Covalent binding of catechol estrogens to glutathione catalyzed by horseradish peroxidase, lactoperoxidase, or rat liver microsomes

Oxidation of catechol estrogens (CE) leads to the reactive electrophilic CE quinones. Reaction of CE-3,4-quinones with DNA has been implicated in tumor initiation. One pathway to prevent this reaction is conjugation of CE quinones with glutathione (GSH). Four CE, 4-hydroxy estrone (4-OHE₁), 4- hydroxyestradiol (4-OHE₂), 2-OHE₁, and 2-OHE₂, were conjugated with GSH after oxidation catalyzed by horseradish peroxidase (HRP), lactoperoxidase (LP), or rat liver microsomal cytochrome P450. This reaction is a free- radical chain autoxidation that produces very high yields of products. Six mono-GSH conjugates, 4-OHE₁(E₂)-2-SG, 2-OHE₁(E₂)-1-SG, and 2-OHE₁(E₂)- 4-SG, and four di-GSH conjugates, 4-OHE₁(E₂)-1,2-bisSG and 2-OHE₁(E₂)1,4- bisSG, were identified and quantified. These di-GSH conjugates were also obtained quantitatively from oxidation of mono-GSH conjugates by the same enzymes. HRP and LP gave very similar product profiles. Phenobarbital- and 3- methylcholanthrene-induced microsomes with either NADPH or cumene hydroperoxide as cofactor oxidized 4-OHE₂ to form similar amounts of GSH conjugates. Enzymatic oxidation of 2-OHE₁(E₂) in the presence of GSH produced more 2-OHE₁(E₂)-4-SG than the 1-isomer. This contrasts with the direct reaction of E₁(E₂)-2,3-Q and GSH, in which the 1-isomer is formed more abundantly than the 4-isomer (Cao, K., Devanesan, P. D., Ramanathan, R., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (1998) Chem. Res. Toxicol. 11, 909-916). Competitive enzymatic oxidation of equimolar 4-OHE₂ and 2- OHE₂ in the presence of an equimolar amount of GSH yielded more 2-OHE₂ conjugates than 4-OHE₂ conjugates, despite E₂-3,4-Q being more reactive with GSH than E₂-2,3-Q. These results suggest that 2-OHE₂ is a better substrate than 4-OHE₂ in the catalytic oxidation to quinones, despite the greater reactivity of E₂-3,4-Q, compared to E₂-2,3-Q, with GSH.