Nucleos(t)ide analogues (NAs) are currently the most important anti-viral treatment option for patients with chronic hepatitis B (cHB). Adefovir dipivoxil (AdV), a diester pro-drug of adefovir, has been widely used for the clinical therapy of hepatitis B virus infection. It has been previously reported that adefovir induced chromosomal aberrations (cAs) in the in vitro human peripheral blood lymphocyte assay, while the genotoxic mechanism remains elusive. To evaluate the possible mechanisms, the genotoxic effects of AdV on the TK6 and DT40 cell lines, as well as DNA repair-deficient variants of dT40 cells, were assessed in the present study. A karyotype assay revealed AdV-induced cAs, particularly chromosomal breaks, in wild-type dT40 and TK6 cells. A γ-H2AX foci formation assay confirmed the presence of DNA damage following treatment with AdV. Furthermore, Brca1 -/-dT40 cells exhibited an increased sensitivity to AdV, while the knockdown of various other dNA damage-associated genes did not markedly affect the sensitivity. These comprehensive genetic studies identified the genotoxic capacity of ADV and suggested that Brca1 may be involved in the tolerance of AdV-induced dNA damage. These results may contribute to the development of novel drugs against cHB with higher therapeutic efficacy and less genotoxicity.

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Genistein (GES), a phytoestrogen, has potential chemopreventive and chemotherapeutic effects on cancer. The anticancer mechanism of GES may be related with topoisomerase II associated DNA double-strand breaks (DSBs). However, the precise molecular mechanism remains elusive. Here, we performed genetic analyses using human lymphoblastoid TK6 cell lines to investigate whether non-homologous DNA end joining (NHEJ) and homologous recombination (HR), the two major repair pathways of DSBs, were involved in repairing GES-induced DNA damage. Our results showed that GES induced DSBs in TK6 cells. Cells lacking Ligase4, an NHEJ enzyme, are hypersensitive to GES. Furthermore, the sensitivity of Ligase4 cells was associated with enhanced DNA damage when comparing the accumulation of γ-H2AX foci and number of chromosomal aberrations (CAs) with WT cells. In addition, cells lacking Rad54, a HR enzyme, also presented hypersensitivity and increased DNA damages in response to GES. Meanwhile, Treatment of GES-lacking enhanced the accumulation of Rad51, an HR factor, in TK6 cells, especially in Ligase4 . These results provided direct evidence that GES induced DSBs in TK6 cells and clarified that both NHEJ and HR were involved in the repair of GES-induced DNA damage, suggesting that GES in combination with inhibition of NHEJ or HR would provide a potential anticancer strategy.

Carnosol is a natural compound with pharmacological action due to its anti‐cancer properties. However, the precise mechanism for its anti‐carcinogenic effect remains elusive. In this study, we used lymphoblastoid TK6 cell lines to identify the DNA damage and repair mechanisms of carnosol. Our results showed that carnosol induced DNA double‐strand breaks (DSBs). We also found that cells lacking tyrosyl‐DNA phosphodiesterase 1 (TDP1), an enzyme related to topoisomerase 1 (TOP1), and tyrosyl‐DNA phosphodiesterase 2 (TDP2), an enzyme related to topoisomerase 2 (TOP2), were supersensitive to carnosol. Carnosol was found to induce the formation of the TOP1‐DNA cleavage complex (TOP1cc) and TOP2‐DNA cleavage complex (TOP2cc). When comparing the accumulation of γ‐H2AX foci and the number of chromosomal aberrations (CAs) with wild‐type (WT) cells, the susceptivity of the TDP1−/− and TDP2−/− cells were associated with an increased DNA damage. Our results provided evidence of carnosol inducing DNA lesions in TK6 cells and demonstrated that the damage induced by carnosol was associated with abnormal topoisomerase activity. We conclude that TDP1 and TDP2 play important roles in the anti‐cancer effect of carnosol.

Apigenin (APG), a flavone sub-class of flavonoids, possesses a diverse range of biological activities, including anti-cancer and anti-inflammatory effects. Previous studies identified the genotoxicity of APG in certain cancer cells, which may be associated with its anticancer effect. However, the DNA damage repair mechanism induced by APG has remained elusive. In order to clarify the molecular mechanisms, the present study determined the toxicity of APG to the wild-type (WT) DT40 chicken B-lymphocyte cell line, as well as to DT40 cells with deletions in various DNA repair genes, and their sensitivities were compared. It was demonstrated that cells deficient of Rad54, a critical homologous recombination gene, were particularly sensitive to APG. Cell-cycle analysis demonstrated that APG caused an increase in the G 2 /M-phase population of Rad54-/cells that was greater than that in WT cells. Furthermore, it was demonstrated by immunofluorescence assay that Rad54-/cells exhibited significantly increased numbers of γ-phosphorylated H2AX variant histone foci and chromosomal aberrations compared to the WT cells in response to APG. Of note, the in vitro complex of enzyme assay indicated that APG induced increased topoisomerase I (Top1) covalent protein DNA complex in Rad54-/cells compared to WT cells. Finally, these results were verified using the TK6 human lymphoblastoid cell line and it was demonstrated that, as for DT40 cells, Rad54 deficiency sensitized TK6 cells to APG. The present study demonstrated that Rad54 was involved in the repair of APG-induced DNA damage, which was associated with Top1 inhibition.