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In the present study 42 polycyclic aromatic compounds (PACs) were investigated for their estrogenic potential using the VM7Luc4E2 transactivation assay. Relative potencies were determined for mass-balance analysis. In addition, compounds were tested in combination with the estrogen receptor (ER) antagonist ICI182,780 (ICI) and the aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor α-naphthoflavone. Luciferase induction and CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity were measured to assess whether the estrogenic activity was elicited by the compound itself and/or by its metabolites. Relative potencies ranged between 10-7 and 10-4 . The ability of ICI to decrease luciferase activity stimulated by all compounds indicated that the induction responses were ER-dependent. The aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor α-naphthoflavone decreased luciferase induction and EROD activity by several compounds, including the methylated chrysenes, suggesting that metabolites of these chemicals contributed to ER activation. Several PACs, such as acridine and its derivatives, appear to directly activate the ER. Furthermore, extracts of soils from industrial areas were examined using this bioassay, and estrogenic activity was detected in all soil samples. Mass-balance analysis using a combination of relative potencies and chemical analysis of the samples suggested that polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs, such as 1- and 3-methylchrysene, are important contributors to the overall estrogenic activity. However, these results revealed that a considerable proportion of the estrogenic activity in the soil remained unexplained, indicating the presence of other significant estrogenic compounds. Environ Toxicol Chem 2018;37:385-397. © 2017 SETAC.

Estrogenic endocrine-disrupting chemicals are found in environmental and biological samples, commercial and consumer products, food, and numerous other sources. Given their ubiquitous nature and potential for adverse effects, a critical need exists for rapidly detecting these chemicals. The authors developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the US Environmental Protection Agency (USEPA) and Organisation for Economic Co-operation and Development (OECD) as a bioanalytical method to detect estrogen receptor (ER) agonists/antagonists. Unfortunately, these cells appear to contain only 1 of the 2 known ER isoforms, ERα but not ERβ, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ERβ expression plasmid and positive clones identified using ERβ-selective ligands (genistein and Br-ERβ-041). A highly responsive clone (BG1LucERβc9) was identified that exhibited greater sensitivity and responsiveness to ERβ-selective ligands than BG1Luc4E2 cells, and quantitative reverse-transcription polymerase chain reaction confirmed the presence of ERβ expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ERβ-dependent reporter gene expression. Together, these results not only demonstrate the utility of this dual-ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ERα- and ERβ-selective chemicals.

In the present study 42 polycyclic aromatic compounds (PACs) were investigated for their estrogenic potential using the VM7Luc4E2 transactivation assay. Relative potencies were determined for mass-balance analysis. In addition, compounds were tested in combination with the estrogen receptor (ER) antagonist ICI182,780 (ICI) and the aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor α-naphthoflavone. Luciferase induction and CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity were measured to assess whether the estrogenic activity was elicited by the compound itself and/or by its metabolites. Relative potencies ranged between 10

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.

Estrogenic endocrine-disrupting chemicals are found in environmental and biological samples, commercial and consumer products, food, and numerous other sources. Given their ubiquitous nature and potential for adverse effects, a critical need exists for rapidly detecting these chemicals. The authors developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the US Environmental Protection Agency (USEPA) and Organisation for Economic Co-operation and Development (OECD) as a bioanalytical method to detect estrogen receptor (ER) agonists/antagonists. Unfortunately, these cells appear to contain only 1 of the 2 known ER isoforms, ERα but not ERβ, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ERβ expression plasmid and positive clones identified using ERβ-selective ligands (genistein and Br-ERβ-041). A highly responsive clone (BG1LucERβc9) was identified that exhibited greater sensitivity and responsiveness to ERβ-selective ligands than BG1Luc4E2 cells, and quantitative reverse-transcription polymerase chain reaction confirmed the presence of ERβ expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ERβ-dependent reporter gene expression. Together, these results not only demonstrate the utility of this dual-ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ERα- and ERβ-selective chemicals.

An aryl hydrocarbon (Ah) receptor-based luciferase cell culture bioassay developed to detect 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other halogenated aromatics was modified and optimized to detect and quantitate polycyclic aromatics (PAHs). Twenty-four PAHs were analyzed, and subsequent EC50 and EC20 concentrations (based on the median and 20% TCDD maximal response, respectively) and appropriate induction equivalency factors (calculated by comparison to the response obtained with TCDD) were determined from dose-response experiments. Six compounds were shown to be active in the system, with benzo[k]fluoranthene > benz[a,h]anthracene indeno[1,2,3-cd]pyrene > benzo[a]pyrene > benzo[b]fluoranthene > chrysene. A complex mixture of 16 PAHs was also analyzed using this system, and overall induction equivalency (or 1-EQ) of the mixture was shown to be very similar to that predicted from the sum of the activity estimated for each individual PAH. Overall, our results strongly support the use of this system for the detection and relative quantitation of Ah receptor-active PAHs.

In a recent survey of pesticide concentrations in aquatic ecosystems of Central America, the antifungal triazole compound propiconazole was found to be the most widely distributed pesticide. Previously, technical grade propiconazole (TGP) has been shown to modulate cytochrome P450 activity in mammals and birds. The present study investigated the concentration- and timedependent effects of TGP on hepatic cytochrome CYP1A gene expression and catalytic activity in rainbow trout (Oncorhyncus mykiss). The TGP produced both a mixed-pattern response and a biphasic response for CYP1A expression following waterborne exposure. Evidence for inhibitory complex formation with cytochrome P450 may explain the occurrence of the mixed-pattern response. To further characterize the influence of TGP on CYP1A expression, a comparison was made between TGP and analytical grade propiconazole (AGP) with in vivo and in vitro assays. This comparison demonstrated that induction of the CYP1A gene resulted from unidentified compounds in TGP. Since TGP can be coapplied with other agricultural pesticides, either inhibition or induction of CYP1A protein activity by TGP and potentially other cytochrome P450 isoenzymes may lead to unexpected toxicological interactions.

Halogenated aromatic hydrocarbons (HAHs) and other chemicals that act as aryl hydrocarbon (Ah) receptor (AhR) agonists cause a variety of toxicity effects. In sac fry of many fish species, these effects include blue-sac disease and mortality. Because HAHs occur in complex mixtures, their toxicity in the environment is difficult to predict. A bioassay useful in predicting AhR-mediated toxicity to fish was developed using the RTH-149 rainbow trout hepatoma cell line. Stable transfection of this cell line with the pGudLuc 1.1 plasmid, which contains a firefly luciferase reporter gene under the transcriptional regulation of dioxin responsive enhancers, has produced a recombinant cell line designated Remodulated Lightning Trout (RLT 2.0). The RLT 2.0 bioassay method detection limit for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is 4 pM. The responses of the RLT 2.0 bioassay to TCDD and several HAH congeners closely matched the responses observed in vivo in fish. The RLT 2.0 bioassay can provide an integrative measure of the total AhR-mediated toxic activity of complex mixtures to fish. The assay will be useful in screening environmental extracts, guiding chemical analysis, and interpreting the AhR-mediated mechanism of toxicity.

A method was developed to isolate lipophilic compounds efficiently from small aliquots of blood plasma and test these for total dioxin-like toxic potency using recombinant rat (H4IIE) and mouse (Hepa1c1c7) hepatoma cell lines, containing the firefly (Photinus pyralis) luciferase gene under trans-activational control of the aryl hydrocarbon receptor (AhR). For this experiment, blood plasma was used originating from eider ducks (Somateria mollissima) that had been dosed with 3,39,4,49-tetrachlorobiphenyl (PCB-77) or with the technical PCB-mixture Clophen A50. For each sample the CALUX (chemical-activated luciferase expression) response of both the fat-containing organic extract and the fat-free, cleaned extract were compared with data from chemical analyses of these samples. The CALUX responses for the extracts were converted into so-called CALUX TEQs (TCDD equivalents), using a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) standard curve. The CALUX TEQs in both fatty and cleaned extracts correlated significantly with PCB-77 or PCB-153 levels (depending on the dosage group) determined in blood plasma using gas chroma- tography-mass spectrometry (GC-MS). For PCB-77 a toxic equivalency factor (TEF) of 1.5 3 10 23 was calculated based on these correlations. In addition, PCB-118 and PCB-156 levels in abdominal fat (assessed with GC with electron capture detection) and hepatic ethoxyresorufin O-deethylase activities correlated well with the CALUX TEQs in both fatty and cleaned blood plasma extracts, suggesting the TEQ levels in blood offer a good measure for internal dose. Plasma cholesterol and triglyceride levels were determined as a measure of lipid content, in 10-ml aliquots of blood plasma using enzymatic spectrophotometric determination. In conclusion, we have demonstrated that the CALUX assay is a rapid, sensitive assay for assessing the toxic potency of (mixtures of) AhR-active compounds in small aliquots of blood plasma. The limit of detection for the CALUX assay is currently less than 0.1 fmol (32 fg) TEQ, which corresponds with the amount of TEQs present in 0.1 to 1 ml of blood plasma in environmentally exposed species or man.

This study was undertaken to evaluate the relationship between mammalian and piscine 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalency factors (TEFs) for PCBs, based on induction of CYP1A enzyme activity, catalytic protein, and mRNA. Rainbow trout administered a single i.p. injection of TCDD had an average ({+-}SD) ED50 of 0.91 {+-} 0.14 {mu}g TCDD/kg for induction of ethoxyresorufin O-deethylase (EROD) activity. Ortho-substituted PCB congeners 2,3,3{prime},4,4{prime}-pentachlorobiphenyl (PCB 105), 2,3{prime},4,4{prime},5-pentachlorobiphenyl (PCB 118), 2,3,3{prime},4,4{prime},5-hexachlorobiphenyl (PCB 156), and 2,2{prime}3,4,4{prime},5-hexachlorobiphenyl (PCB 138) did not induce CYP1A activity in rainbow trout. Only three non-ortho-substituted PCBs, i.e., 3,3{prime},4,4{prime}-tetrachlorobiphenyl (PCB 77), 3,3{prime},4,4{prime},5-pentachlorobiphenyl (PCB 126), and 3,3{prime},4,4{prime},5,5{prime}-hexachlorobiphenyl (PCB 169) induced CYP1A enzyme activity, protein, and mRNA. The ED50s for induction of EROD activity were calculated as 134, 5.82, and 93.7 {mu}g/kg for PCB 77, PCB 126, and PCB 169, respectively. The TCDD-TEFs based on EROD activity were 0.0006, 0.0014, and 0.0003 for PCB 77, PCB 126, and PCB 169, respectively. Binary mixtures of TCDD and three PCBs were also evaluated. Based on EROD activity and CYP1A protein, mixtures of TCDD and PCB 77 were slightly greater than additive. Mixtures of TCDD-PCB 156 and TCDD-PCB 126 were slightly less than additive. Results from these studies indicate that mammal-derived TEFs will underestimate the potencymore » of planar chlorinated hydrocarbon mixtures to induce the CYP1A catalytic activity in rainbow trout. Also, while interactions among PCB congeners and TCDD were somewhat equivocal, they did not greatly differ from predicted additive responses.« less

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. Environ. Toxicol. Chem. 2011;30:1915-1925. # 2011 SETAC

An aryl hydrocarbon (Ah) receptor-based luciferase cell culture bioassay developed to detect 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other halogenated aromatics was modified and optimized to detect and quantitate polycyclic aromatics (PAHs). Twenty-four PAHs were analyzed, and subsequent EC50 and EC20 concentrations (based on the median and 20% TCDD maximal response, respectively) and appropriate induction equivalency factors (calculated by comparison to the response obtained with TCDD) were determined from dose-response experiments. Six compounds were shown to be active in the system, with benzo[k]fluoranthenebenz[a,h]anthracene indeno[1,2,3-cd]pyrenebenzo[a]pyrenebenzo[b]fluoranthenechrysene. A complex mixture of 16 PAHs was also analyzed using this system, and overall induction equivalency (or 1-EQ) of the mixture was shown to be very similar to that predicted from the sum of the activity estimated for each individual PAH. Overall, our results strongly support the use of this system for the detection and relative quantitation of Ah receptor-active PAHs.

Migrating white sturgeon (Acipenser transmontamus) may be subject to agricultural, municipal, and industrial wastewater effluents that likely contain different classes of endocrine-disrupting contaminants. Concern is mounting about the negative effects of environmental estrogens on fish reproduction; however, in environmental mixtures, the affects from estrogenic compounds may be suppressed by aryl hydrocarbon receptor (AhR) ligands. Indeed, reductions in 17beta-estradiol-induced (0.01 and 1 mg/kg) vitellogenin (VTG) levels were observed in white sturgeon coinjected with beta-naphthoflavone (BNF; 50 mg/kg), a model for contaminants that activate the AhR. Variation in the time of injection was used to attempt to correlate VTG inhibition to ethoxyresorufin-O-deethylase activity. No evidence was found to suggest that the inhibition of VTG is a direct result of enhanced estrogen metabolism by BNF-induced enzymes. Results of the present study are relevant for monitoring programs that measure VTG, because these results show that AhR-active environmental contaminants can repress VTG synthesis, which commonly is used as an indicator of estrogen-mimicking contaminants. Furthermore, suppression of natural estrogen signaling by AhR agonists may have significant effects on fish reproduction.