Immunochemical identification of hepatic protein adducts derived from estragole

Hepatic protein adducts derived from the allylbenzene food flavor estragole, which is hepatocarcinogenic when given to rodents at high doses, have been identified using immunochemical approaches. Male Fischer 344 rats were given estragole orally and hepatic protein adducts were detected by immunoblotting, using antisera raised by immunizing rabbits with 4-methoxycinnamic acid-modified rabbit serum albumin. A major 155-kDa adduct was expressed in livers of animals that had been treated with estragole at 100, 300, or 500 mg/kg. Levels of expression of the adduct increased disproportionately with respect to dose, and other adducts (170, 100, 44, and 35 kDa) were detected also in the high-dose group. Rats given estragole for 5 days, at 300 mg/kg/day, expressed predominantly 155- and 44-kDa adducts. The 155-, 100-, 44-, and 35-kDa adducts were detected in greatest abundance in liver microsomal fractions, while the 170-kDa adduct was most abundant in the nuclear fraction. Interestingly, whereas the 170-, 155-, 100-, and 35-kDa adducts were detected in cytosolic fractions, relatively low levels of the 44-kDa adduct were detected in nuclear fractions but not in cytosolic fractions. The various adducts were solubilized when microsomal fractions were extracted with sodium carbonate and were digested by trypsin. This implies that the target proteins are peripheral membrane proteins bound to the outer surface of microsomal membranes. Experiments undertaken with isolated rat hepatocytes and with V79 cells transfected with human monoamine phenol sulfotransferase cDNA revealed that adduct formation required 1'-hydroxylation of estragole, followed by sulfation. The pattern of adducts expressed when the transfected V79 cells were incubated with 1'-hydroxyestragole was very similar to that expressed in livers of estragole-treated rats. These cells should constitute a valuable in vitro model system for investigation of toxicological consequences arising from estragole-induced protein adduct formation.