Gene expression patterns associated with PFOA exposure in Czech young men and women.

Perfluorooctanoic acid (PFOA), a member of per- and polyfluoroalkyl substances (PFASs), has been widely used in manufacturing for decades. Currently, PFOA is strictly regulated, but due to its high stability and persistence, it is detected in both environmental as well as in human matrices. To elucidate mechanisms of PFOA toxicity in humans, we determined the genome-wide transcriptomic changes of peripheral blood mononuclear cells (PBMC) responding to PFOA exposure in a sex-stratified analysis. This work employed samples from 145 female and 143 male participants of the CELSPAC: YA study to characterize PFOA-associated transcripts in a broader context using computational analysis. PFOA-associated gene expression differed significantly between men and women, as only 2 % of mapped genes were expressed in both sexes. Disease-specific enrichment analysis revealed cancer and immune-related disease terms as those most enriched in male and female populations. Patterns of enriched terms within the gene set enrichment analysis indicated three main targets of PFOA toxicity: i) lipid metabolism for women; ii) cell cycle regulation for men; and iii) immune system response for both sexes. In summary, our genome-wide transcriptomics analysis described sex-specific differences in PFOA-associated gene expression and provided evidence about biological pathways underlying PFOA toxicity in humans.

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.

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.

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.

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.

Estrogenicity of chemical mixtures revealed by a panel of bioassays.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

Endocrine disrupting potential of replacement flame retardants - Review of current knowledge for nuclear receptors associated with reproductive outcomes.

BACKGROUND AND AIM: Endocrine disrupting chemicals (EDCs) constitute a major public health concern because they can induce a large spectrum of adverse effects by interfering with the hormonal system. Rapid identification of potential EDCs using in vitro screenings is therefore critical, particularly for chemicals of emerging concerns such as replacement flame retardants (FRs). The review aimed at identifying (1) data gaps and research needs regarding endocrine disrupting (ED) properties of replacement FRs and (2) potential EDCs among these emerging chemicals. METHODS: A systematic search was performed from open literature and ToxCast/Tox21 programs, and results from in vitro tests on the activities of 52 replacement FRs towards five hormone nuclear receptors (NRs) associated with reproductive outcomes (estrogen, androgen, glucocorticoid, progesterone, and aryl hydrocarbon receptors) were compiled and organized into tables. Findings were complemented with information from structure-based in silico model predictions and in vivo information when relevant. RESULTS: For the majority of the 52 replacement FRs, experimental in vitro data on activities towards these five NRs were either incomplete (15 FRs) or not found (24 FRs). Within the replacement FRs for which effect data were found, some appeared as candidate EDCs, such as triphenyl phosphate (TPhP) and tris(1,3-dichloropropyl)phosphate (TDCIPP). The search also revealed shared ED profiles. For example, anti-androgenic activity was reported for 19 FRs and predicted for another 21 FRs. DISCUSSION: This comprehensive review points to critical gaps in knowledge on ED potential for many replacement FRs, including chemicals to which the general population is likely exposed. Although this review does not cover all possible characteristics of ED, it allowed the identification of potential EDCs associated with reproductive outcomes, calling for deeper evaluation and possibly future regulation of these chemicals. By identifying shared ED profiles, this work also raises concerns for mixture effects since the population is co-exposed to several FRs and other chemicals.

Flame Retardants-Mediated Interferon Signaling in the Pathogenesis of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a growing concern worldwide, affecting 25% of the global population. NAFLD is a multifactorial disease with a broad spectrum of pathology includes steatosis, which gradually progresses to a more severe condition such as nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and eventually leads to hepatic cancer. Several risk factors, including exposure to environmental toxicants, are involved in the development and progression of NAFLD. Environmental factors may promote the development and progression of NAFLD by various biological alterations, including mitochondrial dysfunction, reactive oxygen species production, nuclear receptors dysregulation, and interference in inflammatory and immune-mediated signaling. Moreover, environmental contaminants can influence immune responses by impairing the immune system's components and, ultimately, disease susceptibility. Flame retardants (FRs) are anthropogenic chemicals or mixtures that are being used to inhibit or delay the spread of fire. FRs have been employed in several household and outdoor products; therefore, human exposure is unavoidable. In this review, we summarized the potential mechanisms of FRs-associated immune and inflammatory signaling and their possible contribution to the development and progression of NAFLD, with an emphasis on FRs-mediated interferon signaling. Knowledge gaps are identified, and emerging pharmacotherapeutic molecules targeting the immune and inflammatory signaling for NAFLD are also discussed.

The efficiency of antineoplastic drug contamination removal by widely used disinfectants-laboratory and hospital studies.

OBJECTIVE: Antineoplastic drugs (ADs) pose risks to healthcare staff. Surface disinfectants are used in hospitals to prevent microbial contamination but the efficiency of disinfectants to degrade ADs is not known. We studied nine disinfectants on ten ADs in the standardized laboratory and realistic in situ hospital conditions. METHODS: A survey in 43 hospitals prioritized nine most commonly used disinfections based on different ingredients. These were tested on inert stainless steel and in situ on contaminated hospital flooring. The effects against ten ADs were studied by LC-MS/MS (Cyclophosphamide CP; Ifosfamide IF; Capecitabine CAP; Sunitinib SUN; Methotrexate MET; Doxorubicin DOX; Irinotecan IRI; Paclitaxel PX; 5-Fluorouracil FU) and ICP-MS (Pt as a marker of platinum-based ADs). RESULTS: Monitoring of the floor contamination in 26 hospitals showed that the most contaminated are the outpatient clinics that suffer from a large turnover of staff and patients and have limited preventive measures. The most frequent ADs were Pt, PX, FU and CP with maxima exceeding the recommended 1 ng/cm2 limit by up to 140 times. IRI, FU, MET, DOX and SUN were efficiently removed by hydrolysis in clean water and present thus lower occupational risk. Disinfectants based on hydrogen peroxide were efficient against PX and FU (> 70% degradation) but less against other ADs, such as carcinogenic CP or IF, IRI and CAP. The most efficient were the active chlorine and peracetic acid-based products, which however release irritating toxic vapors. The innovative in situ testing of ADs previously accumulated in hospital flooring showed highly problematic removal of carcinogenic CP and showed that alcohol-based disinfectants may mobilize persistent ADs contamination from deeper floor layers. CONCLUSION: Agents based on hydrogen peroxide, peracetic acid, quaternary ammonium salts, glutaraldehyde, glucoprotamine or detergents can be recommended for daily use for both disinfection and AD decontamination. However, they have variable efficiencies and should be supplemented by periodic use of strong chlorine-based disinfectants efficient also against the carcinogenic and persistent CP.

Ready to go 3D? A semi-automated protocol for microwell spheroid arrays to increase scalability and throughput of 3D cell culture testing.

3-dimensional (3D) cell cultures are being increasingly recognized as physiologically more relevant in vitro models than traditional monolayer cultures, because they better mimic in vivo-like microenvironment, cell-cell and cell-extracellular matrix interactions. Nevertheless, the broader use of 3D models might be limited by requirements for special consumables, equipment, or skills for 3D cell cultures, and by their limited throughput and scalability. In this study, we optimized and adapted a commercially available agarose-micromolding technique to produce scaffold-free spheroid cultures. Brightfield microscopy was used for routine nondestructive and noninvasive evaluation of spheroid formation and growth. The workflow is compatible with manual, as well as high speed automated microscopic image acquisition, and it is supplemented with an in-house developed macro 'Spheroid_Finder' for open source software Fiji to facilitate rapid automated image analysis. This protocol was used to characterize and quantify spheroid formation and growth of two different hepatic cell lines, hTERT immortalized, but non-cancerous, adult human liver stem cell line HL1-hT1, and human hepatocellular carcinoma cell line HepG2, as well as their responses to a model antiproliferative and cytotoxic agent, 5-fluorouracil. The complete protocol provides a simple and ready-to-use solution to initiate scaffold-free spheroid cultures in any laboratory with standard equipment for mammalian in vitro cell culture work. Thus, it allows to increase throughput and scale of spheroid culture experiments, which can be greatly utilized in different areas of biomedical, pharmaceutical and toxicological research.

Dietary Intake of Acrylamide and Risk of Breast, Endometrial, and Ovarian Cancers: A Systematic Review and Dose-Response Meta-analysis.

Acrylamide is a probable human carcinogen. Aside from occupational exposures and smoking, diet is the main source of exposure in humans. We performed a systematic review of the association between estimated dietary intake of acrylamide and risk of female breast, endometrial, and ovarian cancers in nonexperimental studies published through February 25, 2020, and conducted a dose-response meta-analysis. We identified 18 papers covering 10 different study populations: 16 cohort and two case-control studies. Acrylamide intake was associated with a slightly increased risk of ovarian cancer, particularly among never smokers. For endometrial cancer, risk was highest at intermediate levels of exposure, whereas the association was more linear and positive among never smokers. For breast cancer, we found evidence of a null or inverse relation between exposure and risk, particularly among never smokers and postmenopausal women. In a subgroup analysis limited to premenopausal women, breast cancer risk increased linearly with acrylamide intake starting at 20 µg/day of intake. High acrylamide intake was associated with increased risks of ovarian and endometrial cancers in a relatively linear manner, especially among never smokers. Conversely, little association was observed between acrylamide intake and breast cancer risk, with the exception of premenopausal women.

Immunomodulatory effects of cyanobacterial toxin cylindrospermopsin on innate immune cells.

Cylindrospermopsin (CYN), a cyanobacterial toxin, is an important water pollutant with broad biological activity. It has been known mainly from tropical areas, but the area of occurrence of its producers is spreading to temperate climates. It can be found in high concentrations in the environment as well as in purified drinking waters. The aim of the study is to bring a basic information on the ability of CYN to interfere with mammalian innate immunity cells and thus increase the understanding of the immunomodulatory potency of CYN. This study investigated whether immune cells can be a target of CYN either alone or in combination with a model immunomodulatory agent, lipopolysaccharide (LPS). We examined the effects on cellular viability and inflammation signaling of CYN on murine macrophage-like RAW 264.7 cells. Macrophages were treated either with pure toxin (1 µM) or together with a known stimulator of immunologically active cells, bacterial or cyanobacterial LPS. CYN has had a significant effect on production on pro-inflammatory mediator tumor necrosis factor α (TNF-α) which correlates with its effect on reactive oxygen species (ROS) production. We found that CYN potentiated the effect of bacterial and cyanobacterial LPS that was documented by activation of inflammatory signaling pathways including mitogen-activated protein kinase p38 as well as consequent expression of inducible nitric oxide synthase (iNOS) and increased production of pro-inflammatory mediators such as nitric oxide (NO), TNF-α, interleukin-6 (IL-6). Our study brings one of the first information that contributes to the elucidation of immunomodulatory role of CYN in macrophages under normal and pro-inflammatory conditions.

Cylindrospermopsin induces cellular stress and activation of ERK1/2 and p38 MAPK pathways in adult human liver stem cells.

Cyanobacterial toxin cylindrospermopsin (CYN) is an emerging freshwater contaminant, whose expanding environmental occurrence might result into increased human health risks. CYN is potent hepatotoxin, with cytotoxicity and genotoxicity documented in primary hepatocytes or hepatoma cell lines. However, there is only limited information about CYN effects on adult human liver stem cells (LSCs), which play an important role in liver tissue development, regeneration and repair. In our study with human liver cell line HL1-hT1 which expresses characteristics of LSCs, CYN was found to be cytotoxic and increasing cell death after 24-48 h exposure to concentrations >1 µM. Subcytotoxic 1 µM concentration did not induce cell death or membrane damage, but inhibited cellular processes related to energy production, leading to a growth stagnation after >72 h. Interestingly, these effects were not associated with increased DNA damage, reactive oxygen species production, or endoplasmic reticulum stress. However, CYN induced a sustained (24-48 h) activation of mitogen-activated protein kinases ERK1/2 and p38, and increased expression of stress-related transcription factor ATF3. Thus, LSCs were not primarily affected by CYN-induced genotoxicity and oxidative stress, but via activation of signaling and transcriptional pathways critical for regulation of cell proliferation, stress responses, cell survival and inflammation. Alterations of LSCs during CYN-induced liver injury, including the role of nongenotoxic mechanisms, should be therefore considered in mechanistic assessments of chronic CYN hepatotoxicity and hepatocarcinogenicity.

Freshwater Cyanotoxin Cylindrospermopsin Has Detrimental Stage-specific Effects on Hepatic Differentiation From Human Embryonic Stem Cells.

Cylindrospermopsin (CYN) has been recognized as a potent waterborne hepatotoxin with an increasing environmental occurrence. However, CYN effects on the specific populations of hepatic cells involved in liver tissue development, renewal, and regeneration, have not been characterized yet. We used human embryonic stem cells to analyze the hepatic differentiation stage-specific effect of CYN. Our results strongly suggest that CYN might contribute to the development of chronic adverse outcomes by disrupting liver tissue homeostasis in terms of (1) cellular stress and damage induced in the mature differentiated hepatocytes, which was associated with a necrotic cell death and thus possibly also inflammatory responses; (2) selective elimination of HNF4α+ cells from populations of progenitor cells and immature hepatocytes during hepatic differentiation, which could possibly lead to an impaired liver renewal and regeneration; (3) impaired hepatic functions of immature hepatocytes, such as decreased albumin secretion or increased lipid accumulation, which could contribute to the development of liver steatosis; and (4) survival of the immature and AFP-expressing cells with the limited ability to further differentiate, which could represent a tumor-promoting condition.

Branched Poly(ethylene imine)s as Anti-algal and Anti-cyanobacterial Agents with Selective Flocculation Behavior to Cyanobacteria over Algae.

Poly(ethylene imine)s (PEIs) have been widely studied for biomedical applications, including antimicrobial agents against potential human pathogens. The interactions of branched PEIs (B-PEIs) with environmentally relevant microorganisms whose uncontrolled growth in natural or engineered environments causes health, economic, and technical issues in many sectors of water management are studied. B-PEIs are shown to be potent antimicrobials effective in controlling the growth of environmentally relevant algae and cyanobacteria with dual-functionality and selectivity. Not only did they effectively inhibit growth of both algae and cyanobacteria, mostly without causing cell death (static activity), but they also selectively flocculated cyanobacteria over algae. Thus, unmodified B-PEIs provide a cost-effective and chemically facile framework for the further development of effective and selective antimicrobial agents useful for control of growth and separation of algae and cyanobacteria in natural or engineered environments.

Immunomodulatory effects of selected cyanobacterial peptides in vitro.

Cyanobacteria produce many biologically active metabolites synthesized via nonribosomal synthetic pathways such as cyclic microcystins (MCs) and linear aeruginosins (Aers). The present study aimed to investigate the effects of different MC variants and the newly isolated aerugenosin Aer-865 on macrophages, which represent one of the key effector cells within the innate immune responses. Specifically, our study included RAW 264.7 macrophage activation associated with production of cytotoxic and cytostatic nitric oxide (NO) as well as pro-inflammatory mediators like tumor necrosis factor α (TNFα) and interleukin 6 (IL-6). From the compounds investigated, commonly occurring MC variants (-RR, -YR) and Aer-865 had no significant effects within the non-cytotoxic concentrations tested, i.e. 0.001-1 µM for MCs and 0.1-50 µM for Aer-865. In contrast to known immunoactive MC-LR, the negligible immunomodulatory potential of tested MC congeners could be related to their differences in structure. The knowledge of MC structure-specific activities contributes to the understanding of complex toxicity of different MC variants and most importantly their mixtures. This study is one of the first study that evaluate the effect of larger set of cyanobacterial peptides on macrophages and compare their immunomodulatory potential.

Tumor-promoting cyanotoxin microcystin-LR does not induce procarcinogenic events in adult human liver stem cells.

HL1-hT1 cell line represents adult human liver stem cells (LSCs) immortalized with human telomerase reverse transcriptase. In this study, HL1-hT1 cells were found to express mesenchymal markers (vimentin, CD73, CD90/THY-1 and CD105) and an early hepatic endoderm marker FOXA2, while not expressing hepatic progenitor (HNF4A, LGR5, α-fetoprotein) or differentiated hepatocyte markers (albumin, transthyretin, connexin 32). In response to microcystin-LR (MC-LR), a time- and concentration-dependent formation of MC-positive protein bands in HL1-hT1 cells was observed. Cellular accumulation of MC-LR occurred most likely via mechanisms independent on organic anion transporting polypeptides (OATPs) or multidrug resistance (MDR) proteins, as indicated (a) by a gene expression analysis of 11 human OATP genes and 4 major MDR genes (MDR1/P-glycoprotein, MRP1, MRP2 and BCRP); (b) by non-significant effects of OATP or MDR1 inhibitors on MC-LR uptake. Accumulation of MC-positive protein bands in HL1-hT1 cells was associated neither with alterations of cell viability and growth, dysregulations of ERK1/2 and p38 kinases, reactive oxygen species formation, induction of double-stranded DNA breaks nor modulations of stress-inducible genes (ATF3, HSP5). It suggests that LSCs might have a selective, MDR1-independent, survival advantage and higher tolerance towards MC-induced cytotoxic, genotoxic or cancer-related events than differentiated adult hepatocytes, fetal hepatocyte or malignant liver cell lines. HL1-hT1 cells provide a valuable in vitro tool for studying effects of toxicants and pharmaceuticals on LSCs, whose important role in the development of chronic toxicities and liver diseases is being increasingly recognized.

Phytoestrogens and sterols in waters with cyanobacterial blooms - Analytical methods and estrogenic potencies.

Compounds with estrogenic potencies and their adverse effects in surface waters have received much attention. Both anthropogenic and natural compounds contribute to overall estrogenic activity in freshwaters. Recently, estrogenic potencies were also found to be associated with cyanobacteria and their blooms in surface waters. The present study developed and compared the solid phase extraction and LC-MS/MS analytical approaches for determination of phytoestrogens (8 flavonoids - biochanin A, coumestrol, daidzein, equol, formononetin, genistein, naringenin, apigenin - and 5 sterols - ergosterol, ß-sitosterol, stigmasterol, campesterol, brassicasterol) and cholesterol in water. The method was used for analyses of samples collected in stagnant water bodies dominated by different cyanobacterial species. Concentrations of individual flavonoids ranged from below the limit of detection to 3.58 ng/L. Sterols were present in higher amounts up to 2.25 µg/L. Biological potencies of these phytoestrogens in vitro were characterized using the hERα-HeLa-9903 cell line. The relative estrogenic potencies (compared to model estrogen - 17ß-estradiol) of flavonoids ranged from 2.25E-05 to 1.26E-03 with coumestrol being the most potent. None of the sterols elicited estrogenic response in the used bioassay. Estrogenic activity was detected in collected field water samples (maximum effect corresponding to 2.07 ng/L of 17ß-estradiol equivalents, transcriptional assay). At maximum phytoestrogens accounted for only 1.56 pg/L of 17ß-estradiol equivalents, contributing maximally 8.5% of the total estrogenicity of the water samples. Other compounds therefore, most likely of anthropogenic origin such as steroid estrogens, are probably the major drivers of total estrogenic effects in these surface waters.