PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of ijerphMDPI Open Access JournalsMDPI Open Access JournalsThis articleThis JournalInstructions for authorsAdd your e-mail address to receive forthcoming issues of this journal
 
Int J Environ Res Public Health. 2010 May; 7(5): 2045–2056.
Published online 2010 May 4. doi:  10.3390/ijerph7052045
PMCID: PMC2898035

Estrogenic Activity of Coumestrol, DDT, and TCDD in Human Cervical Cancer Cells

Abstract

Endogenous estrogens have dramatic and differential effects on classical endocrine organ and proliferation. Xenoestrogens are environmental estrogens that have endocrine impact, acting as both estrogen agonists and antagonists, but whose effects are not well characterized. In this investigation we sought to delineate effects of xenoestrogens. Using human cervical cancer cells (HeLa cells) as a model, the effects of representative xenoestrogens (Coumestrol-a phytoestrogen, tetrachlorodioxin (TCDD)-a herbicide and DDT-a pesticide) on proliferation, cell cycle, and apoptosis were examined. These xenoestrogens and estrogen inhibited the proliferation of Hela cells in a dose dependent manner from 20 to 120 nM suggesting, that 17-β-estrtadiol and xenoestrogens induced cytotoxic effects. Coumestrol produced accumulation of HeLa cells in G2/M phase, and subsequently induced apoptosis. Similar effects were observed in estrogen treated cells. These changes were associated with suppressed bcl-2 protein and augmented Cyclins A and D proteins. DDT and TCDD exposure did not induce apoptosis. These preliminary data taken together, suggest that xenoestrogens have direct, compound-specific effects on HeLa cells. This study further enhances our understanding of environmental modulation of cervical cancer.

Keywords: xenoestrogens, Coumestrol, DDT, TCDD, Cell Cycle

1. Introduction

Endogenous estrogens, especially 17-β-estradiol, have significant impact on cell mediated and humoral immune and autoimmune responses [15]. Derived from plant or industrial synthesis, environmental xenobiotics with potential estrogenic or hormonal activities are known as xenoestrogens. These compounds are ubiquitous, exhibit bioaccumulation, and act as estrogen agonists or antagonists, disrupting normal endocrine axes [615]. Xenoestrogens have significantly weaker binding affinities than endogenous estrogens to traditional steroid receptors [9,14,15] and their medical, environmental, and societal impact is the frequent subject of debate [12,13]. Representative xenoestrogens include compounds such as coumestrol, a phytoestrogen found in high levels in legumes that acts as an estrogen agonist. Coumestrol has been shown to modulate production of thymic hormones [17]. DDT (o,p-dichlorodiphenyltrichloroethane), a synthetic organochlorine pesticide, has a weak estrogenic agonist activity (as well as androgen antagonist activity) [21] and has been associated with immunosuppression in murine models [2224] and modulation of cell cycle and apoptosis [2527], but its effects on other diseases such as cervical cancer has not been characterized. TCDD (tetrachlorodibenzo-p-dioxin), a polychlorinated biphenyl dioxin, has been widely studied with variable results, having both estrogen agonist and antagonist activity [6,14,21]. It has also been found to modulate cell cycle proteins [29], induce thymic involution [30], and modulate cytokine expression [31,32]. Xenoestrogens may act at the cellular and molecular levels, binding to both steroid and aryl hydrocarbon receptors exhibiting both dependent and independent receptor modulations of specific gene transcriptional elements [29,3235]. As a result, xenoestrogens have the potential to variably modulate cell proliferation, cell cycle progression, apoptosis and cytokine production in much the same way as 17-β-estradiol does [3640]. This modulation is likely to occur in association with alterations in bcl-2 or p53 protein levels [29,38,39]. In this investigation, using the HeLa cell line as a model we explored xenoestrogen-specific effects.

2. Material and Methods

Reagents and Cell Culture

Human cervical cancer (HeLa) cells were purchased from American Type Culture Collection (Rockville, MD), maintained in logarithmic growth, and cultured in DMEM. Cells were cultured in a density between 0.1 and 1.0 × 106/mL. Medium was deemed complete when supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were cultured in suspension at 37°C and 5% CO2 in a humidified incubator and carried at 0.1–2.0 × 106 cells/mL, passaging two to three times weekly as needed. Cells were pelleted and resuspended in fresh complete medium in tissue culture plates 24 h before use in experiments to avoid any confounding gene expression that might occur because of handling. Xenoestrogens were dissolved in 1,4-dioxane or DMSO with final concentrations of solvent in control or treated cultures < 0.1%.

Proliferation Assay

Cells were cultured in triplicate at 1.0 × 106/mL for 72 h, treated with different concentrations (0, 20, 40, 60, 80, 90 nM) of 17-β-estradiol, coumestrol, DDT and TCDD. Proliferation was measured by determination of total viable cell mass using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay Kit (Promega, Madison, WI) according to the manufacturer’s instructions. Absorbance at 490 nm was determined on a Bio-Rad (Hercules, CA) plate reader.

Cell Cycle Analysis

Cell cycle analysis was performed as previously described [38,39]. Briefly, 1 × 106 cells /mL were grown in suspension, harvested by centrifugation, washed, and fixed in 1% paraformaldehyde. After washing, the cells were permeabilized in 70% ethanol, washed, and re-suspended in PBS. RNase (Sigma) was added at a final concentration of 5 U/mL. Cells were stained with propidium iodide (PI). Flow microfluorometry was performed and DNA histograms were generated and analyzed using a Becton Dickinson Flowscan (Franklin Lakes, NJ). This method correlates closely with other measures of apoptosis including TUNEL and Annexin V staining while providing additional cell cycle information [41]. This method also allows for enumeration of the percentages of cells in G0/G1 (resting phase), S (DNA synthesis phase), G2M (mitotic phase), and hypodiploid or apoptotic percentages (those cells containing less than the normal amount of DNA) [41].

Western Blot Analysis

For Western analysis, 5 × 106 cells were cultured, treated with various concentrations of estrogen, coumestrol, DDT and TCDD compounds. Total protein concentration was determined by the method of Bradford using Bio-Rad Protein Assay reagents (Bio-Rad) in a microtiter assay plate. Total cellular protein (30ug) was electrophoresed on 12.5% SDS-PAGE gel, transferred to a polyvinylidine difluoride membrane (Amersham, Arlington Heights, IL) by electroblotting overnight. Membranes were blocked with 10% electrophoresis grade biotin-depleted non-fat dry milk (BioRad) in 1 X PBS (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween-20), rinsed in PBS, probed with monoclonal mouse anti-human bcl-2, Cyclin A and D (BD Bioscience San Diego, CA) using 1:1,000 dilution, and washed 3 times in PBS. The secondary antibody was HRP-conjugated goat anti-mouse whole IgG used at 1:1,000 (Transduction Laboratories). All antibodies were diluted in 1% milk in TBS. Membranes were washed three times. Detection of membrane-bound proteins was carried out by enhanced chemiluminesence with an ECL reagent kit (using 0.06 mL/cm2 of reagent) and Hybond autoradiography film (both Amersham). Biotinylated standards were used for molecular weight determination and were detected with 1:3,000 streptavidin-horseradish peroxidase (Amersham).

3. Results

Xenoestrogen Effects on HeLa Cell Proliferation

Study results show compound-specific effects on accumulation of viable HeLa cell mass. 17-β-estradiol and TCDD were used as representative controls of endogenous and environmental anti-estrogen compounds. In Figure 1, coumestrol, DDT and TCDD exhibited concentration dependent (20 to 120 nM) suppression on HeLa cell proliferation, indicating the cytotoxic effects of 17-β-estradiol [3639]. Viable cell mass was assessed by MTT assay (see Methods). Change in total cell number was confirmed in cell cultures by enumeration (not shown). * p < 0.05 was determined using ANOVA with Bonferroni correction for multiple comparisons.

Figure 1.
HeLa cell viability over 72 h exposure to 20 to 120nM 17-β-estradiol (E), DDT (D), Coumestrol, TCDD vs. control/solvents (C). 17-β-estradiol, DDT, Coumestrol and TCDD had profound effects on HeLa cell proliferation.

Estrogen Suppresses Bcl-2 in a Dose-Dependent Manner

Members of the bcl-2 family are crucial regulators of apoptosis in mammalian cells. The bcl-2 family includes antiapoptotic proteins, such as Bcl-2, and proapoptotic proteins, such as Bax. Since estrogen induced apoptosis is dependent on time and dose, we first tested bcl-2 response to estrogen. HeLa cells demonstrated a dose-dependent decrease (20, 40, 60, 80, 90 nM) in the expression of the antiapoptotic Bcl-2 protein upon estrogen treatment (Figure 2).

Figure 2.
Expression of bcl-2 in HeLa cells exposed to17-β-estradiol (20, 40, 60, 80, and 90 nM) for 24 h. Western blot analysis of bcl-2 expression was performed as indicated in the Materials and Methods. Beta actin expression was used to assess equal ...

Xenoestrogen Modulation of Cell Cycle Phase Distribution

Cell accumulation or growth is a homeostatic balance between proliferation and apoptosis [42,43]. Questions have been raised regarding the MTT assay as a measure of viable cell mass, especially for xenoestrogens [44]. Therefore, xenoestrogen effects on cell cycle phase distribution in HeLa cells was also assessed by propidium iodide (PI) staining [38,39]. Representative cell cycle histograms for 17-β-estradiol, coumestrol, DDT and TCDD are shown in Figure 3, 17-β-estradiol and coumestrol had significant cell cycle phase effect on actively growing HeLa cells, causing redistribution from G0/G1 to apoptosis (p < 0.01), whereas TCDD and DDT had minimal effect, when analyzed by PI staining.

Figure 3.
Histograms showing representative cell cycle phases of β-estradiol, coumestrol, DDT and TCDD treated HeLa cells for 24 H. Flow microfluorometry was performed as indicated in the Materials and Methods. Percent of apoptotic cells (labeled M4) is ...

Xenoestrogen Suppression of Bcl-2 and Stimulation of Cyclin A and D

Bcl-2, Cyclin A and D cell regulatory proteins modulate cell cycle progression and apoptosis [42,43,46]. Given the observed effects of xenoestrogens on HeLa cell proliferation and cell cycle distribution, examination of bcl-2, Cyclin A and D protein levels was performed. As shown in representative Western blots (Figure 4; n = 3), xenoestrogens DDT, coumestrol, and 17-β-estradiol suppressed bcl-2 protein whereas, anti-estrogen, TCDD did not have a significant effect on bcl-2 expression as compared to the control. DDT, coumestrol and TCDD increased Cyclin A and D protein levels to variable degrees in HeLa cells (Figures 5 and and66 respectively). These preliminary results are consistent with recent reports of potential modulation of bcl-2 by xenoestrogens [4750], supporting the concept that xenoestrogens may modulate cancer cell biology through associated changes in bcl-2.

Figure 4.
Expression of bcl-2 protein in β-estradiol, DDT, coumestrol, and TCDD treated HeLa cells for 24 h. Western blot analysis of bcl-2 expression was performed as indicated in the Materials and Methods. β actin expression was used to assess ...
Figure 5.
Expression of cyclin A protein in β-estradiol, DDT, coumestrol, and TCDD treated HeLa cells for 24 h. Western blot analysis of cyclin A expression was performed as indicated in the Materials and Methods. β actin expression was used to ...
Figure 6.
Expression of cyclin D protein in β-estradiol, DDT, coumestrol, and TCDD treated HeLa cells for 24 h. Western blot analysis of cyclin D expression was performed as indicated in the Materials and Methods. β actin expression was used to ...

4. Discussion

In this study we examined xenoestrogen’s direct actions on proliferation, cell cycle phase distribution and apoptosis in Hela cells. While the HeLa cells used in this study are transformed and may not accurately reflect primary cervical cells in vivo, they serve as a useful model and a basis for further examination of direct effects of xenoestrogens. As others have reported [51,52], and we confirmed in this investigation, xenoestrogens have a marked overall suppression effect on HeLa cell proliferation.

Xenoestrogen concentration used in this study, while supraphysiological, were comparable to those known to maximally stimulate estrogen receptor transcriptional activity [53]. Based on previous in vitro studies, xenoestrogens in the range of 20 nM to 120 nM demonstrate maximal effects on cell apoptosis and cell differentiation [28,54] and these high concentrations may be required in transformed cell lines. Although the concentrations in this investigation are some what higher that those found in the environment, long-term bioacccumulative actions may be anticipated to affect cancer cell biology. With receptor binding affinities up to 10,000 fold weaker than endogenous estrogens, the environmental impact of xenoestrogens is the subject of scientific and societal debate [51,55]. These preliminary studies of representative xenoestrogens have been designed to detect and dissect potential effects on HeLa cells.

Xenoestrogens are environmental hormones or compounds that exhibit estrogenic activity. They may interact with or disrupt endogenous estrogenic activity, and, as suggested by some investigations, may have implications for health and disease [27,56]. In the present study, estrogen, coumestrol and DDT, but not TCDD were shown to variably but significantly suppress bcl-2 protein expression in HeLa cells. However, DDT, coumestrol and TCDD upregulated cyclin A and D protein expression. The variability is likely due to xenoestrogenic potency with respect to estrogenic activity [57,58], as well as possible differences in mechanisms of action [2325]. Nevertheless, our experimental data suggest that these specific xenoestrogens, at high concentrations, have specific effects on HeLa cells.

While concentrations of xenoestrogens used in this study may exceed those detected in the environment and the general population, chronic, low level of exposure is known to have biological effects [58,60]. The purpose of this investigation was to identify possible mechanisms of HeLa cell modulation and not necessarily establish environmental exposure-based cause-and-effect evidence. Furthermore, acute in vitro effects cannot be adequately extrapolated to chronic, low dose exposure in vivo effects. Hence, results in the current study should be interpreted with utmost caution. Observed effects occurred only in selected xenoestrogens, implying that effects may be compound-specific and that broad generalization for individual compounds are not appropriate. Data in the current study suggest a direct induction of apoptosis by estrogen and coumestrol, but not DDT AND TCDD. While verification and extension of our results is needed, the apoptosis induced in estrogen and coumestrol suggests at least one of potential effects, by which some xenoestrogens affect cell viability and induce cell death.

The effects observed in the current study may be estrogen receptor dependent or independent [61]. Jeon and Esser have shown that TCDD elicits its biological function through binding of the AHR to distal DNA motifs [62]. However, xenoestrogens may also have AHR receptor independent effects on hela cells and xenoestrogen mechanisms of action are likely pleiotropic [63]. Delineating xenoestrogen-mediated effects through the ER or AHR is pivotal to understanding molecular mechanisms of xenoestrogens, but is beyond the scope of this initial study.

Acknowledgments

This research was financially supported in part by NIH-RCMI grant # G12RR013459.

References

1. Whitacre CC, Reingold SC, O’Looney PA. Task force on gender, multiple sclerosis and autoimmunity. A gender gap in autoimmunity Science 1999. 2831277–1278.1278 Supplementary material available online: www.sciencemag.org/feature/data/983519.shl (accessed on January 2, 2010). [PubMed]
2. Verthelyi D. Sex hormones as immunomodulators in health and disease. Int. Immunopharmacol. 2001;1:983–993. [PubMed]
3. Olsen NJ, Kovacs WJ. Gonadal steroids and immunity. Endocrine. Rev. 1996;17:369–384. [PubMed]
4. Fox HS. Sex steroids and the immune system. Ciba Foundation Symposium. 1995;191:203–211. [PubMed]
5. McMurray RW. Estrogen, prolactin, and autoimmunity: actions and interactions. Int. Immunopharmacol. 2001;1:995–1008. [PubMed]
6. Wolff MS. Environmental estrogens. Environ. Health Perspect. 1995;103:784–785. [PMC free article] [PubMed]
7. Safe SH. Endocrine disrupters and human health—is there a problem? An update. Environ. Health Perspect. 2000;108:487–93. [PMC free article] [PubMed]
8. Kaiser J. Endocrine disrupters. Panel cautiously confirms low-dose effects. Science. 2000;290:695–697. [PubMed]
9. Neubert D. Vulnerability of the endocrine system to xenobiotic influence. Reg. Toxicol. Pharmacol. 1997;26:9–29. [PubMed]
10. Ahmed SA, Hissong BD, Verthelyi D, Donner K, Becker K, Karpuzoglu-Sahin E. Gender and risk of autoimmune diseases: possible role of estrogenic compounds. Environ. Health Perspect. 1999;5:681–686. [PMC free article] [PubMed]
11. Ashby J. Testing for endocrine disruption post-EDSTAC: extrapolation of low dose rodent effects to humans. Toxicol. Lett. 2001;120:233–242. [PubMed]
12. Crinnion WJ. Environmental medicine, part one: the human burden of environmental toxins and their common health effects. Altern. Med. Rev. 2000;5:52–63. [PubMed]
13. Ziegler J. Environmental “endocrine disrupters” get a global look. J. Natl. Cancer Inst. 1997;89:1184–1187. [PubMed]
14. Barton HA, Andersen ME. Endocrine active compounds: from biology to dose response assessment. Crit. Rev. Toxicol. 1998;28:363–423. [PubMed]
15. Barton HA, Andersen ME. Dose-response assessment strategies for endocrine-active compounds. Regul. Toxicol. Pharmacol. 1997;25:292–305. [PubMed]
16. Domon OE, McGarrity LJ, Bishop M, Yoshioka M, Chen JJ, Morris SM. Evaluation of the genotoxicity of the phytoestrogen, coumestrol, in AHH-1 TK(+/−) human lymphoblastoid cells. Mutat. Res. 2001;474:129–137. [PubMed]
17. Sakabe K, Okuma M, Karaki S, Matsuura S, Yoshida T, Aikawa H, Izumi S, Kayama F. Inhibitory effect of natural and environmental estrogens on thymic hormone production in thymus epithelial cell culture. Int. J. Immunopharmacol. 1999;21:861–868. [PubMed]
18. Hiroi H, Tsutsumi O, Momoeda M, Takai Y, Osuga Y, Taketani Y. Differential interactions of bisphenol A and 17beta-estradiol with estrogen receptor alpha (ERalpha) and ERbeta. Endocr. J. 1999;46:773–778. [PubMed]
19. Howdeshell KL, Hotchkiss AK, Thayer KA, Vandenbergh JG, vom Saal FS. Exposure to bisphenol A advances puberty. Nature. 1999;401:763–764. [PubMed]
20. Sakazaki H, Ueno H, Nakamuro K. Estrogen receptor alpha in mouse splenic lymphocytes:possible involvement in immunity. Toxicol. Lett. 2002;133:221–229. [PubMed]
21. Tapiero H, Ba GN, Tew KD. Estrogens and environmental estrogens. Biomed. Pharmacother. 2002;56:36–44. [PubMed]
22. Street JC, Sharma RP. Alteration of induced cellular and humoral immune responses by pesticides and chemicals of environmental concern: quantitative studies of immunosuppression by DDT, aroclor 1254, carbaryl, carbofuran, and methylparathion. Toxicol. Appl. Pharmacol. 1975;32:587–602. [PubMed]
23. Banerjee BD, Koner BC, Ray A. Influence of stress on DDT-induced humoral immune responsiveness in mice. Environ. Res. 1997;74:43–47. [PubMed]
24. Koner BC, Banerjee BD, Ray A. Organochlorine pesticide-induced oxidative stress and immune suppression in rats. Indian J. Exp. Biol. 1998;36:395–398. [PubMed]
25. Dees C, Askari M, Foster JS, Ahamed S, Wimalasena J. DDT mimicks estradiol stimulation of breast cancer cells to enter the cell cycle. Mol. Carcinog. 1997;18:107–114. [PubMed]
26. Diel P, Olff S, Schmidt S, Michna H. Effects of the environmental estrogens bisphenol A, o,p'-DDT, p-tert-octylphenol and coumestrol on apoptosis induction, cell proliferation and the expression of estrogen sensitive molecular parameters in the human breast cancer cell line MCF-7. J. Steroid. Biochem. Mol. Biol. 2002;80:61–70. [PubMed]
27. Ziegler J. Environmental “endocrine disrupters” get a global look. J. Natl. Cancer Inst. 1997;89:1184–1187. [PubMed]
28. Ndebele K, Tchounwou PB, McMurray RW. Effects of xenoestrogens on T lymphocytes: Modulation of Bcl2, p53, and apoptosis. Int. J. Mol. Sci. 2003;4:45–61.
29. Silverstone AE, Frazier DE, Jr, Fiore NC, Soults JA, Gasiewicz TA. Dexamethasone, beta-estradiol, and 2,3,7,8-tetrachlorodibenzo-p-dioxin elicit thymic atrophy through different cellular targets. Toxicol. Appl. Pharmacol. 1994;126:248–259. [PubMed]
30. Kamath AB, Xu H, Nagarkatti PS, Nagarkatti M. Evidence for the induction of apoptosis in thymocytes by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin in vivo. Toxicol. Appl. Pharmacol. 1997;142:367–377. [PubMed]
31. Prell RA, Oughton JA, Kerkvliet NI. Effect of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin on anti-CD3-induced changes in T-cell subsets and cytokine production. Int. J. Immunopharmacol. 1995;17:951–961. [PubMed]
32. Lai ZW, Hundeiker C, Gleichmann E, Esser C. Cytokine gene expression during ontogeny in murine thymus on activation of the aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-pdioxin. Mol. Pharmacol. 1997;52:30–37. [PubMed]
33. Jeon MS, Esser C. The murine IL-2 promoter contains distal regulatory elements responsive to the Ah receptor, a member of the evolutionarily conserved bHLH-PAS transcription factor family. J. Immunol. 2000;165:6975–6983. [PubMed]
34. Kharat I, Saatcioglu F. Antiestrogenic effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin are mediated by direct transcriptional interference with the liganded estrogen receptor. Cross-talk between aryl hydrocarbon- and estrogen-mediated signaling. J. Biol. Chem. 1996;271:10533–10537. [PubMed]
35. Hossain A, Tsuchiya S, Minegishi M, Osada M, Ikawa S, Tezuka FA, Kaji M, Konno T, Watanabe M, Kikuchi H. The Ah receptor is not involved in 2,3,7,8-tetrachlorodibenzo- pdioxin-mediated apoptosis in human leukemic T cell lines. J. Biol. Chem. 1998;273:19853–19858. [PubMed]
36. Blagosklonny MV, Neckers LM. Cytostatic and cytotoxic activity of sex steroids against human leukemia cell lines. Cancer Letters. 1994;76:81. [PubMed]
37. Kincade PW, Medina KL, Smithson G. Sex hormones as negative regulators of lymphopoiesis. Immunol. Rev. 1994;137:119–134. [PubMed]
38. Jenkins JK, Suwannaroj S, Elbourne KB, Ndebele K, McMurray RW. 17-β-estradiol alters Jurkat lymphocyte cell cycling and induces apoptosis through suppression of bcl-2 and cyclin A. Internat. J. Immunopharmacol. 2001;11:1897–1911. [PubMed]
39. McMurray RW, Suwannaroj S, Ndebele K, Jenkins JK. Differential effects of sex steroids on T and B lymphocytes: modulation of cell cycling, apoptosis, and bcl-2. Pathobiol. 2001;69:44–58. [PubMed]
40. McMurray RW, Ndebele K, Jenkins JK. 17-β-estradiol suppresses IL-2 and IL-2 receptor. Cytokine. 2001;14:324–333. [PubMed]
41. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods. 1991;139:271. [PubMed]
42. Cohen JJ. Programmed cell death in the immune system. Adv. Immunol. 1991;50:55. [PubMed]
43. King KL, Cidlowski JA. Cell cycle and apoptosis: common pathways to life and death. J. Cell Biochem. 1995;58:175. [PubMed]
44. Pagliacci MC, Spinozzi F, Migliorati G, Fumi G, Smacchia M, Grignani F, Riccardi C, Nicoletti I. Genistein inhibits tumour cell growth in vitro but enhances mitochondrial reduction of tetrazolium salts: a further pitfall in the use of the MTT assay for evaluating cell growth and survival. Eur. J. Cancer. 1993;29A:1573–1577. [PubMed]
45. Neumann CM, Oughton JA, Kerkvliet NI. Anti-CD3-induced T-cell activation—II. Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) Intenat. J. Immunopharmacol. 1993;15:543–550. [PubMed]
46. Huang DC, O’Reilly LA, Strasser A, Cory S. The anti-apoptosis function of Bcl-2 can be genetically separated from its inhibitory effect on cell cycle entry. EMBO Journal. 1997;16:4628–4635. [PubMed]
47. Kannan K, Holcombe RF, Jain SK, Alvarez-Hernandez X, Chervenak R, Wolf RE, Glass J. Evidence for the induction of apoptosis by endosulfan in a human T-cell leukemic line. Mol. Cell. Biochem. 2000;205:53–66. [PubMed]
48. Roy D, Palangat M, Chen CW, Thomas RD, Colerangle J, Atkinson A, Yan ZJ. Biochemical and molecular changes at the cellular level in response to exposure to environmental estrogen-like chemicals. J. Toxicol. Environ. Health. 1997;50:1–29. [PubMed]
49. Rininger JA, Stoffregen DA, Babish JG. Murine hepatic p53, RB, and CDK inhibitory protein expression following acute 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. Chemosphere. 1997;34:1557–1568. [PubMed]
50. Burrow ME, Tang Y, Collins-Burow BM, Krajewski S, Reed JC, McLachlan JA, Beckman BS. Effects of environmental estrogens on tumor necrosis factor alpha-mediated apoptosis in MCF-7 cells. Carcinogenesis. 1999;20:2057–2061. [PubMed]
51. Schimpl A, Berberich I, Kneitz B, Kramer S, Santner-Nanan B, Wagner S, Wolf M, Hunig T. IL-2 and autoimmune disease. Cytokine Growth Factor Rev. 2002;13:369–378. [PubMed]
52. Nohara K, Fujimaki H, Tsukumo S, Inouye K, Sone H, Tohyama C. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on T cell-derived cytokine production in ovalbumin (OVA)-immunized C57Bl/6 mice. Toxicology. 2002;172:49–58. [PubMed]
53. Karin M, Delhase M. The I kappa B kinase and NF-kB: key elements of proinflammatory signalling. Semin. Immunol. 2000;12:85–98. [PubMed]
54. Landegren U, Andersson J, Wigzell H. Analysis of human T lymphocyte activation in a T cell tumor model system. Eur. J. Immunol. 1985;15:308–311. [PubMed]
55. Ray A, Ray P. Down modulation of interleukin 6 gene expression by 17B estradiol in the absence of high affinity DNA binding by the estrogen receptor. J. Biol. Chem. 1994;269:12940–12946. [PubMed]
56. Wolff MS. Environmental estrogens. Environ. Health Perspect. 1995;103:784–785. [PMC free article] [PubMed]
57. Safe SH. Endocrine disrupters and human health—is there a problem? An update. Environ. Health Perspect. 2000;108:487–493. [PMC free article] [PubMed]
58. Kaiser J. Endocrine disrupters. Panel cautiously confirms low-dose effects. Science. 2000;290:695–697. [PubMed]
59. Sohoni P, Sumpter JP. Several environmental oestrogens are also anti-androgens. J. Endocrinol. 1998;158:327–339. [PubMed]
60. Ulrich EM, Caperell-Grant A, Jung SH, Hites RA, Bigsby RM. Environmentally relevant xenoestrogen tissue concentrations correlated to biological responses in mice. Environ. Health Perspect. 2000;108:973–977. [PMC free article] [PubMed]
61. Frigo DE, Burow ME, Mitchell K, Chiang TC, McLachlan JA. DDT and its metabolites alter gene expression in human uterine cell lines through estrogen receptor-independent mechanisms. Environ. Health Perspect. 2002;110:1239–1245. [PMC free article] [PubMed]
62. Jeon MS, Esser C. The murine IL-2 promoter contains distal regulatory elements responsive to the Ah receptor, a member of the evolutionarily conserved bHLH-PAS transcription factor family. J. Immunol. 2000;165:6975–6983. [PubMed]
63. Kharat I, Saatcioglu F. Antiestrogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin are mediated by direct transcriptional interference with the liganded estrogen receptor. Cross talk between aryl hydrocarbon- and estrogen-mediated signaling. J. Biol. Chem. 1996;271:10533–10537. [PubMed]

Articles from International Journal of Environmental Research and Public Health are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)