ADV (purity, ≥99%) and camptothecin (CPT; purity, ≥99%) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). These chemicals were dissolved in dimethyl sulfoxide (DMSO). Stock solutions of ADV (10 mM) and CPT (100 μM) were stored at -20°C in aliquots. In each experiment, the final concentration of DMSO never exceeded 0.1%.

All of the cell lines used in the present study were provided by Professor Shunichi Takeda (Kyoto University, Kyoto, Japan). The DT40 cell lines (25-30) with different phenotypes used in the present study are summarized in Table I. The DT40 cells were incubated in RPMI-1640 medium containing 10% newborn calf serum, 1% chicken serum, 1% penicillin streptomycin (all Wisent, Inc., St. Bruno, QC, Canada), 200 mM L-glutamine and 50 μM β-mercaptoethanol (both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 5% CO₂ at 39.5°C. The TK6 cells were routinely maintained in RPMI-1640 medium (Wisent, Inc.) including 10% horse serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Wisent Inc.) at 37°C in the presence of 5% CO₂. These sera (except chicken serum) were heat-deactivated at 56°C for 30 min prior to use.

The anti-proliferative effects of ADV and CPT on cells was determined with MTT assay (31-33). Briefly, the cells (1×10⁴ cells/ml) were seeded into 96-well plates in complete medium, followed by incubation in the presence of various concentrations of ADV or CPT for 72 h. The concentrations of ADV were 0, 0.06, 0.12, 0.18, 0.24 and 0.3 μM for DT40 cells or 0, 1, 2, 3, 4 and 5 μM for TK6 cells. The CPT concentrations were 0, 10, 20, 30, 40 and 50 nM for DT40 cells, or 0, 2, 4, 6 and 8 nM for TK6 cells. The concentrations of CPT were 0, 1, 2, 3, 4 and 5 nM, or 0, 10, 20, 30, 40 and 50 nM for DT40 cells specifically for sensitivity experiments. DMSO (<0.1%) was applied as a solvent and vehicle control with 3 replicates for each concentration of the drugs. Following 69 h treatment at 39.5°C for DT40 and at 37°C for TK6 cells, 20 μl of 5 mg/ml MTT (Amresco, LLC, Solon, OH, USA) was added to the cells for 3 h at 39.5°C for DT40 and at 37°C for TK6 cells, and subsequently, the formazan dye crystals were dissolved by 50 μl of 20% sodium dodecyl sulfate overnight at 39.5°C for DT40 and at 37°C for TK6 cells. The absorbance was detected at a wavelength of 570 nm by a microplate reader. The wells without cells served as a blank control. All the experiments were repeated at least 3 times. The 50% inhibitory concentration (IC₅₀) of the drugs on the cells was calculated with SPSS 25.0 software (IBM Corp, Armonk, NY, USA).

Preparation of chromosome samples was performed as described previously (34-37), with certain modifications. Briefly, cells (2×10⁵ cells/ml) were seeded in 6-well plates and were treated with ADV or CPT in complete medium. DT40 cells were treated with 0.2 or 0.4 μM ADV for 9-30 h and 5 or 25 nM CPT for 6-24 h at 39.5°C. TK6 cells were performed with 5 μM ADV for 12-36 h and 2.5 μM ADV for 12 h at 37°C. Colcemid (0.2 μg/ml; Gibco; Thermo Fisher Scientific, Inc.) was added 3 h prior to harvesting in order to enrich mitotic cells. The cells were then incubated in 1 ml hypotonic KCl solution (75 mM) for 25 min at room temperature. Subsequently, the cells were fixed with 3 ml Carnoy's solution [methanol/acetic acid, 3:1, (v/v)] for 35 min. A drop of this suspension was placed onto ethanol-washed glass slides and immediately dried by a flame. Finally, the dried slides were dyed with 5% Giemsa solution for 5 min at room temperature and carefully rinsed with water prior to air-drying. In the present study, 50 metaphase cells per each experiment were analyzed under a light microscope (magnification, ×1,000). All assays were performed 3 times. A break or gap in a chromosome was evaluated and defined according to The International System for Human Cytogenetic Nomenclature (ISCN) (37).

γ-H2A histone family member X (H2AX) formation is a rapid and sensitive cellular response to DSBs (38). Immunofluorescent staining was performed as reported previously (39). Briefly, cells (2×10⁵ cells/ml) cultured on 12-well-plates. DT40 cells were treated with 0.1 μM ADV or 0.1 μM CPT for 3, 6 and 9 h at 39.5°C, and TK6 cells were treated with 1 μM ADV or 20 nM CPT for 3, 6 and 9 h at 37°C and then harvested on glass slides. The cells were fixed with 3% paraformaldehyde for 10 min at room temperature followed by washing with PBS 3 times. The fixed cells were permeabilized with 0.1% Nonidet P-40 for 20 min at room temperature and washed with PBS again. Following blocking with 3% bovine serum albumin (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 30 min at room temperature, the cells were probed with anti-γ-H2AX mouse monoclonal antibody (cat. no. 05-636; 1:1,000 dilution; EMD Millipore, Billerica, MA, USA) in a humidified environment at 4°C overnight. Following washing with PBS, the cells were incubated for 1 h at 37°C with Alexa Fluor 488-labeled goat anti-mouse IgG (H+L) (cat. no. A0428; 1:1,000 dilution; Beyotime Institute of Biotechnology, Wuhan, China). The nuclei were stained with 4′,6-diamidino-2-phenylindole for 10 min at room temperature. Finally, a fluorescence microscope (IX81; Olympus Corporation, Tokyo, Japan) was used to visualize the γ-H2AX foci (magnification, ×1,000). The foci in 100 nuclei were counted. The visible foci as described in previous reports were counted by eye using Photoshop (version 12.0.3; Adobe Systems, Inc., San Jose, CA, USA) (40). The experiments were performed 3 times.

Statistical analysis was performed with SPSS 25.0 software (IBM Corp.). The statistically significant differences were determined with a Student's t-test or two-way analysis of variance with Tukey's post hoc test. Values are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Adefovir was previously demonstrated to induce CAs by a human peripheral blood lymphocyte assay in vitro without metabolic activation (19). As a diester prodrug of adefovir (19), it cannot be excluded that ADV may also has mutagenic effects. DSBs, one type of DNA damage, may be reliably identified by γ-H2AX foci detection (41). In the present study, DT40 and TK6 cells were continuously exposed to various concentrations of ADV for 72 h and CPT was used as a positive control. The results of the MTT assay indicated that ADV exerts a notable cytotoxic effect in WT DT40 and TK6 cells (Fig. 1). Furthermore, DT40 cells were treated with 0.1 μM ADV for 3, 6 or 9 h to dynamically investigate the changes in the number of γ-H2AX foci. In addition, TK6 cells were also exposed to 1 μM ADV for the same durations. As presented in Figs. 2 and S1, the number of γ-H2AX foci was significantly increased following treatment with ADV and exhibited a peak at 6 h. According to the quantitative distribution of γ-H2AX foci in WT DT40 and TK6 cells (Fig. S1), ADV induced a greater percentage of γ-H2AX-positive cells.

In addition, DNA damage was analyzed by measuring cytologically detectable CAs in mitotic cells. WT DT40 cells were exposed to 0.2 or 0.4 μM ADV, and CAs were determined at 9, 12, 15, 24 and 30 h. The maximum number of CAs was observed at 12 h of ADV treatment (Fig. 3A and B). Similarly, TK6 cells were exposed to 5 μM ADV for 12-36 h and with 2.5 μM ADV for 12 h. CAs were determined at 12, 24 and 36 h. The maximum number of chromosomal breaks was observed at 12 h (Fig. 3C-E). These results confirmed that ADV was able to generate DNA DSBs in DT40 and TK6 cells.

To study the genotoxic mechanisms of ADV, an MTT assay using WT and mutant DT40 cells was performed. The sensitivity of ADV was assessed in a panel of DT40 clones, each of which was deficient in a different DNA repair pathway (Table I) (25-30). DT40 cells were treated with ADV at various concentrations for 72 h. It was revealed that ADV inhibited cell growth in a dose-dependent manner (Fig. 4). As presented in Fig. 4A-C, only Brca1^−/− (28,30) cells exhibited significant sensitivity to ADV. The present study also exposed WT and Brca1^−/− DT40 cells to ADV and evaluated their cellular response using a colony survival assay (Data S1). Compared with WT DT40 cells, the colony survival of Brca1^−/− cells was significantly reduced at 14 days (Fig. S2). The results of the clonogenic assays also confirmed this sensitivity (Fig. S2). The cell variants deficient in other DNA repair genes, including poly (adenosine diphosphate-ribose) polymerase 1 (Parp1^−/−) (27), REV3-like DNA directed polymerase ζ catalytic subunit (Rev3^−/−) (25), Ku autoantigen 70 kDa (Ku70^−/−) (29) and xeroderma pigmentosum group A-complementing protein (Xpa^−/−) (26), were not more sensitive to ADV than WT cells (Fig. 4). In the present experiments, CPT was selected as a positive control (Fig. 4D-F).

In the present study, CAs were classified as chromosome or chromatid gaps, breaks and exchanges according to The ISCN (37). WT or Brca1^−/− DT40 cells were exposed to 0.2 or 0.4 μM ADV for 9, 12, 15, 24 or 30 h. Compared with WT DT40 cells, Brca1^−/− cells exhibited significantly increased CAs at 15, 24 and 30 h, and the higher the dose the greater the increase in CAs observed (Fig. 5A and B). Notably, a monophasic pattern of CAs was induced in WT and Brca1^−/− cells. In these 2 cell lines, a peak was detectable at 12 h for WT and 24 h for Brca1^−/− cells, respectively, and it was much higher in Brca1^−/− cells than in WT cells (Fig. 5A and B). As a positive control, WT and Brca1^−/− DT40 cells were treated with 5 nM CPT for 6, 12, 18 and 24 h (Fig. 5C). At the same time, WT DT40 cells were treated with 25 nM CPT (Fig. 5D). The results indicated that CPT induced more DNA damage in Brca1^−/− cells when compared with WT DT40 cells. These results further confirmed that Brca1 participates in repairing ADV-induced DNA damage.