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Estrogen-Induced Generation of Reactive Oxygenand Nitrogen Species, Gene Damage, and Estrogen-Dependent CancersDeodutta Roy a , Qiuyin Cai b , Quentin Felty a & Satya Narayan ca Department of Environmental and Occupational Health, Florida International University,Miami, Florida, USAb Department of Medicine and Vanderbilt–Ingram Cancer Center, Vanderbilt University,Nashville, Tennessee, USAc Department of Anatomy and Cell Biology and UF Shands Cancer Center, University ofFlorida, College of Medicine, Gainesville, Florida, USAVersion of record first published: 09 Jul 2007.

To cite this article: Deodutta Roy , Qiuyin Cai , Quentin Felty & Satya Narayan (2007): Estrogen-Induced Generationof Reactive Oxygen and Nitrogen Species, Gene Damage, and Estrogen-Dependent Cancers, Journal of Toxicology andEnvironmental Health, Part B: Critical Reviews, 10:4, 235-257

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Journal of Toxicology and Environmental Health, Part B, 10:235–257, 2007Copyright © Taylor & Francis Group, LLCISSN: 1093-7404 print / 1521-6950 onlineDOI: 10.1080/15287390600974924

ESTROGEN-INDUCED GENERATION OF REACTIVE OXYGEN AND NITROGEN SPECIES, GENE DAMAGE, AND ESTROGEN-DEPENDENT CANCERS

Deodutta Roy1, Qiuyin Cai2, Quentin Felty1, Satya Narayan3

1Department of Environmental and Occupational Health, Florida International University, Miami, Florida, 2Department of Medicine and Vanderbilt–Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee, 3Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, College of Medicine, Gainesville, Florida, USA

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-1b) and tumor necrosis factor alpha (TNF-a), alsogenerate reactive oxygen and nitrogen species (RO/NS). Different cellular signaling pathways may operate inresponse 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. Thereis increasing evidence supporting that estrogen-induced alterations in the genome of cells is produced by oxidativeattack. Furthermore, ROS generated by estrogen exposure and/or active metabolites of estrogen in combination withreceptor-mediated proliferation of genetically damaged cells may be involved in tumor development. This view issupported by the findings of DNA modifications produced in vitro or in vivo by natural and synthetic estrogens inthe target organs of cancer both in experimental models and in humans. Interaction of estrogen-induced oxidantsand estrogen metabolites with DNA was shown to generate mutations in genes. Cotreatment with an inhibitor of IL-1b and TNF-a synthesis, pentoxifylline, decreased stilbene estrogen-induced levels of myeloperoxidase (MPO),8-hydroxydeoxyguanosine formation, and gene mutations, and prevented stilbene estrogen-induced lesions. StableMCF-7 clones overexpressing IL-1b resulted in a high level of IL-1b peptide secretion undergoing cell apoptosis, andan elevated level of p53 protein in response to high oxidative stress when compared to nontransfected cells,whereas MCF-7 clones overexpressing IL-1b that resulted in a moderate level of IL-1b secretion stimulated the clonalexpansion of MCF-7 and TM3 cells. Estrogen-induced MCF-7 cell growth and cyclin D1 expression were suppressedby antioxidants and mitochondrial blockers. These studies support that in addition to ovarian estrogen-mediated ERsignaling, mitogenic signals may also come from estrogen-induced RO/NS. Further validation of this concept that theconcentration of the RO/NS within the cellular microenvironment determines its stimulatory or inhibitory growthsignals as well as its genotoxic effects regulating the growth of estrogen-dependent tumors may result in novelpreventive strategies.

Physiological concentrations of estrogens are essential for growth of hormone responsiveorgans, estrogen receptor-mediated cell signaling, and several other biochemical and molecularactivities. Supraphysiological or pharmacological concentrations of both natural and synthetic estro-gens are known to produce adverse effects, such as immunotoxicty, teratogenicity, and carcinoge-nicity (Roy & Cai, 2002). Neoplasia of hormone-responsive tissues currently accounts for >35% ofall newly diagnosed cancers in men and >40% of all newly diagnosed cancers in women in theUnited States (Henderson & Feigelson, 2000). Over the years, stilbene estrogen, diethylstilbestrol(DES), and 17β-estradiol (E2) have been shown to induce mammary, bladder, ovarian, testicular,lymphatic, uterine, and prostatic tumors in mice and rats; ovarian and mammary tumors in dogs;endometrial carcinomas in rabbits; and kidney, testicular, and uterine tumors in hamsters (IARC,1979). Elevated level of estrogen is a risk factor for cancer in hormone-dependent organs, particu-larly breast, endometrium, ovary, prostate, and testis (Roy & Singh, 2004). In 1999, the Interna-tional Agency for Research on Cancer (IARC) categorized combined oral contraceptives consistingof the steroid hormone estrogen in combination with a progestogen and postmenopausal estrogentherapy as human carcinogens (IARC, 1999). In 2002, the U.S. National Toxicology Program (NTP)listed steroidal estrogens used in estrogen replacement therapy and oral contraceptives as human

This work was supported by NIH grant ES10851 to D. R. Y.Address correspondence to Deodutta Roy, PhD, Department of Environmental and Occupational Health, Robert Stempel School of

Public Health, Florida International University, 11200 SW 8th Street, HLS 591, Miami, FL 33199, USA. E-mail: Droy@fiu.edu

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carcinogens (NTP, 2002; NCI, 2003). A recent Women’s Health Initiative study of estrogen aloneand estrogen plus progestin showed an increased risk of breast cancer among women taking thesehormones (NCI, 2003). The neoplastic transformation of human breast epithelial cells by E2 clearlysuggests the role of estrogen in the initiation of breast cancer (Russo et al., 2002). The exact mecha-nisms of initiation and progression of estrogen-related cancers are not clear.

The relationship between endocrinologic and immunologic phenomena has long been of inter-est. Immune responses differ between males and females. These observations are true in many spe-cies, including humans, and apply to both humoral and cellular responses (Olsen & Kovacs, 1996).Sex hormones play an important role in immunomodulation. Estrogens are potent stimulators of thereticuloendothelial system and certain subsets of macrophages in adult animals, and DES and E2suppress the specific activation of T cells in a reversible manner (Kalland, 1982). The immunomod-ulatory effects of DES and E2 are highly cell type, tissue, and species specific. Many of the immuno-modulatory actions of estrogen are mediated through the local synthesis of cytokines andchemokines. For example, recruitment of macrophages into the uterus at proestrous is largely underthe regulation of E2-induced uterine epithelial synthesis of the mononuclear phagocytic growth fac-tor colony-stimulating factor-1 (Miller & Hunt, 1996). Similarly, eosinophil recruitment appears tobe due to the E2-induced expression of eotaxin, an eosinophil chemoattractant (Lee et al., 1989).The uterine synthesis of other immune cytokines (lymphokines) also appears to be under the regu-lation of E2. Estrogen responsiveness is a complex genetic trait, and the loci associated with this traitare only now becoming amenable to gene mapping techniques. For example, recently, Est1, whichregulates E2-induced eosinophilic recruitment in Fischer rats, was localized on rat chromosome 5(Ropper et al., 1999). Mapping of quantitative trait loci (QTL) for estrogen responsiveness hashelped to identify QTL responsible for estrogen responsiveness in the pituitary gland of the rat, butnot those responsible for estrogen responsiveness in the uterus (Wendell & Gorski, 1997). Ourrecent data implicated inflammatory responses in the etiopathogenesis of pubertal uterine and tes-ticular lesions, including cancer, induced by perinatal exposure to estrogen (Roy, 2000, reviewed inRoy & Cai, 2002). It was also shown that estrogen-induced growth stimulation of macrophage cellsand MCF7 cells occurs in part through reactive oxygen species (ROS) (Sharga et al., 2003; Venkatet al., 2003; Felty & Roy, 2004; Felty et al., 2005a). Together, these studies indicate that reactiveoxygen/nitrogen species (RO/NS) can produce genotoxic effects as well as support the growth ofestrogen-dependent tumors.

It appears that estrogen exposure produces genetic alterations by several mechanisms (Roy &Singh, 2004). In addition to estrogen receptor (ER)-mediated signaling, E2 may support the growthof tumor cells by alternative pathways. In the present study, it was proposed to focus our efforts onreviewing the highly novel concept that estrogen exposure through directly acting on the mitochon-dria, immunoactivation leading to the production of proinflammatory cytokines, and/or metabolicredox cycling is involved in the induction of instability in the genome. It was envisioned that this isan important mechanism that drives the carcinogenesis process, but that it occurs in the context ofother processes such as ER-mediated signaling and estrogen-reactive metabolite-associated geno-toxicity that may also contribute to the process. First, estrogen’s ability to produce RO/NS andredox cycling of hydroxylated estrogen was reviewed. Then the roles of mitochondria and immuno-activation in (1) generation of oxidants, (2) induction of instability in the genome through oxidativeattack of purine and pyrimidine bases, and (3) emerging data concentrating on the ability of estrogen-induced RO/NS to produce instability in the genome of target organs of cancer are discussed.

ESTROGEN METABOLISM AND COVALENT BINDING OF ESTROGEN-REACTIVE METABOLITES TO DNA

Cells are protected from estrogen-mediated mitogenicity and genotoxicity through conjugationof parent estrogens to sulfate and glucuronide moieties. Studies have shown that endogenous andsynthetic estrogen, that is, estrone (E1), 17β-estradiol (E2), estriol (E3), 17α-ethinyl estradiol (EE2),and equilenin (Eq), through aromatic hydroxylation by specific cytochrome P-450 isoforms/peroxidaseenzymes are converted into catechol estrogens, that is, 2-hydroxyestrogens (2-OH-E1/E2/E3/EE2)

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and 4-hydroxyestrogens (4-OH-E1/E2/E3/EE2/Eq (Roy & Singh, 2004; Sarabia et al., 1997; Lee et al.,2003; Zhang et al., 1999; Hanna et al., 2000). The catechol estrogens are inactivated by methyla-tion catalyzed by catechol methyltransferases as well as by glucuronidation and sulfation catalyzedby glucuronosyltransferases and sulfotransferases, respectively (Raftogianis et al., 2000). As shown inFigure 1, catalytic oxidation of catecholestrogens, 2-OH-E1/2/3 and 4-OH-E1/2/3/EE2/Eq, gives riseto corresponding estrogen 2,3-quinone (E-2,3-Q) and estrogen 3,4-quinone (E-3,4-Q) (Roy et al.,1992; Zhang et al., 1999). Estrogen quinones are conjugated with glutathione both in vivo and invitro (Chang et al., 1998; Devanesan et al., 2001), and this conjugation is catalyzed by glutathionetransferases (Chen & Roy, 1993).

The genes encoding the enzymes responsible for the formation and metabolism of androgensand estrogens are expressed in a large number of peripheral tissues. There has been a recent resur-gence of interest in studying the association between polymorphism in estrogen metabolism genes(i.e., COMT, CYP1A1, CYP1B1, CYP19, GST, and SULT) that may account for a proportion of enzy-matic variability and estrogen-related cancers (Hanna et al., 2000; Lavigne et al., 2001; Raftogianiset al., 2000). It has been reviewed by several investigators (Thompson & Ambrosone, 2000) and istherefore briefly discussed here. Some of the recent studies have linked polymorphic variants of theestrogen-metabolizing genes with higher risk for developing estrogen-related cancers. For example,sulfotransferase (SULT) 1A1 is involved in the inactivation of estrogens. A G → A transition at codon213 (CGC/Arg to CAC/His) of the SULT1A1 gene has been reported recently, and individualshomozygous for the His allele possess a substantially lower activity of this enzyme than those withother genotypes (Zheng et al., 2001). It was reported recently that the His allele of sulfotransferase(SULT) 1A1 may be a risk factor for breast cancer, particularly among women who had risk factorsrelated to higher endogenous estrogen exposure (Zheng et al., 2001). The allele encoding low activ-ity catechol O-methyltransferase (COMT) may be a risk factor for the postmenopausal developmentof breast cancer in certain women (Lavigne et al., 2001). Analysis of the methylation status andexpression of two COMT isoforms, membrane-bound COMT (MB-COMT) and soluble COMT(S-COMT), suggests that MB-COMT is inactivated and methylated, although S-COMT is activatedand unmethylated in all endometrial cancer cell lines. Methylation of multiple promoters of theCOMT gene may selectively inactivate MB-COMT and contribute to endometrial carcinogenesis(Sasaki et al., 2003). The mean level of 8-OHdG in DNA of breast cancer tissues was shown to behigher among patients with genotype of high catechol O-methyltransferase (COMT) activity or lowglutathione S-transferase (GST)P1 activity (Matsui et al., 2000). There are also a large number ofreports that do not show any association between genetic variants of these estrogen metabolizinggenes and the risk of estrogen-dependent cancer. In men 40–50% of androgen synthesis and in

FIGURE 1. Redox cycling of estrogens generating mutagenic lesions. DES, diethylstilbestrol; CE, catechol estrogen; SQ, semiquinone; Q,quinine; Q-SG, quinone–glutathione conjugate.

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women 75% of estrogen synthesis before menopause and close to 100% after menopause occur inperipheral target tissues from precursor steroids of adrenal origin (Labrie, 1991). The extent towhich individual variability in estrogen exposure to the target tissues may be explained by allelicvariation and associated risk of estrogen-related cancer is not yet clearly understood. The concen-trations of estrogens and their metabolites in the target tissue determine their carcinogenicity.Therefore, allelic variations in estrogen metabolizing genes alone may not reflect the level of estrogenexposure to the target tissues. Interactions between other endogenous and exogenous factors-mediatedsignaling pathways that impact parent hormones and their metabolites along activation and conju-gation pathways are not clear. Unless case-control or other epidemiologic comparison studies ofallelic variation take into account these interactions, genetic variants of estrogen metabolizing genesmay not shed significant light in an understanding of their risk in estrogen-related cancer.

The oxidation products of catechol estrogens, the major metabolites of steroidal estrogens, bindcovalently to bases of DNA and to nucleophilic sites of the proteins to form adducts, whereas theparent hormones estrone and estradiol are not able to form covalent bonds with nucleotide basesand amino acids (Figure 1; Roy & Liehr, 1999). These adducts may be stable DNA adducts thatremain in DNA unless removed by repair or may form depurinating adducts that are released fromDNA by destabilizetion of the glycosyl bond (Cavalieri et al., 2000). The covalent addition of estro-gens to DNA was investigated most thoroughly with the stilbene estrogen DES, followed by that of17β-estradiol, equilenin, and 17alpha-ethinylestradiol.

Considering the unstable nature of estrogen-reactive intermediates and the presence of abun-dant amounts of nucleophilic molecules capable of scavenging reactive intermediates in the mainorganelle of metabolic activation (endoplasmic reticulum) and in the cytoplasm, it seems unlikelythat estrogen genotoxic metabolites will traverse the distance from the endoplasmic reticulum tothe nucleus or mitochondrion, the sites of genotoxicity. Studies showed that nuclei are capable ofcatalyzing redox cycling of estrogens, and estrogen-reactive metabolites generated during nuclearredox cycling covalently bind to nuclear DNA along with histone and nonhistone nuclear proteins(Roy & Thomas, 1994; Roy & Pathak, 1993, 1995; Roy et al., 1997, 1998). This provides support tothe concept that in the cell the metabolic activation of estrogens into genotoxic metabolites occursin close proximity to the site of genotoxicity. In support of this concept, data demonstrated thatmitoplasts (i.e., mitochondria without outer layer of membrane) are able to convert stilbene estro-gen and 2-hydroxyestradiol to reactive metabolites, which covalently bind to mitochondrial DNA(Thomas & Roy, 2001).

DNA adducts induced by quinone and semiquinone forms of catechol estrogens are found invarious target tissues of cancer (Roy & Singh, 2004; Cavalieri et al., 2000). Liquid Chromatography(LC)–mass spectrometry (MS)–MS analysis of mammary tissue extract from rat showed theformation of an alkylated depurinating guanine adduct induced by equine estrogen metabolite 4-hydroxyequilenin (Zhang et al, 2001). Estrogen DNA adducts (catechol ethinyl E2–DNA andcatechol equilenin–DNA) were detected in human breast tumor tissues and healthy tissues by combinednano LC–nano ES tandem mass spectrometry (Embrechts et al., 2003). Recently, 4-hydroxy cate-chol estrogen conjugates with glutathione or its hydrolytic products (cysteine and N-acetylcysteine)were detected in picomole amounts in both tumors and hyperplastic mammary tissues from ERKO/Wnt-1 mice, demonstrating the formation of CE-3,4-quinones (Devanesan et al., 2001). A higherlevel of mitochondrial DNA adducts than nuclear DNA adducts was observed in the target organ ofcancer (liver) of male Sprague Dawley rats treated with stilbene estrogen (Thomas & Roy, 2001).Experimental evidence also indicate that these estrogen-induced DNA adducts are the site of muta-tions. For example, 2-OHE quinone-derived DNA adducts were found to be mutagenic generatingprimarily G → T and A → T mutations in simian kidney (COS-7) cells (Terashima et al., 2001). E(2)-3,4-Q induced the rapidly depurinating 4-hydroxy estradiol (4-OHE(2))-1-N3Ade adduct, andabundant A to G mutations in H-ras DNA were observed in SENCAR mouse skin treated with estra-diol 3,4-quinone (E(2)-3,4-Q) (Chakravarti et al, 2001). In vivo formation of stilbene estrogenquinone, covalent binding of estrogen reactive metabolites to transcriptionally active nuclear pro-teins (Roy & Pathak, 1995; Roy & Palangat, 1994), and chromosomal abnormalities (Banerjee &Roy, 1996; Banerjee et al., 1994; Roy et al., 1997) support this mechanism of estrogen-induced

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DNA damage. These studies indicate that estrogen metabolites react with DNA to form adducts,and these adducts generate mutations that may contribute to tumor initiation.

There are other reports that also clearly indicate that estrogen exposure induces mutations.Recently, it was shown that the generation of mutations were attributed to physiological concentra-tions of estrogens (Singh et al., 2005). The DES-reactive metabolite quinone increases homologousrecombination in Escherichia coli (Korah & Humayun, 1993); both DES and E2 are mutagenic in thegpt+ Chinese hamster G12 cell line (Klein, 1995); and covalent DNA adducts formed by both DESquinone and E2 quinone arrest the progression of DNA synthesis (Roy & Abul-Hajj, 1997). Muta-tions in the catalytic domain of an important DNA repair enzyme, DNA polymerase beta, and inthe microsatellite repeats were reported in estrogen-induced kidney tumors (Yan & Roy; 1995,Hodgson et al., 1998). Somatic mutation of microsatellite repeats was widespread in the genome ofclear-cell adenocarcinoma of the lower reproductive tract in young women prenatally exposed tothe synthetic estrogen diethylstilbestrol (Endo et al., 1994). Using the RAPD technique, studiesrecently identified several loci mutated in the DES-induced hamster kidney tumors. Further charac-terization of some of the mutated loci resulted in the identification of a novel gene, as well as acDNA sequence showing significant homology with CYP 1A1 gene (Singh & Roy, unpublished). Itwas recently been shown that catechol estrogens may induce aldehydic DNA lesions in calf thymusDNA (Lin et al., 2003). Equilin and equilenin are the major components of the widely prescribeddrug used for estrogen replacement therapy. These equine estrogens are metabolized primarily to4-hydroxyequilin and 4-hydroxyequilenin, respectively, which are autoxidized to react with DNA,resulting in various DNA lesions. Mutagenic events induced by 4-hydroxyequilin in supF shuttlevector plasmid propagated in human cells were identified (Yasui et al., 2003).

ESTROGEN-MEDIATED GENERATION OF RO/NS

Free radical generation by redox cycling of estrogen has already been reviewed by Roy andLiehr (1999) and therefore is discussed in brief here. Liehr and Roy (1990) were first to demonstratemicrosomal enzymes cytochromes P-450 catalyze redox cycling of stilbene estrogen and catecholestrogen and free radical generation by metabolic redox cycling between quinone and hydro-quinone forms of estrogens (Roy & Liehr, 1999). Redox cycling generated by enzymatic reductionof CE-Q to CE-SQ and subsequent autoxidation back to CE-Q by oxygen forms superoxide radicalsand hydroxy radicals (Figure 1). Recently, Seacat et al. (1997) showned that oxidation of the cate-chols of estradiol and 17α-ethinylestradiol by Cu2+ produces hydroxy radicals. Free radicals gener-ated during redox cycling of estrogens by microsomal, mitochondrial, or nuclear enzymatic systemsor nonenzymatically by Cu+ or Fe2+ are capable of damaging cellular macromolecules, includingDNA, proteins, and lipids. Estrogen-induced free radical damage to DNA such as strand breakage,8-hydroxylation of purine bases of DNA, and lipid hydroperoxide-mediated DNA modification wasreviewed by Roy and Liehr (1999) and therefore not discussed here.

Secondarily, estrogens produce macrophage proliferation and activation (Venkat et al., 2003)and this will be discussed in detail in the following section . Yoshie and Ohshima (1998) demon-strated that DNA strand breakage was induced synergistically in the presence of both a NO-releasingcompound [2-(N,N-diethylamino)-diazenolate-2-oxide.diethylammonium salt] and a CE. Estrogensalso affect the function of polymorphonuclear leukocytes (PMNs) resulting in the release of oxidants,including hypochlorite/hypochlorous acid (HOCl/OCl–) (Cavalieri et al., 2000). 2-Hydroxylatedestrogens, however, act as powerful inhibitors of PMN activity, possibly one of the protective propertiesof the 2-hydroxylated CE.

Mitochondria are significant targets for estrogen (Chen & Yager, 2004; Felty & Roy, 2004,2005; Roy et al., 2004). Recently, estrogen-induced mitochondrial oxidants playing an importantrole in carcinogenesis were reviewed (Felty & Roy 2004, 2005) and therefore are discussed here inbrief. Felty et al. (2005) reported that physiological concentrations of E2 stimulate a rapid produc-tion of intracellular reactive oxygen species (ROS), and ROS formation in epithelial cells dependson cell adhesion, the cytoskeleton, and integrins. In our studies of E2-induced ROS generation inMCF7 and other cells, it was not possible to find any hydroxylated estrogen metabolites or their

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adducts immediately after addition of E2, and this rules out the possibility of ROS generation byredox cycling of hydroxylated estrogens. These events occur earlier than ER-mediated genomicactions. E2-stimulated ROS production does not depend on the presence of the ER in breast cancercells, as the ER-negative cell line MDA-MB 468 produced ROS equal to that of ER-positive cell linesMCF7, T47D, and ZR75. ROS formation upon E2 exposure might explain oxidative damage tohormone-dependent tumors and subsequent genetic alterations (Roy & Cai, 2002; Roy & Singh,2004). E2-induced ROS generation also provides mechanistic support to the generation ofmutations by physiological concentrations of estrogens (Kong et al., 2000; Singh et al., 2005).

ESTROGEN, IMMUNOACTIVATION/INFLAMMATION, GENERATION OF OXIDANTS, AND OXIDATIVE DNA DAMAGES

Influence of Estrogens on Immune CellsGenerally, females have a more active immune response and a concomitantly higher incidence

of autoimmune diseases as compared to males (Cannon & St. Pierre, 1997). Growing evidence hasshown that female sex hormones play a major role in this heightened immune response. Gonadalhormones exert significant effects on proinflammatory cytokine IL-1 production. Plasma IL-1 bioac-tivity is low or undetectable in blood samples of men and from women during the follicular phase,whereas IL-1 bioactivity is fourfold higher in luteal phase plasma (Cannon & Danrello, 1985). Thespontaneous secretion of IL-1 bioactivity by isolated blood monocytes is 10-fold higher in the lutealphase compared to the follicular phase (Polan et al., 1990). A questionaire-based epidemiologicalstudy involving 1700 persons who were exposed to DES (520 mothers, 1079 daughters, and 94sons) indicated that respiratory-tract infections, asthma, arthritis, and lupus were reported more fre-quently among the persons with DES exposure (Wingard & Turiel, 1988). Autoimmune diseases areincreased among persons exposed to DES in utero compared with unexposed controls. The rate ofany autoimmune disease was 28.6 cases/1000 persons in the DES-exposed daughters and 16.9cases/1000 persons in the unexposed women (Noller et al., 1988). An epidemiological study com-pared self-reports of immune-related diseases in diethylstilbestrol (DES) daughters and controls,DES daughters were at higher risk of developing immune-related diseases (Vingerhoets et al., 1998).

Sex hormones, estrogen, progesterone, and androgen are known to exert their influence onboth immature and mature immune cells. Gonadal steroids modulate immune function by produc-ing effects on developmental processes as well as effects on differentiated cells. Estrogens exerteffects on thymus and bone marrow, as well as mature peripheral immune cells (Figure 1) (Olsen &Kovacs, 1996). Sex steroids act on the immune system in a variety of ways by altering function andphenotype of T and B cell, immunoglobulin levels and possibly the kinetics of Ig synthesis, orsynthesis of cytokines (Lahita, 1993).

The demonstration that neonatal DES or E2 exposure exerts strong imprinting effects on theimmune system in immunoprivileged tissues emphasizes the need for research into the possible roleof an altered immune system in the etiopathology of DES- and E2-related diseases. The possiblerole of the immune system in the development of tumors of the urogenital tract in experimentalanimals exposed perinatally has not been studied in detail. Neonatally DES-exposed female micepossessing impaired natural killer (NK) cell function showed increased susceptibility to transplanted,as well as primary carcinogen, 3-methylcholanthrene-induced tumors (Kalland, 1982). DES-treatedneonatal mice of susceptible strains also show a higher incidence of mammary tumor virus-inducedtumors correlating with lack of virus-specific cytotoxic T lymphocytes. The incidence of DMBA-induced mammary tumors in the rat was increased significantly following prenatal exposure to DES,and inhibited by neonatal DES exposure. Transplacental treatment of hamsters with DES increasedthe susceptibility to chemical carcinogens in adult life (Kalland, 1982). These studies demonstrateclearly the biologic consequences of perinatal DES exposure on tumor development. Whetherthese effects are mediated through suppression of T-helper/NK cells and/or activation of macroph-ages remains to be elucidated. Here, is discussed the influence of estrogen on the individual type ofimmune cells.

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Monocyte–macrophages Sex steroid hormones, estrogen, progesterone, and testosterone,exert profound influences on macrophage biology and function (Miller & Hunt, 1996). Macroph-ages are found throughout the female and male reproductive tracts, including the ovary and tes-tis, and are abundant within the endometrial stroma and myometrial connective tissue of thecycling uterus (Miller & Hunt, 1996). Acute exposure to DES in adult mice produces markedalterations in the cellularity of lymphoid and hemopoietic tissues and in the number of circulatingleukocytes (Boorman et al., 1980). The number of circulating mouse blood monocytes and peri-toneal macrophages are largely enhanced (Miller & Hunt, 1996; Boorman et al., 1980). Popula-tions of macrophages and neutrophils in the uterus are under the control of the female sexsteroids estrogen and progesterone (P4). Regulation of leukocytes was implicated for changes inuterine immune responses during the estrous cycle, pregnancy, and implantation (Tibbets et al.,1999). The influx of macrophages and neutrophils is induced by estrogen, while P4 may bothstimulate and inhibit leukocyte influx, depending on the timing of P4 with respect to estrogen.Estrogen treatment results in recruitment of macrophages and neutrophils into the mouse uterus.Using progesterone receptor knockout (PRKO) mice, it was shown that this effect is dependenton progesterone receptors (PR) (Tibbets et al., 1999). In humans, peripheral blood monocytecounts increase during the periods of high circulating estradiol levels, such as the ovulatoryperiod in healthy women (Maoz et al., 1985).

T cells Treatment with E2 results in relative increases in mature thymocytes (Screpanti et al.,1991). Estrogen treatment also activates the process of extrathymic T-cell maturation, especiallyin the liver (Okuyama et al., 1992). Expression of the estrogen receptor (ER) in the thymus hasbeen demonstrated. It seems that both developing thymocytes and thymic epithelial cells expressER (Olsen & Kovacs, 1996). Expression of ER in the peripheral T cells was also reported (Cohenet al., 1983). In vivo administration of estrogen alters total peripheral T-cell activity in differentmodel systems, suggesting either enhancement of helper/inducer or reduction in suppressor/cytotoxic cellular activity (Olsen & Kovacs, 1996). Physiological concentrations of estradiol blockthe negative regulatory effects of a subset of T lymphocytes that suppress immunoglobulin pro-duction by B lymphocytes in humans (Paavonen et al., 1981). Aged mice exposed subacutely toDES produce significant alteration in the thymus (Smith & Holladay, 1997). Following 5 consecu-tive days of intraperitoneal injection with 1.5 or 6 mg/kg DES, severe thymic hypocellularity wasobserved. Moreover, cell maturation within the thymus is also affected, as indicated by a signifi-cant decrease in CD4+8+ cells and a concomitant increase in CD4−8− cells (Smith & Holladay,1997). In utero, DES exposure is associated with a hyperreactive immune response during thereproductive years. Peripheral blood lymphocyte’s response to the T-cell mitogen PHA (phytohe-magglutinin) was significantly higher in cells of in utero DES-exposed women than controls (Wayset al., 1987).

B cells Sex steroids participate in steady-state regulation of B lymphopoiesis (Smithson et al.,1998; Medina et al., 2000). Although pregnancy in normal mice does not alter numbers of earlypro-B cells, all of the subpopulations developing after this stage are reduced relative to other bonemarrow cells (Medina et al., 1993, 2000). The alteration in B lymphopoiesis during pregnancy is theresult of hormonal action. The supporting evidence comes from the finding that ovariectomy innonpregnant female mice results in expansion of the numbers of bone-marrow B cells, and thesechanges are reversed by estrogen replacement (Masuzawa et al., 1994). Moreover, changes in rela-tive numbers of developing B cells in these estrogen-treated nonpregnant animals are generally par-allel to those observed in pregnant mice (Kincade et al., 1994). In general, females have betterB-cell-mediated immunity than age-matched males (Walker et al., 1999). Estrogen is a potentimmunomodulator and enhances production of antibodies by B cells (Olsen & Kovacs, 1996).Exposure of normal C57Bl/6 mice to estrogen alone, in the absence of any deliberate stimulation,promotes B-cell hyperactivity by enhancing the numbers and activity of plasma cells producing anti-bodies to both self- and non-self-antigens (Paavonen et al., 1981). Estrogen induces B-cell hyperac-tivity, including promoted autoantibodies to double-stranded DNA and phospholipids, andincreases plasma cell number and autoantibody yields per B cell (Verthelyi & Ahmed, 1998; Walkeret al., 1999; Ahmed et al., 1999).

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Activation of Immune Cells by EstrogenExposure of the developing immune system to estrogen results in persistent alterations in

immune reactivity. DES exposure was shown to interfere with the development of the NK/T lym-phocyte progenitor. The prenatal exposure in DES sons and daughters was reported to be associ-ated with a higher rate of infections and increased susceptibility to pathogens (Kalland, 1982;Kalland & Holmdahl, 1984). In hamsters, neonatal DES exposure produces extensive leukocyteinfiltration of the uterus (Hendry et al., 1997). As DES and E2 are capable of interfering withhematopoetic progenitors (Kalland, 1982), one does not rule out the contribution of immunosup-pression in pathologic development through functional impairment of the T lymphocytes/NK.Although the inflammatory early reversible responses in the uterus, ovary, cervix, kidney, prostate,and testes in response to estrogen exposure in neonates or immature rodents (rats, mice, and ham-sters) or chickens have been recognized for more than 25 yr, little information exists on either themechanisms controlling the macrophage infiltration in uterus or other estrogen-dependent tissues inresponse to estrogen or the biologic functions of these cells in these organ systems. Evidence in sup-port of estrogen-mediated immunoactivation is provided next, based on markers of activation ofsome immune cells.

Influence of Estrogens on the Indicators of Immunoactivation There are a large number ofbiomarkers of activated immune cells or inflammation; however, here, data focus on selected indi-cators of immunoactivation/inflammation that are induced in response to estrogen.

Myeloperoxidase (MPO) MPO is considered a classic marker for leukocyte activation andinflammation. Micromolar concentrations of E2, estrone, 16-alpha-hydroxyestrone, and estriolenhance the oxidative metabolism of activated human polymorphic neutrophils. These estrogensand their metabolites are considered to induce MPO release from the resting (inactivated) cells andstimulate generation of oxidants in the absence of pathogens (Jansson, 1991). Treatment of rats withE2 during postnatal days 22 to 32 resulted in a significant increase in MPO activity in the adult lat-eral prostate (Stoker et al., 1999b). Recently, it was reported that people with high estradiol levelsshow an increase in myeloperoxidase (MPO) protein in the plasma (Santanam et al., 1998). More-over, MPO intracellular activity and the amount of MPO release in women during menopauseincrease significantly after hormone replacement therapy (Beksi et al., 1999, 2001). Law et al.(1993) localized human estrogen-responsive finger protein (EFP) gene (ZNF-147) with a YAC contigcontaining the myeloperoxidase (MPO) gene. EFP expression is induced by estrogen via an estro-gen-responsive element (ERE) in the 3′-untranslated region of the gene. MPO expression may alsobe induced by estrogen. The fragments located on introns 7 and 9 of MPO gene enhance theexpression of MPO (Yamada et al., 1993). These two regions consist of a consensus half-siteGGTCA common to estrogen response elements (EREs).

Proinflammatory cytokines In some tissues, estrogens are known to suppress proinflammatorycytokine production, while in other tissue types, proinflammatory cytokine expression is enhancedby estrogen exposure (Ruh et al., 1998). Conflicting results have been published regarding the influ-ence of specific steroidal hormones on the release of proinflammatory cytokines, such as tumornecrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, in humans, because most of the studies didnot take into the account of overall effect of the phase of the menstrual cycle, when various hor-mones exert their effects simultaneously, and other confounding factors, such as use of alcohol andoral contraceptives. In a recent study using healthy premenopausal women with and without intakeof contraceptives, it was reported that the concentration of estradiol in plasma correlated with therelease of TNF-α and IL-6 during the luteal phase (Schwartz et al., 2000). The modulatory effects ofDES and E2 on proinflammatory cytokines shown in Figure 2 are highly cell type, tissue, and speciesspecific.

1. Interleukin-1 beta (IL-1β). Estrogen increases IL-1 synthesis. Ovariectomy leads to decreasedlevels of IL-1 synthesis, and this effect is reversed by estrogen replacement (Hu et al., 1988).Estradiol treatment increases the number of peritoneal macrophages and the peritoneal mac-rophages became more sensitive to LH to produce IL-1 in hamsters (Yoshida et al., 1996).

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Human peripheral monocyte IL-1 activity is modulated by physiologic levels of gonadal steroids(Polan et al., 1988). Low concentrations of both E2 (10−9 M–10−10 M) and progesterone (P, 10−8

M–10−9 M) result in maximal IL-1 stimulation. At higher concentrations of both E2 (10−7 M) and P(10−7 M−10−5 M) there is a significant reduction in IL-1 activity. Treatment of male Wistar ratswith E2 results in an upregulation of the IL-1β transcripts (Harris et al., 2000).

2. Interleukin-6 (IL-6). E2 stimulates IL-6 secretion by lipopolysaccharide (LPS)-activated humanperipheral blood mononuclear cells. However, this effect requires supraphysiological concentra-tions of hormone (10−6 M) (Li et al., 1993). Physiological levels of E2 inhibit IL-6 production inbone–marrow-derived stromal cells (Girasole et al., 1992). The estrogen agonist estriol exerts sig-nificant effects on IL-6 in response to an endotoxin challenge (Zuckman et al., 1996). Pretreat-ment of mice with pharmacological doses of estriol (0.4–2 mg/kg) results in a more rapidelevation in IL-6 levels in serum following LPS challenge compared to control. Some of the effectattributed to IL-6 is mediated through JAK2 signaling pathways (Narazaki et al., 1994). Treatmentof male Wistar rats with E2 results in a significant upregulation of the IL-6 transcripts (Harris et al.,2000).

3. TNF-alpha. E2 increases TNF-α both in the murine macrophage cell line J774 cultured inmedium alone, and when this cell line is stimulated with lipopolysaccharide (LPS) (D’Agostinoet al., 1999; Felty et al., unpublished). Estriol also exerts significant effects on TNF produced inresponse to an endotoxin challenge (Zuckman et al., 1996). Pretreatment of mice with pharma-cological doses of estriol, 0.4–2 mg/kg, resulted in a significant increase in serum TNF levels inboth control and autoimmune MRL/lpr mice, following LPS challenge. The effect on TNF wasblocked by the estrogen antagonist tamoxifen (Zuckman et al., 1996). TNF-α also stimulates aro-matase expression in adipose stromal cells in the presence of dexamethasone (Simpson et al.,1997; Zhao et al., 1996). On the other hand, progesterone decreases steady-state levels of TNF-α mRNA in LPS-activated mouse macrophages in vitro. Progesterone may achieve this resultthrough effects via an inhibitor of NF-κB (Miller & Hunt, 1998).

4. Interferon-gamma (IFNγ). Expression of the IFNγ gene may be directly controlled by estrogen. Ina transient expression assay, E2 markedly increases activity of the IFNγ promoter in lymphoidcells that express the estrogen receptor. This effect is mediated by sequences in the 5′-flankingregion of the gene, and may augment the effect of T cell-activating agents (Fox et al., 1991). E2enhances the secretion of IFNγ by CD4+T cells using neuroantigen-specific T-cell clones iso-lated from normal control subjects and patients with the demyelinating disease multiple sclerosis(Gilmore et al., 1997).

FIGURE 2. Activation of macrophage enzymatic pathways by estrogen leading to generation of oxidants.

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5. Granulocyte–macrophage colony stimulating factors (GM-CSF). The expression of GM-CSF in ratuterine stromal cells is regulated by ovarian steroids. Ovariectomy significantly reduces the appear-ance of GM-CSF-mRNA-positive cells and the levels of expression for GM-CSF mRNA in the wholerat uterus (Tamura et al., 1993). E2 and progesterone (P) cotreatment stimulates the expression forGM-CSF mRNA and the production of immunoreactive GM-CSF in the stromal tissues. Uterineepithelial cells were shown to be a potent source of the GM-CSF by in vitro studies. GM-CSF syn-thesis and/or release by uterine epithelial cells are stimulated by estrogen, whereas P exerts a mod-erate inhibitory effect (Robertson et al., 1996). Analysis of GM-CSF mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR) in uterine epithelial cell cultures and in intactuteri from steroid hormone-treated ovariectomized mice indicated that the effects of E2 and P onGM-CSF release are mediated at least in part at the transcriptional level (Robertson et al., 1996).

Inflammation During Pregnancy During pregnancy the number of human macrophages andtheir phagocytic capability increase with rising concentrations of E2 (Miller & Hunt, 1996; Baranaoet al., 1992). During pregnancy, uterine macrophages undergo further differentiation and displayan elevated expression of Ia molecules, which increase the efficiency of antigen presentation andheighten phagocytic capacity (Miller & Hunt, 1996).

Mechanisms of Estrogen-Induced Immunoactivation Macrophage-secreted cytokines, suchas IL-1β and TNF-α induce significant levels of directed infiltration of leukocytes. Leukocyte infiltra-tion in the uterus and testis may be in response to neonatal DES exposure, as observed in ourrecent studies that show an inflammation in the uterus strikingly similar to that elicited by tissueinjury or LPS (Roy, 2000). In the case of women undergoing menopause, treatment with hormonereplacement therapy results in a significant increase in the levels of intracellular MPO activity andMPO release (Bekesi et al., 1999, 2001). Our recent studies are the first to establish that neonatalexposure of hamsters to DES leads to a severalfold induction of MPO activity in the uterus and tes-tis, and this was accompanied by hyperproduction of the proinflammatory cytokines TNF-α and IL-1β in the uterus and testis in pubertal animals (Roy & Cai, 2002). These effects were not temporaryand persisted after 150 of DES exposure of neonates. Ruh et al. (1998) reported that E2 and LPS actsynergistically to activate an IL-1β promoter–CAT construct in the RAW264.7 macrophage cell line.This is an ER-mediated processes, since it is necessary to introduce the ER into the cell line in orderto stimulate the enhanced promoter activity, and the response is blocked by antiestrogenic com-pounds such as tamoxifen and ICI 182 780. Monocytes–macrophages are not simply phagocyticcells of the immune system but are also involved in important endocrine processes during develop-ment, express estrogen receptors, and convert androgen to estrogen by aromatase. Akoum et al.(2000) showed that E2 enhances endometrial cell responsiveness to the proinflammatory cytokineIL-1 β by upregulating interleukin-1-induced monocyte chemotactic protein-1 (MCP-1) expression.Leukocyte infiltration in the endometrium of neonatally DES-exposed hamsters was seen as early as9 of life and clearly is an early event (Hendry et al., 1997). Transplantation of neonatally DES-treated uteri of 7-old hamsters into the cheek pouch of an ovariectomized hamster receiving E2leading to the development of preneoplastic lesions clearly established that this effect is an inherentproperty of neonatally DES-exposed uteri and not a consequence of DES-induced changes in thehost environment (Hendry et al., 1997). It appears that an imbalance of the in utero sex hormoneenvironment critically influences the fetal and perinatal uterine environment. Exposure to IL-1β orTNF-α of human fibroblast cells releases significant levels of superoxide (5 nmol/h/106 cells) (Burdon,1997). Based on these findings, it was postulated that persistent inflammation leading to macroph-age activation is involved in the generation of oxidants, such as O2−,·OH, and H2O2. The mecha-nisms underlying the persistent hyperproduction of the proinflammatory cytokines TNF-α and IL-1βin the uterus, breast, testis, and ovary as a result of E2 exposure is not clear. ROS are considered tobe important mediators of immunoactivation/inflammation. H2O2 leads to the activation of tran-scription factors such as HSF, activation protein (AP)-1, and NF-κB. Data suggest that oxyradicalsgenerated as a result of immunoactivation in the uterus and testis of hamsters exposed perinatally toE2 may be responsible for signaling the hyperproduction of TNF-α and IL-1β through activation oftranscriptional regulators of oxidative stress inducible genes.

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Ovarian estrogen synthesis only starts to occur in the pubertal period (25 d after birth in thehamster). Immunoactivation was seen in the hamster uterus as early as 9 d after neonatal exposureto estrogen (Hendry et al., 1997). Therefore, delayed responses (i.e., the pubertal response) may bea result of imprinting of known or unknown specific transcription coactivators, co-repressors, ortranscriptional factors associated with expression of TNF-α and IL-1β genes, which appear tobecome highly sensitive to ovarian steroids. These factors may be controlled or recruited by the signaltransducer H2O2 produced by immunoactivation of macrophages and/or genotoxic metabolitesproduced by redox cycling of elevated levels of ovarian estrogens.

Estrogen and Generation of Oxidants by Activated Immune CellsInflammatory reaction results from the recruitment, to the site of inflammation, of phagocytic

cells (monocytes–macrophages, eosinophils, and neutrophils), and from their activation and theirinteractions with specific immune cells (T and B lymphocytes) in a given tissue. Reactive oxygenspecies (ROS) such as superoxide, OH, HOCl, NO, and O2 are generated by phagocytes (Figure 2).Upon stimulation, macrophages rapidly produce oxidants such as O2

•− and H2O2 in an NADPH oxi-dase-catalyzed reduction of molecular oxygen. Inducible nitric oxide synthase (iNOS) may also besynthesized by the cells and produce nitric oxide (NO). NO and O2

•− may rapidly interact to form amuch more potent oxidant, peroxynitrite (ONOO−). Much of the observed toxicity of O2

− andH2O2 has been attributed to intracellular reduction of these species to ·OH. H2O2 freely diffusesthrough cellular membrane and is a precursor of ·OH, an actual DNA-damaging species, as well asof other potentially damaging species such as hypochlorous acid (HOCl). During inflammatoryresponses, HOCl is produced by neutrophils through the MPO-catalyzed oxidation of chlorideanions by H2O2. HOCl is a potent one- and two-electron oxidant that reacts with numerous biolog-ical substrates including DNA. At physiological pH, about 50% of HOCl exists as the hypochloriteanion (OCl−) (Burcham, 1999). NADPH oxidase activity in the uterus is positively modulated by17β-Estradiol (Garcia-Duran et al., 1999). Estradiol markedly augments the superoxide radical pro-files in uterine cells compared to control. Treatment of male Wistar rats with E2 results in an upreg-ulation of iNOS transcripts in the prostate (Harris et al., 2000). 17β-estradiol stimulates neuronaliNOS expression in human neutrophils and thus increases the ability of human neutrophils toproduce NO· (Garcia-Duran et al., 1999; Duran et al., 2000). Thus, in addition to redox cycling ofcatechol estrogens, E2 also generates oxidants through activating immune cells, such as monocytes,macrophages, eosinophils, and neutrophils (Figure 2). The clinical observations have consistentlyshown that females exhibit higher immune reactivity compared to males (Cannon & St. Pierre, 1997).Experimental model studies also indicate that gender specific hormones have profound effects onimmune cell activation and their biological activity. The counts of circulatory neutrophils are approxi-mately 20% higher in women, and there is a significant correlation between the counts of neutrophilsand urinary estrogens measured during the different stages of the menstrual cycle (Bain & England,1975). Furthermore, the generation of ROS is 40% higher in cells isolated from women and varies in acyclic manner through the menstrual cycle (Mallery et al., 1986). In the final trimester of pregnancy,generation of ROS by neutrophils is reduced approximately 50% (Crouch et al, 1995).

TNF-α exposure of cells was shown to result in single-strand DNA breaks and other types ofDNA damage (Delaney et al., 1997). It was recently shown that exposure to TNF-α (20 ng/ml) ofUN37 cells induced a two- to fivefold rise in several species of oxidized bases, including 8-OH-G(Nathan et al., 2000). TNF-α was found shown to increase mitochondrial production of oxygen rad-icals. The magnitude of TNF-α induced rises in 8-OH-adenosine was comparable to the levelinduced by H2O2 and far exceeded that induced by ionizing radiation (Nathan et al., 2000). Thus,TNF-α is a potent inducer of base oxidations in DNA, and genotoxicity of TNF-α pathway impli-cates it as a potential mutagen. From these studies, it is clear that inflammation or activation ofmonocytes–macrophages/esosinophilsneutrophils through the induction of proinflammatory cytok-ines might generate oxidative damage in the genome, which, in turn, may produce mutations. Asdiscussed earlier, E2 is able to produce inflammation in the target organs and activates phagocyticimmune cells. E2 remained available and mediated a delayed induction of oxidative DNA damageand mutations in prepubertal hamster uteri and testes. Ovarian estrogen synthesis only starts to

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occur in the pubertal period (25 d after birth in the hamster). Immunoactivation was seen in thehamster uterus as early as 9 d after neonatal exposure to estrogen (Hendry et al., 1997). Therefore,delayed responses of DNA damage and mutations (i.e., the pubertal response) may be a result ofimmunological imprinting. As discussed earlier, cell exposure to IL-1β or TNF-α produces a signifi-cant level of superoxide in the mitochondria. Cell exposure to TNF-α induces formation of oxidizedbases, including 8-OH-dG. Data recently showed that a single neonatal DES treatment producedsignificant increases in hydrogen peroxide concentration when compared to control testes. Cotreat-ment with an inhibitor of synthesis of IL-1β and TNF-α, pentoxifylline, inhibited DES-inducedincreases in MPO activities, 8-OHdG formation, and mutations in the testicular genome and pre-vented the DES-induced testicular lesions. Similar effects were observed in the uterus of animalsexposed neonatally to DES. Our data support the concept that E2-induced immunoactivationresults in the generation of mutations in the testicular and uterine genome through oxidative damagesto purine and pyrimidine bases (Figure 3).

EVIDENCE INDICATING OXIDATIVE DNA DAMAGES IN ESTROGEN-DEPENDENT TARGET ORGANS OF CANCER

As described earlier, the activated immune system/inflammation has the capacity to produce oxi-dative damage and mutations in the genome of E2-dependent tissues in humans and experimental

FIGURE 3. The concentration of the estrogen-induced RO/NS within the cellular microenvironment determines its stimulatory orinhibitory signals as well as its genotoxic effects regulating the growth of estrogen-dependent cells. Toxicological concentrations of estro-gen producing high (H) concentrations of reactive oxygen and nitrogen species (RO/NS) may be directly toxic to the cells by activatingapoptosis, whereas exposure of physiological doses of estrogen to the same cells under this condition generating moderate or low (L)levels of reactive oxygen and nitrogen species (RO/NS) may stimulate growth signals. Alternatively, estrogen exposure may also enhancethe expression of NOS ( nitric oxidase synthase), leading to the formation of NO (nitric oxide). NO after reacting with superoxide, result-ing in the production of peroxinitrate (ONOO−), may also participate in growth signaling receptors (Rs), IL-1β R, TNF-α-R, cytokine (Cyt),or chemokine (Chem).

E2

Cyt orChem

R

OtherCytokines orChemokines

O2

PM

IL-1β IL-1βR

TNF-αTNF-α

RO2

• H2O2

- SOD

nNOS

NO⋅ ONOO–

Highdamage Cell

ApoptosisUn-repaired

Un-repaired

Inter-mediatedamage

Normal CellsRepaired

Chroniclow

damage

Genemutations

Preneoplastic Cells

Cell Growth

Mitochondria Gene Damages Fate of Cells

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animals, which, in turn, may play a role in the induction of cancer. Here, evidence in support ofimmunoactivation/inflammation and oxidative DNA damage in the estrogen-dependent target organof cancers is provided.

BreastIt was reported that macrophages are a major component of the normal breast’s stroma and

comprise a substantial cellular component of the cell mass (up to 50%) of breast carcinomas (Kellyet al., 1988). Breast macrophages constitute an in situ source of estradiol. E2 production by mac-rophages in breast tissue appears sufficient to stimulate the proliferation of adjacent epithelial cellsand to autoregulate cytokine production (Mor et al., 1998). Breast tumor cells are known to secretea number of chemokines, such as IL-8 and MCP-1, which attract macrophages and lymphocytes(Reed & Purohit, 1997). On the other hand, it is evident that cytokines secreted by infiltrated mac-rophage and lymphocytes, such as IL-6 and TNF-α, play important roles in stimulating E2 synthesisin breast cancer cells. Recently, inflammation in invasive breast cancer was reported (Lee et al.,1997). The main pattern of inflammation was a diffuse infiltrate of macrophages, and to a lesserextent T cells (Lee et al, 1997). Recently, it was shown that breast tumor cells express IL-1α, IL-1β,and IL-1R in most specimens tested (Miller et al., 2000).

Breast carcinoma DNA contains significantly higher concentrations of base modifications,such as 8-hydroxyguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyade-nine (Malins & Haimanot, 1991). The concentration of total identified base modifications repre-sented a more than ninefold increase over the control value. It was also found by Malins and hisassociates (1993) that breast cancer tissues contain a statistically higher level of 8-hydroxygua-nine than normal breast tissue. Determination of 8-OHdG in DNA isolated from 75 humanbreast tissue specimens and from normal and transformed human breast cell lines revealed thatthe amount of 8-hydroxy-2′-deoxyguanosine (8-OHdG) are 0.25 pmol/g in normal breast tissuefrom reduction mammoplasty, 0.98 pmol/g in benign tumors, and 2.44±0.49 pmol/g DNA inmalignant breast tissue with invasive ductal carcinoma (Musarrat et al., 1996). The endogenousformation of 8-OHdG in cultured breast cancer cells is significantly higher than that of normalbreast epithelial cells. A significantly elevated level (3.35-fold higher) of 8-OHdG was observedin ER-positive compared with ER-negative malignant tissues. 8-OHdG level in ER-positive cellline (MCF-7) was significantly higher (9.3-fold higher) than ER-negative cell line (MDA-MB 231)(Musarrat et al., 1996). This suggests a positive relationship between 8-OHdG formation andestrogen responsiveness. Analysis of 8-OHdG in DNA of breast cancer tissues and noncancerousbreast tissues in 61 Japanese patients showed that the 8-OHdG levels in DNA of breast cancer tis-sues are significantly higher than those of their corresponding noncancerous breast tissues (2.07vs. 1.34 8-OHdG/105 dG, p < .0001) (Matsui et al., 2000). There was a higher level of 8-OHdGin the DNA of early-stage cancer tissue than that of later stage cancer tissue, which suggests thatROS may play an important role in the early phase of carcinogenesis. Genetic polymorphism inenzymes involved in ROS metabolism may also play a role in individual susceptibility to oxidant-related breast cancer. For example, the mean level of 8-OHdG in DNA of breast cancer tissueswas higher among patients with genotype of high catechol O-methyltransferase (COMT) activityor low glutathione S-transferase (GST)P1 activity (Matsui et al., 2000).

OvaryThere are several epidemiological studies that suggest that factors producing epithelial inflam-

mation are involved in ovarian carcinogenesis (Ness & Cottreau, 1999). Several types of exposurethat do not directly affect ovulation or steroid hormone levels but that do enhance local inflamma-tion have been implicated as ovarian cancer risk factors. For example, asbestos and talc exposure,endometriosis (i.e., ectopic implantation of uterine lining tissue), and pelvic inflammatory diseasecannot be directly linked to ovulation or to hormones but do produce local pelvic inflammation.Inflammation entails cell damage, oxidative stress, and elevation of cytokines and prostaglandins, allof which may be mutagenic. The fact that anti-inflammatory medications reduce the occurrence ofovarian cancer supports the hypothesis that inflammation is involved in ovarian tumorigenesis

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(Cramer et al., 1998). In a early study, Haskill et al. (1982) reported infiltration of the inflammatorycells (lymphocyte and macrophages) in human ovarian cancers.

There is increasing evidence that cytokines may play an important role in the progression ofovarian cancer (Nash et al., 1999). Many cytokines are expressed in the normal ovary. Multiplecytokines are produced by ovarian cancer cells. Five different human ovarian epithelial tumor celllines and tumor cells isolated from the ascitic fluid of four cancer patients expressed IL-1α and IL-1βgenes constitutively (Li et al., 1992). Ovarian cancer cells also secrete IL-6 and macrophage colony-stimulating factor, constitutively. The mechanism by which cytokines enhance tumor cell growth isnot full understood. Cytokines may enhance tumor growth directly by functioning as growth factorsor by promoting metastasis through enhancing tumor angiogenesis (Nash et al., 1999). Cytokinesproduced by tumors may modulate immune responses that favor tumor progression. IL-6 enhancestumor cell growth by promoting tumor cell attachment and migration. IL-1β and TNF-α may pro-mote tumor growth by increasing production of IL-6. Serum IL-6 levels were shown to be higher inovarian cancer patients than in healthy controls. Moreover, higher serum IL-6 levels were found inovarian cancer compared with other gynecological malignancies. Scambia et al. (1994) reportedthat 53% of primary epithelial ovarian tumors were found to be IL-6-positive, whereas 35% and10% of endometrial and cervical cancer patients were found to be IL-6-positive, respectively. Thegenes superoxide dismutase (SOD2), myeloperoxidase (MPO), and NAD(P)H:quinone oxidoreduc-tase 1 (NQO1) are involved in inflammation and oxidative stress. For SOD2, women with the TC(val/ala) or CC (ala/ala) genotypes were at increased risk [odds ratio (OR) 2.1, 95% confidence inter-val (CI) 1.1–4.0] (Olson et al, 2004). For NQO1, the TT (ser/ser) genotype was associated withsomewhat increased risk (OR=2.3, 95% CI 0.69–7.6). Though these results need to be confirmedby other studies, they point to a possible role for genes involved in oxidative stress in the developmentof ovarian cancer.

ProstateNeonatal exposure of male Wistar rats to E2 in the presence or absence of dihydrotestosterone

propionate (DHT) was previously shown to result in prostate inflammation (Robinette, 1988). Peri-natal exposure to estrogenic compounds may increase the incidence of prostatitis in adult animals.Rats treated from gestation d 18 to postnatal d 5 with E2 resulted in inflammatory infiltrate in 45%in ventral prostates of 90-d-old rats (Stoker et al., 1999b). Rats treated with E2 or bisphenol A dur-ing postnatal d 22 to 32 show inflammation and a significant increase in MPO activity in the laterallobes of the prostate in adult animals (Stoker et al., 1999a). Interestingly, tamoxifen treatment alsoinduces inflammatory infiltrate in 27.8% of the animal prostates. A neonatal single injection of beta-estradiol 17-cypionate induces inflammation in all prostate lobes and in the seminal vesicles(Kawamura et al., 2000). Lymphocytes infiltrated the stroma and epithelium of ventral prostates.Moreover, lymphocytes penetrated through smooth muscle cells into the epithelium. Smooth mus-cle cells may be the targets for E2 action in the prostate of estrogenized animals. Upregulation ofseveral proinflammtory genes was reported in the rat prostate shortly after exposure to E2 and wellbefore any inflammatory cells were observed in the prostate (Harris et al., 2000)

Using RT-PCR analysis, IL-1β transcripts were found in the androgen-insensitive prostate cancerPC-3 cell line (Hoosein, 1998). Moreover, IL-1β was present in both stromal and epithelial com-partments in human prostate carcinoma specimens. IL-6 stimulates the growth of prostate cancercell lines LNCaP and pcDNA3 cells (Qiu et al., 1998). IL-6 may function as a growth factor for pro-static carcinoma cells either by autocrine and/or paracrine mechanisms (Okamoto et al., 1997).Chronic inflammatory lesions in the prostate resulting in hyperactive prostate epithelium and anincrease in proliferative rate were reported (De Marzo et a., 1999). The inflammatory responsecoupled with increased proliferation may put cells at high risk for DNA damage and developmentof neoplasia.

TestisThe testes and sperm appear to be particularly susceptible to immunological damage (Hedger,

1987). Although prone to immunological damage, there is considerable evidence that the testis is

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an “immunologically privileged” tissue, in the sense that foreign tissue grafts into the testes of someexperimental animals can survive for extended periods (Hedger, 1987; Head et al., 1983; Head &Billingham, 1985). There have been several developmental studies in the rat testis indicating thatthe number of macrophages increases dramatically around the time of puberty (Mendis-Handagema et al., 1987; Hutson, 1990). Neutrophils are almost never observed in normal testes(Hedger et al., 1995). Several studies established that the Leydig cell is important for the develop-ment and maintenance of the large resident macrophage population of the rat testis (Wang et al.,1994; Raburn et al, 1993). Circulating leukocytes and antibodies appear to have almost unre-stricted access to the testicular interstitial tissue (Hedger, 1987). The testicular lymphatic vessels areextensive, large, and drain directly to local lymph nodes (Fawcett et al., 1973). The interstitial tis-sues of the rat and mouse are similar to the human and contain large numbers of macrophages andlymphocytes (Fawcett et al., 1973). Macrophages residing within the rat testis have a novel cytokinesecretion profile and an altered responsiveness to inflammatory activators compared with perito-neal macrophages. Basal productions of IL-1β, TNF-α, and IL-6 by testicular macrophages aresimilar to peritoneal macrophages, but testicular macrophages produce eightfold greater levels ofGM-CSF than peritoneal macrophages (Kern et al., 1995). A bilateral synchronous germ-cell tumorwas reported in a 68-yr-old man who received E2 therapy for 11 mo (Hem et al., 1988). Exposureof CD-1 mice to DES on d 9–16 of gestation was shown to result in an almost 100% frequency ofcryptorchidism and impairment of spermatogenesis as well as a lower incidence of testicular fibrosisand inflammatory lesions (Bosland, 1996; Arai et al., 1983, Newbold et al., 1987; McLachlan et al.,1975). Marked inflammation of the testis was seen in 8% of prenatally exposed DES mice (Newbold,1998).

An early study by Bacon and Kirkman (1955) showed that subcutaneously implanted com-pressed pellets consisting of 20 mg estrogen (DES, 17-estradiol, ethinyl estradiol, or estrone) resultin chronic inflammation in hamster testes by the evidence of masses of lymphoid cells or of largephagocytic elements usually containing lipids. The inflammation was most frequently observed inthe lower portions of the seminiferous tubules, the tubuli recti, and the rete testis (Ferry et al.,1997). Neutrophilic infiltration was observed only in the epididymis and other accessory organs.Scattered intratubular phagocytic cells were commonly found in the testes of all treated groups. Thistreatment also produced testis adenoma and Sertoli-cell tumors. Recently, Ferry et al. (1997)showed expression of MPO in human granulocytic sarcomas of the testis by using immunohis-tochemical staining. Studies showed that neonatal subcutaneous single administration of DES toSyrian hamster induced abnormal testicular structure and tumors (Roy & Cai, 2002). The pathologi-cal study showed infiltration of neutrophils and macrophages in the testis. Testis inflammation wasalso indicated by increased activities of MPO. Hydrogen peroxide concentration was significantlyincreased in the DES-treated group. The mRNA levels of the proinflammatory cytokines IL-1β andTNF-α were significantly increased in the neonatal DES-treated group when compared to control.Neonatal DES treatment also significantly increased oxidative DNA damage (8-OHdG) levels andproduced mutations in a novel gene in the testis. Cotreatment with an inhibitor of synthesis of IL-1βand TNF-α, pentoxifylline, decreased DES-induced levels of MPO, 8-OHdG formation, and genemutaions, and prevented DES-induced lesions (Roy, 2000; Roy & Cai, 2002). Our data support thehypothesis that E2-induced inflammation resulting in the oxidative damages and mutations in testic-ular genome is involved in the initiation and promotion of testicular cancer.

UterusE2 exerts proinflammatory effects in the uterus, where it produces an influx of neutrophils and

macrophages, tissue edema, and proliferation of uterine epithelial cells (De & Wood, 1990;Quarmby & Korach, 1984). E2-induced uterine inflammation is specific to certain types of leuko-cytes. E2 treatment results in a massive influx of neutrophils and macrophages in mice while num-bers of B lymphocytes in the uterus are unaffected (Tibbetts et al., 1999; De & Wood, 1990).Hendry and his associates (1997) reported leukocyte infiltration in the endometrium of neonatalDES-exposed hamsters. Most of the infiltrating cells are eosinophils, and the infiltration is observedas soon as d 9 of life. It was recently shown that (1) neonatal sc single administration of DES to

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Syrian hamster increases 8-OHdG formation in the uterine genome; (2) MPO activity and proin-flammatory cytokines IL-1β and TNF-α mRNA transcripts were higher in the DES-treated group;and (3) DES treatment significantly increased the levels of hydrogen peroxide (Roy, 2000; Roy &Cai, 2002). These studies suggest that E2 exposure induces inflammatory response in the targetorgans, and this, in turn, produces oxidative damages to the genome.

E2-INDUCED CELL SIGNALING THROUGH ROS

Liposomes containing SOD or catalase inhibit in vitro estrogen-induced proliferation of Syrianhamster renal proximal tubular cells (Oberly et al., 1991). E2-induced stimulation of macrophagecells and MCF7 cells in part occurs through ROS (Venkat et al., 2003; Felty et al., 2005a). Studiesfound inhibition of estrogen-induced MCF7 cell growth by ROS scavengers such as N-acetylcysteine,ebselen, and catalase (Felty et al., 2005b). ROS may modulate effector molecules such as PKC, p53,extracellular regulated kinase (ERK), nuclear factor-κB (NF-κB), and the c-fos/c-jun heterodimer(AP-1), and these effector molecules are known to participate in growth signal transduction (Felty &Roy, 2004). Recently data demonstrated evidence for the involvement of redox signaling withestrogen-induced cell proliferation (Felty et al., 2005a, 2005b). Physiological concentrations of E2stimulate a rapid production of intracellular ROS, which led to the phosphorylation of c-jun andCREB, and increased activity of redox sensitive transcription factors Nrf-1, c-jun, and CREB, knownto be involved in the regulation of cell cycle genes. Although direct ER transcription factor effectsare required to promote DNA synthesis, our recent data showed that MCF7 cell growth and cyclinD1 expression are suppressed by antioxidants and mitochondrial blockers, which supports ournovel finding that nongenomic E2-induced mitochondrial ROS modulate the early stage of cellcycle progression (Felty et al., 2005b).

These studies suggest that in addition to ovarian estrogens, mitogenic signals may also comefrom E2 directly acting on mitochondria of epithelial or immune cells through TNF-α- and IL-1β-generated superoxide and hydrogen peroxide. This, in turn, might help to fix the genotoxic effect ofthe estrogen and/or inflammation; and the production of mutational changes in the genome. In theabsence of mitogenic stimuli, DNA damaged viable cells might undergo senescence or apoptosis(Figure 3). Therefore, G1 arrested cells waiting for the repair of DNA damage may receive pressurefrom the mitogenic signals produced by estrogens and/or TNF-α and IL-1β generated low concen-trations of superoxide and H2O2, which may force cells to exit out of G1 arrest. This mitogenic pres-sure may allow cells to enter into the S phase and proceed through G1/S checkpoint in order tocomplete cell division. This would result in an increased rate of fixation of DNA damage leading tomutation (Figure 3). Since E2-induced ROS exert mitogenic effects in the cells, this may also con-tribute to the potential for fixation of damage to bases of DNA leading to a probability of highermutational frequency. These studies suggest that the estrogen generated oxidants together with anE2-driven increase in epithelial cell proliferation may initiate and promote neoplastic lesions inE2-sensitive tissues.

SUMMARY

Induction of ER upon E2 exposure is not sufficient for the development of carcinogenesis. Untilrecently, it has been argued that hydroxylated metabolites of estrogen (catechol estrogens) partici-pate in redox cycling and during the redox cycling of estrogen reactive oxygen species (ROS) aregenerated. This redox reactions may occur in vivo, if free hydroxylated metabolites are in closeproximity to activating enzymes within the target cells. Studies recently showed that estrogens orestrogen metabolites directly acting on mitochondria of epithelial or immune cells generate reactiveoxygen and nitrogen species, which in turn may induce oxidative damage in the genome of targetcancer cells. Data from our laboratory and others provide evidence in the support of this concept inbreast, ovary, uterus, prostate, and testicular tumors. Since secretion of proinflammatory cytokine,such as IL-1 agonists and antagonists, is up to 10-fold higher in women and appears to be modu-lated by E2, any defects in the regulation of the proinflammatory cytokines may result in more

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prominent manifestations of E2-dependent diseases, including cancer, in women than in men. It isenvisioned that this is an important mechanism that drives the carcinogenesis process, but that itoccurs in the context of other processes such as ER-mediated signaling and estrogen-reactivemetabolite-associated genotoxicity under investigations that may also contribute to the process. Insome cases (for example, acute response after estrogen exposure), the immunostimulatory effectson immune cells by E2 leading to production of reactive oxygen and nitrogen species may be bene-ficial for host defense or other purposes in the target tissues. However, the persistent activation orinactivation of some immune cells by estrogen in the wrong circumstances leading to the generationfor a longer period of time of elevated levels of oxidants, such as reactive oxygen and nitrogen spe-cies, may produce gene mutations through oxidative damages in the genome and thus lead todevelopment of E2-dependent diseases. Whether such imbalance in E2 milieu and immune systemis an underlying explanation for preponderance of E2-dependent cancers in women and men is aquestion that needs attention for further research. Studies are in progress in our laboratory tocritically evaluate the influence of antiestrogens on immune activation, and the influence of anti-inflammatory compounds on E2-induced carcinogenesis.

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