Detection of DNA damage induced by human carcinogens in acellular assays: potential application for determining genotoxic mechanisms

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.     

Cogan syndrome associated with mesenteric vascular insufficiency

Nonsyphilitic interstitial keratitis and deafness was first described as a distinct entity by Cogan in 1945. For years thereafter, it was believed to involve only the ocular and aural systems. It is gradually becoming apparent that the syndrome can also include other organ systems by affecting their blood supply. A patient with diagnosed Cogan syndrome subsequently developed mesenteric vascular insufficiency that was surgically corrected. This report lends more support to the belief that nonsyphilitic interstitial keratitis with deafness is only one manifestation of a more generalized vascular disease.