Altered Transcriptome Response in PBMCs of Czech Adults Linked to Multiple PFAS Exposure: B Cell Development as a Target of PFAS Immunotoxicity.

While the immunomodulation effects of per- and polyfluoroalkyl substances (PFASs) are described on the level of clinical signs in epidemiological studies (e.g., suppressed antibody response after vaccination), the underlying mechanism has still not been fully elucidated. To reveal mechanisms of PFAS exposure on immunity, we investigated the genome-wide transcriptomic changes of peripheral blood mononuclear cells (PBMCs) responding to PFAS exposure (specifically, exposure to PFPA, PFOA, PFNA, PFDA, PFUnDA, PFHxS, and PFOS). Blood samples and the chemical load in the blood were analyzed under the cross-sectional CELSPAC: Young Adults study. The overall aim of the study was to identify sensitive gene sets and cellular pathways conserved for multiple PFAS chemicals. Transcriptome networks related to adaptive immunity were perturbed by multiple PFAS exposure (i.e., blood levels of at least four PFASs). Specifically, processes tightly connected with late B cell development, such as B cell receptor signaling, germinal center reactions, and plasma cell development, were shown to be affected. Our comprehensive transcriptome analysis identified the disruption of B cell development, specifically the impact on the maturation of antibody-secreting cells, as a potential mechanism underlying PFAS immunotoxicity.

Anti-androgenic activity of novel flame retardants in mixtures: Newly identified contribution from tris(2,3-dibromopropyl) isocyanurate (TDBP-TAZTO).

In recent decades, male infertility has been on the rise, largely attributed to exposure to chemicals with endocrine-disrupting properties. The adverse effects of disrupting androgen actions on the development and reproductive health of children and adolescents have been extensively studied. Flame retardants (FRs), used in consumer products to delay flammability, have been identified as antagonists of the androgen receptor (AR), potentially leading to adverse outcomes in male reproductive health later in life. This study examined the interaction of eight novel FRs with the AR, employing an in vitro AR-dependent luciferase reporter gene assay utilizing MDA-kb2 cells. The investigation revealed the anti-androgenic activity of tris(2,3-dibromopropyl) isocyanurate (TDBP-TAZTO), a frequently detected FR in the environment. Furthermore, TDBP-TAZTO contributed to anti-androgenic activity when combined with six other anti-androgenic FRs. The mixture effects were predicted by three commonly employed models: concentration addition (CA), generalized CA, and independent action, with the CA model showcasing the highest accuracy. This suggests that all FRs act through a similar mechanism, as further confirmed by in silico molecular docking, indicating limited synergy or antagonism. Importantly, in the mixtures, each FR contributed to the induction of anti-androgenic effects at concentrations below their individual effective concentrations in single exposures. This raises concern for public health, especially considering the co-detection of these FRs and their potential co-occurrence with other anti-androgenic chemicals like bisphenols. Therefore, our findings, along with previous research, strongly support the incorporation of combined effects of mixtures in risk assessment to efficiently safeguard population health.

Impact of PFAS exposure on prevalence of immune-mediated diseases in adults in the Czech Republic.

BACKGROUND: Per- and polyfluoroalkyl substances (PFASs) are emerging environmental contaminants with multiple hazardous properties including immunomodulation potency. Human exposure to PFASs has been associated with various immune-mediated diseases and outcomes. This study aimed to investigate the association between PFAS exposure and immune-mediated diseases such as allergies, eczemas, and autoimmune diseases in a population of adults in the Czech Republic. METHODS: This study included 309 adults from the Central European Longitudinal Study of Parents and Children: Young Adults (CELSPAC: YA). 12 PFASs were measured in participants' serum by HPLC-MS/MS, 3 PFASs were removed from the subsequent analyses due to low detection frequency. The associations of 9 PFASs with 9 immune-mediated diseases were assessed by logistic regression. Furthermore, Bayesian kernel machine regression (BKMR) was used to estimate the effect of the PFAS mixture on immune-mediated diseases. All analyses were adjusted for sex, age, BMI, smoking, education, and family history of immune-mediated diseases. In cases of a statistically significant interaction of PFASs and sex, stratified analyses were performed for men and women. RESULTS: Perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) were negatively associated with both atopic eczema (OR per IQR increase 0.58 (95% CI 0.37-0.90) for PFOA and 0.56 (0.32-0.95) for PFOS) and contact dermatitis (0.37 (0.16-0.85) for PFOA and 0.33 (0.11-0.94) for PFOS). Perfluoroundecanoate (PFUnDA) was negatively associated with pollen, dust, and mite allergy (0.62 (0.43-0.89)). BKMR modelling showed a negative tendency in the overall effect of PFAS mixture on immune-health outcomes. Based on the stratified analysis, sex was suggested to be an effect modifier in the association of PFOS and atopic eczema. CONCLUSION: Our results contribute to the body of literature that observes the immunosuppressive effect of PFAS exposure during eczemas and allergies, both for PFASs individually and as a mixture.

Rapid extraction and analysis of oxidative stress and DNA damage biomarker 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine: Application to a study with pregnant women.

Oxidative stress is an important toxicity and genotoxicity mechanism of many chronic adverse health outcomes. This study developed a sensitive extraction method for urine matrix (based on lyophilization, without the need for pre-cleaning by solid phase extraction), coupled to LC-MS/MS analysis of the biomarker 8-hydroxy-2'-deoxyguanosine (8-OHdG). The methodology was validated in urine samples from a cohort of Spanish pregnant women collected during the first, second and third trimester of pregnancy, and urine samples collected within 24 h after delivery (n = 85). A detection and quantification limit of 0.01 and 0.05 µg/L, respectively, were established. The median 8-OHdG concentration was 2.18 µg/L (range 0.33-7.79); and the corresponding creatinine-adjusted concentrations ranged from 1.04 to 13.12 with median of 4.48 µg 8-OHdG/g creatinine. The concentrations of non-adjusted 8-OHdG significantly decreased (p < 0.05) in the 3rd trimester and post-delivery urine samples when compared to the 1st trimester levels. 8-OHdG concentrations were further studied in placenta samples matching the same urine samples (n = 26), with a median value of 1.3 ng 8-OHdG/g of tissue. Placental 8-OHdG concentrations were correlated with urinary levels of non-adjusted 8-OHdG in the 3rd trimester. Considering the small cohort size, results must be interpreted with caution, however statistical analyses revealed elevated urinary non-adjusted 8-OHdG levels in the 1st trimester of mothers that delivered boys compared to those who delivered girls (p < 0.01). Increased urinary non-adjusted 8-OHdG concentrations at the time of delivery were significantly associated with clinical records (any type of clinical record during pregnancy; p < 0.05). The novel extraction and analytical method for the assessment of 8-OHdG is applicable for sensitive analysis of multiple analytes or biomarkers in urine matrix. This method could also be applied for other matrices such as blood or tissues. Our findings show that 8-OHdG in urine of pregnant women could predict oxidative stress in placenta and can be related to characteristics such as maternal obesity, mode of delivery and newborn sex.

Implementation of effect biomarkers in human biomonitoring studies: A systematic approach synergizing toxicological and epidemiological knowledge.

Human biomonitoring (HBM) studies have highlighted widespread daily exposure to environmental chemicals. Some of these are suspected to contribute to adverse health outcomes such as reproductive, neurological, and metabolic disorders, among other developmental and chronic impairments. One of the objectives of the H2020 European Human Biomonitoring Initiative (HBM4EU) was the development of informative effect biomarkers for application in a more systematic and harmonized way in large-scale European HBM studies. The inclusion of effect biomarkers would complement exposure data with mechanistically-based information on early and late adverse effects. For this purpose, a stepwise strategy was developed to identify and implement a panel of validated effect biomarkers in European HBM studies. This work offers an overview of the complete procedure followed, from comprehensive literature search strategies, selection of criteria for effect biomarkers and their classification and prioritization, based on toxicological data and adverse outcomes, to pilot studies for their analytical, physiological, and epidemiological validation. We present the example of one study that demonstrated the mediating role of the effect biomarker status of brain-derived neurotrophic factor BDNF in the longitudinal association between infant bisphenol A (BPA) exposure and behavioral function in adolescence. A panel of effect biomarkers has been implemented in the HBM4EU Aligned Studies as main outcomes, including traditional oxidative stress, reproductive, and thyroid hormone biomarkers. Novel biomarkers of effect, such as DNA methylation status of BDNF and kisspeptin (KISS) genes were also evaluated as molecular markers of neurological and reproductive health, respectively. A panel of effect biomarkers has also been applied in HBM4EU occupational studies, such as micronucleus analysis in lymphocytes and reticulocytes, whole blood comet assay, and malondialdehyde, 8-oxo-2'-deoxyguanosine and untargeted metabolomic profile in urine, to investigate, for example, biological changes in response to hexavalent chromium Cr(VI) exposure. The use of effect biomarkers in HBM4EU has demonstrated their ability to detect early biological effects of chemical exposure and to identify subgroups that are at higher risk. The roadmap developed in HBM4EU confirms the utility of effect biomarkers, and support one of the main objectives of HBM research, which is to link exposure biomarkers to mechanistically validated effect and susceptibility biomarkers in order to better understand the public health implications of human exposure to environmental chemicals.

Replacement Flame-Retardant 2-Ethylhexyldiphenyl Phosphate (EHDPP) Disrupts Hepatic Lipidome: Evidence from Human 3D Hepatospheroid Cell Culture.

The present study aims to evaluate the effects of repeated exposure to 2-ethylhexyldiphenyl phosphate (EHDPP) on human liver cells. In vitro three-dimensional (3D) hepatospheroid cell culture was utilized to explore the potential mechanisms of EHDPP-mediated metabolic disruption through morphological, transcriptional, and biochemical assays. Lipidomics analysis was performed on the individual hepatospheroids to investigate the effects on intracellular lipid profiles, followed by hepatospheroid morphology, growth, functional parameters, and cytotoxicity evaluation. The possible mechanisms were delineated using the gene-level analysis by assessing the expression of key genes encoding for hepatic lipid metabolism. We revealed that exposure to EHDPP at 1 and 10 µM for 7 days alters the lipid profile of human 3D hepatospheroids. Dysregulation in several lipid classes, including sterol lipids (cholesterol esters), sphingolipids (dihydroceramide, hexosylceramide, ceramide, sphingomyelin), glycerolipids (triglycerides), glycerophospholipids, and fatty acyls, was noted along with alteration in genes including ACAT1, ACAT2, CYP27A1, ABCA1, GPAT2, PNPLA2, PGC1α, and Nrf2. Our study brings a novel insight into the metabolic disrupting effects of EHDPP and demonstrates the utility of hepatospheroids as an in vitro cell culture model complemented with omics technology (e.g., lipidomics) for mechanistic toxicity studies.

Application of AOPs to assist regulatory assessment of chemical risks - Case studies, needs and recommendations.

While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA.

Associations between Per- and Polyfluoroalkyl Substances (PFAS) and Cardiometabolic Biomarkers in Adults of Czechia: The Kardiovize Study.

Even though there is evidence of decreasing trends of per- and polyfluoroalkyl substances (PFAS) in Czechia, there are still major sources of PFAS pollution. Regarding the still-inconsistent results of the relationship between cardiometabolic health and PFAS, the present study sought to determine the association between PFAS levels and the presence of cardiometabolic biomarkers, including blood pressure and dysglycemia drivers in the Czech population. A cross-sectional study with 479 subjects (56.4% women, median: 53 years, range: 25-89) was conducted. Four PFAS were measured in serum: perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and perfluorooctane sulfonate (PFOS). The associations between natural log (ln)-transformed PFAS and cardiometabolic biomarkers were assessed through generalized additive models using linear regression and smoothing thin plate splines, adjusted for potential confounders. There were positive and significant (p < 0.05) associations between the ln-transformed PFOA and glucose (ß = 0.01), systolic (ß = 0.76) and diastolic blood pressure (ß = 0.65); total cholesterol (ß = 0.07) and LDL-c (ß = 0.04); and PFOS with glucose (ß = 0.03), BMI (ß = 2.26), waist circumference (ß = 7.89), systolic blood pressure (ß = 1.18), total cholesterol (ß = 0.13), and HDL-c (ß = 0.04). When significant, the correlations of PFNA and PFDA were negative. Of the four PFAS, only PFOA and PFOS showed a positive association, even in serum levels not as high as the values from the literature.

Pyrethroids and developmental neurotoxicity - A critical review of epidemiological studies and supporting mechanistic evidence.

BACKGROUND: Pyrethroid metabolites are widely detectable in urine from the general population, including pregnant women and children. Pyrethroids are neurotoxic and suggested endocrine disruptors. Exposure during vulnerable developmental time windows may have long-term impacts on neurodevelopment. OBJECTIVE: To evaluate the epidemiological evidence for neurodevelopmental effects related to prenatal and childhood pyrethroid exposure in a systematic review and to assess biological plausibility by evaluating mechanistic evidence. METHODS: We searched PubMed and Web of Science up to September 1, 2021 and included original studies published in English in which pyrethroid exposure was measured or estimated during pregnancy or childhood and associations with neurodevelopmental outcomes in the children were investigated. The Navigation Guide Systematic Review Methodology was used to evaluate the epidemiological evidence. For mechanistic evidence, we focused on relevant key events (KEs) suggested in Adverse Outcome Pathways (AOPs) using the OECD-supported AOP-wiki platform. A systematic search combining the KEs with pyrethroids, including 26 individual compounds, was performed in the ToxCast database. RESULTS: Twenty-five epidemiological studies met the inclusion criteria, 17 presented findings on prenatal exposure, 10 on childhood exposure and two on both exposure windows. The overall body of evidence was rated as "moderate quality" with "sufficient evidence" for an association between prenatal pyrethroid exposure and adverse neurodevelopment. For childhood exposure, the overall rating was "low quality" with "limited evidence" because of cross-sectional study design. Regarding mechanistic evidence, we found that pyrethroids are able to interfere with neurodevelopmental KEs included in established AOPs for adverse neurodevelopmental. The evidence was strongest for interference with thyroid hormone (TH) function. CONCLUSION: Pyrethroids are probably human developmental neurotoxicants and adverse impacts of pyrethroid exposure on neurodevelopment are likely at exposure levels occurring in the general population. Preventive measures to reduce exposure among pregnant women and children are warranted.

Treatment of cylindrospermopsin by hydroxyl and sulfate radicals: Does degradation equal detoxification?

Drinking water treatment ultimately aims to provide safe and harmless drinking water. Therefore, the suitability of a treatment process should not only be assessed based on reducing the concentration os a pollutant concentration but, more importantly, on reducing its toxicity. Hence, the main objective of this study was to answer whether the degradation of a highly toxic compound of global concern for drinking water equals its detoxification. We, therefore, investigated the treatment of cylindrospermopsin (CYN) by •OH and SO4-• produced in Fenton and Fenton-like reactions. Although SO4-• radicals removed the toxin more effectively, both radical species substantially degraded CYN. The underlying degradation mechanisms were similar for both radical species and involved hydroxylation, dehydrogenation, decarboxylation, sulfate group removal, ring cleavage, and further fragmentation. The hydroxymethyl uracil and tricyclic guanidine moieties were the primary targets. Furthermore, the residual toxicity, assessed by a 3-dimensional human in vitro liver model, was substantially reduced during the treatment by both radical species. Although the results indicated that some of the formed degradation products might still be toxic, the overall reduction of the toxicity together with the proposed degradation pathways allowed us to conclude: "Yes, degradation of CYN equals its detoxification!".

AOP-helpFinder webserver: a tool for comprehensive analysis of the literature to support adverse outcome pathways development.

MOTIVATION: Adverse outcome pathways (AOPs) are a conceptual framework developed to support the use of alternative toxicology approaches in the risk assessment. AOPs are structured linear organizations of existing knowledge illustrating causal pathways from the initial molecular perturbation triggered by various stressors, through key events (KEs) at different levels of biology, to the ultimate health or ecotoxicological adverse outcome. RESULTS: Artificial intelligence can be used to systematically explore available toxicological data that can be parsed in the scientific literature. Recently, a tool called AOP-helpFinder was developed to identify associations between stressors and KEs supporting thus documentation of AOPs. To facilitate the utilization of this advanced bioinformatics tool by the scientific and the regulatory community, a webserver was created. The proposed AOP-helpFinder webserver uses better performing version of the tool which reduces the need for manual curation of the obtained results. As an example, the server was successfully applied to explore relationships of a set of endocrine disruptors with metabolic-related events. The AOP-helpFinder webserver assists in a rapid evaluation of existing knowledge stored in the PubMed database, a global resource of scientific information, to build AOPs and Adverse Outcome Networks supporting the chemical risk assessment. AVAILABILITY AND IMPLEMENTATION: AOP-helpFinder is available at http://aop-helpfinder.u-paris-sciences.fr/index.php. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Innovative electrochemical biosensor for toxicological investigations on algae and cyanobacteria.

An electrochemical biosensor is presented that directly reflects the metabolic activity of prokaryotic and eukaryotic cells. This biosensor can be used measure the biological activity of bacteria, yeasts and mammalian cells. This makes the sensor interesting for various applications in industry and science. A possible application is bioprocess control, monitor activities from yeasts, bacteria and fungi to increase the yield. Other applications are starter culture quality studies in the food industry and cytoxicological evaluation with mammalian cells. Our latest investigations additionally indicate the applicability of the electrochemical biosensor to measure algae and cyanobacteria. In our investigations, we were able to show that it was also possible to detect photosynthetic organisms with the electrochemical measurement method, used for investigations on prokaryotic and eukaryotic organisms before. Therefore, this the present study demonstrates an alternative to using this electrochemical biosensor equipped with alga and cyanobacteria for toxicological investigations based on selected test chemicals. The results of this study show a good correlation with those from reference methods, such as the Algal Growth Inhibition Test and the Microtox Test. The advantages of the new electrochemical biosensor are easy handling and shorter measurement time by using different types of test organisms. The evaluation of the sensor signal is based on the current-time curves of a potentiostatic measurement produced by the detection of microbially reduced mediator molecules immobilized in a gel structure. The mediator molecules are reduced during the measurement process. The reduced mediator molecules produce a current signal, which rapidly provides information about the vigor and vitality of living bacteria, yeasts, fungi or cells.

The Exposome and Toxicology: A Win-Win Collaboration.

The development of the exposome concept has been one of the hallmarks of environmental and health research for the last decade. The exposome encompasses the life course environmental exposures including lifestyle factors from the prenatal period onwards. It has inspired many research programs and is expected to influence environmental and health research, practices, and policies. Yet, the links bridging toxicology and the exposome concept have not been well developed. In this review, we describe how the exposome framework can interface with and influence the field of toxicology, as well as how the field of toxicology can help advance the exposome field by providing the needed mechanistic understanding of the exposome impacts on health. Indeed, exposome-informed toxicology is expected to emphasize several orientations including (1) developing approaches integrating multiple stressors, in particular chemical mixtures, as well as the interaction of chemicals with other stressors, (2) using mechanistic frameworks such as the adverse outcome pathways to link the different stressors with toxicity outcomes, (3) characterizing the mechanistic basis of long-term effects by distinguishing different patterns of exposures and further exploring the environment-DNA interface through genetic and epigenetic studies, and (4) improving the links between environmental and human health, in particular through a stronger connection between alterations in our ecosystems and human toxicology. The exposome concept provides the linkage between the complex environment and contemporary mechanistic toxicology. What toxicology can bring to exposome characterization is a needed framework for mechanistic understanding and regulatory outcomes in risk assessment.

Levels and risks of surface contamination by thirteen antineoplastic drugs in the Czech and Slovak hospitals and pharmacies.

The consumption of hazardous antineoplastic drugs (ADs) used in anticancer chemotherapies is steadily increasing representing thus risks to both human health and the environment. Hospitals may serve as a contamination source, and pharmacists preparing the antineoplastic drugs (ADs) as well as nurses administering chemotherapy and caring for oncology patients are among the healthcare professionals being highly exposed. Here, we present the results of systematic monitoring (2018-2020) of surface contamination by 13 ADs in the pharmacies and hospitals in the Czech Republic (CZ; large-scale monitoring, 20 workplaces) and Slovak Republic (SK; pilot study at 4 workplaces). The study evaluated contamination by three commonly monitored ADs, i.e., 5-fluorouracil (FU), cyclophosphamide (CP), and platinum (total Pt representing cis-, carbo-, and oxaliplatin) together with ten less explored ADs, i.e., gemcitabine (GEM), ifosfamide (IF), paclitaxel (PX), irinotecan (IRI), docetaxel (DOC), methotrexate (MET), etoposide (ETOP), capecitabine (CAP), imatinib (IMAT), and doxorubicin (DOX). Floors and desktop surfaces in hospitals (chemotherapy application rooms, nurse working areas) were found to be more contaminated, namely with CP and Pt, in both countries when compared to pharmacies. Comparison between the countries showed that hospital surfaces in SK are generally more contaminated (e.g., CP median was 20 times higher in SK), while some pharmacy areas in the CZ were more contamined in comparison with SK. The newly studied ADs were detected at lower concentrations in comparison to FU, CP, and Pt, but some markers (GEM, IF, PX, and IRI) were frequently observed, and adding these compounds to routine monitoring is recommended.

Instrumental and bioanalytical assessment of pharmaceuticals and hormone-like compounds in a major drinking water source-wastewater receiving Zayandeh Rood river, Iran.

Zayandeh Rood river is the most important river in central Iran supplying water for a variety of uses including drinking water for approximately three million inhabitants. The study aimed to investigate the quality of water concerning the presence of pharmaceutical active compounds (PhACs) and hormonelike compounds, which have been only poorly studied in this region. Sampling was performed at seven sites along the river (from headwater sites to downstream drinking water source, corresponding drinking water, and treated wastewater) affected by wastewater effluents, specific drought conditions, and high river-water demand. The targeted and nontargeted chemical analyses and in vitro bioassays were used to evaluate the presence of PhACs and hormonelike compounds in river water. In the samples, 57 PhACs and estrogens were detected with LC-MS/MS with the most common and abundant compounds valsartan, carbamazepine, and caffeine present in the highest concentrations in the treated wastewater in the concentrations of 8.4, 19, and 140 µg/L, respectively. A battery of in vitro bioassays detected high estrogenicity, androgenicity, and AhR-mediated activity (viz., in treated wastewater) in the concentrations 24.2 ng/L, 62.2 ng/L, and 0.98 ng/L of 17ß-estradiol, dihydrotestosterone and 2,3,7,8-TCDD equivalents, respectively. In surface water samples, estrogenicity was detected in the range of <0.42 (LOD) to 1.92 ng/L of 17ß-estradiol equivalents, and the drinking water source contained 0.74 ng/L of 17ß-estradiol equivalents. About 19% of the estrogenicity could be explained by target chemical analyses, and the remaining estrogenicity can be at least partially attributed to the potentiation effect of detected surfactant residues. Drinking water contained several PhACs and estrogens, but the overall assessment suggested minor human health risk according to the relevant effect-based trigger values. To our knowledge, this study provides some of the first comprehensive information on the levels of PhACs and hormones in Iranian waters.

Occurrence of cylindrospermopsin, anatoxin-a and their homologs in the southern Czech Republic - Taxonomical, analytical, and molecular approaches.

Water bloom-forming cyanobacteria have a severe impact on freshwater quality. Although some cyanobacterial toxins such as microcystins have been studied extensively, other toxins like anatoxin-a (ATX) and their structural analogs - as well as cyanobacterial taxa producing these toxins remain to be explored in detail. The present study investigated levels of ATX, CYN and their homologs along with the occurrence of anaC and cyrJ genes in water blooms in 16 sites in the Czech Republic that were pre-selected concerning the presence of potential toxin producers. Besides, we also studied toxins and genes in a series of strains available in our laboratories. ATX and its congener HATX were detected in 5 natural biomass samples from the Czech Republic (maximum concentration 2.8 micrograms per gram d.w.). Interestingly, the anaC gene coding for ATX production was not detected in any of these toxin-positive biomass samples. The concentrations of ATX congeners in cyanobacterial laboratory strains were about 10-times higher than those of the original ATX, which calls for further research addressing levels and hazards of ATX analogs. Regarding the CYN and 7-deoxyCYN (other CYN congeners were not analyzed in this study) - these toxins were identified in a single small pond in the Czech Republic at concentrations 4.3 and 2.7 micrograms per gram of biomass d.w., respectively (corresponded to dissolved concentrations higher than 1 microgram per liter). The CYN-positive sample was dominated by CYN-producing taxa Raphidiopsis (basionym Cylindrospermopsis) and Cuspidothrix. We also confirmed the presence of a specific cyrJ gene in this natural bloom sample. To our knowledge, this is the first study pointing to Raphidiopsis (Cylindrospermopsis) and Cuspidothrix as producers of CYN in Europe. This observation calls for further research because of their increasing occurrence in (Central) Europe along with the global change. The present study demonstrates the importance of using combined (taxonomical, analytical, and molecular) approaches in the assessment of hazardous cyanobacteria and their toxins in freshwaters.

Cyanobacteria Microcystis aeruginosa Contributes to the Severity of Fish Diseases: A Study on Spring Viraemia of Carp.

Fish are exposed to numerous stressors in the environment including pollution, bacterial and viral agents, and toxic substances. Our study with common carps leveraged an integrated approach (i.e., histology, biochemical and hematological measurements, and analytical chemistry) to understand how cyanobacteria interfere with the impact of a model viral agent, Carp sprivivirus (SVCV), on fish. In addition to the specific effects of a single stressor (SVCV or cyanobacteria), the combination of both stressors worsens markers related to the immune system and liver health. Solely combined exposure resulted in the rise in the production of immunoglobulins, changes in glucose and cholesterol levels, and an elevated marker of impaired liver, alanine aminotransferase (ALT). Analytical determination of the cyanobacterial toxin microcystin-LR (MC-LR) and its structurally similar congener MC-RR and their conjugates showed that SVCV affects neither the levels of MC in the liver nor the detoxification capacity of the liver. MC-LR and MC-RR were depurated from liver mostly in the form of cysteine conjugates (MC-LR-Cys, MC-RR-Cys) in comparison to glutathione conjugates (LR-GSH, RR-GSH). Our study brought new evidence that cyanobacteria worsen the effect of viral agents. Such inclusion of multiple stressor concept helps us to understand how and to what extent the relevant environmental stressors co-influence the health of the fish population.

An adverse outcome pathway based in vitro characterization of novel flame retardants-induced hepatic steatosis.

A wide range of novel replacement flame retardants (nFRs) is consistently detected in increasing concentrations in the environment and human matrices. Evidence suggests that nFRs exposure may be associated with disruption of the endocrine system, which has been linked with the etiology of various metabolic disorders, including nonalcoholic fatty liver disease (NAFLD). NAFLD is a multifactorial disease characterized by the uncontrolled accumulation of fats (lipids) in the hepatocytes and involves multiple-hit pathogenesis, including exposure to occupational and environmental chemicals such as organophosphate flame retardants (OPFRs). In the present study we aimed to investigate the potential mechanisms of the nFRs-induced hepatic steatosis in the human liver cells. In this study, we employed an in vitro bioassay toolbox to assess the key events (KEs) in the proposed adverse outcome pathways (AOP) (s) for hepatic steatosis. We examined nine nFRs using AOP- based in vitro assays measuring KEs such as lipid accumulation, mitochondrial dysfunction, gene expression, and in silico approach to identify the putative molecular initiating events (MIEs). Our findings suggest that several tested OPFRs induced lipid accumulation in human liver cell culture. Tricresyl phosphate (TMPP), triphenyl phosphate (TPHP), tris(1,3-dichloropropyl) phosphate (TDCIPP), and 2-ethylhexyl diphenyl phosphate (EHDPP) induced the highest lipid accumulation by altering the expression of genes encoding hepatic de novo lipogenesis and mitochondrial dysfunction depicted by decreased cellular ATP production. Available in vitro data from ToxCast and in silico molecular docking suggests that pregnane X receptor (PXR) and peroxisome proliferator-activated receptor gamma (PPARγ) could be the molecular targets for the tested nFRs. The study identifies several nFRs, such as TMPP and EHDPP, TPHP, and TDCIPP, as potential risk factor for NAFLD and advances our understanding of the mechanisms involved, demonstrating the utility of an AOP-based strategy for screening and prioritizing chemicals and elucidating the molecular mechanisms of toxicity.

Pesticide mixture toxicity assessment through in situ and laboratory approaches using embryo-larval stages of the pacific oyster (Magallana gigas).

Worsened state of oysters in French Arcachon Bay, demand an investigation of possible causes. This study evaluated the effects of an environmentally relevant mixture of five common pesticides on the early-life stages of the Pacific oyster (Magallana gigas). Laboratory assays with artificial mixture and in situ transplantation were complementarily used to investigate a series of sublethal endpoints. The laboratory exposure revealed developmental toxicity at 0.32 µg/L, which corresponds to mixture concentrations in Arcachon Bay. Downregulation of some gene transcriptions was observed at environmental level. No difference in larvae development was revealed among the three sites in Arcachon Bay. This study was the first to evaluate locomotion of oyster larvae exposed in situ. Suspected poor water quality in the inner part of Arcachon Bay was reflected by impairment at the molecular level. In conclusion, current concentrations of the tested pesticides in Arcachon Bay hinder larval development and affect several biological functions.