Developmental exposure to a mixture of unconventional oil and gas chemicals: A review of experimental effects on adult health, behavior, and disease.

Unconventional oil and natural gas extraction (UOG) combines directional drilling and hydraulic fracturing and produces billions of liters of wastewater per year. Herein, we review experimental studies that evaluated the potential endocrine-mediated health impacts of exposure to a mixture of 23 UOG chemicals commonly found in wastewater. The purpose of this manuscript is to synthesize and summarize a body of work using the same UOG-mix but with different model systems and physiological endpoints in multiple experiments. The studies reviewed were conducted in laboratory animals (mice or tadpoles) and human tissue culture cells. A key feature of the in vivo studies was the use of four environmentally relevant doses spanning three orders of magnitude ranging from concentrations found in surface and ground water in UOG dense areas to concentrations found in UOG wastewater. This UOG-mix exhibited potent antagonist activity for the estrogen, androgen, glucocorticoid, progesterone, and thyroid receptors in human tissue culture cells. Subsequently, pregnant mice were administered the UOG-mix in drinking water and offspring were examined in adulthood or to tadpoles. Developmental exposure profoundly impacted pituitary hormone concentrations, reduced sperm counts, altered folliculogenesis, and increased mammary gland ductal density and preneoplastic lesions in mice. It also altered energy expenditure, exploratory and risk-taking behavior, the immune system in three immune models in mice, and affected basal and antiviral immunity in frogs. These findings highlight the diverse systems affected by developmental EDC exposure and the need to examine human and animal health in UOG regions.

Environmental signatures and effects of an oil and gas wastewater spill in the Williston Basin, North Dakota.

Wastewaters from oil and gas development pose largely unknown risks to environmental resources. In January 2015, 11.4ML (million liters) of wastewater (300g/L TDS) from oil production in the Williston Basin was reported to have leaked from a pipeline, spilling into Blacktail Creek, North Dakota. Geochemical and biological samples were collected in February and June 2015 to identify geochemical signatures of spilled wastewaters as well as biological responses along a 44-km river reach. February water samples had elevated chloride (1030mg/L) and bromide (7.8mg/L) downstream from the spill, compared to upstream levels (11mg/L and <0.4mg/L, respectively). Lithium (0.25mg/L), boron (1.75mg/L) and strontium (7.1mg/L) were present downstream at 5-10 times upstream concentrations. Light hydrocarbon measurements indicated a persistent thermogenic source of methane in the stream. Semi-volatile hydrocarbons indicative of oil were not detected in filtered samples but low levels, including tetramethylbenzenes and di-methylnaphthalenes, were detected in unfiltered water samples downstream from the spill. Labile sediment-bound barium and strontium concentrations (June 2015) were higher downstream from the Spill Site. Radium activities in sediment downstream from the Spill Site were up to 15 times the upstream activities and, combined with Sr isotope ratios, suggest contributions from the pipeline fluid and support the conclusion that elevated concentrations in Blacktail Creek water are from the leaking pipeline. Results from June 2015 demonstrate the persistence of wastewater effects in Blacktail Creek several months after remediation efforts started. Aquatic health effects were observed in June 2015; fish bioassays showed only 2.5% survival at 7.1km downstream from the spill compared to 89% at the upstream reference site. Additional potential biological impacts were indicated by estrogenic inhibition in downstream waters. Our findings demonstrate that environmental signatures from wastewater spills are persistent and create the potential for long-term environmental health effects.

Altered gene expression profile in vaginal polypoid endometriosis resembles peritoneal endometriosis and is consistent with increased local estrogen production.

BACKGROUND: In a university hospital setting, a 25-year-old woman presented with large vaginal and cervical polyps. Past medical history was significant for stage IV endometriosis. Polypectomy was performed and the polyps were histologically consistent with endometriosis. Gene expression was compared with control vaginal tissue to assess if the altered gene expression profile was similar to peritoneal endometriosis. METHODS AND RESULTS: Using quantitative reverse transcription, real-time PCR, estrogen receptor-ß expression was found to be upregulated 10-fold while estrogen receptor-α expression was downregulated 5-fold in the vaginal polyp relative to control vaginal tissue. The estrogen-synthesizing enzyme aromatase was upregulated 8-fold and 3ß-hydroxysteroid dehydrogenase was upregulated 400-fold in the polyp. Immunohistochemical staining revealed altered cell type localization for progesterone receptor in the polyp and increased cell proliferation in polyp stromal cells relative to control. CONCLUSIONS: Increased proliferation in the vaginal polypoid endometriotic tissue may be due to increased local estrogen production. The altered gene expression profile was very similar to the altered gene expression profile seen in peritoneal endometriosis.

Development of an ER action indicator mouse for the study of estrogens, selective ER modulators (SERMs), and Xenobiotics.

We have developed a transgenic mouse that functions as a reporter of ER activity, termed ER action indicator (ERIN), by incorporating a transgene with an estrogen-responsive promoter (three copies of the vitellogenin estrogen response element with a minimal thymidine kinase promoter) linked to the reporter gene beta-galactosidase. Evaluation of ER activity in female ERIN mice demonstrated estrogen-inducible expression of the reporter gene in the uterus, pituitary, and hypothalamus; established targets of estrogen action. Importantly, we also identified ER activity in a number of nonclassical estrogen target tissues, including kidney, liver, adrenal, and thyroid gland. ERIN provides a system to measure the same end point (transgene regulation) in different target tissues, permitting separation of the contributions of cell- and promoter-specific factors in determining ER pharmacology. In this regard we observed that on this specific promoter the pituitary gland was 25-fold more sensitive than the uterus to the estrogen diethylstilbestrol, implying the existence of cell-specific factors that influence ligand sensitivity. Our studies also identified considerable difference in the efficacy and potency of ER ligands in the uterus when ER transcriptional activity was assayed vs. uterine weight gain. Specifically, we observed that the environmental estrogen bisphenol A was a potent agonist in stimulating ER transcriptional activity, whereas it exhibited little uterotropic activity. In contrast to bisphenol A, tamoxifen significantly increased uterine weight, but minimally induced ER reporter activity in this tissue. Given the results of these studies, we believe that ERIN will be a useful model to evaluate ER ligand pharmacology and will assist in defining the cellular and molecular mechanisms that determine agonist and antagonist activity.

Comparative analyses of mechanistic differences among antiestrogens.

Antiestrogens such as tamoxifen are one of the most effective methods of treating estrogen receptor (ERalpha) positive breast cancers; however, the effectiveness of this therapy is limited by the almost universal development of resistance to the drug. If antiestrogens are recognized differently by the cell as it has been suggested, then in disease conditions where tamoxifen fails to function effectively, a mechanistically different antiestrogen might yield successful results. Although many antiestrogens have been developed, a direct comparison of their mechanisms of action is lacking, thus limiting their utility. Therefore, to determine if there are mechanistic differences among available antiestrogens, we have carried out a comprehensive analysis of the molecular mechanisms of action of 4-hydroxy-tamoxifen (40HT), idoxifene, raloxifene, GW7604, and ICI 182,780. Using a novel set of peptides that recognize different surfaces on ERalpha, we have found that following binding to ERalpha, each ligand induces a distinct ERalpha-ligand conformation. Furthermore, transcriptional assays indicate that each ERalpha-ligand complex is recognized distinctly by the transcription machinery, and consequently, antiestrogens vary in their ability to inhibit estradiol- and 40HT-mediated activities. Relative binding assays have shown that the affinity of these ligands for ERalpha is not always representative of their inhibitory activity. Using this assay, we have also shown that the pharmacology of each antiestrogen is influenced differently by hormone binding proteins. Furthermore, GW7604, like ICI 182,780, but unlike the other antiestrogens evaluated, decreases the stability of the receptor. Overall, our results indicate that there are clear mechanistic distinctions among each of the antiestrogens studied. However, GW7604 and ICI 182,780 differ more significantly from tamoxifen than idoxifene and raloxifene. These data, which reveal differences among antiestrogens, should assist in the selection of compounds for the clinical regulation of ERalpha function.

Developmental effects of estrogenic chemicals are predicted by an in vitro assay incorporating modification of cell uptake by serum.

Many estrogenic chemicals found in the environment (xenoestrogens) show a lower affinity for plasma estrogen binding proteins relative to the natural estrogens such as estradiol. These binding proteins, which include alphafetoprotein in rats and mice, sex hormone binding globulin in humans, and albumin in all species, regulate estrogen uptake into tissues. Therefore, the in vivo estrogenic potency relative to estradiol of xenoestrogens that show lower binding to these serum proteins will thus be underestimated in assays that compare the potency of xenoestrogens to estradiol and do not take serum binding into account. We have examined the effects of the binding components in serum on the uptake of a number of xenoestrogens into intact MCF-7 human breast cancer cells. Since most estrogenic chemicals are not available in radiolabeled form, their uptake is determined by competition with [3H]estradiol for binding to estrogen receptors (ER) in an 18-h assay. Serum modified access (SMA) of cell uptake of xenoestrogens is calculated as the RBA in serum-free-medium divided by the RBA in serum, and the bioactive free fraction of xenoestrogen in serum is then also calculated. We predicted the concentration of two xenoestrogens, bisphenol A and octylphenol, required to alter development of the prostate in male mouse fetuses. Whereas octylphenol was predicted to be a more potent estrogen than bisphenol A when tested in serum-free medium, our assay predicted that bisphenol A would be over 500-times more potent than octylphenol in fetal mice. The finding that administration of bisphenol A at a physiologically relevant dose predicted from our in vitro assay to pregnant mice from gestation day 11 to 17 increased adult prostate weight in male offspring relative to controls (similar to the effect of estradiol), while the same doses of octylphenol did not alter prostate development, provided support for our hypothesis.

Low-dose bioactivity of xenoestrogens in animals: fetal exposure to low doses of methoxychlor and other xenoestrogens increases adult prostate size in mice.

The hormonal activity of natural estrogens is influenced by the degree to which they bind to serum proteins. In the pregnant female and in the fetus, greater than 99% of estradiol may be bound by serum binding proteins. Therefore, even though total serum levels of estradiol appear very high in fetuses, we have found that in rodent fetuses, there is a very low free concentration of estradiol (0.2 pg/ml). Naturally occurring variation in fetal serum estradiol predicts differences in numerous postnatal traits, including prostate size. In addition, when this low level of free estradiol was experimentally increased from 0.2 to 0.3 pg/ml during the last third of fetal life, treated male mice showed an increase in adult prostate weight. Fetal exposure to low doses of xenobiotic estrogens by feeding to pregnant females, including the compounds methoxychlor (20 and 2000 micrograms/kg body weight), DES (0.02 to 2 micrograms/kg body weight) and bisphenol A (2 and 20 micrograms/kg body weight), also led to increased prostate weight in adulthood. In contrast, fetal doses of natural estradiol and DES above the physiological range of estrogenic activity, and within a toxicological dose range, led to the opposite outcome, a reduction in subsequent adult prostate weight. This indicates that it may be impossible to assess endocrine-disrupting activities in response to low doses within a physiological range of activity by using high, toxic doses of xenoestrogens in testing procedures. We have developed approaches in vitro to predict the potential estrogenic bioactivity of compounds in the physiologically relevant range in animals and humans. We address the following factors in predicting the final observed endocrine-disrupting effect in the animal: (1) the intrinsic estrogenic activity of a given molecule, (2) the effective free concentration determined by how the molecule is carried in serum, (3) partitioning between aqueous and lipid compartments in body and cell lipids, and (4) absorption and metabolism relative to the route of exposure. The studies and strategies we describe are important in developing criteria for a tiered testing system for the detection of estrogenic chemicals as well as endocrine-disrupting chemicals with different modes of action.

The effective free fraction of estradiol and xenoestrogens in human serum measured by whole cell uptake assays: physiology of delivery modifies estrogenic activity.

The biological activity of natural estrogens is influenced by the degree to which they bind to serum proteins. To determine directly how serum affected the uptake of estradiol, we compared the whole cell uptake of [3H]estradiol in intact MCF-7 human breast cancer cells from serum-free medium with the uptake from 100% serum from adult men. In estrogen receptor saturation assays, 28.9 times more estradiol was required in serum to occupy the same number of estrogen receptors as was required in serum-free medium (SFM), suggesting that the effective free fraction of estradiol in adult male serum was 3.46% (1/28.9). Since most xenoestrogens are not available in tritium-labeled form, the cell uptake of unlabeled xenoestrogens could not be measured directly with saturation analysis. Therefore, we developed the relative binding affinity-serum modified access (RBA-SMA) assay to determine the effect of serum on the access of nonradioactive xenoestrogens to estrogen receptors within intact MCF-7 cells. Serum modified access (SMA) was calculated by dividing the relative binding affinity (RBA, relative to estradiol) measured in 100% serum, by the RBA measured in serum-free medium. An SMA > 1 indicated that the xenoestrogen had greater access to estrogen receptors than estradiol from serum. In contrast, an SMA < 1 indicated that the xenoestrogen had less access to estrogen receptors from serum than did estradiol. The synthetic estrogen diethylstilbestrol (DES) binds poorly to sex hormone binding globulin (SHBG), and DES showed enhanced access in serum, SMA = 6.2. Additional calculations through the Ki (inhibition constant) indicated that this corresponded to an effective free fraction of 26.9% for DES in serum. The phytoestrogens, coumestrol, genistein, and equol, showed substantial enhanced access in serum, over 10-fold relative to estradiol (SMA = 12.1, 10.3, and 11.3, respectively), and effective free fractions in serum of 47.8, 45.8, and 49.7%, respectively. Since most in vitro assays of xenoestrogens do not address how serum influences their bioactivity, the estrogenic activity of these phytoestrogens would be underestimated. Conversely, biochanin A showed decreased access from serum (SMA = 0.44) and had an effective free fraction of 2.4%; its estrogenic activity would be overestimated in serum-free assays.

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.

Two chemicals previously shown to have estrogenic activity, bisphenol A and octylphenol, were examined for their effects on accessory reproductive organs and daily sperm production in male offspring of mice fed these chemicals during pregnancy. These chemicals are used in the manufacture of plastics and other products, and have been detected in food and water consumed by animals and people. From gestation day 11-17 female mice were fed an average concentration (dissolved in oil) of bisphenol A or octylphenol of 2 ng/g body weight (2 ppb) and 20 ng/g (20 ppb). The 2 ppb dose of bisphenol A is lower than the amount reported to be swallowed during the first hour after application of a plastic dental sealant (up to 931 micrograms; 13.3 ppb in a 70 kg adult). We found that the 2 ng/g dose of bisphenol A permanently increased the size of the preputial glands, but reduced the size of the epididymides; these organs develop from different embryonic tissues. At 20 ng/g, bisphenol A significantly decreased efficiency of sperm production (daily sperm production per g testis) by 20% relative to control males. The only significant effect of octylphenol was a reduction in daily sperm production and efficiency of sperm production at the 2 ng/g dose. A new approach to studying physiologically relevant doses of environmental endocrine disruptors is discussed, particularly with regard to the development of the reproductive organs, the brain, and behavior.

Prostate enlargement in mice due to fetal exposure to low doses of estradiol or diethylstilbestrol and opposite effects at high doses.

On the basis of results of studies using high doses of estrogens, exposure to estrogen during fetal life is known to inhibit prostate development. However, it is recognized in endocrinology that low concentrations of a hormone can stimulate a tissue, while high concentrations can have the opposite effect. We report here that a 50% increase in free-serum estradiol in male mouse fetuses (released by a maternal Silastic estradiol implant) induced a 40% increase in the number of developing prostatic glands during fetal life; subsequently, in adulthood, the number of prostatic androgen receptors per cell was permanently increased by 2-fold, and the prostate was enlarged by 30% (due to hyperplasia) relative to untreated males. However, as the free serum estradiol concentration in male fetuses was increased from 2- to 8-fold, adult prostate weight decreased relative to males exposed to the 50% increase in estradiol. As a model for fetal exposure to man-made estrogens, pregnant mice were fed diethylstilbestrol (DES) from gestation days 11 to 17. Relative to controls, DES doses of 0.02, 0.2, and 2.0 ng per g of body weight per day increased adult prostate weight, whereas a 200-ng-per-g dose decreased adult prostate weight in male offspring. Our findings suggest that a small increase in estrogen may modulate the action of androgen in regulating prostate differentiation, resulting in a permanent increase in prostatic androgen receptors and prostate size. For both estradiol and DES, prostate weight first increased then decreased with dose, resulting in an inverted-U dose-response relationship.

Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol.

We have developed a relative binding affinity-serum modified access (RBA-SMA) assay to determine the effect of serum on the access of xenoestrogens to estrogen receptors within intact cultured MCF-7 human breast cancer cells. We used this assay to predict low dose activity of two xenoestrogens in mice. In serum-free medium, bisphenol A, a component of polycarbonates and of resins used to line metal food cans, showed a lower relative binding affinity (RBA; 0.006%) than octylphenol (0.072%) and nonylphenol (0.026%), which are used as surfactants in many commercial products (all RBAs are relative to estradiol, which is equal to 100%). In 100% serum from adult men, bisphenol A showed a higher RBA (0.01%) than in serum-free medium and thus enhanced access to estrogen receptors relative to estradiol. In contrast, octylphenol showed a 22-fold decrease in RBA (0.0029%) and nonylphenol showed a 5-fold decrease in RBA (0.0039%) when measured in adult serum. This indicates that, relative to estradiol, serum had less of an inhibitory effect on the cell uptake and binding in MCF-7 cells of bisphenol A, while serum had a greater inhibitory effect on octylphenol and nonylphenol relative to estradiol. Extrapolation of these relative activities in adult serum predicted that the estrogenic bioactivity of bisphenol A would be over 500-fold greater than that of octylphenol in fetal mouse serum. Bisphenol A and octylphenol were fed to pregnant mice at 2 and 20 micrograms/kg/day. Exposure of male mouse fetuses to either dose of bisphenol A, but to neither dose of octylphenol, significantly increased their adult prostate weight relative to control males, which is consistent with the higher predicted bioactivity of bisphenol A than octylphenol in the RBA-SMA assay. In addition, our findings show for the first time that fetal exposure to environmentally relevant parts-per-billion (ppb) doses of bisphenol A, in the range currently being consumed by people, can alter the adult reproductive system in mice.

Estrogenic pesticides: binding relative to estradiol in MCF-7 cells and effects of exposure during fetal life on subsequent territorial behaviour in male mice.

Numerous chemicals released into the environment by man are able to disrupt the functioning of the endocrine system by binding to estrogen receptors in estrogen-responsive cells. The ability of o,p'-dichlorodiphenyl trichloroethane (DDT) and methoxychlor to compete with estradiol for binding to estrogen receptors in MCF-7 cells (relative binding affinity; RBA) was examined in both serum-free medium and 100% serum; this is referred to as a relative binding affinity-serum modified access (RBA-SMA) assay. RBA's ranged from 0.04% for o,p'-DDT (which showed enhanced access to cells in serum relative to serum-free medium) to 0.004% for methoxychlor (which did not show enhanced access in serum). Based on these findings, these pesticides, along with diethylstilbestrol (DES) as a positive control, were fed to pregnant mice from days 11-17 of pregnancy. When the male offspring were examined in adulthood for their rate of urine marking in a novel territory (territorial behaviour), the rate of urine marking increased dramatically with low doses of DES (relative to controls) and then decreased significantly at the highest dose administered prenatally. Relative binding in MCF-7 cells accurately predicted the doses of o,p'-DDT and methoxychlor that produced the same results, providing support for the hypothesis that effects on behaviour were mediated by binding to estrogen receptors in the developing brain.

Cellulose: its regeneration in the native lattice.

The regeneration of cellulose from solutions in 85 percent phosphoric acid into glycerol at 170 degrees C resulted in its recovery wholly in the native or cellulose I lattice. Identification of the structure of the regenerated material is based on comparisons of its x-ray diffractogram and its Raman spectrum with those of the native fiber, cellulose II, and cellulose IV.