Further characterization of benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects.
The present study aims to further characterize benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects. Therefore, we measured DNA effects by the comet assay and adduct levels by high-performance liquid chromatography (HPLC) in human lymphocytes and A549 cells exposed to (±)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(±)-anti-BPDE] or (+)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(+)-anti-BPDE]. Both, the racemic form and (+)-anti-BPDE, which is the most relevant metabolite with regard to mutagenicity and carcinogenicity, induced DNA migration in cultured lymphocytes in the same range of concentrations to a similar extent in the alkaline comet assay after exposure for 2h. Nevertheless, (+)-anti-BPDE induced significantly enhanced DNA migration after 16 and 18h post-cultivation which was not seen in response to (±)-anti-BPDE. Combination of the comet assay with the Fpg (formamidopyrimidine-DNA glycosylase) protein did not enhance BPDE-induced effects and thus indicated the absence of Fpg-sensitive sites (oxidized purines, N7-guanine adducts, AP-sites). The aphidicolin (APC)-modified comet assay suggested significant excision repair activity of cultured lymphocytes during the first 18h of culture after a 2 h-exposure to BPDE. In contrast to these repair-related effects measured by the comet assay, HPLC analysis of stable adducts did not reveal any significant removal of (+)-anti-BPDE-induced adducts from lymphocytes during the first 22h of culture. On the other hand, HPLC measurements indicated that A549 cells repaired about 70% of (+)-anti-BPDE-induced DNA-adducts within 22h of release. However, various experiments with the APC-modified comet assay did not indicate significant repair activity during this period in A549 cells. The conflicting results obtained with the comet assay and the HPLC-based adduct analysis question the real cause for BPDE-induced DNA migration in the comet assay and the reliability of the APC-modified comet assay for the determination of DNA excision repair activity in response to BPDE in different cell types.
Julia
J
Bausinger
Petra
P
Schütz
Department of Food Chemistry and Toxicology, Karlsruhe Institute of Technology (KIT), Adenauerring 20a, D-76131 Karlsruhe, Germany.
Ann Liza
AL
Piberger
Günter
G
Speit
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity*; D045744:Cell Line, Tumor; D020552:Comet Assay; D018736:DNA Adducts; D004249:DNA Damage / Q000187:drug effects; D004785:Environmental Pollutants / Q000633:toxicity; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D009153:Mutagens / Q000633:toxicity*
D018736:DNA Adducts; D004785:Environmental Pollutants; D009153:Mutagens; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide