Genetic Evidence for Genotoxic Effect of Entecavir, an Anti-Hepatitis B Virus Nucleotide Analog

Abstract

Nucleoside analogues (NAs) have been the most frequently used treatment option for chronic hepatitis B patients. However, they may have genotoxic potentials due to their interference with nucleic acid metabolism. Entecavir, a deoxyguanosine analog, is one of the most widely used oral antiviral NAs against hepatitis B virus. It has reported that entecavir gave positive responses in both genotoxicity and carcinogenicity assays. However the genotoxic mechanism of entecavir remains elusive. To evaluate the genotoxic mechanisms, we analyzed the effect of entecavir on a panel of chicken DT40 B-lymphocyte isogenic mutant cell line deficient in DNA repair and damage tolerance pathways. Our results showed that Parp1-/- mutant cells defective in single-strand break (SSB) repair were the most sensitive to entecavir. Brca1-/-, Ubc13-/- and translesion-DNA-synthesis deficient cells including Rad18-/- and Rev3-/- were hypersensitive to entecavir. XPA-/- mutant deficient in nucleotide excision repair was also slightly sensitive to entecavir. γ-H2AX foci forming assay confirmed the existence of DNA damage by entecavir in Parp1-/-, Rad18-/- and Brca1-/- mutants. Karyotype assay further showed entecavir-induced chromosomal aberrations, especially the chromosome gaps in Parp1-/-, Brca1-/-, Rad18-/- and Rev3-/- cells when compared with wild-type cells. These genetic comprehensive studies clearly identified the genotoxic potentials of entecavir and suggested that SSB and postreplication repair pathways may suppress entecavir-induced genotoxicity.

Fig 1. Mutant cells defective in DNA repair pathways were sensitive to entecavir.

(A) The X-axis represents the concentration of entecavir and the Y-axis represents the relative number of surviving cells at 72 hours. Survival data were log-transformed giving approximate normality. Analysis of covariance (ANCOVA) was used to test for differences in the linear dose-response curves between wild-type and a series of mutant cells. A p-value < 0.05 was considered to be significant. (B) Relative IC50 values of cell survival results in wild-type and their mutants exposed to entecavir or CPT. Each IC50 value was calculated from results of cell survival data shown in Fig 1A and S1 Fig Relative IC50 values were normalized according to the IC50 value of parental wild-type cells. The IC50 was calculated by SPSS software version13.0. Data shown are the means of three experiments. Values shown are mean ± SD.

Fig 2. Entecavir induced the accumulation of γ-H2AX in nuclei of DT40 cells.

(A) Immuno-staining of wild-type (WT) and mutant DT40 clones using anti-γ-H2AX antibody and DAPI. Cells were fixed 6 hours after treated with entecavir 100nM. ETV, entecavir. (B) Quantification of γ-H2AX foci in individual cells of the indicated genotype. Cells were treated with entecavir 100nM for 6h. Data shown are the means of three experiments. Values shown are mean ± SD. ** P < 0.01, * P < 0.05 compared to WT. More than 100 cells were analyzed for each data point.

Fig 3. DNA repair-deficient cells showed a marked increase in entecavir-induced chromosome breaks.

Increased frequency of chromosomal aberrations (CAs) in DNA repair-deficient cells and WT treated with entecavir (200nM) from 3 hours to 24 hours. Data are derived from 50 metaphase cells for each treatment. The experiments were independently repeated three times for statistical analysis. Values shown are mean ± SD. * P < 0.05 compared to WT. The differences between the WT and DNA repair-deficient cell lines were tested for statistical significance using t-test.

Fig 4. Representative karyotype analysis of entecavir pretreated Rad18^-/- cells.

(A) Representative karyotype of untreated Rad18^-/- cells. (B) Chromosomal aberrations (CAs) in Rad18^-/- cells following 200nM entecavir pretreatment for 15 h. Macrochromosomes 1–5 and Z are identified. Chromosome gaps are shown by arrow.

Fig 5. Model of entecavir-induced genotoxicity related to single-strand break (SSB) repair and postreplication repair (PRR) pathway.

The triphosphate of entecavir is incorporated into DNA strand by host replication or repair polymerases, which blocking extension of the nascent strand and inducing DNA SSB and Parp1 dependent repair. The entecavir-induced DNA lesions could also be repaired by PRR to avoid the replication fork collapse and chromosomal breaks when cells enter into S phase.