John A. McLachlan

Synergistic Activation of Estrogen Receptor with Combinations of Environmental Chemicals

Certain chemicals in the environment are estrogenic. The low potencies of these compounds, when studied singly, suggest that they may have little effect on biological systems. The estrogenic potencies of combinations of such chemicals were screened in a simple yeast estrogen system (YES) containing human estrogen receptor (hER). Combinations of two weak environmental estrogens, such as dieldrin, endosulfan, or toxaphene, were 1000 times as potent in hER-mediated transactivation as any chemical alone. Hydroxylated polychlorinated biphenyls shown previously to synergistically alter sexual development in turtles also synergized in the YES. The synergistic interaction of chemical mixtures with the estrogen receptor may have profound environmental implications. These results may represent a previously uncharacterized level of regulation of estrogen-associated responses.

Reproductive tract lesions in male mice exposed prenatally to diethylstilbestrol

Sixty percent of the male offspring from pregnant mice treated with diethylstilbestrol during gestation were sterile. The affected animals had gonadal changes which included intra-abdominal or fibrotic testes, or both. Additionally, nodular masses in the ampullary region of the reproductive tract were observed in 6 of 24 animals; one of these appeared to be preneoplastic.

Female reproductive disorders: the roles of endocrine-disrupting compounds and developmental timing

OBJECTIVE To evaluate the possible role of endocrine-disrupting compounds (EDCs) on female reproductive disorders emphasizing developmental plasticity and the complexity of endocrine-dependent ontogeny of reproductive organs. Declining conception rates and the high incidence of female reproductive disruptions warrant evaluation of the impact of EDCs on female reproductive health. DESIGN Publications related to the contribution of EDCs to disorders of the ovary (aneuploidy, polycystic ovary syndrome, and altered cyclicity), uterus (endometriosis, uterine fibroids, fetal growth restriction, and pregnancy loss), breast (breast cancer, reduced duration of lactation), and pubertal timing were identified, reviewed, and summarized at a workshop. CONCLUSION(S) The data reviewed illustrate that EDCs contribute to numerous human female reproductive disorders and emphasize the sensitivity of early life-stage exposures. Many research gaps are identified that limit full understanding of the contribution of EDCs to female reproductive problems. Moreover, there is an urgent need to reduce the incidence of these reproductive disorders, which can be addressed by correlative studies on early life exposure and adult reproductive dysfunction together with tools to assess the specific exposures and methods to block their effects. This review of the EDC literature as it relates to female health provides an important platform on which womens health can be improved.

Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: The case of Bisphenol A

Background In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. Objectives We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. Discussion Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., “good science”). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. Conclusions Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

The estrogenic and antiestrogenic activities of phytochemicals with the human estrogen receptor expressed in yeast

We have used the expression of the human estrogen receptor (hER) and two estrogen response elements linked to the lacZ gene in yeast (YES) to study the estrogenic and antiestrogenic activities of various phytochemicals. Coumestrol, alpha-zearalenol, or genistein could produce beta-galactosidase activity comparable to estradiol, but these required concentrations 100 to 1000-fold greater than estradiol. These compounds did not possess antiestrogenic activity. Narigenin, kaempferide, phloretin, biochanin A, flavone, or chrysin only partially induced beta-galactosidase activity in the YES at any concentration tested. When narigenin, kaempferide, or phloretin was given concurrently with estradiol, the estradiol-dependent beta-galactosidase activity was not inhibited by more than 50%. However, biochanin A, flavone, or chrysin could inhibit the activity of estradiol in a dose-response manner with IC50 values of 500 nM, 2 microM, and 10 microM, respectively. Combinations of biochanin A, chrysin, and flavone decreased estradiol-dependent beta-galactosidase activity in an additive fashion. Similar to the antiestrogens tamoxifen or ICI 182, 780, the antiestrogenic activity of these compounds with the exception of chrystin involved the disruption of hER dimerization, as demonstrated in the yeast two-hybrid system. Biochanin A, chrysin, or flavone were less effective in inhibiting the activity of an estrogenic polychlorinated biphenyl than they were inhibiting the activity of estradiol. Interestingly, this latter group of antiestrogenic phytocompounds did not inhibit the estrogenic activity of such phytochemicals as coumestrol or genistein. These results suggest that the antiestrogenic activity of biochanin A and flavone occurs by a mechanism similar to tamoxifen or ICI 182,780. Moreover, it seems that phytochemicals functioning as antiestrogens do not inhibit the activity of all estrogenic chemicals to the same extent. This suggests that conformational changes induced by different estrogens bound to the hER may regulate the antiestrogenic activity of a compound.

Environmental Exposure, DNA Methylation, and Gene Regulation

Abstract: DNA methylation is an epigenetic mechanism that regulates chromosomal stability and gene expression. Abnormal DNA methylation patterns have been observed in many types of human tumors, including those of the breast, prostate, colon, thyroid, stomach, uterus, and cervix. We and others have shown that exposure to a wide variety of xenobiotics during critical periods of mammalian development can persistently alter the pattern of DNA methylation, resulting in potentially adverse biological effects such as aberrant gene expression. Thus, this epigenetic mechanism may underlie the observed increased risk in adulthood of several chronic diseases, including cancer, in response to xenobiotic exposures early in life. We present here the lessons learned from studies on the effects of perinatal diethylstilbesterol (DES) exposure on the methylation pattern of the promoters of several estrogen‐responsive genes associated with the development of reproductive organs. Perinatal DES exposure, which induces epithelial tumors of the uterus in mice and is associated with several reproductive tract abnormalities and increased vaginal and cervical cancer risk in women, provides a clear example of how estrogenic xenobiotic exposure during a critical period of development can abnormally demethylate DNA sequences during organ development and possibly increase cancer risk later in life. In addition, nutritional factors and stress may also alter DNA methylation during early life and modulate the risk of cancer and other chronic diseases in adulthood. We suggest that DNA methylation status may be influenced by environmental exposures in early life, leading to increased risk of cancer in adulthood.

Fathead minnow (Pimephales promelas) vitellogenin: purification, characterization and quantitative immunoassay for the detection of estrogenic compounds

The egg yolk precursor protein, vitellogenin (VTG), was purified from blood plasma of 17beta-estradiol (E2)-treated male fathead minnows (Pimephales promnelas) by anion-exchange chromatography on DEAE-agarose. A rabbit antiserum was raised against their blood plasma and then adsorbed with plasma from untreated (control) males to render the antiserum specific to VTG. The adsorbed antiserum was used to detect fathead minnow VTG (fVTG) in Western and dot blotting experiments and in an enzyme-linked immunosorbent assay (ELISA). The antiserum recognised fVTG as a approximately 156 kDa protein in plasma from vitellogenic females and E2-injected males but not untreated males. Its identity was confirmed by analysis of: (1) amino acid composition; (2) an internal amino acid sequence; (3) reactivity to the homologous antiserum; and (4) recognition by monoclonal antibodies prepared against the VTG from common carp (Cyprinus carpio) and brown bullhead (Ameiurus nebulosus). Specificity of the homologous antiserum to fVTG was confirmed by Western blotting of serially diluted plasma from vitellogenic females. Utility of the antiserum and purified fVTG for detecting exposure of male fathead minnows to estrogenic compounds was verified using a dot blotting immunoassay of fVTG and detected by chemiluminescence. Adult male fish were exposed to various concentrations of E2 (10(-8), 10(-9) and 10(-10) M) in their rearing water and plasma assayed for the presence of VTG at different time points (2, 7, 14 and 21 days). A competitive, antibody-capture, quantitative ELISA was then developed based on the purified fVTG and its respective antiserum. The ELISA was validated by demonstrating parallel binding slopes of dilution curves prepared with plasma from E2-injected males, vitellogenic females, and aqueous egg extracts as compared with purified fVTG standard. Plasma concentrations of VTG as low as 3 ng ml(-1) were detected in the ELISA, for which inter- and intra-assay coefficients of variation were both less than 5%. Furthermore, plasma from control males was unreactive with the fVTG antiserum. The VTG ELISA could be useful for the detection of estrogenic properties associated with certain compounds and could be easily incorporated into standard laboratory toxicity assays using this species.

Pesticides reduce symbiotic efficiency of nitrogen-fixing rhizobia and host plants.

Unprecedented agricultural intensification and increased crop yield will be necessary to feed the burgeoning world population, whose global food demand is projected to double in the next 50 years. Although grain production has doubled in the past four decades, largely because of the widespread use of synthetic nitrogenous fertilizers, pesticides, and irrigation promoted by the “Green Revolution,” this rate of increased agricultural output is unsustainable because of declining crop yields and environmental impacts of modern agricultural practices. The last 20 years have seen diminishing returns in crop yield in response to increased application of fertilizers, which cannot be completely explained by current ecological models. A common strategy to reduce dependence on nitrogenous fertilizers is the production of leguminous crops, which fix atmospheric nitrogen via symbiosis with nitrogen-fixing rhizobia bacteria, in rotation with nonleguminous crops. Here we show previously undescribed in vivo evidence that a subset of organochlorine pesticides, agrichemicals, and environmental contaminants induces a symbiotic phenotype of inhibited or delayed recruitment of rhizobia bacteria to host plant roots, fewer root nodules produced, lower rates of nitrogenase activity, and a reduction in overall plant yield at time of harvest. The environmental consequences of synthetic chemicals compromising symbiotic nitrogen fixation are increased dependence on synthetic nitrogenous fertilizer, reduced soil fertility, and unsustainable long-term crop yields.

Reduced fertility in female mice exposed transplacentally to diethylstilbestrol (DES)

Prenatal exposure to diethylstilbestrol (DES), a synthetic estrogen, has been associated with a low incidence of vaginal adenocarcinoma as well as a variety of more numerous benign abnormalities in the reproductive tract of human beings and experimental animals. For the purpose of assessing the effects of prenatal exposure to DES on postnatal reproduction tract function, timed pregnant CD-1 mice were treated subcutaneously with doses of DES ranging from 0.01 to 100 microgram/kg/day on days 9 through 16 gestation. The fertility of the female offspring was determined postnatally by a repetitive forced breeding technique. The most striking effect observed was a dose-related decrease in reproductive capacity ranging from minimal subfertility at the lower DES doses to a high frequency of total sterility at the highest DES doses. Reduced reproductive capacity appeared to be a reflection of both a decrease in the total number of litters and smaller litter sizes. A major component of the sterility seen in those females given higher doses of DES was oviductal/ovarian, since the number of ova recovered from the oviductal ampullae after induced ovulation was less than 30% that of controls. In addition, structural abnormalities of the oviduct, uterus, cervix, and vagina were observed, and contributed to infertility. These data suggest that in utero exposure to DES results in permanent impairment of female mouse reproductive capacity. Recent reports of altered pregnancy outcomes in young women who were exposed in utero to DES demonstrate the clinical importance of the findings obtained in mice.

Estrogenic and antiestrogenic activities of flavonoid phytochemicals through estrogen receptor binding-dependent and -independent mechanisms.

Members of the flavonoid class of phytochemicals have previously been demonstrated to possess estrogenic activity in a number of hormonally responsive systems. We have performed the present study to characterize the estrogenic and antiestrogenic activity of flavonoids in the estrogen receptor (ER)-positive MCF-7 human breast cancer cell line. Using an ER-dependent reporter gene assay and an ER competition binding assay, we have identified phytochemicals possessing estrogenic and antiestrogenic activities, which appeared to correlate directly with their capacity to displace [3H]estradiol from ER. Several flavonoids, including kaempferide, apigenin, and flavone, were distinct, in that their antiestrogenic activity did not appear to correlate with binding to ER, and therefore their suppression of estrogen-mediated gene transactivation and proliferation may occur independent of direct antagonism of the receptor. Further examination in HEK-293 cells transfected with ERα or ERβ demonstrated potent antagonism with kaempferide and apigenin, while flavone was weakly antagonistic only toward ERβ. These results suggest that the receptor binding-independent antiestrogenic chemicals may function through alternate signaling pathways as indirect ER modulators in a receptor- and cell type-specific manner. We conclude that antiestrogenic activities of flavonoid phytochemicals may occur through ER binding-dependent and -independent mechanisms and that the binding-independent antiestrogen activity of certain flavonoids is biologically significant in regulation of breast cancer cell proliferation.