Exposure of mammalian cell cultures to benzo[a] and light results in oxidative DNA damage as measured by 8-hydroxydeoxyguanosine formation

Abstract

Carcinogenic polycyclic aromatic hydrocarbons induce DNA damage through direct covalent interactions with nucleotides of the DNA in cells in which they are activated to 'ultimate carcinogenic metabolites'. To determine whether they also induce oxidative damage to DNA under the same circumstances, early passage Syrian hamster embryo and human mammary carcinoma cell line MCF-7 cultures were treated for 24 h with 0-5 micrograms/ml benzo[a]pyrene (BaP) or for 1 h with 0-100 microM methylene blue (as a positive control for oxidative damage). The cells were then exposed to fluorescent light for 1 or 4 h or retained in darkness. After cell harvest, DNA isolation and enzymatic digestion of the DNA to deoxyribonucleosides, the amounts of 8-hydroxy-2'deoxyguanosine (8-OH-dGuo) and unmodified deoxyguanosine present were determined by reverse-phase HPLC with electrochemical and UV detection respectively. Cultures treated with methylene blue for 1 h followed by light exposure for 1 h contained 5-fold (10 microM) and 8- to 28-fold (100 microM) higher 8-OH-dGuo levels than cells treated with methylene blue not exposed to light or untreated cells with methylene blue not exposed to light or untreated cells exposed to light. There was no significant change in 8-OH-dGuo levels in cultures treated with 1-5 micrograms/ml BaP for 24 h in the absence of light. However, both the human and hamster cell cultures treated with BaP and then exposed to fluorescent light for 4 h contained 3-fold (1 micrograms/ml) and 8- to 10-fold (5 micrograms/ml) higher 8-OH-dGuo levels than those not exposed to light or not treated with BaP. These results indicate that BaP treatment does not cause 8-OH-dGuo formation in DNA of cells maintained in darkness. Exposure of BaP-treated cells to fluorescent light causes formation of significant amounts of oxidative DNA damage as measured by 8-OH-dGuo formation. These findings suggest that oxidative damage of DNA could be involved in tumor induction by BaP in tissues, such as skin, in which exposure to BaP can occur in the presence of light.