In vitro toxicological assessment of PhSeZnCl in human liver cells

Abstract

Phenylselenenylzinc chloride (PhSeZnCl) is an air-stable selenolate, easily synthesizable through oxidative insertion of elemental zinc into the Se-halogen bond of the commercially available phenylselenyl chloride. PhSeZnCl was shown to possess a marked GPx-like activity both in NMR and in vitro tests, and to effectively react with cellular thiols, and was supposed for a potential use in the chemotherapy of drug-resistant cancers. However, activity of PhSeZnCl in hepatic cells has never been tested before now. In this in vitro approach, we evaluated the cytotoxic, genotoxic, and apoptotic activities, as well as the effects on cell cycle of PhSeZnCl in two preclinical hepatic models, namely HepG2 and HepaRG cells. Results showed that cell viability of HepG2 and HepaRG cells decreased in a dose-dependent manner, with a more marked effect in HepG2 tumour cells. Moreover, treatment with 50 µg/mL PhSeZnCl caused an increase of primary DNA damage (4 h) and a statistically significant increase of HepG2 cells arrested in G₂/M phase. In addition, it altered mitochondrial membrane potential and induced chromosomal DNA fragmentation (24 h). In HepaRG cells, PhSeZnCl was able to determine a cell cycle-independent induction of apoptosis. Particularly, 50 µg/mL induced mitochondrial membrane depolarization after 24 h and apoptosis after 4 h treatment. Futhermore, all PhSeZnCl concentrations tested determined a significant increase of apoptotic cells after 24 h. Apoptosis was also highlighted by the detection of active Caspase-3 by Western Blot analysis after 24 h exposure. In conclusion, this first toxicological assessment provides new insights into the biological activity of PhSeZnCl in preclinical hepatic models that will be useful in future safety assessment investigation of this compound as a potential pharmaceutical.

Supplementary information: The online version contains supplementary material available at 10.1007/s43188-022-00148-y.

Keywords: Apoptosis; Cell cycle; Comet assay; HepG2; HepaRG; Phenylselenenylzinc chloride.

Fig. 1

PhSeZnCl chemical data

Fig. 2

Effects of PhSeZnCl on cell viability in HepG2 [A] and HepaRG [B] cells after 4- or 24-h exposure. Concentration and time-dependent effects of PhSeZnCl on cell viability were assessed by the Trypan blue dye exclusion assay and AO/DAPI double staining. Results are summarized as mean ± SEM. IC₅₀ was estimated by considering logarithm transformed PhSeZnCl concentrations

Fig. 3

Effects of PhSeZnCl on HepG2 [A] and HepaRG [B] cell cycle determined using the three highest concentrations which did not show cytotoxic effects. Statistical analysis: p values indicate statistically significant different proportion of cells in the cell cycle phases, compared with the negative control, one-way ANOVA followed by Dunnett’s post hoc analysis

Fig. 4

Western Blot analysis for the detection of pro- and activated Caspase-3. [A] 24 h treated HepaRG cells: representative image; [B] results (normalized to β-Actin) are presented as arbitrary units relative to control, taken the latter as a unit. Statistical analysis: one-way ANOVA followed by Dunnett’s post hoc analysis; Student’s t-test was used for positive controls; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. [C, D] microphotographs of HepaRG cells: untreated (control) and 50 µg/mL treated cells, respectively