368 research outputs found
Effect of Benzene and Ethylbenzene on the Transcription of methyl-\u3cem\u3etert\u3c/em\u3e-butyl Ether Degradation Genes of \u3cem\u3eMethylibium petroleiphilum\u3c/em\u3e PM1
Methyl-tert-butyl ether (MTBE) and its degradation by-product, tert-butyl alcohol (TBA), are widespread contaminants detected frequently in groundwater in California. Since MTBE was used as a fuel oxygenate for almost two decades, leaking underground fuel storage tanks are an important source of contamination. Gasoline components such as BTEX (benzene, toluene, ethylbenzene and xylenes) are often present in mixtures with MTBE and TBA. Investigations of interactions between BTEX and MTBE degradation have not yielded consistent trends, and the molecular mechanisms of BTEX compounds’ impact on MTBE degradation are not well understood. We investigated trends in transcription of biodegradation genes in the MTBE-degrading bacterium, Methylibium petroleiphilum PM1 upon exposure to MTBE, TBA, ethylbenzene and benzene as individual compounds or in mixtures. We designed real-time quantitative PCR assays to target functional genes of strain PM1 and provide evidence for induction of genes mdpA (MTBE monooxygenase), mdpJ (TBA hydroxylase) and bmoA (benzene monooxygenase) in response to MTBE, TBA and benzene, respectively. Delayed induction of mdpA and mdpJ transcription occurred with mixtures of benzene and MTBE or TBA, respectively. bmoA transcription was similar in the presence of MTBE or TBA with benzene as in their absence. Our results also indicate that ethylbenzene, previously proposed as an inhibitor of MTBE degradation in some bacteria, inhibits transcription of mdpA, mdpJ and bmoAgenes in strain PM1
Gene \u3cem\u3emdpC\u3c/em\u3e Plays a Regulatory Role in the Methyl-\u3cem\u3etert\u3c/em\u3e-butyl Ether Degradation Pathway of \u3cem\u3eMethylibium petroleiphilum\u3c/em\u3e Strain PM1
Among the few bacteria known to utilize methyl tert-butyl ether (MTBE) as a sole carbon source, Methylibium petroleiphilum PM1 is a well-characterized organism with a sequenced genome; however, knowledge of the genetic regulation of its MTBE degradation pathway is limited. We investigated the role of a putative transcriptional activator gene, mdpC, in the induction of MTBE-degradation genes mdpA (encoding MTBE monooxygenase) and mdpJ (encoding tert-butyl alcohol hydroxylase) of strain PM1 in a gene-knockout mutant mdpC−. We also utilized quantitative reverse transcriptase PCR assays targeting genes mdpA, mdpJ and mdpC to determine the effects of the mutation on transcription of these genes. Our results indicate that gene mdpC is involved in the induction of both mdpA and mdpJ in response to MTBE and tert-butyl alcohol (TBA) exposure in PM1. An additional independent mechanism may be involved in the induction of mdpJ in the presence of TBA
Chemicals having estrogenic activity can be released from some bisphenol a-free, hard and clear, thermoplastic resins
Background: Chemicals that have estrogenic activity (EA) can potentially cause adverse health effects in mammals
including humans, sometimes at low doses in fetal through juvenile stages with effects detected in adults.
Polycarbonate (PC) thermoplastic resins made from bisphenol A (BPA), a chemical that has EA, are now often
avoided in products used by babies. Other BPA-free thermoplastic resins, some hypothesized or advertised to be
EA-free, are replacing PC resins used to make reusable hard and clear thermoplastic products such as baby bottles.
Methods: We used two very sensitive and accurate in vitro assays (MCF-7 and BG1Luc human cell lines) to quantify
the EA of chemicals leached into ethanol or water/saline extracts of fourteen unstressed or stressed (autoclaving,
microwaving, UV radiation) thermoplastic resins. Estrogen receptor (ER)-dependent agonist responses were confirmed
by their inhibition with the ER antagonist ICI 182,780.
Results: Our data showed that some (4/14) unstressed and stressed BPA-free thermoplastic resins leached chemicals
having significant levels of EA, including one polystyrene (PS), and three Tritan™ resins, the latter reportedly
EA-free. Exposure to UV radiation in natural sunlight resulted in an increased release of EA from Tritan™ resins.
Triphenyl-phosphate (TPP), an additive used to manufacture some thermoplastic resins such as Tritan™, exhibited
EA in both MCF-7 and BG1Luc assays. Ten unstressed or stressed glycol-modified polyethylene terephthalate
(PETG), cyclic olefin polymer (COP) or copolymer (COC) thermoplastic resins did not release chemicals with detectable
EA under any test condition.
Conclusions: This hazard survey study assessed the release of chemicals exhibiting EA as detected by two sensitive,
widely used and accepted, human cell line in vitro assays. Four PC replacement resins (Tritan™ and PS) released
chemicals having EA. However, ten other PC-replacement resins did not leach chemicals having EA (EA-free-resins).
These results indicate that PC-replacement plastic products could be made from EA-free resins (if appropriate EA-free
additives are chosen) that maintain advantages of re-usable plastic items (price, weight, shatter resistance) without
releasing chemicals having EA that potentially produce adverse health effects on current or future generations.This work was supported by the following NIH/NIEHS grants: R44 ES011469, 01–03 (CZY);
1R43/44 ES014806, 01–03 (CZY); subcontract (CZY, PI) on an NIH Grant 01–03
43/44ES018083-01. This work was also supported by NIH grants to MSD (P42
ES004699), and DJK and SIY (1R43ES018083-01-03, NIEHS 1R44ES019442-01-03
and NIEHS 2R44ES016964-01-03).Neuroscienc
Ginsenosides are novel naturally-occurring aryl hydrocarbon receptor ligands.
The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of structurally diverse chemicals. In this study, we examined the ability of a series of ginsenosides extracted from ginseng, a traditional Chinese medicine, to bind to and activate/inhibit the AHR and AHR signal transduction. Utilizing a combination of ligand and DNA binding assays, molecular docking and reporter gene analysis, we demonstrated the ability of selected ginsenosides to directly bind to and activate the guinea pig cytosolic AHR, and to stimulate/inhibit AHR-dependent luciferase gene expression in a recombinant guinea pig cell line. Comparative studies revealed significant species differences in the ability of ginsenosides to stimulate AHR-dependent gene expression in guinea pig, rat, mouse and human cell lines. Not only did selected ginsenosides preferentially activate the AHR from one species and not others, mouse cell line was also significantly less responsive to these chemicals than rat and guinea pig cell lines, but the endogenous gene CYP1A1 could still be inducted in mouse cell line. Overall, the ability of these compounds to stimulate AHR signal transduction demonstrated that these ginsenosides are a new class of naturally occurring AHR agonists
Comparative analysis of homology models of the Ah receptor ligand binding domain: Verification of structure-function predictions by site-directed mutagenesis of a nonfunctional receptor
The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the biological and toxic effects of a wide variety of structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While significant interspecies differences in AHR ligand binding specificity, selectivity, and response have been observed, the structural determinants responsible for those differences have not been determined, and homology models of the AHR ligand-binding domain (LBD) are available for only a few species. Here we describe the development and comparative analysis of homology models of the LBD of 16 AHRs from 12 mammalian and nonmammalian species and identify the specific residues contained within their ligand binding cavities. The ligand-binding cavity of the fish AHR exhibits differences from those of mammalian and avian AHRs, suggesting a slightly different TCDD binding mode. Comparison of the internal cavity in the LBD model of zebrafish (zf) AHR2, which binds TCDD with high affinity, to that of zfAHR1a, which does not bind TCDD, revealed that the latter has a dramatically shortened binding cavity due to the side chains of three residues (Tyr296, Thr386, and His388) that reduce the amount of internal space available to TCDD. Mutagenesis of two of these residues in zfAHR1a to those present in zfAHR2 (Y296H and T386A) restored the ability of zfAHR1a to bind TCDD and to exhibit TCDD-dependent binding to DNA. These results demonstrate the importance of these two amino acids and highlight the predictive potential of comparative analysis of homology models from diverse species. The availability of these AHR LBD homology models will facilitate in-depth comparative studies of AHR ligand binding and ligand-dependent AHR activation and provide a novel avenue for examining species-specific differences in AHR responsiveness. © 2013 American Chemical Society
Detection of xenoestrogens in serum after immunoprecipitation of endogenous steroidal estrogens.
In this article we report a simple and efficient method for detecting nonsteroidal estrogens in a biologic sample. This method uses polyclonal antibodies to estradiol (E2) to immunoprecipitate these major biologically active steroidal estrogens, leaving behind the nonsteroidal estrogens, which are then detected in a cell-based transcriptional activation bioassay for estrogen receptor agonist. The immunoprecipitation method efficiently removed 99% of radiolabeled E2 and estrone (E1) from human serum. In experiments in which supraphysiologic concentrations of E2 and E1 to human serum, all of the immunoreactive estrogens were still removed by the immunoprecipitation protocol. We carried out an in vivo validation study of this method in which we treated female macaques with the xenoestrogen nonylphenol (NP), during the late follicular phase of the menstrual cycle. We used blood samples collected before and after treatment to evaluate and characterize endogenous and exogenous serum estrogens. An immunoassay for E2 did not detect the NP in treated monkeys. The cell-based bioassay also did not detect the estrogenic activity of NP because of its saturation by the endogenous serum steroidal estrogens. However, when steroidal estrogens were removed by immunoprecipitation, we detected the estrogenic activity of NP in the bioassay. Thus, this approach is appropriate for detecting exogenous, nonsteroidal estrogens in serum samples
2,3-cis-2R,3R-(−)-epiafzelechin-3-O-p-coumarate, a novel flavan-3-ol isolated from Fallopia convolvulus seed, is an estrogen receptor agonist in human cell lines
BACKGROUND: The plant genus Fallopia is well-known in Chinese traditional medicine and includes many species that contain bioactive compounds, namely phytoestrogens. Consumption of phytoestrogens may be linked to decreased incidence of breast and prostate cancers therefore discovery of novel phytoestrogens and novel sources of phytoestrogens is of interest. Although phytoestrogen content has been analyzed in the rhizomes of various Fallopia sp., seeds of a Fallopia sp. have never been examined for phytoestrogen presence. METHODS: Analytical chemistry techniques were used with guidance from an in vitro estrogen receptor bioassay (a stably transfected human ovarian carcinoma cell line) to isolate and identify estrogenic components from seeds of Fallopia convolvulus. A transiently transfected human breast carcinoma cell line was used to characterize the biological activity of the isolated compounds on estrogen receptors (ER) α and β. RESULTS: Two compounds, emodin and the novel flavan-3-ol, (−)-epiafzelechin-3-O-p-coumarate (rhodoeosein), were identified to be responsible for estrogenic activity of F. convolvulus seed extract. Absolute stereochemistry of rhodoeosein was determined by 1 and 2D NMR, optical rotation and circular dichroism. Emodin was identified by HPLC/DAD, LC/MS/MS, and FT/ICR-MS. When characterizing the ER specificity in biological activity of rhodoeosein and emodin, rhodoeosein was able to exhibit a four-fold greater relative estrogenic potency (REP) in breast cells transiently-transfected with ERβ as compared to those transfected with ERα, and emodin exhibited a six-fold greater REP in ERβ-transfected breast cells. Cell type-specific differences were observed with rhodoeosein but not emodin; rhodoeosein produced superinduction of reporter gene activity in the human ovarian cell line (> 400% of maximum estradiol [E2] induction) but not in the breast cell line. CONCLUSION: This study is the first to characterize the novel flavan-3-ol compound, rhodoeosein, and its ability to induce estrogenic activity in human cell lines. Rhodoeosein and emodin may have potential therapeutic applications as natural products activating ERβ, and further characterization of rhodoeosein is necessary to evaluate its selectivity as a cell type-specific ER agonist
Aryl hydrocarbon receptor-mediated activity of particulate organic matter from the Paso del Norte airshed along the U.S.-Mexico border.
In this study, we determined the biologic activity of dichloromethane-extracted particulate matter < 10 micro m in aerodynamic diameter (PM10) obtained from filters at three sites in the Paso del Norte airshed, which includes El Paso, Texas, USA; Juarez, Chihuahua, Mexico, and Sunland Park, New Mexico, USA. The extracts were rich in polycyclic aromatic hydrocarbons (PAHs) and had significant biologic activity, measured using two in vitro assay systems: ethoxyresorufin-(O-deethylase (EROD) induction and the aryl hydrocarbon-receptor luciferase reporter system. In most cases, both EROD (5.25 pmol/min/mg protein) and luciferase activities (994 relative light units/mg) were highest in extracts from the Advance site located in an industrial neighborhood in Juarez. These values represented 58% and 55%, respectively, of induction associated with 1 micro M ss-naphthoflavone exposures. In contrast, little activity was observed at the Northeast Clinic site in El Paso, the reference site. In most cases, luciferase and EROD activity from extracts collected from the Tillman Health Center site, situated in downtown El Paso, fell between those observed at the other two sites. Overall, a statistically significant correlation existed between PM10 and EROD and luciferase activities. Chemical analysis of extracts collected from the Advance site demonstrated that concentrations of most PAHs were higher than those reported in most other metropolitan areas in the United States. Calculations made with these data suggest a cancer risk of 5-12 cases per 100,000 people. This risk estimate, as well as comparisons with the work of other investigators, raises concern regarding the potential for adverse health effects to the residents of this airshed. Further work is needed to understand the sources, exposure, and effects of PM10 and particulate organic material in the Paso del Norte airshed
Time-integrated monitoring of dioxin-like polychlorinated biphenyls (dl-PCBs) in aquatic environments using the ceramic toximeter and the CALUX bioassay
a b s t r a c t Ceramic passive samplers or toximeters (packed with active carbon 1%, w/w, on celite), in combination with the CALUX bioassay have been used as a time-integrated monitoring technique for dissolved dioxinlike PCBs in urban and industrial wastewaters. The technique showed to be reliable during laboratory experiments: (1) PCB-126 amounts extracted from the passive samplers increased linearly with the time of exposure and (2) PCB-126 concentrations calculated from the amounts accumulated by the passive samplers were in agreement with their concentrations in the testing solution. Afterwards the toximeters were applied in the field. Two sampling sites located in Egypt were chosen: the Belbeis drainage canal, and the EMAK paper mill. A total of 18 ceramic toximeters were exposed to the wastewater in both sampling sites for a maximum period of 4 weeks. Two samplers were collected weekly from each site to monitor the increase in target analytes over time. Extracts were analyzed using the CALUX bioassay and the total dioxin-like PCB toxicity was reported for the aqueous phase (water column), as well as the solid phase (sediment and sludge) in both sampling sites. The time-weighted average concentration (TWA) of dl-PCBs in wastewater of the paper mill during the sampling period ranged between 7.1 and 9.1 pg-BEQ L À 1 , while that of the drainage canal ranged between 9.5 and 12.2 pg-BEQ L À 1 . The dl-PCBs in the fibrous sludge (paper mill) and bottom sediment (drainage canal) were 0.5 and 0.4 pg-BEQ g À 1 dryweight, respectively. The organic-carbon normalized partition coefficients between sediment and water (log K oc ) for the paper mill and the canal were 2.4 and 4.3, respectively
Gas-Phase Ambient Air Contaminants Exhibit Significant Dioxin-like and Estrogen-like Activity in Vitro
Several adverse health effects, such as respiratory and cardiovascular morbidity, have been linked to exposure to particulate matter in ambient air; however, the biologic activity of gas-phase ambient organic air contaminants has not been examined as thoroughly. Using aryl hydrocarbon receptor (AHR)–based and estrogen receptor (ER)–based cell bioassay systems, we assessed the dioxin-like and estrogenic activities of gas-phase organic ambient air contaminants compared with those of particulate-phase contaminants using samples collected between seasons over 2 years from an urban and a rural location in the Greater Toronto Area, Canada. The concentration of the sum (∑) of polycyclic aromatic hydrocarbons, which was highest in the gas phase, was 10–100 times more abundant than that of ∑polychlorinated biphenyls, ∑nitro-polycyclic aromatic hydrocarbons, and ∑organochlorine pesticides, and 10(3) to 10(4) times more abundant than ∑polychlorinated dibenzo-p-dioxins/dibenzofurans. Gas-phase samples induced significant AHR- and ER-dependent gene expression. The activity of the gas-phase samples was greater than that of the particulate-phase samples in the estrogen assay and, in one case, in the AHR assay. We found no strong associations between either summer or winter seasons or urban or rural locations in the relative efficacy of the extracts in either the ER or AHR assay despite differences in chemical composition, concentrations, and abundance. Our results suggest that mechanistic studies of the health effects of ambient air must consider gas and particulate phases because chemicals present in both phases can affect AHR and ER signaling pathways
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