An investigation of the DNA‐damaging ability of benzene and its metabolites in human lymphocytes, using the comet assay

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Abstract
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 assay, and conditions were optimised to determine responses. Metabolic activation (S‐9 mix) was included in the assay for varying times to try to enhance effects. In addition, the effects of catalase were investigated as it is known to be present in S‐9 mix reducing oxidative damage, and some benzene metabolites are known to react through oxygen radical mechanisms. Effects were also examined in cycling cells to determine whether they were more sensitive to damage then noncycling cells. Comets were measured either by eye or by image analysis. Data have been presented according to length of treatments. When Comets were measured by eye after treatment with hydrogen peroxide (H2O2), the positive control, and each compound for 0.5 hr, only H lymphocytes. When comets were measured by image analysis, a 0.5‐hr treatment with H2O2 and benzenetriol and catechol confirmed results analysed by eye, with S‐9 mix greatly reducing responses. When treatments were increased to 1 hr in the presence and absence of S‐9 mix, benzene at a 5‐fold increased dose produced a significant positive response 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 positive time and dose‐related responses, and at the highest dose of benzoquinone the morphology of the nucleus was affected. Effects tended to become more pronounced at high doses and after longer exposures, although this was not always consistent from experiment to experiment. In conclusion, benzene and all metabolites investigated gave positive responses. Where altered responses were observed, they were significantly different from the corresponding controls.

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