Toxicity to bronchial cells and endocrine disruptive potentials of indoor air and dust extracts and their association with multiple chemical classes.

Pollution of indoor environment, where people spend much of their time, comprises complex mixtures of compounds with vastly understudied hazard potential. This study examined several important specific toxic effects and pollutant levels (177 compounds) of indoor samples (air gas phase, PM10 and dust) from different microenvironments after two extractions with focus on their gas/particle/dust distribution and polarity. The endocrine disruptive (ED) potential was assessed by human cell-based in vitro bioassays addressing anti-/estrogenicity, anti-/androgenicity, aryl hydrocarbon, thyroid and peroxisome proliferator-activated receptor-mediated activities. Potential toxicity to respiratory tract tissue was assessed using human bronchial cell line. The toxicological analyses pointed out the relevance of both inhalation and ingestion exposure, with significant effects detected after exposure to extracts from all three studied matrices with distinct gas/particle distribution patterns. Chemical analyses document the high complexity of indoor pollutant mixtures with greatest levels of phthalates, their emerging alternatives, and PAHs in dust. Despite the detection of up to 108 chemicals, effects were explained only to low extent. This emphasizes data gaps regarding ED potencies of many detected abundant indoor contaminants, but also potential presence of other unidentified ED compounds. The omnipresent ED potentials in indoor environment rise concern regarding associated human health risk.

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.

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.

Cytotoxic and Genotoxic Effects of Cyanobacterial and Algal Extracts-Microcystin and Retinoic Acid Content.

In the last decade, it has become evident that complex mixtures of cyanobacterial bioactive substances, simultaneously present in blooms, often exert adverse effects that are different from those of pure cyanotoxins, and awareness has been raised on the importance of studying complex mixtures and chemical interactions. We aimed to investigate cytotoxic and genotoxic effects of complex extracts from laboratory cultures of cyanobacterial species from different orders (Cylindrospermopsis raciborskii, Aphanizomenon gracile, Microcystis aeruginosa, M. viridis, M. ichtyoblabe, Planktothrix agardhii, Limnothrix redekei) and algae (Desmodesmus quadricauda), and examine possible relationships between the observed effects and toxin and retinoic acid (RA) content in the extracts. The cytotoxic and genotoxic effects of the extracts were studied in the human hepatocellular carcinoma HepG2 cell line, using the MTT assay, and the comet and cytokinesis-block micronucleus (cytome) assays, respectively. Liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) was used to detect toxins (microcystins (MC-LR, MC-RR, MC-YR) and cylindrospermopsin) and RAs (ATRA and 9cis-RA) in the extracts. Six out of eight extracts were cytotoxic (0.04-2 mgDM/mL), and five induced DNA strand breaks at non-cytotoxic concentrations (0.2-2 mgDM/mL). The extracts with genotoxic activity also had the highest content of RAs and there was a linear association between RA content and genotoxicity, indicating their possible involvement; however further research is needed to identify and confirm the compounds involved and to elucidate possible genotoxic effects of RAs.

Resazurin assay for assessment of antimicrobial properties of electrospun nanofiber filtration membranes.

We developed a simple and fast microplate assay for evaluation of the antimicrobial activity of electrospun nanofiber filtration membranes or similar porous materials for water treatment technologies. Resazurin (alamarBlue®) was used as an indicator of the amount of viable experimental microorganisms Gram-negative Escherichia coli, Gram-positive Enterococcus faecalis, and natural wastewater treatment plant effluent bacteria. A bacterial inoculum of concentration 1-3 × 105 CFU mL-1 was pipetted onto the surface of assessed both functionalized and respective control membranes and incubated in 12-well plates for 4 h at 37 °C. Kinetics of resazurin metabolization, i.e. its reduction to fluorescent resorufin, was evaluated fluorimetrically (λex520/λem590 nm). A number of viable bacteria on the membranes expressed as CFU mL-1 was calculated from the kinetic curves by using calibration curves that were constructed for both experimental bacterial species. Antimicrobial activities of the membranes were evaluated by either resazurin assay or modified ISO 20743 plate count assay. Results of both assays showed the significant antimicrobial activity of membranes functionalized with silver nanoparticles for both bacterial species and wastewater treatment plant effluent bacteria as well (log CFU reduction compared to control membrane > 4), while membranes containing specific quaternary ammonium salts were inefficient (log CFU reduction < 1). The suitability of resazurin microplate assay for testing nanofiber filtration membranes and analogous matrices has proven to be a faster and less demanding alternative to the traditionally used approach providing comparable results.

In vitro assessment of sex steroids and related compounds in water and sediments - a critical review.

Detection of endocrine disrupting compounds in water and sediment samples has gained much importance since the evidence of their effects was reported in aquatic ecosystems in the 1990s. The aim of this review is to highlight the advances made in the field of in vitro analysis for the detection of hormonally active compounds with estrogenic, androgenic and progestogenic effects in water and sediment samples. In vitro assays have been developed from yeast, mammalian and in a few cases from fish cells. These assays are based either on the hormone-mediated proliferation of sensitive cell lines or on the hormone-mediated expression of reporter genes. In vitro assays in combination with various sample enrichment methods have been used with limits of detection as low as 0.0027 ng L-1 in water, and 0.0026 ng g-1 in sediments for estrogenicity, 0.1 ng L-1 in water, and 0.5 ng g-1 in sediments for androgenicity, and 5 ng L-1 in water for progestogenicity expressed as equivalent concentrations of standard reference compounds of 17ß-estradiol, dihydrotestosterone and progesterone, respectively. The experimental results and limits of quantification, however, are influenced by the methods of sample collection, preparation, and individual laboratory practices.

Yeast Biosensors for Detection of Environmental Pollutants: Current State and Limitations.

Yeast biosensors have become suitable tools for the screening and detection of environmental pollutants because of their various advantages compared to other sensing technologies. On the other hand, many limitations remain with regard to their optimal performance and applicability in several contexts, such as low-concentration samples and on-site testing. This review summarizes the current state of yeast biosensors, with special focus on screening and assessment of environmental contaminants, discusses both pros and cons, and suggests steps towards their further development and effective use in the environmental assessment.

Freeze-drying as suitable method to achieve ready-to-use yeast biosensors for androgenic and estrogenic compounds.

Recombinant yeast assays (RYAs) have been proved to be a suitable tool for the fast screening of compounds with endocrine disrupting activities. However, ready-to-use versions more accessible to less equipped laboratories and field studies are scarce and far from optimal throughputs. Here, we have applied freeze-drying technology to optimize RYA for the fast assessment of environmental compounds with estrogenic and androgenic potencies. The effects of different cryoprotectants, initial optical density and long-term storage were evaluated. The study included detailed characterization of sensitivity, robustness and reproducibility of the new ready-to-use versions, as well as comparison with the standard assays. Freeze-dried RYAs showed similar dose-responses curves to their homolog standard assays, with Lowest Observed Effect Concentration (LOEC) and Median effective Concentration (EC50) of 1 nM and 7.5 nM for testosterone, and 0.05 nM and 0.5 nM for 17ß-estradiol, respectively. Freeze-dried cells stored at 4 °C retained maximum sensitivity up to 2 months, while cells stored at -18 °C showed no decrease in sensitivity throughout the study (10 months). This ready-to-use RYA is easily accessible and may be potentially used for on-site applications.

Polymer-immobilized ready-to-use recombinant yeast assays for the detection of endocrine disruptive compounds.

Recombinant yeast assays (RYAs) constitute a suitable tool for the environmental monitoring of compounds with endocrine disrupting activities, notably estrogenicity and androgenicity. Conventional procedures require yeast reconstitution from frozen stock, which usually takes several days and demands additional equipment. With the aim of applying such assays to field studies and making them more accessible to less well-equipped laboratories, we have optimized RYA by the immobilization of Saccharomyces cerevisiae cells in three different polymer matrices - gelatin, Bacto agar, and Yeast Extract Peptone Dextrose agar - to obtain a ready-to-use version for the fast assessment of estrogenic and androgenic potencies of compounds and environmental samples. Among the three matrices, gelatin showed the best results for both testosterone (androgen receptor yeast strain; AR-RYA) and 17ß-estradiol (estrogen receptor yeast strain; ER-RYA). AR-RYA was characterized by a lowest observed effect concentration (LOEC), EC50 and induction factor (IF) of 1nM, 2.2nM and 51, respectively. The values characterizing ER-RYA were 0.4nM, 1.8nM, and 63, respectively. Gelatin immobilization retained yeast viability and sensitivity for more than 90d of storage at 4°C. The use of the immobilized yeast reduced the assay duration to only 3h without necessity of sterile conditions. Because immobilized RYA can be performed either in multiwell microplates or glass tubes, it allows multiple samples to be tested at once, and easy adaptation to existing portable devices for direct in-field applications.

Interaction of temperature and an environmental stressor: Moina macrocopa responds with increased body size, increased lifespan, and increased offspring numbers slightly above its temperature optimum.

For organisms, temperature is one of the most important environmental factors and gains increasing importance due to global warming, since increasing temperatures may pose organisms close to their environmental tolerance limits and, thus, they may become more vulnerable to environmental stressors. We analyzed the temperature-dependence of the water-soluble antioxidant capacity of the cladoceran Moina macrocopa and evaluated its life trait variables with temperature (15, 20, 25, 30°C) and humic substance (HS) concentrations (0, 0.18, 0.36, 0.90, 1.79 mM DOC) as stressors. Temperatures below and above the apparent optimum (20°C) reduced the antioxidative capacity. Additions of HSs increased body length, but decreased mean lifespan at 15 and 20°C. There was no clear HS-effect on offspring numbers at 15, 20, and 30°C. At 25°C with increasing HS-concentration, lifespan was extended and offspring numbers increased tremendously, reaching 250% of the control. Although the applied HS preparation possesses estrogenic and antiandrogenic activities, a xenohormone mechanism does not seem plausible for the reproductive increase, because comparable effects did not occur at other temperatures. A more convincing explanation appears to be the mitohormesis hypothesis which states that a certain increase of reactive oxygen production leads to improved health and longevity and, with Moina, also to increased offspring numbers. Our results suggest that at least with the eurythermic M. macrocopa, a temperature above the optimum can be beneficial for several life trait variables, even when combined with a chemical stressor. Temperatures approximately 10°C above its optimum appear to adversely affect the lifespan and reproduction of M. macrocopa. This indicates that this cladoceran species seems to be able to utilize temperature as an ecological resource in a range slightly above its thermal optimum.

Antiandrogenic activity of humic substances.

For long, natural organic matter (NOM) composed mainly of humic substances (HS) were regarded as inert in the ecosystems with respect to their possible chemical interaction with exposed organisms. However recently, NOM have been shown to elicit various adverse effects generally attributed to synthetic xenobiotics, including estrogenic effects translating into shifts of the gender ratios in populations. However, the anti/androgenic pathway was not yet evaluated. Here, we applied an anti/androgenic sensitive cell line MDA stably transfected with the firefly luciferase gene under transcriptional control of the androgen responsive element. With five out of twelve tested NOM preparations, particularly with soil and coal isolates, we identified a relatively high, concentration-dependent antiandrogenic effect. This appears to be the first study to show this endocrine disrupting pathway for a ubiquitous biogeochemical matrix, a potential activity which should not be neglected in forthcoming studies on synthetic endocrine disruptors in the environment.

Enhancement of AhR-mediated activity of selected pollutants and their mixtures after interaction with dissolved organic matter.

Dissolved organic matter (DOM) in freshwaters is present at concentrations ranging from 0.5 to 50 mg L⁻¹, and consists of various organic compounds, including humic substances (HS). HS exert a variety of direct and indirect biological effects, including interaction with the aryl hydrocarbon receptor (AhR). AhR is a cytosolic receptor that binds various hydrophobic organic compounds (HOCs) and mediates some of their toxic effects. In vitro effects of binary mixtures of various DOM (mainly HS) with various HOCs on AhR-mediated responses were studied by use of H4IIE-luc cells. Six out of 12 DOM activated the AhR even at environmentally relevant concentrations (17 mg L⁻¹). In simultaneous exposures of H4IIE-luc cells to DOM (17 mg L⁻¹) and each of the model compounds, 2,3,7,8-TCDD, PCB126, PCB169, benzo[a]pyrene, benzo[a]anthracene, dibenz[a,h]anthracene, fluoranthene, a mixture of persistent organic pollutants (POPs), a mixture of polycyclic aromatic hydrocarbons (PAHs), and a mixture of all HOCs, either significant additive or facilitative effects were observed when compared to activities of single HOCs. No significant decrease of effects due to possible sorption of HOCs to DOM was observed, even in subsequent experiments when HOCs+DOM mixtures were preincubated for six days before exposure to H4IIE-luc. Thus, DOM does not seem to protect organisms against AhR-mediated toxic effects of HOCs (as usually predicted due to sorption of HOCs on DOM), but it can actually enhance their potency for AhR-mediated effects in some situations.

Humic substances. Part 2: Interactions with organisms.

GOAL, SCOPE AND BACKGROUND: Freshwater bodies which chemistry is dominated by dissolved humic substances (HS) seem to be the major type on Earth, due to huge non-calcareous geological formations in the Northern Hemisphere and in the tropics. Based on the paradigm of the inertness of being organic, direct interactions of dissolved HS with freshwater organisms are mostly neglected. However, dissolved organic carbon, the majority of which being HS, are natural environmental chemicals and should therefore directly interact with organisms. Major results that widened our perspective on humic substance ecology come from experiments with the compost nematode, Caenorhabditis elegans, which behaved contradictorily to textbook knowledge and provoked an in-depth re-consideration of some paradigms. APPROACH: To overcome old paradigms on HS and their potential interactions with organisms, we reviewed recent international literature, as well as 'grey' literature. We also include results from own ongoing studies. RESULTS: This review focuses on direct interactions of dissolved HS with freshwater organisms and disregards indirect effects, such as under-water light quenching. Instead we show with some macrophyte and algal species that HS adversely interfere with photosynthesis and growth, whereby closely related algal species show different response patterns. In addition to this, HS suppress cyanobacteria more than eukaryotic algae. Quinones in the HS appear to be the effective structure. Furthermore, HS can modulate the offspring numbers in the nematode C. elegans and cause feminization of fish and amphibians--they possess hormone-like properties. The ecological consequences of this potential remain obscure at present. HS also have the potential to act as chemical attractants as shown with C. elegans and exert a mild chemical stress upon aquatic organisms in many ways: induction of molecular chaperons (stress proteins), induction and modulation of biotransformation and anti-oxidant enzymes. Furthermore, they produce an oxidative stress with lipidperoxidation as one clear symptom or even stress defense strategy. Stronger chemical stresses by HS may even lead to teratogenic effects as shown with fish embryos; all physiological responses to HS-mediated stress require energy, which were compensated on the expense of yolk as shown with zebra fish embryos. One Finnish field survey supports the view of a strong chemical stress, as the weight yield in fish species decreases with increasing HS content in the lakes. DISCUSSION: HS exert a variety of stress symptoms in aquatic and compost organisms. According to current paradigms of ecotoxicology, these symptoms have to be considered adverse, because their compensation consumes energy which is deducted from the main metabolism. However, the nematode C. elegans looks actively for such stressful environments, and this behavior is only understandable in the light of new paradigms of aging mechanisms, particularly the Green Theory of Aging. In this respect, we discuss the mild HS-mediated stress to aquatic and compost organisms. New empirical findings with HS themselves and HS building blocks appear to be consistent with this emerging paradigm and show that the individual lifespan may be expanded. At present the ecological consequences of these findings remain obscure. However, a multiple-stress resistance may be acquired which improves the individual fitness in a fluctuating environment. CONCLUSIONS: It appears that dissolved HS have to be considered abiotic ecological driving forces, somewhat less obvious than temperature, nutrients, or light. PERSPECTIVES: The understanding of the ecological control by dissolved humic substances is still fragmentary and needs to be studied in more details.

A high-power laser-driven source of sub-nanosecond soft X-ray pulses for single-shot radiobiology experiments.

A large-scale, double-stream gas puff target has been illuminated by sub-kJ, near-infrared (NIR) focused laser pulses at the PALS facility (Prague Asterix Laser System) to produce high-energy pulses of soft X rays from hot, dense plasma. The double-puff arrangement ensures high gas density and conversion efficiency from NIR to X rays approaching that typical for solid targets. In addition, its major advantage over solid targets is that it is free of debris and has substantially suppressed charged-particle emission. The X-ray emission characteristics of the source were determined for a range of gases that included krypton, xenon, N(2), CO and N(2)-CO. A demonstrated application of the xenon-based source is a single-shot damage induction to plasmid DNA. The yields of single-strand breaks (SSBs) and double-strand breaks (DSBs) were determined as a function of energy fluence adjusted by varying distance of sample from the source and thickness of aluminum filters.

Optical and X-ray emission spectroscopy of high-power laser-induced dielectric breakdown in molecular gases and their mixtures.

Large-scale plasma was created in molecular gases (CO, CO2, N2, H2O) and their mixtures by high-power laser-induced dielectric breakdown (LIDB). Compositions of the mixtures used are those suggested for the early earth's atmosphere of neutral and/or mildly reducing character. Time-integrated optical spectra emitted from the laser spark have been measured and analyzed. The spectra of the plasma generated in the CO-containing mixtures are dominated by emission of both C2 and CN radicals. A vibrational temperature of approximately 10(4) K was determined according to an intensity distribution in a vibronic structure of the CN (B2Sigma(+)u-X2Sigma(+)g) violet band. For comparison, the NH3-CH4-H2-H2O mixture has been irradiated as a model of the strongly reducing version of the early earth's atmosphere. In this mixture, excited CN seems to be significantly less abundant than C2. The LIDB experiments were in the molecular gases carried out not only in the static cell but also using a large, double stream pulse jet (gas puff target) placed in the vacuum interaction chamber. The obtained soft X-ray emission spectra indicate the presence of highly charged atomic ions in the hot core of high-power laser sparks.

Activation of Ah receptor by pure humic acids.

Humic substances (HS) are ubiquitous in the environment. However, some studies indicate that HS could induce direct adverse effects on human health and hormone-like effects in fish, amphibians, and invertebrates. In this study we investigated a possible biochemical mechanism of HS toxicity via activation of the aryl hydrocarbon receptor (AhR). AhR mediates the toxic and biological effects of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but a number of structurally diverse compounds has also been found to activate AhR. Alkali solutions of humic acids (HA) were prepared, and subsequently, lipophilic compounds (including parts of HA) were extracted by liquid-liquid extraction with hexane/dichloromethane. Organic extract of HA was further treated with sulfuric acid to study the role of possible trace persistent contaminants. In vitro dioxin-like activities of obtained extract and HA solutions have been evaluated using H4IIE.luc cells by determining the ethoxyresorufin-O-deethylase (EROD) activity and induction of AhR-dependent reporter luciferase. Traces of nonpersistent residues in HA with known AhR activity were identified and quantified by GC-MS. Our results show that an alkali solution as well as organic extract of HA were active in both EROD and luciferase assays, while H2SO4-treated extract activity was negligible. Only nonsignificant levels of AhR-inducing contaminants (PAHs and PCBs) were found in the HA samples. Our results indicate that HA or their fragments can elicit significant inductions of AhR-mediated effects in vitro. To our best knowledge, this study is the first in providing direct evidence of dioxin-like effects of HA. Further efforts should focus on detailed characterization of potential toxic effects of various HSs.