Strong Evidence from Studies with Brachymorphic Mice and Pentachlorophenol That 1́-Sulfoöxysafrole Is the Major Ultimate Electrophilic and Carcinogenic Metabolite of 1́-Hydroxysafrole in Mouse Liver

The role of sulfation of 1́-hydroxysafrole in the formation of hepatic macromolecular adducts and in hepatic tumor formation in mice given 1́-hydroxysafrole was investigated by the use of: (a) mice treated with the specific sulfotransferase inhibitor pentachlorophenol; and (b) brachymorphic mice, which are characterized by a deficiency in the hepatic synthesis of 3́-phosphoadenosine 5́-phosphosulfate. Cytosolic sulfotransferase activity for 1́-hydroxysafrole in both mouse and rat liver was significantly inhibited by 10 µM pentachlorophenol, usually by 90%. Prior administration of nontoxic amounts of pentachlorophenol, either in the diet of adult female CD-1 mice or by i.p. injection of 12-day-old male C57BL/6 × C3H F₁ (hereafter called B6C3F₁) mice, resulted in an 85% decrease in the level of adducts formed from 1́-hydroxysafrole in hepatic DNA and RNA as compared to those of non-pentachlorophenol-treated animals. Likewise, the chronic administration of a nontoxic level of pentachlorophenol in the diet of adult female CD-1 mice strongly inhibited hepatic tumor induction by long-term dietary administration of either safrole or 1́-hydroxysafrole. Initiation of hepatic tumors by a single i.p. injection of 1́-hydroxysafrole to 12-day-old male B6C3F₁ mice was strongly inhibited by prior treatment with pentachlorophenol. Under these conditions, the hepatocarcinogenicity of diethylnitrosamine was not inhibited by pentachlorophenol. Supplementation with adenosine triphosphate and sulfate of hepatic cytosols from adult female or 12-day-old brachymorphic progeny of a B6C3 background outbred to B6C3F₁ mice (B6C3F₂), of either sex, resulted in 5- to 10-fold less binding of 1́-hydroxysafrole to added RNA than when cytosols from phenotypically normal B6C3F₂ mice were used. On administration of [³H]-1́-hydroxysafrole to adult female or 12-day-old brachymorphic B6C3F₂ mice of either sex, the levels of hepatic DNA and RNA adducts were 7- to 12-fold lower than those obtained in phenotypically normal B6C3F₂ mice of the same age and sex. Brachymorphic mice were also much less responsive than their phenotypically normal littermates to the induction of liver tumors by 1́-hydroxysafrole; lower incidences were observed both when the carcinogen was fed chronically to adult females and when it was administered to males only prior to weaning. Thus, all of these data strongly support the conclusion that 1́-sulfoöxysafrole is the major ultimate electrophilic and tumor-initiating metabolite of 1́-hydroxysafrole.