DNA Damage,gadd153Expression, and Cytotoxicity in Plateau-Phase Renal Proximal Tubular Epithelial Cells Treated with a Quinol Thioether

2-Bromo-bis-(glutathion-S-yl)hydroquinone [2-Br-bis-(GSyl)HQ] causes DNA single-strand breaks (SSB), causes growth arrest, induces the expression ofgadd153(a gene inducible by growth arrest and DNA damage), and decreases histoneH2BmRNA in log-phase renal proximal tubular epithelial cells (LLC-PK1). Renal epithelial cellsin vivonormally exhibit a low mitotic index, therefore experiments in both plateau- and log-phase cells are necessary for a comprehensive understanding of the stress response to 2-Br-bis-(GSyl)HQ. In the present article we demonstrate that not all features of the stress response in log-phase cells are reproduced in plateau-phase cells. Thus, although 2-Br-bis-(GSyl)HQ causes concentration and time-dependent increases in DNA SSB, and increases the expression ofgadd153,histoneH2BmRNA levels are unaltered in plateau-phase cells. The relationship between reactive oxygen species, DNA damage, gene expression, and cytotoxicity was also investigated. Our findings suggest that (i) 2-Br-bis-(GSyl)HQ-mediated DNA damage in LLC-PK1cells is mediated by the generation of H2O2; (ii) DNA damage, either directly or indirectly, contributes to cell death; and (iii) DNA damage, either directly or indirectly, provides the initial signal forgadd153expression. In addition, DNA repair is rapid in LLC-PK1cells, and the DNA-repair inhibitors 1-β-d-arabinofuranosylcytosine and hydroxyurea have no effect on the amount of DNA SSB. Although the addition of 3-aminobenzamide following 2-Br-bis-(GSyl)HQ exposure has no effect on the removal of DNA SSB, it causes a slight but significant increase ingadd153expression and cell viability, indicating that activation of poly(ADP-ribose)polymerase may exacerbate toxicity. Finally, aurintricarboxylic acid did not prevent DNA SSB or cytotoxicity in 2-Br-bis-(GSyl) HQ-treated LLC-PK1cells, implying that activation of endonucleases does not play a role in these processes.