Two-step metabolic activation to ortho-benzoquinone intermediate and its role in 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside-induced liver injury in mice.

2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) is the most abundant constituent of Polygonum multiflorum and is exclusively found in this herb. This renowned herbal medicine has been documented to lead to liver damage in humans. The present study demonstrated that TSG underwent 2-step metabolic activation to generate a reactive metabolite, involving both intestinal and hepatic metabolisms. TSG was hydrolyzed to its aglycone 2,3,5,4'-tetrahydroxystilbene (TS) in the intestine, and then, the 2,3 catechol of 2,3,5,4'-tetrahydroxystilbene was metabolized to an ortho-benzoquinone intermediate in the liver. The reactive metabolite was characterized as the N-acetyl-cysteine conjugate both in vivo and in vitro. Its structure was verified by a combined isotope-labeling strategy using the ¹⁴N/¹⁵N, H/D, and ⁷⁹Br/⁸¹Br isotope pattern-based mass shifts. Intestinal β-glucosidase and hepatic CYP3A4 and CYP2C9 contributed to the reactive metabolite formation. The reactive intermediate could covalently modify the hepatic proteins through cysteine in mice. Combined with the treatment with β-glucosidase, a single oral administration of 400 mg/kg TSG caused liver centrilobular necrosis and degeneration in mice. Selective CYP3A inhibitor ketoconazole protected TSG-induced liver injury, concurrently attenuating protein adduct formation modified by reactive metabolites. The results indicate that TSG does not exert hepatotoxic effects but that the reactive ortho-benzoquinone metabolite from the oxidation of the 2,3 catechol of aglycone is responsible for TSG-induced liver injury. The study also facilitates a better understanding of the principal hepatotoxic chemicals in Polygonum multiflorum. SIGNIFICANCE STATEMENT: 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) undergoes intestinal deglycosylation to generate its aglycone, and then, the 2,3 catechol of aglycone was metabolized to an ortho-benzoquinone intermediate in the liver. β-glucosidase potentiates TSG-induced liver injury and protein adduction by the reactive metabolite. The results indicate that the reactive metabolite of TSG exerts hepatotoxic effects rather than the parent compound.