DNA–Protein Crosslinks Induced by Nickel Compounds in Isolated Rat Renal Cortical Cells and Its Antagonism by Specific Amino Acids and Magnesium Ion

Abstract

Suspensions of isolated renal cortical cells in modified Krebs-Henseleit buffer (pH 7.4) were incubated with nickel chloride, nickel acetate, nickel sulfate, and nickel subsulfide (0–2 mM) at 37°C for 2 h. A significant increase (63%) in DNA–protein crosslinks was observed at 2 mM nickel sulfate, whereas nickel subsulfide induced a significant increase in such crosslinks beginning at 0.5 mM concentration and a maximum increase of 200% of the control value reached at 2 mM concentration. No significant reduction in viability of renal cortical cells (as measured by trypan blue exclusion) was observed due to these nickel compounds at any concentration used. In the second series of experiments, coincubation of nickel subsulfide (2 mM) withl-histidine (8 or 16 mM),l-cysteine (4 or 8 mM), orl-aspartic acid (8 or 24 mM) significantly reduced the DNA–protein crosslinks induced by 2 mM nickel subsulfide. Similarly Mg2+(24 mM), but not Ca2+(24 mM), was able to antagonize nickel subsulfide-induced increase in DNA–protein crosslinks. High extracellular levels of Mg2+and these amino acids significantly decreased the accumulation of Ni2+from nickel subsulfide in renal cortical cells. Furthermore, these amino acids at high concentrations significantly inhibited the binding of Ni2+from nickel subsulfide to deproteinized DNA from renal cortical cells, whereas such inhibition due to Mg2+was close to significant (0.1 >p> 0.05).In vitroexposures of renal cortical cells to nickel subsulfide (0–2 mM) increased the formation of reactive oxygen species in concentration-dependent manner. Furthermore, coincubation of 2 mM nickel subsulfide with either catalase, dimethylthiourea, mannitol, or vitamin C at 37°C for 2 h resulted in a significant decrease of nickel subsulfide-induced formation of DNA–protein crosslinks, suggesting that nickel subsulfide-induced DNA–protein crosslink formation in isolated rat renal cortical cells is caused by the formation of reactive oxygen species. The potent protective effects of these specific amino acids and Mg2+against nickel subsulfide-induced DNA–protein crosslink formation in isolated renal cortical cells are due to reduction of cellular uptake of Ni2+and inhibition of the binding of Ni2+to deproteinized DNA.