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Ercole L Cavalieri - One of the best experts on this subject based on the ideXlab platform.

  • Estrogen metabolism and formation of Estrogen dna adducts in estradiol treated mcf 10f cells the effects of 2 3 7 8 tetrachlorodibenzo p dioxin induction and Catechol o methyltransferase inhibition
    The Journal of Steroid Biochemistry and Molecular Biology, 2007
    Co-Authors: Muhammad Zahid, Ercole L Cavalieri, Muhammad Saeed, Eleanor G Rogan
    Abstract:

    Formation of Estrogen metabolites that react with DNA is thought to be a mechanism of cancer initiation by Estrogens. The Estrogens estrone (E(1)) and estradiol (E(2)) can form Catechol Estrogen (CE) metabolites, Catechol Estrogen quinones [E(1)(E(2))-3,4-Q], which react with DNA to form predominantly depurinating adducts. This may lead to mutations that initiate cancer. Catechol-O-methyltransferase (COMT) catalyzes an inactivation (protective) pathway for CE. This study investigated the effect of inhibiting COMT activity on the levels of depurinating 4-OHE(1)(E(2))-1-N3Ade and 4-OHE(1)(E(2))-1-N7Gua adducts in human breast epithelial cells. MCF-10F cells were treated with TCDD, a cytochrome P450 inducer, then with E(2) and Ro41-0960, a COMT inhibitor. Estrogen metabolites and depurinating DNA adducts in culture medium were analyzed by HPLC with electrochemical detection. Pre-treatment of cells with TCDD increased E(2) metabolism to 4-OHE(1)(E(2)) and 4-OCH(3)E(1)(E(2)). Inclusion of Ro41-0960 and E(2) in the medium blocked formation of methoxy CE, and depurinating adducts were observed. With Ro41-0960, more adducts were detected in MCF-10F cells exposed to 1 microM E(2), whereas without the inhibitor, no increases in adducts were detected with E(2) < or =10 microM. We conclude that low COMT activity and increased formation of depurinating adducts can be critical factors leading to initiation of breast cancer.

  • Cytochrome P450 isoforms catalyze formation of Catechol Estrogen quinones that react with DNA.
    Metabolism: clinical and experimental, 2007
    Co-Authors: Yan Zhang, Ercole L Cavalieri, Nilesh W. Gaikwad, Kevin Olson, Muhammad Zahid, Eleanor G Rogan
    Abstract:

    Abstract Accumulating evidence suggests that specific metabolites of Estrogens, namely, Catechol Estrogen quinones, react with DNA to form adducts and generate apurinic sites, which can lead to the mutations that induce breast cancer. Oxidation of estradiol (E 2 ) produces 2 Catechol Estrogens, 4-hydroxyestradiol (4-OHE 2 ) and 2-OHE 2 among the major metabolites. These, in turn, are oxidized to the quinones, E 2 -3,4-quinone (E 2 -3,4-Q) and E 2 -2,3-Q, which can react with DNA. Oxidation of E 2 to 2-OHE 2 is mainly catalyzed by cytochrome P450 (CYP) 1A1, and CYP3A4, whereas oxidation of E 2 to 4-OHE 2 in extrahepatic tissues is mainly catalyzed by CYP1B1 as well as some CYP3As. The potential involvement of CYP isoforms in the further oxidation of Catechols to semiquinones and quinones has, however, not been investigated in detail. In this project, to identify the potential function of various CYPs in oxidizing Catechol Estrogens to quinones, we used different recombinant human CYP isoforms, namely, CYP1A1, CYP1B1, and CYP3A4, with the scope of oxidizing the Catechol Estrogens 2-OHE 2 and 4-OHE 2 to their respective Estrogen quinones, which then reacted with DNA. The depurinating adducts 2-OHE 2 -6-N3Ade, 4-OHE 2 -1-N3Ade, and 4-OHE 2 -1-N7Gua were observed in the respective reaction systems by ultraperformance liquid chromatography/tandem mass spectrometry. Furthermore, more than 100-fold higher levels of Estrogen-glutathione (GSH) conjugates were detected in the reactions. Glutathione conjugates were observed, in much smaller amounts, when control microsomes were used. Depurinating adducts, as well as GSH conjugates, were obtained when E 2 -3,4-Q was incubated with CYP1B1 or control microsomes in a 30-minute reaction, further demonstrating that GSH is present in these recombinant enzyme preparations. These experiments demonstrated that CYP1A1, CYP1B1, and CYP3A4 are able to oxidize Catechol Estrogens to their respective quinones, which can further react with GSH, protein, and DNA, the last resulting in depurinating adducts that can lead to mutagenesis.

  • Catechol Estrogen quinones as initiators of breast and other human cancers implications for biomarkers of susceptibility and cancer prevention
    Biochimica et Biophysica Acta, 2006
    Co-Authors: Ercole L Cavalieri, Ryszard Jankowiak, Eleanor G Rogan, Dhubajyoti Chakravarti, Joseph B Guttenplan, Elizabeth Hart, James N Ingle, Paola Muti, Jose Russo, Richard J Santen
    Abstract:

    Abstract Exposure to Estrogens is associated with increased risk of breast and other types of human cancer. Estrogens are converted to metabolites, particularly the Catechol Estrogen-3,4-quinones (CE-3,4-Q), that can react with DNA to form depurinating adducts. These adducts are released from DNA to generate apurinic sites. Error-prone base excision repair of this damage may lead to the mutations that can initiate breast, prostate and other types of cancer. The reaction of CE-3,4-Q with DNA forms the depurinating adducts 4-hydroxyestrone(estradiol) [4-OHE 1 (E 2 )-1-N3Ade and 4-OHE 1 (E 2 )-1-N7Gua. These two adducts constitute more than 99% of the total DNA adducts formed. Increased levels of these quinones and their reaction with DNA occur when Estrogen metabolism is unbalanced. Such an imbalance is the result of overexpression of Estrogen activating enzymes and/or deficient expression of the deactivating (protective) enzymes. This unbalanced metabolism has been observed in breast biopsy tissue from women with breast cancer, compared to control women. Recently, the depurinating adduct 4-OHE 1 (E 2 )-1-N3Ade has been detected in the urine of prostate cancer patients, but not in urine from healthy men. Mutagenesis by CE-3,4-Q has been approached from two different perspectives: one is mutagenic activity in the lacI reporter gene in Fisher 344 rats and the other is study of the reporter Harvey- ras gene in mouse skin and rat mammary gland. A → G and G → A mutations have been observed in the mammary tissue of rats implanted with the CE-3,4-Q precursor, 4-OHE 2 . Mutations have also been observed in the Harvey- ras gene in mouse skin and rat mammary gland within 6–12 h after treatment with E 2 -3,4-Q, suggesting that these mutations arise by error-prone base excision repair of the apurinic sites generated by the depurinating adducts. Treatment of MCF-10F cells, which are Estrogen receptor-α-negative immortalized human breast epithelial cells, with E 2 , 4-OHE 2 or 2-OHE 2 induces their neoplastic transformation in vitro, even in the presence of the antiEstrogen ICI-182,780. This suggests that transformation is independent of the Estrogen receptor. The transformed cells exhibit specific mutations in several genes. Poorly differentiated adenocarcinomas develop when aggressively transformed MCF-10F cells are selected and injected into severe combined immune depressed (SCID) mice. These results represent the first in vitro/in vivo model of Estrogen-induced carcinogenesis in human breast epithelial cells. In other studies, the development of mammary tumors in Estrogen receptor-α knockout mice expressing the Wnt-1 oncogene (ERKO/Wnt-1) provides direct evidence that Estrogens may cause breast cancer through a genotoxic, non-Estrogen receptor-α-mediated mechanism. In summary, this evidence strongly indicates that Estrogens can become endogenous tumor initiators when CE-3,4-Q react with DNA to form specific depurinating adducts. Initiated cells may be promoted by a number of processes, including hormone receptor stimulated proliferation. These results lay the groundwork for assessing risk and preventing disease.

  • the greater reactivity of estradiol 3 4 quinone vs estradiol 2 3 quinone with dna in the formation of depurinating adducts implications for tumor initiating activity
    Chemical Research in Toxicology, 2006
    Co-Authors: Muhammad Zahid, Eleanor G Rogan, Ekta Kohli, Muhammad Saeed, Ercole L Cavalieri
    Abstract:

    Strong evidence supports the idea that specific metabolites of Estrogens, mainly Catechol Estrogen-3,4-quinones, can react with DNA to become endogenous initiators of breast, prostate, and other human cancers. Oxidation of the Catechol Estrogen metabolites 4-hydroxyestradiol (4-OHE2) and 2-OHE2 leads to the quinones, estradiol-3,4-quinone (E2-3,4-Q) and estradiol-2,3-quinone (E2-2,3-Q), respectively. The reaction of E2-3,4-Q with DNA affords predominantly the depurinating adducts 4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua, whereas the reaction of E2-2,3-Q with DNA yields the newly synthesized depurinating adduct 2-OHE2-6-N3Ade. The N3Ade adducts are lost from DNA by rapid depurination, while the N7Gua adduct is lost from DNA with a half-life of ∼3 h at 37 °C. To compare the relative reactivity of E2-3,4-Q and E2-2,3-Q, the compounds were reacted individually with DNA for 0.5−20 h at 37 °C, as well as in mixtures (3:1, 1:1, 1:3, and 5:95) for 10 h at 37 °C. Depurinating and stable adducts were analyzed. In similar ...

  • spectral characterization of Catechol Estrogen quinone ceq derived dna adducts and their identification in human breast tissue extract
    Chemical Research in Toxicology, 2003
    Co-Authors: Yuri Markushin, Ercole L Cavalieri, Gerald J. Small, Wenwan Zhong, Eleanor G Rogan, Edward S Yeung, Ryszard Jankowiak
    Abstract:

    Estrogens, including the natural hormones estrone (E(1)) and estradiol (E(2)), are thought to be involved in tumor induction. Catechol Estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE(1)) and 4-hydroxyestradiol (4-OHE(2)) react with DNA and form depurinating N7Gua and N3Ade adducts that might be responsible for tumor initiation (Cavalieri, E. L., et al. (2000) J. Natl. Cancer Inst. Monogr. 27, 75). Current detection limits for the CEQ-derived DNA adducts by high-performance liquid chromatography with multichannel electrochemical detection are in the picomole range. To improve the limit of detection (LOD) for CEQ-derived DNA adducts, spectrophotometric monitoring was investigated. Spectroscopic studies of 4-OHE(1)-1-N3Ade, 4-OHE(1)-1-N7Gua, 4-OHE(2)-1-N3Ade, and 4-OHE(2)-1-N7Gua adduct standards were performed at 77 and 300 K. Upon laser excitation at 257 nm, the 4-OHE(1)- and 4-OHE(2)-derived N7Gua and N3Ade adducts are strongly phosphorescent at T = 77 K. No phosphorescence was observed at 300 K. Both N3Ade and N7Gua adduct types have weak phosphorescence origin bands near 383 and 385 nm, respectively. The corresponding phosphorescence lifetimes are 1.11 +/- 0.05 and 0.37 +/- 0.05 s. The LOD, based on phosphorescence measurements, is in the low femtomole range. The concentration LOD is approximately 10(-9) M, i.e., similar to that recently obtained for CEQ-derived N-acetylcysteine conjugates (Jankowiak, R., et al. (2003) Chem. Res. Toxicol. 16, 304). The LOD in capillary electrophoresis (CE) with field-amplified sample stacking and absorbance detection is about 3 x 10(-8) M. To verify whether CEQ-derived DNA adducts are formed in humans or not, tissue extracts from two breast cancer patients were analyzed by CE interfaced with room temperature absorption and low temperature (laser-excited) phosphorescence spectroscopies. For the first time, formation of CEQ-derived DNA adducts is shown in humans. For example, the level of 4-OHE(1)-1-N3Ade in the breast tissue extract from a patient with breast carcinoma (8.40 +/- 0.05 pmol/g of tissue) is larger by a factor of about 30 than that in the breast tissue sample from a woman without breast cancer (0.25 +/- 0.05 pmol/g of tissue). In contrast, similar amounts of 4-OHE(2)-1-N3Ade were observed in both types of tissue. Although more breast tissue samples from women with and without breast cancer need to be studied, these results suggest that the N3Ade adducts could serve as biomarkers to predict the risk of breast cancer.

Eleanor G Rogan - One of the best experts on this subject based on the ideXlab platform.

  • Estrogen metabolism and formation of Estrogen dna adducts in estradiol treated mcf 10f cells the effects of 2 3 7 8 tetrachlorodibenzo p dioxin induction and Catechol o methyltransferase inhibition
    The Journal of Steroid Biochemistry and Molecular Biology, 2007
    Co-Authors: Muhammad Zahid, Ercole L Cavalieri, Muhammad Saeed, Eleanor G Rogan
    Abstract:

    Formation of Estrogen metabolites that react with DNA is thought to be a mechanism of cancer initiation by Estrogens. The Estrogens estrone (E(1)) and estradiol (E(2)) can form Catechol Estrogen (CE) metabolites, Catechol Estrogen quinones [E(1)(E(2))-3,4-Q], which react with DNA to form predominantly depurinating adducts. This may lead to mutations that initiate cancer. Catechol-O-methyltransferase (COMT) catalyzes an inactivation (protective) pathway for CE. This study investigated the effect of inhibiting COMT activity on the levels of depurinating 4-OHE(1)(E(2))-1-N3Ade and 4-OHE(1)(E(2))-1-N7Gua adducts in human breast epithelial cells. MCF-10F cells were treated with TCDD, a cytochrome P450 inducer, then with E(2) and Ro41-0960, a COMT inhibitor. Estrogen metabolites and depurinating DNA adducts in culture medium were analyzed by HPLC with electrochemical detection. Pre-treatment of cells with TCDD increased E(2) metabolism to 4-OHE(1)(E(2)) and 4-OCH(3)E(1)(E(2)). Inclusion of Ro41-0960 and E(2) in the medium blocked formation of methoxy CE, and depurinating adducts were observed. With Ro41-0960, more adducts were detected in MCF-10F cells exposed to 1 microM E(2), whereas without the inhibitor, no increases in adducts were detected with E(2) < or =10 microM. We conclude that low COMT activity and increased formation of depurinating adducts can be critical factors leading to initiation of breast cancer.

  • Cytochrome P450 isoforms catalyze formation of Catechol Estrogen quinones that react with DNA.
    Metabolism: clinical and experimental, 2007
    Co-Authors: Yan Zhang, Ercole L Cavalieri, Nilesh W. Gaikwad, Kevin Olson, Muhammad Zahid, Eleanor G Rogan
    Abstract:

    Abstract Accumulating evidence suggests that specific metabolites of Estrogens, namely, Catechol Estrogen quinones, react with DNA to form adducts and generate apurinic sites, which can lead to the mutations that induce breast cancer. Oxidation of estradiol (E 2 ) produces 2 Catechol Estrogens, 4-hydroxyestradiol (4-OHE 2 ) and 2-OHE 2 among the major metabolites. These, in turn, are oxidized to the quinones, E 2 -3,4-quinone (E 2 -3,4-Q) and E 2 -2,3-Q, which can react with DNA. Oxidation of E 2 to 2-OHE 2 is mainly catalyzed by cytochrome P450 (CYP) 1A1, and CYP3A4, whereas oxidation of E 2 to 4-OHE 2 in extrahepatic tissues is mainly catalyzed by CYP1B1 as well as some CYP3As. The potential involvement of CYP isoforms in the further oxidation of Catechols to semiquinones and quinones has, however, not been investigated in detail. In this project, to identify the potential function of various CYPs in oxidizing Catechol Estrogens to quinones, we used different recombinant human CYP isoforms, namely, CYP1A1, CYP1B1, and CYP3A4, with the scope of oxidizing the Catechol Estrogens 2-OHE 2 and 4-OHE 2 to their respective Estrogen quinones, which then reacted with DNA. The depurinating adducts 2-OHE 2 -6-N3Ade, 4-OHE 2 -1-N3Ade, and 4-OHE 2 -1-N7Gua were observed in the respective reaction systems by ultraperformance liquid chromatography/tandem mass spectrometry. Furthermore, more than 100-fold higher levels of Estrogen-glutathione (GSH) conjugates were detected in the reactions. Glutathione conjugates were observed, in much smaller amounts, when control microsomes were used. Depurinating adducts, as well as GSH conjugates, were obtained when E 2 -3,4-Q was incubated with CYP1B1 or control microsomes in a 30-minute reaction, further demonstrating that GSH is present in these recombinant enzyme preparations. These experiments demonstrated that CYP1A1, CYP1B1, and CYP3A4 are able to oxidize Catechol Estrogens to their respective quinones, which can further react with GSH, protein, and DNA, the last resulting in depurinating adducts that can lead to mutagenesis.

  • Catechol Estrogen quinones as initiators of breast and other human cancers implications for biomarkers of susceptibility and cancer prevention
    Biochimica et Biophysica Acta, 2006
    Co-Authors: Ercole L Cavalieri, Ryszard Jankowiak, Eleanor G Rogan, Dhubajyoti Chakravarti, Joseph B Guttenplan, Elizabeth Hart, James N Ingle, Paola Muti, Jose Russo, Richard J Santen
    Abstract:

    Abstract Exposure to Estrogens is associated with increased risk of breast and other types of human cancer. Estrogens are converted to metabolites, particularly the Catechol Estrogen-3,4-quinones (CE-3,4-Q), that can react with DNA to form depurinating adducts. These adducts are released from DNA to generate apurinic sites. Error-prone base excision repair of this damage may lead to the mutations that can initiate breast, prostate and other types of cancer. The reaction of CE-3,4-Q with DNA forms the depurinating adducts 4-hydroxyestrone(estradiol) [4-OHE 1 (E 2 )-1-N3Ade and 4-OHE 1 (E 2 )-1-N7Gua. These two adducts constitute more than 99% of the total DNA adducts formed. Increased levels of these quinones and their reaction with DNA occur when Estrogen metabolism is unbalanced. Such an imbalance is the result of overexpression of Estrogen activating enzymes and/or deficient expression of the deactivating (protective) enzymes. This unbalanced metabolism has been observed in breast biopsy tissue from women with breast cancer, compared to control women. Recently, the depurinating adduct 4-OHE 1 (E 2 )-1-N3Ade has been detected in the urine of prostate cancer patients, but not in urine from healthy men. Mutagenesis by CE-3,4-Q has been approached from two different perspectives: one is mutagenic activity in the lacI reporter gene in Fisher 344 rats and the other is study of the reporter Harvey- ras gene in mouse skin and rat mammary gland. A → G and G → A mutations have been observed in the mammary tissue of rats implanted with the CE-3,4-Q precursor, 4-OHE 2 . Mutations have also been observed in the Harvey- ras gene in mouse skin and rat mammary gland within 6–12 h after treatment with E 2 -3,4-Q, suggesting that these mutations arise by error-prone base excision repair of the apurinic sites generated by the depurinating adducts. Treatment of MCF-10F cells, which are Estrogen receptor-α-negative immortalized human breast epithelial cells, with E 2 , 4-OHE 2 or 2-OHE 2 induces their neoplastic transformation in vitro, even in the presence of the antiEstrogen ICI-182,780. This suggests that transformation is independent of the Estrogen receptor. The transformed cells exhibit specific mutations in several genes. Poorly differentiated adenocarcinomas develop when aggressively transformed MCF-10F cells are selected and injected into severe combined immune depressed (SCID) mice. These results represent the first in vitro/in vivo model of Estrogen-induced carcinogenesis in human breast epithelial cells. In other studies, the development of mammary tumors in Estrogen receptor-α knockout mice expressing the Wnt-1 oncogene (ERKO/Wnt-1) provides direct evidence that Estrogens may cause breast cancer through a genotoxic, non-Estrogen receptor-α-mediated mechanism. In summary, this evidence strongly indicates that Estrogens can become endogenous tumor initiators when CE-3,4-Q react with DNA to form specific depurinating adducts. Initiated cells may be promoted by a number of processes, including hormone receptor stimulated proliferation. These results lay the groundwork for assessing risk and preventing disease.

  • the greater reactivity of estradiol 3 4 quinone vs estradiol 2 3 quinone with dna in the formation of depurinating adducts implications for tumor initiating activity
    Chemical Research in Toxicology, 2006
    Co-Authors: Muhammad Zahid, Eleanor G Rogan, Ekta Kohli, Muhammad Saeed, Ercole L Cavalieri
    Abstract:

    Strong evidence supports the idea that specific metabolites of Estrogens, mainly Catechol Estrogen-3,4-quinones, can react with DNA to become endogenous initiators of breast, prostate, and other human cancers. Oxidation of the Catechol Estrogen metabolites 4-hydroxyestradiol (4-OHE2) and 2-OHE2 leads to the quinones, estradiol-3,4-quinone (E2-3,4-Q) and estradiol-2,3-quinone (E2-2,3-Q), respectively. The reaction of E2-3,4-Q with DNA affords predominantly the depurinating adducts 4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua, whereas the reaction of E2-2,3-Q with DNA yields the newly synthesized depurinating adduct 2-OHE2-6-N3Ade. The N3Ade adducts are lost from DNA by rapid depurination, while the N7Gua adduct is lost from DNA with a half-life of ∼3 h at 37 °C. To compare the relative reactivity of E2-3,4-Q and E2-2,3-Q, the compounds were reacted individually with DNA for 0.5−20 h at 37 °C, as well as in mixtures (3:1, 1:1, 1:3, and 5:95) for 10 h at 37 °C. Depurinating and stable adducts were analyzed. In similar ...

  • spectral characterization of Catechol Estrogen quinone ceq derived dna adducts and their identification in human breast tissue extract
    Chemical Research in Toxicology, 2003
    Co-Authors: Yuri Markushin, Ercole L Cavalieri, Gerald J. Small, Wenwan Zhong, Eleanor G Rogan, Edward S Yeung, Ryszard Jankowiak
    Abstract:

    Estrogens, including the natural hormones estrone (E(1)) and estradiol (E(2)), are thought to be involved in tumor induction. Catechol Estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE(1)) and 4-hydroxyestradiol (4-OHE(2)) react with DNA and form depurinating N7Gua and N3Ade adducts that might be responsible for tumor initiation (Cavalieri, E. L., et al. (2000) J. Natl. Cancer Inst. Monogr. 27, 75). Current detection limits for the CEQ-derived DNA adducts by high-performance liquid chromatography with multichannel electrochemical detection are in the picomole range. To improve the limit of detection (LOD) for CEQ-derived DNA adducts, spectrophotometric monitoring was investigated. Spectroscopic studies of 4-OHE(1)-1-N3Ade, 4-OHE(1)-1-N7Gua, 4-OHE(2)-1-N3Ade, and 4-OHE(2)-1-N7Gua adduct standards were performed at 77 and 300 K. Upon laser excitation at 257 nm, the 4-OHE(1)- and 4-OHE(2)-derived N7Gua and N3Ade adducts are strongly phosphorescent at T = 77 K. No phosphorescence was observed at 300 K. Both N3Ade and N7Gua adduct types have weak phosphorescence origin bands near 383 and 385 nm, respectively. The corresponding phosphorescence lifetimes are 1.11 +/- 0.05 and 0.37 +/- 0.05 s. The LOD, based on phosphorescence measurements, is in the low femtomole range. The concentration LOD is approximately 10(-9) M, i.e., similar to that recently obtained for CEQ-derived N-acetylcysteine conjugates (Jankowiak, R., et al. (2003) Chem. Res. Toxicol. 16, 304). The LOD in capillary electrophoresis (CE) with field-amplified sample stacking and absorbance detection is about 3 x 10(-8) M. To verify whether CEQ-derived DNA adducts are formed in humans or not, tissue extracts from two breast cancer patients were analyzed by CE interfaced with room temperature absorption and low temperature (laser-excited) phosphorescence spectroscopies. For the first time, formation of CEQ-derived DNA adducts is shown in humans. For example, the level of 4-OHE(1)-1-N3Ade in the breast tissue extract from a patient with breast carcinoma (8.40 +/- 0.05 pmol/g of tissue) is larger by a factor of about 30 than that in the breast tissue sample from a woman without breast cancer (0.25 +/- 0.05 pmol/g of tissue). In contrast, similar amounts of 4-OHE(2)-1-N3Ade were observed in both types of tissue. Although more breast tissue samples from women with and without breast cancer need to be studied, these results suggest that the N3Ade adducts could serve as biomarkers to predict the risk of breast cancer.

Thomas R. Sutter - One of the best experts on this subject based on the ideXlab platform.

  • Induction of cytochrome P450 1B1 and Catechol Estrogen metabolism in ACHN human renal adenocarcinoma cells.
    The Journal of steroid biochemistry and molecular biology, 1997
    Co-Authors: David C. Spink, Barbara C. Spink, John F Gierthy, Carrie L Hayes, Ying Li, Thomas R. Sutter
    Abstract:

    The Catechol Estrogen metabolites of 17beta-estradiol (E2), 2-hydroxyestradiol (OHE2) and 4-OHE2, differ in hormonal properties and carcinogenic potential. In Syrian hamster kidney, 4-OHE2 induces clear-cell carcinoma whereas 2-OHE2 does not, and an E2 4-hydroxylase appears to be involved in E2-induced carcinogenesis in these animals. Specific E2 4-hydroxylase activity has been observed in extrahepatic tissues from several species. In humans, cytochrome P450 1B1 (CYP1B1) appears to be an extrahepatic E2 4-hydroxylase under the regulatory control of the aromatic hydrocarbon receptor (AhR). As an initial approach to investigating CYP1B1 expression and E2 4-hydroxylase activity in human kidney, we used the ACHN cell line, derived from a human renal adenocarcinoma. In untreated ACHN cells, a very low level of CYP1B1 mRNA expression was observed and CYP1B1 protein could not be detected; however, in ACHN cells exposed to the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), CYP1B1 mRNA levels were elevated 28-fold, and the CYP1B1 protein was detected by immunoblot analysis. Exposure of ACHN cells to TCDD resulted in minimal induction of the CYP1A1 mRNA, and the CYP1A1 protein was not detectable prior to or after exposure to TCDD. E2 hydroxylase activity could not be detected with microsomes from untreated ACHN cells, although activities at C-4 and, to a lesser extent, at C-2 of E2 were observed with microsomes from TCDD-treated ACHN cells. In experiments with intact ACHN cells, elevated rates of formation of 4-methoxyestradiol (MeOE2) and 2-MeOE2 were observed in response to treatment with TCDD. The EC50 for induction of the CYP1B1 mRNA was 1.5 nM TCDD; EC50s for the stimulation of 2- and 4-MeOE2 formation were 0.68 and 1.1 nM TCDD. These results indicate that the ACHN cell line may be a useful in vitro model system to study the regulation of CYP1B1 expression and the cytotoxic effects associated with E2 4-hydroxylation.

  • induction of cytochrome p450 1b1 and Catechol Estrogen metabolism in achn human renal adenocarcinoma cells
    The Journal of Steroid Biochemistry and Molecular Biology, 1997
    Co-Authors: David C. Spink, Barbara C. Spink, John F Gierthy, Carrie L Hayes, Ying Li, Thomas R. Sutter
    Abstract:

    The Catechol Estrogen metabolites of 17β-estradiol (E2), 2-hydroxyestradiol (OHE2) and 4-OHE2, differ in hormonal properties and carcinogenic potential. In Syrian hamster kidney, 4-OHE2 induces clear-cell carcinoma whereas 2-OHE2 does not, and an E2 4-hydroxylase appears to be involved in E2-induced carcinogenesis in these animals. Specific E2 4-hydroxylase activity has been observed in extrahepatic tissues from several species. In humans, cytochrome P450 1B1 (CYP1B1) appears to be an extrahepatic E2 4-hydroxylase under the regulatory control of the aromatic hydrocarbon receptor (AhR). As an initial approach to investigating CYP1B1 expression and E2 4-hydroxylase activity in human kidney, we used the ACHN cell line, derived from a human renal adenocarcinoma. In untreated ACHN cells, a very low level of CYP1B1 mRNA expression was observed and CYP1B1 protein could not be detected; however, in ACHN cells exposed to the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), CYP1B1 mRNA levels were elevated 28-fold, and the CYP1B1 protein was detected by immunoblot analysis. Exposure of ACHN cells to TCDD resulted in minimal induction of the CYP1A1 mRNA, and the CYP1A1 protein was not detectable prior to or after exposure to TCDD. E2 hydroxylase activity could not be detected with microsomes from untreated ACHN cells, although activities at C-4 and, to a lesser extent, at C-2 of E2 were observed with microsomes from TCDD-treated ACHN cells. In experiments with intact ACHN cells, elevated rates of formation of 4-methoxyestradiol (MeOE2) and 2-MeOE2 were observed in response to treatment with TCDD. The EC50 for induction of the CYP1B1 mRNA was 1.5 nM TCDD; EC50s for the stimulation of 2- and 4-MeOE2 formation were 0.68 and 1.1 nM TCDD. These results indicate that the ACHN cell line may be a useful in vitro model system to study the regulation of CYP1B1 expression and the cytotoxic effects associated with E2 4-hydroxylation.

Ryszard Jankowiak - One of the best experts on this subject based on the ideXlab platform.

  • Integrated microfluidic device for the separation and electrochemical detection of Catechol Estrogen-derived DNA adducts
    Analytical and bioanalytical chemistry, 2010
    Co-Authors: Abdulilah Dawoud Bani-yaseen, Toshikazu Kawaguchi, Alexander K. Price, Christopher T. Culbertson, Ryszard Jankowiak
    Abstract:

    Catechol Estrogen-derived DNA adducts are formed as a result of the reaction of Catechol Estrogen metabolites (e.g., Catechol Estrogen quinones) with DNA to form depurinating adducts. Developing a new methodology for the detection of various DNA adducts is essential for medical diagnostics, and to this end, we demonstrate the applicability of on-chip capillary electrophoresis with an integrated electrochemical system for the separation and amperometric detection of various Catechol Estrogen-derived DNA adducts. A hybrid PDMS/glass microchip with in-channel amperometric detection interfaced with in situ palladium decoupler is utilized and presented. The influence of buffer additives along with the effect of the separation voltage on the resolving power of the microchip is discussed. Calibration plots were constructed in the range 0.4-10 μM with r(2) ≥ 0.999, and detection limits in the attomole range are reported. These results suggest that on-chip analysis is applicable for analyzing various DNA adducts as potential biomarkers for future medical diagnostics.

  • Catechol Estrogen quinones as initiators of breast and other human cancers implications for biomarkers of susceptibility and cancer prevention
    Biochimica et Biophysica Acta, 2006
    Co-Authors: Ercole L Cavalieri, Ryszard Jankowiak, Eleanor G Rogan, Dhubajyoti Chakravarti, Joseph B Guttenplan, Elizabeth Hart, James N Ingle, Paola Muti, Jose Russo, Richard J Santen
    Abstract:

    Abstract Exposure to Estrogens is associated with increased risk of breast and other types of human cancer. Estrogens are converted to metabolites, particularly the Catechol Estrogen-3,4-quinones (CE-3,4-Q), that can react with DNA to form depurinating adducts. These adducts are released from DNA to generate apurinic sites. Error-prone base excision repair of this damage may lead to the mutations that can initiate breast, prostate and other types of cancer. The reaction of CE-3,4-Q with DNA forms the depurinating adducts 4-hydroxyestrone(estradiol) [4-OHE 1 (E 2 )-1-N3Ade and 4-OHE 1 (E 2 )-1-N7Gua. These two adducts constitute more than 99% of the total DNA adducts formed. Increased levels of these quinones and their reaction with DNA occur when Estrogen metabolism is unbalanced. Such an imbalance is the result of overexpression of Estrogen activating enzymes and/or deficient expression of the deactivating (protective) enzymes. This unbalanced metabolism has been observed in breast biopsy tissue from women with breast cancer, compared to control women. Recently, the depurinating adduct 4-OHE 1 (E 2 )-1-N3Ade has been detected in the urine of prostate cancer patients, but not in urine from healthy men. Mutagenesis by CE-3,4-Q has been approached from two different perspectives: one is mutagenic activity in the lacI reporter gene in Fisher 344 rats and the other is study of the reporter Harvey- ras gene in mouse skin and rat mammary gland. A → G and G → A mutations have been observed in the mammary tissue of rats implanted with the CE-3,4-Q precursor, 4-OHE 2 . Mutations have also been observed in the Harvey- ras gene in mouse skin and rat mammary gland within 6–12 h after treatment with E 2 -3,4-Q, suggesting that these mutations arise by error-prone base excision repair of the apurinic sites generated by the depurinating adducts. Treatment of MCF-10F cells, which are Estrogen receptor-α-negative immortalized human breast epithelial cells, with E 2 , 4-OHE 2 or 2-OHE 2 induces their neoplastic transformation in vitro, even in the presence of the antiEstrogen ICI-182,780. This suggests that transformation is independent of the Estrogen receptor. The transformed cells exhibit specific mutations in several genes. Poorly differentiated adenocarcinomas develop when aggressively transformed MCF-10F cells are selected and injected into severe combined immune depressed (SCID) mice. These results represent the first in vitro/in vivo model of Estrogen-induced carcinogenesis in human breast epithelial cells. In other studies, the development of mammary tumors in Estrogen receptor-α knockout mice expressing the Wnt-1 oncogene (ERKO/Wnt-1) provides direct evidence that Estrogens may cause breast cancer through a genotoxic, non-Estrogen receptor-α-mediated mechanism. In summary, this evidence strongly indicates that Estrogens can become endogenous tumor initiators when CE-3,4-Q react with DNA to form specific depurinating adducts. Initiated cells may be promoted by a number of processes, including hormone receptor stimulated proliferation. These results lay the groundwork for assessing risk and preventing disease.

  • spectral characterization of Catechol Estrogen quinone ceq derived dna adducts and their identification in human breast tissue extract
    Chemical Research in Toxicology, 2003
    Co-Authors: Yuri Markushin, Ercole L Cavalieri, Gerald J. Small, Wenwan Zhong, Eleanor G Rogan, Edward S Yeung, Ryszard Jankowiak
    Abstract:

    Estrogens, including the natural hormones estrone (E(1)) and estradiol (E(2)), are thought to be involved in tumor induction. Catechol Estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE(1)) and 4-hydroxyestradiol (4-OHE(2)) react with DNA and form depurinating N7Gua and N3Ade adducts that might be responsible for tumor initiation (Cavalieri, E. L., et al. (2000) J. Natl. Cancer Inst. Monogr. 27, 75). Current detection limits for the CEQ-derived DNA adducts by high-performance liquid chromatography with multichannel electrochemical detection are in the picomole range. To improve the limit of detection (LOD) for CEQ-derived DNA adducts, spectrophotometric monitoring was investigated. Spectroscopic studies of 4-OHE(1)-1-N3Ade, 4-OHE(1)-1-N7Gua, 4-OHE(2)-1-N3Ade, and 4-OHE(2)-1-N7Gua adduct standards were performed at 77 and 300 K. Upon laser excitation at 257 nm, the 4-OHE(1)- and 4-OHE(2)-derived N7Gua and N3Ade adducts are strongly phosphorescent at T = 77 K. No phosphorescence was observed at 300 K. Both N3Ade and N7Gua adduct types have weak phosphorescence origin bands near 383 and 385 nm, respectively. The corresponding phosphorescence lifetimes are 1.11 +/- 0.05 and 0.37 +/- 0.05 s. The LOD, based on phosphorescence measurements, is in the low femtomole range. The concentration LOD is approximately 10(-9) M, i.e., similar to that recently obtained for CEQ-derived N-acetylcysteine conjugates (Jankowiak, R., et al. (2003) Chem. Res. Toxicol. 16, 304). The LOD in capillary electrophoresis (CE) with field-amplified sample stacking and absorbance detection is about 3 x 10(-8) M. To verify whether CEQ-derived DNA adducts are formed in humans or not, tissue extracts from two breast cancer patients were analyzed by CE interfaced with room temperature absorption and low temperature (laser-excited) phosphorescence spectroscopies. For the first time, formation of CEQ-derived DNA adducts is shown in humans. For example, the level of 4-OHE(1)-1-N3Ade in the breast tissue extract from a patient with breast carcinoma (8.40 +/- 0.05 pmol/g of tissue) is larger by a factor of about 30 than that in the breast tissue sample from a woman without breast cancer (0.25 +/- 0.05 pmol/g of tissue). In contrast, similar amounts of 4-OHE(2)-1-N3Ade were observed in both types of tissue. Although more breast tissue samples from women with and without breast cancer need to be studied, these results suggest that the N3Ade adducts could serve as biomarkers to predict the risk of breast cancer.

  • Spectroscopic characterization of the 4-hydroxy Catechol Estrogen quinones-derived GSH and N-acetylated Cys conjugates.
    Chemical research in toxicology, 2003
    Co-Authors: Ryszard Jankowiak, Ercole L Cavalieri, Yuri Markushin, Gerald J. Small
    Abstract:

    Estrogens, including the natural hormones estrone (E1) and estradiol (E2), are thought to be involved in tumor induction. Specifically, Catechol Estrogen quinones (CEQs) derived from the Catechol Estrogens 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) react with DNA and form DNA adducts (Cavalieri, E. L., et al. (1997) Proc. Natl Acad. Sci. U.S.A. 94, 10037). CEQs are also conjugated with GSH, a reaction that prevents damage to DNA, providing biomarkers of exposure to CEQs. Current detection limits for these analytes by HPLC with multichannel electrochemical detection are in the picomole range (Devanesan, P., et al. (2001) Carcinogenesis 22, 489). To improve the detection limit of CEQ-derived conjugates, spectrophotometric monitoring was investigated. Fluorescence and/or phosphorescence spectra of the 4-OHE1, 4-OHE2, Cys, N-acetylcysteine (NAcCys), 4-OHE1-2-SG, and 4-OHE2-2-SG conjugates and their decomposition products 4-OHE1-2-NAcCys and 4-OHE2-2-NAcCys were obtained at 300 and 77 K. It is s...

  • spectral characterization of fluorescently labeled Catechol Estrogen 3 4 quinone derived n7 guanine adducts and their identification in rat mammary gland tissue
    Chemical Research in Toxicology, 1998
    Co-Authors: Ryszard Jankowiak, Ercole L Cavalieri, Douglas E. Stack, Dan Zamzow, Rosa Todorovic, G J Small
    Abstract:

    The oxidation of carcinogenic 4-hydroxyCatechol Estrogens (CE) of estrone (E1) and estradiol (E2) to Catechol Estrogen 3,4-quinones (CE-3,4-Q) results in electrophilic intermediates that covalently bind to DNA to form depurinating adducts [Cavalieri et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 10937]. These DNA adducts, 4-OHE1-1-N7Gua and 4-OHE2-1-N7Gua, are nonfluorescent. To utilize laser-excited fluorescence methods, the Catechol Estrogen-derived metabolites and adducts were labeled with a fluorescent marker. The 4-OHEi-1-N7Gua adduct standards (i = 1, 2) and 4-OHEi metabolites have been derivatized with 1-pyrenesulfonyl chloride and investigated by low-temperature spectroscopy under non-line-narrowing and line-narrowing conditions. Molecular modeling studies assisted in interpretation of the fluorescence spectra; energetically favored structures of the 4-OHE2-1-N7Gua-dipyrene adduct and 4-OHE2-dipyrene metabolite reveal unique conformations which, in agreement with fluorescence data, show a significant pi-pi interaction of pyrene labels with guanine and/or the aromatic ring of Catechol Estrogen. The conformation obtained for the 4-OHE2-1-N7Gua-dipyrene adduct appears to be conducive to mixing of its pipi state with pyrene-guanine charge-transfer states, consistent with the experimentally observed strong electron-phonon coupling. Non-line-narrowed and line-narrowed spectra obtained at 77 and 4.2 K, respectively, are shown to distinguish 4-OHE2-1-N7Gua-dipyrene adducts from 4-OHE2-dipyrene metabolites. These standards have subsequently been used for the spectroscopic identification of depurinating DNA adducts formed in a tissue culture experiment where rat mammary gland tissue was treated with the Estrogen quinone E2-3,4-Q. The depurinating adduct formed is 4-OHE2-1-N7Gua.

Judy L. Bolton - One of the best experts on this subject based on the ideXlab platform.

  • Catechol Estrogen 4 hydroxyequilenin is a substrate and an inhibitor of Catechol o methyltransferase
    Chemical Research in Toxicology, 2003
    Co-Authors: Jiaqin Yao, Minsun Chang, Xuemei Liu, Xiaofeng Yang, Julie E Goodman, Hong Liu, Andrew D Mesecar, Richard B Van Breemen, James D Yager, Judy L. Bolton
    Abstract:

    Redox and/or electrophilic metabolites formed during Estrogen metabolism may play a role in Estrogen carcinogenesis. 4-Hydroxyequilenin (4-OHEN) is the major phase I Catechol metabolite of the equine Estrogens equilenin and equilin, which are components of the most widely prescribed Estrogen replacement formulation, Premarin. Previously, we have found that 4-OHEN rapidly autoxidized to an o-quinone in vitro and caused toxic effects such as the inactivation of human detoxification enzymes. 4-OHEN has also been shown to be a substrate for Catechol-O-methyltransferase (COMT) in human breast cancer cells. In the present study, we demonstrated that 4-OHEN was not only a substrate of recombinant human soluble COMT in vitro with a K(m) of 2.4 microM and k(cat) of 6.0 min(-)(1) but it also inhibited its own methylation by COMT at higher concentrations in the presence of the reducing agent dithiothreitol. In addition, 4-OHEN was found to be an irreversible inhibitor of COMT-catalyzed methylation of the endogenous Catechol Estrogen 4-hydroxyestradiol with a K(i) of 26.0 microM and a k(2) of 1.62 x 10(-)(2) s(-)(1). 4-OHEN in vitro not only caused the formation of intermolecular disulfide bonds as demonstrated by gel electrophoresis, but electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry also showed that 4-OHEN alkylated multiple residues of COMT. Peptide mapping experiments further indicated that Cys33 in recombinant human soluble COMT was the residue most likely modified by 4-OHEN in vitro. These data suggest that inhibition of COMT methylation by 4-OHEN might reduce endogenous Catechol Estrogen clearance in vivo and further enhance toxicity.

  • inhibition of cellular enzymes by equine Catechol Estrogens in human breast cancer cells specificity for glutathione s transferase p1 1
    Chemical Research in Toxicology, 2002
    Co-Authors: Jiaqin Yao, Emily Pisha, Minsun Chang, Xuemei Liu, Dan Yao, Ebrahim C Elguindi, Sylvie Y Blond, Judy L. Bolton
    Abstract:

    Glutathione S-transferases (GSTs) are a family of detoxification isozymes that protect cells by conjugating GSH to a variety of toxic compounds, and they may also play a role in the regulation of both cellular proliferation and apoptosis. We have previously shown that human GST P1-1, which is the most widely distributed extrahepatic isozyme, could be inactivated by the Catechol Estrogen metabolite 4-hydroxyequilenin (4-OHEN) in vitro [Chang, M., Shin, Y. G., van Breemen, R. B., Blond, S. Y., and Bolton, J. L. (2001) Biochemistry 40, 4811-4820]. In the present study, we found that 4-OHEN and another Catechol Estrogen, 4,17beta-hydroxyequilenin (4,17beta-OHEN), significantly decreased GSH levels and the activity of GST within minutes in both Estrogen receptor (ER) negative (MDA-MB-231) and ER positive (S30) human breast cancer cells. In addition, 4-OHEN caused significant decreases in GST activity in nontransformed human breast epithelial cells (MCF-10A) but not in the human hepatoma HepG2 cells, which lack GST P1-1. We also showed that GSH partially protected the inactivation of GST P1-1 by 4-OHEN in vitro, and depletion of cellular GSH enhanced the 4-OHEN-induced inhibition of GST activity. In addition, 4-OHEN GSH conjugates contributed about 27% of the inactivation of GST P1-1 by 4-OEHN in vitro. Our in vitro kinetic inhibition experiments with 4-OHEN showed that GST P1-1 had a lower K(i) value (20.8 microM) compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 52.4 microM), P450 reductase (PR, 77.4 microM), pyruvate kinase (PK, 159 microM), glutathione reductase (GR, 230 microM), superoxide dismutase (SOD, 448 microM), catalase (562 microM), GST M1-1 (620 microM), thioredoxin reductase (TR, 694 microM), and glutathione peroxidase (GPX, 1410 microM). In contrast to the significant inhibition of total GST activity in these human breast cancer cells, 4-OHEN only slightly inhibited the cellular GAPDH activity, and other cellular enzymes including PR, PK, GR, SOD, catalase, TR, and GPX were resistant to 4-OHEN-induced inhibition. These data suggest that GST P1-1 may be a preferred protein target for equine Catechol Estrogens in vivo.

  • p-Quinone methides are the major decomposition products of Catechol Estrogen o-quinones.
    Carcinogenesis, 1996
    Co-Authors: Judy L. Bolton, Li Shen
    Abstract:

    The mechanism of Catechol Estrogen-induced carcinogenesis could involve alkylation of critical cellular macromolecules by electrophilic quinoids. The o-quinones formed from peroxidase/P450-catalyzed oxidation of Catechol Estrogens have previously been implicated as the ultimate carcinogens. In the present study, we have shown that additional reactive intermediates can be produced from isomerization of the Catechol Estrogen o-quinones to highly electrophilic p-quinone methides (QMs). The o-quinones of the Catechol Estrogens were incubated at 37 degrees C (pH 7.4) in the absence of GSH. Aliquots were removed at various times and combined with GSH. The GSH adducts were isolated and characterized by 1H-NMR, UV, and electrospray mass spectrometry. The o-quinone of 2-hydroxyestrone isomerized to two QMs; a QM stabilized by one alkyl substituent in the B ring, 2-OHE-QM1 (3-hydroxy-1-(10),3(4),5(6)-oestratrien-2,17-dione) and one having two alkyl substituents on the methylene group in the C ring, 2-OHE-QM2 (2-hydroxy-1(2),4(5),9(10)-oestratrien-3,17-dione). Only one QM was observed from the o-quinone of 4-hydroxyestrone, 4-OHE-QM2 (4-hydroxy-1(2),4(5),9(10)- oestratrien-3,17-dione) which is analogous to the C ring analog (2-OHE-QM2) from the o-quinone of 2-hydroxyestrone. The GSH adduct of 4-OHE-QM2 decomposed at pH 7.4 to give 9(11)-dehydro-4-hydroxyestrone as the major product. Finally, the disappearance of the Estrogen o-quinone GSH adducts correlated with the formation of the GSH conjugates of the QMs. These data suggest that in cells with low levels of GSH, the formation of these potent electrophiles represents the major reaction pathway for Estrogen o-quinones. The implications of the o-quinone/QM pathway for the in vivo effects of Catechol Estrogens are not known; however, given the direct link between excessive exposure to endogenous Estrogens and the enhanced risk of breast cancer, the potential for formation of additional reactive intermediates needs to be explored.