ERCC1 and ERCC2 haplotype modulates induced BPDE-DNA adducts in primary cultured lymphocytes

Abstract

Background: Benzo[a]pyrene(B[a]P), and its ultimate metabolite Benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE), are classic DNA damaging carcinogens. DNA damage caused by BPDE is normally repaired by Nucleotide Excision Repair (NER), of which ERCC1 and ERCC2/XPD exert an indispensable role. Genetic variations in ERCC1 and ERCC2 have been related to DNA repair efficiency. In this study we used lymphocytes from healthy individuals to show that polymorphisms in ERCC1 and ERCC2 are directly associated with decreased DNA repair efficiency.

Methods: ERCC1 (rs3212986 and rs11615) and ERCC2 (rs13181, rs1799793 and rs238406) were genotyped in 818 healthy Han individuals from the northeast of China. BPDE induced DNA adducts in lymphocytes were assessed by high performance liquid chromatography (HPLC) in 282 randomly selected participants. The effect of ERCC1 rs3212986 and ERCC2 rs238406 on DNA damage caused by B[a]P was assessed with a modified comet assay.

Results: We found that the variant genotypes of ERCC1 rs3212986 and ERCC2 rs238406 were associated with the high levels of BPDE-DNA adducts. Especially ERCC1 rs3212986 A-allele variant was significantly associated with the high BPDE-DNA adducts. Haplotype analysis showed that the ERCC1 haplotype AC (OR = 2.36, 95% CI = 1.84-2.97), ERCC2 haplotype AGA (OR = 1.51, 95% CI = 1.06-2.15) and haplotype block AGAAC (OR = 5.28, 95% CI = 2.95-9.43), AGCAC (OR = 1.35 95% CI = 1.13-1.60) were linked with high BPDE-DNA adducts. In addition, we found that the combined minor alleles of ERCC1 rs3212986 and ERCC2 rs238406 were associated with a reduced DNA repair capacity.

Conclusions: Our results suggest that the variant genotypes of ERCC1 rs3212986 and ERCC2 rs238406 are associated with decreased repair efficiency of BPDE induced DNA damage, and may be predictive for an individual's DNA repair capacity in response to environmental carcinogens.

Figure 1. Chromatograms of blank, DNA, BPDE and DNA+BPDE solutions using HPLC-UV detection.

Figure 1 reflects the representive HPLC readouts. We used the Chromatograms to detect BPDE-DNA adducts level. The retention time of 9.38 minute was identified to be BPDE-DNA adducts.

Figure 2. DNA damage caused by B[a]P exposure detected by a modified comet assay.

Figure 2 shows a typical image to reflect the damage levels caused by BPDE-DNA adducts in a randomly selected sample. BPDE covalently binds to cellular DNA and forms interactive complexes. 50 µmol H₂O₂ was used to induce DNA fragmentation, resulting in long tails after electrophoreses in control lymphocytes (see Fig. 2A. control or non-exposed group). BPDE-DNA adducts will capture the short H₂O₂ induced DNA fragments, and consequently, a shorter tail olive (or tail area) will be found in BPDE-exposed cells compared to non-exposed cells (see Fig. 2B. BPDE-exposed group).

Figure 3. Comparison of DNA damage caused by B[a]P in different combined minor alleles of ERCC1 rs3212986 and ERCC2 rs238406.

We selected the participants carrying different ERCC1 rs3212986 and ERCC2 rs238406 genotypes and analyzed their induced DNA damage levels induced by B[a]P using the modified comet assay. The damage levels were evaluated by the tail olive moment ratio, tail area ratio and the combined holistic marking respectively. The relationship between the combined minor alleles of ERCC1 rs3212986 and ERCC2 rs238406 and the effect on the repair efficacy of the DNA damage level caused by B[a]P were evaluated. Interestingly, we found following the increasing copies of the combined minor alleles, a reduced DNA repair capacity had been found in the tail olive moment ratio, tail area ratio and the combined holistic marking. (P<0.01).