Comparative 32P-postlabeling analysis of benzo[a]pyrene--DNA adducts formed in vitro upon activation of benzo[a]pyrene by human, rabbit and rodent liver microsomes.
An interspecies comparison was made of the DNA-adducts formed in vitro upon incubation of rat liver DNA (RL-DNA) with benzo[a]pyrene (BP) in the presence of liver microsomes. Incubations were carried out with RL-DNA, BP (100 microM) and liver microsomes from hamsters, mice, rabbits, rats, 3-methylcholanthrene (3MC) pretreated rats and from humans. To analyse the adduct profiles, the 32P-postlabeling technique with the nuclease P1-enhancement procedure was used. The total amount of adduct formed varied greatly with the species; also the number of adduct spots detected was different, ranging from 1 to 5. In all incubations the BP-N2-deoxyguanosine adduct was formed. Relative to the total adduct level, the level of this adduct varied from 26% with rat, 54% with hamster, 56% with 3MC-pretreated rat, 58% with mouse and 75% with rabbit, to 100% with human liver microsomes. In human liver microsomes both the total amount of cytochrome P-450 per mg microsomal protein and the ethoxyresorufin O-deethylation (EROD) activity were low compared to that in animal liver microsomes. In microsomes from 3MC-pretreated rats the EROD activity was strongly induced. There was no correlation between EROD activity in non-induced microsomes and total adduct level. To compare BP-DNA adduct formation in human white blood cells (WBC) with that in RL-DNA, WBC were incubated with BP and 3MC-pretreated rat microsomes. The adduct profile in WBC-DNA differed from that observed after incubation of RL-DNA: the BP-N2-deoxyguanosine adduct in WBC-DNA accounted for 97% of the total adduct level. It is concluded that the 32P-postlabeling method is a suitable technique to investigate both qualitative and quantitative differences in BP-DNA adduct formation between species. Furthermore, the incubation of microsomes from the liver (or other sources) with a genotoxic agent and isolated DNA or cells can be a useful approach to study the formation and stability of reactive intermediates that are able to bind to DNA, also with respect to differences between species or tissue.
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TNO Medical Biological Laboratory, Department of Genetic Toxicology, Rijswijk The Netherlands.
Wolterbeek
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Rutten
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Baan
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D000818:Animals; D001564:Benzo(a)pyrene / Q000032:analysis* / Q000378:metabolism; D006224:Cricetinae; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000378:metabolism; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D018736:DNA Adducts; D005260:Female; D006801:Humans; D008297:Male; D008748:Methylcholanthrene; D051379:Mice; D008862:Microsomes, Liver / Q000737:chemistry* / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D013045:Species Specificity
D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; C056612:benzo(a)pyrene-DNA adduct; D001564:Benzo(a)pyrene; D008748:Methylcholanthrene; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1