Premium
An investigation of the DNA‐damaging ability of benzene and its metabolites in human lymphocytes, using the comet assay
Author(s) -
Anderson D.,
Yu T.W.,
Schmezer P.
Publication year - 1995
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.2850260406
Subject(s) - muconic acid , hydroquinone , catechol , chemistry , benzene , comet assay , dna damage , catalase , hydrogen peroxide , benzoquinone , oxidative stress , biochemistry , dna , organic chemistry
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 (H 2 O 2 ), the positive control, and each compound for 0.5 hr, only H 2 C 2and benzenetriol induced pronounced DNA damage without metabolic activation. The effect of catechol was moderate compared with that of benzenetriol. There was a very weak effect of benzene 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 reduced by the external metabolising system, but p ‐benzoquinone became activated to some extent. Catalase abolished the effect of benzenetriol, suggesting that H 2 O 2formed during autoxidation may be responsible for the DNA‐damaging ability of this metabolite. The presence of catalase in S‐9 mix may explain the detoxification of benzenetriol and the failure to detect consistent benzene responses. Mitogen‐stimulated cycling cells were less sensitive to H 2 O 2and benzenetriol than unstimulcted G 0lymphocytes. When comets were measured by image analysis, a 0.5‐hr treatment with H 2 O 2 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. © 1995 Wiley‐Liss, Inc.