Td corrigé bisphenol a pdf

bisphenol a

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This is posted on the web at:  HYPERLINK "http://endocrinedisruptors.missouri.edu/vomsaal/vomsaal.html" http://endocrinedisruptors.missouri.edu/vomsaal/vomsaal.html

CURRENT NUMBER OF PUBLISHED STUDIES REPORTING SIGNIFICANT ADVERSE EFFECTS OR NO EVIEDNCE OF HARM DUE TO ADMINISTRATION OF LOW DOSES OF BISPHENOL A (BELOW 50 MG/KD/DAY) TO EXPERIMENTAL ANIMALS

176 STUDIES ON LOW DOSE EFFECTS OF BISPHENOL A IN ANIMALS
149 published studies reporting significant, and in many cases clearly adverse, effects
27 published studies reporting no evidence of harm
There are 2 major factors that predict the finding of no evidence of harm in these studies:
Strain of rat used was a predictor of finding harm or no harm:
13 / 27 studies reporting no harm used the CD-SD (Crl:CD) rat
– all 13 (100%) of the CD-SD rat studies reported no harm
(3 / 13 negative studies funded by chemical corporations used the CD-SD rat)
(10 / 14 negative studies funded by governments used the CD-SD rat)
The Charles River Sprague-Dawley (CD-SD) rat colony was subjected to selection over a 40-year period by Charles River for very large litters and large body size, and CD-SD rats are no longer similar in phenotype to the Sprague-Dawley rats that were purchased by Charles River in 1950. CD-SD rats require high doses of potent estrogenic drug ethinylestradiol to show responses. The CD-SD strain of rat is insensitive to any estrogenic chemical, not just bisphenol A.

2. Source of funding:
13 / 27 studies reporting no harm were funded by chemical corporations

SOURCE OF STUDY OUTCOME
FUNDING HARM NO HARM TOTAL
-----------------------------------------------------------------------------------------------------
Government 149 (93%) 14 (7%) 163 (92%)

Chemical Corporations 0 (0%) 13 (100%) 13 (8%)
-----------------------------------------------------------------------------------------------------
149 27 176

This is a highly biased distribution of outcomes based on source of funding.

CONTENTS

I. BISPHENOL A: LOAEL AND REFERENCE DOSE

II. REVIEW OF THE “LOW DOSE” ISSUE, INCLUDING LOW DOSE STUDIES OF BISPHENOL A, AS OF OCTOBER, 2000 BY THE US-NIH

III. ARTICLES DISCUSSING IMPLICATIONS FOR RISK ASSESSMENT OF FINDINGS: 1. AT DOSES BELOW THE PRIOR LOAEL (50 MG/KG/DAY), 2. OF NON-MONOTONIC (INVERTED-U) FUNCTIONS, AND 3. NO THRESHOLD

IV. REPORT IN 1936 THAT BISPHENOL A HAD FULL ESTROGENIC ACTIVITY IN A STUDY OF ESTROGENIC DRUGS

V. DEFINITION OF “LOW DOSE”

VI. EPIDEMIOLOGICAL STUDIES OF THE RELATIONSHIP BETWEEN BISPHENOL A AND DISEASE IN HUMANS

VII. IN VIVO STUDIES PUBLISHED IN PEER-REVIEWED JOURNALS
REPORTING THAT BISPHENOL A CAUSES SIGNIFICANT EFFECTS IN ANIMALS
WITH DOSES AT AND BELOW THE PUBLISHED LOAEL OF 50 mg/kg/day;
SIGNIFICANT EFFECTS AT DOSES IN THE PPB RANGE FOR AQUATIC ANIMALS ARE ALSO INCLUDED

VIII. FINDINGS OF SIGNIFICANT EFFECTS FROM THE ABOVE LIST USING DOSES AT AND BELOW THE REFERENCE DOSE OF 50 µg/kg/day

IX. INVERTED-U DOSE-RESPONSE CURVES

X. DES SHOWS SAME EFFECTS AS BISPHENOL A

XI. INSENSITIVITY OF THE UTEROTROPHIC ASSAY TO LOW DOSES OF BISPHENOL A WHEN USING SOME STRAINS OF RATS AND MICE

XII. CHEMICAL INDUSTRY FUNDED STUDY OF BISPHENOL REPORTED TO THE US-EPA AND THE PUBLIC AS HAVING NEGATIVE RESULTS, BUT WITH POSITIVE RESULTS AS DETERMINED BY A NIH REVIEW PANEL

XIII. CHEMICAL INDUSTRY FUNDED STUDIES INCORRECTLY DESCRIBED AS REPLICATING PROCEDURES BY VOM SAAL (DIFFERENT FEED WAS USED) THAT REPORTED NEGATTIVE RESULTS FOR BOTH BISPHENOL A AND THE POSITIVE CONTROL CHEMICAL, DES

XIV. STUDIES REPORTING NO IN VIVO EFFECTS OF LOW DOSES OF BISPHENOL THAT USED AN INSENSITIVE RAT, THE CHARLES RIVER SPRAGUE-DAWLEY (CD-SD) RAT

XV. FINDINGS OF NO SIGNIFICANT EFFECTS IN EXPERIMENTAL LABORATORY ANIMALS AT DOSES OF BISPHENOL A BELOW 50 MG/KG/DAY OR AT PPB DOSES IN AQUATIC ANIMALS

XVI. IN VITRO STUDIES OF MOLECULAR MECHANISMS

XVII. BISPHENOL A BINDING AND RESPONSES WITH ER-alpha AND ER-beta

XVIII. EXPOSURE LEVELS IN HUMAN ADULTS AND FETUSES

XIX. BISPHENOL A METABOLISM STUDIES IN MICE AND RATS, INCLUDING EXPOSURE DURING POSTNATAL LIFE AND OF FETUSES AFTER MATERNAL EXPOSURE

XX. EXPOSURE AND METABOLISM OF BISPHENOL A IN OTHER SPECIES

XXI. ENVIRONMENTAL MONITORING AND ANALYTICAL METHODS

XXII. LEACHING OF BISPHENOL A FROM PRODUCTS

XXIII. ESTROGENIC ACTIVITY OF BISPHENOL A DIMETHACRYLATE (BIS-DMA), BISPHENOL A GLYCEROLATE DIMETHACRYLATE (BIS-GMA) AND BISPHENOL A DIGLYCIDYL ETHER (BADGE), WHICH IS A PPAR GAMMA-SELECTIVE ANTAGONIST, IN DENTAL SEALANTS

XXIV. BISPHENOL A AND CANCER

XXV. BROMINATED BISPHENOL A (FLAME RETARDANT)

XXVI. BISPHENOL A EFFECTS IN PLANTS




I. BISPHENOL A: LOAEL AND REFERENCE DOSE

CASRN (80-05-7)
1 nM = 228 ppt (molecular weight = 228)
LOAEL = 50 mg/kg/day, Based on studies in the 1980s that used only very high doses, the lowest dose that was examined (50 mg/kg/day) caused adverse effects, and this is termed the LOAEL
Acceptable Daily Intake level (also called the Reference Dose) = 50 µg/kg/day. The amount that is predicted based on models to be safe for humans is calculated by dividing the LOAEL by 1000 (see IRIS, US-EPA). Until the 1990’s the assumption that 50 µg/kg/day bisphenol A was safe was not challenged by directly examining this dose to see if it actually caused effects.
 ADDIN EN.REFLIST IRIS (1988). Bisphenol A. (CASRN 80-05-7), US-EPA Integrated Risk Information System Substance file.  HYPERLINK "http://www.epa.gov/iris/subst/0356.htm" http://www.epa.gov/iris/subst/0356.htm. Accessed, 2002.


II. US-NATIONAL TOXICOLOGY PROGRAM WITHIN THE NIH: PEER REVIEW OF THE “LOW DOSE” LITERATURE, INCLUDING BISPHENOL A, OCTOBER, 2000

NTP, N. T. P. (2001). Endocrine Disruptors Low Dose Peer Review, Raleigh, NC. http://ntp.niehs.nih.gov/index.cfm?objectid=06F5CE98-E82F-8182-7FA81C02D3690D47

III. REVIEW ARTICLES CONCERNING BISPHNENOL A IN RELATION TO:
1. DOSES BELOW THE PRIOR LOAEL (50 mg/kg/day)
2. NON-MONOTONIC (INVERTED-U) DOSE-RESPONSE FUNCTIONS
3. NO THRESHOLD FOR ENDOCRINE DISRUPTING CHEMICALS
4. RECYCLING OF PLASTIC PRODUCTS
5. IMPLICATIONS FOR RISK ASSESSMENT
6. HUMAN EXPOSURE

Report of the Berkeley Plastics Task Force on the Manufacture and Recycling of Plastics. The Berkeley Ecology Center, Berkeley, California.
 HYPERLINK "http://www.mindfully.org/Berkeley/Berkeley-Plastics-Task-Force.htm" http://www.mindfully.org/Berkeley/Berkeley-Plastics-Task-Force.htm
 HYPERLINK "http://www.ecologycenter.org/iptf/plastic_types/index.html" http://www.ecologycenter.org/iptf/plastic_types/index.html

Calafat, AM, Ye, X, Silva, MJ, Kuklenyik, Z and Needham, LL (2006). Human exposure assessment to environmental chemicals using biomonitoring. Int. J. Androl. 29: 166-171; discussion 181-185.
In modern societies, humans may be exposed to a wide spectrum of environmental chemicals. Although the health significance of this exposure for many chemicals is unknown, studies to investigate the prevalence of exposure are warranted because of the chemicals' potential harmful health effects, as often indicated in animal studies. Three tools have been used to assess exposure: exposure history/questionnaire information, environmental monitoring, and biomonitoring (i.e. measuring concentrations of the chemicals, their metabolites, or their adducts in human specimens). We present an overview on the use of biomonitoring in exposure assessment using phthalates, bisphenol A and other environmental phenols, and perfluorinated chemicals as examples. We discuss some factors relevant for interpreting and understanding biomonitoring data, including selection of both biomarkers of exposure and human matrices, and toxicokinetic information. The use of biomonitoring in human risk assessment is not discussed.

Maffini, MV, Rubin, BS, Sonnenschein, C and Soto, AM (2006). Endocrine disruptors and reproductive health: The case of bisphenol A. Mol. Cell Endocrinol. 254-255: 179-186.
Epidemiological studies have reported that during the last 60 years the quantity and quality of human sperm has decreased and the incidence of male genital tract defects, testicular, prostate and breast cancer has increased. During the same time period, developmental, reproductive and endocrine effects have also been documented in wildlife species. The last six decades have witnessed a massive introduction of hormonally active synthetic chemicals into the environment leading some to postulate that the diverse outcomes documented in human and wildlife populations might be the result of extemporaneous exposure to xenoestrogens during development. The estrogen-mimic bisphenol-A (BPA) is used as a model agent for endocrine disruption. BPA is used in the manufacture of polycarbonate plastics and epoxy resins from which food and beverage containers and dental materials are made. Perinatal exposure to environmentally relevant BPA doses results in morphological and functional alterations of the male and female genital tract and mammary glands that may predispose the tissue to earlier onset of disease, reduced fertility and mammary and prostate cancer.

Sheehan, D. M., Willingham, E., Gaylor, D., Bergeron, J. M. and Crews, D. (1999). No threshold dose for estradiol-induced sex reversal of turtle embryos: how little is too much? Environ. Health Perspect. 107:155-159.

Sheehan, D. M. (2000). Activity of environmentally relevant low doses of endocrine disruptors and the bisphenol A controversy: Initial results confirmed. Proc Soc Exp Biol Med 224:57-60.
This article by Sheehan discusses the importance to science and regulators of the publication by Gupta reporting that she replicated our findings with bisphenol A in a series of studies, validating prior published findings in the eyes of unbiased scientists.

Sheehan, D.M. (2006). No-threshold dose-response curves for nongenotoxic chemicals: Findings and applications for risk assessment. Environ Res. 100:93-99.
We tested the hypothesis that no threshold exists when estradiol acts through the same mechanism as an active endogenous estrogen. A Michaelis-Menten (MM) equation accounting for response saturation, background effects, and endogenous estrogen level fit a turtle sex-reversal data set with no threshold and estimated the endogenous dose. Additionally, 31 diverse literature dose-response data sets were analyzed by adding a term for nonhormonal background; good fits were obtained but endogenous dose estimations were not significant due to low resolving power. No thresholds were observed. Data sets were plotted using a normalized MM equation; all 178 data points were accommodated on a single graph. Response rates from approximately 1% to >95% were well fit. The findings contradict the threshold assumption and low-dose safety. Calculating risk and assuming additivity of effects from multiple chemicals acting through the same mechanism rather than assuming a safe dose for nonthresholded curves is appropriate.

vom Saal, F.S. and Sheehan, D.M. Challenging risk assessment. Forum for Appllied Research and Public Policy, 13(3):11-18, 1998.

vom Saal, F.S. and Welshons, W.V. NIH panel confirms that endocrine disrupting chemicals cause effects at very low doses. Risk Policy Report 7(11):47-50, November 30, 2000. Inside Washington Publishers. Source: Risk Policy Report via InsideEPA.com

vom Saal, F. S. V.; Richter, C. A.; Ruhlen, R. R.; Nagel, S. C.; Timms, B. G., and Welshons, W. V. The importance of appropriate controls, animal feed, and animal models in interpreting results from low-dose studies of bisphenol A. Birth Defects Research Part A-Clinical & Molecular Teratology. 2005; 73(3):140-145. ISSN: 1542-0752. Interpreting results of studies that report only negative effects is problematic. A number of published studies to determine whether chemicals with estrogenic activity can cause effects at low doses have not taken into account the possibility that the commercial animal feed being used can mask effects of even potent estrogenic drugs such as diethylstilbestrol (DES). In addition, the sensitivity of the strain of animal being used for the specific category of chemical being tested has not always been described. For environmental chemicals, such as the estrogenic polycarbonate plastic monomer bisphenol A, DES is an appropriate positive control for estrogenic effects, and using an appropriate low dose of DES can eliminate the possibility of false-negative conclusions of safety when the above or other variables contribute to the negative outcome. Only when simultaneous positive effects of low doses of a positive control chemical such as DES and negative effects of environmentally relevant low doses of the test chemical are demonstrated within the same experiment are conclusions of no effect of the test chemical warranted, and this has not been reported for bisphenol A in any study. Instead, more than 90 refereed journal publications have reported effects due to exposure to low doses of bisphenol A in a wide variety of animals (for references see: http://rcp.missouri.edu/endocrinedisruptors/vomsaal/vomsaal.html). However, due to lack of attention to the importance of appropriate positive controls, a small number of studies reporting negative effects of bisphenol A have created a false sense of controversy regarding low-dose effects of bisphenol A.
vom Saal, F.S. and Hughes, C. (2005). An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment. Environ. Health Perspect. 113:926-933.
Bisphenol A (BPA) is the monomer used to manufacture polycarbonate plastic, the resin lining of cans, and other products, with global capacity in excess of 6.4 billion lb/year. Because the ester bonds in these BPA-based polymers are subject to hydrolysis, leaching of BPA has led to widespread human exposure. A recent report prepared by the Harvard Center for Risk Analysis and funded by the American Plastics Council concluded that evidence for low-dose effects of BPA is weak on the basis of a review of only 19 studies; the report was issued after a delay of 2.5 years. A current comprehensive review of the literature reveals that the opposite is true. As of December 2004, there were 115 published in vivo studies concerning low-dose effects of BPA, and 94 of these report significant effects. In 31 publications with vertebrate and invertebrate animals, significant effects occurred below the predicted "safe" or reference dose of 50 microg/kg/day BPA. An estrogenic mode of action of BPA is confirmed by in vitro experiments, which describe disruption of cell function at 10(superscript)- 12(/superscript) M or 0.23 ppt. Nonetheless, chemical manufacturers continue to discount these published findings because no industry-funded studies have reported significant effects of low doses of BPA, although > 90% of government-funded studies have reported significant effects. Some industry-funded studies have ignored the results of positive controls, and many studies reporting no significant effects used a strain of rat that is inappropriate for the study of estrogenic responses. We propose that a new risk assessment for BPA is needed based on a) the extensive new literature reporting adverse effects in animals at doses below the current reference dose; b) the high rate of leaching of BPA from food and beverage containers, leading to widespread human exposure; c) reports that the median BPA level in human blood and tissues, including in human fetal blood, is higher than the level that causes adverse effects in mice; and d) recent epidemiologic evidence that BPA is related to disease in women.

vom Saal, F.S. and Welshons, W.V. Large effects from small exposures: II. The importance of positive controls in low-dose research on bisphenol A. Environmental Research 100:50-76, 2006.
Over six-billion pounds per year of the monomer bisphenol A (BPA) are used to manufacture polycarbonate plastic products, resins lining cans, dental sealants, and polyvinyl chloride plastic products. There are 109 published studies as of July 2005 that report significant effects of low doses of BPA in experimental animals, with many adverse effects occurring at blood levels in animals within and below average blood levels in humans; 40 studies report effects below the current reference dose of 50mug/kg/day that is still assumed to be safe by the US-FDA and US-EPA in complete disregard of the published findings. The extensive list of significant findings from government-funded studies is compared to the 11 published studies that were funded by the chemical industry, 100% of which conclude that BPA causes no significant effects. We discuss the importance of appropriate controls in toxicological research and that positive controls are required to determine whether conclusions from experiments that report no significant effects are valid or false.

 ADDIN EN.REFLIST vom Saal, F.S., Nagel, S.C., Timms, B.G. and Welshons, W.V. (2005). Implications for human health of the extensive bisphenol A literature showing adverse effects at low doses: a response to attempts to mislead the public. Toxicology 212:244-52, author reply 253-4.

Welshons, W.V., Thayer, K.S., Taylor, J., Judy, B. and vom Saal, F.S. (2003). Large effects from small exposures: I. Mechanisms for endocrine-disrupting chemicals with estrogenic activity. Environ. Health Perspect. 111(8): 994-1006.

Welshons, W.V., Nagel, S.C. and vom Saal, F.S. Large effects from small exposures: III. Mechanisms mediating responses to the low doses of the plastic monomer bisphenol A. Endocrinol. 147:S56-S69, 2006.
Over 6 billion pounds per year of the estrogenic monomer bisphenol A (BPA) are used to manufacture polycarbonate plastic products, in resins lining metal cans, in dental sealants, and in blends with other types of plastic products. The ester bond linking BPAmolecules in polycarbonate and resins undergoes hydrolysis, resulting in the release of free BPA into food, beverages, and the environment, and numerous monitoring studies now show almost ubiquitous human exposure to biologically active levels of this chemical. BPA exerts estrogenic effects through the classical nuclear estrogen receptors, and BPA acts as a selective estrogen receptor modulator. However, BPA also initiates rapid responses via estrogen receptors presumably associated with the plasma membrane. Similar to estradiol, BPA causes changes in some cell functions at concentrations between 1 pM and 1 nM, and the mean and median range of unconjugatedBPAmeasured by multiple techniques in human pregnant maternal, fetal, and adult blood and other tissues exceeds these levels. In contrast to these published findings, BPA manufacturers persist in describing BPA as a weak estrogen and insist there is little concern with human exposure levels. Our concern with human exposure to BPA derives from 1) identification of molecular mechanisms mediating effects in human and animal tissues at very low doses, 2) in vivo effects in experimental animals caused by low doses within the range of human exposure, and 3) widespread human exposure to levels of BPA that cause adverse effects in animals.


IV. REPORT IN 1936 THAT BISPHENOL A HAD FULL ESTROGENIC ACTIVITY IN A STUDY OF ESTROGENIC CHEMICALS, AND THEN IN 1938, REPORT ON THE SYNTHESIS OF A SIMILAR MOLEDULE, DES
(The 1936 publication was 17 years prior to synthesis of polycarbonate from bisphenol A in 1952)

Dodds, E.C. and Lawson, W. Synthetic oestrogenic agents without the phenanthrene nucleus. Nature 137:996, 1936.
Approximately 70 mg/kg/day bisphenol A was injected into ovariectomized adult rats, and cornification of the vaginal epithelium, similar to that caused by estradiol, was observed.

Dodds, E. C., W. Lawson and R. L. Noble (1938). Biological effects of the synthetic oestrogenic substance 4: 4'-dihydroxy- a: B-dimethylstilbene. Lancet 234:1389-1391.
Sir Charles Dodds received the Nobel prize for discovering the extremely potent estrogenic drug diethylstilbestrol (DES) in 1938, which was more powerful than any of the estrogenic chemicals, including bisphenol A, examined in this 1936 article. Bisphenol A was thus never used as a drug, and, instead, in 1957 bisphenol A was used as the monomer to make polycarbonate plastic and resins used to line cans.


V. DEFINITION OF “LOW DOSE”
Studies in which doses below the currently accepted LOAEL reported by the US-EPA (IRIS) of 50 mg/kg/day showed significant effects are included here in the list of low-dose positive effects, since they would impact the LOAEL used to calculate the acceptable daily intake value.

An alternative approach suggested by the NTP Low Dose Peer Review Panel (2001) was that doses 10-fold below the LOAEL (5 mg/kg/day) were recommended for inclusion in the new “low dose” range, based on the assumption that doses 10-fold below the LOAEL can be considered the no adverse effect level (NOAEL). This assumption has now shown to be false by the large number of studies cited below that show effects not only below 5 mg/kg/day, but below the predicted safe dose of 50 µg/kg/day.


VI. EPIDEMIOLOGICAL STUDIES OF THE RELATIONSHIP BETWEEN BISPHENOL A AND DISEASE IN HUMANS

Chu, CY, Ponten, A, Sun, CC and Jee, SH (2006). Concomitant contact allergy to the resins, reactive diluents and hardener of a bisphenol A/F-based epoxy resin in subway construction workers. Contact Dermatitis 54: 131-139.
An outbreak of suspected contact dermatitis among subway construction workers was suspected to be due to a new bisphenol A/F-based epoxy resin system (ERS). The construction workers used ERSs during the insertion of iron bars into concrete walls. The objective of the study was to determine the components (if any) of the ERS responsible for the contact allergy. Patch testing was performed on 20 of the 22 construction workers who had had contact with the ERS, and to the various subcomponents of component A on 5 of the 7 who reacted to this component. 9 patients (9/22, 40.9%) had clinical symptoms and signs of suspected contact dermatitis at presentation. 7 of these 9, but none of the 11 asymptomatic individuals, were positive to component A, while all were negative to component B. Of the 5 cases receiving further patch testing, all reacted to m-xylylene diamine, 4 to 1,6-hexanediol diglycidyl ether, 3 to epoxy resins of the bisphenol F-type and trimethylolpropane triglycidyl ether 0.25% petrolatum, and only 1 to epoxy resins of the bisphenol A-type. Contact allergy to ERSs may involve hardeners and diluents as well as resins, and patch testing for reaction to all components should be performed.

 ADDIN EN.REFLIST Hiroi, H., Tsutsumi, O., Takeuchi, T., Momoeda, M., Ikezuki, Y., Okamura, A., Yokota, H. and Taketani, Y. (2004). Differences in serum bisphenol A concentrations in premenopausal normal women and women with endometrial hyperplasia. Endocr J 51:595-600.
 Exposure to endocrine disrupting chemicals (EDCs) has been raised in relation to its potential for adverse health outcomes. Bisphenol A (BPA) is an estrogenic EDC widely found in plastic products. We determined BPA concentrations in premenopausal women by an enzyme-linked immunosorbent assay and evaluated possible linkage between its contamination levels and endometrial hyperplasia, an estrogen-related disorder of the uterus. It has been implied that higher levels of BPA, which binds to estrogen receptor and plays estrogenic roles may, enhance endometrial hyperplasia. Serum BPA was detectable in all subjects and its concentrations in healthy controls with normal endometrium were 2.5 +/- 1.5 ng/ml (mean +/- SD). BPA levels in patients with simple endometrial hyperplasia with benign nature were 2.9 +/- 2.0 ng/ml and were not significantly different from the controls. Unexpectedly, BPA levels in patients with complex endometrial hyperplasia with malignant potential were 1.4 +/- 0.4 ng/ml and significantly lower compared to both control and simple endometrial hyperplasia groups. In addition, we measured the serum BPA levels in postmenopausal endometrial cancer patient (1.4 +/- 0.5 ng/ml), which were also significantly lower than control and simple endometrial hyperplasia groups. These findings suggest the presence of associations between BPA exposure and complex endometrial hyperplasia and endometrial cancer. The mode of action of BPA may be more complex than expected and the contradictory results may serve as a clue to addressing the mechanisms of linkage between occurrence of estrogen-dependent diseases and endocrine disruption.

Sugiura-Ogasawara, M., Ozaki, Y., Sonta, S., Makino, T. and Suzumori, K. (2005). Exposure to bisphenol A is associated with recurrent miscarriage. Human Reproduction Online, June 9.
BACKGROUND: Little is known about the influence of high exposure to bisphenol A on recurrent miscarriage and immunoendocrine abnormalities. METHODS: Serum bisphenol A, antiphospholipid antibodies (aPLs), antinuclear antibodies (ANAs), natural killer cell (NK) activity, prolactin, progesterone, thyroid-stimulating hormone (TSH) and free T4 were examined in 45 patients with a history of three or more (3–11) consecutive first-trimester miscarriages and 32 healthy women with no history of live birth and infertility. Subsequent pregnancy outcome and embryonic karyotype of abortuses were examined prospectively. RESULTS: The mean 6 SD values for bisphenol A in patients were 2.59 6 5.23 ng/ml, significantly higher than the 0.77 6 0.38 ng/ml found for control women (P 5 0.024). High exposure to bisphenol A was associated with the presence of ANAs but not hypothyroidism, hyperprolactinaemia, luteal phase defects, NK cell activity or aPLs. A high level of bisphenol A in itself did not predict subsequent miscarriage. CONCLUSION: Exposure to bisphenol A is associated with recurrent miscarriage.

Takeuchi T, Tsutsumi O, Ikezuki Y, Takai Y, Taketani Y. 2004. Positive relationship between androgen and the endocrine disruptor, bisphenol A, in normal women and women with ovarian dysfunction. Endocr J 51:165-9. This study was performed to investigate the serum levels of bisphenol A (BPA), an endocrine disruptor, in women with ovarian dysfunction and obesity. Fasting serum samples were obtained from 19 non-obese and 7 obese women with normal menstrual cycles: 7 patients with hyperprolactinemia, 21 patients with hypothalamic amenorrhea, and 13 non-obese and 6 obese patients with polycystic ovary syndrome (PCOS). BPA was measured by an enzyme-linked immunosorbent assay. BPA was detected in all human sera. Serum BPA concentrations were significantly higher in both non-obese and obese women with polycystic ovary syndrome (1.05 +/- 0.10 ng/ml, 1.17 +/- 0.16 ng/ml; p500 microg/L. When BPA was reacted with sodium hypochlorite (24 hours; residual chlorine at 1 ppm), however, complete decomposition of BPA and its chlorinated derivatives was observed. The decrease in BPA and its chlorinated derivatives paralleled the decrease in estrogenic potency evaluated by the induction of vitellogenin (VTG) in the serum of mature male Japanese medaka. Induction of VTG by BPA occurred at 200 ppb.

 ADDIN EN.REFLIST Takahashi, O. and S. Oishi (2003). Testicular toxicity of dietarily or parenterally administered bisphenol A in rats and mice. Food Chem Toxicol 41:1035-44.
Male Crj:Wistar rats, HsdHot:Holtzman SD rats, Crj:CD-1(ICR) mice and C57BL/6CrSlc mice were administered bisphenol A (BPA) in the diet at a level of 0 (control) and 0.25% for 8 weeks. Daily BPA intake was about 200 and 400 mg/kg for rats and mice, respectively. No conspicuous signs of general or reproductive toxicity were observed after administration in any strain of these animals. Serum testosterone concentrations were not decreased in BPA-fed rats and mice. Successive subcutaneous administration of BPA at a dose of 200 mg/kg/day for 4 weeks significantly decreased the testis, epididymis, prostate and seminal vesicle weights, and the testicular daily sperm production in Jcl:Wistar rats. Successive intraperitoneal administration of BPA at a dose of 20 mg/kg/day for 4 weeks decreased the prostate and seminal vesicle weights (but not the testis or epididymis weights) and also decreased serum testosterone and both liver and kidney weight. An intraperitoneal dose of 2 mg BPA/kg/day to Wistar rats did not cause any toxicity. These results indicate that dietarily administered BPA is less toxic to most strains of rats and mice, and the maximum non-toxic dose and/or minimum toxic dose may be about 200 mg/kg/day. Subcutaneous or intraperitoneal BPA is much more toxic on male reproductive and sex accessory organs than dietary.

Takai, Y., Tsutsumi, O., Ikezuki, Y., Hiroi, H., Osuga, Y., Momoeda, M., Yano, T. and Taketani, Y. (2000). Estrogen receptor-mediated effects of a xenoestrogen, bisphenol A, on preimplantation mouse embryos. Biochem Biophys Res Commun 270:918-921.
Bisphenol A at doses of 228 ppt and 684 ppt accelerated development of 2-cell mouse embryos to the 8-cell stage, while a 2.3 ppm dose of bisphenol A inhibited development. The stimulatory effects of low doses of bisphenol A were inhibited by the antiestrogen tamoxifen. These findings provide more evidence for inverted-U dose-response relationships for bisphenol A.

Takai, Y., Tsutsumi, O., Ikezuki, Y., Kamei, Y., Osuga, Y., Yano, T. and Taketan, Y. (2000). Preimplantation exposure to bisphenol A advances postnatal development. Reproductive Toxicology 15:71-74.
At the 2 cell stage mouse embryos were cultured with a 230 ppt dose of bisphenol A, and rate of development to the blastocyst stage was accelerated. The control and bisphenol A-treated embryos were implanted into and then nursed by control females. At weaning, the animals treated with bisphenol A as embryos were 37% heavier than controls. Embryonic exposure to BPA thus increases postnatal body weight.

 ADDIN EN.REFLIST Takao, T., W. Nanamiya, I. Nagano, K. Asaba, K. Kawabata and K. Hashimoto (1999). Exposure with the environmental estrogen bisphenol A disrupts the male reproductive tract in young mice. Life Sci 65:2351-7.
Here we examine plasma hormone levels and histology in the testis of C57BL/6 mice following either 4- or 8-week oral administration of bisphenol A. Bisphenol A was administered via drinking water at 0.5 and 50 µg//ml. Average water intake was about 6 ml per day and body weights were about 25 g. The daily doses were thus about 120 µg/kg/day and 12 mg/kg/day. Plasma free testosterone levels were dramatically decreased following 8 weeks of 12 mg/kg/day bisphenol A treatment compared with control group, and morphologically multinucleated giant cells having greater than three nuclei were found in seminiferous tubules in the testis following the 8-week bisphenol A treatment at both doses while no control showed this. No differences in plasma corticosterone and luteinizing hormone levels were seen between bisphenol A and control groups. Thus, exposure with bisphenol A around pubertal period may directly disrupt the male reproductive tract. These facts suggest that more detailed studies will warrant the assessment of the risk to the developing human testis from exposure to bisphenol A and other environmental estrogens in prepubertal and pubertal period.

Takao, T., W. Nanamiya, H. P. Nazarloo, R. Matsumoto, K. Asaba and K. Hashimoto (2003). Exposure to the environmental estrogen bisphenol A differentially modulated estrogen receptor-alpha and -beta immunoreactivity and mRNA in male mouse testis. Life Sci. 72(10): 1159-69.
Bisphenol A at concentrations of 0.5 or 50 µg/ml in the drinking water was fed to young male C57BL/6 mice (doses are likely in the range of 0.2 and 20 mg/kg/day). Effects on estrogen receptor (ER) alpha and beta proteins and mRNA in the testis following 8-weeks of oral administration of bisphenol A. ERß was localized in the nuclei of spermatogonia and/or spermatocytes, and the number of ERß containing cells (and mRNA) per testis were significantly decreased in the 50 microg/ml bisphenol A-treated group compared with controls. In contrast, ERað immunopositive cells (and mRNA) per testis were markedly increased in the 50 microg/ml bisphenol A-treated group compared with the controls. The existence of ER alpha and beta in the testis suggests that estrogens directly affect germ cells during testicular development and spermatogenesis, and differential modulation of ER alpha and beta in the testis could be involved in the effects of bisphenol A.

Talsness, C., O. Fialkowski, C. Gericke, H.-J. Merker and I. Chahoud (2000). The effects of low and high doses of bisphenol A on the reproductive system of female and male rat offspring. Congenital Anomalies 40:S94-S107.
Bisphenol A was fed by gavage to Spragd-Dawley rats at doses of 0.1 or 50 mg/kg/day, and females were fed 0.2 mg/kg/day ethinylestradiol (a positive control) from gestation day 6-21. A wide range of outcomes on the reproductive organs and reproductive function in male and female offspring were examined. An interesting feature of the extensive findings reported is the occurrence of non-monotonic, inverted-U dose-response curves. For example, body weight of pups at weaning was depressed at the low, but not high dose of bisphenol A, and vaginal opening was delayed by the low dose and accelerated by the high dose of bisphenol A. Disruption of the estrous cycle in female offspring occurred at the low but not high dose of bisphenol A. Anogenital distance at birth, a marker of prenatal masculinization, was significantly reduced in males at the low but not high dose of bisphenol A. On postnatal day 70, prostate weight was significantly increased and daily sperm production was decreased at the low dose but not by the high dose of bisphenol A. This latter finding replicates the findings in mice reported by Gupta (2000) and vom Saal et al. 1998).

Thuillier, R., Wang, Y. and Culty, M. (2003). Prenatal Exposure to Estrogenic Compounds Alters the Expression Pattern of Platelet-Derived Growth Factor Receptors alpha and beta in Neonatal Rat Testis: Identification of Gonocytes as Targets of Estrogen Exposure. Biol. Reprod. 68:867-880.
We examined the effects of maternal exposure to estrogens on platelet-derived growth factor (PDGF) receptor (PDGFR) expression in newborn rat testis. Pregnant rats were treated from gestation Day 14 to birth with corn oil containing diethylstilbestrol (0.01 – 2 µg/kg/day), bisphenol A (0.1 – 200 mg/kg/day), genistein, or coumestrol by gavage or subcutaneous injection. These treatments induced a dose-dependent increase in the expression of PDGFR alpha and beta mRNAs, determined by semiquantitative reverse transcription polymerase chain reaction, though diethylstilbestrol had a biphasic effect on both mRNAs. A significant effect of BPA occurred at doses of 1 – 200 mg/kg/day and for DES at doses of 0.01, 0.1 and 1 µg/kg/day, but at a DES dose 2 µg/kg/day, the response decreased back to control levels, forming an inverted-U dose-response curve. In situ hybridization analysis showed that PDGFRalpha mRNA increased mostly in the interstitium, while PDGFRbeta mRNA increased both in the interstitium and seminiferous cords. Immunohistochemical studies of PDGFRalpha and beta proteins revealed that both receptors were present in testis before and after birth and that they were upregulated upon treatment with estrogens in 3-day-old rats, with PDGFRbeta increasing dramatically in gonocytes. PDGFRalpha and beta mRNAs and proteins were also found in purified gonocytes. Our previous finding that PDGF and 17beta-estradiol induce gonocyte proliferation in vitro, together with the present finding that in vivo exposure to estrogens upregulates PDGF receptors in testis, suggest that PDGF pathway is a target of estrogens in testis. In addition, these data identify PDGFRbeta in gonocytes as a major target of gestational estrogen exposure, suggesting that estrogen may have a physiological interaction with PDGF during gonocyte development. These results, however, do not exclude the possibility that the effects of the compounds examined in this study might be due to estrogen receptor-independent action(s).

Timms, B. G., K. L. Howdeshell, L. Barton, S. Bradley, C. A. Richter and F. S. vom Saal (2005). Estrogenic chemicals in plastic and oral contraceptives disrupt development of the mouse prostate and urethra. Proc. Natl. Acad. Sci. 102:7014-7019.
Exposure of human fetuses to manmade estrogenic chemicals can occur through several sources. For example, fetal exposure to ethinylestradiol occurs because each year approximately 3% of women taking oral contraceptives become pregnant. Exposure to the estrogenic chemical bisphenol A occurs through food and beverages because of significant leaching from polycarbonate plastic products and the lining of cans. We fed pregnant CD-1 mice ethinylestradiol (0.1 µg/kg/day) and bisphenol A (10 µg/kg/day), which are doses below the range of exposure by pregnant women. In male mouse fetuses both ethinylestradiol and bisphenol A produced an increase in the number and size of dorsolateral prostate ducts and an overall increase in prostate duct volume. Histochemical staining of sections with proliferating cell nuclear antigen and mouse keratin 5 antibodies indicated that this was due to a marked increase in proliferation of basal epithelial cells located in the primary ducts. The urethra was malformed in the colliculus region and significantly constricted where it enters the bladder, which could contribute to urine flow disorders. These effects were identical to those caused by a similar 0.1 µg/kg/day dose of the estrogenic drug, diethylstilbestrol (DES), a known human developmental teratogen and carcinogen. In contrast, a 2000-fold higher DES dose completely inhibited dorsolateral prostate duct formation, revealing opposite effects of high and low doses of estrogen. Acceleration in the rate of proliferation of prostate epithelium during fetal life by small amounts of estrogenic chemicals could permanently disrupt cellular control systems and predispose the prostate to disease in adulthood.

Tohei, A., S. Suda, K. Taya, T. Hashimoto and H. Kogo (2001). Bisphenol A inhibits testicular functions and increases luteinizing hormone secretion in adult male rats. Exp. Biol. Med. 226:216-221.
Adult male Wistar rats were given subcutaneous injections of bisphenol A for 2 weeks at a dose of about 3 mg/kg/day. Bisphenol A treatment resulted in significant decrease in plasma testosterone, which was associated with an increase in plasma LH. Testicular content, but not plasma levels, of inhibin were also decreased. When challenged with an iv injection of 10 IU human chorionic gonadotropin (hCG), bisphenol A exposed males showed significantly depressed levels of plasma progesterone and testosterone, demonstrating a direct inhibitory effect of bisphenol A on the testicular response to gonadotropin stimulation.

Toyama, Y. and S. Yuasa (2004). Effects of neonatal administration of 17beta-estradiol, beta-estradiol 3-benzoate, or bisphenol A on mouse and rat spermatogenesis. Reprod Toxicol 19:181-8.
Bisphenol A (BPA) is a global environmental contaminant that has been implicated as a potential endocrine disruptor. In the present study, newborn rats and mice were injected subcutaneously with BPA (doses approximately 30, 300, 1500 and 3000 µg/kg/day) to determine the potential developmental effects on the testis. Testes were examined by light and electron microscopy at 15 weeks of age. Other groups of newborn mice and rats were injected with 17beta-estradiol (E(2)) or beta-estradiol 3-benzoate (E(2)B) in a similar manner. BPA, E(2), and E(2)B had similar effects on testes. When treated animals reached puberty and spermiogenesis began, the first sign of the effects was detected in the steps 2-3 spermatids: the acrosomal granule and nucleus were deformed. Henceforth, abnormalities in the acrosome and nucleus were observed in older spermatids and spermatozoa. Ectoplasmic specialization between the Sertoli cell and spermatids was also affected: some specializations were partially or totally deleted. These abnormalities occurred beginning at 300 µg/kg/day bisphenol A and 1 µg/kg/day E2 and E2B. When animals fully matured, the effects of the agents were not found in the testes, and the animals were found to be fertile. The results of the present study show that BPA acts as an estrogen with approximately 300-fold lower potency that estradiol.

Toyama, Y.; Suzuki-Toyota, F.; Maekawa, M.; Ito, C., and Toshimori, K. (2004). Adverse effects of bisphenol A to spermiogenesis in mice and rats. Archives of Histology & Cytology 67(4):373-381. ISSN: 0914-9465. Either a 20 or 200 µg/kg body weight/injection of bisphenol A (BPA) was subcutaneously administered to adult mice and rats for 6 days, and the effects on the testes were investigated by electron and light microscopy. Abnormalities were observed in the spermatids: acrosomal vesicles, acrosomal caps, acrosonies and nuclei of the spermatids were severely, deformed. The ectoplasmic specialization between the Sertoli cell and spermatids were also affected: incomplete specialization, redundant ectopic specialization and aplasia were observed. Rats and mice responded similarly to BPA. There were no dose dependencies between the 20- and 200 mug/kg body weight/injection groups. The ectoplasmic specialization between adjoining Sertoli cells, or blood-testis barrier, was not affected. Since similar adverse effects were observed when adult mice were treated with beta-estradiol 3-benzoate, the effects of BPA reported here seem to reflect the estrogenic effects on the testes. Animals kept for an additional two months after cessation of the administration were shown to be fertile and the testes showed normal histology, indicating that the adverse effects were transitory.
(Trudeau, V.L., Turque, N., Le Mevel, S., Alliot, C., Gallant, N., Coen, L., Pakdel, F. and Demeneix, B. (2005). Assessment of estrogenic endocrine-disrupting chemical actions in the brain using in vivo somatic gene transfer. Environ Health Perspect 113:329-334.
Estrogenic endocrine-disrupting chemicals abnormally stimulate vitellogenin gene expression and production in the liver of many male aquatic vertebrates. However, very few studies demonstrate the effects of estrogenic pollutants on brain function. We have used polyethylenimine-mediated in vivo somatic gene transfer to introduce an estrogen response element-thymidine kinase-luciferase (ERE-TK-LUC) construct into the brain. To determine if waterborne estrogenic chemicals modulate gene transcription in the brain, we injected the estrogen-sensitive construct into the brains of Nieuwkoop-Faber stage 54 Xenopus laevis tadpoles. Both 0.5 nM ethinylestradiol (EE2; p < 0.002) and 50 nM (11.4 ppb) bisphenol A (BPA; p < 0.03) increased luciferase activity by 1.9- and 1.5-fold, respectively. In contrast, low physiologic levels of 17ss-estradiol had no effect (p > 0.05). The mixed antagonist/agonist tamoxifen was estrogenic in vivo and increased (p < 0.003) luciferase activity in the tadpole brain by 2.3-fold. There have been no previous reports of somatic gene transfer to the fish brain; therefore, it was necessary to optimize injection and transfection conditions for the adult goldfish (Carassius auratus). Following third brain ventricle injection of cytomegalovirus (CMV)-green fluorescent protein or CMV-LUC gene constructs, we established that cells in the telencephalon and optic tectum are transfected. Optimal transfections were achieved with 1 microg DNA complexed with 18 nmol 22 kDa polyethylenimine 4 days after brain injections. Exposure to EE2 increased brain luciferase activity by 2-fold in males (p < 0.05) but not in females. Activation of an ERE-dependent luciferase reporter gene in both tadpole and fish indicates that waterborne estrogens can directly modulate transcription of estrogen-responsive genes in the brain. We provide a method adaptable to aquatic organisms to study the direct regulation of estrogen-responsive genes in vivo.

Vandenberg, L.N., Maricel V. Maffini, Perinaaz R. Wadia, Carlos Sonnenschein, Beverly S. Rubin, and Ana M. Soto. (2006). Exposure to environmentally relevant doses of the xenoestrogen bisphenol-A alters development of the fetal mouse mammary gland. Endocrinol. Online: October 5, 2006. doi:10.1210/en.2006-0561.
Humans are routinely exposed to bisphenol-A (BPA), an estrogenic compound that leaches from dental materials, food and beverage containers and other plastic consumer products. Effects of perinatal BPA exposure on the mouse mammary gland have been observed in puberty and adulthood, long after the period of exposure has ended. The aim of this study was to examine fetal mammary gland development at embryonic day (E)18 and assess changes in the tissue organization and histoarchitecture following exposure to an environmentally relevant dose of BPA. In unexposed fetuses, the relative position of the fetus with respect to its female and male siblings in the uterus influenced growth of the ductal tree, which was more developed in females placed between two males than in females placed between two females. Exposure of dams to 250ng BPA/kg body weight/day from E8 to E18 significantly increased ductal area and ductal extension in exposed fetuses and obliterated positional differences. In the stroma, BPA exposure promoted maturation of the fat pad and altered the localization of collagen. Within the epithelium, BPA exposure led to a decrease in cell size and delayed lumen formation. Because mammary gland development is dependent on reciprocal interactions between these compartments, the advanced maturation of the fat pad and changes in the extracellular matrix may be responsible for the altered growth, cell size and lumen formation observed in the epithelium. These results suggest that alterations in mammary gland phenotypes observed at puberty and adulthood in perinatally exposed mice have their origins in fetal development.

vom Saal, F. S., Cooke, P. S., Buchanan, D. L., Palanza, P., Thayer, K. A., Nagel, S. C., Parmigiani, S. and Welshons, W. V. (1998). A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm production, and behavior. Toxicol Ind Health 14(1-2):239-60.
Bisphenol A was administered orally to pregnant CF-1 mice. A 2 µg/kg/day dose resulted in enlarged prostate and preputial glands, but smaller seminal vesicles and epididymides, while a 20 µg/kg/day dose resulted in a decrease in daily sperm production per g testis in male offspring. The increase in prostate weight and decrease in epididymis weight was replicated by Gupta (2000) using a 50 µg/kg/day dose of bisphenol A. The effect of bisphenol A on the prostate in these male mice was reported in Nagel et al. 1997.

Wang, Y., R. Thuillier and M. Culty (2004). Prenatal estrogen exposure differentially affects estrogen receptor-associated proteins in rat testis gonocytes. Biol Reprod 71:1652-64.
We previously reported that gonocytes from 3-day-old Sprague-Dawley rat testes proliferate in response to estradiol. In the present study, we found that purified gonocytes contained the mRNAs of estrogen receptor beta (ERbeta) and the chaperones Hsp90, p23, and Cyp40, but no inducible Hsp70. Immunoblot analysis showed high levels of ERbeta, Hsp90, p23, Cyp40, and the constitutive Hsc70 in gonocytes. Prenatal (maternal) exposure of Sprague-Dawley rats from GD 14-parturition to the estrogenic compounds diethylstilbestrol (DES) (0.01-1 µg/kg/day by injection), and by gavage bisphenol A (0.1-200 mg/kg/day), genistein (0.1-10 mg/kg/day), and coumestro (1-100 mg/kg/day), led to significantly increased Hsp90 mRNA levels in testis (BPA significant at 10 mg/kda/day), but not p23 and Cyp40 (significant inhibition by BPA at 1 mg/kg/day). Effects were seen in response to BPA at doses of 1 –200 mg/kg/day and to DES at doses of 0.01 –1 µg/kg/day. In situ hybridization analysis indicated that Hsp90 mRNA was prominent in gonocytes, where it was increased following phytoestrogen exposure, whereas bisphenol A induced a more generalized increase throughout the testis. Immunoblot analysis of testicular extracts demonstrated that Hsp90 protein levels were significantly increased following estrogen exposure, and immunohistochemical analysis indicated that this increase occurred predominantly in gonocytes. By contrast, no change was observed in the expression of Cyp40, p23, and ERbeta, whereas Hsc70 was increased by bisphenol A only. Using an antibody and reverse transcriptase-polymerase chain reaction probes specific for Hsp90alpha, we subsequently confirmed that Hsp90alpha was primarily expressed in gonocytes, and that it was increased following estrogen exposure. Hsp90 immunolocalization in fetal and prepubertal testes showed an increased expression in fetal gonocytes upon estrogen exposure, but no difference in the subsets of Hsp90-positive germ cells in prepubertal testes. These results demonstrate that prenatal estrogen exposure specifically affects Hsp90 expression in gonocytes. Considering the interaction of Hsp90 with several signaling molecules, changes in its expression levels may lead to subsequent changes in gonocyte development.

(Watanabe, M., Mitani, N., Ishii, N. and Miki, K. (2005). A mutation in a cuticle collagen causes hypersensitivity to the endocrine disrupting chemical, bisphenol A, in Caenorhabditis elegans. Mutat Res 570:71-80.
A novel mutant gene, bis-1 (bisphenol A sensitive) has been isolated in the nematode, Caenorhabditis elegans, that affects the response to endocrine disrupting chemicals (EDC). The bis-1(nx3) allele is hypersensitive to bisphenol A (BPA), is allelic to a collagen gene (col-121), and is expressed in hypodermal cells. Among the collagen mutants so far studied, bis-1(nx3), dpy-2(e8), dpy-7(e88) and dpy-10(e128) showed BPA sensitivity. The isolated mutant may work as a useful tool for the assay of EDC toxicity since the physiological effect of the collagen mutation (glycine substitution) indicates an increased sensitivity to BPA.

(Watts, M. M., Pascoe, D. and Carroll, K. (2001). Chronic exposure to 17alpha-ethinylestradiol and bisphenol A - Effects on development and reproduction in the freshwater invertebrate Chironomus riparius (Diptera: Chironomidae). Aquat. Toxicol. 55:113-124.
The freshwater invertebrate (the nonbiting midge) Chironomus riparius was examined. At doses ranging from 78 ng/L to 750 µg/L (78 ppt to 750 ppb) the emergence of male and female adults in the second generation of exposed animals was significantly delayed.

(Watts, M. M., D. Pascoe and K. Carroll (2003). Exposure to 17 alpha-ethinylestradiol and bisphenol A--effects on larval moulting and mouthpart structure of Chironomus riparius. 54(2): 207-15.
The effects of the endocrine-disrupting chemicals 17alpha-ethinylestradiol and bisphenol A at doses ranging from 10 ng/L-1mg/L (10 ppt – 1 ppm) on the development of the aquatic life-cycle stages (eggs to pupa) of Chironomus riparius were investigated. In addition, three mouthpart structures (mentum, mandibles, and pecten epipharyngis) present on the head capsules of fourth-instar larvae were examined for structural deformities, which were observed at very lowest exposure concentration of 10 ng/L (10 ppt); the incidence of deformities was greater in the chironomids exposed to EE than BPA. Effects were mainly associated with the mentum, with statistically significant differences in median deformity score (Kruskal-Wallis P dienestrol > 4-OH-tamoxifen > 17 beta-estradiol > coumestrol, ICI-164384 > estrone, 17 alpha-estradiol > nafoxidine, moxestrol > clomifene > estriol, 4-OH-estradiol > tamoxifen, 2-OH-estradiol, 5-androstene-3 beta, 17 beta-diol, genistein for the ER alpha protein and dienestrol > 4-OH-tamoxifen > diethylstilbestrol > hexestrol > coumestrol, ICI-164384 > 17 beta-estradiol > estrone, genistein > estriol > nafoxidine, 5-androstene-3 beta, 17 beta-diol > 17 alpha-estradiol, clomifene, 2-OH-estradiol > 4-OH-estradiol, tamoxifen, moxestrol for the ER beta protein. The rat tissue distribution and/or the relative level of ER alpha and ER beta expression seems to be quite different, i.e. moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ER alpha and prostate, ovary, lung, bladder, brain, uterus, and testis for ER beta. The described differences between the ER subtypes in relative ligand binding affinity and tissue distribution could contribute to the selective action of ER agonists and antagonists in different tissues.

Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, Van Der Saag PT, van der Burg B, Gustafsson J-A 1998 Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139:4252-4263.
The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ER alpha or ER beta protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ER alpha or ER beta complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ER alpha and ER beta protein revealed a single binding component for [3H]-17beta-estradiol (E2) with high affinity [dissociation constant (Kd) = 0.05 - 0.1 nM]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ER beta than to ER alpha. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and 2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity of ER alpha and ER beta at concentrations of 100-1000 nM. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 >> zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ER alpha and E2 >> genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ER beta. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ER alpha and a mixed agonist-antagonist for ER beta. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ER beta, and they may trigger many of the biological responses that are evoked by the physiological estrogens.

Kurosawa, T., H. Hiroi, O. Tsutsumi, T. Ishikawa, Y. Osuga, T. Fujiwara, S. Inoue, M. Muramatsu, M. Momoeda and Y. Taketani (2002). The activity of bisphenol A depends on both the estrogen receptor subtype and the cell type. Endocr. J. 49: 465-71.
Bisphenol A (BPA), a monomer of plastic used in consumer products, is abundant in the environment and enters the body by ingestion or adsorption. In order to characterize the estrogenic effect of BPA, we performed luciferase assay on three independent cell lines derived from different tissues transfected with either human ERalpha cDNA or ERbeta cDNA. The estrogenic activities of BPA were detectable in all cell lines via both ERalpha and ERbeta. In 293T cells and Hec-1 cells, the estrogenic activities were significantly decreased when cells expressing ERalpha were incubated with 10(-6) M BPA in the presence of 10(-8) M 17beta-estradiol (E2) while the activities via ERbeta were essentially unchanged in the same conditions. Interestingly, no reduction of estrogenic activity was detected in HOS-TE85 cells via either ERalpha or ERbeta. Our results indicate that BPA only acts as an agonist of estrogen via ERbeta whereas it has dual actions as an agonist and antagonist in some types of cells via ERalpha. Thus, the activity of BPA may depend on the ER subtype and the tissue involved.

Lee, B.-C., Kamata, M., Akatsuka, Y., Takeda, M., Ohno, K., Kamei, T. and Magara, Y. (2004). Effects of chlorine on the decrease of estrogenic chemicals. Water Research 38:733-739.
The effects of chlorination on the elimination of three estrogenic chemicals such as 17beta-estradiol, nonylphenol and bis-phenol A were investigated using yeast two-hybrid assay (YTA), estrogen receptor (ER) competition assay (ER-CA), and high-performance liquid chromatography/mass spectrometry (LC/MS). The results of YTA, ER-CA and the analysis of LC/MS indicated that the estrogenic activity of the above-mentioned three endocrine disruptors were significantly reduced as a result of chlorination. The decrease in estrogenic activity paralleled a decrease in estrogenic chemicals under the influence of free chlorine. One common characteristic of estrogenic chemicals is the presence of a phenolic ring. Considering that a phenolic ring is likely to undergo some sort of transformation in an aqueous chlorination solution, the above-mentioned results may be applied to the rest of the estrogenic chemicals in natural waters.

Lee, H. J., S. Chattopadhyay, E. Y. Gong, R. S. Ahn and K. Lee (2003). Antiandrogenic effects of bisphenol A and nonylphenol on the function of androgen receptor. Toxicol. Sci. 75:40-6.
This study used the ARhLBD-activating signal cointegrator 1 (ASC1) yeast two-hybrid system, which reflects the androgen-dependent interaction between androgen receptor (AR) and its coactivator, ASC1. Both bisphenol A and nonylphenol acted as potent AR antagonists comparable to a known strong antagonist, cyproterone acetate. Ligand competition assays revealed that [3H]5alpha-dihydroxytestosterone (DHT) binding to AR is inhibited a maximum of 30 and 40% at approximately 5 nM of nonylphenol and 50 nM 11.5 ng/ml; ppb) of bisphenol A, respectively (50% competition, or the IC50, was 80 nM bisphenol A). In addition, the nuclear translocation of green fluorescent protein (GFP)-AR fusion protein in the presence of testosterone was affected by the addition of bisphenol A and nonylphenol, which cause rather dispersed distribution of GFP-AR between the nuclear and the cytoplasmic compartments. Furthermore, in transient transfection assays, both chemicals inhibited androgen-induced AR transcriptional activity. Taken together, the results suggest that bisphenol A and nonylphenol affect multiple steps of the activation and function of AR, thereby inhibiting the binding of native androgens to AR, AR nuclear localization, AR interaction with its coregulator, and its subsequent transactivation. These effects of bisphenol A are occurring in the low ng/ml (ppb) range and are thus of concern based on the ppb levels being reported in human blood. Bisphenol A also has anti-thyroid hormone activity (Moriyama et al., 2002; Ishihara et al., 2003). Thus, in addition to acting as an estrogem-mimicking chemical, bisphenol A can block the action of testosterone and thyroid hormone in cells. Paris et al (22002) also showed that BPA had antiandrogenic activity.

Lee, M.S., Hyun, S.H., Lee, C.K., Im, K.S., Hwang, I.T. and Lee, H.J. (2003). Impact of xenoestrogens on the growth of human endometrial epithelial cells in a primary culture system. Fertil Steril 79:1464-1465.

 ADDIN EN.REFLIST Lee, M.H., Chung, S.W., Kang, B.Y., Park, J., Lee, C.H., Hwang, S.Y. and Kim, T.S. (2003). Enhanced interleukin-4 production in CD4+ T cells and elevated immunoglobulin E levels in antigen-primed mice by bisphenol A and nonylphenol, endocrine disruptors: involvement of nuclear factor-AT and Ca2+. Immunol. 109:76-86.
 Bisphenol A (BPA) and p-nonylphenol (NP) are representative endocrine disruptors (EDs) that may have adverse effects on human health. The influence of these compounds on allergic immune responses remains unclear. In this study, we have examined the effects of BPA and NP on production of interleukin-4 (IL-4), a pro-inflammatory cytokine closely associated with allergic immune responses. Both BPA and NP significantly enhanced IL-4 production in keyhole limpet haemocyanin (KLH)-primed CD4+ T cells in a concentration-dependent manner. Treatment with BPA or NP in vivo resulted in significant increase of IL-4 production in CD4+ T cells and of antigen-specific immunoglobulin E (IgE) levels in the sera of KLH-primed mice. Furthermore, BPA and NP enhanced the activation of IL-4 gene promoter in EL4 T cells transiently transfected with IL-4 promoter/reporter constructs, and the enhancing effect mapped to a region in the IL-4 promoter containing binding sites for nuclear factor (NF)-AT. Activation of T lymphocytes by phorbol 12-myristate 13-acetate/ionomycin resulted in markedly enhanced binding activities to the NF-AT site, which significantly increased upon addition of BPA or NP, as demonstrated by the electrophoretic mobility shift assay, indicating that the transcription factor NF-AT was involved in the enhancing effect of BPA and NP on IL-4 production. The enhancement of IL-4 production by BPA or NP was significantly reduced by nitrendipine, a blocker of Ca2+ influx, and by FK506, a calcineurin inhibitor. FK506 inhibited the NF-AT-DNA binding activity and IL-4 gene promoter activity enhanced by BPA or NP. These results represent the first report describing possible enhancement of allergic response by EDs through increasing IL-4 production in CD4+ T cells and antigen-specific IgE levels in the sera via the stimulation of Ca2+/calcineurin-dependent NF-AT activation.

Letcher, R.J., Sanderson, J.T., Bokkers, A., Giesy, J.P. and van den Berg, M. (2005). Effects of bisphenol A-related diphenylalkanes on vitellogenin production in male carp (Cyprinus carpio) hepatocytes and aromatase (CYP19) activity in human H295r adrenocortical carcinoma cells. Toxicol. Appl. Pharmacol. Online: May 2005.
The present study investigated the effects of the known xenoestrogen bisphenol A (BPA) relative to eight BPA-related diphenylalkanes on estrogen receptor (ER)-mediated vitellogenin (vtg) production in hepatocytes from male carp (Cyprinus carpio), and on aromatase (CYP19) activity in the human adrenocortical H295R carcinoma cell line. Of the eight diphenylalkanes, only 4,4'-(hexafluoropropylidene)diphenol (BHF) and 2,2'-bis(4-hydroxy-3-methylphenyl)propane (BPRO) induced vtg, i.e., to a maximum of 3% to 4% (at 100 muM) compared with 8% for BPA relative to the maximum induction by 17beta-estradiol (E2, 1 muM). Bisphenol A diglycidyl ether (BADGE) was a potent antagonist of vtg production with an IC50 of 5.5 muM, virtually 100% inhibition of vtg at 20 muM, and an inhibitive (IC50) potency about one-tenth that of the known ER antagonist tamoxifen (IC50, 0.6 muM). 2,2'-Diallyl bisphenol A, 4,4'-(1,4-phenylene-diisopropylidene)bisphenol, BPRO, and BHF were much less inhibitory with IC50 concentrations of 20-70 muM, and relative potencies of 0.03 and 0.009 with tamoxifen. Bisphenol ethoxylate showed no anti-estrogenicity (up to 100 muM), and 4,4'-isopropylidene-diphenol diacetate was only antagonistic at 100 muM. When comparing the (anti)estrogenic potencies of these bisphenol A analogues/diphenylalkanes, anti-estrogenicity occurred at lower concentrations than estrogenicity. 4,4'-Isopropylidenebis(2,6-dimethylphenol) (IC50, 2.0 muM) reduced E2-induced (EC50, 100 nM) vtg production due to concentration-dependent cytotoxicity as indicated by a parallel decrease in MTT activity and vtg, whereas the remaining diphenylalkanes did not cause any cytotoxicity relative to controls. None of the diphenylalkanes (up to 100 muM) induced EROD activity indicating that concentration-dependent, CYP1A enzyme-mediated metabolism of E2, or any Ah-receptor-mediated interaction with the ER, was not a likely explanation for the observed anti-estrogenic effects. At concentrations as great as 100 muM, none of the diphenylalkanes directly inhibited aromatase (CYP19) activity in H295R cells. Environmental exposure of fish to BPA and related diphenylalkanes, depending on the structure, may pose anti-estrogenic, and to a lesser extent estrogenic, risks to development and reproduction.

Masuno, H., T. Kidani, K. Sekiya, K. Sakayama, T. Shiosaka, H. Yamamoto and K. Honda (2002). Bisphenol A in combination with insulin can accelerate the conversion of 3T3-L1 fibroblasts to adipocytes. J. Lipid Res. 43: 676-684.
Bisphenol A induced fibroblasts (3T3-L1 cells) to differentiate into adipocytes in cell culture when tested at a dose of 20 µg/ml. In combination with insulin (5 µg/ml), bisphenol A dramatically increased lipoprotein lipase activity and increased triacylglycerol (TG) content in cells (uptake of TG is regulated by lipoprotein lipase). These findings suggest that bisphenol A can increase body fat mass, which is also suggested by the findings of Howdeshell et al. (1999) in mice exposed prenatally to a very low dose of bisphenol A, as well as findings by others: Ashby et al., 1999; Takai et al., 2000; Rubin et al. 2001; Akingbemi et al., 2004.

Masuno, H., Iwanami, J., Kidani, T., Sakayama, K. and Honda, K. (2005). Bisphenol A accelerates terminal differentiation of 3T3-L1 cells into adipocytes through the phosphatidylinositol 3-kinase pathway. Toxicol Sci 84:319-27.
In order to identify whether bisphenol A (BPA) acts as an adipogenic agent, following the hormonal induction of differentiation into adipocytes, 3T3-L1 cells were treated for six days with BPA alone. Treatment with BPA increased the triacylglycerol (TG) content of the cultures, increased the percentage of Oil Red O-staining cells in the cultures, and increased the levels of lipoprotein lipase (LPL) and adipocyte-specific fatty acid binding protein (aP2) mRNAs. These findings indicate that BPA was able to accelerate terminal differentiation of 3T3-L1 cells into adipocytes. LY294002, a chemical inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), blocked completely the increasing effect of BPA on TG accumulation and expression of LPL and aP2 mRNAs. Western blot analysis revealed that BPA increased the level of phosphorylated Akt kinase. Based on these findings, we concluded that BPA acted through the PI 3-kinase and Akt kinase pathway, resulting in increased TG accumulation and expression of adipocyte genes. The structure-activity relationship for BPA-related chemicals was examined. Eight derivatives of BPA (three diphenylalkanes with different substituents at the central carbon atom, three diphenylalkanes with ester bonds on hydroxyl groups in the phenolic rings, one bisphenol consisting of a sulphur atom at the central position, one chemical with cyanic groups, instead of hydroxyl groups, in the phenolic rings) accelerated terminal adipocyte differentiation and their potencies to increase TG accumulation were 73-97% of that of BPA. Two diphenylalkanes with ether bonds on hydroxyl groups and two alkylphenols (4-nonylphenol and 4-tert-octylphenol) did not have the ability to accelerate terminal adipocyte differentiation.

 ADDIN EN.REFLIST Masuyama, H. and Hiramatsu, Y. (2004). Involvement of suppressor for Gal 1 in the ubiquitin/proteasome-mediated degradation of estrogen receptors. J Biol Chem 279:12020-12026.
 The proteasome-mediated pathway involves the degradation of several nuclear receptors. Previously we demonstrated that the interaction between the suppressor for Gal 1 (SUG1) and nuclear receptors, the vitamin D receptor, or the pregnane X receptor was involved in proteasome-mediated degradation. In our recent experiments, we examined the potential role of SUG1 in the proteasome-mediated degradation of estrogen receptors (ER)alpha and -beta. Both ERs interacted with SUG1 in a ligand-dependent manner. Functionally, the overexpression of SUG1 inhibited both ERalpha- and ERbeta-mediated transcription in the presence of ligands. Transient expression studies demonstrated that the overexpression of wild-type SUG1 generated proteolytic fragments of both ERs and that these products were blocked by a proteasome inhibitor. The overexpression of SUG1 also enhanced the formation of ubiquitinated proteins of both ERs in the presence of ligand. On the other hand, bisphenol A (BSA), which activated ER-mediated transcription, did not enhance the interaction between ERbeta and SUG1. Furthermore, the degradation of ERbeta was much slower in the presence of BSA than in the presence of estradiol or phthalate, which is another endocrine-disrupting chemical. Also, BSA had no effect on the formation of proteolytic fragments of ERbeta, and neither did it have any effect on the ubiquitination of ERbeta. These findings indicate that the ubiquitin/proteasome-mediated degradation of both ER proteins may involve the interaction of SUG1 with both ERs. Moreover, BSA strongly blocked the ubiquitination and degradation of ERbeta compared with estradiol, suggesting that BSA may affect the ERbeta-mediated transcription of target genes by inhibiting ERbeta degradation.

Matthews, J. B., K. Twomey and T. R. Zacharewski (2001). In vitro and in vivo interactions of bisphenol A and its metabolite, bisphenol A glucuronide, with estrogen receptors alpha and beta. Chemical Research in Toxicology 14(2): 149-157.
Bisphenol A ERß binding affinity 38-fold greater than ERað.ð

Meerts, I. A. T. M., R. J. Letcher, S. Hoving, G. Marsh, A. Bergman, J. G. Lemmen, B. van der Burg and A. Brouwer (2001). In vitro estrogenicity of polybrominated diphenyl ethers, hydroxylated PBDEs and polybrominated bisphenol A compounds. Environ. Health Perspect. 109:399-407.

Moriyama, K., T. Tagami, T. Akamizu, T. Usui, M. Saljo, N. Kanamoto, Y. Hataya, A. Shimatsu, H. Kuzuya and K. Nakao (2002). Thyroid hormone action is disrupted by bisphenol A as an antagonist. Jounral of Clinical Endocrinology and Metabolism 87:5185-5190.
Bisphenol A (BPA), a monomer of polycarbonate plastics, has been shown to possess estrogenic properties and act as an agonist for the estrogen receptors. Although an epidemiologically based investigation has suggested that some chemicals could disrupt thyroid function in animals, the effects on thyroid hormone receptors (TRs) are unknown. We show here that BPA inhibits TR-mediated transcription by acting as an antagonist. In the transient gene expression experiments, BPA suppressed transcriptional activity that is stimulated by thyroid hormone (T(3)) in a dose-dependent manner. The inhibitory effects were observed in the presence of physiological concentrations of T(3). In contrast, in the case of negatively regulated TSHalpha promoter, BPA activated the gene transcription that is suppressed by T(3). To elucidate possible mechanisms of the antagonistic action of BPA, the effects on T(3) binding and cofactor interaction with TR were examined. The K(i) value for BPA was 200 micro M when assessed by inhibition of [(125)I]T(3) binding to rat hepatic nuclear TRs. In a mammalian two-hybrid assay, BPA recruited the nuclear corepressor to the TR. These results suggest that BPA could displace T(3) from the TR and recruit a transcriptional repressor, resulting in gene suppression. This is the first report that BPA can antagonize T(3) action at the transcriptional level. BPA may disrupt the function of various types of nuclear hormone receptors and their cofactors to disturb our internal hormonal environment.

Mueller, S.O., Kling, M., Arifin Firzani, P., Mecky, A., Duranti, E., Shields-Botella, J., Delansorne, R., Broschard, T. and Kramer, P.J. (2003). Activation of estrogen receptor alpha and ERbeta by 4-methylbenzylidene-camphor in human and rat cells: comparison with phyto- and xenoestrogens. Toxicol Lett 142:89-101.
4-Methylbenzylidene-camphor (4-MBC) is an organic sunscreen that protects against UV radiation and may therefore help in the prevention of skin cancer. Recent results on the estrogenicity of 4-MBC have raised concerns about a potential of 4-MBC to act as an endocrine disruptor. Here, we investigated the direct interaction of 4-MBC with estrogen receptor (ER) alpha and ERbeta in a series of studies including receptor binding, ER transactivation and functional tests in human and rat cells. 4-MBC induced alkaline phosphatase activity, a surrogate marker for estrogenic activity, in human endometrial Ishikawa cells. Interestingly, 4-MBC induced weakly ERalpha and with a higher potency ERbeta mediated transactivation in Ishikawa cells at doses more than 1 microM, but showed no distinct binding affinity to ERalpha or ERbeta. In addition, 4-MBC was an effective antagonist for ERalpha and ERbeta. In an attempt to put 4-MBC's estrogenic activity into perspective we compared binding affinity and potency to activate ER with phyto- and xenoestrogens. 4-MBC showed lower estrogenic potency than genistein, coumestrol, resveratrol, bisphenol A and also camphor. Analysis of a potential metabolic activation of 4-MBC that could account for 4-MBC's more distinct estrogenic effects observed in vivo revealed that no estrogenic metabolites of 4-MBC are formed in primary rat or human hepatocytes. In conclusion, we were able to show that 4-MBC is able to induce ERalpha and ERbeta activity. However, for a hazard assessment of 4-MBC's estrogenic effects, the very high doses of 4-MBC required to elicit the reported effects, its anti-estrogenic properties as well as its low estrogenic potency compared to phytoestrogens and camphor has to be taken into account.

 ADDIN EN.REFLIST Nakagawa, Y., Suzuki, T., Nakagawa, Y. and Suzuki, T. (2001). Metabolism of bisphenol A in isolated rat hepatocytes and oestrogenic activity of a hydroxylated metabolite in MCF-7 human breast cancer cells. Xenobiotica 31:113-123.
1. The metabolites of bisphenol A (BPA; 2, 2-bis(4-hydroxyphenyl)propane) in freshly isolated rat hepatocytes and the oestrogenic activities of BPA and its metabolites, particularly 3-hydroxybisphenol A (3-OH-BPA), in MCF-7 cells and competitive binding assays have been studied, respectively.2. During a 2-h incubation, almost all of the BPA (0.25 mM) added to the hepatocyte suspensions was rapidly converted to a major conjugate, monoglucuronide (approximately 75% of total metabolites), and two minor conjugates, which were tentatively identified as monosulphates of BPA and a hydroxylated intermediate, 3-OH-BPA, as determined by mass spectroscopy coupled with HPLC or GC/MS. On the other hand, free 3-OH-BPA was identified as a trace metabolite, whose level was approximately 1 or 2 muM at 1 h in hepatocyte suspensions treated with 0.25 or 0.5 mM BPA, respectively.3. In another experiment, 3-OH-BPA as well as BPA displaced competitively 17 beta -oestradiol bound to the recombinant human oestrogen receptor alpha in a concentration dependent-manner: IC50 of diethylstilbestrol, BPA and 3-OH-BPA were approximately 2.5 x 10(-8), 10(-5) and 5 x 10(-5) M, respectively. Further, BPA and 3-OH-BPA at intermediate concentrations (10(-7)-10(-6) M) caused proliferation of MCF-7 human breast cancer cells, whereas the effect of BPA was more potent than that of 3-OH-BPA. At higher concentrations, both BPA (> 10(-4) M) and 3-OH-BPA (> 10(-5) M) were cytotoxic.4. Based on the proliferative potency in MCF-7 cells and the IC50 for the competitive binding, the oestrogenic activity of 3-OH-BPA was less than that of BPA. These results indicate that BPA itself rather than its metabolite acts as a xeno-oestrogen and that 3-OH-BPA is cytotoxic, possibly acting via reactive semiquinone and/or quinone metabolites, rather than a xeno-oestrogenic mechanism, in MCF-7 cells.

Nagel, S.C., vom Saal, F.S., Thayer, K.A., Dhar, M.G., Boechler, M. And Welshons, W.V. (1997). Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol. Environ. Health Perspect. 105:70-76.
The lower binding of bisphenol A in plasma relative to estradiol predicted a greater potency of bisphenol A in vivo than predicted, particularly in fetuses when plasma binding restricts estrogen entry into tissues. As predicted, administration to pregnant CF-1 mice of low doses (2 and 20 µg/kg/day) of bisphenol A resulted in prostate enlargement in male offspring.

Nikula, H., T. Talonpoika, M. Kaleva and J. Toppari (1999). Inhibition of hCG stimulated steroidogenesis in cultured mouse leydig cells by bisphenol A and octylphenols. Toxicol. Appl. Pharmacol. 157: 166-173.
Niwa, T., M. Fujimoto, K. Kishimoto, Y. Yabusaki, F. Ishibashi and M. Katagiri (2001). Metabolism and interaction of bisphenol A in human hepatic cytochrome P450 and steroidogenic CYP17. Biol. Pharm. Bull. 24(9): 1064-7.
The metabolism of bisphenol A (BPA) was determined for 11 forms of human hepatic cytochromes P450 (CYPs) expressed in the yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli. Additionally, the effect of BPA on the progesterone 17alpha-chydroxylase activity of CYP17 was investigated. CYP2C18 catalyzed BPA metabolism most efficiently, followed by CYP2C19 and CYP2C9. CYP2C9 and CYP2C18 exhibited the highest affinity (Km=3.9 microM) for BPA metabolism. The Vmax of CYP2C18 (8.10 nmol x min(-1) x nmol CYP(-1)) was 5 times higher than that of CYP2C9. Although the Vmax of CYP2C19 was 1.5 times higher than that of CYP2C18, the affinity of CYP2C19 was 12 times lower than that of CYP2C9 and CYP2C18. Therefore the intrinsic clearance (Vmax/Km) of CYP2C18 was more than 5 times higher than that of CYP2C9 and CYP2C19. On the other hand, BPA exhibited a competitive-type inhibition of the progesterone 17alpha-hydroxylase activity of CYP17 with a Ki value of 71 microM, whereas no metabolism of BPA by CYP17 was detected. These results suggest that BPA is mainly metabolized by the CYP2C subfamily in human liver, and that BPA inhibits human steroidogenic CYP17 activities.

Niwa, T., M. Tsutsui, K. Kishimoto, Y. Yabusaki, F. Ishibashi and M. Katagiri (2000). Inhibition of drug-metabolizing enzyme activity in human hepatic cytochrome P450s by bisphenol A. Biol. Pharm. Bull. 23:498-501.
Effect of bisphenol A on drug-metabolizing enzyme activities by human hepatic cytochrome P450s (CYP) was investigated. We measured aminopyrine N-demethylation by eleven kinds of cDNA-expressed CYPs. CYP2C19 and CYP2B6 catalyzed most efficiently the aminopyrine N-demethylation, followed by CYP2C8 and CYP2D6. Bisphenol A (1 mM) most efficiently inhibited aminopyrine N-demethylation by CYP2C8 and CYP2C19 by 82% and 85%, respectively, whereas inhibition of the activities by CYP 2B6 and 2D6 was less than 40%. Bisphenol A exhibited a noncompetitive-type inhibition of aminopyrine N-demethylase activity by CYP2C8 with Ki value of 97 microM. Additionally, we investigated the inhibitory effect of bisphenol A on CYP2C19-mediated S-mephenytoin 4-hydroxylation. Bisphenol A exhibited a mixed-type inhibition with Ki value of 113 microM. These results suggest that bisphenol A inhibits human hepatic CYP activities, especially CYP2C8 and CYP2C19.

Paris, F., P. Balaguer, B. Terouanne, N. Servant, C. Lacoste, J. P. Cravedi, J. C. Nicolas and C. Sultan (2002). Phenylphenols, biphenols, bisphenol-A and 4-tert-octylphenol exhibit alpha and beta estrogen activities and antiandrogen activity in reporter cell lines. Mol. Cell Endocrinol. 193(1-2): 43-49.
We previously demonstrated the interactions of different chemical compounds with estrogen receptors ERalpha and ERbeta and the androgen receptor (AR) using different reporter cell lines. In this study, we characterize the ERalpha, ERbeta and AR activity of different biphenyls using the same tools. We provide evidence that several phenyl derivatives present both estrogenic and antiandrogenic activity. The extent of hydroxylation and the position of the hydroxyl function were important in determining their estrogenicity and antiandrogenicity. Of the tested compounds, bisphenol-A and 4,4' biphenol had very high estrogenic activity, although it was lower than that of the strong estrogenic alkylphenol, 4-tert-octylphenol. Bisphenol-A and 4,4' biphenol were able to activate ERs at concentrations lower than 1 microM, whereas the other compounds only activated at concentrations above 1 microM. Interestingly, 4,4' biphenol was a better agonist for ERbeta than for ERalpha. No androgenic activity was detected for any of these compounds. Bisphenol-A, 3-OH phenylphenol, 4-OH phenylphenol and 4,4' biphenol exhibited antiandrogenic activity close to that of 4-tert-octylphenol (IC(50) approximately 5 microM). In whole cell binding assays, these compounds displaced [3H] R1881 with Ki = 10 microM. Although these Ki values seem high in comparison with that of hydroxyflutamide (0.4 microM), one must keep in mind that environmental chemicals can accumulate in adipose tissues for several years. In conclusion, these environmental chemicals may have a negative impact on androgen action during fetal and post-natal life.

Quesada, I., E. Fuentes, M. C. Viso-Leon, B. Soria, C. Ripoll and A. Nadal (2002). Low doses of the endocrine disruptor bisphenol-A and the native hormone 17beta-estradiol rapidly activate transcription factor CREB. FASEB. J. 16(12): 1671-3.
Pancreatic ß cells were examined using 228 pg/ml (1nM) bisphenol A and estradiol (282 pg/ml, 1nM) administered for 5 min for effects on phosphorylation of the transcription factor CREB following depolarization of the cell membrane and opening of Ca++ channels and a rapid influx into the cell of calcium. Bisphenol A and estradiol had the same stimulating effect on phosphorylation of CREB and calcium uptake, in that they potentiated the effect of glucose as a stimulator of CREB phosphorylation. The estrogen receptor antagonist ICI 182,780 had no effect on this response while estradiol conjugated to peroxidase (with limited capacity to enter cells) caused the response, suggesting a membrane-receptor mediates the response rather than the classical genomic ER.

Recchia AG, Vivacqua A, Gabriele S, Carpino A, Fasanella G, Rago V, Bonofiglio D, Maggiolini M. 2004. Xenoestrogens and the induction of proliferative effects in breast cancer cells via direct activation of oestrogen receptor alpha. Food Additives & Contaminants 21:134-144. Environmental contamination with a variety of industrial products has been associated with developmental and reproductive abnormalities in wildlife species. Increasing evidence has suggested that bisphenol A (BPA) and 4-nonylphenol (NPH), two major endocrine-disrupting chemicals, might be responsible for adverse effects on humans as a consequence of ubiquitous use together with potential oestrogen-like activity. To provide insight into the oestrogen-like nature of BPA and NPH, their ability to activate a reporter gene construct via an oestrogen response element in the hormone-dependent breast cancer cell lines MCF7 and T47D was ascertained. Both compounds transactivated the endogenous oestrogen receptor (ER) alpha in a direct fashion since the anti-oestrogen 4-hydroxytamoxifen abolished the response. In addition, using steroid-receptor-negative HeLa cells engineered to express ERalpha and ER beta and the hormone-binding domains of both ERalpha and ER beta , BPA and NPH confirmed the direct transcriptional activity. Interestingly these properties were supported in MCF7 cells by the ability to autoregulate ERalpha expression as well as to induce its nuclear compartmentalization. We therefore evaluated by reverse transcriptase polymerase chain reaction the expression of oestrogen-controlled genes such as cathepsin D and TFF1 (formerly pS2), which were increased by both chemicals tested. The agonistic effects exhibited in all assays performed prompted the evaluation of a more complex biological response such as the proliferation of MCF7 and T47D cells. The same concentration of xenoestrogens eliciting substantial transcriptional activity significantly stimulated the proliferation of both breast cancer cell lines, although with a reduced effectiveness with respect to the natural hormone 17beta-oestradiol. The results indicate that the biological action of environmental oestrogen such as BPA and NPH should be taken into account for the potential impact on human disease-like hormone-dependent breast cancer. However, further studies are needed to clarify their bioavailability and metabolism as well as whether compound mixtures could produce noticeable effects by synergistic activity.
Routhledge, E.J., White, R., Parker, M.G. and Sumpter, J.P. (2000). Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) alpha and ERß. J. Biol. Chem. 46:35986-35993.
It has been proposed that tissue-specific estrogenic and/or antiestrogenic actions of certain xenoestrogens may be associated with alterations in the tertiary structure of estrogen receptor (ER) alpha and/or ERbeta following ligand binding; changes which are sensed by cellular factors (coactivators) required for normal gene expression. However, it is still unclear whether xenoestrogens affect the normal behavior of ERalpha and/or ERbeta subsequent to receptor binding. In view of the wide range of structural forms now recognized to mimic the actions of the natural estrogens, we have assessed the ability of ERalpha and ERbeta to recruit TIF2 and SRC-1a in the presence of 17beta-estradiol, genistein, diethylstilbestrol, 4-tert-octylphenol, 2',3',4', 5'-tetrachlorobiphenyl-ol, and bisphenol A. We show that ligand-dependent differences exist in the ability of ERalpha and ERbeta to bind coactivator proteins in vitro, despite the similarity in binding affinity of the various ligands for both ER subtypes. The enhanced ability of ERbeta (over ERalpha) to recruit coactivators in the presence of xenoestrogens was consistent with a greater ability of ERbeta to potentiate reporter gene activity in transiently transfected HeLa cells expressing SRC-1e and TIF2. We conclude that ligand-dependent differences in the ability of ERalpha and ERbeta to recruit coactivator proteins may contribute to the complex tissue-dependent agonistic/antagonistic responses observed with certain xenoestrogens. In summary, HeLa cells were transfected with ERað and ERß, and bisphenol A showed a 10-fold greater binding affinity (relative to estradiol) for ERß than ERað. Binding of bisphenol A to ERß but not ERað also resulted in recruitment of the ER coactivator TIF2 with a potency relative to estradiol of 0.05.

Roy, P., H. Salminen, P. Koskimies, J. Simola, A. Smeds, P. Saukko and I. T. Huhtaniemi (2004). Screening of some anti-androgenic endocrine disruptors using a recombinant cell-based in vitro bioassay. J. Steroid Biochem. Mol. Biol. 88:157-66.
The Chinese hamster ovarian cell line (CHO K1) in the 96-well format were cotransfected with plasmids encoding mouse mammary tumour virus-neomycin-luciferase and human androgen receptor (hAR), and a stable cell line was established which stably expressed both the hAR and the androgen-responsive luciferase reporter. Stimulation of the cells with androgens for 24h resulted in about 15-fold stimulation of luciferase activity, with the minimum effective dose of testosterone being 0.1nmol/l. Potent steroidal and non-steroidal anti-androgens, such as hydroxyflutamide and cyproterone acetate, significantly inhibited the androgen-induced transactivation. Non-androgenic steroids like estradiol, progesterone, dexamethasone and cortisol showed weak activity at high concentrations. About 60 different chemicals (mostly pesticides or their metabolites, and common industrial chemicals) were screened with the cell line for their ability to stimulate luciferase activity or inhibit that evoked by 0.1nmol/l R1881, used as a positive androgenic control. About 10 highly potent anti-androgenic chemicals were identified. The most potent anti-androgenic compounds identified included bisphenol A (IC-50 = 19.6 µmol/l), alpha-hexachlorocyclohexane (7.7 µmol/l), vinclozolin (3.9 µmol/l) and 4,4-DDE (20.9 µmol/l). These compounds had alone either no effect or were weak agonists (with cytotoxic effects at very high concentrations), but none showed any significant agonistic activity.

Sakurai, K., M. Kawazuma, T. Adachi, T. Harigaya, Y. Saito, N. Hashimoto and C. Mori (2004). Bisphenol A affects glucose transport in mouse 3T3-F442A adipocytes. Br. J. Pharmacol. 141(2): 209-214.
At doses between 1-100 µM bisphenol A stimulated an increase in the glucose transporter (GLUT-4) and glucose uptake into 3T3-F442A adiposities in cell culture. Interestingly, this effect of bisphenol A was not inhibited by the estrogen receptor antagonist ICI 182,780, revealing that this effect is not mediated by nuclear estrogen receptors.

Sato, K., Matsuki, N., Ohno, Y. and Nakazzawa, K. (2002). Effects of 17beta-estradiol and xenoestrogens on the neuronal survival in an organotypic hippocampal culture. Neuroendocrinol. 76:223-234.
Bisphenol A a showed a markedly lower affinity to estrogen receptors relative to estradiol, ethinylestradiol or DES, but all chemicals showed the same maximal effects on neuronal survival at 1 nM (230 ppt bisphenol A), suggesting that effects are not mediated through the classical estrogen receptors. This hypothesis is supported by the finding that at 1 nM estradiol and bisphenol A equally increased expression of N-methyl-D-aspartate receptors and increased dendritic spine density in the hippocampal CA3 neurons.

 ADDIN EN.REFLIST Schrader, T.J., Langlois, I., Soper, K. and Cherry, W. (2002). Mutagenicity of bisphenol A (4,4'-isopropylidenediphenol) in vitro: effects of nitrosylation. Teratog. Carcinog. Mutagen. 22:425-441.
 Bisphenol A (4,4'-isopropylidenediphenol) is a common component of polycarbonate plastics and epoxy resins. Since bisphenol A-containing plastics and resins have found uses in food-contact items, its potential migration into foodstuffs and possible health consequences have been the focus of many recent studies. However, the potential mutagenic activation of bisphenol A by nitrosylation has received little attention. Incubation of bisphenol A with sodium nitrite under acidic conditions produced a yellow-brown product. When nitrosylated bisphenol A was tested in the Ames Salmonella/microsome assay at 100 ng to 1 mg/plate, dose-dependent increases in mutagenicity were found in both TA98 and TA100 Salmonella strains. These results indicated the presence of a direct-acting mutagenic activity causing both frameshift and base pair mutations, respectively. When compared to colony formation in untreated controls, the addition of rat liver S9 for metabolic activation had little influence on revertant colony formation. Unreacted bisphenol A dissolved in DMSO, acidic buffer, or inactivated nitrosylation solution showed negligible mutagenicity. When the nature of the mutagenic changes was examined using the Ames II trade mark Assay, a variety of base pair changes was found including T:A to A:T - S9, G:C to A:T +/- S9,C:G to A:T +/- S9 and C:G to G:C +/- S9. Bisphenol A also induced frameshift mutations at G:C sites. In addition, the presence of electrophiles was shown by the production of an intensely coloured orange-red product upon incubation of nitrosylated bisphenol A with the nucleophile 4-(4'-nitrobenzyl)pyridine. These findings suggest that migration of bisphenol A into nitrite containing foodstuffs, or its ingestion in the presence of nitrite, could lead to the formation of mutagenic compounds.

Seidlova-Wuttke, D., H. Jarry and W. Wuttke (2004). Pure estrogenic effect of benzophenone-2 (BP2) but not of bisphenol A (BPA) and dibutylphtalate (DBP) in uterus, vagina and bone. Toxicology 205:103-12.
Contradictory results whether the endocrine disrupters (ED) benzophenone-2 (BP2), bisphenol A (BPA) and dibutylphtalate (DBP) exert estrogenic effects have been published. Selective estrogen receptor modulators (SERMs) exert estrogenic effects in some but not in all organs and ED may be SERMs. Therefore, we studied their binding properties to recombinant ERalpha and ERbeta protein and their effects in the uterus, vagina and bone of ovariectomized rats. BP2 bound to both receptor subtypes, while BPA had a relatively high ERbeta selectivity. DBP did not bind to ERalpha but with a low affinity to ERbeta. In the uterus, only E(2) and BP2 increased uterine weight and the complement C3 but decreased ERbeta gene expression. Discrete effects of BPA and DBP in the uterus were found upon histological examination. In the vagina, BP2 but not BPA and DBP had clear estrogenic effects. E(2) and BP2 had antiosteoporotic effects in the metaphysis of the tibia. The serum surrogate parameters of bone metabolism, i.e. osteocalcin and the cross (rat) laps were significantly reduced by E(2), an effect shared with BP2 but not by the two other EDs. The conclusion: BP2 acts as ERalpha and ERbeta agonist mimicking effects of E(2), while the effects of BPA and DBP are not pure estrogenic.

Seiwa, C., J. Nakahara, T. Komiyama, Y. Katsu, T. Iguchi and H. Asou (2004). Bisphenol A exerts thyroid-hormone-like effects on mouse oligodendrocyte precursor cells. Neuroendocrinology 80:21-30.
We report studies on the mechanism of action of bisphenol A (BPA) on the differentiation of oligodendrocyte precursor cells (OPCs). Our results show that: (1) BPA inhibits the differentiation of OPCs induced by exposure to thyroid hormone (T3). (2) The effect is mediated through various mechanisms via the thyroid hormone receptor (TRbeta1) which is considered to be responsible for OPC differentiation. (3) The action of BPA on OPC differentiation does not involve the FcRgamma-Fyn-myelin basic protein (MBP) cascade as an inducer of OPC differentiation nor does it suppress CREB phosphorylation, which is considered to be induced by the T3-TR complex. (4) The presence of MBP isoforms (21.5, 18.5, 17.0 and 14.0 kDa) was detected in OPCs, and the expression of exon 2-containing isoforms (i.e. 17.0 and 21.5 kDa) was upregulated upon treatment with T3. In contrast, expression of MBP was inhibited by BPA.

Singleton, D. W., Y. Feng, Y. Chen, S. J. Busch, A. V. Lee, A. Puga and S. A. Khan (2004). Bisphenol-A and estradiol exert novel gene regulation in human MCF-7 derived breast cancer cells. Mol Cell Endocrinol 221:47-55.
Xenoestrogens such as bisphenol-A (BPA) can mimic endogenous 17beta-estradiol (E2) in vitro and in vivo through binding the estrogen receptor (ER), and modulating target gene expression. In the present study, we compared global gene regulation by BPA and E2 in estrogen responsive (ERalpha-HA) human breast cancer cells derived from the MCF-7 cell line. The ERalpha-HA cells (stably over-expressing ERalpha) were exposed to E2 (10(-8)M) or BPA (10(-6)M), for 3h followed by analysis of global gene expression. More than 40 transcripts were significantly changed in ERalpha-HA cells, with many being unique to BPA. At least 15 genes were modulated by BPA in the ER-null C4-12 cell line, indicating ER independent activity. Utilizing quantitative reverse transcription-polymerase chain reaction (RT-PCR), we confirmed BPA and E2 mediated regulation of four selected genes. A consensus Alu-type estrogen responsive element (ERE) was found in the Wiskott-Aldrich syndrome protein (WASP) gene, which conferred responsiveness to BPA and E2 in a reporter gene assay. Significant stimulation was seen only in ERalpha expressing cells, thus indicating a functional ERE. Taken together these data illustrate novel gene regulation by BPA and E2, which has implications for in vivo actions and previous reports of additive and synergistic effects on breast cancer cell growth.

Singleton, D.W., Feng, Y., Yang, J., Puga, A., Lee, A.V. and Khan, S.A. (2005). Gene expression profiling reveals novel regulation by bisphenol-A in estrogen receptor-alpha-positive human cells. Environ Res. Online: Nov 2005 www.sciencedirect.com.
Bisphenol-A (BPA) shows proliferative actions in uterus and mammary glands and may influence the development of male and female reproductive tracts in utero or during early postnatal life. Because of its ability to function as an estrogen receptor (ER) agonist, BPA has the potential to disrupt normal endocrine signaling through regulation of ER target genes. Some genes are regulated by both estradiol (E2) and BPA, but those exclusive to either agent have not been described. Using a yeast strain incorporating a vitellogenin A2 ERE-LacZ reporter gene into the genome, we found that BPA (BPA was examined at 1 µM and E2 at 10 nM concentrations) induced expression of the reporter in colonies transformed with the ERalpha expression plasmid, illustrating BPA-mediated regulation within a chromatin context. Additionally, a reporter gene transiently transfected into the endometrial cancer (Ishikawa) cell line also showed BPA activity, although at 100-fold less potency than E2. To compare global gene expression in response to BPA and E2, we used a variant of the MCF-7 breast cancer cell line stably expressing HA-tagged ERalpha. Cultures were treated for 3h with an ethanol vehicle, E2 (10(-8)M), or BPA (10(-6)M), followed by isolation of RNA and microarray analysis with the human U95A probe array (Affymetrix, Santa Clara, CA, USA). More than 300 genes were changed 2-fold or more by either or both agents, with roughly half being up-regulated and half down-regulated. A number of growth- and development-related genes, such as HOXC1 and C6, Wnt5A, Frizzled, TGFbeta-2, and STAT inhibitor 2, were found to be affected exclusively by BPA. We used quantitative real-time PCR to verify regulation of the HOXC6 gene, which showed decreased expression of approximately 2.5-fold by BPA. These results reveal novel effects by BPA and E2, raising interesting possibilities regarding the role of endocrine disruptors in sexual development.

Sohoni, P. and Sumpter, J..P. (1998). Several environmental oestrogens are also anti-androgens. Journal of Endocrinology. 158:327-339.
Bisphenol A has an efficacy similar to the antiandrogenic drug Flutamide in inhibiting binding of DHT to androgen receptors in a yeast reporter assay.

Takemura, H., Ma, J., Sayama, K., Terao, Y., Zhu, B.T. and Shimoi, K. (2005). In vitro and in vivo estrogenic activity of chlorinated derivatives of bisphenol A. Toxicology 207:215-221.
The estrogenic activity of bisphenol A (BPA) and its chlorinated derivatives, 2-(3-chloro-4-hydroxyphenyl)-2-(4-hydroxyphenyl)propane (3-ClBPA) and 2,2-bis(3-chloro-4-hydroxyphenyl)propane (3,3'-diClBPA) was assessed by determining their relative binding affinity for the human estrogen receptor-alpha and -beta (ERalpha and ERbeta) and also their uterotrophic activity in ovariectomized female rats. BPA and its chlorinated derivatives were active in competing with [3H]17beta-estradiol for their binding to the human ERalpha and ERbeta proteins. While 3-ClBPA and 3,3'-diClBPA competed more effectively for ERalpha binding than BPA (IC50 values of 2.48x10(-5), 1.28x10(-5), and 1.08x10(-4)M, respectively), they had similar activity as BPA for competing the binding to ERbeta (IC50 values of 1.43x10(-5), 1.87x10(-5), and 2.59x10(-5)M, respectively). To determine the uterotropic activity, three doses (10, 50 and 100 mg/kg/day) of BPA and its derivatives were given to mature ovariectomized Sprague-Dawley rats for 3 consecutive days. Treatment of animals with 50 and 100 mg/kg/day of BPA or its chlorinated derivatives caused a significant increase in the uterine wet weight and the endometrial area. The results of our present study demonstrated that the affinities of 3-ClBPA and 3,3'-diClBPA for ERalpha were higher than the affinity of BPA, although the in vivo estrogenic activity of the two chlorinated BPAs in ovariectomized female Sprague-Dawley rats appeared to be comparable to that of BPA.

Tanabe, N., T. Kimoto and S. Kawato (2006). Rapid Ca2+ signaling induced by Bisphenol A in cultured rat hippocampal neurons. Neuro Endocrinol Lett 27(1-2): 97-104.
OBJECTIVES: Bisphenol A (BPA) is a typical endocrine disrupter. We investigated the mechanisms of rapid Ca2+ signaling induced by a low dose BPA application in cultured hippocampal neurons. MATERIALS AND METHODS: The primary culture of hippocampal neurons were prepared from postnatal 3 to 5-day-old rats. Cells were loaded with Calcium Green-1 fluorophore. Ca2+ imaging and analysis were performed by Argus system. RESULTS: The application of BPA at 10-100 nM induced a transient increase in the intracellular Ca2+ of N-methyl-D-aspartate (NMDA)-responsive neurons. The Ca2+ transient occurred within 30 sec after the BPA application. The proportion of BPA-responsive neurons was 9.6 % and 8.5 % of the total NMDA-responsive neurons, respectively, upon 10 nM and 100 nM BPA application. The pre-treatment of neurons with Ca2+ channel blockers, thapsigargin and nifedipine, considerably decreased the proportion of BPA-responsive neurons to 0.7 % and 3.7%, respectively. The treatment of neurons with an antagonist of estrogen receptor, ICI 182,780, also significantly decreased the proportion of BPA-responsive neurons down to 1.1 %. CONCLUSION: These results suggest that a low dose BPA application rapidly drives the Ca2+ signaling system via activation of non-genomic pathway including estrogen receptors.

 ADDIN EN.REFLIST Tarumi, H., Imazato, S., Narimatsu, M., Matsuo, M. and Ebisu, S. (2000). Estrogenicity of fissure sealants and adhesive resins determined by reporter gene assay. J Dent Res 79:1838-1843.
 It is controversial whether the dental resinous materials containing 2,2-bis[4-(2-hydroxy-3-methacryloyloxypropoxy)phenyl]propane (Bis-GMA), which is synthesized from the estrogenic compound bisphenol A (BPA), include unreacted BPA and/or can mimic the effects of natural steroid hormones. In the present study, the estrogenic activities of 3 fissure sealants and 5 adhesive resins, which were all unpolymerized, were determined by means of a reporter gene assay, and the relevance of the components to the estrogenicity was investigated. Two commercially available sealants were confirmed to have estrogenic activity, although none of the tested materials contained BPA. In contrast, hydrophobic monomer bisphenol A dimethacrylate (BPA-DMA), which is also estrogenic, was found to be included in these estrogenic sealants in an amount greater than the minimum concentration to show estrogenicity. This suggests that the estrogenicity of the two proprietary sealants was associated with BPA-DMA rather than with BPA.

Toyohira, Y., K. Utsunomiya, S. Ueno, K. Minami, Y. Uezono, R. Yoshimura, M. Tsutsui, F. Izumi and N. Yanagihara (2003). Inhibition of the norepinephrine transporter function in cultured bovine adrenal medullary cells by bisphenol A. Biochem. Pharmacol. 65:2049-54.
Bisphenol A and estradiol altered norepinephrine (NE) transporter function in cultured bovine adrenal medullary cells. Bisphenol A significantly inhibited [3H]NE uptake by the cells in a concentration-dependent manner (1-100 microM). Kinetic analysis revealed that bisphenol A, as well as 17beta-estradiol, noncompetitively inhibited [3H]NE uptake. Bisphenol A and 17beta-estradiol inhibited the specific binding of [3H]desipramine to plasma membranes isolated from bovine adrenal medulla. As shown by Scatchard analysis of [3H]desipramine binding, bisphenol A increased the dissociation constant (K(d)) and decreased the maximal binding (B(max)), indicating a mixed type of inhibition. 17beta-Estradiol increased the K(d) without altering the B(max), thereby indicating competitive inhibition. The present findings suggest that bisphenol A inhibits the function of the NE transporter by acting on a site different from that of 17beta-estradiol in the adrenal medulla and probably in the brain noradrenergic neurons.

 ADDIN EN.REFLIST Tsutsui, T., Tamura, Y., Yagi, E., Hasegawa, K., Takahashi, M., Maizumi, N., Yamaguchi, F. and Barrett, J.C. (1998). Bisphenol-A induces cellular transformation, aneuploidy and DNA adduct formation in cultured Syrian hamster embryo cells. Int, J, Cancer 75:290-294.
 Bisphenol-A (BP-A) is a major component of epoxy, polycarbonate and other resins. For an assessment of in vitro carcinogenicity and related activity of BP-A, the abilities of this compound to induce cellular transformation and genetic effects were examined simultaneously using the Syrian hamster embryo (SHE) cell model. Cellular growth was reduced by continuous treatment with BP-A at doses > or = 100 microM. However, colony-forming efficiencies were not decreased significantly following treatment with up to 200 microM BP-A for 48 hr. Morphological transformation of SHE cells was induced by treatment of cells with BP-A at 50 to 200 microM for 48 hr. BP-A exhibited transforming activity at doses > or = 50 microM but was less active than the benzo[alpha]pyrene used as a positive control. Over the dose range that resulted in cellular transformation, treatment of SHE cells with BP-A failed to induce gene mutations at the Na+/K+ ATPase locus or the hprt locus. No statistically significant numbers of chromosomal aberrations were detected in SHE cells treated with BP-A. However, treatment of cells with BP-A induced numerical chromosomal changes in the near diploid range at doses that induced cellular transformation. 32P-Postlabeling analysis revealed that exposure of cells to BP-A also elicited DNA adduct formation in a dose-dependent fashion. Our results indicate that BP-A has cell-transforming and genotoxic activities in cultured mammalian cells and potential carcinogenic activity.

 ADDIN EN.REFLIST Tsutsui, T., Tamura, Y., Suzuki, A., Hirose, Y., Kobayashi, M., Nishimura, H., Metzler, M. and Barrett, J.C. (2000). Mammalian cell transformation and aneuploidy induced by five bisphenols. Int. J. Cancer 86:151-154.
 Bisphenol-A (BP-A), a monomer of plastics used in numerous consumer products and a xenoestrogen, induces cellular transformation and aneuploidy in Syrian hamster embryo (SHE) cells. In this study, the abilities of 4 other bisphenols to induce cellular transformation and genetic effects in SHE cells were examined and compared to BP-A. Cellular growth was inhibited by all bisphenols in a concentration-related manner. The growth inhibitory effect of the bisphenols ranked: BP-5 > BP-4 > BP-3 > BP-2 or BP-A. Morphological transformation of SHE cells was induced by BP-A, BP-3, BP-4 and BP-5, and the induced-transformation frequencies were highest with BP-4. None of the bisphenols induced gene mutations at the Na(+)/K(+) ATPase locus or the hprt locus, or chromosomal aberrations in SHE cells. By contrast, aneuploidy induction in the near-diploid range was exhibited by BP-A, BP-3, BP-4 or BP-5, corresponding to the transforming activity of each compound. The results indicate that BP-A, BP-3, BP-4 and BP-5 exhibit transforming activity in SHE cells, while BP-2 does not, and that aneuploidy induction may be a causal mechanism of the transforming activity.

 ADDIN EN.REFLIST Wada, H., Tarumi, H., Imazato, S., Narimatsu, M. and Ebisu, S. (2004). In vitro estrogenicity of resin composites. J. Dent. Res. 83:222-226.
 Previously, we have reported that sealants incorporating bisphenol A dimethacrylate showed estrogenicity by a reporter gene assay. This study tested the hypothesis that commercial composites, which contain various monomers and additives, exhibit estrogenic activity in vitro. The estrogenic activities of eluates obtained from 24 composites and 18 chemicals identified from the composites tested were examined with the use of the reporter gene assay. Among the 24 composites, 6 products were estrogenic, and among the 18 constituents, 1 photostabilizer, 2-hydroxy-4-methoxy-benzophenone (HMBP), 1 photoinitiator, 2,2-dimethoxy-2-phenyl-acetophenone (DMPA), and 1 inhibitor, 2,6-di-tert-butyl-p-cresol (BHT) had significant estrogenic activity. The concentration of HMBP in 4 estrogenic eluates was greater than the minimum concentration required for estrogenicity, and DMPA was found at a higher level than the minimum estrogenic concentration in the remaining 2 estrogenic specimens. These results suggest that the observed estrogenic activity of 6 composites is associated with the elution of either HMBP or DMPA.

 ADDIN EN.REFLIST Walsh, D. E., P. Dockery and C. M. Doolan (2005) Estrogen receptor independent rapid non-genomic effects of environmental estrogens on [CA++]i in human breast cancer cells. Mol. Cell Endocrinol. 230, 23-30.
 The aim of this study was to identify and characterize an alternative pathway through which environmental estrogenic compounds may mediate their intracellular effects. Three human breast cancer cell lines were employed including MCF-7 cells, which express both ERalpha and ERbeta; MDA-MB-231 cells, which express ERbeta but not ERalpha; and SKBR-3 cells, which express neither ERalpha nor ERbeta. The effect of environmental estrogenic compounds on intracellular calcium ion concentration ([Ca(2+)](i)) was measured and compared to that of 17beta-estradiol (E2). A rapid and maintained increase in [Ca(2+)](i) was observed following the application of nanomolar concentrations of environmental estrogens and E2 regardless of the expression of ERalpha and ERbeta. Removal of extracellular Ca(2+) completely abolished the steroid-induced [Ca(2+)](i) increase. Pre-treatment of cells with the estrogen receptor (ER) antagonist ICI 182,780 had no effect on either basal [Ca(2+)](i) or the steroid-triggered [Ca(2+)](i) response. In summary, we have demonstrated ER independent rapid non-genomic effects of environmental estrogenic compounds, at nanomolar concentrations, on [Ca(2+)](i). The results of this study demonstrate an alternative pathway to explain potent intracellular effects of endocrine disrupting chemicals. Regarding bisphenol A, rapid (within 1.5 min) influx of calcium was observed in human MCF-7 breast cancer cells in response to both oestradiol and bisphenol A that was significant at the lowest dose tested, which was 0.1 nM (23 ppt bisphenol A); for oestradiol, the EC50 was 0.11 nM) and for bisphenol A the EC50 was 0.15 nM or 342 ppt.

Wetherill, Y. B., Petra, C. E., Monk, K. R., Puga, A. and Knudsen, K. E. (2002). The xenoestrogen bisphenol A induces inappropriate androgen receptor activation and mitogenesis in prostate adenocarcinoma cells. Molecular Cancer Therapeutics 7:515-524.
Bisphenol A stimulated proliferation of human prostate cancer (LNCaP) cells. There was an inverted-U dose-response curve, with maximum stimulation at 230 ppt, lower stimulation at 23 ppt and 2.3 ppb, and no stimulation at either 2.3 ppt (NOAEL) and 23 ppb (which also would have been erroneously thought to be the NOAEL if this was the lowest dose tested.

 ADDIN EN.REFLIST Wetherill, Y. B., N. I. Fisher, A. Staubach, M. Danielsen, R. W. de Vere White and K. E. Knudsen (2005) Xenoestrogen action in prostate cancer: Pleiotropic effeccts dependent of androgen receeptor status. Cancer Res. 65, 54-65.
Androgen is critical for prostate development, growth, and survival. Therapies for advanced prostate cancer aim to block androgen receptor (AR) action. However, recurrent tumors ultimately arise, which harbor restored AR activity. One mechanism of such reactivation occurs through AR mutations, rendering the receptor responsive to noncanonical ligands. We have shown previously that a known xenoestrogen, bisphenol A (BPA), activates a tumor-derived AR mutant (T877A), leading to androgen-independent prostate cancer cell proliferation. Here, we show that BPA cooperates with androgen to activate AR-T877A as shown by both reporter assays and increased levels of prostate-specific antigen expression. Further investigations using both yeast and mammalian model systems revealed that multiple AR alleles are responsive to BPA, thus expanding the potential influence of xenoestrogens on prostate cancer. Moreover, in vitro radioligand binding assay revealed that BPA alters 5a-dihydrotestosterone binding to AR-T877A likely through noncompetitive inhibition. We also show that higher concentrations of BPA block proliferation of AR-positive, androgen-dependent prostate adenocarcinoma cells (LNCaP and LAPC-4), with a more modest inhibitory effect on androgen-independent cells (22Rv-1). By contrast, AR-negative prostate cancer cells failed to show growth inhibition after exposure to high BPA dose. Together, these data show that BPA can serve as a potential ‘‘hormone sensitizer’’ of the mutant ARs present in advanced prostate adenocarcinomas, thereby possibly contributing toward therapeutic relapse in advanced prostate cancer patients and supporting the notion that nonsteroidal environmental compounds can alter the function of nuclear receptor complexes

 ADDIN EN.REFLIST Wozniak, A. L., N. N. Bulayeva and C. S. Watson (2005) Xenoestrogens at picomolar to nanomolar concentrations trigger membrane estrogen receptor-a mediated Ca++ fluxes and prolactin release in GH3/B6 pituitary tumor cells. Environ. Health Perspect. 113:431-439.
 Xenoestrogens (XEs) are widespread in our environment, and known to have deleterious effects in animal (and perhaps human) populations. Acting as inappropriate estrogens, XEs are thought to interfere with endogenous estrogens such as estradiol (E2) to disrupt normal estrogenic signaling. We investigated the effects of E2 vs. several XEs representing organochlorine pesticides (dieldrin, endosulfan, DDE), plastics manufacturing byproducts/detergents (nonylphenol, bisphenol A), a phytoestrogen (coumestrol), and a synthetic estrogen (DES) on the pituitary tumor cell subline GH3/B6/F10, previously selected for expression of high levels of membrane estrogen receptor-alpha. Picomolar to nanomolar concentrations of both E2 and XEs caused intracellular Ca++ changes within 30 sec of administration. Each XE produced a unique temporal pattern of Ca++ elevation. Removing Ca++ from the extracellular solution abolished both spontaneous and XE-induced intracellular Ca++ changes, as did 10µM nifedipine. This suggests that XEs mediate their actions via voltage dependent L-type Ca++ channels in the plasma membrane. None of the Ca++ fluxes came from intracellular Ca++ stores. E2 and each XE also caused unique time- and concentration dependent patterns of prolactin (PRL) secretion that were largely complete within 3 minutes of administration. PRL secretion was also blocked by nifedipine, demonstrating a correlation between Ca++ influx and PRL secretion. These data indicate that at very low concentrations, XEs mediate membrane-initiated intracellular Ca++ increases resulting in PRL secretion via a mechanism similar to that for E2, but with distinct patterns and potencies which could explain their abilities to disrupt endocrine functions. With regard to bisphenol A, it significantly stimulated a rapid (within 30 sec) influx of calcium at the lowest dose that was examined (0.23 ppt or 10-12 M); the greatest response occurred at 230 ppt, while the magnitude of the response decreased at 2.3 ppb, forming an inverted-U dose-response curve. The calcium influx response to bisphenol A at 230 ppt was actually greater than that for oestradiol or DES. Prolactin release, which is triggered by calcium influx in these cells, was detected within 1 min at 0.23 ppt bisphenol A, similar to the response to oestradiol.

Yanagihara N, Toyohira Y, Ueno S, Tsutsui M, Utsunomiya K, Liu M, Tanaka K. 2004 Oct 14. Stimulation of catecholamine synthesis by environmental estrogenic pollutants. Endocrinology, on line, 10/14. Environmental estrogenic pollutants are compounds that have been shown to have estrogenic effects on fetal development and reproductive systems. Less attention, however, has been paid to their influence on neuronal functions. We report here the effects of estrogenic pollutants on catecholamine synthesis in bovine adrenal medullary cells used as a model system of noradrenergic neurons. Treatment of cultured bovine adrenal medullary cells with p-nonylphenol and bisphenol A at 10 nM for 3 days stimulated (14)C-catecholamine synthesis from [(14)C]tyrosine and tyrosine hydroxylase activity, an effect that was not inhibited by ICI 182,780, an antagonist of estrogen receptors. Significant effects of p-nonylphenol on (14)C-catecholamine synthesis were observed at 0.1 nM that is 45 times lower than that of the international regulatory standard (4.5 nM) and the maximum effects were around at 10 - 100 nM. The concentrations (0.1 - 10 nM) used in the present study are similar to the range observed in rivers in the U.S. or Europe. On the other hand, short-term treatment of cells with 10 nM p-nonylphenol for 10 min also activated tyrosine hydroxylase, which was suppressed by U0126, an inhibitor of mitogen-activated protein kinase (MAPK) kinase. Furthermore, treatment of cells with p-nonylphenol for 5 min increased the phospho-p44/42MAPK in a concentration (1 - 1000 nM)-dependent manner while p-nonylphenol (100 nM, 2 days) enhanced both levels of nonphospho- and phospho-p44/42MAPK. These findings suggest that short-term and long-term treatment of cells with estrogenic pollutants at environmental concentrations stimulates catecholamine synthesis and MAPK through an estrogen receptor-independent pathway.

Yoneda, T.; Hiroi, T.; Osada, M.; Asada, A., and Funae, Y. (2003). Non-genomic modulation of dopamine release by bisphenol-A in PC12 cells. Journal of Neurochemistry. 87(6):1499-1508. An endocrine disruptor chemical, bisphenol-A (BPA), is reported to have several short-term actions in various tissues and/or cells; however, the mechanisms of these actions have not been fully elucidated. We investigated short-term actions evoked by BPA in pheochromocytoma PC12 cells. BPA elicited dopamine release in PC12 cells in a dose-dependent manner. A selective N-type calcium channel antagonist (omega-conotoxin GVIA) and a ryanodine receptor blocker (ryanodine) inhibited the BPA-induced dopamine release. The expression of ryanodine receptor mRNA was detected by RT-PCR in PC12 cells. Subsequently, in order to prove whether membrane receptors participate in BPA-evoked dopamine release, a guanine nucleotide-binding protein inhibitor [guanosine 5'-(beta-thio) diphosphate], cyclic AMP antagonist (Rp-cAMPS) or protein kinase A inhibitor (H7 or H89) was added to PC12 cells prior to BPA-treatment. All of these agents suppressed BPA-evoked dopamine release, indicating that multiple signaling pathways may be involved in BPA-evoked dopamine release in PC12 cells. In conclusion, we demonstrated that BPA induced dopamine release in a non-genomic manner through guanine nucleotide-binding protein and N-type calcium channels. These findings illustrate a novel function of BPA and suggest that exposure to BPA influences the function of dopaminergic neurons.
Yu Z, Zhang L, Wu D. 2004 Jul. [Effects of three environmental estrogens on expression of proliferation and apoptosis-associated genes in PEO4 cells]. Wei Sheng Yan Jiu 33:404-6. This study was designed to investigate the molecular mechanisms of proliferation and apoptosis by environmental estrogens (n-4-noniphenol, NP; Bisphenol A, BisA; Dibutylphthalate, DBP) in ovarian cancer PEO4 cells. METHODS: PEO4 cells were maintained in DMEM medium with 10% neonatal bovine serum. Five days before the beginning of experiments, the cells were seeded in phenol red-free DMEM medium containing 5% charcoal dextran-treated FBS. The cells were harvested and seeded in 6-well culture plates or in 75ml flacks. After various concentration of NP, BisA and DBP treatment for 72h, the cells were harvested and detected mRNA and protein expression of PCNA, bcl-2 and bax by reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. RESULTS: 32 x 10(-7) mol/L NP and 32 x 10(-7) mol/L BisA could significantly up-regulate PCNA and bcl-2 mRNA expression and down-regulate the bax mRNA expression, and 32 x 10(-6) mol/L DBP could up-regulate PCNA mRNA expression, but had no effect on bax and bcl-2 mRNA expression. These results were further confirmed by following immunohistochemistry. CONCLUSION: PCNA, bcl-2 and bax pathway might involve in cell proliferation and apoptosis events by environmental estrogens in ovarian cancer PEO4 cells.


XVII. BISPHENOL A BINDING AND RESPONSES WITH ER-alpha AND ER-beta

There is now considerable evidence that bisphenol A acts as a SERM, and relative to estradiol: 1) interacts differently within the ligand-binding domain of estrogen receptors  ADDIN EN.CITE Gould1998909017Gould, J.C.Leonard, L.S.Maness, S.C.Wagner, B.L.Conner K.Zacharewski, T.Safe, S.McDonnell, D.P.Gaido, K.W.Bisphenol A interacts with the estrogen receptor alpha in a distinct manner from estradiolMol. Cell Endocrinol.Mol. Cell Endocrinol.203-2141421998(Gould et al. 1998), 2) shows a different binding affinity for and regulation of ERað ðand ERß in target cells  ADDIN EN.CITE Kuiper1997969617Kuiper, G. G.Carlsson, B.Grandien, K.Enmark, E.Haggblad, J.Nilsson, S.Gustafsson, J. A.Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and betaEndocrinologyEndocrinol.863-701383Amino Acid SequenceAnimalComparative StudyFemaleHumanIsomerismLigandsMaleMolecular Sequence DataRats*Receptors, Estrogen/ge [Genetics]*Receptors, Estrogen/me [Metabolism]*RNA, Messenger/me [Metabolism]Support, Non-U.S. Gov'tTissue Distribution1997Routhledge200089017Routhledge, E.J.White, R.Parker, M.G.Sumpter, J.P.Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) a and ERßJ. Biol. Chem.J. Biol. Chem.35986-35993462000(Kuiper et al. 1997; Routhledge et al. 2000), and 3) interacts differently with transcriptional co-regulators  ADDIN EN.CITE Routhledge200089017Routhledge, E.J.White, R.Parker, M.G.Sumpter, J.P.Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) a and ERßJ. Biol. Chem.J. Biol. Chem.35986-35993462000(Routhledge et al. 2000). Additional studies concerning this issue are also listed below.
Cappelletti, V., Saturno, G., Miodini, P., Korner, W. and Daidone, M.G. (2003). Selective modulation of ER-beta by estradiol and xenoestrogens in human breast cancer cell lines. 60:567-576.
In the last decades, substances with estrogenic activity have been dispersed into the environment. Xenoestrogens act by binding to estrogen receptors, ligand-regulated transcription factors, for which two subtypes have been described, ER-alpha and ER-beta, which are often coexpressed at variable amounts in different tissues. We investigated variations in the expression of ER-alpha and ER-beta mRNAs following treatment with four xenoestrogens (bisphenol A, 4-tert octylphenol, 2-hydroxybiphenyl, 4-hydroxybiphenyl) and with 17beta-estradiol in estrogen-sensitive (T47D) and estrogen-insensitive (BT20) breast cancer cell lines. Although to a variable extent, both estradiol and the tested xenoestrogens increased the expression of ER-beta mRNA, whereas a slight effect on ER-alpha was observed only in T47D cells. Upregulation of ER-beta expression by estradiol and xenoestrogens was observed only in the presence of detectable ER-alpha protein levels. These findings indicate a regulatory role for ER-beta in ER-alpha-mediated transcription and a role for ER-beta in mediating xenoestrogen toxicity.

Kurosawa, T., H. Hiroi, O. Tsutsumi, T. Ishikawa, Y. Osuga, T. Fujiwara, S. Inoue, M. Muramatsu, M. Momoeda and Y. Taketani (2002). The activity of bisphenol A depends on both the estrogen receptor subtype and the cell type. 49: 465-71.
Bisphenol A (BPA), a monomer of plastic used in consumer products, is abundant in the environment and enters the body by ingestion or adsorption. In order to characterize the estrogenic effect of BPA, we performed luciferase assay on three independent cell lines derived from different tissues transfected with either human ERalpha cDNA or ERbeta cDNA. The estrogenic activities of BPA were detectable in all cell lines via both ERalpha and ERbeta. In 293T cells and Hec-1 cells, the estrogenic activities were significantly decreased when cells expressing ERalpha were incubated with 10(-6) M BPA in the presence of 10(-8) M 17beta-estradiol (E2) while the activities via ERbeta were essentially unchanged in the same conditions. Interestingly, no reduction of estrogenic activity was detected in HOS-TE85 cells via either ERalpha or ERbeta. Our results indicate that BPA only acts as an agonist of estrogen via ERbeta whereas it has dual actions as an agonist and antagonist in some types of cells via ERalpha. Thus, the activity of BPA may depend on the ER subtype and the tissue involved.

 ADDIN EN.REFLIST Masuyama, H. and Hiramatsu, Y. (2004). Involvement of suppressor for Gal 1 in the ubiquitin/proteasome-mediated degradation of estrogen receptors. J Biol Chem 279:12020-12026.
 The proteasome-mediated pathway involves the degradation of several nuclear receptors. Previously we demonstrated that the interaction between the suppressor for Gal 1 (SUG1) and nuclear receptors, the vitamin D receptor, or the pregnane X receptor was involved in proteasome-mediated degradation. In our recent experiments, we examined the potential role of SUG1 in the proteasome-mediated degradation of estrogen receptors (ER)alpha and -beta. Both ERs interacted with SUG1 in a ligand-dependent manner. Functionally, the overexpression of SUG1 inhibited both ERalpha- and ERbeta-mediated transcription in the presence of ligands. Transient expression studies demonstrated that the overexpression of wild-type SUG1 generated proteolytic fragments of both ERs and that these products were blocked by a proteasome inhibitor. The overexpression of SUG1 also enhanced the formation of ubiquitinated proteins of both ERs in the presence of ligand. On the other hand, bisphenol A (BSA), which activated ER-mediated transcription, did not enhance the interaction between ERbeta and SUG1. Furthermore, the degradation of ERbeta was much slower in the presence of BSA than in the presence of estradiol or phthalate, which is another endocrine-disrupting chemical. Also, BSA had no effect on the formation of proteolytic fragments of ERbeta, and neither did it have any effect on the ubiquitination of ERbeta. These findings indicate that the ubiquitin/proteasome-mediated degradation of both ER proteins may involve the interaction of SUG1 with both ERs. Moreover, BSA strongly blocked the ubiquitination and degradation of ERbeta compared with estradiol, suggesting that BSA may affect the ERbeta-mediated transcription of target genes by inhibiting ERbeta degradation.

Matthews, J. B., K. Twomey and T. R. Zacharewski (2001). In vitro and in vivo interactions of bisphenol A and its metabolite, bisphenol A glucuronide, with estrogen receptors alpha and beta. Chemical Research in Toxicology 14(2): 149-157.
The estrogenic activities of bisphenol A (BPA) and its major metabolite BPA glucuronide (BPA-G) were assessed in a number of in vitro and in vivo assays. BPA competed with [H-3]-17 beta -estradiol (E2) for binding to mouse uterine cytosol ER, a glutathione S-transferase (GST)-human ER D, E, and F domain fusion protein (GST-hER alpha def) and full-length recombinant hER beta. The IC50 values for E2 were similar for all three receptor preparations, whereas BPA competed more effectively for binding to hER beta (0.96 muM) than to either mouse uterine cytosol ER (26 muM) or GST-hERadef (36 CIM) In contrast, BPA-G did not competitively displace [H-3]E2 from any of the ER preparations. In MCF-7 cells transiently transfected with Gal4-hER alpha def or Gal4-hER beta def, BPA induced reporter gene activity with comparable EC50 values (71 and 39 muM, respectively). No significant induction of reporter gene activity was seen for BPA-G. Cotreatment studies showed that concentrations of (10 muM) BPA and BPA-G did not antagonize EB-induced luciferase mediated through either Gal4-hER alpha def or Gal4-hER beta def. In vivo, the uterotropic effect of gavage or subcutaneous (sc) administration of 0.002-800 mg of BPA/kg of body weight/day for three consecutive days was examined in immature rats. Dose-related estrogenic effects on the rat uterus were observed at oral doses of 200 and 800 mg/kg and at sc doses of 10, 100, and 800 mg/kg. These results demonstrate that BPA competes more effectively for binding to ER beta, but induces ER alpha- and ER beta -mediated gene expression with comparable efficacy. In contrast, BPA-G did not exhibit any in vitro estrogenic activity. In addition, there was a clear route dependency on the ability of BPA to induce estrogenic responses in vivo. In summary, bisphenol A ERß binding affinity was 38-fold greater than for ERað.ð

Paris, F., P. Balaguer, B. Terouanne, N. Servant, C. Lacoste, J. P. Cravedi, J. C. Nicolas and C. Sultan (2002). Phenylphenols, biphenols, bisphenol-A and 4-tert-octylphenol exhibit alpha and beta estrogen activities and antiandrogen activity in reporter cell lines. Mol. Cell Endocrinol. 193(1-2): 43-9.
This study provides evidence that several phenyl derivatives present both estrogenic and antiandrogenic activity. The extent of hydroxylation and the position of the hydroxyl function were important in determining their estrogenicity and antiandrogenicity. Of the tested compounds, bisphenol-A and 4,4' biphenol had very high estrogenic activity, although it was lower than that of the strong estrogenic alkylphenol, 4-tert-octylphenol. Bisphenol-A and 4,4' biphenol were able to activate ERs at concentrations lower than 1 microM, whereas the other compounds only activated at concentrations above 1 microM. Interestingly, 4,4' biphenol was a better agonist for ERbeta than for ERalpha. No androgenic activity was detected for any of these compounds. Bisphenol-A, 3-OH phenylphenol, 4-OH phenylphenol and 4,4' biphenol exhibited antiandrogenic activity close to that of 4-tert-octylphenol (IC(50) approximately 5 microM). In whole cell binding assays, these compounds displaced [3H] R1881 with Ki = 10 microM. Although these Ki values seem high in comparison with that of hydroxyflutamide (0.4 microM), one must keep in mind that environmental chemicals can accumulate in adipose tissues for several years. In conclusion, these environmental chemicals may have a negative impact on androgen action during fetal and post-natal life.

Pennie, W. D., T. C. Aldridge and A. N. Brooks (1998). Differential activation by xenoestrogens of ER alpha and ER beta when linked to different response elements. J. Endocrinol. 158: R11-R14.

Routhledge, E.J., White, R., Parker, M.G. and Sumpter, J.P. (2000). Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) að and ERß. J. Biol. Chem. 46:35986-35993.
HeLa cells were transfected with ERað and ERß, and bisphenol A showed a 10-fold greater binding affinity (relative to estradiol) for ERß than ERað. Binding of bisphenol A to ERß but not ERað also resulted in recruitment of the ER coactivator TIF2 with a potency relative to estradiol of 0.05.

Seidlova-Wuttke, D., H. Jarry and W. Wuttke (2004). Pure estrogenic effect of benzophenone-2 (BP2) but not of bisphenol A (BPA) and dibutylphtalate (DBP) in uterus, vagina and bone. Toxicology 205:103-12.
Contradictory results whether the endocrine disrupters (ED) benzophenone-2 (BP2), bisphenol A (BPA) and dibutylphtalate (DBP) exert estrogenic effects have been published. Selective estrogen receptor modulators (SERMs) exert estrogenic effects in some but not in all organs and ED may be SERMs. Therefore, we studied their binding properties to recombinant ERalpha and ERbeta protein and their effects in the uterus, vagina and bone of ovariectomized rats. BP2 bound to both receptor subtypes, while BPA had a relatively high ERbeta selectivity. DBP did not bind to ERalpha but with a low affinity to ERbeta. In the uterus, only E(2) and BP2 increased uterine weight and the complement C3 but decreased ERbeta gene expression. Discrete effects of BPA and DBP in the uterus were found upon histological examination. In the vagina, BP2 but not BPA and DBP had clear estrogenic effects. E(2) and BP2 had antiosteoporotic effects in the metaphysis of the tibia. The serum surrogate parameters of bone metabolism, i.e. osteocalcin and the cross (rat) laps were significantly reduced by E(2), an effect shared with BP2 but not by the two other EDs. The conclusion: BP2 acts as ERalpha and ERbeta agonist mimicking effects of E(2), while the effects of BPA and DBP are not pure estrogenic.

Takao, T., W. Nanamiya, H. P. Nazarloo, R. Matsumoto, K. Asaba and K. Hashimoto (2003). Exposure to the environmental estrogen bisphenol A differentially modulated estrogen receptor-alpha and -beta immunoreactivity and mRNA in male mouse testis. 72(10): 1159-1169.
Bisphenol A at concentrations of 0.5 or 50 µg/ml in the drinking water was fed to young male mice (doses are likely in the range of 0.2 and 20 mg/kg/day). Effects on estrogen receptor (ER) alpha and beta proteins and mRNA in the testis following 8-weeks of oral administration of bisphenol A. ERß was localized in the nuclei of spermatogonia and/or spermatocytes, and the number of ERß containing cells (and mRNA) per testis were significantly decreased in the 50 microg/ml bisphenol A-treated group compared with controls. In contrast, ERað immunopositive cells (and mRNA) per testis were markedly increased in the 50 microg/ml bisphenol A-treated group compared with the controls. The existence of ER alpha and beta in the testis suggests that estrogens directly affect germ cells during testicular development and spermatogenesis, and differential modulation of ER alpha and beta in the testis could be involved in the effects of bisphenol A.


XVIII. EXPOSURE LEVELS IN HUMAN ADULTS AND FETUSES

Calafat, A.M., Kuklenyik, Z., Reidy, J.A., Caudill, S.P, Ekong, J. and Needham, L.L. (2005). Urinary concentrations of bisphenol A and 4-nonyl phenol in a human reference population. Environ. Health Perspect. 113:391-395.
Bisphenol A (BPA) is used to manufacture polycarbonate plastic and epoxy resins, which are used in baby bottles, as protective coatings on food containers, and for composites and sealants in dentistry. 4-Nonyl phenol (NP) is used to make nonylphenol ethoxylates, nonionic surfactants applied as emulsifying, wetting, dispersing, or stabilizing agents in industrial, agricultural, and domestic consumer products. The potential for human exposure to BPA and NP is high because of their widespread use. We measured BPA and NP in archived urine samples from a reference population of 394 adults in the United States using isotope-dilution gas chromatography mass spectrometry. The concentration ranges of BPA and NP were similar to those observed in other human populations. BPA was detected in 95% of the samples examined at concentrations at or above 0.1 micrograms per liter of urine (µg/L); the geometric mean and median concentrations were 1.33 µg/L (1.36 µg per gram of creatinine [µg/g creatinine]) and 1.28 µg/L (1.32 µg/g creatinine), respectively; the 95th percentile concentration was 5.18 µg/L (7.95 µg/g creatinine). NP was detected in 51% of the samples examined at or above 0.1 µg/L. The median and 95th percentile concentrations were less than 0.1 µg/L and 1.57 µg/L (1.39 µg/g creatinine), respectively. The frequent detection of BPA suggests widespread exposure to this compound in residents of the United States. The lower frequency of detection of NP than of BPA could be explained by a lower exposure of humans to NP, by different pharmacokinetic factors (i.e., absorption, distribution, metabolism, elimination), by the fact that 4-n-nonyl phenol —the measured NP isomer— represents a small percentage of the NP used in commercial mixtures, or a combination of all of the above. Additional research is needed to determine the best urinary biomarker(s) to assess exposure to NP. Despite the sample population’s non-representativeness of the U.S. population (although sample weights were used to improve the extent to which the results represent the U.S. population) and relatively small size, this study provides the first reference range of human internal dose levels of BPA and NP in a demographically diverse human population.

Engel, S.M., Levy, B., Liu, Z., Kaplan, D. and Wolff, M.S. (2006). Xenobiotic phenols in early pregnancy amniotic fluid. Reprod. Toxicol. 21: 110-112.
We found detectable levels of three phytoestrogens (enterolactone, daidzein and genistein) and bisphenol A (BPA) in 21 residual amniotic fluid specimens that were collected before 20 weeks gestation. Samples were obtained by amniocentesis from women who were referred to the Mount Sinai Medical center because of advanced maternal age. Phytoestrogens were present in higher concentrations than BPA. Enterolactone was detected at the highest concentration (median 95.9mug/L), followed by daidzein and genistein (9.5 and 1.4 mug/L, respectively). BPA was present at very low concentrations (10%>LOD of 0.5mug/L). The relative concentration of the chemicals measured in amniotic fluid were identical to those in urine reported by other studies, i.e. enterolactone>daidzein>genistein>>BPA. Amniotic fluid is a source of fetal exposure to polar xenobiotics that come from the mother.

Fujimaki, K, Arakawa, C, Yoshinaga, J, Watanabe, C, Serizawa, S, Imai, H, Shiraishi, H and Mizumoto, Y (2004). [Estimation of intake level of bisphenol A in Japanese pregnant women based on measurement of urinary excretion level of the metabolite]. Nippon Eiseigaku Zasshi 59: 403-408.
[Article in Japanese, only abstract in English]
OBJECTIVE: The daily bisphenol A (BPA) intake level of Japanese pregnant women was surveyed based on the measurement of the urinary excretion level of a BPA metabolite. METHODS: Spot urine samples were collected from 56 pregnant women who visited the gynecology division of a hospital for a routine health check between June and October 2003. The urinary concentrations of the BPA metabolite and creatinine were measured by GC/MS/MS and spectrophotometry, respectively. Daily BPA intake was assumed to be equal to daily excretion. RESULTS: The daily intake of BPA among Japanese pregnant women was estimated to be in the range of < 0.3 to 7.9 microg/day (median < 2.0 microg/day), being consistent with the levels in previous studies for non-pregnant Japanese women. This level was far below the current Acceptable Daily Intake (0.01 mg/kg/day) which was set by the European Commission. The maximum estimated intake per body weight (0.16 microg/kg/day) reached 1/10 of the Lowest Adverse Effect Level of BPA for pregnant mice for a reproductive effect on the offspring (2 microg/kg/day). CONCLUSION: It is desirable to lessen BPA intake from a precautionary viewpoint, particularly in pregnant women.

 ADDIN EN.REFLIST Hiroi, H., Tsutsumi, O., Takeuchi, T., Momoeda, M., Ikezuki, Y., Okamura, A., Yokota, H. and Taketani, Y. (2004). Differences in serum bisphenol A concentrations in premenopausal normal women and women with endometrial hyperplasia. Endocr J 51:595-600.
 Exposure to endocrine disrupting chemicals (EDCs) has been raised in relation to its potential for adverse health outcomes. Bisphenol A (BPA) is an estrogenic EDC widely found in plastic products. We determined BPA concentrations in premenopausal women by an enzyme-linked immunosorbent assay and evaluated possible linkage between its contamination levels and endometrial hyperplasia, an estrogen-related disorder of the uterus. It has been implied that higher levels of BPA, which binds to estrogen receptor and plays estrogenic roles may, enhance endometrial hyperplasia. Serum BPA was detectable in all subjects and its concentrations in healthy controls with normal endometrium were 2.5 +/- 1.5 ng/ml (mean +/- SD). BPA levels in patients with simple endometrial hyperplasia with benign nature were 2.9 +/- 2.0 ng/ml and were not significantly different from the controls. Unexpectedly, BPA levels in patients with complex endometrial hyperplasia with malignant potential were 1.4 +/- 0.4 ng/ml and significantly lower compared to both control and simple endometrial hyperplasia groups. In addition, we measured the serum BPA levels in postmenopausal endometrial cancer patient (1.4 +/- 0.5 ng/ml), which were also significantly lower than control and simple endometrial hyperplasia groups. These findings suggest the presence of associations between BPA exposure and complex endometrial hyperplasia and endometrial cancer. The mode of action of BPA may be more complex than expected and the contradictory results may serve as a clue to addressing the mechanisms of linkage between occurrence of estrogen-dependent diseases and endocrine disruption.

Ikezuki, Y., O. Tsutsumi, Y. Takai, Y. Kamei and Y. Taketani (2002). Determination of bisphenol A concentrations in human biological fluids reveals significant early prenatal exposure. Human Reprod. 17:2839-2841.
Using an enzyme-linked immunosorbent assay, Ikezuki et al. determined bisphenol A in serum of non-pregnant women, ovarian follicular fluid obtained during in vitro fertilization procedures, serum from early and late pregnancy women, fetal umbilical cord blood at delivery, and amniotic fluid at 15-18 weeks gestation and at late pregnancy. The human sera showed average bisphenol A at 1.4 to 2.4 ng/ml levels, while the 15-18 week fetal amniotic fluid showed higher levels averaging 8.3 ng/ml, the highest level of exposure in the study coinciding with a period of great fetal sensitivity. The authors concluded that there was accumulation of bisphenol A in early fetuses, and significant prenatal exposure. Human ovarian follicular fluid contained bisphenol A at an average concentration of 2.4 ng/ml, which is of particular concern following the publication of effects of the chemical on ovarian meiotic congression failure and aneuploidy in mice at levels of bisphenol A that are presumably well below this value  ADDIN EN.CITE Hunt2003959517Hunt, P.A.Koehler, K.E.Susiarjo, M.Hodges, C.A.Hagan, A.Voigt, R.C.Thomas, S.Thomas, B.F.Hassold, T.J.Bisphenol A causes meiotic aneuploidy in the female mouseCurrent BiologyCurrent Biol.546-553132003(Hunt et al. 2003) based on the findings of Zalko et al. (2003).

Inoue, K., K. Kato, Y. Yoshimura, T. Makino and H. Nakazawa (2000). Determination of bisphenol A in human serum by high-performance liquid chromatography with multi-electrode electrochemical detection. J. Chromat. B 749:17-23.
Inoue et al. described HPLC linked to multielectrode electrochemical detection that yielded highly sensitive detection of bisphenol A at 0.01 ng/ml in solvent and 0.05 ng/ml from serum. The authors also described the preparation and importance of water free of bisphenol A at this level of detection. Bisphenol A was detected in five healthy human volunteers at a mean value of 0.32 ng/ml serum.

Kang, JH, Kondo, F and Katayama, Y (2006). Human exposure to bisphenol A. Toxicol.: Online June 16.
Bisphenol A (BPA), 2,2-bis(4-hydroxyphenyl)propane, is made by combining acetone and phenol. It has estrogenic activity and is acutely toxic to aquatic organisms. BPA is used mainly as a material for the production of epoxy resins and polycarbonate plastics. Due to an increase in products based on epoxy resins and polycarbonate plastics, human exposure to BPA has increased. The environment (aquatic environment, air and soil) can be one source of human BPA exposure, but the primary route of human exposure is foods. The daily human intake of BPA is < 1 mug/kg body weight/day on the basis of several studies, and whether these doses can have an adverse endocrine disruptive effect on humans, especially fetuses, needs to be studied carefully. [FvS NOTE: The statement that human intake of BPA is < 1 microgram /kg /day is not consistent with the levels of BPA detected in human blood and other tissues in many other studies]

Kuroda, N., Y. Kinoshita, Y. Sun, M. Wada, N. Kishikawa, K. Nakashima, T. Makino and H. Nakazawa (2003). "Measurement of bisphenol A levels in human blood serum and ascitic fluid by HPLC using a fluorescent labeling reagent." Journal of Pharmaceutical and Biomedical Analysis 30(6): 1743-1749.
Matsumoto, A., N. Kunugita, K. Kitagawa, T. Isse, T. Oyama, G. L. Foureman, M. Morita and T. Kawamoto (2003). "Bisphenol A levels in human urine." Environmental Health Perspectives 111(1): 101-104.

Miyamoto, K and Kotake, M (2006). Estimation of daily bisphenol a intake of Japanese individuals with emphasis on uncertainty and variability. Environ. Sci. 13: 15-29.
The purpose of this study was to comprehensively assess the exposure of Japanese individuals to bisphenol A (BPA) with emphasis on uncertainty and variability in available information. The uncertainty and variability in parameters were numerically analyzed using Monte Carlo simulation. The uncertainty in the functional relationship between sources and exposure was treated by comparing two approaches: one was to aggregate ingestion and inhalation through all possible exposure pathways and the other was to estimate the intake from urinary excretion by backward calculation. For individuals aged 6 months or above, food was the most significant source of intake. The alteration of the method used in inactivating the inside surface of drink cans slightly contributed to the decrease in daily intake. By the backward calculation approach based on urinary excretion, 95% confidence intervals for the daily intake for high-exposure populations were estimated to be 0.037-0.064 microg/kg/day for males and 0.043-0.075 microg/kg/day for females. Even conservatively estimated daily intakes were lower than the EU's temporary tolerable daily intake (TDI) of 10 microg/kg/day as well as the U.S. Environmental Protection Agency (US EPA)'s reference dose (RfD) of 50 microg/kg/day. Thus, it is unlikely that humans, including infants and young children, are at unacceptable risk from possible BPA exposure.

Ouchi, K. and S. Watanabe (2002). "Measurement of bisphenol A in human urine using liquid chromatography with multi-channel coulometric electrochemical detection." Journal of Chromatography B: Analytical Technologies in the Biomedical & Life Sciences 780(2): 365-370.

Sajiki, J., K. Takahashi and J. Yonekubo (1999). Sensitive method for the determination of bisphenol-A in serum using two systems of high-performance liquid chromatography. J. Chromat. B 736:255-261.
Sajiki et al. described HPLC linked to either electrochemical detection or mass spectrometry/electrospray ionization, with detection limits for bisphenol A of 0.2 and 0.1 ng/ml, respectively. UV-linked detection may be limited to 10 to 100 ng/ml. These authors reported bisphenol A at levels of 0 to 1.6 ng/ml serum in 20 healthy human samples.

Sasaki, N., Okuda, K., Kato, T., Kakishima, H., Okuma, H., Abe, K., Tachino, H., Tuchida, K. and Kubono, K. (2005). Salivary bisphenol-A levels detected by ELISA after restoration with composite resin. J Mater Sci Mater Med 16:297-300.
Bisphenol-A diglycidylether methacrylate (Bis-GMA), which is synthesized from bisphenol-A (BPA), a compound with exogenous endocrine disrupter action, is widely used as a dental material. During clinical filling with sealants and composite resins, the compounds are solidified by polymerization and then used. However, it has been noted that unpolymerized monomers may become dissolved in saliva. In this study using a competitive ELISA system, we investigated the changes in the BPA concentration in saliva after restoration with composite resins. Commercial composite resins from nine companies were tested. Mixed saliva was collected from 21 subjects. Based on the dynamics of salivary BPA detected by this ELISA system, we concluded that several tens to 100 ng/ml of BPA were contained in saliva after filling teeth with composite resin but that sufficient gargling can remove it from the oral cavity. Our data suggest that sufficient gargling after treatment is important for risk management.

Schonfelder, G. Wittfoht, W. Hopp, H., Talsness, C., Paul, I and Chahoud, I. (2002). Parent bisphenol A accumulation in human maternal-fetal-placental unit. Environ. Health Perspect. 110:A703-A707.
Bisphenol A (BPA), an endocrine disruptor, is employed in the manufacture of a wide range of consumer products. The suggestion that BPA, at amounts to which we are exposed, alters the reproductive organs of developing rodents has caused concern. At present, no information exists concerning the exposure of human pregnant women and their fetuses to BPA. We therefore investigated blood samples from mothers (n = 37) between weeks 32 and 41 of gestation. Afer the births, we also analyzed placental tissue and umbilical cord blood from the same subjects. We developed a novel chemical derivatization–gas chromatography/mass spectrometry method to analyze parent BPA at concentrations < 1 µg/mL in plasma and tissues. Concentrations of BPA ranged from 0.3 to 18.9 ng/mL (median = 3.1 ng/mL) in maternal plasma, from 0.2 to 9.2 ng/mL (median = 2.3 ng/mL) in fetal plasma, and from 1.0 to 104.9 ng/g (median = 12.7 ng/g) in placental tissue. BPA blood concentrations were higher in male than in female fetuses. Here we demonstrate parent BPA in pregnant women and their fetuses. Exposure levels of parent BPA were found within a range typical of those used in recent animal studies and were shown to be toxic to reproductive organs of male and female offspring. We suggest that the range of BPA concentrations we measured may be related to sex differences in metabolization of parent BPA or variable maternal use of consumer products leaching BPA.

 ADDIN EN.REFLIST Sugiura-Ogasawara, M., Ozaki, Y., Sonta, S., Makino, T. and Suzumori, K. (2005). Exposure to bisphenol A is associated with recurrent miscarriage. Human Reproduction 20:2325-2329.
 BACKGROUND: Little is known about the influence of high exposure to bisphenol A on recurrent miscarriage and immunoendocrine abnormalities. METHODS: Serum bisphenol A, antiphospholipid antibodies (aPLs), antinuclear antibodies (ANAs), natural killer cell (NK) activity, prolactin, progesterone, thyroid-stimulating hormone (TSH) and free T4 were examined in 45 patients with a history of three or more (3-11) consecutive first-trimester miscarriages and 32 healthy women with no history of live birth and infertility. Subsequent pregnancy outcome and embryonic karyotype of abortuses were examined prospectively. RESULTS: The mean 6 SD values for bisphenol A in patients were 2.59 6 5.23 ng/ml, significantly higher than the 0.77 6 0.38 ng/ml found for control women (P 5 0.024). High exposure to bisphenol A was associated with the presence of ANAs but not hypothyroidism, hyperprolactinaemia, luteal phase defects, NK cell activity or aPLs. A high level of bisphenol A in itself did not predict subsequent miscarriage. CONCLUSION: Exposure to bisphenol A is associated with recurrent miscarriage.

Sun Y, Irie M, Kishikawa N, Wada M, Kuroda N, Nakashima K. 2004 Oct. Determination of bisphenol A in human breast milk by HPLC with column-switching and fluorescence detection. Biomed Chromatogr 18:501-7. A highly sensitive HPLC method was developed for the determination of xenoestrogenic compound, bisphenol A (BPA) in human breast milk samples. After a two-step liquid-liquid extraction, BPA was derivatized with fluorescent labeling reagent, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The excess fluorescent reagent could be removed effectively using a column-switching system. The separation of DIB-BPA from endogenous materials in milk was carried out on two C(18) columns and fluorescence intensity was monitored at 475 nm with the excitation of 350 nm. A good linearity (r = 0.994) was observed of BPA in the concentration range of 0.2-5.0 ng mL(-1) in breast milk, and the detection limit was 0.11 ng mL(-1) at a signal-to-noise ratio of 3. Intra- and inter-day precision (RSD, %) were less than 8.7 and 10.4, respectively. Twenty-three breast milk samples of healthy lactating women were analyzed for the BPA concentration; the mean value was 0.61 +/- 0.20 ng mL(-1), with no correlation to the lipid content of milk samples.

Takeuchi, T. and O. Tsutsumi (2002). Serum bisphenol A concentrations showed gender differences, possibly linked to androgen levels. Biochem. Biophys. Res. Communic. 291:76-78.
Takeuchi described an antibody-linked detection with sensitivity of approximately 0.3 ng/ml serum. This assay detected bisphenol A at 1.49 ng/ml in human male serum, and 0.64 in serum from women during the mid-follicular stage of the cycle and 1.04 ng/ml in human females with polycystic ovary syndrome. A positive correlation between serum BPA and both total and free testosterone was discovered in all subjects. The authors point out that these levels were above the level that affected preimplantation development.

Takeuchi T, Tsutsumi O, Ikezuki Y, Takai Y, Taketani Y. 2004. Positive relationship between androgen and the endocrine disruptor, bisphenol A, in normal women and women with ovarian dysfunction. Endocrin. J. 51:165-169.
This study was performed to investigate the serum levels of bisphenol A (BPA), an endocrine disruptor, in women with ovarian dysfunction and obesity. Fasting serum samples were obtained from 19 non-obese and 7 obese women with normal menstrual cycles: 7 patients with hyperprolactinemia, 21 patients with hypothalamic amenorrhea, and 13 non-obese and 6 obese patients with polycystic ovary syndrome (PCOS). BPA was measured by an enzyme-linked immunosorbent assay. BPA was detected in all human sera. Serum BPA concentrations were significantly higher in both non-obese and obese women with polycystic ovary syndrome (1.05 +/- 0.10 ng/ml, 1.17 +/- 0.16 ng/ml; p 0.05 in each case). We conclude that the impurities in industrial grade BPA, although some are of much higher estrogenic activity than BPA itself, do not significantly increase the estrogenicity of the industrial compound and therefore do not increase possible adverse health effects from such activity.

Thompson, T. and P. P. Klemchuk (1996). Light stabilization of bisphenol A polycarbonate. Polymer Durability: Degradation, Stabilization, and Lifetime Prediction. R. L. Clough, N. C. Billingham and K. T. Gillen. Washington, D.C., American Chemical Society. 303: 303-317.
Thompson and Klemchuk report that polycarbonate as an ester is susceptible to hydrolysis and base-catalyzed hydrolysis, mainly at elevate d temperatures, while it is more resistant to hydrolysis at ambient temperatures. However, while the findings of Howdeshell et al. (2003) confirm that this applies to new polycarbonate cages, these findings show that as polycarbonate cages age, associated with discoloration and cracking, there is a marked increase in leaching of free BPA into water at room temperature.

Thomson, B.M. and Grounds, P.R. (2005). Bisphenol A in canned foods in New Zealand: an exposure assessment. Food Addit Contam 22:65-72.
Exposure to bisphenol A (BPA) from the consumption of canned and bottled food has been determined for New Zealand adults. Eighty different canned foods purchased from retail outlets in Christchurch, New Zealand, between November 2003 and February 2004 were analysed for BPA concentration by gas chromatography/mass spectrometry. BPA was detected in all foods analysed except for soft drinks. Concentrations ranged from < 10 to 29 microg kg(-1), except for individual samples of tuna, corned beef and coconut cream, which were 109, 98 and 191 microg kg(-1) , respectively. The limit of quantitation was