4-Hydroxyestradiol is conjugated with thiols primarily at C-2: evidence from regiospecific displacement of tritium by rat liver microsomes or tyrosinase.
4-Hydroxyestradiol bearing a 3H label specifically at C-2 was prepared chemically and incubated with male rat liver microsomes or mushroom tyrosinase. A very high proportion (80-90%) of the 3H was displaced from the labeled steroid when either glutathione or N-acetylcysteine was present, and tyrosinase was shown not to require NADPH as cofactor for this reaction. In either case, only negligible amounts (less than 3%) of the 3H radioactivity were found associated with water-soluble adducts in contrast to 3H-labeled 2-hydroxyestradiol, which gave rise to about 25% of such products. The effect of ascorbic acid on the microsomal reaction with regiospecifically labeled estradiol, 2-hydroxyestradiol, and 4-hydroxyestradiol was also investigated, and the results are discussed in terms of the reactivity at different carbon atoms in ring A of the catechol estrogens. All the evidence points to conjugation of 4-hydroxyestradiol with glutathione or N-acetylcysteine at C-2 but not C-1 of this highly reactive catechol estrogen. Measuring the displacement of 3H as 3H2O from specific positions in the steroid ring provides a useful and sensitive method to assess the formation of adducts in cases where their isolation and characterization is particularly difficult.
Jellinck
P H
PH
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
D000818:Animals; D004156:Catechol Oxidase / Q000378:metabolism*; D004958:Estradiol / Q000031:analogs & derivatives* / Q000032:analysis; D002393:Estrogens, Catechol / Q000032:analysis*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D051381:Rats; D013438:Sulfhydryl Compounds / Q000032:analysis*; D014316:Tritium
D002393:Estrogens, Catechol; D013438:Sulfhydryl Compounds; D014316:Tritium; D004958:Estradiol; C014036:4-hydroxyestradiol; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase