Presence of benzo[a]pyrene diol epoxide adducts in target DNA leads to an increase in UV-induced DNA single strand breaks and supF gene mutations

Abstract

Exposure to DNA damaging agents and mutagens often occurs as combinations of agents, or as complex mixtures of chemicals. We found that plasmid DNA adducted with benzo[a]pyrene diol epoxide (BPDE) was more susceptible to UV-induced single strand breaks than was control DNA. To determine whether the increase in DNA damage also applied to mutagenic lesions, the supF gene forward mutation assay was used to compare mutations induced by BPDE alone, UVB, UVC, BPDE followed by UVB and BPDE followed by UVC. It was found that the mutation frequency for BPDE + UVB (1167 in 10(4) transformants) was higher than BPDE alone (12 in 10(4) transformants) or UVB alone (446 in 10(4) transformants), and the mutation frequency for BPDE + UVC (197 in 10(4) transformants) was higher than BPDE alone or UVC alone (26 in 10(4) transformants). For BPDE + UVB and BPDE + UVC there was a significant increase in plasmids with multiple mutations. Whilst these indicate error prone repair due to the single strand breaks, the different mutation frequencies in plasmids treated to give similar levels of strand breaks suggest other mechanisms for the mutations in plasmids with single mutation events. The spectrum of non-multiple mutations in the two combined treatments included both UV signature mutations (GC-->AT as the most common mutation) and BPDE signature mutations (GC-->TA and GC-->CG as the most common mutations). However, the increase in absolute mutation frequency of BPDE signature mutations between BPDE treatment and BPDE + UV treatment was greater than the increase in absolute mutation frequency of UV signature mutations, even though the level of BPDE adducts was identical in each case. These results suggest two possibilities: (i) the BPDE adducts are photoactivated to a more mutagenic lesion, or (ii) the presence of UV lesions lead to the BPDE adducts becoming more mutagenic.

Binary exposure of A549 cells to benzo[a]pyrene and UVC radiation yields enhanced DNA damage in the comet assay but no enhancement of 8‐oxo‐deoxyguanosine

“…Only when that adducted DNA was UVC-irradiated were strand breaks induced, at a much greater frequency than in unadducted UVC-irradiated plasmid. As in this report we have not found evidence of the enhancement of oxidative DNA damage in cells exposed to BP and UVC, it may be that the additional strand breaks and mutations previously observed in plasmid DNA [Routledge et al, 2001] are due to sensitization of the DNA to UVinduced strand breakage.…”

“…Strand breaks induced by BP in the alkaline comet assay are probably induced during the repair of BPDE adducts by the cells. In a previous study [Routledge et al, 2001], it was found that BPDE adduction of naked plasmid DNA did not itself induce DNA strand breaks as measured by the plasmid strand-break assay, which occurs in the absence of DNA repair. Only when that adducted DNA was UVC-irradiated were strand breaks induced, at a much greater frequency than in unadducted UVC-irradiated plasmid.…”

“…In our previous study of mutations induced by UV irradiation of BPDE adducted DNA [Routledge et al, 2001], it was noted that UV irradiation increased the absolute levels of G:C3 T:A mutations, the type of mutations commonly induced by BPDE adducts. This occurred even though the number of BPDE adducts was the same in both cases.…”

Development of a novel site-specific mutagenesis assay using MALDI-ToF MS (SSMA-MS)

“…This assay is, however, not site specific as it involves treating a double stranded plasmid with a reactive compound to generate an array of adducts at a variety of positions throughout the plasmid. The assay does detect 97% of possible base substitutions within the 85 bp supF gene (22) as well as deletions and insertions and because the plasmid is treated in vitro , aliquots of the treated DNA can be analysed for adduct quantification in parallel to the mutation assay. The supF gene is however, a non-essential sequence, so any mutations which are induced would not affect the cell survival or other pathways of carcinogenesis.…”

Polycyclic aromatic hydrocarbons in porcine and bovine organs and tissues

“…It can be deduced, from the few data available on hydrocarbon body burdens, that PAH are rapidly metabolised and excreted from the body, and therefore PAH themselves do not persist for long time periods. During metabolism, PAH moieties can become covalently bound to tissue constituents such as proteins and nucleic acids (Routledge et al, 2001). Protein-bound metabolites are likely to persist, therefore, for periods that do not exceed the normal lifetime of the protein itself.…”