Enrichment of nucleobase adducts from genomic DNA in the cytoplasm by solid-phase extraction

The exact levels of some DNA adducts, like N7-deoxyguanosine (N7-dG), can be under-calculated since these adducts may depurinate due to their chemical instability, leading to corresponding nucleobase adducts being released into the cytoplasm. To accurately quantify the levels of DNA adducts, it is necessary to consider those modified nucleobases. However, high levels and diversity of cytoplasmic small molecule metabolites (SMMs) can strongly interfere with the detection of adducts, and it is almost impossible to remove them with nucleobase adducts being well retained. Therefore, we aimed to establish an optimized enrichment method based on solid-phase extraction (SPE) to reduce the co-elution of SMMs with target analytes. In this vein, we employed three bisphenols (BPA, BPF, and BPAF) as examples, prepared corresponding N7-guanine (N7-Gua) adducts, loaded on an Oasis hydrophilic-lipophilic balance ® (HLB) cartridge, used a series of mobile phases containing different percentage of methanol for elution, and evaluated the levels of these adducts in each eluent. First, we found that neutral samples led to the best retention for all three adducts compared with acidified or basified ones. We next employed normal distribution fitting model to characterize the elution of analytes from H₂O/methanol with different pHs and observed that neutral mobile phases resulted in more hydrophobic elution for all three adducts. Besides, N7-BPA-Gua and N7-BPF-Gua obtained narrow fitted peaks at neutral pH, while N7-BPAF-Gua had minimized elution windows at low pH. After optimization, we exposed 293T cells to the aforementioned bisphenols and quantified the N7-Gua adducts in the cytoplasm and the corresponding N7-dG adducts in genomic DNA. The results revealed that with the same levels of BPs exposure, BPAF led to the highest levels of adducts in both cytoplasm and genomic DNA samples, followed by BPA and BPF in order. In summary, our research established an appropriate model to describe the elution of DNA adducts in the SPE, applied it to optimize the loading and elution conditions, and discussed the genotoxicity of bisphenols by accurate quantification of both cleaved and uncleaved N7-dG adducts.