- Email: [email protected]
Biogenesis and Inactivation of Catecholestrogens
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J. Weisz et 01.
References 1. Liehr, J. G., and Sirbasku, D. A. (1985). Estrogen-dependent kidney tumors. In Tissue Culture of Epithelial Cells. (M. Taub, ed.), pp. 205-234. Plenum Press, New York. 2. Yager, J. D., and Liehr, J. G. (1996). Molecular mechanisms of estrogen carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 36, 203-232. 3. Liehr, J. G. (1997). Hormole-associated cancer: Mechanistic similarities between human breast cancer and estrogen-induced kidney carcinogenesis in hamsters. Environ. Health Perspect. 105, 565-569. 4. Liehr, J. G., and Roy, D. (1990). Free radical generation by redox cycling of estrogens. Free Radic. Biol. Med. 8, 415-423. 5. Liehr, J. G., and Ricci, M. J. (1996). 4-Hydroxylation of estrogens as marker of human mammary tumors. l'roc. Natl. Acad. Sci. U.S.A. 93, 3294-3296.
J. Weisz," G. A. Clawson,t and C. R. Crevelingt Departments of *Obstetrics and Gynecology and tPathology The M. S. Hershey Medical Center Pennsylvania State University Hershey, Pennsylvania I7033
*National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892
Biogenesis and Inactivation of Catecholestrogens That catecholestrogens (CEs) may provide a functional link between estrogens (Es)and catecholamines was first suggested over three decades ago. It was based on the discovery that 2-hydroxylated CEs (2-OH-CEs), products of a major pathway of metabolic inactivation of estrone and estradiol in liver, were not only inactivated by catechol-0-methyltransferase (COMT), but also, by virtue of their high affinity for COMT, could inhibit inactivation of catecholamines by this enzyme (1).Interest in 2-hydroxylation of estrogens (Es) as a potential pathway of metabolic activation of Es was rekindled a decade later following reports that 2-OH-CEs may be generated in the central nervous system (CNS) (1).This finding led to the suggestion that CEs may provide a direct biochemical link between Es and catecholaminergic function restricted to sites in the CNS where they are produced. The demonstration that 2-OHCEs can inhibit not only COMT, but also tyrosine hydroxylase lent further support to this hypothesis (1, 2). However, the initial enthusiasm for this Advances in Pharmacology, Vohme 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved. 1054-3589/98 $25.00
Biogenesis and Inactivation of Catecholestrogens
829
postulate waned as difficulties were encountered in obtaining conclusive, reproducible evidence for any essential role for CEs in the E-dependent neuronal functions selected for study (1).In retrospect, the frustrations caused by these early studies are not surprising considering how little was known at that time about the biogenesis and metabolism of CEs, the mechanism by which they may act, and the lack of specificity and precision of the indirect assays used in many studies. It is now clear that CEs can be generated by several discrete enzymatic pathways, that these can result in the formation of both 2- and 4hydroxylated CEs (4-OH-CEs), and that these two classes of CEs differ in their properties, both as catechols and as Es (2). The value of these insights, availability of specific and sensitive assays for measuring CE synthase activities, and molecular biological tools now available for probing the role of CEs as local mediators of the physiological actions of Es are highlighted in this symposium by S. K. Day. A new dimension to the potential significance of CE formation within E-target organs was added by evidence, reviewed in this symposium by Liehr and Cavalieri, implicating CEs in the pathopysiology of E-action, notably carcinogenesis and neuronal degeneration.
1. Biogenesis of CEs Hydroxylation of Es at C-2 and C-4 can be catalyzed by two distinct biochemical mechanisms (2). The two differ in their cofactor requirement and in the profile of CEs they generate. One utilizes NADPH as the cofactor, molecular oxygen as the oxygen source, and a cytochrome P450 as the catalyst. It generates predominantly either 2- O Y 4-OH-CEs, depending on the form of P450 involved. The second utilizes an organic hydroperoxide (OHP)as cofactor and oxygen source, can be catalyzed by some hemoprotein peroxidases as well as by some as yet unidentified cytochrome P450, and generates 2- and 4-OHCEs in similar amounts. For reasons presented in this chapter, these differences in cofactor requirement and profile of CEs generated have functional implications for both the physiology and the pathophysiology of E action.
A. NADPH-Dependent 2-Hydroxylation of Es Studies of NADPH-dependent aromatic hydroxylation of Es have until recently focused almost exclusively on 2-hydroxylation. Several forms of P450 have been identified that can catalyze 2-hydroxylation of Es. Specifically, these are two members of the P4501A subfamily (P4501A1 and -1A2), members of the P4503A subfamily, and, in rats, also the male-specific P4502Cll. These P4SOs are expressed in many tissues besides the liver and, as shown in cytochemical studies, their expression in different organs is restricted to anatomically defined cell populations. Because P4501A1, P4501A2, and P4503A1 are also responsible for the phase I metabolism (metabolic activation-inactivation) of many prevalent lipophilic zenobiotics and drugs, and are inducible by them, these P450s provide an interface between environmental chemicals and Es and, by implication, also catecholaminergic function.
830
j. Weisz eta/.
NADPH-dependent 4-hydroxylation of Es was first identified in rat anterior pituitary and then in hamster kidney (2). However, only recently has a form of P450 been identified that can catalyze selective 4-hydroxylation Es (3). It was discovered by investigators interested in mechanisms responsible for the apparent antiestrogenicity of TCDD, and in determining if altered metabolism of Es could contribute to the phenomenon. On treating MCF-7 breast cancer cells with TCDD, they noted a marked increase in both 2- and 4-hydroxylation of Es. The increase in 2-hydroxylation could be accounted for by induction of members of the P4501A subfamily. The catalyst for the 4-hydroxylation proved to be a previously unidentified member of the P450 superfamily. Based on its sequence homology with P4501A1 and -1A2, it was designated P4501B1. Because 4-OH-CEs are potent long-acting Es, the discovery of P4501B1 did not contribute to understanding TCDD’s apparent antiestrogenic effects. It did, however, direct attention to 4-OH-CEs as potential local mediators of E-action. Although much remains to be learned about the properties of 4-OH-CEs, it is already evident that these are likely to differ in many important respects from those of 2-OH-CEs, both as Es and as catechols, differences with implications for both physiology and pathophysiology (discussion follows). P4501B1 appears to be expressed constitutively in many extrahepatic tissues, and its expression, like that of P450s mediating 2-hydroxylation of Es, can be shown to be restricted to defined anatomical loci and cell populations, a finding consistent with the concept that CEs act as autocrine-paracrine mediators of the action of primary Es (4).
B. Peroxidative CE Formation Aromatic hydroxylation of ES can be catalyzed in the presence of an OHP by certain hemoprotein peroxidases as well as microsomes (2, 4). The mechanism involved is analogous to the OHP-dependent metabolism of polycyclic aromatic hydrocarbon. Cytochrome P4.50-dependence of microsomal OHPdependent CE formation has been demonstrated using CO and pharmacological inhibitors of P450. However, the form(s) of P450 mediating this pathway CE formation is not known. None of the major, inducible hepatic forms of P450 appear to be responsible, because neither inducers of P450 administered in vivo nor inhibitory antibodies in vitro affected peroxidative activity of hepatic microsomes. Whether peroxidative activity is expressed under normal physiological conditions, and what the nature or source of OHPs might be under such conditions are also not known. However, OHPs can be generated in virtually unlimited amounts under conditions of oxidative stress. Under these conditions, peroxidative CE formation could be triggered and sustained unchecked and contribute to geno- and cytotoxicity by mechanisms described by others in this symposium. In particular, semiquinone and quinone E metabolites of CEs are highly reactive and can form DNA adducts. CEs can also become a source of reactive oxygen species (ROS) by entering into redox cycling (i.e., repeated cycles of oxidation and reduction between catechol and quinone Es). Because peroxidative activity generates 2- and 4-OH-CEs in similar amounts, and 2-OH-CEs can inhibit the inactivation of 4-OH-CEs by COMT, the peroxi-
Biogenesis and Inactivation of Catecholestrogens
83 I
dative pathway of CE formation could be especially relevant to pathophysiology. Enzymatic demethylation of 0-methylated CEs by specific isoforms of P450 could provide an additional mechanism by which CEs could be generated at specific sites. This postulate is supported by evidence for demethylation of 0-methylated CEs administered in vivo and reports of remarkably high potency of 0-methylated CEs in some in vitro bioassays (4). This potential mechanism for targeting of CEs to specific cell populations has remained unexplored.
II. Differences in Properties of 2- and 4-OH-CEs; Estrogenic Potency and Inactivation There is good evidence that 4-OH-CEs are potent long-acting Es ( 2 , 4). In contrast, 2-OH-CEs have been generally considered to be weak Es based on the marked decrease in affinity for the E receptor caused by 2-hydroxylation and the weak estrogenicity of 2-OH-Es in in vivo bioassays. This conclusion may, however, have to be revised in light of the unexpectedly high potency of 2-OH-CEs in in vitro bioassays, in which target cells are exposed directly to the steroids. These differences between in vivo and in vitro potency could be due to differences in pharmacokinetics of the two classes of CEs and/or their further metabolism. The 2- and 4-OH-CEs differ also in their properties as catechols. Thus, although 2- and 4-OH-CEs have similar apparent affinity for COMT and similar maximal velocities of 0-methylation, 2-OH-CEs can inhibit the 0methylation of 4-OH-CEs (2,4). Whether 2- and 4-OH-CEs differ also in their ability to inhibit tyrosine hydroxylase remains to be determined. Systematic comparisons of 2- and 4-OH-CEs as catechols are needed in light of evidence that 4-OH-CEs are not simply minor by-products of 2-hydroxylation, but that they may be generated in significant amounts in many extrahepatic tissues, either by some forms of P450 (eg., P4501B1) or by a peroxidative mechanism.
111. Metabolism of CEs: The COMT Paradigm Because CEs are highly reactive and are potential sources of excess ROS, there is an obvious need for their prompt inactivation. CEs’ postulated role as local mediators of E-action also implies a need for inactivating enzymes close to sites of formation of CEs in order to restrict their actions spatially and temporally. Understanding of how CEs are inactivated, however, has lagged behind that of their biogenesis. Only COMT has been investigated to any extent within this context. COMT in liver and red blood cells plays a major role in bulk inactivation of catechols, including CEs generated in liver ( 2 , 4). In extrahepatic organs, such as brain, uterus, kidney, and mammary gland, COMT has been shown to be expressed only in a few anatomically defined loci and cell populations ( 5 ) .Such selective expression is consistent with the notion that COMT participates in regulation of local concentrations of catechols, including
832
J. Weisz et a/.
CEs. Studies of expression of COMT in uterus in relationship to implantation also support this notion ( 6 ) . Implantation is a critical physiological function in which locally generated CEs have been postulated to play an essential role. In rat uterus, COMT has neen shown to be induced at the time of implantation, specifically on the antimesometrial side where implantation is known to occur, and its induction has been shown to depend on progesterone, a key regulator and coordinator of implantation. We have examined the effect of Es on the expression of COMT in hamster kidney, a model for E-induced cancer in which CEs generated in situ are postulated to play an essential, initiating role. Using immunocytochemistry, we have shown COMT to be localized in control animals in epithelial cells of proximal convoluted tubules (PCTs), mainly in the deep juxtamedullary cortex. E treatment not only induced COMT in PCTs throughout the cortex, but also caused the appearance of nuclear COMT. By immunoblotting, the nuclear COMT was identified to be the smaller soluble form of COMT. The physiological role of COMT in PCTs is presumably to regulate levels of locally generated dopamine and, thereby, the rate of natruresis (7).The induction of COMT caused by E treatment, in particular its transport into the nucleus, implies a threat to the genome by increased catechol load, resulting, presumably, from CEs generated in situ. Interestingly, we also observed nuclear localization of COMT in human breast cancer cells, whereas in normal mammary ductal epithelial cells COMT is localized exclusively in cytoplasm. Clearly, such correlations, although suggestive, do not prove participation of CEs in either implantation or carcinogenesis. However, they underscore the need for a systematic examination of enzymes that can inactivate CEs, and they provide model systems for such studies. Ultimately, defining the role of CEs will require observing, and modulating their generation, using specific pharmacological inhibitors and molecular biological strategies. Acknowledgment Supported by National Institutes of Health grants R55 CA58739-01 and CA21141-17.
References 1. Catchol Estrogens. Proceedings of Fogarthy International Conference, Bethesda, MD. (G. R. Meriam and M. B. Lipsett, eds.). Raven Press, New York, 1983. 2. Weisz, J. (1994).Biogenesis of catecholestrogens: Metabolic activation of estrogens by phase I enzymes. Polycyclic Arorn. Compd. 6, 241-251. 3. Hayes, C. L., Spink, D. C., Spink, B., Cao, J. Q., Walker, N. J., and Suter, T. R. (1996). 17P-Estradiol hydroxylation by human cytochrome P450 1B1. Froc. Nutl. Acad. Sci. U.S.A. 93, 9776-9781. 4. Weisz, J. (1991). Metabolism of estrogens in target cells. Diversification and amplification of hormone action and the catecholestrogen hypothesis. I n New Biology of Steroid Hormones. (R. Hochberg and F. Naftolin, eds.), pp. 201-212. Raven Press, New York. 5 . Creveling, C., and Hartman, B. K. (1982). Relationship between the cellular localization and physiological function catechol-0-methyltransferase. In Biochemistry of D-Adenosyl
Catecholestrogens as Procarcinogens
833
L-Methionine and Related Compounds. (E. Usdin, R. T. Burchard, and C. R. Creveling, eds.). pp. 479-486. Macmillan Press, London. 6 . Creveling, C. R., and Inoue K. (1995). Induction of catechol-0-methylrransferase in the luminal epithelium of rat uterus by progesterone. Drug Metab. Dispos. 23, 430-432. 7. Meister, B., Bean, A. J., and Aperia, A. (1993). Catechol-0-methyltransferase mRNA in kidney and its appearance during ontogeny. Kidney Znt. 44, 726-733.
D. E. Stack, E. L. Cavalieri, and E. G. Rogan Eppley Cancer Research Institute University of Nebraska Medical Center Omaha, Nebraska 68 I98
Catecholestrogens as Procarcinogens: Depurinating Adducts and Tumor Initiation Excessive exposure to endogenous estrogens has been linked to cancer in both rodent models and human studies. While many studies have been aimed at understanding the relationship between estrogen exposure and cancer, the mechanism of estrogen-induced cancer is still poorly understood. The role of estrogens in receptor-mediated processes leading to the stimulation of cell proliferation has been widely studied to better understand the etiology of estrogen-induced cancers. More recently, the genotoxic properties of electrophilic estrogen metabolites have been investigated to help shed light on the mechanism of hormonal carcinogenesis (1). Estradiol (El) and estrone (El),when hydroxylated at the 2- or 4-position, form catecholestrogens (CEs),which are among the major metabolites of El and EZ.CEs are typically conjugated by catechol-0-methyltransferases (COMTs)to afford 0-methylated CEs. Nonmethylated CEs can be oxidized to the reactive electrophilic o-quinones (CE-Qs)by peroxidases and cytochrorne P450. Therefore, COMTs act as protective enzymes. With elevated rates of CE formation and/or deficient COMT activity, increased levels of CEs can occur, leading to the formation of CE-Qs. Once formed, CE-Qs can act as Michael acceptors, binding to cellular macromolecules, including DNA. Malignant renal tumors are induced in Syrian golden hamsters by treatment with 4-OH-El or 4-OH-E2, whereas the corresponding 2-OH isomers are inactive (2, 3 ) . Furthermore, elevated levels of the 4-OH isomer (relative to the 2OH isomer) have been detected in tissues prone to estrogen-induced cancers, such as rat pituitary, mouse uterus, human MCF-7 breast cancer cells, human Aduanres in Phannacology, Volume 42
Copyright 0 1998 by Academic Press. All rights ot reproducrron in any form reserved 1054-3589/98$25.00
J. Weisz et 01.
References 1. Liehr, J. G., and Sirbasku, D. A. (1985). Estrogen-dependent kidney tumors. In Tissue Culture of Epithelial Cells. (M. Taub, ed.), pp. 205-234. Plenum Press, New York. 2. Yager, J. D., and Liehr, J. G. (1996). Molecular mechanisms of estrogen carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 36, 203-232. 3. Liehr, J. G. (1997). Hormole-associated cancer: Mechanistic similarities between human breast cancer and estrogen-induced kidney carcinogenesis in hamsters. Environ. Health Perspect. 105, 565-569. 4. Liehr, J. G., and Roy, D. (1990). Free radical generation by redox cycling of estrogens. Free Radic. Biol. Med. 8, 415-423. 5. Liehr, J. G., and Ricci, M. J. (1996). 4-Hydroxylation of estrogens as marker of human mammary tumors. l'roc. Natl. Acad. Sci. U.S.A. 93, 3294-3296.
J. Weisz," G. A. Clawson,t and C. R. Crevelingt Departments of *Obstetrics and Gynecology and tPathology The M. S. Hershey Medical Center Pennsylvania State University Hershey, Pennsylvania I7033
*National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892
Biogenesis and Inactivation of Catecholestrogens That catecholestrogens (CEs) may provide a functional link between estrogens (Es)and catecholamines was first suggested over three decades ago. It was based on the discovery that 2-hydroxylated CEs (2-OH-CEs), products of a major pathway of metabolic inactivation of estrone and estradiol in liver, were not only inactivated by catechol-0-methyltransferase (COMT), but also, by virtue of their high affinity for COMT, could inhibit inactivation of catecholamines by this enzyme (1).Interest in 2-hydroxylation of estrogens (Es) as a potential pathway of metabolic activation of Es was rekindled a decade later following reports that 2-OH-CEs may be generated in the central nervous system (CNS) (1).This finding led to the suggestion that CEs may provide a direct biochemical link between Es and catecholaminergic function restricted to sites in the CNS where they are produced. The demonstration that 2-OHCEs can inhibit not only COMT, but also tyrosine hydroxylase lent further support to this hypothesis (1, 2). However, the initial enthusiasm for this Advances in Pharmacology, Vohme 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved. 1054-3589/98 $25.00
Biogenesis and Inactivation of Catecholestrogens
829
postulate waned as difficulties were encountered in obtaining conclusive, reproducible evidence for any essential role for CEs in the E-dependent neuronal functions selected for study (1).In retrospect, the frustrations caused by these early studies are not surprising considering how little was known at that time about the biogenesis and metabolism of CEs, the mechanism by which they may act, and the lack of specificity and precision of the indirect assays used in many studies. It is now clear that CEs can be generated by several discrete enzymatic pathways, that these can result in the formation of both 2- and 4hydroxylated CEs (4-OH-CEs), and that these two classes of CEs differ in their properties, both as catechols and as Es (2). The value of these insights, availability of specific and sensitive assays for measuring CE synthase activities, and molecular biological tools now available for probing the role of CEs as local mediators of the physiological actions of Es are highlighted in this symposium by S. K. Day. A new dimension to the potential significance of CE formation within E-target organs was added by evidence, reviewed in this symposium by Liehr and Cavalieri, implicating CEs in the pathopysiology of E-action, notably carcinogenesis and neuronal degeneration.
1. Biogenesis of CEs Hydroxylation of Es at C-2 and C-4 can be catalyzed by two distinct biochemical mechanisms (2). The two differ in their cofactor requirement and in the profile of CEs they generate. One utilizes NADPH as the cofactor, molecular oxygen as the oxygen source, and a cytochrome P450 as the catalyst. It generates predominantly either 2- O Y 4-OH-CEs, depending on the form of P450 involved. The second utilizes an organic hydroperoxide (OHP)as cofactor and oxygen source, can be catalyzed by some hemoprotein peroxidases as well as by some as yet unidentified cytochrome P450, and generates 2- and 4-OHCEs in similar amounts. For reasons presented in this chapter, these differences in cofactor requirement and profile of CEs generated have functional implications for both the physiology and the pathophysiology of E action.
A. NADPH-Dependent 2-Hydroxylation of Es Studies of NADPH-dependent aromatic hydroxylation of Es have until recently focused almost exclusively on 2-hydroxylation. Several forms of P450 have been identified that can catalyze 2-hydroxylation of Es. Specifically, these are two members of the P4501A subfamily (P4501A1 and -1A2), members of the P4503A subfamily, and, in rats, also the male-specific P4502Cll. These P4SOs are expressed in many tissues besides the liver and, as shown in cytochemical studies, their expression in different organs is restricted to anatomically defined cell populations. Because P4501A1, P4501A2, and P4503A1 are also responsible for the phase I metabolism (metabolic activation-inactivation) of many prevalent lipophilic zenobiotics and drugs, and are inducible by them, these P450s provide an interface between environmental chemicals and Es and, by implication, also catecholaminergic function.
830
j. Weisz eta/.
NADPH-dependent 4-hydroxylation of Es was first identified in rat anterior pituitary and then in hamster kidney (2). However, only recently has a form of P450 been identified that can catalyze selective 4-hydroxylation Es (3). It was discovered by investigators interested in mechanisms responsible for the apparent antiestrogenicity of TCDD, and in determining if altered metabolism of Es could contribute to the phenomenon. On treating MCF-7 breast cancer cells with TCDD, they noted a marked increase in both 2- and 4-hydroxylation of Es. The increase in 2-hydroxylation could be accounted for by induction of members of the P4501A subfamily. The catalyst for the 4-hydroxylation proved to be a previously unidentified member of the P450 superfamily. Based on its sequence homology with P4501A1 and -1A2, it was designated P4501B1. Because 4-OH-CEs are potent long-acting Es, the discovery of P4501B1 did not contribute to understanding TCDD’s apparent antiestrogenic effects. It did, however, direct attention to 4-OH-CEs as potential local mediators of E-action. Although much remains to be learned about the properties of 4-OH-CEs, it is already evident that these are likely to differ in many important respects from those of 2-OH-CEs, both as Es and as catechols, differences with implications for both physiology and pathophysiology (discussion follows). P4501B1 appears to be expressed constitutively in many extrahepatic tissues, and its expression, like that of P450s mediating 2-hydroxylation of Es, can be shown to be restricted to defined anatomical loci and cell populations, a finding consistent with the concept that CEs act as autocrine-paracrine mediators of the action of primary Es (4).
B. Peroxidative CE Formation Aromatic hydroxylation of ES can be catalyzed in the presence of an OHP by certain hemoprotein peroxidases as well as microsomes (2, 4). The mechanism involved is analogous to the OHP-dependent metabolism of polycyclic aromatic hydrocarbon. Cytochrome P4.50-dependence of microsomal OHPdependent CE formation has been demonstrated using CO and pharmacological inhibitors of P450. However, the form(s) of P450 mediating this pathway CE formation is not known. None of the major, inducible hepatic forms of P450 appear to be responsible, because neither inducers of P450 administered in vivo nor inhibitory antibodies in vitro affected peroxidative activity of hepatic microsomes. Whether peroxidative activity is expressed under normal physiological conditions, and what the nature or source of OHPs might be under such conditions are also not known. However, OHPs can be generated in virtually unlimited amounts under conditions of oxidative stress. Under these conditions, peroxidative CE formation could be triggered and sustained unchecked and contribute to geno- and cytotoxicity by mechanisms described by others in this symposium. In particular, semiquinone and quinone E metabolites of CEs are highly reactive and can form DNA adducts. CEs can also become a source of reactive oxygen species (ROS) by entering into redox cycling (i.e., repeated cycles of oxidation and reduction between catechol and quinone Es). Because peroxidative activity generates 2- and 4-OH-CEs in similar amounts, and 2-OH-CEs can inhibit the inactivation of 4-OH-CEs by COMT, the peroxi-
Biogenesis and Inactivation of Catecholestrogens
83 I
dative pathway of CE formation could be especially relevant to pathophysiology. Enzymatic demethylation of 0-methylated CEs by specific isoforms of P450 could provide an additional mechanism by which CEs could be generated at specific sites. This postulate is supported by evidence for demethylation of 0-methylated CEs administered in vivo and reports of remarkably high potency of 0-methylated CEs in some in vitro bioassays (4). This potential mechanism for targeting of CEs to specific cell populations has remained unexplored.
II. Differences in Properties of 2- and 4-OH-CEs; Estrogenic Potency and Inactivation There is good evidence that 4-OH-CEs are potent long-acting Es ( 2 , 4). In contrast, 2-OH-CEs have been generally considered to be weak Es based on the marked decrease in affinity for the E receptor caused by 2-hydroxylation and the weak estrogenicity of 2-OH-Es in in vivo bioassays. This conclusion may, however, have to be revised in light of the unexpectedly high potency of 2-OH-CEs in in vitro bioassays, in which target cells are exposed directly to the steroids. These differences between in vivo and in vitro potency could be due to differences in pharmacokinetics of the two classes of CEs and/or their further metabolism. The 2- and 4-OH-CEs differ also in their properties as catechols. Thus, although 2- and 4-OH-CEs have similar apparent affinity for COMT and similar maximal velocities of 0-methylation, 2-OH-CEs can inhibit the 0methylation of 4-OH-CEs (2,4). Whether 2- and 4-OH-CEs differ also in their ability to inhibit tyrosine hydroxylase remains to be determined. Systematic comparisons of 2- and 4-OH-CEs as catechols are needed in light of evidence that 4-OH-CEs are not simply minor by-products of 2-hydroxylation, but that they may be generated in significant amounts in many extrahepatic tissues, either by some forms of P450 (eg., P4501B1) or by a peroxidative mechanism.
111. Metabolism of CEs: The COMT Paradigm Because CEs are highly reactive and are potential sources of excess ROS, there is an obvious need for their prompt inactivation. CEs’ postulated role as local mediators of E-action also implies a need for inactivating enzymes close to sites of formation of CEs in order to restrict their actions spatially and temporally. Understanding of how CEs are inactivated, however, has lagged behind that of their biogenesis. Only COMT has been investigated to any extent within this context. COMT in liver and red blood cells plays a major role in bulk inactivation of catechols, including CEs generated in liver ( 2 , 4). In extrahepatic organs, such as brain, uterus, kidney, and mammary gland, COMT has been shown to be expressed only in a few anatomically defined loci and cell populations ( 5 ) .Such selective expression is consistent with the notion that COMT participates in regulation of local concentrations of catechols, including
832
J. Weisz et a/.
CEs. Studies of expression of COMT in uterus in relationship to implantation also support this notion ( 6 ) . Implantation is a critical physiological function in which locally generated CEs have been postulated to play an essential role. In rat uterus, COMT has neen shown to be induced at the time of implantation, specifically on the antimesometrial side where implantation is known to occur, and its induction has been shown to depend on progesterone, a key regulator and coordinator of implantation. We have examined the effect of Es on the expression of COMT in hamster kidney, a model for E-induced cancer in which CEs generated in situ are postulated to play an essential, initiating role. Using immunocytochemistry, we have shown COMT to be localized in control animals in epithelial cells of proximal convoluted tubules (PCTs), mainly in the deep juxtamedullary cortex. E treatment not only induced COMT in PCTs throughout the cortex, but also caused the appearance of nuclear COMT. By immunoblotting, the nuclear COMT was identified to be the smaller soluble form of COMT. The physiological role of COMT in PCTs is presumably to regulate levels of locally generated dopamine and, thereby, the rate of natruresis (7).The induction of COMT caused by E treatment, in particular its transport into the nucleus, implies a threat to the genome by increased catechol load, resulting, presumably, from CEs generated in situ. Interestingly, we also observed nuclear localization of COMT in human breast cancer cells, whereas in normal mammary ductal epithelial cells COMT is localized exclusively in cytoplasm. Clearly, such correlations, although suggestive, do not prove participation of CEs in either implantation or carcinogenesis. However, they underscore the need for a systematic examination of enzymes that can inactivate CEs, and they provide model systems for such studies. Ultimately, defining the role of CEs will require observing, and modulating their generation, using specific pharmacological inhibitors and molecular biological strategies. Acknowledgment Supported by National Institutes of Health grants R55 CA58739-01 and CA21141-17.
References 1. Catchol Estrogens. Proceedings of Fogarthy International Conference, Bethesda, MD. (G. R. Meriam and M. B. Lipsett, eds.). Raven Press, New York, 1983. 2. Weisz, J. (1994).Biogenesis of catecholestrogens: Metabolic activation of estrogens by phase I enzymes. Polycyclic Arorn. Compd. 6, 241-251. 3. Hayes, C. L., Spink, D. C., Spink, B., Cao, J. Q., Walker, N. J., and Suter, T. R. (1996). 17P-Estradiol hydroxylation by human cytochrome P450 1B1. Froc. Nutl. Acad. Sci. U.S.A. 93, 9776-9781. 4. Weisz, J. (1991). Metabolism of estrogens in target cells. Diversification and amplification of hormone action and the catecholestrogen hypothesis. I n New Biology of Steroid Hormones. (R. Hochberg and F. Naftolin, eds.), pp. 201-212. Raven Press, New York. 5 . Creveling, C., and Hartman, B. K. (1982). Relationship between the cellular localization and physiological function catechol-0-methyltransferase. In Biochemistry of D-Adenosyl
Catecholestrogens as Procarcinogens
833
L-Methionine and Related Compounds. (E. Usdin, R. T. Burchard, and C. R. Creveling, eds.). pp. 479-486. Macmillan Press, London. 6 . Creveling, C. R., and Inoue K. (1995). Induction of catechol-0-methylrransferase in the luminal epithelium of rat uterus by progesterone. Drug Metab. Dispos. 23, 430-432. 7. Meister, B., Bean, A. J., and Aperia, A. (1993). Catechol-0-methyltransferase mRNA in kidney and its appearance during ontogeny. Kidney Znt. 44, 726-733.
D. E. Stack, E. L. Cavalieri, and E. G. Rogan Eppley Cancer Research Institute University of Nebraska Medical Center Omaha, Nebraska 68 I98
Catecholestrogens as Procarcinogens: Depurinating Adducts and Tumor Initiation Excessive exposure to endogenous estrogens has been linked to cancer in both rodent models and human studies. While many studies have been aimed at understanding the relationship between estrogen exposure and cancer, the mechanism of estrogen-induced cancer is still poorly understood. The role of estrogens in receptor-mediated processes leading to the stimulation of cell proliferation has been widely studied to better understand the etiology of estrogen-induced cancers. More recently, the genotoxic properties of electrophilic estrogen metabolites have been investigated to help shed light on the mechanism of hormonal carcinogenesis (1). Estradiol (El) and estrone (El),when hydroxylated at the 2- or 4-position, form catecholestrogens (CEs),which are among the major metabolites of El and EZ.CEs are typically conjugated by catechol-0-methyltransferases (COMTs)to afford 0-methylated CEs. Nonmethylated CEs can be oxidized to the reactive electrophilic o-quinones (CE-Qs)by peroxidases and cytochrorne P450. Therefore, COMTs act as protective enzymes. With elevated rates of CE formation and/or deficient COMT activity, increased levels of CEs can occur, leading to the formation of CE-Qs. Once formed, CE-Qs can act as Michael acceptors, binding to cellular macromolecules, including DNA. Malignant renal tumors are induced in Syrian golden hamsters by treatment with 4-OH-El or 4-OH-E2, whereas the corresponding 2-OH isomers are inactive (2, 3 ) . Furthermore, elevated levels of the 4-OH isomer (relative to the 2OH isomer) have been detected in tissues prone to estrogen-induced cancers, such as rat pituitary, mouse uterus, human MCF-7 breast cancer cells, human Aduanres in Phannacology, Volume 42
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