Involvement of oxygen free radicals in the serum-mediated increase of benzoquinone genotoxicity.
The genotoxicity of benzoquinone (BQ), a toxic benzene metabolite, is greatly enhanced by the presence of fetal calf serum (FCS) in the incubation medium. The FCS effect is abolished by heat denaturation of serum proteins and is slightly decreased by dialysis. In the present study, we have further investigated the serum effect on BQ genotoxicity by measuring DNA damage produced in peripheral blood mononuclear cells (PBMCs) using the Comet assay. We have also evaluated the effect of human serum and rat liver post-mitochondrial fraction (S9) on the DNA damage produced by BQ. Both human serum and a rat liver S9 enhanced the genotoxicity of BQ in a manner similar to FCS. Gel filtration experiments showed that all the enhancing activity of the serum eluted with the high molecular weight fractions, suggesting that low molecular weight serum constituents do not play an important role in modulating genotoxicity. The genotoxicity-enhancing activity of serum was inhibited by the iron chelator deferoxamine and by superoxide dismutase and catalase. Incubating PBMCs with BQ in the presence of FCS also resulted in the accumulation of intracellular peroxides as demonstrated by loading the cells with 2',7'-dichlorofluorescin diacetate and analyzing for peroxide formation by flow cytometry. These results indicate that oxygen free radicals are involved in the enhancement of BQ-induced DNA damage by serum. We hypothesize that enzyme activities that reduce BQ by transferring single electrons could be the source of the oxygen free radicals.
Fabiani
Roberto
R
Dipartimento di Scienze Biochimiche e Biotecnologie Molecolari, Sezione di Scienze Igienistiche e Ambientali, Università di Perugia, Perugia, Italy. fabiro@unipg.it
De Bartolomeo
Angelo
A
Morozzi
Guido
G
D000818:Animals; D016227:Benzoquinones / Q000633:toxicity*; D002273:Carcinogens / Q000633:toxicity*; D002374:Catalase / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D020552:Comet Assay; D003470:Culture Media; D018736:DNA Adducts; D004249:DNA Damage; D003676:Deferoxamine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D005434:Flow Cytometry; D005452:Fluoresceins / Q000494:pharmacology; D006801:Humans; D007501:Iron / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism; D008970:Molecular Weight; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D010545:Peroxides / Q000737:chemistry; D051381:Rats; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism
D016227:Benzoquinones; D002273:Carcinogens; D002614:Chelating Agents; D003470:Culture Media; D018736:DNA Adducts; D005452:Fluoresceins; D010545:Peroxides; D017382:Reactive Oxygen Species; C029569:diacetyldichlorofluorescein; C004532:quinone; D007501:Iron; D002374:Catalase; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D003676:Deferoxamine; D010100:Oxygen