No abstract

Raloxifene was approved in 1997 by the FDA for the treatment of osteoporosis in postmenopausal women, and it is currently in clinical trials for the chemoprevention of breast cancer. Before widespread use as a chemopreventive agent in healthy women, the potential cytotoxic mechanisms of raloxifene should be investigated. In the current study, raloxifene was incubated with GSH and either rat or human liver microsomes, and the metabolites and GSH conjugates were characterized using liquid chromatography-tandem mass spectrometry. Raloxifene was converted to raloxifene diquinone methide GSH conjugates, raloxifene o-quinone GSH conjugates, and raloxifene catechols. For comparison, three raloxifene catechols were synthesized and characterized. In particular, 7-hydroxyraloxifene was found to oxidize to the 6,7-o-quinone. As compared with raloxifene diquinone methide, which has a half-life of less than 1 s in phosphate buffer, the half-life of raloxifene 6,7-o-quinone was much longer at t 1/2 ) 69 ( 2.5 min. The stability offered by raloxifene 6,7-o-quinone implies that it may be more toxic than raloxifene diquinone methide. Cytotoxicity studies in the human breast cancer cell lines S30 and MDA-MB-231 showed that 7-hydroxyraloxifene was more toxic than raloxifene in both cell lines. These results suggest that raloxifene could be metabolized to electrophilic and redox active quinoids, which have the potential to cause toxicity in vivo.

Premarin (Wyeth-Ayerst) is the estrogen replacement treatment of choice and continues to be one of the most widely dispensed prescriptions in the United States. In addition to endogenous estrogens, Premarin contains unsaturated estrogens including equilenin. We synthesized the catechol metabolite of equilenin, 4-hydroxyequilenin (4-OHEN), and found that the semiquinone radical of 4-OHEN reacted with 2'-deoxynucleosides generating very unusual adducts. 2'-Deoxyguanosine (dG), 2'-deoxyadenosine (dA), or 2'-deoxycytosine (dC) all gave four isomers, but no product was observed for thymidine under similar physiological conditions. The structures of these adducts were determined by electrospray mass spectrometry and NMR experiments including 1H, 13C, DQF-COSY, ROESY, HOHAHA, HMQC, and HMBC. The spectral data show that dG forms a cyclic adduct with the 4-OHEN producing 2-N1,3-N2-deoxyguanosyl-1,3-dihydroxy-5,7,9(10)-estratriene-4,17-d ione. Similarly, reaction with dA produced 1-N6,3-C2-deoxyadenosyl-2,3-dihydroxy-5,7,9(10)-estratriene-4,17-d ione, and incubations with dC resulted in 1-N3,3-N4-deoxycytosyl-2,3-dihydroxy-5,7,9(10)-estratriene-4,17-di one. We found that care needed to be taken during the isolation of the dA adducts in particular, as any exposure to acidic environments caused hydrolysis of the sugar moiety leaving alkylated adenine. In mixtures of the deoxynucleosides treated with 4-OHEN, reaction occurred primarily with dG followed by dC and dA. With DNA significant apurinic sites were produced as 4-OHEN-adenine adducts were detected in the ethanol wash prior to hydrolysis. When the DNA was hydrolyzed to deoxynucleosides and analyzed by electrospray mass spectrometry, only one isomer of 4-OHEN-dG and one isomer of 4-OHEN-dC were observed. Our data suggest that several different types of DNA lesions could be expected from 4-OHEN including apurinic sites and bulky stable adducts, in addition to the published oxidized damage to DNA caused by 4-OHEN. The production of these semiquinone radical-derived DNA adducts could play a role in the carcinogenic effects of Premarin estrogens.

Tamoxifen is widely prescribed for the treatment of hormone-dependent breast cancer, and it has recently been approved by the Food and Drug Administration for the chemoprevention of this disease. However, long-term usage of tamoxifen has been linked to increased risk of developing endometrial cancer in women. One of the suggested pathways leading to the potential toxicity of tamoxifen involves its oxidative metabolism to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. The resulting quinone methide has the potential to alkylate DNA and may initiate the carcinogenic process. To further probe the chemical reactivity and toxicity of such an electrophilic species, we have prepared the 4-hydroxytamoxifen quinone methide chemically and enzymatically, examined its reactivity under physiological conditions, and quantified its reactivity with GSH. Interestingly, this quinone methide is unusually stable; its half-life under physiological conditions is approximately 3 h, and its half-life in the presence of GSH is approximately 4 min. The reaction between 4-hydroxytamoxifen quinone methide and GSH appears to be a reversible process because the quinone methide GSH conjugates slowly decompose over time, regenerating the quinone methide as indicated by LC/MS/MS data. The tamoxifen GSH conjugates were detected in microsomal incubations with 4-hydroxytamoxifen; however, none were observed in breast cancer cell lines (MCF-7) perhaps because very little quinone methides is formed. Toremifene, which is a chlorinated analogue of tamoxifen, undergoes similar oxidative metabolism to give 4-hydroxytoremifene, which is further oxidized to the corresponding quinone methide. The toremifene quinone methide has a half-life of approximately 1 h under physiological conditions, and its rate of reaction in the presence of excess GSH is approximately 6 min. More detailed analyses have indicated that the 4-hydroxytoremifene quinone methide reacts with two molecules of GSH and loses chlorine to give the corresponding di-GSH conjugates. The reaction mechanism likely involves an episulfonium ion intermediate which may contribute to the potential cytotoxic effects of toremifene. Similar to what was observed with 4-hydroxytamoxifen, 4-hydroxytoremifene was metabolized to di-GSH conjugates in microsomal incubations at about 3 times the rate of 4-hydroxytamoxifen, although no conjugates were detected with MCF-7 cells. Finally, these data suggest that quinone methide formation may not make a significant contribution to the cytotoxic and genotoxic effects of tamoxifen and toremifene.

The risk factors for women developing breast and endometrial cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy in particular has been correlated with a slight increased cancer risk. Previously, we showed that equilenin, a minor component of Premarin (Wyeth-Ayerst), was metabolized to highly cytotoxic quinoids which caused oxidative stress and alkylation of DNA in vitro [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1127]. In this study, we have compared the chemistry of the major catechol metabolite of equilin (4-hydroxyequilin), which is found in several estrogen replacement formulations, to the equilenin catechol (4-hydroxyequilenin). Unlike endogenous catechol estrogens, both equilin and equilenin were primarily converted by rat liver microsomes to 4-hydroxylated rather than 2-hydroxylated o-quinone GSH conjugates. With equilin, a small amount of 2-hydroxyequilin GSH quinoids were detected (4-hydroxyequilin:2-hydroxyequilin ratio of 6:1); however, no peaks corresponding to 2-hydroxyequilenin were observed in incubations with equilenin. These data suggest that unsaturation in the B ring alters the regiochemistry of P450-catalyzed hydroxylation from primarily 2-hydroxylation for endogenous estrogens to 4-hydroxylation for equine estrogens. 4-Hydroxyequilenin-o-quinone reacts with GSH to give two mono-GSH conjugates and one di-adduct. The behavior of 4-hydroxyequilin was found to be more complex than 4-hydroxyequilenin as conjugates resulting from 4-hydroxyequilenin were detected in addition to the 4-hydroxyequilin-GSH adducts. The mechanism of decomposition of 4-hydroxyequilin likely involves isomerization to a quinone methide which readily aromatizes to 4-hydroxyequilenin followed by autoxidation to 4-hydroxyequilenin-o-quinone. Similar results were obtained with 2-hydroxyequilin, although, in contrast to 4-hydroxyequilenin, 2-hydroxyequilenin does not autoxidize and the reaction stops at the catechol. Since 4-hydroxyequilin is converted to 4-hydroxyequilenin and 4-hydroxyequilenin-o-quinone, similar effects were observed for this equine catechol, including consumption of NAD(P)H likely by the 4-hydroxyequilenin-o-quinone, depletion of molecular oxygen by 4-hydroxyequilenin or its semiquinone radical, and alkylation of deoxynucleosides and DNA by 4-hydroxyequilenin quinoids. Finally, preliminary studies conducted with the human breast tumor cell line MCF-7 demonstrated that the cytotoxic effects of the catechol estrogens from estrone, equilin, and 2-hydroxyequilenin were similar, whereas 4-hydroxyequilenin was a much more potent cytotoxin ( approximately 30-fold). These results suggest that the catechol metabolites of equine estrogens have the ability to cause alkylation/redox damage in vivo primarily through formation of 4-hydroxyequilenin quinoids.