Chemical-activated Luciferase Gene Expression Research Articles

Non-target analysis of crude oil photooxidation products at high latitudes and their biological effects

With new oil and gas lease sales in high-latitude regions, there exists a need to better understand the chemical fate of spilled oil and its effects on biological life. To address this need, laboratory simulations of crude oil spills under sub-Arctic conditions were conducted using artificial seawater and exposure to solar irradiation to create Hydrocarbon Oxidation Products (HOPs). HOPs characterization and their biological effects were assessed using ultra high-performance liquid chromatography (UHPLC) with high resolution mass Orbitrap spectrometry and the aryl hydrocarbon receptor (AhR) chemically activated luciferase gene expression (CALUX) assay. Non-target UHPLC-Orbitrap mass spectrometry analysis identified 251 HOPs that were in greater abundance in light-exposed samples than dark controls. Oxidized polycyclic aromatic hydrocarbons were also detected, including phenanthrene quinone, anthraquinone, hydroxyanthraquinone, and 9-fluoreneone. The composition of HOPs were consistent with photo-products of alkylated two to four ring PAHs, primarily compounds between 1 and 3 aromatic rings and 1–3 oxygens. The HOP mixture formed during photochemical weathering of Cook Inlet crude oil induced greater AhR activity than parent petroleum products solubilized in dark controls, indicating that HOPs, as a complex mixture, may contribute to petroleum toxicity more than the parent petroleum compounds. These non-targeted approaches provide the most comprehensive analysis of hydrocarbon oxidation products to date, highlighting the diversity of the complex mixture resulting from the photooxidation of crude oil and the limitations of targeted analyses for adequately monitoring HOPs in the environment. Taken together, these data identify a critical “blind spot” in environmental monitoring and spill clean-up strategies as there is a diverse pool of HOPs that may negatively impact human and ecosystem health.

Brominated Dioxins in Egg, Broiler, and Feed Additives: Significance of Bioassay-Directed Screening for Identification of Emerging Risks in Food.

Food authorities aim to safeguard our food. This requires sensitive analyses to guarantee detection of both banned and regulated substances at low concentrations. At the same time, broad screening methods are needed to identify new emerging risks. For this purpose, effect-based bioassays combined with mass spectrometric analyses offer an advantage. During the regular monitoring of dioxins in agricultural products, a discrepancy was observed between the results of the DR CALUX (Dioxin-Responsive Chemical Activated Luciferase gene Expression) bioassay and the confirmatory gas chromatographic high resolution mass spectrometric (GC-HRMS) analysis in egg and broiler fat samples. The response in the bioassay was high, suggesting a clear exceedance of the maximum limits of dioxins in these samples, yet regulated dioxins or dl-PCBs were not detected by GC/HRMS analysis. Ultimately, a broad screening analysis using GC-HRMS resulted in the identification of 2,3,7,8-tetrabromo-dibenzofuran (2,3,7,8-TBDF) in both egg and broiler fat. To investigate the potential source of this brominated furan contaminant, different samples were analyzed: bedding material, poultry feed, feed additives (choline chloride and l-lysine), and seaweed. The poultry feed and feed additives all contained 2,3,7,8-TBDF. Using a feed-to-food transfer model, it became clear that the poultry feed was probably the source of 2,3,7,8-TBDF in broilers and eggs through a feed additive like L-lysine or choline chloride. This study underlines the importance of using a combination of effect-based screening assays with sensitive analytical methods to detect potential new and emerging risks.

Impact of inorganic and organic pollutants from a Belgian wastewater treatment plant on adjacent surface and groundwaters

Under the pressure of global droughts and water shortage, it is essential to evolve toward a sustainable and robust water system. One possible avenue is the maximum reuse of treated wastewater, but the quality of which determines its reuse. Therefore, inorganic (Cd, Pb, Cr, Ni, Cu, and As) and organic (xenoestrogens and polycyclic aromatic contaminants, PACs) contaminants were monthly monitored in an effluent of the wastewater treatment plant (WWTP), the surrounding surface waters and the local groundwater in Belgium. Dissolved and particulate concentrations of inorganic contaminants in these water bodies were analyzed. In addition, Diffusive Gradients in Thin-films (DGT) was used in situ to obtain bioavailable metal fractions. In the WWTP effluent and surface waters, only Ni exceeds the Annual Average-Environmental Quality Standard (AA-EQS), while in the groundwater, dissolved As was the predominant element. Moreover, in the surface and effluent waters the highest lability degrees were observed for Cd and Ni. The concentrations of these metal species in the effluent water were lower than in the other water bodies. Micro-organic pollutants, xenoestrogens and PACs were analyzed by dual Estrogen and Aryl hydrocarbon Receptor - Chemical Activated LUciferase gene eXpression (ER & AhR-CALUX) assays. Since the annual averaged (AA) bioequivalent concentration of E2 (0.18 ng/L) is below the AA-EQS standard (0.4 ng/L), and the bioequivalent concentration of benzo[a]pyrene never exceeded the maximum admissible concentration (MAC), the reclamation and reuse of treated wastewater for groundwater replenishment and agricultural irrigation should pose no environmental problems, at least in a short-term.

Deciphering the origin of total estrogenic activity of complex mixtures.

Identifying compounds with endocrine properties in food is getting increasingly important. Current chemical analysis methodology is mainly focused on the identification of known substances without bringing insight for biological activity. Recently, the application of bioassays has been promoted for their potential to detect unknown bioactive substances and to provide information on possible interactions between molecules. From the toxicological perspective, measuring endocrine activity cannot inform on endocrine disruption and/or health risks without sufficient knowledge on the nature of the responsible factors. The present study addresses a promising approach using High Performance Thin-Layer Chromatography (HPTLC) coupled to bioassays were analyzed using the Liquid Chromatography Mass-Spectrometry (LC-MS). The estrogen receptor activation was assessed using the transcription activation Estrogen Receptor Alpha Chemical Activated LUciferase gene eXpression assay (ERα- CALUX) and the HPTLC coupled to the Estrogen Screen Yeast assay (p-YES). Seven isoflavones were identified in the soy isolates. Estrogen receptor activation was assessed for both, the identified isoflavones and the soy isolates with ERα-CALUX test. Correlation between the soy isolates extracts and the identified isoflavones was shown. Moreover, p-YES revealed the presence of an estrogenic bioactive zone. Analysis of the bioactive zone through LCHRMS highlighted signals corresponding to several isoflavones already detected in the isolates as well as two additional ones. For all detected isoflavones, an estrogenic activity dose-response was established in both bioassays. Finally, genistein, daidzein, and naringenin were found as the most active substances. A concordance analysis integrating the analytical and bioassay data indicated that genistein and daidzein were the drivers of the estrogenic activity of these soy protein isolates. Altogether, these data suggest that the integration of HPTLC-bioassay together with chemical analysis is a powerful approach to characterize the endocrine activity of complex mixtures.

The role of receptor-mediated activities of 4- and 5-ring unsubstituted and methylated polycyclic aromatic hydrocarbons (PAHs) in developmental toxicity.

The present study evaluated the aryl hydrocarbon receptor (AhR), estrogen receptor-α (ER-α), and retinoic acid receptor (RAR) mediated activities of nine 4- and 5-ring unsubstituted and monomethylated polycyclic aromatic hydrocarbons (PAHs) using a series of Chemical-Activated LUciferase gene eXpression (CALUX) assays. The potential role of these aforementioned receptors in relation to the developmental toxicity of these PAHs was further assessed in the zebrafish embryotoxicity test (ZET). The results show that all nine tested PAHs were AhR agonists, benz[a]anthracene (BaA) and 8-methyl-benz[a]anthracene (8-MeBaA) were ER-α agonists, and none of the tested PAHs induced ER-α antagonistic or RAR (ant)agonistic activities. In the AhR CALUX assay, all the methylated PAHs showed higher potency (lower EC50) in activating the AhR than their respective unsubstituted PAHs, implying that the addition of a methyl substituent on the aromatic ring of PAHs could enhance their AhR-mediated activities. Co-exposure of zebrafish embryos with each individual PAH and an AhR antagonist (CH223191) counteracted the observed developmental retardations and embryo lethality to a certain extent, except for 8-methyl-benzo[a]pyrene (8-MeBaP). Co-exposure of zebrafish embryos with either of the two estrogenic PAHs (i.e., BaA and 8-MeBaA) and an ER-α antagonist (fulvestrant) neutralized embryo lethality induced by 50 μM BaA and the developmental retardations induced by 15 μM 8-MeBaA. Altogether, our findings suggest that the observed developmental retardations in zebrafish embryos by the PAH tested may partially be AhR- and/or ER-α-mediated, whereas the RAR seems not to be relevant for the PAH-induced developmental toxicity in the ZET.

Dynamics of estrogenic activity in an urban river receiving wastewater effluents: effect-based measurements with CALUX

Estrogenic substances (ES) in an urban river Zenne (BE) dominated by wastewater effluents were assessed over the course of one year. To measure the bioequivalent (BEQ) 17 β-estradiol (E2) concentrations of ES, the biological effect-based methodology - the Chemical-Activated LUciferase gene eXpression (CALUX) bioassay was used. Daily water discharges were collected from January 2015 to February 2016 at or near the sampling stations in the Brussels Capital Region. An annual water budget shows that approximately 50% of the Zenne River flow downstream is from wastewater effluent. The estrogenic activity and yearly average ES load in influents and effluents of wastewater treatment plants (WWTPs) located in the North and South, combined sewer overflows (CSOs) and the Zenne River, were assessed for upstream and downstream of two WWTPs of Brussels. Both WWTPs with activated sludge treatment remove more than 90% of the ES. The influent concentrations of ES at the South and North WWTPs ranged from 30-359 and 18-55 ng E2 eq./L, respectively. The effluent concentrations of ES ranged from 1.0-2.1 and 1.1-6.6 ng E2 eq./L at WWTP-S and -N, respectively. The yearly average ES loads were 0.05-0.14 and 0.39-1.5 g E2 eq./d for WWTP-S and -N, respectively. The temporal variation of E2-eq concentrations at the river stations Z3 and Z5 (upstream) ranged from 1 to 2 ng E2 eq./L, while the ES activity at sites Z9 and Z11 (downstream) varied from 2-17 ng E2 eq./L and from 1-8 ng/L ng E2 eq./L, respectively. The relative ES loads to the Zenne River are as follows: WWTPs (31%), CSOs (27%), upstream Zenne (15%), a missing source (14%), and local tributaries (13%). ES in the Zenne River behave in a pseudo-persistent manner because of continuous input from the WWTPs and slow degradation in the 18 km river stretch. The BEQ concentration of E2 exceeds the EU environmental quality standards (EQS) of 0.4 ng E2/L throughout the Zenne River.

Estrogenic, androgenic, and glucocorticoid activities and major causative compounds in river waters from three Asian countries.

Estrogen, androgen, and glucocorticoid receptors (ER, AR, and GR) agonist activities in river water samples from Chennai and Bangalore (India), Jakarta (Indonesia), and Hanoi (Vietnam) were evaluated using a panel of chemical-activated luciferase gene expression (CALUX) assays and were detected mainly in the dissolved phase. The ER agonist activity levels were 0.011-55ng estradiol (E2)-equivalent/l, higher than the proposed effect-based trigger (EBT) value of 0.5ng/l in most of the samples. The AR agonist activity levels were < 2.1-110ng dihydrotestosterone (DHT)-equivalent/l, and all levels above the limit of quantification exceeded the EBT value of 3.4ng/l. GR agonist activities were detected in only Bangalore and Hanoi samples at dexamethasone (Dex)-equivalent levels of < 16-150ng/l and exceeded the EBT value of 100ng/l in only two Bangalore samples. Major compounds contributing to the ER, AR, and GR agonist activities were identified for water samples from Bangalore and Hanoi, which had substantially higher activities in all assays, by using a combination of fractionation, CALUX measurement, and non-target and target chemical analysis. The results for pooled samples showed that the major ER agonists were the endogenous estrogens E2 and estriol, and the major GR agonists were the synthetic glucocorticoids Dex and clobetasol propionate. The only AR agonist identified in major androgenic water extract fractions was DHT, but several unidentified compounds with the same molecular formulae as endogenous androgens were also found.

Photolytic degradation of novel polymeric and monomeric brominated flame retardants: Investigation of endocrine disruption, physiological and ecotoxicological effects

Ecotoxicological effects of photolytic degradation mixtures of the two brominated flame retardants PolymericFR and Tetrabromobisphenol A-bis (2,3-dibrom-2-methyl-propyl) Ether (TBBPA-BDBMPE) have been studied in vitro and in vivo. Both substances were experimentally degraded separately by exposure to artificial UV-light and the resulting degradation mixtures from different time points during the UV-exposure were applied in ecotoxicological tests. The in vitro investigation showed no effects of the degraded flame retardants on the estrogenic and androgenic receptors via the CALUX (chemically activated luciferase gene expression) assay. Short-term exposures (up to 96 h) of Lumbriculus variegatus lead to temporary physiological reactions of the annelid. The exposure to degraded PolymericFR lead to an increased activity of Catalase, while the degradation mixture of TBBPA-BDBMPE caused increases of Glutathione-S-transferase and Acetylcholine esterase activities. Following a chronic exposure (28 d) of L. variegatus, no effects on the growth, reproduction, fragmentation and energy storage of the annelid were detected. The results indicate that the experimental degradation of the two flame retardants causes changes in their ecotoxicological potential. This might lead to acute physiological effects on aquatic annelids, which, however, do not affect the animals chronically according to our results.

Bioanalytical screening of low levels of dioxins and dioxin-like PCBs in pig meat (pork) for checking compliance with EU maximum and action levels using highly sensitive \u201cthird generation\u201d recombinant H4L7.5c2 rat hepatoma cells

BackgroundLow maximum and action levels set by the European Union for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) in pig meat (pork) have led to a demand for reliable and cost-effective bioanalytical screening methods implemented upstream of gas chromatography/high-resolution mass spectrometry confirmatory technology, that can detect low levels of contamination in EU-regulated foods with quick turn-around times.ResultsBased on the Chemically Activated LUciferase gene eXpression (CALUX) bioassay, extraction and clean-up steps were optimized for recovery and reproducibility within working ranges significantly lower than in current bioassays. A highly sensitive “3rd generation” recombinant rat hepatoma cell line (H4L7.5c2) containing 20 dioxin responsive elements was exposed to pork sample extracts, and their PCDD/Fs and DL-PCBs levels were evaluated by measuring luciferase activity. The method was validated according to the provisions of Commission Regulation (EU) 2017/644 of 5 April 2017 with spiking experiments performed selectively for PCDD/Fs and DL-PCBs and individual calibration for PCDD/Fs, DL-PCBs and the calculated sum of PCDD/Fs and DL-PCBs. The resulting performance parameters met all legal specifications as confirmed by re-calibration using authentic samples. Cut-off concentrations for assessing compliance with low maximum levels and action levels set for PCDD/Fs and DL-PCBs within a range of 0.50–1.25 pg WHO-TEQ/g fat were derived, ensuring low rates of false-compliant results (ß-error < 1%) and keeping the rate of false-noncompliant results well under control (α-error < 12%).ConclusionsWe present a fast and efficient bioanalytical routine method validated according to the European Union’s legal requirements on the basis of authentic samples, allowing the analyst to reliably identify pork samples and any other EU-regulated foods of animal origin suspected to be noncompliant with a high level of performance and turn-around times of 52 h. This was facilitated in particular by a quick and efficient extraction step followed by selective clean-up, use of a highly sensitive “3rd generation” H4L7.5c2 recombinant rat hepatoma cell CALUX bioassay, and optimized assay performance with improved calibrator precision and reduced lack-of-fit errors. New restrictions are proposed for the calibrator bias and the unspecific background contribution to reportable results. The procedure can utilize comparably small sample amounts and allows an annual throughput of 840–1000 samples per lab technician. The described bioanalytical method contributes to the European Commission's objective of generating accurate and reproducible analytical results according to Commission Regulation (EU) 2017/644 across the European Union.

Development of a framework to derive effect-based trigger values to interpret CALUX data for drinking water quality

Bioassays are increasingly being implemented for water quality monitoring as targeted chemical analyses are not always sufficient for the detection of all emerging chemicals or transformation products. However, the interpretation of bioassay results remains challenging, in particular because a positive response does not necessarily indicate that there may be an increased risk. For this purpose, effect-based trigger (EBT) values have been introduced as thresholds above which action needs to be undertaken to determine the cause of the response. The goals of this study were to (i) evaluate various approaches used to determine EBT values and (ii) based on the findings, derive human health EBT values for Chemical Activated LUciferase gene eXpression (CALUX) in vitro bioassays used for routine monitoring of water quality in the Netherlands. Finally, (iii) an uncertainty analysis was carried out to determine the protective power of the derived EBT values and the chance that potentially harmful substances might not be detected. EBT values that can be implemented in routine monitoring could be determined for four of eight selected bioassays. These EBT were compared to bioassay results from routine water quality monitoring carried out in the Netherlands. Furthermore, a framework for the calculation and evaluation of derived EBT values for routine application to monitor drinking water and its sources is proposed.

Storm Event-Driven Occurrence and Transport of Dissolved and Sorbed Organic Micropollutants and Associated Effects in the Ammer River, Southwestern Germany.

Storm events lead to agricultural and urban runoff, to mobilization of contaminated particulate matter, and to input from combined sewer overflows into rivers. We conducted time-resolved sampling during a storm event at the Ammer River, southwest Germany, which is representative of small river systems in densely populated areas with a temperate climate. Suspended particulate matter (SPM) and water from 2 sampling sites were separately analyzed by a multi-analyte liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for 97 environmentally relevant organic micropollutants and with 2 in vitro bioassays. Oxidative stress response (AREc32) may become activated by various stressors covering a broad range of physicochemical properties and induction of aryl hydrocarbon receptor-chemical-activated luciferase gene expression (AhR-CALUX) by hydrophobic compounds such as dioxins and dioxin-like molecules. Compound numbers, concentrations, their mass fluxes, and associated effect fluxes increased substantially during the storm event. Micropollutants detected in water and on SPM pointed toward inputs from combined sewer overflow (e.g., caffeine, paracetamol), urban runoff (e.g., mecoprop, terbutryn), and agricultural areas (e.g., azoxystrobin, bentazone). Particle-facilitated transport of triphenylphosphate and tris(1-chloro-2-propyl) phosphate accounted for up to 34 and 33% of the total mass flux even though SPM concentrations were <1 g L-1 . Effect fluxes attributed to SPM were similar or higher than in the water phase. The important role of SPM-bound transport emphasizes the need to consider not only concentrations but also mass and effect fluxes for surface water quality assessment and wastewater/stormwater treatment options. Environ Toxicol Chem 2021;40:88-99. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Bisphenols emerging in Norwegian and Czech aquatic environments show transthyretin binding potency and other less-studied endocrine-disrupting activities

Bisphenols are increasingly recognized as environmental pollutants with endocrine-disrupting potential. Nonetheless, the study of environmental occurrence and some endocrine-disrupting activities of some bisphenols came widely into focus of research only recently. The aims of the present study were to: 1) determine the predominant bisphenols in Norwegian sewage sludge and sediment and in Czech surface waters, and 2) characterize the binding of bisphenols to a transport protein transthyretin (TTR) and their (anti-)thyroid, (anti-)progestagenic, and (anti-)androgenic activities.High-performance liquid chromatography with atmospheric pressure chemical ionization or photoionization coupled with high resolution mass spectrometry (HPLC-APCI/APPI-HRMS) and Chemically Activated LUciferase gene eXpression (CALUX) in vitro reporter gene bioassays were used to detect the target compounds and to determine endocrine-disrupting activities, respectively.Bisphenol A (BPA), 4,4′-bisphenol F (BPF), bisphenol S (BPS), and bisphenol E (BPE) were the most frequently found compounds in municipal sewage sludge. Furthermore, bisphenol TMC (BPTMC) and bisphenol AF (BPAF) frequently occurred in sediment and surface waters, respectively. BPA was the major contributor to Ʃ of bisphenols in Norwegian sewage sludge with exception of one sample where BPF predominated. We also monitored a few bisphenols in sediment but only BPTMC was found. BPA, BPAF and BPF were the dominant bisphenols in Czech surface waters.Some bisphenols have shown TTR binding potency (BPAF = BPF > BPA = BPE) and some have displayed the following endocrine-disrupting activities: anti-thyroid (BPAF), anti-progestagenic (BPTMC > BPA = BPAF), and anti-androgenic (BPAF > BPE > BPA > BPTMC > BPF > BPS). It is noteworthy that BPAF exhibited stronger or similarly potent endocrine-disrupting activities compared to BPA. Our results provide new insights into these less-studied endocrine-disrupting activities of environmentally relevant bisphenols and may be useful in prioritizing those compounds that deserve further attention in environmental monitoring and eco-toxicological research.

Plasma glucocorticogenic activity, race/ethnicity and alcohol intake among San Francisco Bay Area women.

Racial and ethnic minorities are at higher risk for a variety of diseases. While sociodemographic and lifestyle factors contribute to racial/ethnic health disparities, the biological processes underlying these associations remain poorly understood. Stress and its biological consequences through the glucocorticoid receptor (GR) have been hypothesized to mediate adverse disease outcomes. In fasting morning samples of 503 control women from the San Francisco Bay Area Breast Cancer Study, we used a sensitive Chemical-Activated LUciferase gene eXpression (CALUX) assay to examine the association of sociodemographic and lifestyle factors with plasma glucocorticogenic (G) activity in three racial/ethnic groups. The G activity is a sensitive measure that reflects biological activity of total plasma glucocorticoids including cortisol and glucocorticoid-like compounds. Associations between G activity and sociodemographic and lifestyle factors were examined using multivariable linear regression models. Latina and non-Latina Black (NLB) women had 9% (P = 0.053) and 14% (P = 0.008) lower morning G activity than non-Latina White (NLW) women, respectively. Additionally, we replicated a previously reported association between G activity and alcohol intake (women who drank >10gms had 19% higher G activity than non-drinkers, P = 0.004) in Latina and NLB women. Further research should assess the association between G activity and health outcomes in a prospective cohort so as to characterize the relationship between total plasma G activity in pre-disease state and disease outcomes across different racial/ethnic populations.

Elucidating the aryl hydrocarbon receptor antagonism from a chemical-structural perspective

ABSTRACTThe aryl hydrocarbon receptor (AhR) plays an important role in several biological processes such as reproduction, immunity and homoeostasis. However, little is known on the chemical-structural and physicochemical features that influence the activity of AhR antagonistic modulators. In the present report, in vitro AhR antagonistic activity evaluations, based on a chemical-activated luciferase gene expression (AhR-CALUX) bioassay, and an extensive literature review were performed with the aim of constructing a structurally diverse database of contaminants and potentially toxic chemicals. Subsequently, QSAR models based on Linear Discriminant Analysis and Logistic Regression, as well as two toxicophoric hypotheses were proposed to model the AhR antagonistic activity of the built dataset. The QSAR models were rigorously validated yielding satisfactory performance for all classification parameters. Likewise, the toxicophoric hypotheses were validated using a diverse set of 350 decoys, demonstrating adequate robustness and predictive power. Chemical interpretations of both the QSAR and toxicophoric models suggested that hydrophobic constraints, the presence of aromatic rings and electron-acceptor moieties are critical for the AhR antagonism. Therefore, it is hoped that the deductions obtained in the present study will contribute to elucidate further on the structural and physicochemical factors influencing the AhR antagonistic activity of chemical compounds.

A mixture of persistent organic pollutants relevant for human exposure inhibits the transactivation activity of the aryl hydrocarbon receptor in vitro

While humans are exposed to mixtures of persistent organic pollutants (POPs), their risk assessment is usually based on a chemical-by-chemical approach. To assess the health effects associated with mixed exposures, knowledge on mixture toxicity is required. Several POPs are potential ligands of the Aryl hydrocarbon receptor (AhR), which involves in xenobiotic metabolism and controls many biological pathways. This study assesses AhR agonistic and antagonistic activities of 29 POPs individually and in mixtures by using Chemical-Activated LUciferase gene eXpression bioassays with 3 transgenic cell lines (rat hepatoma DR-H4IIE, human hepatoma DR-Hep G2 and human mammary gland carcinoma DR-T47-D). Among the 29 POPs, which were selected based on their abundance in Scandinavian human blood, only 4 exerted AhR agonistic activities, while 16 were AhR antagonists in DR-H4IIE, 5 in DR-Hep G2 and 7 in DR-T47-D when tested individually. The total POP mixture revealed to be AhR antagonistic. It antagonized EC50 TCDD inducing AhR transactivation at a concentration of 125 and 250 and 500 fold blood levels in DR-H4IIE, DR-T47-D and DR-Hep G2, respectively, although each compound was present at these concentrations lower than their LOEC values. Such values could occur in real-life in food contamination incidents or in exposed populations. In DR-H4IIE, the antagonism of the total POP mixture was due to chlorinated compounds and, in particular, to PCB-118 and PCB-138 which caused 90% of the antagonistic activity in the POP mixture. The 16 active AhR antagonists acted additively. Their mixed effect was predicted successfully by concentration addition or generalized concentration addition models, rather than independent action, with only two-fold IC50 underestimation. We also attained good predictions for the full dose-response curve of the antagonistic activity of the total POP mixture.

Chemical and bioassay assessment of waters related to hydraulic fracturing at a tight gas production site

Publicly available chemical assessments of hydraulic fracturing related waters are generally based on shale gas practices in the U.S. There is a lack of information on hydraulic fracturing related gas development from EU countries and more generally on other types of extractions. This research fills this knowledge gap by presenting chemical and bioassay assessments of hydraulic fracturing related waters from a tight gas development in the Netherlands. Fracturing fluid, flowback water and groundwater from surrounding aquifers before and after the actual fracturing were analysed by means of high resolution liquid chromatography tandem mass spectrometry, the Ames test and three chemical activated luciferase gene expression bioassays aimed at determining genotoxicity, oxidative stress response and polyaromatic hydrocarbon contamination.After sample enrichment a higher number of peaks can be found in both fracturing fluid and flowback samples. No clear differences in chemical composition were shown in the groundwater samples before and after hydraulic fracturing. Preliminary environmental fate data of the tentatively identified chemicals points towards persistence in water. Clear genotoxic and oxidative stress responses were found in the fracturing fluid and flowback samples. A preliminary suspect screening resulted in 25 and 36 matches in positive and negative ionisation respectively with the 338 possible suspect candidates on the list.Extensive measures relating to the handling, transport and treatment of hydraulic fracturing related waters are currently in place within the Dutch context. The results of the present study provide a scientific justification for such measures taken to avoid adverse environmental and human health impacts.

Toxicological and ecotoxicological evaluation of the water quality in a large and eutrophic freshwater lake of China

Effect-based methods (EBMs) are recommended as holistic approach for diagnosis and monitoring of water quality; however, the application of EBMs is still scare in China. In the present study, water quality of the freshwater lake Taihu (China) was investigated by EBMs. Different types of water samples were collected from three bays of the lake during 2015, 2016 and 2017. A battery of seven effect-based bioassays, including both specific and non-specific toxicity assays, was used. The bioassay battery was recently suggested based on joint activities of the EU project SOLUTIONS and the NORMAN network on emerging pollutants and is also under discussion for being implemented into monitoring activities in the context of the European Water Framework Directive (WFD). Adverse effects were observed towards the primary producer, primary consumer and fish, indicating the potential ecotoxicity of water in Taihu Lake. Mutagenic and estrogenic effects were found in the Ames fluctuation assay and ERα CALUX (Chemically Activated Luciferase Gene-eXpression) assay, respectively, highlighting the potential risks on human health. Algal growth inhibition and mutagenic effects can be observed during each of the three years. Acute toxicity towards Daphnia magna and estrogen receptor agonistic effects were found in at least one of the samples collected in 2016 and 2017, but not in 2015. The endpoints for fish toxicity in the Danio rerio fish embryo test included both lethal and additionally several sublethal effects (only for samples from 2017) and were not compared between years. Algal growth inhibition, fish embryo toxicity, mutagenic effect and estrogenicity were observed in each of the three bays, while Daphnia acute toxicity was only found in Zhushan Bay. Taking together, this study provides a big picture on the water quality of Taihu Lake. The battery of effect-based tools is promising to be a routine for water quality monitoring in China.

Abstract PR05: Estrogenic activity is associated with race/ethnicity and Indigenous American ancestry among San Francisco Bay Area women

Abstract The incidence of breast cancer in the U.S. varies by race/ethnicity. Furthermore, in Hispanic women, breast cancer risk is higher among women with low Indigenous American ancestry compared to those with high Indigenous American ancestry and in U.S.-born compared to foreign-born women. In a previous pilot study among foreign-born and U.S.-born Mexican women, we found that plasma estrogenic (E) activity was associated with genetic ancestry and years of U.S. residence, suggesting the possibility of a hormone-related pathway for the observed differences in breast cancer risk. In the present study, we examined the association between E activity and genetic ancestry in a larger sample of Hispanic women. We also evaluated the association between E activity, demographic factors, and breast cancer risk in non-Hispanic Black (NHB) and non-Hispanic White (NHW) women. We utilized a receptor-mediated Chemical-Activated Luciferase gene eXpression (CALUX) assay for the assessment of total activity profiles against estrogen receptors (ER) in human plasma, which captures levels of both endogenous and exogenous estrogenic compounds. Results were expressed in relative light units (RLUs), with higher RLU values reflecting greater E activity in plasma. ER activation was measured in plasma samples of 329 Hispanic, 100 NHBs, and 74 NHW women who participated as controls in the San Francisco Bay Area Breast Cancer Study (SFBCS). The participants ranged in age from 35 to 79 years, though the study was composed predominantly of postmenopausal women. Multivariable regression models included race/ethnicity, age at blood draw, height, body mass index (BMI), neighborhood socioeconomic status (SES), and alcohol intake. In the univariate model that included all women (n=503) and race/ethnicity as the independent predictor, Hispanics had 24% lower E activity (p=0.061) and NHBs had 36% higher activity (p=0.047) compared to NHWs. In the multivariable model, Hispanics still showed lower E activity levels (34%, p=0.009), but the difference with NHBs and NHWs was no longer statistically significant (p=0.493). After adjustment for levels of two endogenous estrogens, estradiol and estrone, in a subset of samples (n=260), Hispanics continued to show 20% lower E activity compared to NHWs. In addition, when BMI is categorized into tertiles consisting of normal (&amp;lt;25 kg/m2), overweight (25-30 kg/m2), and obese (BMI &amp;gt;30 kg/m2) women, we found that obese women had 73% higher E activity compared to those with normal BMI (p&amp;lt;0.001) and overweight women had 46% higher activity (p=0.006). Among the Hispanic women, we confirmed the previously observed negative association between E activity and Indigenous American ancestry, which is consistent with the observation that Hispanic women with high Indigenous American ancestry have lower risk of developing breast cancer. Overall, our study suggests that observed associations between race/ethnicity, genetic ancestry and BMI with breast cancer risk could be partly due to the mediating effect of endogenous estrogens as well as to the effect of exogenous estrogen-like compounds. Citation Format: Sylvia S. Sanchez, Phum Tachachartvanich, Heather Ruiz, Frank Z. Stanczyk, Scarlett Lin Gomez, Esther M. John, Martyn T. Smith, Laura Fejerman. Estrogenic activity is associated with race/ethnicity and Indigenous American ancestry among San Francisco Bay Area women [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr PR05.

Abstract A53: Plasma glucocorticogenic activity differs by race/ethnicity and alcohol intake among San Francisco Bay Area women

Abstract Breast cancer mortality is higher among non-Hispanic Black (NHB) and Hispanic women than non-Hispanic White (NHW) women in the United States. While various sociodemographic and lifestyle factors contribute to racial/ethnic disparities in breast cancer, the biologic processes underlying these associations remain poorly understood. Cortisol, the predominant endogenous glucocorticoid present in humans, is secreted in a diurnal pattern with the highest concentration occurring shortly after waking followed by a steady decline throughout the day. A flattened diurnal cortisol pattern (e.g., due to lower morning and/or elevated evening cortisol levels) is often observed among chronically stressed individuals and has been linked to poorer survival among breast cancer patients. We examined the association between race/ethnicity and other breast cancer risk factors with glucocorticogenic (G) activity, a measure that reflects plasma cortisol levels, in 503 controls from the San Francisco Bay Area Breast Cancer Study (SFBCS, 329 Hispanic, 100 NHB and 74 NHW women) using a low-cost Chemical-Activated LUciferase gene eXpression (CALUX) assay. Associations between G activity and sociodemographic and lifestyle factors were examined using multivariable linear regression models. Hispanic and NHB women had 14% (P = 0.016) and 16% (P = 0.007) lower morning G activity than NHW women, respectively. Additionally, we replicated our previously reported association between G activity and alcohol intake (women who drank &amp;gt;10 gm had 22% higher G activity than non-drinkers, P = 0.003). This association was only present in Hispanics and NHB. No statistically significant associations were observed between G activity and Indigenous American ancestry, body mass index, or neighborhood socioeconomic status. Our results indicate that NHB and Hispanic women may have a blunted cortisol awakening response potentially due to chronic stress. The increase in morning G activity observed with higher alcohol intake in Hispanics and NHB might reflect the use of alcohol as a stress-coping mechanism. Further research should assess the association between G activity and breast cancer survival in biospecimens from a prospective cohort so as to characterize the relationship between prediagnosis chronic stress and breast cancer outcome across different racial/ethnic groups. Citation Format: Rosemarie M. de la Rosa, Sylvia S. Sanchez, Phum Tachachartvanich, Heather Ruiz, Scarlett Lin Gomez, Esther M. John, Martyn T. Smith, Laura Fejerman. Plasma glucocorticogenic activity differs by race/ethnicity and alcohol intake among San Francisco Bay Area women [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr A53.

The Role of Endocrine and Dioxin-Like Activity of Extracts of Petroleum Substances in Developmental Toxicity as Detected in a Panel of CALUX Reporter Gene Assays.

Recent evidence suggests that the interaction of polycyclic aromatic hydrocarbons (PAHs), present in some petroleum substances (PS), with particular nuclear-hormone-receptors and/or the dioxin (aryl hydrocarbon receptor [AhR]) receptor, may play a role in the prenatal developmental toxicity (PDT) induced by these substances. To address this hypothesis, we evaluated the possible endocrine and dioxin-like activity of the dimethylsulfoxide (DMSO)-extracts of 9 PS, varying in PAH content, and 2 gas-to-liquid (GTL) products, containing no PAHs but having similar other properties as PS, using a series of Chemical Activated LUciferase gene eXpression (CALUX) assays. The results show that the extracts of PS tested in this study possess various endocrine and dioxin-like activities and these in vitro potencies are associated with the quantity and type of PAHs they contain. All tested DMSO-extracts of PS show a strong AhR agonist activity and rather weak antiprogesterone, antiandrogen, and estrogenic activities. In the assays that evaluate thyroid-related and antiestrogen activity, only minor effects of specific extracts, particularly those with a substantial amount of 4–5 ring PAHs, ie, sample No. 34, 98, and 99, were observed. None of the GTL extracts interacted with the selected receptors. Of all assays, the AhR agonist activity correlates best (R2 = 0.80) with the in vitro PDT of the substances as quantified previously in the embryonic stem cell test, suggesting an important role of the AhR in mediating this effect. Hierarchic clustering of the combined CALUX data clustered the compounds in line with their chemical characteristics, suggesting a PS class-specific effects signature in the various CALUX assays, depending on the PAH profile. To conclude, our findings indicate a high potential for endocrine and dioxin-like activity of some PS extracts which correlates with their in vitro PDT and is driven by the PAHs present in these substances.