Search

Life-long estrogen exposure is one of the major risk factors in the development and progression of breast cancer. However, little is known about the molecular mechanisms, by which chronic exposure to estrogen contributes to breast carcinogenesis. The aim of the present study was to investigate the effects of long-term exposure with 4-hydroxyestradiol (4-OHE 2 ) on acquired cancer characteristics of human mammary epithelial MCF-10A cells. The possible regulators were further studied in chronic 4-OHE 2 -treated MCF-10A cells. We observed that MCF-10A cells long-term exposed to 4-OHE 2 acquire the characteristics of cancer cells, such as enhanced cell growth, EMT properties, and increased migration and invasiveness. Moreover, the expression of CYP1B1 was significantly elevated in long-term 4-OHE 2 -treated MCF-10A cells. Block of CYP1B1 significantly reduced the cancer cell characteristics in long-term 4-OHE 2 -treated MCF-10A cells. Our results indicated that 4-OHE 2 mediated enhanced cancer cell characteristics in mammary epithelial cells are an important key event for breast carcinogenesis process. CYP1B1 partially contributes to the 4-OHE 2 induced cancer cell characteristics in MCF-10A cells. Targeting CYP1B1 might offer a new strategy for the treatment of estrogen-induced breast cancer.

The cytochrome P450 (P450) 1 family is an important phase I enzyme involved in carcinogen activation. Nitidine chloride (NC) is a pharmacologically active alkaloid with polyaromatic hydrocarbon found in the roots of Zanthoxylum nitidum (Roxb.) DC, a traditional medicinal herb widely used in China. We examined the inhibitory effects of NC on CYP1A1, 1B1, and 1A2. NC significantly inhibited CYP1A1- and 1B1-catalyzed ethoxyresorufin O -deethylation activity (IC 50 = 0.28 ± 0.06 and 0.32 ± 0.02 μ M, respectively) in a concentration-dependent manner, but only showed slight inhibition of CYP1A2 activity (IC 50 > 50 μ M). Kinetic analysis revealed that NC competitively inhibited CYP1B1 with a K i value of 0.47 ± 0.05 μ M, whereas NC caused a mixed type of inhibition on CYP1A1 with K i and K I values of 0.14 ± 0.04 and 0.19 ± 0.09 μ M, respectively. The observed enzyme inhibition neither required NADPH nor revealed time dependency. Molecular docking manifested the generation of strong hydrogen-bonding interactions of Ser116 in CYP1A1 and Ser127 in CYP1B1 with methoxy moiety of NC. Additionally, NC-induced alteration of estradiol (E2) metabolism was also investigated in the present study. Hydroxyestradiols, including 2-hydroxyestradiol [(2-OHE2) nontoxic] and 4-hydroxyestradiol [(4-OHE2) genotoxic] generated in recombinant enzyme incubation systems and cultured MCF-7 cells were analyzed, and NC was found to preferentially inhibit the nontoxic 2-hydroxylation activity of E2 mediated by CYP1A1. In conclusion, NC was a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. The remarkable inhibition on E2 2-hydroxylation might increase the risk of 4-OHE2-induced genotoxicity. SIGNIFICANCE STATEMENT: CYP1 enzymes catalyze oxidative metabolism of a variety of compounds and are known to play a crucial role in the development of cancer. CYP1A1 and CYP1A2 are responsible for hydroxylation of estradiol (E2) at the C-2 position, resulting in the formation of 2-OHE2, which is proposed to be a detoxification pathway. However, CYP1B1-mediated hydroxylation of E2 at the C-4 position has been suggested to be a tumor initiator. The present study found that nitidine chloride is a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. NC preferentially inhibited the nontoxic E2 2-hydroxylation pathway mediated by CYP1A1, which might increase the risk of 4-OHE2-induced genotoxicity and cause severe drug-drug interactions., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Human cytochrome P450 1B1 (CYP1B1)-mediated formation of 4-hydroxyestradiol (4-OHE2) from 17β-estradiol plays an important role in the progression of human breast cancer, while the biotransformation of 17β-estradiol to 2-hydroxyestradiol mediated by cytochrome P450 1A1 (CYP1A1) is considered as a less harmful pathway. In this study, inhibitory effects of flavonoids baicalein and oroxylin A, a metabolite of baicalein in human body, on CYP1A1 and 1B1 activities were investigated in vitro. The inhibition intensities of baicalein and oroxylin A towards CYP1B1 were greater than towards CYP1A1 with a mixed mechanism. In addition, oroxylin A showed a stronger inhibitory effect than baicalein towards the CYP1B1-mediated 17β-estradiol 4-hydroxylation, with the IC 50 values of 0.0146 and 2.27 μM, respectively. Docking studies elucidated that oroxylin A had a stronger binding affinity than baicalein for CYP1B1. In MCF-7 cells, compared with baicalein-treated groups, oroxylin A with lower doses decreased and increased the formation of 4-OHE2 and 2-hydroxyestradiol, respectively, with a preferential induction of mRNA of CYP1A1 over CYP1B1. In conclusion, this study demonstrated that oroxylin A showed a stronger inhibitory effect than baicalein on CYP1B1-mediated 4-OHE2 formation in MCF-7 cells, providing crucial implications for their possibly preventive/therapeutic potential against breast cancer via inhibition of CYP1B1, particularly of oroxylin A., (© 2019 John Wiley & Sons, Ltd.)

Background: The development of hormone-dependent cancers, including endometrial carcinomas, in great part may be mediated by the genotoxic effects of estrogen metabolites, among which 4-hydroxyestradiol (4OHE2) is characterized by the most prominent DNA-damaging properties. It is assumed that the individual sensitivity to the 4OHE2 may determine the predisposition to endometrial cancer (EС)., Aim: To analyze the sensitivity of peripheral blood lymphocytes (PBLs) of EC patients to the 4OHE2 and to evaluate the repair efficiency of 4OHE2-induced DNA damage., Materials and Methods: The study was performed on the PBLs of 53 EC patients and 20 healthy women. The level of DNA damage was measured using the comet assay and was expressed as % tail DNA. The DNA repair efficiency (%) was evaluated by determining the ratio between the amount of repaired DNA damage and the level of 4OHE2-induced damage that appeared after incubation of PBLs with 4OHE2., Results: In PBLs of EC patients, a higher level of 4OHE2-induced DNA damage (32.0 ± 2.2% tail DNA) and lower DNA repair efficiency (34.0 ± 4.5%) was observed compared to PBLs of healthy women (22.3 ± 2.3% tail DNA and 48.8 ± 4.5%, respectively). PBLs of EC patients with deep tumor invasion of myometrium were characterized by more prominent decrease of DNA repair than those with less invasive tumor (< ½ of myometrium) (20.9 ± 7.8 and 43.7 ± 6.7%, respectively). Furthermore, lower DNA repair efficiency was detected in the PBLs of EC patients with a family history of cancer compared to this parameter in patients with sporadic tumors (20.9±7.8 and 47.1 ± 5.5%, respectively)., Conclusion: The PBLs of EC patients are characterized by increased sensitivity to the genotoxic effect of 4OHE2 and reduced repair efficiency regarding 4OHE2-induced DNA damage. A lower level of DNA repair is observed in EC patients with deep tumor myometrial invasion and a family history of cancer.

The 2- and 4-hydroxy metabolites of 17beta-estradiol (E2) and estrone (E1) are important for E2-mediated carcinogenesis due to the formation of genotoxic ortho-quinone metabolites. To assess the importance of metabolic conjugation for their genotoxicity, the DNA strand-breaking activity of the four catechol estrogens was determined in three cell lines with different activities of catechol-O-methyltransferase (COMT) and UDP-glucuronosyltransferase (UGT). Most DNA strand breaks were observed in V79 cells, which lack these metabolic activities. 2- and 4-hydroxy-E2 were 2.5 times more genotoxic than 2- and 4-hydroxy-E1. MCF-7 cells exhibit COMT activity, and the incidence of DNA strand breaks decreased with increasing methylation; only the 4-hydroxy metabolites of E1 and E2, which were poor substrates of COMT, exhibited low genotoxicity. HepG2 cells converted the catechol and methoxy metabolites of E2 to the respective E1 metabolites by 17beta-hydroxysteroid dehydrogenase (HSD). Moreover, methylation and glucuronidation took place. Only 4-hydroxy-E1 elicited a weak genotoxic response in these cells. The extensive metabolism in HepG2 cells is proposed to account for the failure of catechol estrogens to induce DNA strand breaks. Thus, metabolism by COMT and UGT and, to a minor extent, by HSD is a major determinant for the genotoxicity of catechol estrogens in target cells.

A growing number of studies indicate that breast cancer initiation is related to abnormal estrogen oxidation to form an excess of estrogen-3,4-quinones, which react with DNA to form depurinating adducts and induce mutations. This mechanism is often called estrogen genotoxicity. 4-Catechol estrogens, precursors of the estrogen-3,4-quinones, were previously shown to account for most of the transforming and tumorigenic activity. We examined whether estrogen-induced transformation can be reduced by inhibiting the oxidation of a 4-catechol estrogen to its quinone. We demonstrate that E6 cells (a normal mouse epithelial cell line) can be transformed by a single treatment with a catechol estrogen or its quinone. The transforming activities of 4-hydroxyestradiol and estradiol-3,4-quinone were comparable. N-Acetylcysteine, a common antioxidant, inhibited the oxidation of 4-hydroxyestradiol to the quinone and consequent formation of DNA adducts. It also drastically reduced estrogen-induced transformation of E6 cells. These results strongly implicate estrogen genotoxicity in mammary cell transformation. Since N-acetylcysteine is well tolerated in clinical studies, it may be a promising candidate for breast cancer prevention.

There is a clear association between the excessive exposure to estrogens and the development of cancer in hormone-sensitive tissues (breast, endometrium). It has become clear that there are likely multiple overlapping mechanisms of estrogen carcinogenesis. One major pathway is the extensively studied hormonal pathway, by which estrogen stimulates cell proliferation through nuclear estrogen receptor (ER)-mediated signaling, thus resulting in an increased risk of genomic mutations during DNA replication. A similar "nongenomic pathway", potentially involving newly discovered membrane-associated ERs, also appears to regulate extranuclear estrogen signaling pathways. This perspective is focused on a third pathway involving the metabolism of estrogens to catechols mediated by cytochrome P450 and further oxidation of these catechols to estrogen o-quinones. Oxidative enzymes, metal ions, and in some cases molecular oxygen can catalyze o-quinone formation, so that these electrophilic/redox-active quinones can cause damage within cells by alkylation and/or oxidation of cellular proteins and DNA in many tissues. It appears that the endogenous estrogen quinones primarily form unstable N3-adenine or N7-guanine DNA adducts, ultimately resulting in mutagenic apurinic sites. In contrast, equine estrogen quinones, formed from estrogens present in popular hormone replacement therapy prescriptions, generate a variety of DNA lesions, including bulky stable adducts, apurinic sites, DNA strand cleavage, and oxidation of DNA bases. DNA damage induced by these equine quinones is significantly increased in cells containing ERs, leading us to hypothesize a mechanism involving ER binding/alkylation by the catchol/quinone, resulting in a "Trojan horse". The "Trojan horse" carries the highly redox-active catechol to estrogen -sensitive genes, where high amounts of reactive oxygen species are generated, causing selective DNA damage. Our data further suggest that other key protein targets for estrogen o-quinones could be redox-sensitive enzymes (i.e, GST P1-1, QR). These proteins are involved in stress response cascades that are known to contribute to the regulation of cell proliferation and apoptosis. Finally, it has been shown that catechol estrogens can transform breast epithelial cells into a tumorigenic phenotype and that these transformed cells had differential gene expression of several genes involved in oxidative stress. Given the direct link between excessive exposure to estrogens, metabolism of estrogens, and increased risk of breast cancer, it is crucial that factors that affect the formation, reactivity, and cellular targets of estrogen quinoids be thoroughly explored.

Estrogens are indicated as being the most important etiological factors for the development and progression of breast cancer. The implication of estrogen in breast cancer has been associated mostly with the estrogen receptors that mediate cell proliferation. Evidence also exists to support the hypothesis of a direct role of estrogens as tumor initiators. However, the role of estrogen genotoxicity in breast cancer is still questionable. In this study the genotoxic activity of catecholestrogens and 16alpha-hydroxy estrone has been investigated by performing Salmonella strain TA98 and TA100 Ames tests, sister chromatide exchange assays (SCE) and micronucleus assays on human peripheral lymphocytes (CBMN and ARA/CBMN). We found a lack of positive results with micronucleus assays, except for 2-hydroxy estradiol (2-OHE(2)), which shows a peculiar "bell shaped" trend of micronucleus number versus concentrations. SCE assay suggests weak genotoxic activity of all tested catechol metabolites, except 4-hydroxy estrone (4-OHE(1)), which also showed negative results by ARA/CBMN. In this open debate, our results support the hypothesis of a weak genotoxicity, not correlated with the carcinogenetic potential of estrogens.

Long-term exposure to steroidal estrogens is a key factor contributing to increases in the risk of developing breast cancer. Proposed mechanisms include receptor-activated increases in the rate of cell proliferation leading to the accumulation of genetic damage resulting from reading errors, and the production of DNA damage by species arising from metabolism of 17beta-estradiol (E2) resulting in mutations. In support of the second mechanism, catechol metabolites of E2 induce DNA damage in vitro. In the present study, utilizing the single-cell gel electrophoresis (Comet) assay, we observed increases in the number of single-strand breaks in estrogen receptor alpha-positive (MCF-7) and -negative (MDA-MB-231) breast cancer cells exposed to E2 (for 24 hr) or 4-hydroxy-17beta-estradiol (4-OH-E2; for 2 hr). The concentrations of 4-OH-E2 sufficient to induce these effects were approximately 100 nM, substantially lower than reported previously. The catechol 2-hydroxy-17beta-estradiol (2-OH-E2) also induced strand breaks. 2-OH-E2, often referred to as an improbable carcinogen in humans, is not a major metabolite of E2 in the breast; however, our findings show that it is as DNA-damaging as 4-OH-E2. Formamidopyrimidine glycosylase posttreatment of E2-, 4-OH-E2-, and 2-OH-E2-exposed MCF-7 cells led to an up to sixfold increase in mean tail moment, suggesting that oxidative DNA damage was formed. Comet formation could be partially attenuated by coincubation with dimethylsulfoxide, attributing a small DNA-damaging role to oxyradicals emanating from catechol redox cycling. Similar findings were obtained with MDA-MB-231 cells, indicating that estrogen receptor status is not relevant to these effects. Our observations show that exposure to E2 adds to the oxidative load of cells, and this may contribute to genomic instability., (Copyright 2005 Wiley-Liss, Inc)

The primary purpose of this research is to examine the hypothesis that reactive oxygen species generated by estrogen quinonoids are the main source for the formation of aldehydic DNA lesions (ADL) in genomic DNA. ADL induced by quinonoid metabolites of 17beta-estradiol (E2), e.g. 4-hydroxyestradiol (4-OH-E2), 2-hydroxyestradiol (2-OH-E2), estrogen-3,4-quinones (E2-3,4-Q) and estrogen- 2,3-quinone (E2-2,3-Q), were investigated in calf thymus DNA (CT-DNA) under physiological conditions. The abasic sites resulting from the spontaneous depurination-depyrimidination of the modified bases and the aldehydic base and sugar lesions resulting from the oxidative damage to deoxyribose moieties in the DNA molecules were measured by an aldehyde reactive probe and were estimated as the number of ADL per 106 nucleotides. With the addition of NADPH (100 micro M) and Cu(II) (20 micro M), nanomolar levels (100 nM) of 4-OH-E2 and 2-OH-E2 induced approximately 10-fold increases in the number of ADL over control (P<0.001). In parallel, increases in 8-oxoguanine were detected in DNA exposed to 4-OH-E2 and 2-OH-E2 (100 nM) plus Cu(II) and NADPH. Further investigation indicated that the ADL induced by estrogen catechols plus Cu(II) and NADPH were causally involved in the formation of hydrogen peroxide and Cu(I). Both E2-2,3-Q and E2-3,4-Q alone induced a 2-fold increase in the number of ADL over control (P<0.05) in CT-DNA at high concentrations (1 mM). Neither neutral thermal hydrolysis nor lower ionic strength of the reaction medium induced further increases in the number of ADL in E2-3,4-Q-modified CT-DNA. Conversely, with the inclusion of Cu(II) and NADPH, both E2-3,4-Q and E2-2,3-Q (1 micro M) induced parallel formation of DNA single strand breaks and approximately 20-fold increases in the number of ADL over control (P < 0.001). The data also demonstrated that the ADL induced by estrogen quinones with and without the presence of Cu(II) and NADPH contain 69 and 78% putrescine-excisable ADL in CT-DNA, respectively. Additionally, results of the ADL cleavage assay indicate that the ADL induced by estrogen quinones plus Cu(II) and NADPH in CT-DNA were predominantly T7 exonuclease-excisable (50%) and exonuclease III- excisable (20%) ADL, whereas the intact ADL, and other ADL accounted for 5 and 25%, respectively. These results suggest that the ADL induced by estrogen quinones in CT-DNA are derived from oxidative events rather than depurination/depyrimidination of labile estrogen quinone-DNA adducts. Overall, our results are at variance with the idea that depurination of estrogen quinone-DNA adducts is the major source for the formation of ADL in genomic DNA. We hypothesize that in addition to DNA adducts and oxidized bases, the ADL induced by estrogen quinonoid-mediated oxidative stress may play a role in estrogen-induced carcinogenicity.

Catechol estrogens and catecholamines are metabolized to quinones, and the metabolite catechol (1,2-dihydroxybenzene) of the leukemogenic benzene can also be oxidized to its quinone. We report here that quinones obtained by enzymatic oxidation of catechol and dopamine with horseradish peroxidase, tyrosinase or phenobarbital-induced rat liver microsomes react with DNA by 1,4-Michael addition to form predominantly depurinating adducts at the N-7 of guanine and the N-3 of adenine. These adducts are analogous to the ones formed with DNA by enzymatically oxidized 4-catechol estrogens (Cavalieri,E.L., et al. (1997) PROC: Natl Acad. Sci., 94, 10937). The adducts were identified by comparison with standard adducts synthesized by reaction of catechol quinone or dopamine quinone with deoxyguanosine or adenine. We hypothesize that mutations induced by apurinic sites, generated by the depurinating adducts, may initiate cancer by benzene and estrogens, and some neurodegenerative diseases (e.g. Parkinson's disease) by dopamine. These data suggest that there is a unifying molecular mechanism, namely, formation of specific depurinating DNA adducts at the N-7 of guanine and N-3 of adenine, that could initiate many cancers and neurodegenerative diseases.

Exposure to estrogens is a risk factor for breast and other human cancers. Initiation of breast, prostate and other cancers has been hypothesized to result from reaction of specific estrogen metabolites, catechol estrogen-3,4-quinones, with DNA to form depurinating adducts at the N-7 of guanine and N-3 of adenine by 1,4-Michael addition. The catechol of the carcinogenic synthetic estrogen hexestrol, a hydrogenated derivative of diethylstilbestrol, is metabolized to its quinone, which reacts with DNA to form depurinating adducts at the N-7 of guanine and N-3 of adenine. The catecholamine dopamine and the metabolite catechol (1,2-dihydroxybenzene) of the leukemogen benzene can also be oxidized to their quinones, which react with DNA to form predominantly analogous depurinating adducts. Apurinic sites formed by depurinating adducts are converted into tumor-initiating mutations by error-prone repair. These mutations could initiate cancer by estrogens and benzene, and Parkinson's disease by the neurotransmitter dopamine. These data suggest a unifying molecular mechanism of initiation for many cancers and neurodegenerative diseases and lay the groundwork for designing strategies to assess risk and prevent these diseases.

Catechol estrogens are considered critical intermediates in estrogen-induced carcinogenesis. We demonstrated previously that 17beta-estradiol (E(2)), estrone (E(1)) and four of their catechol estrogens, 2- and 4-hydroxyestradiols (2- and 4-OHE(2)), and 2- and 4-hydroxyestrones (2- and 4-OHE(1)) induce morphological transformation in Syrian hamster embryo (SHE) fibroblasts, and the transforming abilities vary as follows: 4-OHE(1) > 2-OHE(1) > 4-OHE(2) > 2-OHE(2) vertical line E(2), E(1). To examine the involvement of catechol estrogens in the initiation of hormonal carcinogenesis, we studied the ability of E(2), E(1) and their catechol estrogens to induce DNA adducts in SHE cells by using a (32)P-post-labeling assay. DNA adducts were detected in cells treated with each of all the catechol estrogens at concentrations of 10 microg/ml for 1 h and more. 2- or 4-OHE(2) formed a single DNA adduct, which was chromatographically distinct from each other. In contrast, 2- or 4-OHE(1) produced one major and one minor adduct, and the two adducts formed by each catechol estrogen exhibited identical mobilities on the chromatograms. Neither E(2) nor E(1) at concentrations up to 30 microg/ml induced DNA adducts. The abilities of the estrogens to induce DNA adducts were ranked as follows: 4-OHE(1) > 2-OHE(1) > 4-OHE(2) > 2-OHE(2) > > E(2), E(1), which corresponds well to the transforming and carcinogenic abilities of the estrogens. In addition, the level of DNA adducts induced by the catechol estrogens was markedly decreased by co-treatment of cells with the antioxidant L-ascorbic acid. The results indicate the possible involvement of oxidative metabolites of catechol estrogens of E(2) and E(1) in the initiation of endogenous estrogen-induced carcinogenesis.

Estrone (E1) and 17beta-estradiol (E2) are metabolized to catechol estrogens (CE), which may be oxidized to semiquinones and quinones (CE-Q). CE-Q can react with glutathione (GSH) and DNA, or be reduced to CE. In particular, CE-3,4-Q react with DNA to form depurinating adducts (N7Gua and N3Ade), which are cleaved from DNA to leave behind apurinic sites. We report the determination of 22 estrogen metabolites, conjugates and adducts in the urine of male Syrian golden hamsters treated with 4-hydroxyestradiol (4-OHE2). After initial purification, urine samples were analyzed by HPLC with multichannel electrochemical detection and by capillary HPLC/tandem mass spectrometry. 4-Hydroxyestrogen-2-cysteine [4-OHE1(E2)-2-Cys] and N-acetylcysteine [4-OHE1(E2)-2-NAcCys] conjugates, as well as the methoxy CE, were identified and quantified by HPLC, whereas the 4-OHE1(E2)-1-N7Gua depurinating adducts and 4-OHE1(E2)-2-SG conjugates could only be identified by the mass spectrometry method. Most of the administered 4-OHE2 was metabolically converted to 4-OHE1. Formation of thioether (GSH, Cys and NAcCys) conjugates and depurinating adducts [4-OHE1(E2)-1-N7Gua] indicates that oxidation of 4-CE to CE-3,4-Q and subsequent reaction with GSH and DNA, respectively, do occur. The major conjugates in the urine were 4-OHE1(E2)-2-NACCYS: The oxidative pathway of 4-OHE1(E2) accounted for approximately twice the level of products compared with those from the methylation pathway. The metabolites and methoxy CE were excreted predominantly (>90%) as glucuronides, whereas the thioether conjugates were not further conjugated. These results provide strong evidence that exposure to 4-OHE1(E2) leads to the formation of E1(E2)-3,4-Q and, subsequently, depurinating DNA adducts. This process is a putative tumor initiating event. The estrogen metabolites, conjugates and adducts can be used as biomarkers for detecting enzymatic oxidation of estrogens to reactive electrophilic metabolites and possible susceptibility to estrogen-induced cancer.

Estrogen replacement therapy has been correlated with an increased risk of developing breast or endometrial cancer. 4-Hydroxyequilenin (4-OHEN) is a catechol metabolite of equilenin which is a minor component of the estrogen replacement formulation marketed under the name of Premarin (Wyeth-Ayerst). Previously, we showed that 4-OHEN autoxidizes to quinoids which can consume reducing equivalents and molecular oxygen, are potent cytotoxins, and cause a variety of damage to DNA, including formation of bulky stable adducts, apurinic sites, and oxidation of the phosphate-sugar backbone and purine/pyrimidine bases [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1127]. All of these deleterious effects could contribute to the cytotoxic and genotoxic effects of equilenin in vivo. In the study presented here, we examined the relative toxicity of 4-OHEN in estrogen receptor (ER) positive cells (MCF-7 and S30) compared to that in breast cancer cells without the estrogen receptor (MDA-MB-231). The data showed that 4-OHEN was 4-fold more toxic to MCF-7 cells (LC(50) = 6.0 +/- 0. 2 microM) and 6-fold more toxic to S30 cells (LC(50) = 4.0 +/- 0.1 microM) than to MDA-MB-231 cells (LC(50) = 24 +/- 0.3 microM). Using the single-cell gel electrophoresis assay (comet assay) to assess DNA damage, we found that 4-OHEN causes concentration-dependent DNA single-strand cleavage in all three cell lines, and this effect could be enhanced by agents which catalyze redox cycling (NADH) or deplete cellular GSH (diethyl maleate). In addition, the ER(+) cell lines (MCF-7 and S30) were considerably more sensitive to induction of DNA damage by 4-OHEN than the ER(-) cells (MDA-MB-231). 4-OHEN also caused a concentration-dependent increase in the amount of mutagenic lesion 8-oxo-dG in the S30 cells as determined by LC/MS-MS. Cell morphology assays showed that 4-OHEN induces apoptosis in these cell lines. As observed with the toxicity assay and the comet assay, the ER(+) cells were more sensitive to induction of apoptosis by 4-OHEN than MDA-MB-231 cells. Finally, the endogenous catechol estrogen metabolite 4-hydroxyestrone (4-OHE) was considerably less effective at inducing DNA damage and apoptosis in breast cancer cell lines than 4-OHEN. Our data suggest that the cytotoxic effects of 4-OHEN may be related to its ability to induce DNA damage and apoptosis in hormone sensitive cells in vivo, and these effects may be potentiated by the estrogen receptor.

Catechol estrogens may mediate estrogen-induced carcinogenesis because 4-hydroxyestradiol induces DNA damage and renal tumors in hamsters, and this metabolite is formed in the kidney and estrogen target tissues by a specific estrogen 4-hydroxylase. We examined the carcinogenic potential of catechol estrogen in an experimental model previously reported to result in a high incidence of uterine adenocarcinoma after neonatal exposure to diethylstilbestrol. Outbred female CD-1 mice were treated with 2- or 4-hydroxyestradiol, 17beta-estradiol, or 17alpha-ethinyl estradiol on days 1-5 of neonatal life (2 microg/pup/day) and sacrificed at 12 or 18 months of age. Mice treated with 17beta-estradiol or 17a-ethinyl estradiol had a total uterine tumor incidence of 7% or 43%, respectively. 2-Hydroxyestradiol induced tumors in 12% of the mice, but 4-hydroxyestradiol was the most carcinogenic estrogen, with a 66% incidence of uterine adenocarcinoma. Both 2- and 4-hydroxylated catechols were estrogenic and increased uterine wet weights in these neonates. These data demonstrate that both 2- and 4-hydroxyestradiol are carcinogenic metabolites. The high tumor incidence induced by 4-hydroxyestradiol supports the postulated role of this metabolite in hormone-associated cancers.

The growth-promoting effects of estrogens in hormone-dependent tumor tissues involve receptor-mediated pathways that are well-recognized; however, the role of estrogens in tumor initiation remains controversial. Estrogen metabolites, primarily the catechol estrogens (CE's), have been implicated in tumor initiation via a redox cycling mechanism. We have developed metabolically stable CE analogues for the study of receptor versus redox cycling effects on DNA damage. Comparisons between hydroxy estradiols (HE2's), methoxy estradiols (ME2's), and hydroxymethyl estradiols (HME2) in potentiometric and DNA damaging studies were made. DNA damage was assessed in calf thymus DNA using 8-oxo-2'-deoxyguanosine (8-oxo-dG) as a genotoxic marker for oxidative stress. Increases in the number of 8-oxo-dG/10(5) dG were significant for each 2-HE2 and 4-HE2. Cu(II)SO4, a transition metal known to catalyze the redox cycling of o-quinones, substantially increased the amount of DNA damage caused by both CE's. However, DNA damage was only observed at concentrations of 10 microM or higher, much greater than what is found under physiologic conditions. Furthermore, the presence of endogenous antioxidants such as glutathione, SOD, and catalase drastically reduced the amount of DNA damage induced by high concentrations of 2-HE2. There was no DNA damage observed for the non-redox cycling HME2's, making these compounds useful probes in the study of receptor-mediated carcinogenesis. Thus, both 2-HE2 and 4-HE2 are capable of producing oxidative DNA damage at micromolar concentrations in vitro. However, since the amount of CE's has not been shown to surpass nanomolar levels in vivo, it is unlikely that free radical production via redox cycling of CE's is a causative factor in human tumorigenesis.

There is a clear association between excessive exposure to estrogens and the development of cancer in several tissues including breast and endometrium. The risk factors for women developing these cancers are all associated with longer estrogen exposure, as may be facilitated by early menses, late menopause and long-term estrogen replacement therapy. Equilenin (1,3,5(10),6,8-estrapentaen-3-ol-17-one) or its 17-hydroxylated analogs make up 15% of the most widely prescribed estrogen replacement formulation, Premarin, and yet there is very little information on the human metabolism of these estrogens. In this study, we synthesized the catechol metabolite of equilenin, 4-hydroxyequilenin, and examined how aromatization of the B ring affects the formation and reactivity of the o-quinone (3,5-cyclohexadien-1,2-dione). 4-Hydroxyequilenin-o-quinone is much more redox-active and longer-lived than the endogenous catechol estrone-o-quinones, which suggests that the mechanism(s) of toxicity of the former could be quite different. Interestingly, the rate of reduction of the 4-hydroxyequilenin-o-quinone is increased at least 13-fold in the presence of NAD(P)H:quinone oxidoreductase (DT-diaphorase). Once NADH is consumed however, the catechol auto-oxidized rapidly to the o-quinone. NADH consumption was accompanied by dicumarol-sensitive oxygen uptake both with the purified enzyme and with cytosol from human melanoma cells with high levels of DT-diaphorase activity. P450 reductase and rat liver microsomes also catalyzed NADPH consumption and oxygen uptake. 4-Hydroxyestrone-o-quinone was also rapidly reduced by NAD(P)H; however, this o-quinone does not auto-oxidize and once the o-quinone is reduced the reaction terminates. Including oxidative enzymes in the incubation completes the redox couple and 4-hydroxyestrone-o-quinone behaves like 4-hydroxyequilenin-o-quinone. These data suggest that reduction of estrogen-o-quinones may not result in detoxification. Instead this could represent a cytotoxic mechanism involving consumption of reducing equivalents (NADH/NADPH) as well as formation of superoxide and other reactive oxygen species leading to oxidative stress. Finally, we have compared the cytotoxicity of 4-hydroxyequilenin with that of the estrone catechols in human melanoma cells. 4-Hydroxyequilenin is 5-fold more toxic in these cells compared with 4-hydroxyestrone (ED50 = 7.8 versus 38 microM, respectively) suggesting that formation of the longer-lived redox-active 4-hydroxyequilenin-o-quinone was responsible for the cytotoxic differences. These results substantiate the conclusion that the involvement of quinoids in catechol estrogen toxicity depends on a combination of the rate of formation of the o-quinone, the lifetime of the o-quinone, and the electrophilic/redox reactivity of the quinoids.

In an animal model of hormone-mediated carcinogenesis, male golden Syrian hamsters develop renal carcinoma following prolonged exposure to 17beta-estradiol. The basis for the species and tissue specificity is unclear. Detailed information on the disposition of 17beta-estradiol in this model is lacking. Because catechol estrogens have been implicated in this model of carcinogenesis, we investigated the metabolism and nephrotoxicity of 17beta-estradiol in golden Syrian hamsters, with emphasis on the formation of catechol estrogen thioethers. 17beta-Estradiol (50 micromol/kg, i.p.) is a mild nephrotoxicant, causing significant elevations in the urinary excretion of gamma-glutamyl transpeptidase (gamma-GT), alkaline phosphatase, glutathione S-transferase (GST) and glucose. Increases in renal protein carbonyls and lipid hydroperoxides, which are markers of oxidative damage, also occur after administration of 17beta-estradiol (50 micromol/kg, i.p.). 17beta-Estradiol-mediated nephrotoxicity is reduced by treating animals with acivicin, an inhibitor of gamma-GT, implying that toxicity is mediated by metabolites requiring metabolism by this enzyme. Following administration of 17beta-[14C]estradiol (100 micromol/kg) to hamsters, 9.7% of the dose is recovered in bile after 5 h, the majority (7.9%) representing aqueous metabolites. Seven catechol estrogen GSH conjugates were identified, 2-hydroxy-1,4-bis-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-estrone, 2-hydroxy-1-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-17beta-estradiol, and 2-hydroxy-1-(glutathion-S-yl)-17beta-estradiol. At 5.4 micromol/kg of 17beta-estradiol, a dose-reflective of daily exposure levels in the hamster model of nephrocarcinogenicity, 12% of the dose is recovered within 5 h as a combination of GSH conjugates of 2- and 4-hydroxy-17beta-estradiol and 2- and 4-hydroxyestrone. In summary, oxidation of catechol estrogens, followed by GSH conjugation, occurs in vivo and 17beta-estradiol is a mild nephrotoxicant in a manner dependent on the activity of gamma-GT.

17 beta-Estradiol is known to induce kidney tumors in male Syrian hamsters when administered chronically, whereas 4-fluoroestradiol does so only after an extended induction period and 2-fluoroestradiol is not carcinogenic; both fluorinated analogs are hormonally active. Because C-4 and C-2 hydroxylations are, respectively, minor and major routes of estrogen metabolism in vivo, these observations suggest mediation of tumorigenesis by catecholestrogen metabolites. However, the analogs were reported to undergo oxidative defluorination in vitro. We have determined the metabolic fates of estradiol, 2-fluoroestradiol, and 4-fluoroestradiol in male hamsters. [6,7-3H]Estradiol was principally C-2 hydroxylated when given intravenously at either 0.1 mumol/kg or 50 mumol/kg; 2-hydroxyestradiol was eliminated in bile and urine, largely as a glucuronide, without undergoing extensive deactivation via O-methylation. Alicyclic alcohol metabolites were minor products. [6,7-3H]2-Fluoroestradiol underwent either glucuronylation or sequential dehydrogenation and alicyclic hydroxylation followed by glucuronylation but neither oxidative defluorination nor compensatory C-4 hydroxylation. [6,7-3H]4-Fluoroestradiol was also considerably dehydrogenated to the keto form and glucuronylated. Nevertheless, only 4-fluoroestradiol yielded appreciable quantities of C-2 hydroxylated metabolite at the lower dose; methylation was an insignificant pathway. No defluorinated products were observed. Dehydrogenation of both analogs and alicyclic hydroxylation of the 2-fluoroestrone metabolite were less extensive at the higher dose; all of the polar metabolites of 2-fluoroestradiol in bile, although not those in urine, declined to trace amounts. C-2 hydroxylation of 4-fluoroestradiol was greater at this dose. Thus, the rank order of catechol formation from estradiol and its fluoro analogs observed in vivo, unlike that found in microsomal incubations, was consistent with the hypothesis that catechols mediate estrogen-dependent renal carcinogenesis in hamsters.

In male Syrian hamsters, an animal model for estradiol-induced carcinogenesis, 2-fluoroestradiol was not carcinogenic, whereas 4-fluoroestradiol induced kidney tumors after a prolonged latency period, compared with estradiol (100% tumor incidence), when the compounds were administered to hamsters in hormonally equipotent doses. Catechol estrogen metabolites have previously been postulated to mediate this estrogen-induced kidney carcinogenesis. To examine this proposed mechanism of tumor induction by estrogens, we investigated the conversion of 2- and 4-fluoroestradiol to catechol metabolites by kidney and liver microsomes of hamsters and the further conversion to methyl ethers by catechol-O-methyltransferase, and we compared the values with those obtained with nonfluorinated estrogens as substrates. The rates of conversion of 2-fluoroestradiol to 2-hydroxyestradiol and 2-fluoro-4-hydroxyestradiol by hepatic microsomes were 30-50% lower than corresponding rates with estradiol as substrate. With renal microsomes the rate of 4-hydroxylation was 10 times faster than that of estradiol, whereas 2-hydroxylation was at best marginal. With 4-fluoroestradiol as substrate the rate of 2-hydroxylation by hepatic microsomes was enhanced 5-fold, compared with values for estradiol, but 4-hydroxyestradiol formation was almost eliminated. In contrast, the conversion of this substrate to 4-fluoro-2-hydroxyestradiol by kidney microsomes occurred at a rate 15 times faster than 2-hydroxylation of estradiol, whereas 4-hydroxyestradiol formation proceeded at a rate of 315 pmol/mg of protein/min. Except for the decrease in both 2- and 4-hydroxylation of 2-fluoroestradiol by liver microsomes, fluorine substitution of estrogenic phenols enhanced microsome-mediated aromatic hydroxylation at sites unoccupied by substituents. At pH 7.5, the highest rates of catechol-O-methyltransferase-mediated methylation were observed with the catechol metabolites of 2-fluoroestradiol, 2-fluoro-4-hydroxyestradiol and 2-hydroxyestradiol (3780 and 2960 pmol/mg of protein/min, respectively). Lower rates were found with those of 4-fluoroestradiol, 4-fluoro-2-hydroxyestradiol and 4-hydroxyestradiol (1670 and 470 pmol/mg of protein/min, respectively). These data are consistent with the postulate that catechol metabolites of estrogens are reactive intermediates in estrogen-induced carcinogenesis. For the noncarcinogenic 2-fluoroestradiol, a high metabolic flux was observed through a pathway of renal 2-fluoro-4-hydroxyestradiol formation and further conversion to methyl ethers. This flux likely results in low steady state concentrations of catechol metabolites in kidneys of hamsters treated with this modified estrogen and therefore in its lack of carcinogenic activity. In contrast, the carcinogenic activity of 4-fluoroestradiol is consistent with its rapid conversion in the kidney to 2- and 4-hydroxylated metabolites and a less rapid methylation of these catechols.

Estradiol-17 beta (E2) and the two catecholestrogens 2-OHE2 and 4-OHE2, when daily administered at low doses of 10-40 ng/rat, were cytotoxic to the seminiferous epithelium. The structural changes seen after seven days exposure included abnormal meiotic type II cells with uneven chromosome distribution, the formation of binucleated and multinucleated giant cells, of which many were sloughed into the lumina of the seminiferous tubules. The effect of the 4-OHE2 metabolites were always more pronounced that that of 2-OHE2 or E2. After 21 daily exposures, 4-OHE2 proved to be very toxic, the seminiferous tubules were markedly denuded and numerous giant cells were present in the lumina. The catecholestrogens also caused a significant lowering (P less than 0.02) of testosterone serum levels after eight days exposure. E2 at 40 ng/rat/day had no effect on testosterone production. At these low doses the catecholestrogens did not affect gonadotropin release after eight days exposure. Our results indicate that the morphological lesions could not exclusively be attributed to testosterone withdrawal and that a direct effect on developing spermatids is also indicated.

Estradiol and other estrogens are not classified as genotoxic carcinogens, but rather as tumor promoters in the multistage process of carcinogenesis. This study is a reexamination of the carcinogenic status of estradiol and the catecholestradiols (2-OHE2 and 4-OHE2) with the recently developed bacterial assays for genotoxic carcinogens, the Chromotest. The bacterial kits revealed estradiol and catecholestradiols as biphasic and potential genotoxic carcinogens with the following SOS inducing potency values: E2 43,265 (SD 8,300); 2-OHE2 30,153 (SD 2,500), and 4-OHE2 68,939 (SD 4,500). The differences between these values are statistically highly significant (p less than 0.0005). These results were confirmed by studies on Escherichia coli, which showed an increase in cell number and a stimulation of DNA content in the presence of the estrogens. It is therefore concluded that estradiol and the catecholestradiols are possible genotoxic carcinogens which probably act as tumor inducers rather than tumor promoters.

P-450-dependent enzyme systems added to media of cultured rat embryos markedly increased the embryotoxicity of estradiol-17 beta. Increases were markedly attenuated by omission of NADPH, omission of enzyme, substitution of female for male rat liver as enzyme source, d) replacement of N2 with CO or replacement of estradiol-17 beta with diethylstilbestrol. Embryotoxicity correlated well (r = 0.84) with catecholestrogen generating activities. Addition of a catechol-methylating system failed to modify embryotoxicity even though large quantities of methoxyestrogens were formed. The results document that endogenous estrogen can be converted by P-450 to embryotoxic intermediates and suggest that reactive proximate metabolites are precatechols, perhaps epoxyenones.

Estradiol and other estrogens induce renal carcinoma in male Syrian hamsters. The mechanism of carcinogenesis still remains unclear. Activation of estrogens to catechol metabolites has in the past been postulated to play a role in estrogen-induced carcinogenesis. Therefore, the carcinogenic activity of catechol estrogens was investigated. After 175 days of treatment, 4-hydroxyestradiol was found to be as carcinogenic as estradiol in male Syrian hamsters (4/5 and 4/5 animals with kidney tumors, respectively). Animals treated with 2-hydroxyestradiol (0/5) or 2-methoxyestradiol (0/6) did not develop renal carcinoma. The catechol estrogens failed to be mutagenic in the Ames test (reversions of his- S. typhimurium to histidine prototrophy in the TA 100 strain). The lack of carcinogenic activity of 2-hydroxyestradiol was not due to a failure to stimulate estrogen-dependent tumor growth. Growth of H-301 cells, an estrogen-dependent hamster kidney tumor cell line, was supported in vivo by estrogens in the following order: estradiol greater than 4-hydroxyestradiol greater than 2-hydroxyestradiol. Stimulation of tumor growth by 2-methoxyestradiol was not detected. It was concluded that the carcinogenic activity of 4-hydroxyestradiol was consistent with a role of catechol metabolites in estrogen-induced carcinogenesis. However, the intrinsic carcinogenic or hormonal activity of 2-hydroxyestradiol probably can not be assessed accurately in vivo because of its rapid methylation and metabolic clearance.