title: Detection of DNA damage induced by human carcinogens in acellular assays: potential application for determining genotoxic mechanisms.
abstract: Positive outcomes of in vitro genotoxicity tests may not always occur as a consequence of direct reaction of a compound or a metabolite with DNA. To follow-up positive responses in in vitro tests, we developed two supplemental, cell-free assays to examine the potential of compounds and metabolites to directly damage DNA. Calf thymus DNA was used as the target for the direct detection of adducts by 32P-postlabeling/TLC and electrochemical detection, and alkaline gel electrophoresis was used to detect single-strand breakage of bacteriophage lambda DNA. To show that these assays would detect damage from relevant compounds, we examined nine human carcinogens (aflatoxin B1, busulfan, chlorambucil, cyclophosphamide, diethylstilbestrol, melphalan, 2-naphthylamine, phenacetin and potassium chromate). Each of the nine compounds produced a positive result for one or both endpoints. Using multifraction contact-transfer TLC, we detected 32P-labeled DNA adducts produced by aflatoxin B1, chlorambucil, diethylstilbestrol, melphalan, 2-naphthylamine, and potassium chromate (plus hydrogen peroxide). Aflatoxin B1, diethylstilbestrol and 2-naphthylamine required metabolic activation (induced rat liver S9) to generate DNA adducts. Although potassium chromate alone induced a slight increase in the content of 8-hydroxydeoxyguanosine (a promutagenic adduct produced by reactive oxygen species), addition of hydrogen peroxide greatly increased 8-hydroxydeoxyguanosine levels. The damage to lambda DNA by each human carcinogen (or metabolites), except diethylstilbestrol, was sufficient to generate single-strand breaks after neutral thermal hydrolysis at 70 degrees C. Chromate was a weak inducer of DNA fragmentation, but adding hydrogen peroxide to the reaction mixtures dramatically increased the DNA strand breakage. Our data suggest that these non-routine, acellular tests for determining direct DNA damage may provide valuable mechanistic insight for positive responses in cell-based genetic toxicology tests.
authors.affiliation: Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: S P
authors.initials: SP
authors.lastname: Adams
authors.forename: G M
authors.initials: GM
authors.lastname: Laws
authors.forename: R D
authors.initials: RD
authors.lastname: Storer
authors.forename: J G
authors.initials: JG
authors.lastname: DeLuca
authors.forename: W W
authors.initials: WW
authors.lastname: Nichols
mesh_terms: D000818:Animals; D002273:Carcinogens / Q000633:toxicity*; D004247:DNA / Q000187:drug effects*; D018736:DNA Adducts / Q000032:analysis; D004249:DNA Damage; D004586:Electrophoresis; D006801:Humans; D051381:Rats
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; D004247:DNA