Differential oxidant potential of carcinogenic and weakly carcinogenic estrogens: Involvement of metabolic activation and cytochrome P450

Different estrogens vary in their carcinogenic potential despite having similar hormonal potencies; however, mechanisms of estrogen-induced carcinogenesis remain to be fully elucidated. It has been hypothesized that generation of reactive estrogen-quinones and oxidative stress, both of which result from metabolic activation of estrogens, play an essential role in estrogen-induced carcinogenesis. This hypothesis was tested using the estrogen-receptor (ER)-alpha-positive hamster kidney tumor (H301) and the human breast cancer (MCF-7) cell lines. Estrogens with differing carcinogenic potentials were compared in terms of their capacities to induce 8-iso-prostaglandin F(2alpha) (8- iso-PGF(2alpha)), a marker of oxidative stress. Tumor cells were treated with either 17beta-estradiol (E2), a carcinogenic estrogen or 17-alpha-ethinylestradiol (EE), a weakly-carcinogenic estrogen. Tumor cells were also treated with alpha-naphthoflavone, a cytochrome P450 inhibitor, or a combination of alpha-naphthoflavone and E2 to study the effect of metabolic activation of E2 on E2-induced oxidative stress. H301 cells treated with E2 displayed time- and dose-dependent increases in 8-iso-PGF(2alpha), compared to controls; treatment with 10 nM E2 resulted in a maximal 4-fold induction following 48 h of treatment. In contrast, H301 cells treated with EE did not display an increase in 8-iso-PGF(2alpha) compared with controls. In H301 cells cotreated with alpha-naphthoflavone and E2, alpha-naphthoflavone inhibited the E2-induced increase in 8-iso-PGF(2alpha). These data indicate that a carcinogenic estrogen shows strong oxidant potential, whereas a weakly-carcinogenic estrogen shows poor oxidant potential. Furthermore, inhibiting metabolic activation of a carcinogenic estrogen blocks its oxidant potential. Our data support the hypothesis that metabolic activation and subsequent generation of oxidative stress may play critical roles in estrogen-induced carcinogenesis.     

Mechanistic studies on the inhibitory action of dietary dibenzoylmethane, a beta-diketone analogue of curcumin, on 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis

Dietary factors play important roles in the carcinogenic process. The results of epidemiological data and some laboratory animal studies indicate that certain naturally occurring and synthetic components are able to block the carcinogenic process and inhibit the development of certain cancers. Dibenzoylmethane (DBM), a curcumin-related beta-diketone analogue has been reported to exhibit a remarkable inhibitory effect on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis in Sencar mice. The present study investigated the possible mechanisms of inhibitory action of DBM on DMBA-induced mammary tumorigenesis in mice. The summarized results indicate that: (1) in in-vitro studies. DBM inhibited DMBA metabolism and the formation of DMBA-DNA adducts in a dose-dependent manner; (2) in the assay of competitive binding to estrogen receptors with [3H]-estradiol in vitro, DBM showed weak binding affinity; (3) in vivo, feeding of 1% DBM in the diet of immature Sencar mice for 4 -5 weeks decreased the uterine and parametrial fat pad weights, and lowered the serum estrogen and triglyceride levels. This study provides insight into the mechanisms involved in the inhibitory action of DBM in mouse mammary tumorigenesis.     

Increased number of BrdU-labeled neurons in the rostral migratory stream of the estrous prairie vole

In the mammalian forebrain, most neurons originate from proliferating cells in the ventricular zone lining the lateral ventricles, including a discrete area of the subventricular zone in which neurogenesis continues into adulthood. The majority of the cells generated in the anterior portion of the subventricular zone (SVZa) are neuronal precursors with progeny that migrate to the olfactory bulb (OB) along a pathway known as the rostral migratory stream (RMS). The list of factors that influence the proliferation and survival of neurons in the adult brain remains incomplete, but previous studies have implicated neurotrophins in mammals and estrogen in birds. This study examined the effect of estrus induction on the proliferation of SVZa neurons in female prairie voles. Prairie voles, unlike many other rodents, are induced into estrus by chemosensory cues from a male. This olfactory-mediated process results in an increase in serum estrogen levels and the consequent induction of behavioral estrus (sexual receptivity). Female prairie voles induced into estrus by male exposure had a 92% increase in BrdU-labeled cells in the SVZa compared to females exposed to a female. Double-label immunocytochemical studies demonstrated that 80% of the BrdU-labeled cells in the RMS displayed a neuronal phenotype. Ovariectomized females exposed to a male did not show an increase in serum estrogen or BrdU labeling in the RMS. Conversely, ovariectomized females injected with estrogen were sexually receptive and had more BrdU-labeled cells in the RMS than oil-injected females. These data suggest that, in female prairie voles, estrus induction is associated with increased numbers of dividing cells in the RMS, possibly via an estrogen-mediated process.     

Metabolism of estrogens in breast cancer.

This extensive literature compilation reviews major studies on estrogen metabolism in cancer, studies which have led to proposed possible etiological roles of estrogens in human breast cancer. Urinary and plasma estrogen excretion patterns and profiles in women with breast cancer are the topics of part 1. Studies of estrogen profiles in women who are at high-risk for breast cancer are critiqued. The estriol hypothesis is presented and criticised in a chapter. The effects of endocrine ablation on urinary estrogen profiles in breast cancer patients are compiled. Production and metabolism of estrogens in women with breast cancer are rendered, including in vivo biotransformation rates and in vitro transformation data. And the search for estrogen metabolites in women with breast cancer is reviewed. In conclusion it is obvious that the question of whether breast cancer patients have an abnormal metabolism of estrogen has not been answered, but further investigations of estrogen metabolism in breast cancer should be continued because: 1) the possibility that estrogens are carcinogenic has not been ruled out; 2) receptors have been discovered which do correlate with hormone dependency of tumors; 3) present evidence suggests that neoplasm may induce abnormal estrogen metabolism; 4) directional changes of estrogen metabolism that occur in pregnancy may also occur in women with target tissue neoplasia; 5) hepatic tissue's relationship to breast cancer has not received attention; and 6) the role of peripheral aromatization in the pathogenesis of mammary cancer is not yet understood.     

Food-borne amines and amides as potential precursors of endogenous carcinogens

This paper reviews the experimental results of our research in the past several years and other related papers that have been directed toward the occurrence, biotransformation and epidemiological significance of carcinogenic N-nitroso compounds in biosphere. Endogenous carcinogens are a group of cancer-causing compounds produced in vivo from harmless precursors. This category has been exemplified by the well-known carcinogens, N-nitroso compounds. The significance of naturally occurring amines and amides as precursors of carcinogenic N-nitroso compounds in vivo and their implication in the incidence of human cancer have been investigated and emphasized. Extremely high levels of trimethylamine-N-oxide and dimethylamine were detected in squids and other seafoods. More than 90% of trimethylamine-N-oxide were converted to dimethylamine and trimethylamine on pyrolysis. Low levels of dimethylamine and methylamine were also detected in the fermented soybean products, wines and sauces. Both dimethylamine and trimethylamine are excellent precursors of dimethylnitrosamine. Several naturally occurring aromatic amines especially 2-carboline derivatives such as harman, norharman, harmaline, harmalol, harmine and harmol are mutagenic and become more mutagenic to Salmonella typhimurium after nitrosation. Appreciable amounts of piperidine were detected in the popular spice white and black pepper powders. Under acidic condition, piperidine reacts readily with nitrite to form carcinogenic N-nitroso-piperidine. N-Nitrosophenacetin was formed from the reaction of nitrite with the amide drug phenacetin. This new compound showed strong mutagenicity to Salmonella typhimurium and Sarcina lutea and strong teratogenic activity to Leghorn chicken embryos. Studies have shown that the majority of N-nitroso compounds in the body come from in vivo conversion. Most investigators believe that this endogenous pool of N-nitroso compounds may prove to be a major exposure route in man. The presence of naturally occurring amines and amides in the diet then becomes one of the crucial limiting steps in the formation of endogenous N-nitroso compounds in vivo.     

Action of some retinol derivatives and their provitamins on microsome-catalyzed formation of benzo[a]pyrene-DNA adduct

Several vitamin A compounds have been tested for their ability to suppress formation of DNA adduct by the carcinogen benzo[a]pyrene (B[a]P) in an in vitro reaction catalyzed by rat liver microsomes. Retinol, retinal, 3-dehydroretinol and 3-hydroxyretinol were found to be effective inhibitors of adduct formation. Certain carotenoids that are precursors of these retinoids also displayed considerable inhibitory capacity. Carotenoids and the 3-substituted retinoids appeared to modulate the DNA adduct formation exclusively through their action on microsomal enzymes, since an effective inhibition in each case was observed on the formation of B[a]P-7,8-diol, a proximate carcinogenic metabolite of B[a]P. Unsubstituted retinoids, on the other hand, had marginal effect on enzymes but were found effective in accelerating inactivation of B[a]P-7,8-diol-9,10-epoxide, the ultimate carcinogenic metabolite that binds to DNA.     

Evidence from ESI-MS for NQO1-catalyzed reduction of estrogen ortho-quinones

Estrogen ortho-quinones have been implicated as ultimate carcinogenic metabolites of estrogens. The present conclusion that estrogen ortho-quinones are not substrates for NAD(P)H:quinone oxidoreductase (NQO1) stems from earlier reports. In this investigation, we were successful in circumventing the problem of nonenzymatic reduction of estrogen quinone by NAD(P)H, which led to the above conclusion, and for the first time we show that NQO1 catalyzes the reduction of estrogen quinones. Mass spectrometric binding studies involving estradiol-3,4-quinone or menadione with NQO1 clearly support the formation of an enzyme-substrate physical complex. However, the NQO1 mass spectrum did not alter after addition of cholesterol, the control. Two different strategies were employed to ascertain the NQO1 activity in estrogen quinone reduction. First, the ping-pong mechanism of NQO1 catalysis was utilized to overcome the problem of nonenzymatic reduction of the substrate by NADH. Second, tetrahydrofolic acid, which has a lower reducing potential, was used as an alternate cofactor. Both of these methods confirmed the reduction of estradiol-3,4-quinone by NQO1, when the assay mixtures were analyzed by UV or liquid chromatography-mass spectrometry. Furthermore, reduction of 9,10-phenanthrene quinone or menadione was observed using the reported assay conditions. Thus, clear evidence for the catalytic reduction of estrogen ortho-quinones by NQO1 has been obtained; its mechanism and implications are discussed.     

Exploring estrogenic activity in lung cancer

It is well known that a connection between xenobiotics inhalation, especially tobacco combustion and Lung Cancer development is strongly significant and indisputable. However, recent studies provide evidence indicating that another factors such as, estrogens are also involved in lung carcinoma biology and metabolism. Although the status of estrogen receptors (ER), in both cancerous and healthy lung tissue has been well documented, there is still inconclusive data with respect of which isoform of the receptor is present in the lungs. However according to several studies, ERβ appears to be predominant form. Apart from ERs, estrogens can work through a recently discovered G-coupled estrogen receptor. Binding with both types of the receptors causes a signal, which leads to i.e. enhanced cell proliferation. There are many published reports which suggest that estrogen can be synthesized in situ in lung cancer. Some disturbances in the activity and expression levels of enzymes involved in estrogen synthesis were proved. This suggests that increased amounts of sex-steroid hormones can affect cells biology and be the reason of the accelerated development and pathogenesis of lung cancer. There also exist phenomena which associate estrogenic metabolism and tobacco combustion and its carcinogenic influence on the lungs. Compounds present in cigarette smoke induce the activity of CYP1B1, the enzyme responsible for estrogenic metabolism and synthesis of their cateholic derivatives. These structures during their redox cycle are able to release reactive oxygen species or form DNA adduct, which generally leads to destruction of genetic material. This process may explain the synergistic effect of smoking and estrogens on estrogen-dependent lung cancer development.     

Reversibility of pre-malignant estrogen-induced epigenetic changes

The development of early detection and prevention strategies of breast cancer relies on defining molecular and cellular events that characterize progressive alterations underlying preneoplastic changes in the mammary epithelium. Studies have shown that estrogen exerts its carcinogenic effects through both genetic and epigenetic pathways to promote imbalances in proliferation and apoptosis, genomic instability and cancer.

The purpose of this study was to identify the earliest epigenetic changes that could be detected in response to estrogen treatment. More importantly, having detected these early pre-malignant epigenetic changes, a follow-up study was designed to address the potential to reverse these estrogen-induced alterations. Using a well-established ACI rat model, morphological and epigenetic changes were identified in the mammary gland tissue as early as 2 days after exposure to constitutively elevated estrogen levels produced by continuous release estrogen mini-pellets.

Progressive hyperproliferative changes were paralleled by epigenetic disturbances, including the upregulation of DNA methyltransferases and hyperacetylation of histone residues. These changes could be detected early, and they continued to persist if estrogen was maintained within a high physiological range. Epigenetic features of short-term estrogen exposure were strikingly similar to hallmarks of cancer promotion and progression. Yet, importantly, these changes exhibited a degree of reversibility if a source of elevated levels of estrogen was removed.

Knowing that operational reversibility during the promotion stage of carcinogenesis provides a window for intervention, the potential to reverse the effects of elevated levels of estrogen prior to tumor development may prove to be a promising avenue to explore.     

Reduction of estrogen-induced transformation of mouse mammary epithelial cells by N-acetylcysteine

A growing number of studies indicate that breast cancer initiation is related to abnormal estrogen oxidation to form an excess of estrogen-3,4-quinones, which react with DNA to form depurinating adducts and induce mutations. This mechanism is often called estrogen genotoxicity. 4-Catechol estrogens, precursors of the estrogen-3,4-quinones, were previously shown to account for most of the transforming and tumorigenic activity. We examined whether estrogen-induced transformation can be reduced by inhibiting the oxidation of a 4-catechol estrogen to its quinone. We demonstrate that E6 cells (a normal mouse epithelial cell line) can be transformed by a single treatment with a catechol estrogen or its quinone. The transforming activities of 4-hydroxyestradiol and estradiol-3,4-quinone were comparable. N-Acetylcysteine, a common antioxidant, inhibited the oxidation of 4-hydroxyestradiol to the quinone and consequent formation of DNA adducts. It also drastically reduced estrogen-induced transformation of E6 cells. These results strongly implicate estrogen genotoxicity in mammary cell transformation. Since N-acetylcysteine is well tolerated in clinical studies, it may be a promising candidate for breast cancer prevention.     

Acceleration of adenovirus replication and increased virion production by treatment with the steroid hormone 17 beta-estradiol.

We report here that concentration of an estrogen known to promote enhanced transformation and to increase oncogenicity of rat embryo cells, accelerate the production and increase the yield of progeny virions in adenovirus type 12 (Ad 12)-infected HEp-2 cells. Further, measurement of the incorporation of radioactive RNA and DNA precursors indicated that macromolecular synthesis in the estrogen-treated, infected cells was accelerated. Possible explanations for this observation are discussed.     

Steroid hormones and carcinogenesis of the prostate: the role of estrogens

Abstract Androgens have long been known to be the major sex hormones that target the prostate during development, maturation, and carcinogenesis. It is now apparent that estrogens, both those synthesized by the body as well as those from our environment, also target the prostate during all stages of development. Little is known about the mechanisms involved in estrogen stimulation of carcinogenesis and less is known about how to prevent or treat prostate cancer through estrogenic pathways. To better understand how estrogens mediate their carcinogenic effects, the respective roles of estrogen receptor (ER)-α and ER-β must be elucidated in the epithelial and stromal cells that constitute the prostate. Lastly, the significance of ER signaling during various ontogenic periods must be determined. Answers to these questions will further our understanding of the mechanisms of estrogen/ER signaling and will serve as a basis for chemopreventive and/or chemotherapeutic strategies for prostate cancer.     

Environmental contaminants in pathogenesis of breast cancer

This review is an attempt to comprehend the diverse groups of environmental chemical contaminants with a potential for pathogenesis of breast cancer, their probable sources and the possible mechanisms by which these environmental contaminants act and interplay with other risk factors. Estrogens are closely related to the pathogenesis of breast cancer. Oxidative catabolism of estrogen, mediated by various cytochrome P450 enzymes, generates reactive free radicals that can cause oxidative damage. The same enzymes of estrogenic metabolic pathways catalyze biological activation of several environmental (xenobiotic) chemicals. Xenobiotic chemicals may exert their pathological effects through generation of reactive free radicals. Breast tissue can be a target of several xenobiotic agents. DNA-reactive metabolites of different xenobiotic compounds have been detected in breast tissue. Many phase I and II xenobiotic metabolizing enzymes are expressed in both normal and cancerous breast tissues. These enzymes play a significant role in the activation/detoxification of xenobiotic and endogenous compounds including estrogens. More than 30 carcinogenic chemicals are present in tobacco smoke; many of them are fat-soluble, resistant to metabolism and can be stored in breast adipose tissue. Similarly, pesticides are also known to cause oxidative stress; while some act as endocrine disruptor, some are shown to suppress apoptosis in estrogen sensitive cell lines. Reports have shown an association of smoking (both active and passive) and pesticides with breast cancer risk. However, the issues have remained controversial. Different mutagenic substances that are generated in the cooking process e.g., heterocyclic amines and polycyclic aromatic hydrocarbons (PAHs) can be a threat to breast tissue. PAHs and dioxins exert their adverse effects through the aryl hydrocarbon receptor (AhR), which activates several genes involved in the metabolisms of xenobiotic compounds and endogenous estrogens. These chemicals also induce AhR-dependent mitochondrial dysfunction. Many of the environmental pollutants suppress the immune system, which are implicated to risk. A better understanding about the biological effects of different environmental carcinogenic compounds and determination of their impact on rising incidence of breast cancer will be beneficial in improving preventive policy against breast cancer.     

Studies on estrogen biosynthesis using radioactive and stable isotopes

The conversion of androgens into estrogen involves three distinct generic reactions which are catalyzed by a single P450 enzyme (aromatase or P450(aromatase)). The first step in the process is the conversion of 19-methyl into a hydroxymethyl group which requires NADPH + O2, thus representing the well-known hydroxylation process. The next stage, converting the -CH2OH into -CHO, also requires NADPH + O2 and may be rationalized either through a second hydroxylation reaction producing a gem-diol, CH(OH)2 (which dehydrates to the aldehyde), or via another route. The final stage in the process again uses NADPH + O2, culminating in the release of C-19 as formate. Our extensive studies using precursors containing 2H, 3H, and 18O have shown that the carbonyl oxygen of the 19-aldehyde group is the one that was introduced in the first step as the hydroxyl group. The aldehydic oxygen along with another, from O2, used in the third step of the process, is incorporated into the released formate. It was found that at each stage of the process, oxygen atoms were introduced or transferred as "whole numbers." In light of these data, mechanisms in which H2O is used to promote the C-10-C-19 bond cleavage or those in which the conversion of the 19-oxoandrostenedione into estrogen is considered to occur via the sequence -CHO----(-)CH(OH)2----estrogen are eliminated. In addition, our mechanistic analysis makes it unlikely that 1 beta-, 2 beta-, or 10 beta-hydroxysteroids serve as intermediates in estrogen biosynthesis. We consider a free radical mechanism for the hydroxylation process.     

A comparison of the gene expression profiles of CRL-1807 colonocytes exposed to endogenous AAPH-generated peroxides and exogenous peroxides from heated oil

Oxidation of PUFAs in the diet has the potential to be genotoxic and hence carcinogenic. Such carcinogenic processes originate within stem cells of the colon. These cells appear to be predisposed to the carcinogenic process. In colon cells (CRL-1807) exposed to chemical reactions simulating exogenous and endogenous peroxidation reactions, we have observed that undifferentiated cells could mount an effective recombinational repair/TCR response to an endogenous peroxidative DNA damage insult, but not to an external exogenous peroxidative insult as one would encounter from a dietary source. This may suggest that defects in such specific DNA repair may play a role in tumour development in undifferentiated colonocytes exposed to a diet-derived lipid peroxides.     

发酵酒中氨基甲酸乙酯的研究进展

对发酵酒中氨基甲酸乙酯（Ethyl carbamate,EC）的致癌机理、检测方法、形成机制、前驱物质及控制方法等方面进行综述。阐述目前研究进展的同时,对存在问题进行分析并提出解决设想及应用展望。氨基甲酸乙酯可由自然反应及添加前驱物质两种方式形成,其致癌机理是通过两种不同途径破坏DNA结构。通常情况,主要通过减少前体物质的添加和形成抑制氨基甲酸乙酯的生成。氨基甲酸乙酯的通用检测方法为色谱与不同检测器结合使用或色谱与质谱联用,其中,检测前的样品前处理有液液萃取,固相萃取等不同形式。目前,我国检测控制工作虽然已经取得一定进展,但是检测方法不够经济有效,控制方法不能够普遍应用。关于控制方法的最新研究中,基因工程技术与系统生物技术的相关应用纳入进程,后基因组研究和蛋白质学等对基因的应用改造处于探索阶段。随着关于氨基甲酸乙酯的危害、形成机理等方面的研究不断深入,不同机制的控制方法不断出现。EC的处理逐步进入更加完善的状态。