AN INVESTIGATION OF THE DNA-DAMAGING ABILITY OF BENZENE AND ITS METABOLITES IN HUMAN-LYMPHOCYTES, USING THE COMET ASSAY

Benzene and five of its known metabolites-muconic acid, hydroquinone, catechol, p-benzoquinone, and benzentriol-were examined For DNA damage in human lymphocytes using the alkaline Comet essay, and conditions w ere optimised to determine responses. Metabolic activation (S-9 mix) w as included in the assay for varying times to try to enhance effects. In addition, the effects of catalase were investigated as if is known to be present in S-9 mix reducing oxidative damage, and some benzene m etabolites are known to react through oxygen radical mechanisms. Effec ts were also examined in cycling cells to determine whether they were more sensitive to damage then noncycling cells. Comets were measured e ither by eye or by image analysis. Data have been presented according to length of treatments. When Comets were measured by eye after treatm ent with hydrogen peroxide (H2O2), the positive control, and each comp ound for 0.5 hr, only H2O2 and benzenetriol induced pronounced DNA dam age without metabolic activation. The effect of catechol was moderate compared with that of benzenetriol. There was a very weak effect of be nzene in the absence of rat liver S-9 mix. In the presence of S-9 mix, benzene was not activated. The effect of benzenetriol was greatly red uced by the external metabolising system, but p-benzoquinone became ac tivated to some extent. Catalase abolished the effect of benzenetriol, suggesting that H2O2 formed during autoxidation may be responsible fo r the DNA-damaging ability of this metabolite. The presence of catalas e in S-9 mix may explain the detoxification of benzenetriol and the fa ilure to defect consistent benzene responses. Mitogen-stimulated cycli ng cells were less sensitive to H2O2 and benzenetriol than unstimulate d G(O) lymphocytes. When comets were measured by image analysis, a 0.5 -hr treatment with H2O2 and benzenetriol and catechol confirmed result s analysed by eye, with S-9 mix greatly reducing responses. When treat ments were increased to 1 hr in the presence and absence of S-9 mix, b enzene at a 5-fold increased dose produced a significant positive resp onse but not at the lower dose. When treatment times were increased to 2 and 4 hr, doses were also increased, and muconic acid, hydroquinone , catechol, and benzoquinone in the presence of S-9 mix showed positiv e time and dose-related responses, and at the highest dose of benzoqui none the morphology of the nucleus was affected. Effects tended to bec ome more pronounced at high doses and after longer exposures, although this was not always consistent from experiment to experiment. In conc lusion, benzene and all metabolites investigated gave positive respons es. Where altered responses were observed, they were significantly dif ferent from the corresponding controls. (C) 1995 Wiley-liss, Inc.