Joachim G. Liehr

Carcinogenicity of catechol estrogens in Syrian hamsters.

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.

Free radical generation by redox cycling of estrogens

Natural and synthetic estrogens elicit normal hormonal responses in concentrations in a clearly defined yet low range. At elevated doses, metabolic reactions of the phenolic moiety, while harmless at low levels, may become the predominant biochemical activity and may exert deleterious effects. These metabolic pathways, such as i) oxidation of estrogens to catechol estrogens and further to their respective quinones, and ii) free radical generation by redox cycling between catechol estrogens or diethylstilbestrol and their quinones, are investigated for their influence in physiological or pathophysiological processes. In this review, the in vitro capacity of various enzymes to oxidize estrogen hydroquinones to quinones or to reduce corresponding quinones to hydroquinones is evaluated. The in vivo activities of enzymes supporting redox cycling of estrogens and free radical generation is correlated with induction of kidney tumors in Syrian hamsters. Concomitant changes in activities in quinone reductase and other detoxifying enzymes in kidneys of hamsters treated with estrogen support a role of free radicals in the induction of tumors by estrogen. Free radical damage to protein and possibly to DNA in kidneys of estrogen-treated hamsters may be used as markers of free radical action in vivo.

Genotoxic effects of estrogens

Estrogens are associated with several cancers in humans and are known to induce tumors in rodents. In this review a mechanism of carcinogenesis by estrogens is discussed which features the following key events: (1) Steroid estrogens are metabolized by estrogen 2- and 4-hydroxylases to catecholestrogens. Target organs of estrogen-induced carcinogenesis, hamster kidney or mouse uterus, contain high levels of estrogen 4-hydroxylase activity. Since the methylation of 4-hydroxyestradiol by catechol-O-methyltransferase is inhibited by 2-hydroxyestradiol, it is proposed that a build up of 4-hydroxyestrogens precedes estrogen-induced cancer. (2) The catecholestrogen or diethylstilbestrol (DES) are oxidized to semiquinones and quinones by the peroxidatic activity of cytochrome P-450. The quinones are proposed to be (the) reactive intermediates of estrogen metabolism. (3) The quinones may be reduced to catecholestrogens and DES and redox cycling may ensue. Redox cycling of estrogens has been shown to generate free radicals which may react to form the organic hydroperoxides needed as cofactors for oxidation to quinones. (4) The quinone metabolites of catechol estrogens and of DES bind covalently to DNA in vitro whereas DNA binding in vivo has only been examined for DES. When DES is administered to hamsters, the resulting DES-DNA adduct profile in liver, kidney, or other organs closely matches that of DES quinone-DNA adducts in vitro. In vitro, DES-DNA adducts are chemically unstable and are generated in incubations with organic hydroperoxide as cofactor. It is proposed that the instability of adducts and the lower sensitivity of previous assay methods contributed to the reported failures to detect adducts. Steroid estrogen-DNA adducts in vivo are currently under investigation. (5) Tumors are postulated to arise in cells rapidly proliferating due to the growth stimulus provided by the estrogenic activity of the primary estrogen or of hormonally potent metabolites such as 4-hydroxyestradiol. The covalent modification of DNA in these cells is temporary because of the chemical instability of adducts and will result in altered genetic messages in daughter cells, whereas in non-proliferating cells there may be no lasting genetic damage. The sequence of events described above is a plausible mechanism for tumor initiation by estrogens and is partially substantiated by experimental evidence obtained in vitro and/or in vivo.

Estrogen, DNA damage and mutations

Estrogen administration to rodents results in various types of DNA damage and ultimately leads to tumors in estrogen-responsive tissues. Yet these hormones have been classified as nonmutagenic, because they did not induce mutations in classical bacterial and mammalian mutation assays. In this review, we have discussed the induction by estrogens of DNA and chromosomal damage and of gene mutations, because the classical assays were designed to uncover mutations only at one specific locus and could not have detected other types of mutations or changes in other genes. Various types of estrogen-induced DNA damage include: (a) direct covalent binding of estrogen quinone metabolites to DNA; (b) enhancement of endogenous DNA adducts by chronic estrogen exposure of rodents; (c) free radical generation by metabolic redox cycling between quinone and hydroquinone forms of estrogens and free radical damage to DNA such as strand breakage, 8-hydroxylation of purine bases of DNA and lipid hydroperoxide-mediated DNA modification. Two different types of chromosomal damage have also been induced by estrogen in vivo and in cells in culture such as numerical chromosomal changes and also structural chromosomal aberrations. Gene mutations have been induced in several cell types in culture either by the parent estrogen or by reactive estrogen quinone metabolites. Furthermore, in estrogen-induced kidney tumors in hamsters, several mutations have been observed in the DNA polymerase beta gene mRNA. Estradiol also induces microsatellite instability in these kidney tumors and in premalignant kidney exposed to estradiol. Although this work is still ongoing, it can be concluded that estrogens are complete carcinogens capable of tumor initiation by mutation potentially in critical genes. The hormonal effects of estrogens may complete the development of tumors.

Inhibition of Catechol O-Methyltransferase-catalyzed O-Methylation of 2- and 4-Hydroxyestradiol by Quercetin POSSIBLE ROLE IN ESTRADIOL-INDUCED TUMORIGENESIS

Catecholestrogens have been postulated to mediate the induction of kidney tumors by estradiol in male Syrian hamsters. In this study, we examined the mechanism of inhibition by quercetin of the catechol O-methyltransferase-catalyzed O-methylation of catecholestrogens as a basis for the previously reported enhancement of estradiol-induced tumorigenesis by this flavonoid. In hamsters treated with 50 μg of [6,7-3H]estradiol, quercetin increased concentrations of 2- and 4-hydroxyestradiol in kidney by 80 and 59%, respectively. In animals treated with two 10-mg estradiol implants, quercetin also decreased by 63-65% the urinary excretion of 2- and 4-hydroxyestradiol monomethyl ethers. Taken together, these results demonstrate the in vivo inhibition of the O-methylation of catecholestrogens by quercetin. S-Adenosyl-L-homocysteine, produced by the methylation of catecholestrogens, noncompetitively inhibited the O-methylation of 2- and 4-hydroxyestradiol by hamster kidney cytosolic catechol O-methyltransferase (IC approximately 10-14 μM). Due to the rapid O-methylation of quercetin itself, quercetin decreased renal concentrations of S-adenosyl-L-methionine by approximately 25% in control or estradiol-treated hamsters and increased concentrations of S-adenosyl-L-homocysteine by 5-15 nmol/g of wet tissue, which was estimated to cause a 30-70% inhibition of the enzymatic O-methylation of catecholestrogens. Quercetin or fisetin (a structural analog) inhibited the O-methylation of 2- and 4-hydroxyestradiol by a competitive plus noncompetitive mechanism (IC approximately 2-5 μM). These results suggest that the in vivo O-methylation of catecholestrogens is inhibited more by S-adenosyl-L-homocysteine than by quercetin. The accumulation of 2- and 4-hydroxyestradiol during co-administration of estradiol and quercetin may enhance metabolic redox cycling of catecholestrogens and thus estradiol-induced kidney tumorigenesis.

Age-dependent covalent DNA alterations (I-compounds) in rodent tissues: species, tissue and sex specificities

I-compounds are non-polar covalent DNA modifications of as yet undetermined structure that tend to accumulate in an age-dependent manner in tissues of untreated animals. They are detectable by 32P-postlabeling assay because of their adduct-like properties and chromatographically resemble DNA nucleotides containing bulky/hydrophobic moieties. To determine which factors may be involved in their formation, I-compounds were examined by 32P-postlabeling in liver and kidney DNA of female and male Sprague-Dawley rats and Syrian hamsters of different ages (1, 4 and 10 months and 1, 2.5 and 9.5 months, respectively). The following results were obtained: (i) Every tissue DNA studied contained characteristic I-compounds. (ii) Patterns and amounts of I-compounds were reproducible among animals of the same kind. (iii) There were pronounced organ and species differences. (iv) I-compound patterns were sex-dependent. (v) I-compound levels increased with age in all tissues studied, except in male hamster kidney, a target organ of estrogen-induced carcinogenesis. The highest levels were observed in liver and kidney of 10-month-old female rats. (vi) The rise of I-compound levels was less steep during the later part of the observation period for female but not male animals. (vii) Gonadectomy decreased I-compound levels in female hamster kidney DNA, while causing a slight increase in male animals later in life. These I-compounds were identical to previously reported DNA modifications that increased in male hamster kidneys after prolonged estrogen treatment. Points, iv, vi and vii strongly implicated sex hormones in I-compound formation. The qualitative effects of species, tissue differentiation, gender and sex hormones on these DNA modifications support the hypothesis that I-compounds are formed by the binding of endogenous electrophiles to DNA. As persistent DNA alterations, they are likely to affect DNA replication and to play a role in spontaneous and chemically induced carcinogenesis and in aging.

Diethylstilbestrol (DES) quinone: A reactive intermediate in DES metabolism

The quinone of E-diethylstilbestrol (DES), a postulated metabolic intermediate derived from DES, has been synthesized by oxidation of DES in chloroform using silver oxide. The reaction product was structurally characterized by infrared, ultraviolet, nuclear magnetic resonance, and mass spectrometry. The product of oxidation of DES by hydrogen peroxide, catalyzed by horseradish peroxidase and also by rat uterine peroxidase, was shown to be identical with synthetic DES quinone based on identical u.v. spectra and on identical decomposition products. DES quinone was stable only in non-protic solvents such as chloroform. In acids, bases or protic solvents, DES quinone rearranged to Z,Z-dienestrol (beta-DIES). The half-life of DES quinone in water was approximately 40 min; in methanol it was approximately 70 min. Bacterial mutagenicity (Ames) tests did not indicate that DES quinone had mutagenic or genotoxic activity. However, DES quinone was found to bind to calf thymus DNA without any enzyme mediation at levels significantly above the binding of DES under the same conditions. Based on the binding of DES quinone to DNA, this intermediate must be considered as a possible carcinogenic metabolite of DES.

Effect of chronic estrogen treatment of syrian hamsters on microsomal enzymes mediating formation of catecholestrogens and their redox cycling: Implications for carcinogenesis

Estrogens have previously been shown to induce DNA damage in Syrian hamster kidney, a target organ of estrogen-induced cancer. The biochemical mechanism of DNA adduction has been postulated to involve free radicals generated by redox cycling of estrogens. As part of an examination of this postulate, we measured the effect of chronic estrogen treatment of hamsters on renal microsomal enzymes mediating catechol estrogen formation and free radical generation by redox cycling of catechol estrogens. In addition, the activities of the same enzymes were assayed in liver in which tumors do not develop under these conditions. At saturating substrate concentration, 2- and 4-hydroxyestradiol were formed in approximately equal amounts (26 and 28 pmol/mg protein/min, respectively), which is 1-2 orders of magnitude higher than reported previously. Estradiol treatment for 2 months decreased 2-hydroxylase activity per mg protein by 75% and 4-hydroxylase activity by 25%. Hepatic 2- and 4-hydroxylase activities were 1256 and 250 pmol/mg protein/min, respectively. Estrogen treatment decreased both activities by 40-60%. Basal peroxidatic activity of cytochrome P-450, the enzyme which oxidizes estrogen hydroquinones to quinones in the redox cycle, was 2.5-fold higher in liver than in kidney and did not change with estrogen treatment. However, when normalized for specific content of cytochrome P-450 the enzyme activity in kidney was 2.5-fold higher than in liver and increased further by 2-3-fold with chronic estrogen treatment. The activity of cytochrome P-450 reductase, which reduces quinones to hydroquinones in the estrogen redox cycle, was 6-fold higher in liver than in kidney of both control and estrogen-treated animals. When normalized for cytochrome P-450, the activity of this enzyme was similar in liver and kidney, but over 4-fold higher in kidney than liver after estrogen treatment. Basal concentrations of superoxide, a product of redox cycling, were 2-fold higher in liver than in kidney. Estrogen treatment did not affect this parameter in liver, but increased it in kidney by 40%. These data provide evidence for a preferential preservation of enzymes involved in estrogen activation.

Modulation of estrogen-induced carcinogenesis by chemical modifications.

The mechanism of carcinogenesis by estrogens is still unknown. Uncontrolled stimulation of cell proliferation, an endocrine imbalance, or metabolic activation of estrogens to reactive intermediates capable of tissue injury have previously been proposed. In an attempt to gain insight into mechanistic details of estrogen-induced carcinogenesis in male Syrian hamsters, fluorine substituted estrogens, which were impaired in their capacity to be transformed into catechols, have been tested for their carcinogenic activity. 2-Fluoroestradiol was found to be non-carcinogenic in Syrian hamsters despite its estrogenic potency. In a second unrelated experiment, ascorbic acid, which reduced diethylstilbestrol quinone to cis- and trans-diethylstilbestrol in vitro, was administered to estradiolor diethylstilbestrol-treated hamsters. A lowered incidence of kidney tumors in vivo was found in animals receiving ascorbic acid vs estrogen-treated control animals. These results were taken as evidence for a role of estrogen metabolites (catechols formed from estradiol or quinone formed from diethylstilbestrol) in estrogen-induced tumorigenesis. A mechanistic model of metabolic activation of estrogens followed by damage to cellular macromolecules is proposed.

Nuclear localization of catechol-O-methyltransferase in neoplastic and nonneoplastic mammary epithelial cells.

Catechol-O-methyltransferase (COMT) plays both a regulatory and protective role in catechol homeostasis. It contributes to the regulation of tissue levels of catecholamines and catecholestrogens (CEs) and, by blocking oxidative metabolism of catechols, prevents endogenous and exogenous catechols from becoming a source of potentially mutagenic electrophiles. Evidence implicating CEs in carcinogenesis, in particular in the hamster kidney model of estrogen-induced cancer, has focused attention on the protective role of COMT in estrogen target tissues. We have previously reported that treating hamsters with estrogens causes translocation of COMT to nuclei of epithelial cells in the renal cortex, the site of CE biosynthesis and where the cancers arise. This finding suggested that nuclear COMT may be a marker of a threat to the genome by catechols, including CEs. It is postulated that CEs play a role in the genesis of breast cancer by contributing to a state of chronic oxidative stress that is presumed to underlie the high incidence of this disease in the United States. Therefore, here we used immunocytochemistry to re-examine human breast parenchyma for nuclear COMT. In addition to confirming previous reports of cytoplasmic COMT in mammary epithelial cells, we identified nuclear COMT in foci of mammary epithelial cells in histologically normal breast tissue of virtually all control (macromastia) and cancer patients and in breast cancer cells. There was no correlation between tissue histology and the numbers of cells with nuclear COMT, the size of foci containing such cells, or intensity of nuclear COMT immunostaining. The focal nature of the phenomenon suggests that nuclear COMT does not serve a housekeeping function but that it reflects a protective response to an increased local catechol load, presumably of CEs and, as such, that it may be a characteristic of the population of women studied who share the same major risk factor for developing breast cancer, that of living in the industrialized West.