DNA–Protein Crosslinks Induced by Nickel Compounds in Isolated Rat Lymphocytes: Role of Reactive Oxygen Species and Specific Amino Acids

Abstract

Isolated rat lymphocytes in salts-glucose medium (pH 7.2) were incubated with nickel chloride, nickel acetate, nickel sulfate, and a soluble form of nickel subsulfide (0–2 mM) at 37°C for 2 h. The soluble form of nickel subsulfide induced a significant increase in DNA–protein crosslinks (DPXLs) (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 sulfate produced only a 65% increase of such crosslinks at the 2 mM concentration only. No significant reduction in viability of rat lymphocytes (as measured by trypan blue exclusion) due to these nickel compounds was observed at any concentration used. Time-course studies of DPXLs and cellular viability due to 2 mM nickel subsulfide indicate that DPXL formation may not be due in 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) significantly reduced the DPXLs induced by 2 mM nickel subsulfide. But Mg2+ even at 24 mM failed to antagonize nickel subsulfide-induced increase in DPXLs. High concentrations of these amino acids significantly decreased the accumulation of Ni2+ from nickel subsulfide in lymphocytes, suggesting that such reduction of cellular uptake of Ni2+ by these amino acids is partly responsible for the potent protective effects of these amino acids against such genotoxicity of nickel subsulfide. In vitro exposure of lymphocytes to nickel subsulfide (0–2 mM) increased the formation of reactive oxygen species (ROS) in a concentration-dependent manner. Furthermore, coincubation of 2 mM nickel subsulfide with catalase, dimethylthiourea, mannitol, or vitamin C at 37°C for 2 h resulted in a significant decrease of nickel subsulfide-induced formation of DPXLs, suggesting that nickel subsulfide-induced DPXLs formation in isolated rat lymphocytes is caused by the formation of ROS. 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+-induced DPXL formation in rat lymphocytes is caused by the induction of Fenton/Haber–Weiss reaction, generating hydroxyl radicals. The potent protective effects of these specific amino acids against nickel subsulfide-induced DPXL formation in isolated rat lymphocytes may be due in part to impaired cellular uptake of Ni2+, inhibition of the binding of Ni2+ to deproteinized DNA, and a reduction in reactive oxygen species.