Investigation of the cytotoxicity and genotoxicity of the active ingredients azithromycin and clarithromycin, macrolide group antibiotics

This study, the cytotoxic effects of the macrolide antibiotics azithromycin and clarithromycin—commonly used in the treatment of infectious diseases—on the HEPG2 cell line were evaluated using the MTT assay, while their apoptotic effects were assessed via flow cytometry. Furthermore, the genotoxic potential of these substances on human peripheral lymphocyte cells was investigated through chromosomal aberration (CA) and micronucleus (MN) assays. In addition, in order to determine the cytotoxic effects of the test substances on cell division, mitotic index (MI) and nuclear division index (NDI) values were calculated. The effects of azithromycin and clarithromycin at concentrations of 0.312, 0.625, 1.25, 2.5, 5, 10, 20, and 40 µg/mL over exposure periods of 24, 48, and 72 hours were evaluated using the MTT assay. Based on the obtained data, IC₅₀ values were calculated, and the effects of the 1.25, 2.5, and 5 µg/mL concentrations at 48 hours of exposure were further analyzed using flow cytometry. Additionally, human peripheral lymphocyte cell cultures were exposed to azithromycin and clarithromycin at the same concentrations (1.25, 2.5, and 5 µg/mL) for 48 hours. Negative and positive control groups were included for comparative evaluation. Azithromycin was observed to reduce cell viability in HEPG2 cells in a concentration-dependent manner, while simultaneously causing a marked increase in the rate of apoptosis. When compared with the control group, these differences were found to be statistically significant (p < 0.05). Similarly, in HEPG2 cells treated with clarithromycin, increasing doses resulted in decreased viable cell rates and elevated total apoptosis rates. Necrotic cell rates remained low across all dose groups (ranging from 0.49% to 3.32%). Statistical analyses revealed that all dose groups differed significantly from the negative control group (p < 0.05). According to the micronucleus (MN) assay results, treatment with azithromycin and clarithromycin led to a dose-dependent and statistically significant increase in the percentage of micronucleated binucleated cells (%MNBN). At the highest concentration of 5 µg/mL, %MNBN rates for both antibiotics were found to be comparable to those observed in the positive control group. Nuclear division index (NDI) analysis demonstrated a significant decrease in antibiotic-treated groups compared to both negative and solvent controls, with this decline becoming more pronounced at higher doses. Similarly, mitotic index (MI) values decreased in a dose dependent manner in the azithromycin and clarithromycin treatment groups, and these reductions were statistically significant when compared to the negative, positive, and solvent control groups (p < 0.05). The findings indicate that both azithromycin and clarithromycin may exert dose-dependent genotoxic and cytostatic effects on human peripheral lymphocytes.