Inhibition of glutathione S-transferase activity by the quinoid metabolites of equine estrogens.
The risk factors for women developing breast and endometrium cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy (ERT) in particular has been correlated with a slight increased cancer risk, although the numerous benefits of ERT may negate this harmful side effect. Equilenin and equilin are equine estrogens which make up between 30% and 45% of the most widely prescribed estrogen replacement formulation, Premarin (Wyeth-Ayerst). In this study we have synthesized the catechol metabolites of equilenin [4-hydroxyequilenin (4-OHEN)] and equilin [4-hydroxyequilin (4-OHEQ)] and examined how changing unsaturation in the B ring affects the formation of o-quinone GSH conjugates and the ability of the o-quinones and/or GSH conjugates to inhibit glutathione S-transferase (GST). Interestingly, both 4-OHEN and 4-OHEQ autoxidized to o-quinones without the need of oxidative enzyme catalysis. 4-OHEN-o-quinone reacts with GSH to give two mono-GSH conjugates and one diadduct. The behavior of 4-OHEQ was found to be more complex than 4-OHEN as conjugates resulting from 4-OHEN were detected in addition to the 4-OHEQ GSH adducts. Both 4-OHEN and 4-OHEQ were found to be potent inhibitors of GST-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene. In contrast, the endogenous catechol estrogens, 4-hydroxyestrone (4-OHE) and 2-hydroxyestrone (2-OHE), were without effect unless tyrosinase was present to convert the catechols to o-quinones. Scavengers of reactive oxygen species and metal chelators had no effect on GST inhibition by catechol estrogens with the exception of the catalase which protected GST activity. Kinetic studies showed that 4-OHEN was a potent irreversible inactivator of GST. Preincubation of the enzyme with 4-OHEN showed a time-dependent increase in inhibitory effect, and gel filtration did not restore GST activity confirming the irreversible nature of the enzyme inactivation. Analysis of the Kitz-Wilson plot gave a dissociation constant of the reversible enzyme-inhibitor complex (Ki = 620 microM) and a rate constant of conversion of the reversible enzyme-inhibitor complex to the irreversibly inhibited enzyme (k2 = 7.3 x 10(-)3 s-1). These data suggest that 4-OHEN is an irreversible inactivator with relatively low affinity for GST; however, once formed the 4-OHEN enzyme complex is rapidly converted to the irreversibly inhibited enzyme. The inhibition mechanism likely involves oxidation of the catechol estrogens to o-quinones and covalent modification and/or oxidation of critical amino acid residues on GST. In addition, hydrogen peroxide generated through redox cycling of the o-quinone and/or semiquinone radical and GSH could cause oxidative damage to GST.
Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231, USA.
M
M
Chang
F
F
Zhang
L
L
Shen
N
N
Pauss
I
I
Alam
R B
RB
van Breemen
S Y
SY
Blond
J L
JL
Bolton
D000818:Animals; D001711:Biotransformation; D002396:Catechols / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004137:Dinitrochlorobenzene; D004855:Equilenin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D004857:Equilin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D045166:Estradiol Congeners / Q000493:pharmacokinetics / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006736:Horses; D007700:Kinetics; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species; D013056:Spectrophotometry, Ultraviolet
D002396:Catechols; D004137:Dinitrochlorobenzene; D045166:Estradiol Congeners; D017382:Reactive Oxygen Species; D004857:Equilin; C065250:4-hydroxy-equilenin; C065248:4-hydroxy-equilin; D005982:Glutathione Transferase; D005978:Glutathione; D004855:Equilenin