Guochun He

Exactly the Same but Different: Promiscuity and Diversity in the Molecular Mechanisms of Action of the Aryl Hydrocarbon (Dioxin) Receptor

The Ah receptor (AhR) is a ligand-dependent transcription factor that mediates a wide range of biological and toxicological effects that result from exposure to a structurally diverse variety of synthetic and naturally occurring chemicals. Although the overall mechanism of action of the AhR has been extensively studied and involves a classical nuclear receptor mechanism of action (i.e., ligand-dependent nuclear localization, protein heterodimerization, binding of liganded receptor as a protein complex to its specific DNA recognition sequence and activation of gene expression), details of the exact molecular events that result in most AhR-dependent biochemical, physiological, and toxicological effects are generally lacking. Ongoing research efforts continue to describe an ever-expanding list of ligand-, species-, and tissue-specific spectrum of AhR-dependent biological and toxicological effects that seemingly add even more complexity to the mechanism. However, at the same time, these studies are also identifying and characterizing new pathways and molecular mechanisms by which the AhR exerts its actions and plays key modulatory roles in both endogenous developmental and physiological pathways and response to exogenous chemicals. Here we provide an overview of the classical and nonclassical mechanisms that can contribute to the differential sensitivity and diversity in responses observed in humans and other species following ligand-dependent activation of the AhR signal transduction pathway.

Third-Generation Ah Receptor–Responsive Luciferase Reporter Plasmids: Amplification of Dioxin-Responsive Elements Dramatically Increases CALUX Bioassay Sensitivity and Responsiveness

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like chemicals are widespread and persistent environmental contaminants that produce diverse toxic and biological effects through their ability to bind to and activate the Ah receptor (AhR) and AhR-dependent gene expression. The chemically activated luciferase expression (CALUX) system is an AhR-responsive recombinant luciferase reporter gene-based cell bioassay that has been used in combination with chemical extraction and cleanup methods for the relatively rapid and inexpensive detection and relative quantitation of dioxin and dioxin-like chemicals in a wide variety of sample matrices. Although the CALUX bioassay has been validated and used extensively for screening purposes, it has some limitations when screening samples with very low levels of dioxin-like chemicals or when there is only a small amount of sample matrix for analysis. Here, we describe the development of third-generation (G3) CALUX plasmids with increased numbers of dioxin-responsive elements, and stable transfection of these new plasmids into mouse hepatoma (Hepa1c1c7) cells has produced novel amplified G3 CALUX cell bioassays that respond to TCDD with a dramatically increased magnitude of luciferase induction and significantly lower minimal detection limit than existing CALUX-type cell lines. The new G3 CALUX cell lines provide a highly responsive and sensitive bioassay system for the detection and relative quantitation of very low levels of dioxin-like chemicals in sample extracts.

Toxicology in the Fast Lane: Application of High-Throughput Bioassays to Detect Modulation of Key Enzymes and Receptors

Background Legislation at state, federal, and international levels is requiring rapid evaluation of the toxicity of numerous chemicals. Whole-animal toxicologic studies cannot yield the necessary throughput in a cost-effective fashion, leading to a critical need for a faster and more cost-effective toxicologic evaluation of xenobiotics. Objectives We tested whether mechanistically based screening assays can rapidly provide information on the potential for compounds to affect key enzymes and receptor targets, thus identifying those compounds requiring further in-depth analysis. Methods A library of 176 synthetic chemicals was prepared and examined in a high-throughput screening (HTS) manner using nine enzyme-based and five receptor-based bioassays. Results All the assays have high Z′ values, indicating good discrimination among compounds in a reliable fashion, and thus are suitable for HTS assays. On average, three positive hits were obtained per assay. Although we identified compounds that were previously shown to inhibit a particular enzyme class or receptor, we surprisingly discovered that triclosan, a microbiocide present in personal care products, inhibits carboxylesterases and that dichlone, a fungicide, strongly inhibits the ryanodine receptors. Conclusions Considering the need to rapidly screen tens of thousands of anthropogenic compounds, our study shows the feasibility of using combined HTS assays as a novel approach toward obtaining toxicologic data on numerous biological end points. The HTS assay approach is very useful to quickly identify potentially hazardous compounds and to prioritize them for further in-depth studies.

The in vivo estrogenic and in vitro anti‐estrogenic activity of permethrin and bifenthrin

Pyrethroids are highly toxic to fish at parts per billion or parts per trillion concentrations. Their intended mechanism is prolonged sodium channel opening, but recent studies reveal that pyrethroids such as permethrin and bifenthrin also have endocrine activity. Additionally, metabolites may have greater endocrine activity than parent compounds. The authors evaluated the in vivo concentration-dependent ability of bifenthrin and permethrin to induce choriogenin (an estrogen-responsive protein) in Menidia beryllina, a fish species known to reside in pyrethroid-contaminated aquatic habitats. The authors then compared the in vivo response with an in vitro assay--chemical activated luciferase gene expression (CALUX). Juvenile M. beryllina exposed to bifenthrin (1, 10, 100 ng/L), permethrin (0.1, 1, 10 µg/L), and ethinylestradiol (1, 10, 50 ng/L) had significantly higher ng/mL choriogenin (Chg) measured in whole body homogenate than controls. Though Chg expression in fish exposed to ethinylestradiol (EE2) exhibited a traditional sigmoidal concentration response, curves fit to Chg expressed in fish exposed to pyrethroids suggest a unimodal response, decreasing slightly as concentration increases. Whereas the in vivo response indicated that bifenthrin and permethrin or their metabolites act as estrogen agonists, the CALUX assay demonstrated estrogen antagonism by the pyrethroids. The results, supported by evidence from previous studies, suggest that bifenthrin and permethrin, or their metabolites, appear to act as estrogen receptor (ER) agonists in vivo, and that the unmetabolized pyrethroids, particularly bifenthrin, act as an ER antagonists in cultured mammalian cells.

Comparative gene responses to collected ambient particles in vitro: endothelial responses.

Epidemiologic studies associate exposure to ambient particulate matter (APM) with increased cardiovascular mortality. Since both pulmonary inflammation and systemic circulation of ultrafine particles are hypothesized as initiating cardiovascular effects, we examined responses of potential target cells in vitro. Human aortic endothelial cells (HAEC) were exposed to 10 μg/ml fine and ultrafine APM collected in an urban setting in summer 2006 or winter 2007 in the San Joaquin Valley, California. RNA isolated after 3 h was analyzed with high-density oligonucleotide arrays. Summer APM treatment affected genes involved in xenobiotic and oxidoreductase activity, transcription factors, and inflammatory responses in HAEC, while winter APM had a robust xenobiotic but lesser inflammatory response. Real-time polymerase chain reaction analysis confirmed that particulate matter (PM)-treated HAEC increased mRNA levels of xenobiotic response enzymes CYP1A1, ALDH1A3, and TIPARP and cellular stress response transcription factor ATF3. Inflammatory response genes included E-selectin, PTGS2, CXCL-2 (MIP-2α), and CCL-2 (MCP-1). Multiplex protein assays showed secretion of IL-6 and MCP-1 by HAEC. Since induction of CYP1A1 is mediated through the ligand-activated aryl hydrocarbon receptor (AhR), we demonstrated APM induced AhR nuclear translocation by immunofluorescence and Western blotting and activation of the AhR response element using a luciferase reporter construct. Inhibitor studies suggest differential influences of polycyclic aromatic hydrocarbon signaling, ROS-mediated responses and endotoxin alter stress and proinflammatory endothelial cell responses. Our findings demonstrate gene responses correlated with current concepts that systemic inflammation drives cardiovascular effects of particulate air pollution. We also demonstrate a unique pattern of gene responses related to xenobiotic metabolism in PM-exposed HAEC.

From ‘Omics to Otoliths: Responses of an Estuarine Fish to Endocrine Disrupting Compounds across Biological Scales

Endocrine disrupting chemicals (EDCs) cause physiological abnormalities and population decline in fishes. However, few studies have linked environmental EDC exposures with responses at multiple tiers of the biological hierarchy, including population-level effects. To this end, we undertook a four-tiered investigation in the impacted San Francisco Bay estuary with the Mississippi silverside (Menidia audens), a small pelagic fish. This approach demonstrated links between different EDC sources and fish responses at different levels of biological organization. First we determined that water from a study site primarily impacted by ranch run-off had only estrogenic activity in vitro, while water sampled from a site receiving a combination of urban, limited ranch run-off, and treated wastewater effluent had both estrogenic and androgenic activity. Secondly, at the molecular level we found that fish had higher mRNA levels for estrogen-responsive genes at the site where only estrogenic activity was detected but relatively lower expression levels where both estrogenic and androgenic EDCs were detected. Thirdly, at the organism level, males at the site exposed to both estrogens and androgens had significantly lower mean gonadal somatic indices, significantly higher incidence of severe testicular necrosis and altered somatic growth relative to the site where only estrogens were detected. Finally, at the population level, the sex ratio was significantly skewed towards males at the site with measured androgenic and estrogenic activity. Our results suggest that mixtures of androgenic and estrogenic EDCs have antagonistic and potentially additive effects depending on the biological scale being assessed, and that mixtures containing androgens and estrogens may produce unexpected effects. In summary, evaluating EDC response at multiple tiers is necessary to determine the source of disruption (lowest scale, i.e. cell line) and what the ecological impact will be (largest scale, i.e. sex ratio).

Development of Species-Specific Ah Receptor-Responsive Third Generation CALUX Cell Lines with Enhanced Responsiveness and Improved Detection Limits.

The Ah receptor (AhR)-responsive CALUX (chemically activated luciferase expression) cell bioassay is commonly used for rapid screening of samples for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), dioxin-like compounds, and AhR agonists/antagonists. By increasing the number of AhR DNA recognition sites (dioxin responsive elements), we previously generated a novel third generation (G3) recombinant AhR-responsive mouse CALUX cell line (H1L7.5c3) with a significantly enhanced response to DLCs compared to existing AhR-CALUX cell bioassays. However, the elevated background luciferase activity of these cells and the absence of comparable G3 cell lines derived from other species have limited their utility for screening purposes. Here, we describe the development and characterization of species-specific G3 recombinant AhR-responsive CALUX cell lines (rat, human, and guinea pig) that exhibit significantly improved limit of detection and dramatically increased TCDD induction response. The low background luciferase activity, low minimal detection limit (0.1 pM TCDD) and enhanced induction response of the rat G3 cell line (H4L7.5c2) over the H1L7.5c3 mouse G3 cells, identifies them as a more optimal cell line for screening purposes. The utility of the new G3 CALUX cell lines were demonstrated by screening sediment extracts and a small chemical compound library for the presence of AhR agonists. The improved limit of detection and increased response of these new G3 CALUX cell lines will facilitate species-specific analysis of DLCs and AhR agonists in samples with low levels of contamination and/or in small sample volumes.

Identification of benzothiazole derivatives and polycyclic aromatic hydrocarbons as aryl hydrocarbon receptor agonists present in tire extracts

Leachate from rubber tire material contains a complex mixture of chemicals previously shown to produce toxic and biological effects in aquatic organisms. The ability of these leachates to induce Ah receptor (AhR)-dependent cytochrome P4501A1 expression in fish indicated the presence of AhR active chemicals, but the responsible chemicals and their direct interaction with the AhR signaling pathway were not examined. Using a combination of AhR-based bioassays, we have demonstrated the ability of tire extract to stimulate both AhR DNA binding and AhR-dependent gene expression and confirmed that the responsible chemicals were metabolically labile. The application of CALUX (chemical-activated luciferase gene expression) cell bioassay-driven toxicant identification evaluation not only revealed that tire extract contained a variety of known AhR-active polycyclic aromatic hydrocarbons but also identified 2-methylthiobenzothiazole and 2-mercaptobenzothiazole as AhR agonists. Analysis of a structurally diverse series of benzothiazoles identified many that could directly stimulate AhR DNA binding and transiently activate the AhR signaling pathway and identified benzothiazoles as a new class of AhR agonists. In addition to these compounds, the relatively high AhR agonist activity of a large number of fractions strongly suggests that tire extract contains a large number of physiochemically diverse AhR agonists whose identities and toxicological/biological significances are unknown.

Novel 2-amino-isoflavones exhibit aryl hydrocarbon receptor agonist or antagonist activity in a species/cell-specific context

The aryl hydrocarbon receptor (AhR) mediates the induction of a variety of xenobiotic metabolism genes. Activation of the AhR occurs through binding to a group of structurally diverse compounds, most notably dioxins, which are exogenous ligands. Isoflavones are part of a family which include some well characterised endogenous AhR ligands. This paper analysed a novel family of these compounds, based on the structure of 2-amino-isoflavone. Initially two luciferase-based cell models, mouse H1L6.1c2 and human HG2L6.1c3, were used to identify whether the compounds had AhR agonistic and/or antagonistic properties. This analysis showed that some of the compounds were weak agonists in mouse and antagonists in human. Further analysis of two of the compounds, Chr-13 and Chr-19, was conducted using quantitative real-time PCR in rat H4IIE and human MCF-7 cells. The results indicated that Chr-13 was an agonist in rat but an antagonist in human cells. Chr-19 was shown to be an agonist in rat but more interestingly, a partial agonist in human. Luciferase induction results not only revealed that subtle differences in the structure of the compound could produce species-specific differences in response but also dictated the ability of the compound to be an AhR agonist or antagonist. Substituted 2-amino-isoflavones represent a novel group of AhR ligands that must differentially interact with the AhR ligand binding domain to produce their species-specific agonist or antagonist activity and future ligand binding analysis and docking studies with these compounds may provide insights into the differential mechanisms of action of structurally similar compounds.

Chemicals having estrogenic activity can be released from some bisphenol a-free, hard and clear, thermoplastic resins

BackgroundChemicals 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.MethodsWe 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.ResultsOur 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.ConclusionsThis 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.