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

A group of enantiomeric nucleoside analogues with beta-D or beta-L configuration, which represent potential candidates for the treatment of hepatitis B virus (HBV) infection, were incubated in human hepatoblastoma HepG2 cells at concentrations between 0.1 and 10 microM for 4-14 days. Then the effect 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 beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (beta-L-FTC), beta-D-2',3'-dideoxy-5-fluoro-3'-thiacytidine (beta-D-FTC), racemic cis 2',3'-dideoxy-5-fluoro-3'thiacytidine [(+/-)-FTC], and 2,4-diamino-7-(2,3-dideoxy-2-fluoro-beta-D-arabinofuranosyl) pyrrolo[2',3'-d]pyrimidine (T70178), whereas a slight increase was associated with beta-D-2-hydroxymethyl-5-(2,6-diaminopurin-9-yl)-1,3-dixolane++ + (beta-D-DAPD) and 4-amino-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimi dine -5-thiocarboxamide (T70182) at 10 microM. A concentration-dependent increase in lactic acid production was observed in cells exposed to beta-D-2',3'-dideoxy-3'-thiacytidine [(+)-BCH-189], racemic cis 2',3'-dideoxy-3'-thiacytidine [(+/-)-BCH-189], beta-D-2',3'-dideoxy-5-fluorocytidine (beta-D-FddC), beta-L-2',3'-dideoxy-5-fluorocytidine (beta-L-FddC), beta-D-2-hydroxymethyl-5-(5-fluorocytosin-I-yl)-1,3,-dioxolane (beta-D-FDOC), 2,4-diamino-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) pyrrolo[2,3-d]pyrimidine (T70080), and 4-amino-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo [2,3-d]pyrimidine (T70179). Inhibition on mtDNA content was demonstrated to be concentration-dependent with (+)-BCH-189, beta-D-FddC, and T70080, whereas 3TC, (+/-)-BCH-189, beta-L-FTC, beta-D-FTC, (+/-)-FTC, beta-L-FddC, beta-D-DAPD, T70178, T70179, and T70182 had no effect. beta-D-FDOC resulted in a marked inhibition of mtDNA synthesis at 10 microM but not at lower concentrations. Cells treated with 3TC, (+/-)-BCH-189, beta-L-FTC, beta-D-FTC, (+/-)-FTC, beta-L-FddC, beta-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 beta-D-FDOC, beta-D-FddC, or T70080, (+)-BCH-189 treatment resulted in loss of cristae in mitochondria. In summary, 3TC, beta-L-FTC, beta-D-FTC, (+/-)-FTC, beta-D-DAPD, T70178, and T70182 exhibited a relatively safe profile, supporting their further development.