Microbial reporter gene assay as a diagnostic and early warning tool for the detection and characterization of toxic pollution in surface waters.

Surface water samples constantly receive a vast mixture of micropollutants mainly originating from wastewater treatment plants (WWTPs). High-throughput live cell arrays provide a promising method for the characterization of the effects of chemicals and the associated molecular mechanisms. In the present study, this test system was evaluated for the first time for the characterization of a set of typical surface water extracts receiving effluent from WWTPs. The extracts containing complex mixtures of micropollutants were analyzed for the expression of 90 stress responsive genes in the Escherichia coli reporter gene assay. The most affected pathways and the genes most sensitive to surface water samples suggested prominent stress-responsive pathways for wastewater-impacted surface water, such as oxidative stress, DNA damage, and drug resistance. Samples strongly affecting particular pathways were identified by statistical analysis of gene expression. Transcription data were correlated with contamination data from chemical screening and percentages of wastewater in the samples. Samples with particular effects and outstanding chemical composition were analyzed. For these samples, hypotheses on the alteration of the transcription of genes involved in drug resistance and DNA repair attributable to the presence of pharmaceuticals were drawn.

Linking mutagenic activity to micropollutant concentrations in wastewater samples by partial least square regression and subsequent identification of variables.

We deployed multivariate regression to identify compounds co-varying with the mutagenic activity of complex environmental samples. Wastewater treatment plant (WWTP) effluents with a large share of industrial input of different sampling dates were evaluated for mutagenic activity by the Ames Fluctuation Test and chemically characterized by a screening for suspected pro-mutagens and non-targeted software-based peak detection in full scan data. Areas of automatically detected peaks were used as predictor matrix for partial least squares projections to latent structures (PLS) in combination with measured mutagenic activity. Detected peaks were successively reduced by the exclusion of all peaks with lowest variable importance until the best model (high R(2) and Q(2)) was reached. Peaks in the best model co-varying with the observed mutagenicity showed increased chlorine, bromine, sulfur, and nitrogen abundance compared to original peak set indicating a preferential selection of anthropogenic compounds. The PLS regression revealed four tentatively identified compounds, newly identified 4-(dimethylamino)-pyridine, and three known micropollutants present in domestic wastewater as co-varying with the mutagenic activity. Co-variance between compounds stemming from industrial wastewater and mutagenic activity supported the application of "virtual" EDA as a statistical tool to separate toxicologically relevant from less relevant compounds.

A toxicokinetic study of specifically acting and reactive organic chemicals for the prediction of internal effect concentrations in Scenedesmus vacuolatus.

The toxic potency of chemicals is determined by using the internal effect concentration by accounting for differences in toxicokinetic processes and mechanisms of toxic action. The present study examines toxicokinetics of specifically acting and reactive chemicals in the green algae Scenedesmus vacuolatus by using an indirect method. Concentration depletion in the exposure medium was measured for chemicals of lower (log KOW < 3: isoproturon, metazachlor, paraquat) and moderate (log KOW 4-5: irgarol, triclosan, N-phenyl-2-naphthylamine) hydrophobicity at 7 to 8 time points over 240 min or 360 min. Uptake and overall elimination rates were estimated by fitting a toxicokinetic model to the observed concentration depletions. The equilibrium of exposure concentrations was reached within minutes to hours or was even not observed within the exposure time. The kinetics of bioconcentration cannot be explained by the chemical's hydrophobicity only, but influential factors such as ionization of chemicals, the ion trapping mechanism, or the potential susceptibility for biotransformation are discussed. Internal effect concentrations associated with 50% inhibition of S. vacuolatus reproduction were predicted by linking the bioconcentration kinetics to the effect concentrations and ranged from 0.0480 mmol/kg wet weight to 7.61 mmol/kg wet weight for specifically acting and reactive chemicals. Knowing the time-course of the internal effect concentration may promote an understanding of toxicity processes such as delayed toxicity, carry-over toxicity, or mixture toxicity in future studies.

Metabolic transformation as a diagnostic tool for the selection of candidate promutagens in effect-directed analysis.

To implement metabolic activation by S9 rat liver homogenate in the selection of candidate promutagens in effect-directed analysis, we critically assessed the capability of LC-HRMS measurements to detect depletion and formation of metabolites by S9 exposure. The exposure of a reference mixture to S9 led to a depletion by >70% for most compounds. Other processes than metabolism were excluded as significant contribution to compound depletion. Metabolites formed by S9 exposure were identified and S9 metabolism was incorporated in the identification of candidate promutagens in a wastewater treatment plant (WWTP) effluent with mutagenic activity only after metabolic activation by S9. The metabolism by S9 in the WWTP effluent was confirmed. Based on a candidate exclusion of all peaks not depleted, thus not activated by the S9 mix, the number of candidate promutagens was reduced by 40%. Selected remaining candidates were evaluated and identified, but could not be confirmed as promutagens.

Identification of novel micropollutants in wastewater by a combination of suspect and nontarget screening.

To detect site-specific, suspected and formerly unknown contaminants in a wastewater treatment plant effluent, we established a screening procedure based on liquid chromatography-high resolution mass spectrometry (LC-HRMS) with stepwise identification schemes. Based on automated substructure searches a list of 2160 suspected site-specific and documented water contaminants was reduced to those amenable to LC-HRMS. After searching chromatograms for exact masses of suspects, presumably false positive detections were stepwise excluded by retention time prediction, the evaluation of isotope patterns, ionization behavior, and HRMS/MS spectra. In nontarget analysis, peaks for identification were selected based on distinctive isotope patterns and intensity. The stepwise identification of nontarget compounds was automated by a plausibility check of molecular formulas using the Seven Golden Rules, an exclusion of compounds with presumably low commercial importance and an automated HRMS/MS evaluation. Six suspected and five nontarget chemicals were identified, of which two have not been previously reported as environmental pollutants.

Biological effects of a dietary omega-3 polyunsaturated fatty acids supplementation in cystic fibrosis patients: a randomized, crossover placebo-controlled trial.

BACKGROUND AND AIMS: Various anti-inflammatory therapies, including dietary omega-3 polyunsaturated fatty acids (PUFA) supplementation, have been investigated in cystic fibrosis (CF) patients. To further explore this nutritional approach, biological effects of an omega-3 PUFA oral liquid supplementation were measured in 17 CF patients in a double-blind, randomized, crossover without a washout period and placebo-controlled study. METHODS: CF patients (age: 18+/-9 year; weight: 43+/-13 kg) received a liquid dietary supplementation either enriched or not in omega-3 PUFA (390-1170 mg/day according to patient weight) during two 6-month periods. RESULTS: Increase in eicosapentaenoic acid was observed in neutrophil membrane following omega-3 PUFA dietary supplementation (from 0.7+/-0.6 to 1.6+/-0.6 micromol%, P<0.01). The leukotriene B(4) (LTB(4))/leukotriene B(5) (LTB(5)) ratio was decreased (from 72+/-27 to 24+/-7, P<0.001) in CF patients taking omega-3 PUFA supplements. In contrast, omega-3 PUFA supplementation affected neither internalization of IL-8 receptors following IL-8 exposure, nor IL-8-induced neutrophil chemotaxis. CONCLUSION: Our results show that omega-3 PUFA are incorporated in neutrophil membranes. The subsequent decrease in LTB(4)/LTB(5) ratio suggests that, in such conditions, neutrophils may produce less pro-inflammatory mediators from the acid arachidonic pathway. These data indicate that omega-3 PUFA intake may have anti-inflammatory effect that still need to be assessed by long-term studies following large groups of patients.