Biomarkers of genotoxicity measured in human lymphocytes exposed to benzo[a]pyrene: Aneugenic effect, and involvement multiple primary DNA lesions

Abstract

Benzo-a-pyrene (B[a]P) is a polycyclic aromatic hydrocarbon classified as carcinogenic to human. Its metabolic activation leads to production of metabolites forming adducts with DNA, being at origin of B[a]P-induced DNA damages, mutagenesis and carcinogenesis. Human blood lymphocytes cultures established from 25 subjects aged 20 to 30 years old, living in Montreal (Quebec, Canada) were exposed to B[a]P (0.4, 4, 20 and 40 µM) for 24 h, then washed and cultivated without B[a]P for an additional 24 h. B[a]P-DNA adducts, DNA single-strand breaks (SSBs), sister chromatid exchanges (SCEs), chromosomal aberrations (CAs) and micronuclei (MN) were analysed. Fluorescent in situ hybridization (FISH) analysis of MN using a pancentromeric probe was also done to assess MN content. Significantly increased formation of B[a]P-DNA adducts, CAs, MN and SCEs were observed starting at [B[a]P]=0.4 µM (p<0.01), with a significant decrease of B[a]P-DNA adducts and MN after exposure to 20 µM B[a]P. For DNA SSBs, no significant increase was observed in all conditions. Besides, FISH analysis showed that B[a]P-induced MN mostly contain centromeres (77.1% vs 68.5% for the control, p<0.01), and specifically three or more centromeres (p<0.01). This study suggests an aneugenic effect of B[a]P in human lymphocytes. Finally, statistical analysis by multiple linear regression showed that two variables (B[a]P exposure level and B[a]P-DNA adducts) significantly explained 53% of observed variability in SCE test, while the percentage of cell presenting a high frequency of SCE (% HFC) was the only variable significantly explaining the observed variability in CA and MN test (34% and 12%, respectively). These observations indicate that DNA breaks, in addition to B[a]P-DNA adducts, contributed to SCEs formation.