Deodutta Roy

Free radical generation by redox cycling of estrogens

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

BIOCHEMICAL AND MOLECULAR CHANGES AT THE CELLULAR LEVEL IN RESPONSE TO EXPOSURE TO ENVIRONMENTAL ESTROGEN-LIKE CHEMICALS

Estrogen-like chemicals are unique compared to nonestrogenic xenobiotics, because in addition to their chemical properties, the estrogenic property of these compounds allows them to act like sex hormones. Whether weak or strong, the estrogenic response of a chemical, if not overcome, will add extra estrogenic burden to the system. At elevated doses, natural estrogens and environmental estrogen-like chemicals are known to produce adverse effects. The source of extra or elevated concentration of estrogen could be either endogenous or exogenous. The potential of exposure for humans and animals to environmental estrogen-like chemicals is high. Only a limited number of estrogen-like compounds, such as diethylstilbestrol (DES), bisphenol A, nonylphenol, polychlorinated biphenyls (PCBs), and dichlorodiphenyltrichloroethane (DDT), have been used to assess the biochemical and molecular changes at the cellular level. Among them, DES is the most extensively studied estrogen-like chemical, and therefore this article is focused mainly on DES-related observations. In addition to estrogenic effects, environmental estrogen-like chemicals produce multiple and multitype genetic and/or nongenetic hits. Exposure of Syrian hamsters to stilbene estrogen (DES) produces several changes in the nuclei of target organ for carcinogenesis (kidney): (1) Products of nuclear redox reactions of DES modify transcription regulating proteins and DNA; (2) transcription is inhibited; (3) tyrosine phosphorylation of nuclear proteins, including RNA polymerase II, p53, and nuclear insulin-like growth factor-1 receptor, is altered; and (4) DNA repair gene DNA polymerase beta transcripts are decreased and mutated. Exposure of Noble rats to DES also produces several changes in the mammary gland: proliferative activity is drastically altered; the cell cycle of mammary epithelial cells is perturbed; telomeric length is attenuated; etc. It appears that some other estrogenic compounds, such as bisphenol A and nonylphenol, may also follow a similar pattern of effects to DES, because we have recently shown that these compounds alter cell cycle kinetics, produce telomeric associations, and produce chromosomal aberrations. Like DES, bisphenol A after metabolic activation is capable of binding to DNA. However, it should be noted that a particular or multitype hit(s) will depend upon the nature of the environmental estrogen-like chemical. The role of individual attack leading to a particular change is not clear at this stage. Consequences of these multitypes of attack on the nuclei of cells could be (1) nuclear toxicity/cell death; (2) repair of all the hits and then acting as normal cells; or (3) sustaining most of the hits and acting as unstable cells. Proliferation of the last type of cell is expected to result in transformed cells.

Estrogen, DNA damage and mutations

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

Role of APC and DNA mismatch repair genes in the development of colorectal cancers

Colorectal cancer is the third most common cause of cancer-related death in both men and women in the western hemisphere. According to the American Cancer Society, an estimated 105,500 new cases of colon cancer with 57,100 deaths will occur in the U.S. in 2003, accounting for about 10% of cancer deaths. Among the colon cancer patients, hereditary risk contributes approximately 20%. The main inherited colorectal cancers are the familial adenomatous polyposis (FAP) and the hereditary nonpolyposis colorectal cancers (HNPCC). The FAP and HNPCC are caused due to mutations in the adenomatous polyposis coli (APC) and DNA mismatch repair (MMR) genes. The focus of this review is to summarize the functions of APC and MMR gene products in the development of colorectal cancers.

Profound effects of the weak environmental estrogen-like chemical bisphenol A on the growth of the mammary gland of Noble rats.

In the present study we have examined the effects of the environmental estrogenic chemical bisphenol A on proliferative activity, cell cycle kinetics and differentiation of the mammary gland of female Noble rats. Differentiation measured by the degree of lobular maturation revealed that the conversion of immature structures to mature structures was significantly increased in response to exposure to both low (0.1 mg/kg/day) and high (54 mg/kg/day) doses of bisphenol A compared to controls. The proliferative activity of epithelial cells was increased by 143% over controls by the exposure of animals to the low dose of bisphenol A, whereas a 220% increase over controls was observed for the high dose of bisphenol A. The labelling index and growth fraction were 19% and 27%, respectively, for a low dose of bisphenol A; and 27% and 45%, respectively, for a high dose of bisphenol A, compared to 18% and 31%, respectively, in controls. A significant increase in the conversion of mammary epithelial cells from G0, to G1, and S-phase cells by 1.8 and 4.5-fold, respectively, was observed in animals exposed to the high dose of bisphenol A compared to that of controls. Based on the previously reported estrogenic activity of an equivalent dose of bisphenol A to that of diethylstilbestrol (DES) (0.1 mg/kg/day), a calculated theoretical dose of the order of 10(6)-fold higher of bisphenol A will be required to produce the same biological effects as DES. A comparison of the proliferative activity of DES and that of an equivalent dose of bisphenol A observed in this study, however, revealed that its influence on proliferative activity in the epithelial cells of the mammary gland was profound. The weak estrogenic activity of bisphenol A does not explain its profound effect on cell proliferation observed in this study. Perturbation of the cell cycle is considered a risk factor for the development of cancer. Bisphenol-mediated perturbation of the cell cycle in epithelial cells may produce adverse effects in the mammary glands of Noble rats.

Estrogen-Induced Generation of Reactive Oxygen and Nitrogen Species, Gene Damage, and Estrogen-Dependent Cancers

In addition to the direct effect of estrogen on mitochondria and the redox cycling of catechol estrogen, estrogen-induced proinflammatory cytokines, such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), also generate reactive oxygen and nitrogen species (RO/NS). Different cellular signaling pathways may operate in response to varying levels of estrogen-induced RO/NS, leading to genotoxic damage, cell apoptosis, or cell growth. At high levels of RO/NS, cells receiving genotoxic insults, if not repaired, may engage the apoptotic pathways. There is increasing evidence supporting that estrogen-induced alterations in the genome of cells is produced by oxidative attack. Furthermore, ROS generated by estrogen exposure and/or active metabolites of estrogen in combination with receptor-mediated proliferation of genetically damaged cells may be involved in tumor development. This view is supported by the findings of DNA modifications produced in vitro or in vivo by natural and synthetic estrogens in the target organs of cancer both in experimental models and in humans. Interaction of estrogen-induced oxidants and estrogen metabolites with DNA was shown to generate mutations in genes. Cotreatment with an inhibitor of IL-1β and TNF-α synthesis, pentoxifylline, decreased stilbene estrogen-induced levels of myeloperoxidase (MPO), 8-hydroxydeoxyguanosine formation, and gene mutations, and prevented stilbene estrogen-induced lesions. Stable MCF-7 clones overexpressing IL-1β resulted in a high level of IL-1β peptide secretion undergoing cell apoptosis, and an elevated level of p53 protein in response to high oxidative stress when compared to nontransfected cells, whereas MCF-7 clones overexpressing IL-1β that resulted in a moderate level of IL-1β secretion stimulated the clonal expansion of MCF-7 and TM3 cells. Estrogen-induced MCF-7 cell growth and cyclin D1 expression were suppressed by antioxidants and mitochondrial blockers. These studies support that in addition to ovarian estrogen-mediated ER signaling, mitogenic signals may also come from estrogen-induced RO/NS. Further validation of this concept that the concentration of the RO/NS within the cellular microenvironment determines its stimulatory or inhibitory growth signals as well as its genotoxic effects regulating the growth of estrogen-dependent tumors may result in novel preventive strategies.

Curcumin inhibits TPA induced expression of c-fos, c-jun and c-myc proto-oncogenes messenger RNAs in mouse skin

Curcumin is a major chemical constituent of turmeric normally eaten by humans. 12-O-Tetradecanoyl phorbol-13-acetate (TPA) is a strong promoter of chemically induced skin cancer. The exact mechanism by which TPA promotes skin cancer is not clear. However, it is known that TPA elevates the expression of oncogenes involved in cell proliferation. Recently, it has been shown that turmeric or curcumin significantly inhibits TPA-induced tumor promotion on mouse skin. However, the mechanism by which curcumin inhibits TPA-induced tumor promotion is not known. In the present studies, we investigated the effect of curcumin on the expression of c-fos, c-jun and c-myc oncogenes in TPA-treated mouse skin in CD-1 mice. A 30-nmol dose of TPA increased the levels of mRNAs for c-fos, c-jun and c-myc oncogenes by 2-3-fold compared with control. Topical application on the dorsal side of the skin with 1 mumol, 10 mumol, 20 mumol or 30 mumol of curcumin 30 min before TPA treatment inhibited the TPA-induced expression of these proto-oncogenes. Inhibition of expression of c-fos and c-jun was more pronounced than that of c-myc. A dose of 10 mumol of curcumin was found to inhibit 90% TPA-induced expression of c-fos and c-jun, and 60% of c-myc. These data strongly suggest that curcumin may inhibit skin cancer through the modulation of expression of these proto-oncogenes.

Estrogen-induced reactive oxygen species-mediated signalings contribute to breast cancer

Elevated lifetime estrogen exposure is a major risk factor for breast cancer. Recent advances in the understanding of breast carcinogenesis clearly indicate that induction of estrogen receptor (ER) mediated signaling is not sufficient for the development of breast cancer. The underlying mechanisms of breast susceptibility to estrogens carcinogenic effect remain elusive. Physiologically achievable concentrations of estrogen or estrogen metabolites have been shown to generate reactive oxygen species (ROS). Recent data implicated that these ROS induced DNA synthesis, increased phosphorylation of kinases, and activated transcription factors, e.g., AP-1, NRF1, E2F, NF-kB and CREB of non-genomic pathways which are responsive to both oxidants and estrogen. Estrogen-induced ROS by increasing genomic instability and by transducing signal through influencing redox sensitive transcription factors play important role (s) in cell transformation, cell cycle, migration and invasion of the breast cancer. The present review discusses emerging data in support of the role of estrogen induced ROS-mediated signaling pathways which may contribute in the development of breast cancer. It is envisioned that estrogen induced ROS mediated signaling is a key complementary mechanism that drives the carcinogenesis process. ROS mediated signaling however occurs in the context of other estrogen induced processes such as ER-mediated signaling and estrogen reactive metabolite-associated genotoxicity. Importantly, estrogen-induced ROS can function as independent reversible modifiers of phosphatases and activate kinases to trigger the transcription factors of downstream target genes which participate in cancer progression.

Estrogen, mitochondria, and growth of cancer and non-cancer cells

In this review, we discuss estrogen actions on mitochondrial function and the possible implications on cell growth. Mitochondria are important targets of estrogen action. Therefore, an in-depth analysis of interaction between estrogen and mitochondria; and mitochondrial signaling to nucleus are pertinent to the development of new therapy strategies for the treatment of estrogen-dependent diseases related to mitochondrial disorders, including cancer.

Estrogen-induced G1/S transition of G0-arrested estrogen-dependent breast cancer cells is regulated by mitochondrial oxidant signaling.

We previously reported that 17-β-estradiol (E2)-induced mitochondrial reactive oxygen species (mtROS) act as signaling molecules. The purpose of this study was to investigate the effects of E2-induced mtROS on cell cycle progression. E2-induced cell growth was reduced by antioxidants N-acetyl-L-cysteine (NAC), catalase, and the glutathione peroxidase mimic ebselen. Flow cytometry showed that mitochondrial blockers of protein synthesis (chloramphenicol), transcription and replication (ethidium bromide), and function (rotenone, rhodamine 6G) blocked E2-induced G1 to S transition. Reduction of E2-induced DNA synthesis in the presence of mitochondrial blockers occurred without influencing the level of ATP. Additionally, the mitochondrial blockers inhibited the E2-induced expression of early cell cycle genes such as cyclins D1, D3, E1, E2, and B2. NAC or rotenone reduced E2-induced cyclin D1 expression. Furthermore, E2-induced binding of AP-1 and CREB to the TRE and CRE response sequences, respectively, in the promoter of cyclin D1 was inhibited by NAC or rotenone. In addition, E2-induced expression of PCNA, PRC1, and bcl-2 were inhibited by mitochondrial blockers. These data indicate that E2-induced mtROS are involved in the regulation of early G1-phase progression. Since neither antioxidants nor mitochondrial blockers used in this study are reported to bind the estrogen receptor (ER), our findings suggest that E2-induced mtROS modulates G1 to S transition and some of the early G1 genes through a nongenomic, ER-independent signaling pathway. Thus, our results suggest (1) a new paradigm that estrogen-induced mitochondrial oxidants control the early stage of cell cycle progression and (2) provide the basis for the discovery of novel antioxidant-based drugs or antioxidant gene therapies for the prevention and treatment of estrogen-dependent breast cancer.