Using the comet assay and lysis conditions to characterize DNA lesions from the acrylamide metabolite glycidamide.

The alkaline comet assay and a cell-free system were used to characterise DNA lesions induced by treatment with glycidamide (GA), a metabolite of the food contaminant acrylamide. DNA lesions induced by GA were sensitively detected when the formamidopyrimidine-DNA-glycosylase (Fpg) enzyme was included in the comet assay. We used LC-MS to characterise modified bases from GA-treated naked DNA with and without subsequent Fpg treatment. N7-GA-Guanine and N3-GA-Adenine aglycons were detected in the supernatant showing some depurination of adducted bases; treatment of naked DNA with Fpg revealed no further increase in the adduct yield nor occurrence of other adducted nucleobases. We treated human lymphocytes with GA and found large differences in DNA lesion levels detected with Fpg, depending on the duration and the pH of the lysis step. These lysis-dependent variations in GA-induced Fpg sensitive sites paralleled those observed after treatment of cells with methyl methane sulfonate (MMS). On the other hand, oxidative lesions (8-oxoGuanine) induced by a photoactive compound (Ro 12-9786) plus light, and also DNA strand breaks induced by X-rays, were detected largely independently of the lysis conditions. The results suggest that the GA-induced lesions are predominantly N7-GA-dG adducts slowly undergoing imidazole ring opening at pH 10 as in the standard lysis procedure; such structures are substrate for Fpg leading to strand breaks. The data suggest that the characteristic alkaline lysis dependence of some DNA lesions may be used to study specific types of DNA modifications. The comet assay is increasingly used in regulatory testing of chemicals; in this context, lysis-dependent variations represent a novel approach to obtain insight in the molecular nature of a genotoxic insult.

In vitro investigations of glycidamide-induced DNA lesions in mouse male germ cells and in mouse and human lymphocytes.

The industrial compound and food contaminant acrylamide (AA) is a probable human carcinogen, also known to induce male-mediated reproductive effects in animals. Most data suggest that its metabolite glycidamide (GA) is involved in the observed toxicity. We have investigated in vitro effects of AA/GA in mouse male germ cells (prior to spermatid elongation) and human and mouse peripheral blood lymphocytes, to assess inter-species and cell-type differences in susceptibility, and to explore the nature of the DNA lesion(s) as well as their potential repair. The comet assay was used in combination with the DNA-repair enzymes Fpg and hOGG1 to measure specific DNA lesions. In contrast to AA, GA induced significant levels of DNA lesions (strand breaks and alkali-labile sites) at millimolar concentrations in mouse testicular cells and human peripheral blood lymphocytes (hPBL). Using Fpg, the GA-induced DNA damage was measured at 20-50-fold higher sensitivity, in all cell types investigated. GA-induced DNA damage could not be recognised by hOGG1, suggesting that, based on the known affinities of these repair enzymes, alkylation of guanine is involved, rather than oxidation. Human lymphocytes appeared to be more susceptible to GA-induced lesions than both types of mouse cells. Mouse testicular cells and lymphocytes seemed to respond similarly to GA-induced Fpg-sensitive DNA lesions. The persistence of lesions was explored with cells from mice either proficient or deficient in Ogg1 (mouse 8-oxoguanine DNA glycosylase). Low in vitro repair of GA-induced Fpg-sensitive lesions was observed in primary male germ cells and lymphocytes from both Ogg1(+/+) and Ogg1(-/-) mice. We conclude that there may be differences between mice and humans in AA/GA-induced genotoxicity, and DNA from mouse male germ cells does not appear to be more sensitive to GA than DNA from peripheral blood lymphocytes in vitro. The usefulness of the comet assay in combination with DNA-repair enzymes is demonstrated.