Is estradiol a genotoxic mutagenic carcinogen?

The natural hormone 17 beta-estradiol (E2) induces tumors in various organs of rats, mice, and hamsters. In humans, slightly elevated circulating estrogen levels caused either by increased endogenous hormone production or by therapeutic doses of estrogen medications increase breast or uterine cancer risk. Several epigenetic mechanisms of tumor induction by this hormone have been proposed based on its lack of mutagenic activity in bacterial and mammalian cell test systems. More recent evidence supports a dual role of estrogen in carcinogenesis as a hormone stimulating cell proliferation and as a procarcinogen inducing genetic damage. Tumors may be initiated by metabolic conversion of E2 to 4-hydroxyestradiol catalyzed by a specific 4-hydroxylase (CYP1B1) and by further activation of this catechol to reactive semiquinone/quinone intermediates. Several types of direct and indirect free radical-mediated DNA damage are induced by E2, 4-hydroxyestradiol, or its corresponding quinone in cell-free systems, in cells in culture, and/or in vivo. E2 also induces various chromosomal and genetic lesions including aneuploidy, chromosomal aberrations, gene amplification, and microsatellite instability in cells in culture and/or in vivo and gene mutations in several cell test systems. These data suggest that E2 is a weak carcinogen and weak mutagen capable of inducing genetic lesions with low frequency. Tumors may develop by hormone receptor-mediated proliferation of such damaged cells.

Characterization of drug metabolism enzymes in estrogen-induced kidney tumors in male Syrian hamsters.

In an attempt to characterize metabolism enzymes of the estrogen-induced kidney tumor in male Syrian hamsters, the activities of enzymes involved in drug and glutathione metabolism were determined in tumor tissue. Kidney tumors were induced in male Syrian hamsters by treatment with estradiol for 8 months. Cytochrome P-450 and cytochrome b5 concentrations in tumors were below detectable levels. However, when cytochrome P-450-mediated oxidation was analyzed by product formation assays, the oxidation of E-diethylstilbestrol to diethylstilbestrol-4',4"-quinone by tumor microsomes was 10-20% of the rate found in control microsomes. In kidney tissue surrounding estrogen-induced tumors, cytochrome P-450 and b5 contents were 50-60% less than those in untreated kidney. Activities of reducing enzymes of drug metabolism (cytochrome P-450, cytochrome b5 and NADH:cytochrome c reductases), glutathione metabolism enzymes (glutathione peroxidase, glutathione transferase, glutathione reductase, and gamma-glutamyl transpeptidase), and free radical scavenging enzymes (superoxide dismutase, catalase, and quinone reductase) in tumor were significantly lower than in untreated kidney tissue. The activities of these enzymes in renal tumor surrounding tissue were between those observed in tumor and control kidney. Glucose-6-phosphate dehydrogenase activity was increased by 50% in surrounding tissue and 430% in tumor compared to values in untreated controls. The decreased enzyme activity levels in hormone-exposed tissue surrounding tumors likely represented an adaptation of this tissue to the neoplastic environment induced by chronic estrogen treatment.

Changes in activities of free radical detoxifying enzymes in kidneys of male Syrian hamsters treated with estradiol.

Target organ-specific estrogen-induced DNA adducts were previously shown to precede renal carcinogenesis in Syrian hamsters. Because estrogens induced these DNA modifications, but were not part of the adduct structure, free radical activation of endogenous electrophiles was postulated as a mechanism of tumor induction by estrogens. In the present study, the activities of enzymes which detoxify reactive intermediates were studied in liver and kidney of hamsters treated with estradiol for 1, 2, and 4 mo and in untreated controls. These studies were done to detect oxidative stress in the target organ of carcinogenesis. In the estrogen-exposed hamster kidney (1, 2, and 4 mo), activities of glutathione peroxidases I and II were significantly increased. The activity of catalase was decreased compared to those in untreated controls. In livers which are not the target organ of carcinogenesis, treatment of hamsters with estrogen for 1, 2, and 4 mo resulted in changes of activities of glutathione peroxidases I and II and catalase, which were opposite to the pattern found in the kidney. Activities of superoxide dismutase, glutathione reductase, glucose-6-phosphate dehydrogenase, gamma-glutamyl transpeptidase, and glutathione transferase in estradiol-treated hamster liver and kidney did not differ significantly from those in either liver or kidney of untreated age-matched controls. Fluorescent products of lipid peroxidation more than doubled in the kidney, but not in the liver of hamsters treated with estradiol for 1 mo. It is concluded that the increases in glutathione, in the activity of glutathione peroxidase, and in products of lipid peroxidation in the kidneys of hamsters treated chronically with estrogen all point towards elevated levels of oxidative stress.

Induction of covalent DNA adducts in rodents by tamoxifen.

The antiestrogen tamoxifen, increasingly used as adjuvant treatment for breast cancer, has been found to covalently modify DNA of rodents. For instance, the liver DNA of female Sprague-Dawley rats treated with a single injection of tamoxifen contained two DNA adducts. Four additional DNA adducts were formed and adduct concentrations increased 5- 7- and 10-15-fold after three and six tamoxifen injections, respectively, from levels observed after a single dose. The accumulation of DNA adducts with repeated administrations of tamoxifen to rodents may make this drug a poor choice for the chronic preventative treatment of breast cancer.

Inhibition of estrogen-induced renal carcinoma in Syrian hamsters by vitamin C.

The ability of vitamin C to inhibit induction of renal carcinoma by estrogens was tested in male Syrian hamsters in vivo. The animals received estrogen (estradiol or diethylstilbestrol) implants s.c. Hamsters which were continuously given vitamin C, administered in the drinking water for estradiol-treated or in the food for diethylstilbestrol-treated animals, were observed to develop renal carcinoma with a significantly lower incidence (10 of 33 animals with estradiol implants; 14 of 29 animals with diethylstilbestrol implants) than animals which did not receive vitamin C supplementation (16 of 23 animals with estradiol implants; 11 of 13 animals with diethystilbestrol implants). Administration of vitamin C to estradiol-treated hamsters for only the first 3 months of the carcinogenesis experiment had no effect on tumor incidence, but vitamin C in drinking water for the last 3 months also lowered incidence. Vitamin C supplementation did not significantly alter the absorption of estrogen from the implant; it did not change the estrogenic effect on the hamsters nor did it significantly influence estrogen-dependent H-301 tumor cell growth. The results were taken as evidence for a mechanism of tumor induction via oxidation of estrogens to reactive metabolites capable of inducing kidney tumors.

8-Hydroxylation of guanine bases in kidney and liver DNA of hamsters treated with estradiol: role of free radicals in estrogen-induced carcinogenesis.

The chronic administration of estradiol induces a high incidence (80-100%) of renal tumors in male Syrian hamsters. As part of our examination of a mechanism of carcinogenesis by free radicals generated during redox cycling of catecholestrogen metabolites, we assayed levels of 8-hydroxy-2'-deoxyguanosine (8-OHdGua), a marker product of hydroxy radical interaction with DNA, in livers and kidneys of hamsters treated with estradiol. Injections of 50 and 100 mg/kg estradiol doubled renal 8-OHdGua levels over controls [10.0 +/- 0.1 (SD) and 5.4 +/- 0.4 8-OHdGua/10(5) dGua, respectively] and raised hepatic 8-OHdGua levels almost 4-fold over control values, respectively. These changes were observed in kidney 4 h and in liver 1 or 2 h after treatment of hamsters with estradiol. Estradiol implants administered to hamsters for 3 days raised renal levels of 8-OHdGua by 50% over control values. Six days after 17 beta-estradiol implantation, 8-OHdGua levels returned to near-normal values. Liver DNA was not affected by estradiol implants. These data support a mechanism of estrogen-induced carcinogenesis by free radicals generated via redox cycling of catecholestrogen metabolites.

Induction of uterine adenocarcinoma in CD-1 mice by catechol estrogens.

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.

Histochemical analysis of the development of estradiol-induced kidney tumors in male Syrian hamsters.

Chronic administration of the estrogen 17 beta-estradiol induces kidney tumors in male Syrian hamsters within 6 months of initial exposure. Although these tumors have previously been studied histologically and histochemically and have been postulated to be derived from proximal tubular and/or interstitial cells, there exists no unambiguous evidence for an epithelial or mesenchymal origin. To elucidate the histogenesis of these neoplasms, kidney sections of hamsters treated with estradiol for 4, 5, and 6 months and age-matched untreated controls were investigated histologically and histochemically. Proliferating foci were observed in kidneys exposed to estradiol for 5 and 6 months. They consisted of clusters of spindle-shaped cells forming solid blocks, cords, or branches located between tubules. These foci were judged to be precursors of larger tumors identified in the latter treatment group. The histological and histochemical profile of foci and tumors matched closely. These lesions were marked by very high activities of alkaline phosphatase, adenyl cyclase, and glucose 6-phosphate dehydrogenase. In contrast, glycogen content and activities of glucose 6-phosphatase, succinate dehydrogenase, and gamma-glutamyl transpeptidase were low or absent. Immunofluorescence of the intermediate filaments revealed that foci and tumors solely expressed vimentin and desmin but not cytokeratin. The morphology, enzyme histochemical pattern, and immunofluorescence strongly support a mesenchymal origin of the estradiol-induced hamster kidney tumors studied. The neoplasms were probably derived from vascular smooth muscle cells of a cell subtype particularly sensitive to hormonal stimulation and transformation.

Elevated 8-hydroxydeoxyguanosine levels in DNA of diethylstilbestrol-treated Syrian hamsters: covalent DNA damage by free radicals generated by redox cycling of diethylstilbestrol.

The generation of free radicals by microsome-mediated redox cycling between catechol estrogens or diethylstilbestrol and their corresponding quinones has previously been demonstrated in vitro. However, the reaction of free radicals with DNA has not yet been detected in animals treated with estrogen and is the subject of this investigation. The reaction of guanine bases of DNA with hydroxyl radicals to form 8-hydroxydeoxyguanosine has been used as a monitor of free radical generation in kidney and liver of Syrian hamsters, a species prone to estrogen-induced carcinogenesis. Prior to in vivo measurements, the in vitro hydroxylation of guanine bases of DNA under conditions of redox cycling of estrogen was investigated. In incubations of DNA or deoxyguanosine with hamster kidney microsomes, NADPH, and diethylstilbestrol 4',4"-quinone, the hydroxylation of guanine bases of free deoxyguanosine or of DNA was 50 to 100% higher than in controls. When incubations were carried out in the presence of iron(III) chloride, the hydroxylation of guanine bases was 2.5- or 10-fold higher than control values. There was a 65% increase from control values in levels of 8-hydroxydeoxyguanosine in liver DNA of hamsters treated with 20 mg/kg/day diethylstilbestrol for 3 days and 100 mg/kg on the 4th day. In hamsters treated chronically with diethylstilbestrol implants for 15 days, 8-hydroxydeoxyguanosine levels more than doubled from control values in kidney but not liver DNA. Treatment of hamsters with estradiol for various time periods did not induce any changes in levels of hydroxylated guanine in either kidney or liver. It was concluded that in vitro and in vivo redox cycling of diethylstilbestrol hydroxylated guanine bases in DNA.

Inhibition of estrogen-induced renal carcinogenesis in male Syrian hamsters by tamoxifen without decrease in DNA adduct levels.

Estrogens have previously been shown to induce covalent DNA modifications specifically in the hamster kidney, the target organ of estrogen-inducible and -dependent renal carcinoma. The DNA adducts, formed by yet unknown mechanisms, have been postulated to mediate hormonal carcinogenesis in this animal model. In an attempt to study a possible involvement of estrogen receptor mechanisms in the formation of DNA adducts, 17 beta-estradiol and the antihormone tamoxifen were concomitantly administered as s.c. implants to male Syrian hamsters. 17 beta-Estradiol-treated and tamoxifen-treated animals served as positive and negative controls, respectively. The tumor incidence decreased from 100% in 17 beta-estradiol-treated controls to 25% in the group receiving tamoxifen in addition to hormone. Tamoxifen-treated animals did not develop kidney tumors and did not show any detectable DNA damage. DNA adduct levels were comparable in hamsters treated with 17 beta-estradiol and 17 beta-estradiol plus tamoxifen for 5 or 7 months. In hamsters inoculated with H-301 cells, which are derived from the estrogen-induced hamster renal carcinoma and are estrogen dependent for growth, tamoxifen decreased estrogen-dependent H-301 tumor growth. However, in cell culture, neither 17 beta-estradiol nor tamoxifen influenced H-301 cell division. It was concluded that tamoxifen inhibited the growth of estrogen-induced renal carcinoma but did not interfere with tumor initiation since it did not inhibit the formation of DNA adducts. Moreover, receptor mechanisms were most probably not involved in the induction of DNA modifications by estrogens.

Mechanism of diethylstilbestrol carcinogenicity as studied with the fluorinated analogue E-3',3",5',5"-tetrafluorodiethylstilbestrol.

E-3',3",5',5"-Tetrafluorodiethylstilbestrol (TF-DES), a structural analogue of diethylstilbestrol synthesized as a possible noncarcinogenic estrogen, was found to induce renal clear-cell carcinoma in Syrian hamster. The tumor induction frequency of TF-DES was the same as that of diethylstilbestrol, although the induction period was longer (approximately 9 months) than that of diethylstilbestrol (approximately 6 months). TF-DES was estrogenic and was found to support in vivo growth of estrogen-dependent H-301 cells, a cell line derived from the primary estrogen-induced and -dependent renal clear-cell carcinoma of male Syrian hamster. These data established the ability of TF-DES not only to induce tumors but also to promote estrogen-dependent tumor growth after the initiation process. Oxidation of TF-DES to TF-DES quinone was catalyzed by horseradish peroxidase. The structure of this metabolic intermediate was confirmed by comparison with synthesized TF-DES quinone. This intermediate was very unstable (half-life in methanol, 24 min; half-life in water, 4 min) and rearranged to Z,Z-3',3",5',5"-tetrafluorodienestrol. Based on the experiments with the fluorinated derivative, it is postulated that stilbestrol estrogens induce tumors via metabolic oxidation to quinone intermediates, which then may interact with DNA or other cellular targets.

Human urinary metabolites of 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftoraful).

Two hydroxylated metabolites were isolated from the urine of a patient who had received ftorafur (5 g/sq m). These metabolites were identified by mass spectrometry and nuclear magnetic resonance spectroscopy as trans-3'- and cis-4'-hydroxyftorafur. The compounds were not converted to 4-fluorouracil when incubated in plasma, base (pH 9), or water. Because of their stability, it is unlikely that these metabolites are in vivo precursors of 5-fluorouracil. There are indications that less stable, unisolatable, hydroxylated ftorafur derivatives are intermediates in the conversion of ftorafur to 5-fluorouracil.

Localization of estrogen-induced DNA adducts and cytochrome P-450 activity at the site of renal carcinogenesis in the hamster kidney.

Renal carcinoma in male Syrian hamsters, induced by chronic administration of estradiol for 5-7 months, is known to arise in the cortex at the cortico-medullary junction. In this in vivo model for hormonal carcinogenesis, estrogen-induced covalent DNA adducts have previously been observed in whole kidney and have been postulated to be involved in tumor induction. In the present study, the intrarenal distribution of estrogen-induced DNA modification and estrogen metabolizing enzymes were investigated in male Syrian hamsters to ascertain a role of metabolism and adduct formation in estrogen-induced carcinogenesis. The highest estrogen-induced DNA adduct concentrations as measured by 32P-postlabeling analysis were found in the renal cortex of hamsters treated with estradiol for 7 months. Total adduct levels in medullary DNA were approximately one-half of those found in cortex. Cytochrome P-450 enzymes were detected only in microsomes of kidney cortex (approximately 0.8 +/- 0.6 nmol P-450/mg protein) but not medulla of untreated male Syrian hamsters. Prostaglandin endoperoxide synthase activity in kidney cortical microsomes was 1/5 of the activity found in medullary microsomes. Thus, microsomal cytochrome P-450 levels and estrogen-induced DNA adduct formation were highest in hamster kidney cortex, the origin of renal tumorigenesis. It is postulated that estrogen metabolism by cytochrome P-450 enzymes leading to covalent DNA modification plays a role in hormonal carcinogenesis in the hamster kidney.

Localization of estrogen receptors in interstitial cells of hamster kidney and in estradiol-induced renal tumors as evidence of the mesenchymal origin of this neoplasm.

The mechanism of estrogen-induced and -dependent kidney carcinogenesis in Syrian hamsters and the cell of origin of the tumor are not well understood; they have been investigated in this study by mapping the cellular locations of estrogen receptor (ER) in estrogen-dependent tumors, in kidney tissue of hamsters treated with estradiol for 0.5 and 5.5 months, and in kidneys of age-matched controls. To validate the methods used, receptors have also been localized in uteri of hamsters and rats and in female hamster kidneys. ERs have been identified in cryostat sections by immunocytochemical techniques using an affinity-purified ER antibody, ER-715. Nuclei of tumors were intensely stained for ERs. In estrogen-treated kidneys and in controls, ER protein was identified in interstitial cells and capillaries, in arteries, and in renal corpuscles, particularly in podocytes and in the parietal layers surrounding the renal corpuscles. There was no ER protein in tubular epithelia even when tubuli were surrounded by tumor cells. The ER distribution in female hamster kidneys closely matched that in male kidneys. However, the staining intensity was stronger in female than in male kidneys. In hamster uteri, there was an intense ER-positive reaction in the nuclei of stroma, in stromal vessels, and in the luminal epithelia as demonstrated previously by others in rat uteri. ER mRNA has also been demonstrated by Northern blot analysis in estrogen-treated kidneys which contained tumors but was undetectable in untreated kidneys. The localization of ERs in estrogen-dependent tumors and in interstitial cell types but not in tubular epithelia supports previous conclusions of an interstitial origin of estrogen-induced hamster kidney tumors.

Correlation of aromatic hydroxylation of 11 beta-substituted estrogens with morphological transformation in vitro but not with in vivo tumor induction by these hormones.

In estrogen-induced cancer, catechol formation from administered steroids has been postulated to be a necessary event for estrogen activation and subsequent damage to cellular macromolecules. In the present study, this hypothesis has been tested using two homologous series of structurally related estrogens: estradiol, 11 beta-methylestradiol, 11 beta-ethylestradiol, 11 beta-methyl-17 alpha-ethinylestradiol, 11 beta-ethyl-17 alpha-ethinylestradiol, 11 beta-methoxy-17 alpha-ethinylestradiol, and 17 alpha-ethinylestradiol. In the Syrian hamster renal carcinoma model, only 11 beta-methylestradiol and 17 alpha-ethinylestradiol were weak carcinogens (2 of 20 and 2 of 24 hamsters with tumors, respectively). The other estrogens tested induced renal carcinoma within 6 to 9 months with an incidence in the 80-100% range. The tumor incidence in vivo did not correlate with the rates of catechol formation by hamster kidney microsomes in vitro. Compared to estradiol (relative rate, 100), catechol formation by the substituted estrogens was significantly lower, ranging from 48 (11 beta-methylestradiol) to 2 (11 beta-methoxy-17 alpha-ethinylestradiol). Kidney DNA of hamsters treated with the four 17 alpha-ethinyl estrogens, when analyzed by 32P postlabeling assay, contained the same set of covalently modified nucleotides the formation of which had previously been found to precede estrogen-induced renal carcinogenesis in vivo. In contrast, relative rates of catechol estrogen formation by BALB/c 3T3 microsomes correlated with induction of morphological transformation of BALB/c 3T3 cells and decreased in the following order: 11 beta-methylestradiol greater than 17 alpha-ethinylestradiol greater than or equal to estradiol greater than 11 beta-ethylestradiol greater than 11 beta-methoxy-17 alpha-ethinylestradiol. The hormonal potencies of several estrogen derivatives studied by various assays did not correlate with in vivo carcinogenic or in vitro cell-transforming activities. It is concluded from these experiments that in cell culture catechol formation and morphological transformation are directly related. In vivo, aromatic hydroxylation of administered estrogens did not correlate with the incidence of estrogen-induced renal carcinoma in Syrian hamsters.

Specific binding of 4-hydroxyestradiol to mouse uterine protein: evidence of a physiological role for 4-hydroxyestradiol.

There are several indications of a possible physiological role for 4-hydroxyestradiol (4-OHE(2)) in hormone-responsive tissues. To examine a hormonal activity of 4-OHE(2), we have studied the binding of (3)H-labeled 4-OHE(2) to mouse uterine cytosolic protein. In uteri of 3-week-old mice, total binding was 319.4 +/- 13.9 fmol/mg protein. Binding in the presence of excess unlabeled 4-OHE(2) dropped to 82.1 +/- 1.7 fmol/mg protein, whereas 214.6 +/- 9.4 fmol/mg protein bound while incubating in an excess of unlabeled 17beta-estradiol (E(2)). The difference between the two binding values in the presence of excess steroid (132.5 +/- 11.1 fmol/mg protein) is taken as selective binding of 4-OHE(2) to a specific protein. In mice older than 4 weeks, the specific 4-OHE(2) binding declined: 32.0 +/- 4.0 fmol/mg protein at 8 weeks, 54.8 +/- 6.3 fmol/mg protein at 12 weeks and 54.6 +/- 5.2 fmol/mg protein at 9 months. Of other organs tested (liver, kidney, lung and whole brain) only lung showed significant selective binding of 4-OHE(2). When E(2)-binding sites are blocked, binding follows first-order kinetics, yielding a dissociation constant (K(d)) value of 11.8 +/- 2.1 nM. The specific binding of 4-OHE(2) was not inhibited by any other steroids or estrogen metabolites that were tested, except for 2-hydroxyestradiol (2-OHE(2)), which displayed competitive inhibition of 4-OHE(2) binding with an inhibition constant (K(i)) value of 98.2 +/- 12.6 nM. These results lead us to conclude that 4-OHE(2) binds to a specific binding protein, distinct and different from binding to estrogen receptors (ERalpha and ERbeta). The physiological role of this binding remains to be elucidated.

The carcinogenic activity of ethinyl estrogens is determined by both their hormonal characteristics and their conversion to catechol metabolites.

Estrogens induce kidney tumors in Syrian hamsters. The mechanism of carcinogenesis is unknown and has been investigated in this study using a weak carcinogen, 17 alpha-ethinyl estradiol (EE), and a strongly carcinogenic estrogen, 17 alpha-ethinyl-11 beta-methoxyestradiol [moxestrol (MOX)]. We investigated rates of conversion of estrogens to catechol metabolites and rates of their methylation to methyl ethers in order to examine the hypothesis that catechol metabolites mediate estrogen-induced carcinogenesis. Rates of conversion of MOX to catechol metabolites by hamster liver or kidney cortex microsomes were 40-50% of those with estradiol as substrate. However, the rate of catechol-O-methyltransferase-catalyzed methylation of 2-hydroxy-MOX was the least of the catechol metabolites examined when incubated with cytosol of hamster kidney. In contrast, EE was converted to catechol metabolites by hamster liver and kidney microsomes at rates 25-35% of those obtained with estradiol. These catechol metabolites of EE were methylated by catechol-O-methyltransferase of hamster kidney cytosol at rates slightly lower than those observed with catechols of estradiol. The progesterone receptor binding of EE and MOX was investigated, because progesterone is known to inhibit estrogen-induced carcinogenesis in the hamster kidney. Neither estradiol nor MOX inhibited the binding of progesterone to its receptor in hamster kidney cytosol. However, in the presence of 20 nM EE, the binding affinity of radiolabeled progesterone to receptor was inhibited (increase in Kd from 0.98 nM in controls to 3.02 nM in the presence of EE). Maximum binding values (5.0 fmol/mg protein in controls and 6.0 fmol/mg protein in the presence of EE) were not significantly altered. These results support the hypothesis that estrogen-induced carcinogenesis is mediated by catechol estrogen metabolites. The carcinogenic estrogen MOX is converted to catechol metabolites at lower rates than estradiol, but their methylation may be sterically hindered by the 11 beta-methoxy substituent. In contrast, the rates of conversion of the weakly carcinogenic EE to catechol metabolites are low, whereas their methylation rates are only marginally lower than those of 2- and 4-hydroxyestradiol. The decreased capacity of EE to form catechol metabolites in conjunction with its partial progestin agonist activity in the target organ of hamsters may contribute to the low tumor incidence.

Elevated 4-hydroxylation of estradiol by hamster kidney microsomes: a potential pathway of metabolic activation of estrogens.

Characterization of enzymes mediating the formation of catecholestrogens (CE) by hamster kidney is of importance because of the proposed role of CE in renal cancer induced in this species by estrogens. We have reexamined the potential of hamster kidney to convert estradiol (E2) to 2- and 4-hydroxylated CE because of recent evidence of the limitations of assays used in previous studies, in particular in measuring 4-hydroxylation of estrogens. Under conditions optimized for NADPH-dependent activity, hamster kidney microsomes exhibited high levels of both E2-2- and E2-4-hydroxylase activities. Evidence that the two activities depend on different forms of cytochrome P-450 was obtained by the demonstration that 2- and 4-hydroxylation of E2 were affected differentially 1) by chronic treatment of hamsters with E2 and 2) by fadrozole hydrochloride, a selective cytochrome P-450 inhibitor. NADPH-dependent 2-hydroxylation of E2 from control and E2-treated hamsters, measured by a direct product isolation assay, was 1 order of magnitude higher (apparent maximum velocity, 24-32 and 6-12.5 pmol/mg protein.min in control and E2-treated hamsters, respectively) than that reported previously using radioenzymatic assays. NADPH-dependent 4-hydroxylation of E2 in controls approached and in E2-treated hamsters exceeded 2-hydroxylation of E2 (apparent maximum velocity, 17-21 and 7.5-19 pmol/mg protein.min in control and E2-treated hamsters, respectively). Thus, estrogen treatment reversed the ratios of NADPH-dependent E2-2-/4-hydroxylase activities by causing a much greater decline in 2- than 4-hydroxylation of E2 (P less than 0.007, by analysis of variance). Fadrozole hydrochloride caused a marked dose-dependent decrease in 2-hydroxylation of E2, in contrast to a small nondose-dependent inhibition of 4-hydroxylation. Under conditions optimized for peroxidatic organic hydroperoxide-dependent activity, hamster kidney microsomes generated 2- and 4-hydroxylated CE in similar amounts. The amounts of the two CE and, consequently, the ratios remained unaffected by estrogen treatment (1:0.9 and 1:1.0 in control and E2-treated hamsters, respectively). Thus, this study establishes that CE can be generated in the same tissue by three different pathways, i.e. NADPH-dependent E2-2-hydroxylase, NADPH-dependent E2-4-hydroxylase, and organic hydroperoxide-dependent E2-2/4-hydroxylase activities. We also show that these three activities can be regulated differentially and are, thus, probably mediated by different forms of cytochrome P-450. In hamster kidney, the potential to generate 4-hydroxylated CE metabolites with distinct properties could be a factor in this tissue's vulnerability to estrogen-induced carcinogenesis.

Conversion of estrone to 2- and 4-hydroxyestrone by hamster kidney and liver microsomes: implications for the mechanism of estrogen-induced carcinogenesis.

As part of an ongoing investigation of the role of metabolic activation of estrogens in the genesis of cancers such as estrogen-induced renal tumors in hamsters, we have 1) determined steroid-17 beta-oxidoreductase activity of microsomes and cytosol prepared from hamster kidney and liver; 2) compared the rates of 2-, 4-, and 16 alpha-hydroxylations of estrone by microsomes from hamster kidney and liver; and 3) determined the rates of inactivation of 2- and 4-hydroxyestrone by catechol-O-methyltransferase from hamster kidney and by purified enzyme. Microsomal steroid-17 beta-oxidoreductase activity in hamster kidney and liver was low and favored the conversion of estrone to estradiol. Cytosolic steroid-17 beta-oxidoreductase activity was only barely detectable in both liver and kidney. Using hepatic microsomes, the rate of 2-hydroxylation of estrone was comparable to that found previously using estradiol as substrate, whereas 4-hydroxylation of estrone was double that of estradiol. Using renal microsomes, the rates of 2- and 4-hydroxylation of estrone were 10- to 20-fold higher than those with estradiol as substrate, and the ratio of 2- to 4-hydroxylation was about 2:1. Fadrozole hydrochloride was an equally good inhibitor of rates of 2- and 4-hydroxylation of estrone (20 microM) by hepatic microsomes (IC50, approximately 25 microM). Corresponding IC50 values with renal microsomes were less than 2 microM, and 2-hydroxylation of estrone was inhibited by Fadrozole hydrochloride up to 15% more than 4-hydroxylation. Treatment of hamsters with estradiol for 2 months decreased rates of 2- and 4-hydroxylation of estrone by renal microsomes by approximately 95%. The rate of conversion of estrone to 16 alpha-hydroxyestrone by hepatic microsomes was 10-20% that of 2-hydroxylation. Renal microsomes catalyzed 16 alpha-hydroxylation of estrone at an even lower rate (approximately 5% of that of 2-hydroxylation). Rates of O-methylation of 2- and 4-hydroxyestrone by hamster kidney cytosol were comparable to those of 2- and 4-hydroxyestradiol. In conclusion, conversion of estrone to its catechol metabolites by microsomes of hamster kidney, a target organ of estrogen-induced carcinogenesis, is quantitatively more important than the conversion to 16 alpha-hydroxyestrone. The findings are consistent with the postulated role of catechol estrogens generated in situ in estrone-induced carcinogenesis.

Estrogens as endogenous genotoxic agents--DNA adducts and mutations.

Estrogens induce tumors in laboratory animals and have been associated with breast and uterine cancers in humans. In relation to the role of estrogens in the induction of cancer, we examine formation of DNA adducts by reactive electrophilic estrogen metabolites, formation of reactive oxygen species by estrogens and the resulting indirect DNA damage by these oxidants, and, finally, genomic and gene mutations induced by estrogens. Quinone intermediates derived by oxidation of the catechol estrogens 4-hydroxyestradiol or 4-hydroxyestrone may react with purine bases of DNA to form depurinating adducts that generate highly mutagenic apurinic sites. In contrast, quinones of 2-hydroxylated estrogens produce less harmful, stable DNA adducts. The catechol estrogen metabolites may also generate potentially mutagenic oxygen radicals by metabolic redox cycling or other mechanisms. Several types of indirect DNA damage are caused by estrogen-induced oxidants, such as oxidized DNA bases, DNA strand breakage, and adduct formation by reactive aldehydes derived from lipid hydroperoxides. Estradiol and the synthetic estrogen diethylstilbestrol also induce numerical and structural chromosomal aberrations and several types of gene mutations in cells in culture and in vivo. In conclusion, estrogens, including the natural hormones estradiol and estrone, must be considered genotoxic carcinogens on the basis of the evidence outlined in this chapter.