conjugates of nephrotoxicants. The cultures were exposed to the conjugates for 12, 24 and 48 h and the toxicity determined using the MTT assay. All four conjugates caused dose-dependent toxicity to RPT cells over the range 50-1,000 μM, the order of toxicity being DCVC > TCVC > TFEC = CDFEC. The inclusion of aminooxyacetic acid (AOAA; 250 μM), an inhibitor of pyridoxal phosphate-dependent enzymes such as C-S lyase, afforded protection, indicating that C-S lyase has a role in the bioactivation of these conjugates. In HPT cultures only DCVC caused significant time- and dose-dependent toxicity. Exposure to DCVC (500 μM) for 48 h decreased cell viability to 7% of control cell values, whereas co-incubation of DCVC (500 μM) with AOAA (250 μM) resulted in cell viability of 71%. Human cultures were also exposed to S-(1,2-dichlorovinyl)-glutathione (DCVG). DCVG was toxic to HPT cells, but the onset of toxicity was delayed compared with the corresponding cysteine conjugate. AOAA afforded almost complete protection from DCVG toxicity. Acivicin (250 μM), an inhibitor of γ-glutamyl transferase (γ-GT), partially protected against DCVG (500 μM)-induced toxicity at 48 h (5% viability and 53% viability in the absence and presence of acivicin, respectively). These results suggest that DCVG requires processing by γ-GT prior to bioactivation by C-S lyase in HPT cells. The activity of C-S lyase, using TFEC as a substrate, and glutamine transaminase K (GTK) was measured in rat and human cells with time in culture. C-S lyase activity in RPT and HPT cells decreased to approximately 30% of fresh cell values by the time the cells reached confluence (120 h), whereas the decline in GTK activity was less marked (50% of the fresh cell values at confluence). Rat cells had threefold higher activity than human cells at each time point. This higher activity may partly explain the differences in toxicity between rat and human proximal tubular cells in culture.