DNA-protein crosslinks induced by nickel compounds in isolated rat lymphocytes: Role of reactive oxygen species and specific amino acids

Isolated rat lymphocytes in salts-glucose medium (pH 7.2) were incubated wi th nickel chloride, nickel acetate, nickel sulfate, and a soluble form of n ickel subsulfide (0-2 mM) at 37 degreesC: for 2 h. The soluble form of nick el subsulfide induced a significant increase in DNA-protein crosslinks (DPX Ls) (111%) beginning at 0.5 mM and a maximum increase of 700% from that of the control value was reached at a 2 mM concentration, whereas nickel sulfa te produced only a 65% increase of such crosslinks at the 2 mM concentratio n only. No significant reduction in viability of rat lymphocytes las measur ed by trypan blue exclusion) due to these nickel compounds was observed at any concentration used. Timecourse studies of DPXLs and cellular viability due to 2 mM nickel subsulfide indicate that DPXL formation may not be due i n part to cellular necrosis, Coincubation of nickel subsulfide (2 mM) with L-histidine (16 mM), L-cysteine (4 or 8 mM), or L-aspartic acid (24 mM) sig nificantly reduced the DPXLs induced by 2 mM nickel subsulfide. But Mg2+ ev en at 24 mM failed to antagonize nickel subsulfide-induced increase in DPXL s, High concentrations of these amino acids significantly decreased the acc umulation of Ni2+ from nickel subsulfide in lymphocytes, suggesting that su ch reduction of cellular uptake of Ni2+ by these amino acids is partly resp onsible for the potent protective effects of these amino acids against such genotoxicity of nickel subsulfide. In vitro exposure of lymphocytes to nic kel subsulfide (0-2 mM) increased the formation of reactive oxygen species (ROS) in a concentration-dependent manner. Furthermore, coincubation of 2 m M nickel subsulfide with catalase, dimethylthiourea, mannitol, or vitamin C at 37 degreesC for 2 h resulted in a significant decrease of nickel subsul fide-induced formation of DPXLs, suggesting that nickel subsulfide-induced DPXLs formation in isolated rat lymphocytes is caused by the formation of R OS, The amino acid treatment also abrogated Ni3S2-induced generation of ROS , Deferoxamine (a highly specific iron chelator) treatment prevented nickel subsulfide-induced DNA-protein crosslink formation, suggesting that Ni2+-i nduced DPXL formation in rat lymphocytes is caused by the induction of Fent on/Haber-Weiss reaction, generating hydroxyl radicals. The potent protectiv e effects of these specific amino acids against nickel subsulfide-induced D PXL formation in isolated rat lymphocytes may be due in part to impaired ce llular uptake of Ni2+ inhibition of the binding of Ni2+ to deproteinized DN A, and a reduction in reactive oxygen species. (C) 2001 Academic Press.