Mercuric ions are highly reactive and form a variety of organic complexes or conjugates in vivo. The renal proximal tubule is a primary target for mercury uptake and toxicity, and circumstantial evidence implicates organic anion transporters in these processes. To test this hypothesis directly, the transport and toxicity of mercuric-thiol conjugates were characterized in a Madin-Darby canine kidney cell line stably transfected with the human organic anion transporter 1 (hOAT1). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-terazolium bromide assays (for mitochondrial dehydrogenase) confirmed that mercuric conjugates of the thiols N-acetylcysteine (NAC), cysteine, or glutathione were more toxic in hOAT1-transfected cells than in the non-transfected cells. The NAC-Hg2+ conjugate was most cytotoxic, inducing greater than 50% cellular death over 18 h at a concentration of 100 muM. The cytotoxic effects were fully reversed by probenecid (an OAT1 inhibitor) and partially reversed by p-aminohippurate (an OAT1 substrate). Toxicity of this conjugate was reduced by the OAT1-exchangeable dicarboxylates alpha-ketoglutarate, glutarate, and adipate, but not by succinate, a nonexchangeable dicarboxylate. 203 Hg-uptake studies showed probenecid-sensitive uptake of mercury-thiol conjugates in the hOAT1-transfected cells. The apparent Km for the NAC-Hg2+ conjugate was 44 +/- 9 muM. Uptake of the NAC-Hg2+ conjugate was cis-inhibited by glutarate, but not by methylsuccinate, paralleling their effects on toxicity. Probenecid-sensitive transport of the NAC-Hg2+ conjugate was also shown to occur in Xenopus laevis oocytes expressing the hOAT1 or the rOAT3 transporters, suggesting that OAT3 may also transport thiol-Hg2+ conjugates. Thus, renal accumulation and toxicity of thiol-Hg2+ conjugates may depend in part on the activity of the organic transport system.
