A. Salama

Catechol-O-Methyltransferase Polymorphism Is Associated With Increased Uterine Leiomyoma Risk in Different Ethnic Groups

Objectives: Uterine leiomyomas (ULMs) are estrogen-dependent tumors that are more common in African American women. The etiology for such ethnic disparity is currently unknown. catechol-O-mythyltransferase (COMT) is an essential enzyme in estrogen metabolism. In the current study, we investigated the association of the functional COMT Val158 Met polymorphism with ULM in different ethnic groups. We also studied the biologic role of COMT in tumor formation in human and rat leiomyoma cell lines and the potential therapeutic utility of COMT inhibitors. Methods: The genotype frequencies of the functional COMT Val158 Met polymorphism among participants with (186 women) or without (142 women) ULMs were compared, as was the differential ethnic distribution of that polymorphism using polymerase chain reaction (PCR) and restriction-fragment linkage polymorphism. Poliferation, Western blot, and reporter transactivation analyses were applied to myometrial and leiomyoma cells representative of different COMT genotypes. Results: Women with the high-activity COMT Val/Val genotype are 2.5 times more likely to develop ULMs than women with other genotypes (confidence interval, 1.017 to 6.151; P < .001). The prevalence of this genotype was significantly higher in African American women (47%) compared with white (19%) or Hispanic (30%) women (P = .003). Myometrial cell lines expressing the Val/Val genotype exhibited significantly enhanced responses to estrogen in proliferation and in estrogen-responsive element reporter assays. COMT-specific inhibitors reversed such a response and induced apoptosis. Myometrial specimens from Val/Val women demonstrated distinct estrogen-regulated gene expression that was consistent with enhanced proliferation and decreased apoptosis. Conclusions: The high-activity COMT Val/Val genotype is associated with increased risk of ULM. Our results provide a possible explanation for the higher prevalence of ULMs among African American women and offer a potential new target for nonsurgical treatment using COMT inhibitors.

Biomonitoring using accessible human cells for exposure and health risk assessment

A major goal for genetic toxicologist is to provide precise information on exposure and health risk assessment for effective prevention of health problems. A frequently used approach for population study has been to utilize readily available blood cells (lymphocytes and red blood cells) as sentinel cell types to detect biological effects from exposure and to provide early warning signals for health risk. However, such approach still cannot be used reliably for developing strategies in risk assessment and disease prevention. It is possible that other available cell types which are more representative of the target cells for disease may be used to overcome the deficiency. In this report, the use of non-blood cells for biomonitoring is briefly reviewed. Their usefulness in certain exposure condition is highlighted and their effectiveness in documenting exposure compared with other cell types such as the traditional blood cells is presented. It is obvious that the decision in using these non-blood cells in biomonitoring is based on the exposure condition and the experimental design. Nevertheless, monitoring studies using non-blood cells should be encouraged with emphasis on providing dose-response information, comparative response with other cell types and effectiveness for health risk assessment.

Effect of Tumor Necrosis Factor-α on Estrogen Metabolism and Endometrial Cells: Potential Physiological and Pathological Relevance

CONTEXT Estrogen and its metabolites play a critical role in the pathophysiology of the endometrium. The bioavailability of estrogen and estrogen metabolites in endometrial tissues depends on the expression of enzymes involved in estrogen biosynthesis and metabolism. Substantial evidence indicates that estrogen-dependent endometrial disorders are also associated with proinflammatory milieu. However, the mechanism whereby inflammation contributes to these conditions is not known. OBJECTIVE The objective of the study was to investigate the effect of TNF-alpha on estrogen metabolism and the expression of estrogen-metabolizing genes in human endometrial glandular epithelial cells (EM1). DESIGN EM1 were treated with 17beta-estradiol (E2) with or without TNF-alpha. Capillary liquid chromatography-tandem mass spectrometry analysis was used for quantitative measurement of estrogens and estrogen metabolites. Western blot analysis, reporter gene assay, and real-time RT-PCR were used to assess the expression of estrogen-metabolizing genes. RESULTS TNF-alpha treatment significantly increased the level of total estrogen and estrogen metabolites and significantly increased the rate of conversion of estrone (E1) into E2. TNF-alpha also enhanced the oxidative metabolism of estrogen into catecholestrogens with concomitant inhibition of their conversion into methoxyestrogens. Gene expression analysis revealed that TNF-alpha induced the expression of genes involved in E2 biosynthesis (steroidogenic factor-1 and aromatase) and activation (17beta- hydroxysteroid dehydrogenase type 1 and cytochrome P-450, 1B1) with simultaneous repression of genes involved in estrogen inactivation (17beta-hydroxysteroid dehydrogenase type 2; catechol O-methyltransferase; and nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase 1). CONCLUSION TNF-alpha increases the local estrogen biosynthesis in human endometrial glandular cells and directs estrogen metabolism into more hormonally active and carcinogenic metabolites. These effects may impact many physiological and pathological processes that occur within the endometrium.

Effect of epoxide hydrolase polymorphisms on chromosome aberrations and risk for lung cancer

Microsomal epoxide hydrolase (mEH) gene is polymorphic and its enzyme is involved in the activation and subsequent detoxification of several tobacco carcinogens, for example polycyclic aromatic hydrocarbons. Therefore, we have investigated the association of two polymorphisms at exons 3 and 4 of the mEH gene with the development of lung cancer in 110 patients and 119 matched controls. In addition, we have investigated the relationship between the different mEH alleles and the frequency of chromosome aberrations (CA), as an approach to understand the role of genetic susceptibility on cancer risk. Our results show that only the homozygous exon 4 fast genotype is significantly associated with increased risk for lung cancer (odds ratio [OR]=6.26; 95% confidence interval [CI]=1.02-38.3). When the exons 3 and 4 polymorphisms are considered together, patients carrying the high enzyme activity genotype have a significantly increased risk for lung cancer (OR=2.46; 95% CI=1.06-5.68). More importantly, the increased risk for this group is confirmed by their having the highest frequency of CA compared to any other genotype groups. In addition, genotypes with higher risk had consistently more CA than those with lower risk. Our CA data also indicates that the low activity genotype may exert a protective role in cigarette smokers, as it was associated with a significant decrease in CA compared to the high and intermediate activity genotypes. In conclusion, the CA data provides evidence to support that susceptibility mEH alleles are significantly involved with the development of lung cancer from cigarette smoking.

Role of polymorphic GSTM1 and GSTT1 genotypes on NNK-induced genotoxicity.

Polymorphisms in chemical metabolizing genes are known to influence individual susceptibility to environmental cancer. We investigated the role of GSTM1 and GSTT1 polymorphisms in modifying the genotoxicity of a tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) using the sister chromatid exchange (SCE), and the tandem-probe fluorescence in situ hybridization chromosome aberration (CA) assays. NNK (0.24, 0.72 or 1.44 mM) induced a significant concentration-dependent increase in the mean number of SCE regardless of genotypes. In comparing the effects between genotypes, significant increase was observed in GSTM1 null cells compared with GSTM1 positive cells only at the low concentration of NNK (0.24 mM). No significant difference was observed between cells with the null and positive GSTT1 genotypes. Using the CA assay, treatment with NNK (0.12, 0.24 or 0.72 mM) induced a significant concentration-dependent increase in the frequency of CA. In addition, cells with the null GSTM1 genotype had significantly increased CA compared with cells with GSTM1 positive genotype at the three concentrations of NNK. Regarding GSTT1 polymorphism, no significant effect was observed between the null and the positive genotypes. Treatment of the cells with 1 mM glutathione monoethyl ester (GSHME) significantly reduced NNK-induced CA in all cells regardless of their genotypes. The effect was clearly more evident in cells with the GSTM1 positive genotype. Therefore, GSHME is protective against NNK-induced CA with more dominant effect in cells with the GSTM1 positive genotype. Our study indicates that GSTM1 may influence NNK-induced genotoxicity and subsequent tobacco-related health effects.

Factors contributing to discrepancies in population monitoring studies

A review of the scientific literature on population monitoring studies (on non-accidentally exposed populations) frequently show that many of these studies using similarly exposed populations and the same laboratory techniques do not produce consistent results. To illustrate the problem, a brief review of studies using well validated techniques (chromosome aberrations and hprt gene mutation) to elucidate genotoxic effects of cigarette smoking is presented. Although many factors can contribute to the generation of discrepant results, two obvious factors are small sample sizes and inadequate experimental data. In addition, a new factor on genetic susceptibility should be considered in population studies whenever appropriate. The new factor is based on recent data showing the influence of polymorphic metabolizing genes on response to environmental mutagens towards biological effects and disease outcome. The common ones include the cytochrome P450 and the glutathione S-transferase genes. The inclusion of susceptibility factors in population monitoring may revolutionize the approach for health risk assessment and for environmental regulations.

Catecholestrogens induce oxidative stress and malignant transformation in human endometrial glandular cells: Protective effect of catechol-O-methyltransferase

Prolonged exposure to unopposed estrogens is a major risk factor for the development of endometrial cancer. Oxidative metabolism of estradiol (E2) into the catecholestrogens (CEs), 4‐hydroxyestradiol (4‐OHE2) and 2‐hydroxyestradiol (2‐OHE2), may play an important role in estrogen carcinogenicity. CEs can be oxidized to the corresponding ortho‐quinone derivatives with concomitant formation of the reactive oxygen species (ROS). Catechol‐O‐methyltransferase (COMT) is the major enzyme involved in the detoxification of CEs in extrahepatic tissues. We investigated the potential of E2, 2‐OHE2 and 4‐OHE2 to induce microsatellite instability (MSI) and neoplastic transformation of immortalized human endometrial glandular (EM) cells. We also investigated the functional significance of COMT gene expression on modulating the effects of E2 and CEs in EM cells. Our data indicated that E2 and 4‐OHE2 induce MSI, ROS and neoplastic transformation in EM cells. The capacity of E2 and its catechol metabolites to induce MSI, ROS and neoplastic transformation in EM cells is ranked as follows: 4‐OHE2 > E2 > 2‐OHE2. Knockdown of COMT expression in EM cells resulted in increased estrogenic milieu and increased estrogen‐induced cell proliferation. More importantly, knockdown of COMT increased the propensity of E2 or CEs to induce ROS, MSI and neoplastic transformation of EM cells. In contrast, overexpression of COMT in EM cells significantly reduced the cellular estrogenic milieu and protected against E2‐ or CEs‐induced, ROS, MSI and neoplastic transformation. The capacity of E2 or CEs to induce neoplastic transformation of human endometrial glandular cells in vitro may suggest that E2‐induced endometrial cancer is mediated by its metabolism into CEs. Our study clearly indicates that COMT gene expression plays a critical role in modulating the hormonal and carcinogenic effects of E2 and CEs and, consequently, modifies the risk for E2‐induced endometrial cancer. To the best of our knowledge, this is the first study to (i) demonstrate the potential capacity of estrogen and its catechol metabolites to induce neoplastic transformation of immortalized human endometrial glandular cells; and (ii) illustrate the important role of COMT gene expression in protecting against E2‐induced endometrial cancer.

Estrogen Metabolite 2-Methoxyestradiol Induces Apoptosis and Inhibits Cell Proliferation and Collagen Production in Rat and Human Leiomyoma Cells: A Potential Medicinal Treatment for Uterine Fibroids

Objective: The current study sought to investigate the effect of the estrogen metabolite 2-methoxyestradiol (2-MeOHE2) on apoptosis, cell proliferation, and collagen synthesis in human and rat leiomyoma cells. Methods: [3H] thymidine and [3H] proline incorporation studies were conducted. The expression of vascular endothelial growth factor (VEGF), cyclin D1, Bcl-2, and Bax were evaluated by Western blot. Flow cytometry analysis was used to study the effect of 2-MeOHE2 on apoptosis and the cell cycle. Results: Compared with untreated controls, treatment of rat leiomyoma (ELT3) cells with 2-MeOHE2 (0.1, 1, 2, 5, or 10 μM) reduced cell proliferation by 17%, 52%, 61%, 73%, and 79%, respectively (P <.05). Similarly, in human uterine leiomyoma cell line (huLM) cells, proliferation was reduced by 4%, 18%, 37%, 41%, and 51%, respectively. 2-MeOHE2 also caused a concentration-dependent inhibition of collagen synthesis by 4%, 16%, 23%, 51%, and 70%, respectively, in huLM cells (P <.05). Cell cycle analysis indicated that 2-MeOHE2 treatment (1 to 5 μM) in huLM cells resulted in G2/M cell cycle arrest and a 45% increase in apoptosis compared with untreated control (P <.05). Western immunoblotting analysis indicated that 2-MeOHE2 induces a concentration-dependent reduction in the expression of cyclin D1, Bcl-2, and VEGF proteins in both rat and human leiomyoma cell lines. Conclusions: This study provides the first evidence that 2-MeOHE2 is a potent antiproliferative/apoptotic and collagen synthesis inhibiting agent in human and rat leiomyoma cells. To the best of our knowledge, this is the first report showing the potential use of 2-methoxyestradiol as a nonsurgical alternative therapy for uterine leiomyomas.

The effect of tamoxifen and raloxifene on estrogen metabolism and endometrial cancer risk

Selective estrogen receptor modulators (SERMs) demonstrate differential endometrial cancer (EC) risk. While tamoxifen (TAM) use increases the risk of endometrial hyperplasia and malignancy, raloxifene (RAL) has neutral effects on the uterus. How TAM increases the risk of EC and why TAM and RAL differentially modulate the risk for EC, however, remain elusive. Here, we tested the hypothesis that TAM increases the risk for EC, at least in part, by enhancing the local estrogen biosynthesis and directing estrogen metabolism towards the formation of genotoxic and hormonally active estrogen metabolites. In addition, the differential effects of TAM and RAL in EC risk are attributed to their differential effect on estrogen metabolism/metabolites. The endometrial cancer cell line (Ishikawa cells) and the nonmalignant immortalized human endometrial glandular cell line (EM1) were used for the study. The profile of estrogen/estrogen metabolites (EM), depurinating estrogen-DNA adducts, and the expression of estrogen-metabolizing enzymes in cells treated with 17β-estradiol (E2) alone or in combination with TAM or RAL were investigated using high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS(2)), ultraperformance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS), and Western blot analysis, respectively. TAM significantly increased the total EM and enhanced the formation of hormonally active and carcinogenic estrogen metabolites, 4-hydroxestrone (4-OHE1) and 16α-hydroxyestrone, with concomitant reduction in the formation of antiestrogenic and anticarcinogenic 2-hydroxyestradiol and 2-methoxyestradiol. Furthermore, TAM increased the formation of depurinating estrogen-DNA adducts 4-OHE1 [2]-1-N7Guanine and 4-OHE1 [2]-1-N3 Adenine. TAM-induced alteration in EM and depurinating DNA adduct formation is associated with altered expression of estrogen metabolizing enzymes CYP1A1, CYP1B1, COMT, NQO1, and SF-1 as revealed by Western blot analysis. In contrast to TAM, RAL has minimal effect on EM, estrogen-DNA adduct formation, or estrogen-metabolizing enzymes expression. These data show that TAM perturbs the balance of estrogen-metabolizing enzymes and alters the disposition of estrogen metabolites, which can explain, at least in part, the mechanism for TAM-induced EC. These results also implicate the differential effect of TAM and RAL on estrogen metabolism/metabolites as a potential mechanism for their disparate effects on the endometrium.

Variant metabolizing gene alleles determine the genotoxicity of benzo[a]pyrene

Understanding the mechanisms involved with genetic susceptibility to environmental disease is of major interest to the scientific community. We have conducted an in vitro study to elucidate the involvement of polymorphic metabolizing genes on the genotoxicity of benzo[a]pyrene (BP). Blood samples from 38 donors were treated with BP and the induction of sister chromatid exchanges (SCE) and chromosome aberrations (CA) were evaluated. The latter is based on the tandem‐probe fluorescence in situ hybridization (FISH) assay. The data indicate that the induction of genotoxicity was clearly determined by the inherited variant genotypes for glutathione‐S‐transferase (GSTM1) and microsomal epoxide hydrolase (EH). In a comparison of the two biomarkers, the CA biomarker shows a more definite association with the genotypes than does SCE. For example, the presence of the GSTM1 null genotype (GSTM1 0/0) is responsible for the highest level and significant induction of CA, irrespective of the presence of other genotypes in the different donors. This effect is further enhanced significantly by the presence of the excessive activation EH gene allele (EH4*) and decreased by the reduced activation EH gene allele (EH3*). Overall, the modulation of genotoxicity by the susceptibility genotypes provides support of their potential involvement in environmental cancer. Furthermore, the data indicate that the variant enzymes function independently by contributing their metabolic capability toward the expression of biologic activities. Therefore, studies like this one can be used to resolve the complexity of genetic susceptibility to environmental disease in human. Environ. Mol. Mutagen. 37:17–26, 2001