Effect of β-enantiomeric and racemic nucleoside analogues on mitochondrial functions in HepG2 cells: Implications for predicting drug hepatotoxicity

Abstract

A group of enantiomeric nucleoside analogues with β- d or β- l configuration, which represent potential candidates for the treatment of hepatitis B virus (HBV) infection, were incubated in human hepato-blastoma HepG2 cells at concentrations between 0.1 and 10 μM for 4–14 days. Then the effects on mitochondrial DNA (mtDNA) content, lactic acid production, lipid droplet formation, and mitochondrial morphology were evaluated. No effect on lactic acid production was detected in cells treated with β- l-2′,3′-dideoxy-3′-thiacytidine (3TC), β- l-2′,3′-dideoxy-5-fluoro-3′-thiacytidine (β- l-FTC), β- d-2′,3′-dideoxy-5-fluoro-3′-thiacytidine (β- d-FTC), racemic cis 2′,3′-dideoxy-5-fluoro-3′-thiacytidine [(±)-FTC], and 2,4- diamino-7-(2,3- dideoxy-2- fluoro-β- d- arabinofuranosyl)pyrrolo[2,3-d] pyrimidine (T70178), whereas a slight increase was associated with β- d-2-hydroxymethyl-5-(2,6-diaminopurin-9-yl)-1,3-dioxolane (β- d-DAPD) and 4- amino-7-(2- deoxy-2- fluoro-β- d- arabinofuranosyl)pyrrolo[2,3-d] pyrimidine-5- thiocarboxamide (T70182) at 10 μM. A concentration-dependent increase in lactic acid production was observed in cells exposed to β- d-2′,3′-dideoxy-3′-thiacytidine [(±)-BCH-189], racemic cis 2′,3′-dideoxy-3′-thiacytidine [(±)-BCH-189], β- d-2′,3′-dideoxy-5-fluorocytidine (β- d-FddC), β- l-2′,3′-dideoxy-5-fluorocytidine (β- l-FddC), β- d-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1, 3,-dioxolane (β- d-FDOC), 2,4- diamino-7-(2- deoxy-2- fluoro-β- d- arabinofuranosyl)pyrrolo[2,3-d] pyrimidine (T70080), and 4- amino-7-(2- deoxy-2-nuoro-β- d- arabinofuranosyl)pyrrolo[2,3-d] pyrimidine (T70179). Inhibition on mtDNA content was demonstrated to be concentration-dependent with (+)-BCH-189, β- d-FddC, and T70080, whereas 3TC, (±)-BCH-189, β- l-FTC, β- d-FTC, (±)-FTC, β- l-FddC, β- d-DAPD, T70178, T70179, and T70182 had no effect. β- d-FDOC resulted in a marked inhibition of mtDNA synthesis at 10 μM but not at lower concentrations. Cells treated with 3TC, (±)- BCH-189, β- l- FTC , β- d-FTC, (±)-FTC, β- l-FddC, β- d-DAPD, T70178, T70179, and T70182 did not show morphological changes compared with the control. In contrast, increased cytoplasmic lipid droplets associated with a loss of cristae in mitochondria were detected in cells treated with either β- d-FDOC, β- d-FddC, or T70080. (+)-BCH-189 treatment resulted in loss of cristae in mitochondria. In summary, 3TC, β- l-FTC, β- d-FTC, (±)-FTC, β- d-DAPD, T70178, and T70182 exhibited a relatively safe profile, supporting their further development.