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Catecholestrogens as Procaroncinogens: Depurinating Adducts and Tumor Initiation
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
a34
D.
E. Stack et of.
uterine myometrial tumors, and breast cancer tissues (1).Because we hypothesize that CE-Qs are the ultimate carcinogenic forms of estrogens, we have investigated the chemical differences in the two CE-Qs generated from the isomeric CEs, estrogen-2,3-quinones [E,(E2)-2,3-Q]and estrogen-3,4-quinones [El(E2)-3,4-Q].The chemical properties of the two CE-Qs in relation to nucleosides and DNA are the subject of this presentation. CE-Qs can be generated chemically by MnOz oxidation of the corresponding CEs in acetonitrile, chloroform, methylene chloride, or acetone. The stability of the two o-quinones, El-2,3-Q and E1-3,4-Q, was measured in aqueous solutions at various pH. In neutral conditions (pH 6.7, no buffer), the two quinones have similar half-lives of 110 min. However, the El-3,4-Q showed an increased stability at lower pH (T% of 190 and 390 min at p H 5 and 3, respectively), whereas the El-2,3-Q showed no increase in stability at lower pH (TYz of 110 and 70 min at pH 5 and 3, respectively). It has been reported that the 2,3quinones of estrogens are less stable and hence more reactive than the 3,4quinones; however, we have found that in competitive studies with glutathione (GSH) at various pH, the 3,4-quinones generate approximately 10 times more of the CE-Q-GSH adduct than the 2,3-quinones (the reaction between CE-Q and GSH occurs rapidly, with complete conversion of CE-Q-GSH adducts). Thus, because the 2,3-quinone and the 3,4-quinone have similar stabilities under physiological conditions, the 3,4-quinones possess more electrophilic character. The reaction of CE-Qs with the nucleosides deoxyguanosine (dG) and deoxyadenosine (dA) was conducted to gain insight into the behavior of these electrophiles with DNA bases. In addition, the synthetic adducts would serve as standard compounds for in vitro and in vivo studies aimed at understanding the genotoxic effects of CE-Qs. When an acetonitrile solution of E1-3,4-Q or E2-3,4-Q was mixed with dG (dissolved in acetic acid-water, 50:50), one adduct, 4-OH-E1(E2)-l(a,P)-N7Gua,was formed. The adduct is a mixture of two conformational isomers resulting from the restricted rotation of the guanine moiety about the N7(Gua)-Cl(estrogen) bond. Attack of the CE-Qs by dG at the N7-position results in loss of the deoxyribose moiety; hence, the 4-OHEl(Ez)-1(a,P)-N7Gua adducts are referred to as depurinating adducts. Reaction of El(E,)-3,4-Q with dA afforded no adducts. Reaction of E1-2,3-Q with dG or dA generated a different adduct profile from that produced by El-3,4-Q. Reaction with dG resulted in the formation of 2-OH-El-6-N2dG, whereas reaction with dA yielded 2-OH-E1-6-N6dA.In this case, the CE-Qs did not react as an o-quinone; instead, tautomerization to a CE-Q methide (CE-QM) occurred, followed by attack of the exocyclic amino group of the nucleoside at the 6-position of the CE-QM. This yields CE adducts with the deoxyribose moiety intact; hence, 2-OH-EI-6-N2dG and 2OH-EI-6-N6dAare referred to as stable adducts that would remain in DNA, unless repaired. The reaction of CE-Qs with DNA was undertaken to determine the type of DNA damage that would occur when these electrophiles bind to DNA. The formation of stable adducts was determined by the 32P-postlabelingtechnique, whereas formation of depurinating adducts was measured by analysis of the reaction medium via high-performance liquid chromatography interfaced to an electrochemical detector. Reaction of E1-2,3-Q and E2-2,3-Q with calf thy-
Catecholestrogens as Procarcinogens
835
mus DNA produced 8.59 and 1.14 pmol stable adducdmol DNA-P, respectively (Table I). Reaction of El-3,4-Q and E2-3,4-Q generated 0.11 and 0.07 pmol stable adduct/mol DNA-P, respectively (4). Thus, as in the chemical reactions, the 2,3-quinones form stable adducts, whereas the 3,4-quinones have a low propensity for stable adduct formation. Reaction of E1-3,4-Q nd E2-3,4-Qalso produced the depurinating adducts 4-OH-E1(E2)-1 (a,P)-N7Gua at 59 and 213 pmol adduct/mol DNA-P, respectively. Thus, when both stable and depurinating adducts are considered, the 3,4-quinones produce a much higher level of binding with DNA. Binding of CE-Qs via activation of the CE precursors is also examined (see Table I). When 4-OH-E1 was activated with horseradish peroxidase (HRP) in the presence of DNA, 4-OH-EI-1(a,/3)-N7Guawas found at 50 pmol adduct/ mol DNA-P. Likewise, HRP activation of 4-OH-E2produced 194 pmol adduct/ mol DNA-P of the 4-OH-E2-1(a,/3)-N7Guaadduct. This level of binding was similar to the direct reaction of the corresponding CE-Qs. However, lactoperoxidase (LP) activation of 4-OH-E, generated approximately 9 times the amount of depurinating adduct compared with CE-Q or HRP activation of CE (see Table I). Phenobarbital-induced rat liver microsomes with cumene hydroperoxide as cofactor also activated 4-OH-E1 to CE-Qs, resulting in 87 pmol adducdmol DNA-P of the 4-OH-E,-1(a,P)-N7Gua adduct. Formation of the 4-OH-E,-1(a,P)-N7Gua depurinating adduct was examined in vivo by treatment of the mammary glands of female Sprague-Dawley rats with 200 nmol/gland of 4-OH-E,. After 24 hr, the rats were killed and the second and third mammary glands on both the right and the left sides were excised. The mammary glands were minced, ground in liquid nitrogen, pooled, and divided into two aliquots, one for analysis of depurinating DNA adducts and one for analysis of the stable DNA adducts. The 4-OH-El-l(a,/3)-N7Gua depurinating adduct was detected at a level of 30 pmol adducdmol DNA-P, whereas the level of stable adducts was less than 0.05 pmol adducdmol DNAP, the limit of detection. These results are in agreement with the properties of E,-3,4-Q in both chemical and biochemical experiments, as reported previously. TABLE I Reaction of CE-Qs and HRP-, LP-. or P-450-Activated CEs with DNA Compound E1-3,4-Q HRP-activated 4-OH-El LP-activated 4-OH-El PB-microsomeiCuOOH-activated 4-OH-E, E,-3,4-Q HRP-activated 4-OH-E2 E,-2,3-Q HRP-activated 2-OH-E, E,-2,3-Q HRP-activated 2-OH-Ez a
Data from Dwivedy et al. (4).
Depurinating adducts ( p d / m o l DNA-P)
Stable adducts (pmollmol DNA-P)
59 50 440 87 213 194
0.11 0.07 0.06 0.01 0.07 0.10 8.59" 3.00" 1.14" 9.26"
836
0.E. Stack et ol.
The CE-Qs derived from 4-OH-El(E2) and 2-OH-E1(E2)display different chemical properties in their reaction with nucleosides and DNA. Notably, 2,3-quinones bind at the exocyclic amino group of dG and dA, allowing the deoxyribose moiety to remain attached to the adduct, whereas the 3,4-quinones form N7Gua adducts in which the glycosidic bond of dG is destabilized and cleaved. The implications for DNA damage are that 2,3-quinones will bind to form stable adducts, whereas the 3,4-quinones, via generation of the 4-OHE1(E2)-l(a,/3)-N7Guaadduct, will form predominantly depurinating adducts. Recent findings have established a strong correlation between the formation of depurinating adducts with polyaromatic hydrocarbons and induction of oncogenic mutations in mouse skin papillomas induced by these compounds (see chapter by Cavalieri and Rogan, this section). Because the 4-hydroxy isomers of El and E2are carcinogenic, whereas the 2-hydroxy isomers are not, we hypothesize that the formation of the 4-OH-E1(E2)-l(a,!,P)-N7Guadepurinating adduct via the E,(E2)-3,4-Qis the critical step in the initiation of cancer by estrogens.
References 1. Stack, D. E., Byun, J., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (1996). Molecular characteristics of catechol estrogen quinones in reaction with deoxyribonucleosides. Cbem. Res. Toxicol. 9, 851-859. 2. Liehr, J. G., Fang, W. F., Sirbasku, D. A., and Ari-Ulubelen, A. (1986). Carcinogenicity of catechol estrogens in Syrian hamsters. J . Steroid Biochem. 24, 353-356. 3. Li, J. J., and Li, S. A. (1987). Estrogen carcinogenesis in Syrian hamster tissues: Role of metabolism. Fed. Proc. 46, 1858-1863. 4. Dwivedy, I., Devanesan, P., Cremonesi, P., Rogan, E., and Cavalieri, E. (1992). Synthesis and characterization of estrogen 2,3-quinones and 3,4-quinones. Comparison of DNA adducts formed by the quinones versus horseradish peroxidase-activated catechol estrogens. Cbem. Res. Toxicol. 5, 828-833.
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
a34
D.
E. Stack et of.
uterine myometrial tumors, and breast cancer tissues (1).Because we hypothesize that CE-Qs are the ultimate carcinogenic forms of estrogens, we have investigated the chemical differences in the two CE-Qs generated from the isomeric CEs, estrogen-2,3-quinones [E,(E2)-2,3-Q]and estrogen-3,4-quinones [El(E2)-3,4-Q].The chemical properties of the two CE-Qs in relation to nucleosides and DNA are the subject of this presentation. CE-Qs can be generated chemically by MnOz oxidation of the corresponding CEs in acetonitrile, chloroform, methylene chloride, or acetone. The stability of the two o-quinones, El-2,3-Q and E1-3,4-Q, was measured in aqueous solutions at various pH. In neutral conditions (pH 6.7, no buffer), the two quinones have similar half-lives of 110 min. However, the El-3,4-Q showed an increased stability at lower pH (T% of 190 and 390 min at p H 5 and 3, respectively), whereas the El-2,3-Q showed no increase in stability at lower pH (TYz of 110 and 70 min at pH 5 and 3, respectively). It has been reported that the 2,3quinones of estrogens are less stable and hence more reactive than the 3,4quinones; however, we have found that in competitive studies with glutathione (GSH) at various pH, the 3,4-quinones generate approximately 10 times more of the CE-Q-GSH adduct than the 2,3-quinones (the reaction between CE-Q and GSH occurs rapidly, with complete conversion of CE-Q-GSH adducts). Thus, because the 2,3-quinone and the 3,4-quinone have similar stabilities under physiological conditions, the 3,4-quinones possess more electrophilic character. The reaction of CE-Qs with the nucleosides deoxyguanosine (dG) and deoxyadenosine (dA) was conducted to gain insight into the behavior of these electrophiles with DNA bases. In addition, the synthetic adducts would serve as standard compounds for in vitro and in vivo studies aimed at understanding the genotoxic effects of CE-Qs. When an acetonitrile solution of E1-3,4-Q or E2-3,4-Q was mixed with dG (dissolved in acetic acid-water, 50:50), one adduct, 4-OH-E1(E2)-l(a,P)-N7Gua,was formed. The adduct is a mixture of two conformational isomers resulting from the restricted rotation of the guanine moiety about the N7(Gua)-Cl(estrogen) bond. Attack of the CE-Qs by dG at the N7-position results in loss of the deoxyribose moiety; hence, the 4-OHEl(Ez)-1(a,P)-N7Gua adducts are referred to as depurinating adducts. Reaction of El(E,)-3,4-Q with dA afforded no adducts. Reaction of E1-2,3-Q with dG or dA generated a different adduct profile from that produced by El-3,4-Q. Reaction with dG resulted in the formation of 2-OH-El-6-N2dG, whereas reaction with dA yielded 2-OH-E1-6-N6dA.In this case, the CE-Qs did not react as an o-quinone; instead, tautomerization to a CE-Q methide (CE-QM) occurred, followed by attack of the exocyclic amino group of the nucleoside at the 6-position of the CE-QM. This yields CE adducts with the deoxyribose moiety intact; hence, 2-OH-EI-6-N2dG and 2OH-EI-6-N6dAare referred to as stable adducts that would remain in DNA, unless repaired. The reaction of CE-Qs with DNA was undertaken to determine the type of DNA damage that would occur when these electrophiles bind to DNA. The formation of stable adducts was determined by the 32P-postlabelingtechnique, whereas formation of depurinating adducts was measured by analysis of the reaction medium via high-performance liquid chromatography interfaced to an electrochemical detector. Reaction of E1-2,3-Q and E2-2,3-Q with calf thy-
Catecholestrogens as Procarcinogens
835
mus DNA produced 8.59 and 1.14 pmol stable adducdmol DNA-P, respectively (Table I). Reaction of El-3,4-Q and E2-3,4-Q generated 0.11 and 0.07 pmol stable adduct/mol DNA-P, respectively (4). Thus, as in the chemical reactions, the 2,3-quinones form stable adducts, whereas the 3,4-quinones have a low propensity for stable adduct formation. Reaction of E1-3,4-Q nd E2-3,4-Qalso produced the depurinating adducts 4-OH-E1(E2)-1 (a,P)-N7Gua at 59 and 213 pmol adduct/mol DNA-P, respectively. Thus, when both stable and depurinating adducts are considered, the 3,4-quinones produce a much higher level of binding with DNA. Binding of CE-Qs via activation of the CE precursors is also examined (see Table I). When 4-OH-E1 was activated with horseradish peroxidase (HRP) in the presence of DNA, 4-OH-EI-1(a,/3)-N7Guawas found at 50 pmol adduct/ mol DNA-P. Likewise, HRP activation of 4-OH-E2produced 194 pmol adduct/ mol DNA-P of the 4-OH-E2-1(a,/3)-N7Guaadduct. This level of binding was similar to the direct reaction of the corresponding CE-Qs. However, lactoperoxidase (LP) activation of 4-OH-E, generated approximately 9 times the amount of depurinating adduct compared with CE-Q or HRP activation of CE (see Table I). Phenobarbital-induced rat liver microsomes with cumene hydroperoxide as cofactor also activated 4-OH-E1 to CE-Qs, resulting in 87 pmol adducdmol DNA-P of the 4-OH-E,-1(a,P)-N7Gua adduct. Formation of the 4-OH-E,-1(a,P)-N7Gua depurinating adduct was examined in vivo by treatment of the mammary glands of female Sprague-Dawley rats with 200 nmol/gland of 4-OH-E,. After 24 hr, the rats were killed and the second and third mammary glands on both the right and the left sides were excised. The mammary glands were minced, ground in liquid nitrogen, pooled, and divided into two aliquots, one for analysis of depurinating DNA adducts and one for analysis of the stable DNA adducts. The 4-OH-El-l(a,/3)-N7Gua depurinating adduct was detected at a level of 30 pmol adducdmol DNA-P, whereas the level of stable adducts was less than 0.05 pmol adducdmol DNAP, the limit of detection. These results are in agreement with the properties of E,-3,4-Q in both chemical and biochemical experiments, as reported previously. TABLE I Reaction of CE-Qs and HRP-, LP-. or P-450-Activated CEs with DNA Compound E1-3,4-Q HRP-activated 4-OH-El LP-activated 4-OH-El PB-microsomeiCuOOH-activated 4-OH-E, E,-3,4-Q HRP-activated 4-OH-E2 E,-2,3-Q HRP-activated 2-OH-E, E,-2,3-Q HRP-activated 2-OH-Ez a
Data from Dwivedy et al. (4).
Depurinating adducts ( p d / m o l DNA-P)
Stable adducts (pmollmol DNA-P)
59 50 440 87 213 194
0.11 0.07 0.06 0.01 0.07 0.10 8.59" 3.00" 1.14" 9.26"
836
0.E. Stack et ol.
The CE-Qs derived from 4-OH-El(E2) and 2-OH-E1(E2)display different chemical properties in their reaction with nucleosides and DNA. Notably, 2,3-quinones bind at the exocyclic amino group of dG and dA, allowing the deoxyribose moiety to remain attached to the adduct, whereas the 3,4-quinones form N7Gua adducts in which the glycosidic bond of dG is destabilized and cleaved. The implications for DNA damage are that 2,3-quinones will bind to form stable adducts, whereas the 3,4-quinones, via generation of the 4-OHE1(E2)-l(a,/3)-N7Guaadduct, will form predominantly depurinating adducts. Recent findings have established a strong correlation between the formation of depurinating adducts with polyaromatic hydrocarbons and induction of oncogenic mutations in mouse skin papillomas induced by these compounds (see chapter by Cavalieri and Rogan, this section). Because the 4-hydroxy isomers of El and E2are carcinogenic, whereas the 2-hydroxy isomers are not, we hypothesize that the formation of the 4-OH-E1(E2)-l(a,!,P)-N7Guadepurinating adduct via the E,(E2)-3,4-Qis the critical step in the initiation of cancer by estrogens.
References 1. Stack, D. E., Byun, J., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (1996). Molecular characteristics of catechol estrogen quinones in reaction with deoxyribonucleosides. Cbem. Res. Toxicol. 9, 851-859. 2. Liehr, J. G., Fang, W. F., Sirbasku, D. A., and Ari-Ulubelen, A. (1986). Carcinogenicity of catechol estrogens in Syrian hamsters. J . Steroid Biochem. 24, 353-356. 3. Li, J. J., and Li, S. A. (1987). Estrogen carcinogenesis in Syrian hamster tissues: Role of metabolism. Fed. Proc. 46, 1858-1863. 4. Dwivedy, I., Devanesan, P., Cremonesi, P., Rogan, E., and Cavalieri, E. (1992). Synthesis and characterization of estrogen 2,3-quinones and 3,4-quinones. Comparison of DNA adducts formed by the quinones versus horseradish peroxidase-activated catechol estrogens. Cbem. Res. Toxicol. 5, 828-833.