Impact of sample acidification and extract storage on hormone receptor-mediated and oxidative stress activities in wastewater.

An underemphasized aspect of sampling strategies in effect-based in vitro testing is to determine suitable collection and preparation techniques. In the current study, the impact of sample acidification on bioactivities was assessed using in vitro bioassays for hormone receptor-mediated effects (estrogen receptor [ER] and androgen receptor [AR]) and the oxidative stress response (Nrf2 activity). Sampling was conducted at a recently upgraded Swedish wastewater treatment plant. Future plans for the treated wastewater include reuse for irrigation or as a potential drinking water source. In the AR and Nrf2 assays, acidification decreased bioactivities in the wastewater influent sample extracts, whereas acidification increased bioactivities following further treatment (disc filtration). In the ER assay, acidification had no impact on the observed bioactivities in the sample extracts. A secondary objective of the study was to assess the stability of the sample extracts over time. Lower activities were detected in the ER and AR assays in all extracts after storage for approximately 1 year. Nrf2 activities did not decrease over time, but rather increased in some of the acidified sample extracts. Overall, the findings suggest that sampling strategies involving acidification may need to be tailored depending on the selected bioassay(s) and the type of wastewater treatments being assessed.

Development, scrutiny, and modulation of transient reporter gene assays of the xenobiotic metabolism pathway in zebrafish hepatocytes.

The "toxicology in the twenty-first century" paradigm shift demands the development of alternative in vitro test systems. Especially in the field of ecotoxicology, coverage of aquatic species-specific assays is relatively scarce. Transient reporter gene assays could be a quick, economical, and reliable bridging technology. However, the user should be aware of potential pitfalls that are influenced by reporter vector geometry. Here, we report the development of an AhR-responsive transient reporter-gene assay in the permanent zebrafish hepatocytes cell line (ZFL). Additionally, we disclose how viral, constitutive promoters within reporter-gene assay cassettes induce squelching of the primary signal. To counter this, we designed a novel normalization vector, bearing an endogenous zebrafish-derived genomic promoter (zfEF1aPro), which rescues the squelching-delimited system, thus, giving new insights into the modulation of transient reporter systems under xenobiotic stress. Finally, we uncovered how the ubiquitously used ligand BNF promiscuously activates multiple toxicity pathways of the xenobiotic metabolism and cellular stress response in an orchestral manner, presumably leading to a concentration-related inhibition of the AhR/ARNT/XRE-toxicity pathway and non-monotonous concentration-response curves. We named such a multi-level inhibitory mechanism that might mask effects as "maisonette squelching." A transient reporter gene assay in zebrafish cell lines utilizing endogenous regulatory gene elements shows increased in vitro toxicity testing performance. Synthetic and constitutive promotors interfere with signal transduction ("squelching") and might increase cellular stress (cytotoxicity). The squelching phenomenon might occur on multiple levels (toxicity pathway crosstalk and normalization vector), leading to a complete silencing of the reporter signal.

An in vitro-based hazard assessment of liquid smoke food flavourings.

Liquid smoke products are widely used as a food additive to create a desired smoke flavour. These products may contain hazardous chemicals generated during the wood-burning process. However, the toxic effects of these types of hazardous chemicals constituting in the commercially available products are largely unknown. Therefore, a test battery of cell-based in vitro methods, covering different modes of actions of high relevance to human health, was applied to study liquid smoke products. Ten liquid smoke flavourings were tested as non-extracted and extracted. To assess the potential drivers of toxicity, we used two different solvents. The battery of in vitro methods covered estrogenicity, androgenicity, oxidative stress, aryl hydrocarbon receptor activity and genotoxicity. The non-extracted samples were tested at concentrations 0.002 to 1 µL liquid smoke flavouring/mL culture medium, while extracted samples were tested from 0.003 to 200 µL/mL. Genotoxicity was observed for nearly all non-extracted and all hexane-extracted samples, in which the former had higher potency. No genotoxicity was observed for ethyl acetate-extracted samples. Oxidative stress was activated by almost all extracted and non-extracted samples, while approximately half of the samples had aryl hydrocarbon receptor and estrogen receptor activities. This study used effect-based methods to evaluate the complex mixtures of liquid smoke flavourings. The increased bioactivities seen upon extractions indicate that non-polar chemicals are driving the genotoxicity, while polar substances are increasing oxidative stress and cytotoxic responses. The differences in responses indicate that non-extracted products contain chemicals that are able to antagonize toxic effects, and upon extraction, the protective substances are lost.

Modeling Bioavailable Concentrations in Zebrafish Cell Lines and Embryos Increases the Correlation of Toxicity Potencies across Test Systems.

Linking cellular toxicity to low-tier animal toxicity and beyond is crucial within the adverse outcome pathway concept and the 3R framework. This study aimed to determine and compare the bioavailable effect concentrations in zebrafish cell lines and embryos. Acute, short-term toxicity (48 h) of eight veterinary pharmaceuticals was measured in two zebrafish cell lines (hepatocytes, fibroblasts) and zebrafish embryos. Seven endpoints of cytotoxicity were recorded. The fish embryo acute toxicity test was modified by adding sublethal endpoints. Chemical distribution modeling (mass balance) was applied to compute the bioavailable compound concentrations in cells (Cfree) and embryos (Cint;aq) based on nominal effect concentrations (Cnom). Effect concentration ratios were calculated (cell effects/embryo effects). A low correlation was observed between cytotoxicity and embryo toxicity when nominal concentrations were used. Modeled bioavailable effect concentrations strongly increased correlations and placed regression lines close to the line of unity and axis origin. Cytotoxicity endpoints showed differences in sensitivity and predictability. The hepatocyte cell line depicted closer proximity to the embryo data. Conclusively, the high positive correlation between the cell- and embryo-based test systems emphasizes the appropriate modulation of toxicity when linked to bioavailable concentrations. Furthermore, it highlights the potential of fish cell lines to be utilized in integrated testing strategies.

Potentials and pitfalls of transient in vitro reporter bioassays: interference by vector geometry and cytotoxicity in recombinant zebrafish cell lines.

The water framework directive re-evaluation proposes the integration of effect-based tools, increasing the need for alternative methods. Especially within aquatic toxicology, coverage of specific toxicity pathways is scarce, and most applications are based on mammalian or bacterial models, not reflecting realistic exposure scenarios. The use of transient reporter gene assays in cells from organisms of interest could be a quick and inexpensive solution. However, interference with cellular homeostasis may impact the system beyond the function of the manipulated gene and thus lead to non-specific results. We describe how varying vector geometry and different regulatory gene elements on plasmids used for transfection in zebrafish hepatocytes and embryonic fibroblasts may lead up to a tenfold difference in potency. Cells were transiently co-transfected with an Nrf2-responsive Firefly luciferase reporter plasmid and eight different Renilla luciferase normalization plasmids. Transfected cells were exposed to two different regimes (0.1-100 µM and 7.8-250 µM) of the oxidative stress-inducing compounds, sulforaphane, tertbutylhydroquinone, and metazachlor. Nrf2 activity was measured in dual-luciferase assays. In parallel, cytotoxicity was assessed for different endpoints (energy metabolism, protein amount, membrane stability, and cell proliferation) in non-transfected cells and cells co-transfected with constructs of increasing size, to be used for normalization. Transfected cells were more susceptible to cytotoxicity in a vector size-dependent manner. Conclusively, we report that vector geometries (size, backbones, gene-regulatory units), cell line (tissue origin), applied transfection methods, and signal normalization may alter the sensitivity of reporter bioassays in a synergistic manner. Further, we propose that thorough bioassay design is needed to ensure reliability and regulatory acceptance.

Relationship between peroxisome proliferator-activated receptor alpha activity and cellular concentration of 14 perfluoroalkyl substances in HepG2 cells.

Peroxisome proliferator-activated receptor alpha (PPARα) is a molecular target for perfluoroalkyl substances (PFASs). Little is known about the cellular uptake of PFASs and how it affects the PPARα activity. We investigated the relationship between PPARα activity and cellular concentration in HepG2 cells of 14 PFASs, including perfluoroalkyl carboxylates (PFCAs), perfluoroalkyl sulfonates and perfluorooctane sulfonamide (FOSA). Cellular concentrations were determined by high-performance liquid chromatography-tandem mass spectrometry and PPARα activity was determined in transiently transfected cells by reporter gene assay. Cellular uptake of the PFASs was low (0.04-4.1%) with absolute cellular concentrations in the range 4-2500 ng mg-1 protein. Cellular concentration of PFCAs increased with perfluorocarbon chain length up to perfluorododecanoate. PPARα activity of PFCAs increased with chain length up to perfluorooctanoate. The maximum induction of PPARα activity was similar for short-chain (perfluorobutanoate and perfluoropentanoate) and long-chain PFCAs (perfluorododecanoate and perfluorotetradecanoate) (approximately twofold). However, PPARα activities were induced at lower cellular concentrations for the short-chain homologs compared to the long-chain homologs. Perfluorohexanoate, perfluoroheptanoate, perfluorooctanoate, perfluorononanoate (PFNA) and perfluorodecanoate induced PPARα activities >2.5-fold compared to controls. The concentration-response relationships were positive for all the tested compounds, except perfluorooctane sulfonate PFOS and FOSA, and were compound-specific, as demonstrated by differences in the estimated slopes. The relationships were steeper for PFCAs with chain lengths up to and including PFNA than for the other studied PFASs. To our knowledge, this is the first report establishing relationships between PPARα activity and cellular concentration of a broad range of PFASs.

Vitamin D analog EB1089 inhibits aromatase expression by dissociation of comodulator WSTF from the CYP19A1 promoter-a new regulatory pathway for aromatase.

The enzyme aromatase, encoded by the CYP19A1 gene, catalyzes the production of estrogens and inhibition of aromatase has therefore become one of the key strategies in breast cancer treatment. We have studied the effects of the vitamin D analog EB1089 on aromatase gene expression and enzyme activity in breast cancer cells. We found that EB1089 was able to decrease the gene expression and enzyme activity as well as inhibit aromatase-dependent cell growth. Furthermore, a low dose of EB1089 combined with low doses of clinically used aromatase inhibitors such as anastrozole, letrozole and exemestane were able to effectively inhibit aromatase-dependent growth of breast cancer cells. The molecular mechanism for this effect of EB1089 on the aromatase gene expression was investigated and we found that it is mediated by the vitamin D receptor (VDR), vitamin D receptor interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF). ChIP and Re-ChIP assays revealed that EB1089 mediates dissociation of WSTF from the CYP19A1 promoter and thereby decreases the gene expression. Regulation of aromatase via WSTF has not been reported previously. Furthermore, gene silencing of WSTF results in decreased gene expression of CYP19A1 and aromatase activity, showing that WSTF is an interesting drug target for development of new anti-cancer drugs. In summary, we report that the vitamin D analog EB1089 is able to decrease the gene expression and enzyme activity of aromatase via a novel regulatory pathway for aromatase and suggest that EB1089 may be a new treatment option for estrogen dependent breast cancer.

Estrogenic, androgenic, and genotoxic activities of zearalenone and deoxynivalenol in in vitro bioassays including exogenous metabolic activation.

Zearalenone (ZEN) and deoxynivalenol (DON) and their derivatives are well-known mycotoxins, which can occur not only in crops but also in water bodies, including drinking water sources. In vitro bioassays can be used to detect biological effects of hazardous compounds in water. To this, when studying biological effects and toxicity in vitro, metabolism is important to consider. In this study, ZEN, α-zearalenol (α-ZEL), DON, 3-acetyl DON, and 15-acetyl DON were evaluated in vitro for hormone receptor-mediated effects (estrogen receptor [ER] and androgen receptor [AR]) and genotoxicity (micronucleus assay) in the presence of an exogenous metabolic activation system (MAS). The ER bioassay proved to be a highly sensitive method to detect low concentrations of the ZEN compounds (EC10 values of 31.4 pM for ZEN, 3.59 pM for α-ZEL) in aqueous solutions. In the presence of the MAS, reduced estrogenic effects were observed for both ZEN compounds (EC10 values of 6.47 × 103 pM for ZEN, 1.55 × 102 pM for α-ZEL). Of the DON compounds, only 3-acetyl DON was estrogenic (EC10 of 0.31 µM), and the effect was removed in the presence of the MAS. Anti-androgenic effects of the ZEN compounds and androgenic effects of the DON compounds were detected in the micromolar range. No induction of genotoxicity was detected for ZEN or DON in the presence of the MAS. Our study highlighted that inclusion of exogenous MAS is a useful tool to detect biological effects of metabolites in in vitro bioassays.

Integrating different tools and technologies to advance drinking water quality exposure assessments.

Contaminants in drinking water are a major contributor to the human exposome and adverse health effects. Assessing drinking water exposure accurately in health studies is challenging, as several of the following study design domains should be addressed as adequately as possible. In this paper, we identify the domains Time, Space, Data Quality, Data Accessibility, economic considerations of Study Size, and Complex Mixtures. We present case studies for three approaches or technologies that address these domains differently in the context of exposure assessment of drinking water quality: regulated contaminants in monitoring databases, high-resolution mass spectrometry (HRMS)-based wide-scope chemical analysis, and effect-based bioassay methods. While none of these approaches address all the domains sufficiently, together they have the potential to carry out exposure assessments that would complement each other and could advance the state-of-science towards more accurate risk analysis. The aim of our study is to give researchers investigating health effects of drinking water quality the impetus to consider how their exposure assessments relate to the above-mentioned domains and whether it would be worthwhile to integrate the advanced technologies presented into planned risk analyses. We highly suggest this three-pronged approach should be further evaluated in health risk analyses, especially epidemiological studies concerning contaminants in drinking water. The state of the knowledge regarding potential benefits of these technologies, especially when applied in tandem, provides more than sufficient evidence to support future research to determine the implications of combining the approaches described in our case studies in terms of protection of public health.

Effects of CYP7B1-related steroids on androgen receptor activation in different cell lines.

The widely expressed steroid hydroxylase CYP7B1 is involved in metabolism of a number of steroids reported to influence estrogen and androgen signaling. Several studies by us and other investigators have linked this enzyme to effects on estrogen receptor activation. In a previous report we examined the effect of CYP7B1-mediated hormone metabolism for estrogen-mediated response in kidney-derived HEK293 cells. In the current study we used an androgen response element (ARE) reporter system to examine androgen-dependent response of some CYP7B1 substrates and CYP7B1-formed metabolites in several cell lines derived from different tissues. The results indicate significantly lower androgen receptor activation by CYP7B1-formed steroid metabolites than by the corresponding steroid substrates, suggesting that CYP7B1-mediated catalysis may decrease some androgenic responses. Thus, CYP7B1-dependent metabolism may be of importance not only for estrogenic signaling but also for androgenic. This finding, that CYP7B1 activity may be a regulator of androgenic signaling by converting AR ligands into less active metabolites, is also supported by real-time RT-PCR experiment where a CYP7B1 substrate, but not the corresponding product, was able to stimulate known androgen-sensitive genes. Furthermore, our data indicate that the effects of some steroids on hormone response element reporter systems are cell line-specific. For instance, despite transfection of the same reporter systems, 5-androstene-3ß,17ß-diol strongly activates an androgen-dependent response element in prostate cancer cells whereas it elicits only ER-dependent responses in kidney HEK293 cells. Potential roles of cell-specific metabolism or comodulator expression for the observed differences are discussed.

Vitamin D-mediated regulation of CYP21A2 transcription - a novel mechanism for vitamin D action.

BACKGROUND: 1α,25-Dihydroxyvitamin D(3) has recently been reported to decrease expression and activity of CYP21A2. In this paper, we have studied the mechanisms for the 1α,25-dihydroxyvitamin D(3)-mediated effect on CYP21A2 transcriptional rate. METHODS: We have studied the effects of 1α,25-dihydroxyvitamin D(3) using luciferase reporter constructs containing different lengths of the CYP21A2 promoter. These constructs were transfected into cell lines derived from human and mouse adrenal cortex. The mechanism for the effects of vitamin D on the CYP21A2 promoter was studied using chromatin immunoprecipitation assay, mutagenesis and gene silencing by siRNA. RESULTS: 1α,25-Dihydroxyvitamin D(3) was found to alter the promoter activity via a VDR-mediated mechanism, including the comodulators VDR interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF). The involvement of comodulator VDIR was confirmed by gene silencing. We identified a vitamin D response element in the CYP21A2 promoter. Interaction between this novel response element and VDR, WSTF and VDIR was shown by chromatin immunoprecipitation assay. When this sequence was deleted, the effect of 1α,25-dihydroxyvitamin D(3) was abolished, indicating that this sequence in the CYP21A2 promoter functions as a vitamin D response element. Interestingly, an altered balance between nuclear receptors and comodulators reversed the suppressing effect of vitamin D to a stimulatory effect. GENERAL SIGNIFICANCE: This paper reports data important for the understanding of the mechanisms for vitamin D-mediated suppression of gene expression as well as for the vitamin D-mediated effects on CYP21A2. We report a novel mechanism for effects of 1α,25-dihydroxyvitamin D(3).

Evaluation of in vitro bioassays as a screening tool to monitor chemical hazards in cow's milk.

Studies on cow's milk have mainly focused on analyzing specific chemical groups and natural components. Therefore, in this study, we evaluated if effect-based in vitro methods could be used as a screening tool to monitor chemical hazards in milk. In total, 32 milk samples were collected from a Swedish dairy company throughout one year. These samples included conventional and organic semi-skimmed as well as raw milk. The milk samples were tested in five in vitro methods covering eight endpoints. These endpoints included cytotoxicity, endocrine disruption (estrogen/androgen induction/inhibition), aryl hydrocarbon receptor activity, oxidative stress and DNA damage. Estrogen and androgen receptor inhibition, in addition to aryl hydrocarbon receptor activity, were the most responsive endpoints, where 10 to 13 out of the 32 milk samples were bioactive. Organic and conventional milk showed no major differences. Overall, no or only low activities were observed in milk samples in the remaining in vitro assays, which is a promising result with regard to applying effect-based methods as a screening tool. Concerning the most responsive assays, more research is needed to understand the normal background variations before they can be used as a screening tool for chemical hazards in milk.

Comparison of the photocatalytic degradability of PFOA, PFOS and GenX using Fe-zeolite in water.

Knowledge on the photocatalytic degradability of the emerging poly- and perfluorinated alkyl substances (PFAS) in water, specifically GenX, is limited. GenX has been detected globally in river water and is considered potentially more toxic than legacy PFAS. In this study, we compared the photocatalytic degradability of GenX with the legacy compounds perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) using Fe-zeolite photocatalysts. After 7 h of irradiation, GenX showed lower removal (79%) and defluorination (33%) as compared to PFOA (100% removal and 69% defluorination) and PFOS (100% removal and 51% defluorination). The quasi-first-order degradation rate of GenX (1.5 h1) was 12 and 1.2 times lower than PFOA (18.4 h-1) and PFOS (1.8 h-1), respectively. Additionally, PFOA's defluorination rate (0.9 h-1) was approximately 2.6 and 9 times higher than GenX (0.35 h-1) and PFOS (0.1 h-1), respectively. These outcomes correlate with GenX's lower hydrophobicity, leading to reduced adsorption (40%) compared to PFOA (99%) and PFOS (87%). Based on identified transformation products, we proposed a GenX degradation pathway, resulting in ultra-short-chain PFASs with a chain length of 2 and 3 carbon atoms, while PFOA and PFOS degraded stepwise, losing 1 carbon-fluorine bond at a time, leading to gradually shorter chain lengths (from 7 to 2 carbon atoms). In conclusion, GenX is more challenging to remove and degrade due to its lower adsorption on the photocatalyst, potential steric hindrance, and higher production of persistent ultra-short-chain transformation products through photocatalysis.

1α,25-Dihydroxyvitamin D3 exerts tissue-specific effects on estrogen and androgen metabolism.

It is well-known that 1α,25-dihydroxyvitamin D(3) and analogs exert anti-proliferative and pro-differentiating effects and these compounds have therefore been proposed to be of potential use as anti-cancer agents. Due to its effects on aromatase gene expression and enzyme activity, 1α,25-dihydroxyvitamin D(3) has been proposed as an interesting substance in breast cancer treatment and prevention. In the present study, we have examined the effects of 1α,25-dihydroxyvitamin D(3) on estrogen and androgen metabolism in adrenocortical NCI-H295R cells, breast cancer MCF-7 cells and prostate cancer LNCaP cells. The NCI-H295R cell line has been proposed as a screening tool to study endocrine disruptors. We therefore studied whether this cell line reacted to 1α,25-dihydroxyvitamin D(3) treatment in the same way as cells from important endocrine target tissues. 1α,25-Dihydroxyvitamin D(3) exerted cell line-specific effects on estrogen and androgen metabolism. In breast cancer MCF-7 cells, aromatase gene expression and estradiol production were decreased, while production of androgens was markedly increased. In NCI-H295R cells, 1α,25-dihydroxyvitamin D(3) stimulated aromatase expression and decreased dihydrotestosterone production. In prostate cancer LNCaP cells, aromatase expression increased after the same treatment, as did production of testosterone and dihydrotestosterone. In summary, our data show that 1α,25-dihydroxyvitamin D(3) exerts tissue-specific effects on estrogen and androgen production and metabolism. This is important knowledge about 1α,25-dihydroxyvitamin D(3) as an interesting substance for further research in the field of breast cancer prevention and treatment. Furthermore, the observed cell line-specific effects are of importance in the discussion about NCI-H295R cells as a model for effects on estrogen and androgen metabolism.

Artificial infiltration in drinking water production: Addressing chemical hazards using effect-based methods.

Artificial infiltration is an established managed aquifer recharge method that is commonly incorporated into drinking water processes. However, groundwater sourced from this type of purification method is prone to contamination with chemical hazards. Such an instance was previously shown at a Swedish DWTP where the river water was contaminated by hazardous chemicals during artificial infiltration. Further, there remains a paucity of research studying the quality of drinking water following this type of treatment from an effect-based bioanalytical perspective. In the current study, an effect-based assessment for chemical hazards was conducted for a Swedish drinking water system comprised of two DWTPs fed artificially-infiltrated river water. In this system, artificial infiltration of the river water takes approximately six to eight months. A sampling event was conducted in the autumn season and the samples were enriched by solid phase extraction. A panel of cell-based reporter gene assays representing several toxicity pathways was selected: oxidative stress response (Nrf2 activity), aryl hydrocarbon receptor (AhR) activation, and hormone receptor-mediated effects (estrogen receptor [ER], androgen receptor [AR]). AhR and ER bioactivities were detected in samples collected from the river intake and in the open-air infiltration basins prior to artificial infiltration. However, the AhR activity decreased and ER activity was effectively removed following artificial infiltration. In the Nrf2 and AR assays, no bioactivities above cut-off levels were detected in any samples collected along the entire treatment process of the drinking water production from source to tap. Using a suite of bioassays, the current study highlighted the effectiveness of artificial infiltration in reducing bioactive compounds in this raw river water. Although artificial infiltration is a common purification method in drinking water production, the limited number of effect-based studies evaluating the effectiveness of this method emphasizes the need for further research to better understand the risks and benefits of this water treatment process.

Profile occurrences and in vitro effects of toxic organic pollutants in metal shredding facilities in Wallonia (Belgium).

End-of-life vehicles and e-waste contain several hazardous substances that can contaminate the environment during treatment processes. Occurrences and adverse effects of toxic organic pollutants emitted from 3 shredder plants located in Wallonia, Belgium, were investigated by chemical and biological analyses of fluff, dust, and scrubbing sludge sampled in 2019. Site 1 showed the highest concentrations of chlorinated compounds in sludge with 7.5 ng/g polychlorinated dibenzo-dioxins/furans and 84.5 µg/g estimated total polychlorinated biphenyls, while site 3 led the brominated flame retardant levels in dust (53.4 µg/g). The level of polycyclic aromatic hydrocarbons was highest in the sludge samples, 78 and 71 µg/g for sites 2 and 3, respectively. The samples induced significant dioxin-like activities in murine and human cells at concentrations of around 0.01-0.1 and 0.5-1 ng (sample) per ml (medium), respectively, with the efficacy similar to 2,3,7,8-tetrachlorodibenzodioxin and EC50 values of around 1 and 10 ng/ml. The samples also displayed high estrogenic activities, already at 1 ng/ml, and several induced a response as efficient as 17ß-estradiol, albeit a low androgenic activity. Shredder workers were estimated to be highly exposed to dioxin-like compounds through dust ingestion and dermal absorption, which is of concern.

Are preserved coastal water bodies in Spanish Mediterranean basin impacted by human activity? Water quality evaluation using chemical and biological analyses.

The Spanish Mediterranean basin is particularly susceptible to climate change and human activities, making it vulnerable to the influence of anthropogenic contaminants. Therefore, conducting comprehensive and exhaustive water quality assessment in relevant water bodies of this basin is pivotal. In this work, surface water samples from coastal lagoons or estuaries were collected across the Spanish Mediterranean coastline and subjected to target and suspect screening of 1,585 organic micropollutants by liquid chromatography coupled to ion mobility separation and high resolution mass spectrometry. In total, 91 organic micropollutants could be confirmed and 5 were tentatively identified, with pharmaceuticals and pesticides being the most prevalent groups of chemicals. Chemical analysis data was compared with data on bioanalysis of those samples (recurrent aryl hydrocarbon receptor (AhR) activation, and estrogenic receptor (ER) inhibition in wetland samples affected by wastewater streams). The number of identified organic contaminants containing aromatic rings could explain the AhR activation observed. For the ER antagonistic effects, predictions on estrogenic inhibition potency for the detected compounds were used to explain the activities observed. The integration of chemical analysis with bioanalytical observations allowed a comprehensive overview of the quality of the water bodies under study.

Effects of CYP7B1-mediated catalysis on estrogen receptor activation.

Most of the many biological effects of estrogens are mediated via the estrogen receptors ERalpha and beta. The current study examines the role of CYP7B1-mediated catalysis for activation of ER. Several reports suggest that CYP7B1 may be important for hormonal action but previously published studies are contradictory concerning the manner in which CYP7B1 affects ERbeta-mediated response. In the current study, we examined effects of several CYP7B1-related steroids on ER activation, using an estrogen response element (ERE) reporter system. Our studies showed significant stimulation of ER by 5-androstene-3beta,17beta-diol (Aene-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-Adiol). In contrast, the CYP7B1-formed metabolites from these steroids did not activate the receptor, indicating that CYP7B1-mediated metabolism abolishes the ER-stimulating effect of these compounds. The mRNA level of HEM45, a gene known to be stimulated by estrogens, was strongly up-regulated by Aene-diol but not by its CYP7B1-formed metabolite, further supporting this concept. We did not observe stimulation by dehydroepiandrosterone (DHEA) or 7alpha-hydroxy-DHEA, previously suggested to affect ERbeta-mediated response. As part of these studies we examined metabolism of Aene-diol in pig liver which is high in CYP7B1 content. These experiments indicate that CYP7B1-mediated metabolism of Aene-diol is of a similar rate as the metabolism of the well-known CYP7B1 substrates DHEA and 3beta-Adiol. CYP7B1-mediated metabolism of 3beta-Adiol has been proposed to influence ERbeta-mediated growth suppression. Our results indicate that Aene-diol also might be important for ER-related pathways. Our data indicate that low concentrations of Aene-diol can trigger ER-mediated response equally well for both ERalpha and beta and that CYP7B1-mediated conversion of Aene-diol into a 7alpha-hydroxymetabolite will result in loss of action.

1alpha,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells.

The current study presents data indicating that 1alpha,25-dihydroxyvitamin D(3) affects the production of hormones and expression of crucial steroidogenic enzymes in the human adrenocortical cell line NCI-H295R. This cell line is widely used as a model for adrenal steroidogenesis. Treatment of the cells with 1alpha,25-dihydroxyvitamin D(3) suppressed the levels of corticosterone, aldosterone, DHEA, DHEA-sulfate and androstenedione in the culture medium. In order to study the mechanisms behind this suppression of hormone production, we investigated the effects of 1alpha,25-dihydroxyvitamin D(3) on important genes and enzymes controlling the biosynthesis of adrenal hormones. The mRNA levels were decreased for CYP21A2 while they were increased for CYP11A1 and CYP17A1. No significant changes were observed in mRNA for CYP11B1, CYP11B2 or 3beta-hydroxysteroid dehydrogenase (3betaHSD). In similarity with the effects on mRNA levels, also the endogenous enzyme activity of CYP21A2 decreased after treatment with 1alpha,25-dihydroxyvitamin D(3). Interestingly, the two CYP17A1-mediated activities were influenced reciprocally - the 17alpha-hydroxylase activity increased whereas the 17,20-lyase activity decreased. The current data indicate that the 1alpha,25-dihydroxyvitamin D(3)-mediated decrease in corticosterone and androgen production is due to suppression of the 21-hydroxylase activity by CYP21A2 and the 17,20-lyase activity by CYP17A1, respectively. In conclusion, the current study reports novel findings on 1alpha,25-dihydroxyvitamin D(3)-mediated effects on hormone production and regulation of genes and enzymes involved in steroidogenesis in the adrenocortical NCI-H295R cell line, a model for human adrenal cortex.

Assessment of source and treated water quality in seven drinking water treatment plants by in vitro bioassays - Oxidative stress and antiandrogenic effects after artificial infiltration.

Drinking water quality and treatment efficacy was investigated in seven drinking water treatment plants (DWTPs), using water from the river Göta Älv, which also is a recipient of treated sewage water. A panel of cell-based bioassays was used, including measurements of receptor activity of aryl hydrocarbon (AhR), estrogen (ER), androgen (AR), peroxisome proliferator-activated receptor alpha (PPARα) as well as induction of oxidative stress (Nrf2) and micronuclei formation. Grab water samples were concentrated by solid phase extraction (SPE) and water samples were analyzed at a relative enrichment factor of 50. High activities of AhR, ER and AR antagonism were present in WWTP outlets along the river. Inlet water from the river exhibited AhR and AR antagonistic activities. AhR activity was removed by DWTPs using granulated activated carbon (GAC) and artificial infiltration. AR antagonistic activity was removed by the treatment plants, except the artificial infiltration plant, which actually increased the activity. Furthermore, treated drinking water from the DWTP using artificial infiltration exhibited high Nrf2 activity, which was not found in any of the other water samples. Nrf2 activity was found in water from eight of the 13 abstraction wells, collecting water from the artificial infiltration. No genotoxic activity was detected at non-cytotoxic concentrations. No Nrf2 or AR antagonistic activities were detected in the inlet or outlet water after the DWTP had been replaced by a new plant, using membrane ultrafiltration and GAC. Neither target chemical analysis, nor chemical analysis according to the drinking water regulation, detected any presence of chemicals, which could be responsible of the prominent effects on oxidative stress and AR antagonistic activity in the drinking water samples. Thus, bioanalysis is a useful tool for detection of unknown hazards in drinking water and for assessment of drinking water treatments.