Preliminary in vitro safety assessment of a novel organoselenium compound: PhSeZnCl

Introduction: A number of novel synthetic organoselenium compounds are currently under development as promising anticancer, antioxidant and antimicrobial agents. However, the toxicity associated with selenium could be a limiting factor for their use in pharmacotherapy. The aim of the present study was to investigate the toxicity of PhSeZnCl, a new synthetic seleniorganic compound with GPx-like activity. Methods: Human hepatoma (HepG2) cells were exposed in vitro to different concentrations of PhSeZnCl. Trypan blue dye exclusion test and acridine orange/DAPI double staining were used to determine cytotoxicity. The three highest non-cytotoxic doses of PhSeZnCl were further analyzed for the effects on DNA damage, apoptosis, cell cycle progression and inflammation. The single-cell-microgel electrophoresis (“comet”) assay and the micronucleus (MN) test were conducted for genotoxicity assessment. Late apoptotic cells and cell cycle progression were evaluated by DNA content analysis using the quantitative DNA-binding dye DAPI. Decrease in mitochondrial membrane potential, which occurs in early apoptotic cells, was detected using the lipophilic cationic dye JC-1. Lastly, levels of pro-inflammatory cytokine IL-8 were quantified using commercially available ELISA kits. Results: Cell viability assays showed that PhSeZnCl produced cytotoxic effects in HepG2 cells up to a concentration of 0.4 mM. Comet assay revealed a significant increase of the extent of DNA damage at the highest non-cytotoxic dose tested (0.2 mM). Conversely, the frequencies of MN in treatment cells were found to be comparable to those of the control. Apoptosis assays and DNA content analysis revealed a time-dependent increase in the proportion of both early and late apoptotic cells and a cell cycle arrest in the G2/M phase following exposure to PhSeZnCl 0.2 mM. For this dose, IL-8 protein levels were also found to be significantly increased with respect to untreated cells. Conclusion: Our results indicate that only the highest non-cytotoxic dose of PhSeZnCl (0.2 mM) cause inflammatory and DNA damaging effects on HepG2 cells. The absence of fixed cytogenetic damage (i.e. micronuclei) could be explained by the induction of cell cycle arrest and apoptotic pathways, which determine the removal of cells with irreparable DNA damage.