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.

Extensive rain events have a more substantial impact than advanced effluent treatment on the endocrine-disrupting activity in an effluent-dominated small river.

Wastewater treatment plants (WWTPs) remain an important primary source of emission for endocrine-disrupting compounds in the environment. As an advanced wastewater treatment process, ozonation is known to reduce endocrine-disrupting activity. However, it remains unclear to which extend improved wastewater treatment may reduce the endocrine-disrupting activity in the receiving water body. The present study investigated possible factors for the endocrine-disrupting activity in a small receiving water body, the Wurm River (North-Rhine Westphalia, Germany), up- and downstream of a local WWTP. The cell-based reporter gene CALUX® assay was applied to identify the endocrine-disrupting activity in the water, sediment, and suspended particulate matter. The water phase and the effluent sampling were primarily driven by applying the full-scale effluent ozonation (sampling campaigns in June 2017 and March 2019). In contrast, the sediment sampling aimed to compare the particle-bound endocrine-disrupting activity during dry (June 2017) and rainy summer (June 2018) seasons. The water phase showed low to moderate estrogenic/antiandrogenic activity. Advanced effluent treatment by ozonation led to a complete reduction of the endocrine-disrupting activity according to the limit of detection of the CALUX® assays. The suspended particulate matter originated from the water phase of the second sampling campaign revealed antiandrogenic activity only. Sediments at the sampling sites along the local WWTP revealed higher estrogenic and antiandrogenic activity after extensive rain events and were not affected by the ozonated effluent. Fluctuation patterns of the endocrine-disrupting activity in sediments were in line with fluctuated concentrations of polycyclic aromatic hydrocarbons. Rainwater overflow basin release was suggested as a vector for particle-bound and dissolved endocrine-disrupting activity in the receiving water body. The present study underlined the necessity for monitoring both water and sediment phases to achieve reliable profiling of the endocrine-disrupting activity. The receptor-mediated CALUX® assays were proven to be suitable for investigating the endocrine-disrupting activity distribution in different river compartments and WWTP effluents.

Effect-directed analysis of estrogenic chemicals in sediments from an electronic-waste recycling area.

Electronic waste (e-waste) pollution is of great concern due to the release of hazardous chemicals during the improper e-waste disposal. Many chemicals leached from e-waste were reported to pose estrogenic effects. To date, little is known regarding the occurrence and biological effects of estrogenic chemicals in sediments near an e-waste area. In this study, an effect-directed analysis (EDA) is applied to determine the estrogenic chemicals in sediments of four sites collected from a typical e-waste recycling city in China. Following screening with the ER-CALUX assay, the extract of sample with the most potent effect was subjected in fractionation using reverse phase liquid chromatography. Based on a target analysis for the active fractions, four compounds, including estrone, 17ß-estradiol, 17α-ethinylestradiol and bisphenol A, were identified, and these contributed to 17% of the total toxic effects in the sample. A further nontarget analysis screened four candidates, namely diethylstilbestrol (DES), hexestrol (HES), nandrolone and durabolin, and the total contribution was found to be 48% from the active sample. Specifically, DES and HES were only detected in the active sample and were found to be the primary drivers of estrogenic effects. An examination of the identified chemicals in the four sites indicated that these estrogenic chemicals may originate from e-waste recycling, livestock excretion and domestic waste. These findings uncovered the estrogenic pollutants in sediments from an e-waste area. Considering single endpoint in biological assay is not abundant to screen chemicals with different toxic effects, further EDA studies with multiple endpoints are required to better understand the occurrence of representative or unknown chemicals in e-waste-polluted areas.

Two synthetic progestins and natural progesterone are responsible for most of the progestagenic activities in municipal wastewater treatment plant effluents in the Czech and Slovak republics.

Vast numbers of xenobiotics are known still to be present in treated municipal wastewater treatment plant (WWTP) effluents. Some of these possess endocrine-disrupting potency and pose risks for exposed aquatic animals. We searched for 17 potential environmental contaminants having affinity to the progesterone receptor. Relative potency values of these progesterone receptor-active chemicals were obtained. On the basis of relative potencies and measured environmental concentrations, the contribution of progestins to measured progestagenic activities was evaluated. Wastewaters (influent and effluent) and surrounding surface waters (upstream and downstream) at six municipal WWTPs were screened using instrumental chemical analysis and in vitro reporter gene bioassay. We showed the presence of target compounds and (anti-)progestagenic activities in municipal wastewater and surface water. Nine and seven progestins were identified in influent and effluent wastewaters, respectively. Only two compounds, progesterone and medroxyprogesterone were found in surface waters. Progestagenic agonistic activities in influents were partially masked by strong anti-progestagenic activities that were detected in all influents and ranged from 2.63 to 83 ng/L of mifepristone equivalents (EQs). Progestagenic activities were detected in all effluents and ranged from 0.06 to 0.47 ng/L of reference compound ORG 2058 EQs (a synthetic progestin equivalents), thus indicating incomplete removal of progestins during wastewater treatment processing. This activity poses a continuing risk for the aquatic environment. By contrast, anti-progestagenic activities showed better removal efficiency in WWTPs compared to progestagenic agonistic activities. Anti-progestagenic activities were found in only three of six effluents and ranged from 0.26 to 2.1 ng/L mifepristone EQs. We explained most of the progestagenic activity in municipal WWTP effluents by the presence of synthetic progestins and progesterone, which contributed 65-96% of such activity in samples where no antagonistic activity was found. The progestins medroxyprogesterone acetate, megestrol acetate and progesterone contributed most to the progestagenic activity detected in municipal effluents. Anti-progestagenic activities were found in some municipal effluents, but no causative agents were revealed because two analysed selective progesterone receptor modulators (SPRMs) with anti-progestagenic activities, mifepristone and ulipristal acetate, were not present in the effluents.

Differentiating true androgen receptor inhibition from cytotoxicity-mediated reduction of reporter-gene transactivation in-vitro.

In vitro effect-based reporter assays are applied as biodetection tools designed to address nuclear receptor mediated-modulation. While such assays detect receptor modulating potential, cell viability needs to be addressed, preferably in the same well. Some assays circumvent this by co-transfecting a second constitutively-expressed marker gene or by multiplexing a cytotoxicity assay. Some assays, such as the CALUX®, lack this feature. The cytotoxic effects of unknown substances can confound in vitro assays, making the interpretation of results difficult and uncertain, particularly when assessing antagonistic activity. It's necessary to determine whether the cause of the reporter signal decrease is an antagonistic effect or a non-specific cytotoxic effect. To remedy this, we assessed the suitability of multiplexing a cell viability assay within the CALUX® transcriptional activation test for anti-androgenicity. Tests of both well-characterized anti-androgens and cytotoxic compounds demonstrated the suitability of this approach for discerning between the molecular mechanisms of action without altering the nuclear receptor assay; though some compounds were both cytotoxic and anti-androgenic. The optimized multiplexed assay was then applied to an uncharacterized set of polycyclic aromatic compounds. These results better characterized the mode of action and the classification of effects. Overall, the multiplexed protocol added value to CALUX test performance.

Incorporation of a metabolizing system in biodetection assays for endocrine active substances.

The use of in vitro assays is important for the biodetection of endocrine active substances (EAS), reducing and replacing the in vivo studies required for regulatory assessment. However, this approach often fails to take into account the role of biotransformation on the activity of the test substances. A method incorporating an S9 metabolic system into the CALUX-reporter gene assays for estrogen receptor α- and anti-androgen receptor -mediated activities has been developed. Methoxychlor, which is known to exhibit increased estrogenic and anti-androgenic activities after biotransformation, was used to set up the method in ERa and anti-AR CALUX. For the anti-androgenic assay, stanozolol was used as a competing agonist not metabolized by S9. The method was first applied in both agonist and antagonist modes to methoxychlor and bisphenol A, as positive and negative controls, respectively. Then, benzo(a)pyrene and flutamide were also tested for their potential of bioactivation. Co-treatment with S9 successfully increased the ERα agonist and AR antagonist potency of methoxychlor; no change was observed for bisphenol A. Incubation with S9 also enhanced the anti-androgenic activity of flutamide. Interestingly, the metabolism of benzo(a)pyrene by the S9 resulted in an increased estrogen receptor-mediated transcriptional activation; any increase in the potency was only minor. It is likely that both enzyme kinetics and metabolite stability have influenced these effects, which would affect the composition of the final metabolite mixture. Together these results demonstrate the relevance of including biotransformation in in vitro bioassays for the detection of EAS.

In vitro observations and in silico predictions of xenoestrogen mixture effects in T47D-based receptor transactivation and proliferation assays.

Within endocrine disruptor research, evaluation and interpretation of mixture effects and the predictive value for downstream responses still warrant more in-depth investigations. We used an estrogen receptor (ER)-mediated reporter gene assay (ER-CALUX®) and a cell proliferation assay (WST-1 assay), both based on the T47D breast cancer cell line, to test mixtures of heterogeneous xenoestrogens. Observed concentration-response curves were compared to those predicted by the concepts of concentration addition (CA), generalized concentration addition (GCA), and a novel full logistic model (FLM). CA performed better regarding mixture potency (EC50 values), whereas GCA was superior in predicting mixture efficacy (maximal response). In comparison, FLM proved to be highly suitable for in silico mixture effect prediction, combining advantages of both CA and GCA. The inter-assay comparison revealed that ER activation is not necessarily predictive for induction of cell proliferation. The results support the use of models like CA, GCA, or FLM in mixture effect evaluation. However, we conclude that reliable estimations regarding the disruptive potential of mixtures of endocrine active substances require an integrative approach considering more than one assay/endpoint to avoid misinterpretations. The formazan-based WST-1 proliferation assay might be a possible alternative to commonly used proliferation assays in endocrine disrupter research.

Pure non-dioxin-like PCB congeners suppress induction of AhR-dependent endpoints in rat liver cells.

The relative potencies of non-ortho-substituted coplanar polychlorinated biphenyl (PCB) congeners to activate the aryl hydrocarbon receptor (AhR) and to cause the AhR-dependent toxic events are essential for their risk assessment. Since some studies suggested that abundant non-dioxin-like PCB congeners (NDL-PCBs) may alter the AhR activation by PCB mixtures and possibly cause non-additive effects, we evaluated potential suppressive effects of NDL-PCBs on AhR activation, using a series of 24 highly purified NDL-PCBs. We investigated their impact on the model AhR agonist-induced luciferase reporter gene expression in rat hepatoma cells and on induction of CYP1A1/1B1 mRNAs and deregulation of AhR-dependent cell proliferation in rat liver epithelial cells. PCBs 128, 138, and 170 significantly suppressed AhR activation (with IC50 values from 1.4 to 5.6 µM), followed by PCBs 28, 47, 52, and 180; additionally, PCBs 122, 153, and 168 showed low but still significant potency to reduce luciferase activity. Detection of CYP1A1 mRNA levels in liver epithelial cells largely confirmed these results for the most abundant NDL-PCBs, whereas the other AhR-dependent events (CYP1B1 mRNA expression, induction of cell proliferation in confluent cells) were less sensitive to NDL-PCBs, thus indicating a more complex regulation of these endpoints. The present data suggest that some NDL-PCBs could modulate overall dioxin-like effects in complex mixtures.

How much do PCB toxic equivalents account for PHAH toxicity in predatory birds?

Various diffuse polyhalogenated aromatic hydrocarbons (PHAHs) exert common toxicity through the aryl hydrocarbon receptor (AhR). Apex predators spatially and temporally integrate diffuse contamination and simultaneous exposure can cause additive toxicity. We investigated the extent to which PCBs, still amongst the most prevalent PHAHs accumulated by predators, accounted for total PHAH toxicity in raptors and fish eating birds from Britain. We analysed egg or liver extracts from six species and compared chemically determined ΣPCB-TEQs concentrations with total AhR-mediated toxicity determined using the chemical-activated luciferase gene expression bioassay (CALUX-TEQ). Dioxin-like PCB profiles in eggs and livers were dominated by congeners 118, 105 and 167. ΣPCB-TEQ and CALUX-TEQ concentrations were positively associated but not in a 1:1 relationship. ΣPCB-TEQ were broadly similar to CALUX-TEQ concentrations in eggs and livers with CALUX-TEQ concentrations >50-80 and 160-320 pg g(-1) lipid respectively, but were lower than CALUX-TEQ concentrations in less contaminated samples.