Influence of α-Naphthoflavone on the Metabolism and Binding of Ethinylestradiol in Male Syrian Hamster Liver Microsomes: Possible Role in Hepatocarcinogenesis¹

The influence of α-naphthoflavone (ANF) on the metabolism and binding of radiolabeled ethinylestradiol (EE₂) has been examined both in vitro and in vivo in hamster liver microsomes. [¹⁴C]EE₂ was metabolized extensively to seven major oxidative metabolites, 7α-hydroxyEE₂, 4-hydroxyEE₂, 2-hydroxyEE₂, d-homoestrone, monohydroxyEE₂, and two dihydroxyEE₂ metabolites identified as catechols with the additional hydroxy group on ring B or C, and a nonpolar fraction. The main EE₂ metabolite found was 2-hydroxyEE₂, and it represented 47% of the total metabolites formed. The total amount of EE₂ catechol metabolites formed in untreated hamster liver microsomes was 65.6%. When ANF was added in vitro to these hepatic microsomes, there was a 27–45% decline in 2-hydroxyEE₂ formation, a 98% reduction in dihydroxyEE₂ (catechol-2), and a 56–66% reduction in the nonpolar fraction. The marked inhibition of EE₂ metabolism by the in vitro addition of ANF is reflected by a corresponding decrease in the irreversible binding of radioactive hormone to hamster liver microsomal proteins. In contrast, a biphasic response of ANF on EE₂ metabolism was observed after in vivo administration for 2.0 and 4.0 months. After 2.0 months of ANF treatment, there was a modest decline in all [¹⁴C]EE₂ metabolites, except 2-hydroxyEE₂ and dihydroxyEE₂ (catechol 1). After 4.0 months of ANF treatment, the reduction in EE₂ metabolism was even lower than after 2.0 months of treatment. Most interestingly, there was a 1.5-fold increase in 2-hydroxyEE₂ after 4.0 months of ANF treatment, representing nearly 69% of the total EE₂ metabolites formed. These results are consistent with an increase in irreversible binding of [¹⁴C]EE₂ metabolites to hamster liver microsomal proteins after 4.0 months of ANF treatment. The relative 1.3-fold and the absolute 1.5-fold increases in 2-hydroxyEE₂ after 2.0 and 4.0 months of ANF treatment in vivo, respectively, suggest that the elevation in this reactive EE₂ metabolite precursor may contribute importantly to hepatotumorigenesis in the hamster following prolonged EE₂ plus ANF treatment.