In-situ monitoring of xenobiotics using genetically engineered whole-cell-based microbial biosensors: recent advances and outlook.

Industrialisation, directly or indirectly, exposes humans to various xenobiotics. The increased magnitude of chemical pesticides and toxic heavy metals in the environment, as well as their intrusion into the food chain, seriously threatens human health. Therefore, the surveillance of xenobiotics is crucial for social safety and security. Online investigation by traditional methods is not sufficient for the detection and identification of such compounds because of the high costs and their complexity. Advancement in the field of genetic engineering provides a potential opportunity to use genetically modified microorganisms. In this regard, whole-cell-based microbial biosensors (WCBMB) represent an essential tool that couples genetically engineered organisms with an operator/promoter derived from a heavy metal-resistant operon combined with a regulatory protein in the gene circuit. The plasmid controls the expression of the reporter gene, such as gfp, luc, lux and lacZ, to an inducible gene promoter and has been widely applied to assay toxicity and bioavailability. This review summarises the recent trends in the development and application of microbial biosensors and the use of mobile genes for biomedical and environmental safety concerns.

Effective processing and evaluation of chemical imaging data with respect to morphological features of the zebrafish embryo.

A workflow was developed and implemented in a software tool for the automated combination of spatially resolved laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) data and data on the morphology of the biological tissue. Making use of a recently published biological annotation software, FishImager automatically assigns the biological feature as regions of interest (ROIs) and overlays them with the quantitative LA-ICP-MS data. Furthermore, statistical tools including cluster algorithms can be applied to the elemental intensity data and directly compared with the ROIs. This is effectively visualized in heatmaps. This allows gaining statistical significance on distribution and co-localization patterns. Finally, the biological functions of the assigned ROIs can then be easily linked with elemental distributions. We demonstrate the versatility of FishImager with quantitative LA-ICP-MS data of the zebrafish embryo tissue. The distribution of natural elements and xenobiotics is analyzed and discussed. With the help of FishImager, it was possible to identify compartments affected by toxicity effects or biological mechanisms to eliminate the xenobiotic. The presented workflow can be used for clinical and ecotoxicological testing, for example. Ultimately, it is a tool to simplify and reproduce interpretations of imaging LA-ICP-MS data in many applications.

A high-resolution MS/MS based strategy to improve xenobiotic metabolites analysis by metabolic pathway extension searching combined with parallel reaction monitoring: Flavonoid metabolism in wound site as a case.

For the analysis of xenobiotic metabolism, metabolites are commonly qualified by high-resolution mass spectrometry such as orbitrap or time-of-flight mass spectrometers, and quantified by triple-quadrupole (QQQ) mass spectrometer based multiple reaction monitoring. While this workflow shows drawback in the difficulty for instrumental parameters transfer, and QQQ provides less specificity. In this work, we constructed a high-resolution MS/MS (HR-MS/MS) based strategy to improve the discovery and quantification of unknown xenobiotic metabolites by metabolic pathway extension (MPE) searching combined with parallel reaction monitoring (PRM). Taking the flavonoid metabolism in diabetes wound S9 incubates as a test case. Firstly, MPE approach was used to screen all potential metabolites. In this step, an m/z value library of all theoretic flavonoid metabolites were constructed based on predefined flavonoid structures through 21 common xenobiotic metabolic reactions, and this library was matched with all features extracted from raw data (MS1 scan) of flavonoid-S9 co-incubate, then the matched features were exported into target list for MS2 fragmentation for structure validation. Secondly, the metabolites were relatively quantified by PRM mode based on their characteristic product ions. As a result, 131 metabolites of 9 different kinds of flavonoids in the skin and muscle were identified. To our best knowledge, this is the first report on the metabolism of flavonoids in the skin or muscle tissue. The results also validated the proposed HR-MS/MS-based strategy provided high specificity throughout both discovery and quantitation process of unknown xenobiotic metabolites without need of instrumental parameter transfer.

First characterization of multixenobiotic activity in Collembola: An approach on cadmium-induced response.

ATP-binding cassette (ABC) efflux pumps mediate the activity of the Multixenobiotic Resistance (MXR) mechanism and have been proposed as a biomarker of environmental pollution mainly in aquatic invertebrates. MXR activity was never investigated in Collembola and represents a potential tool for soil biomonitoring. This study aimed to characterize for the first time the activity of ABC efflux pumps in the gut of collembolan species, and investigate its responsiveness to cadmium (Cd), a common stressor found in polluted soils. We performed in vitro rhodamine-B accumulation assays in the presence of model inhibitors of ABC efflux pumps: verapamil hydrochloride as P-gp (P-glycoprotein) inhibitor, and MK571, as MRPs (multidrug resistance-related proteins) inhibitor. We also performed rhodamine-B accumulation assays under Cd-exposure (209 µg/L;1 µM). Our results showed that all species presented basal (noninduced) level of MXR activity in their gut. Efflux pumps P-gp and/or MRPs activity were confirmed in Cyphoderus innominatus, Cyphoderus similis, and Folsomia candida, the standard species. The rhodamine-B accumulation assays performed with Cd, applied as soil pollutant, showed that the gut of non-standard species C. similis and Trogolaphysa sp. presented an increase of MXR activity for both P-gp and MRP transporters, indicating the potential of these species as test organisms for soil ecotoxicology studies in Neotropical region. Our findings suggest a functional role of ABC transporters in the collembolan gut and their cellular involvement in Cd defense response, corroborating that MXR phenotype in Collembola can be a promising tool for bioindication of soil contamination.

Is It Realistic to Propose Determination of a Lifetime Internal Exposome?

Biomonitoring of xenobiotics has been performed for many years in occupational and environmental medicine. It has revealed hidden exposures and the exposure of workers could be reduced. Although most of the toxic effects of chemicals on humans were discovered in workers, the scientific community has more recently focused on environmental samples. In several countries, urinary and blood samples have been collected and analyzed for xenobiotics. Health, biochemical, and clinical parameters were measured in the biomonitoring program of the Unites States. The data were collected and evaluated as group values, comparing races, ages, and gender. The term exposome was created in order to relate chemical exposure to health effects together with the terms genome, proteome, and transcriptome. Internal exposures were mostly established with snapshot measurements, which can lead to an obvious misclassification of the individual exposures. Albumin and hemoglobin adducts of xenobiotics reflect the exposure of a larger time frame, up to 120 days. It is likely that only a small fraction of xenobiotics form such adducts. In addition, adduct analyses are more work intensive than the measurement of xenobiotics and metabolites in urine and/or blood. New technology, such as high-resolution mass spectrometry, will enable the discovery of new compounds that have been overlooked in the past, since over 300,000 chemicals are commercially available and most likely also present in the environment. Yet, quantification will be challenging, as it was for the older methods. At this stage, determination of a lifetime internal exposome is very unrealistic. Instead of an experimental approach with a large number of people, which is economically and scientifically not feasible, in silico methods should be developed further to predict exposure, toxicity, and potential health effects of mixtures. The computer models will help to focus internal exposure investigations on smaller groups of people and smaller number of chemicals.

Focused ultrasound-based extraction for target analysis and suspect screening of organic xenobiotics in fish muscle.

The development of multitarget and/or suspect screening methods for the analysis of xenobiotics in fish samples is compulsory due to the lack of works in the literature where a deep evaluation of the variables affecting extraction and clean-up steps is performed. The aim of the present work was to optimize and validate a multitarget (180 compounds) method for the analysis of priority and emerging xenobiotics in fish muscle using focused ultrasound-assisted solid-liquid extraction. From the different extraction solvents studied, a single extraction in cold acetonitrile rendered the best consensus results in terms of absolute recoveries and the number of target compounds extracted. Matrix effect was minimized using commercially available Captiva ND-Lipid filters, which provided clean extracts and satisfactory repeatability compared to other approaches. Absolute recoveries were corrected using matrix-matched calibration and apparent recoveries in the 43%-105%, 73%-131% and 78%-128% ranges were obtained at low (20 ng g-1), medium (100 ng g-1), and high (200 ng g-1) spiking levels, respectively. A 60% of the xenobiotics showed limits of identification lower than 20 ng g-1. The developed method was successfully applied to the quantification and suspect screening of samples bought in a local market (hake, gilt-head bream, sea bass and prawn) and fished (thicklip grey mullet) at the Urdaibai estuary (north of Spain). Food additives, antiparasitic drugs and PFOS were quantified at ng g-1 level. Moreover, the targeted method was extended to the suspect screening, revealing the presence of plastic related products (caprolactam, phthalates, polyethylenglycols), pharmaceutical products (albendazole, mebendazole, valpromide) and pesticides or insect repellents (icaridin, myristyl sulfate, nootkatone). Therefore, FUSLE in cold acetonitrile combined with Captiva ND-Lipid filters and liquid chromatography tandem high-resolution mass spectrometry (LC-q-Orbitrap) were successfully applied to both multitarget quantitative analysis and suspect screening of approx. 17,800 compounds.

Revisited: Therapeutic and toxic blood concentrations of more than 1100 drugs and other xenobiotics.

In order to assess the significance of drug/substance levels measured in intensive care medicine and clinical and forensic toxicology as well as for therapeutic drug monitoring, it is essential that a comprehensive collection of data is readily available. We revisited and expanded our 2012 compilation of therapeutic and toxic plasma concentration ranges as well as half-lives of now more than 1100 drugs and other xenobiotics.Data have been abstracted from original papers, text books, and previous compilations and have been completed with data collected in our own forensic and clinical toxicology laboratories. We compiled the data presented in the table and the corresponding annotations over the past 30+ years. A previous compilation was completely double-checked, revised, and updated, if necessary. In addition, more than 200 substances, especially drugs who have been introduced since 2012 to the market as well as illegal drugs and other xenobiotics which became known to cause intoxications were added. We carefully referenced all data. Moreover, the annotations providing details were updated and revised, when necessary.For more than 1100 drugs and other xenobiotics, therapeutic ("normal") and, if data was available, toxic, and comatose-fatal plasma/blood concentrations as well as elimination half-lives were compiled in a table.In case of intoxications, the blood concentration of the substance and/or metabolite better predicts the clinical severity of the case when compared to the assumed amount and time of ingestion. Comparing and contrasting the clinical case against the data provided, including the half-life, may support the decision for or against further intensive care. In addition, the data provided are useful for the therapeutic monitoring of pharmacotherapies, to facilitate the diagnostic assessment and monitoring of acute and chronic intoxications as well as to support forensic and clinical expert opinions.

Synergy between Experiments and Computations: A Green Channel for Revealing Metabolic Mechanism of Xenobiotics in Chemical Toxicology.

Xenobiotic metabolism can lead to metabolites with altered physicochemical and biological properties, which may differ markedly from those of their parent compounds. Thus, xenobiotic metabolism has great implication for chemical safety evaluation, which has become one of the central research areas in chemical toxicology. A plethora of analytical and in vitro methods are now available for investigating the metabolic fate of xenobiotics, especially by cytochrome P450 (CYP), at a high level of detail. However, the interpretations of metabolic reactions often face some mechanistic challenges, for example, the mechanism of the initial and rate-determining step is not easily distinguished due to the transient nature of active species of CYP, and some reactive intermediates are difficult to identify. Alternatively, computational chemistry methodologies such as quantum chemical calculations have the capacity to calculate the electronic structures for enzymatic models with hundreds of atoms, thus to be able to characterize intermediates and transition states during whole metabolic reaction course from both structural and energetics aspects, which can confront some major limitations of experimental methods. In this perspective, I first introduce state of the art experimental and computational approaches for investigating xenobiotic metabolism catalyzed by CYP, respectively. Then the strategies to harvest the synergy between experiments and computations are highlighted, which can be conducted through comparison of their analytical, kinetic, or isotope effect data at a qualitative, semiquantitative, or quantitative level to determine the metabolic mechanism. Two examples are chosen to demonstrate the synergy advantage to elucidate the metabolic mechanism of triphenyl phosphate and atrazine catalyzed by CYP, respectively, which show that the interplay between experiments and computations allows greater insight to be gained than with the isolated methods.

Utilizing Drift Tube Ion Mobility Spectrometry for the Evaluation of Metabolites and Xenobiotics.

Metabolites and xenobiotics are small molecules with a molecular weight that often falls below 600 Da. Over the last few decades, multiple small molecule databases have been curated listing structures, masses, and fragmentation spectra possible in metabolomic and exposomic measurements. To date only a small portion of the spectra in these databases are experimentally derived due to the high expense of obtaining, synthesizing, and analyzing standards. A vast majority of spectra have thus been created using theoretical programs to fit the available experimental data. The errors associated with theoretical data have however caused problems with current small molecule identifications, and accurate quantitation as searching the databases using just one or two analysis dimensions (i.e., chromatography retention times and mass spectrometry (MS) m/z values) results in numerous annotations for each experimental feature. Additional analysis dimensions are therefore needed to better annotate and identify small molecules. Drift tube ion mobility spectrometry coupled with MS (DTIMS-MS) is a promising technique to address this challenge as it is able to perform rapid structural evaluations of small molecules in complex matrices by assessing the collision cross section values for each in addition to their m/z values. The use of IMS in conjunction with other separation techniques such as gas or liquid chromatography and MS has therefore enabled more accurate identifications for the small molecules present in complex biological and environmental samples. Here, we present a review of relevant parameter considerations for DTIMS application with emphasis on xenobiotics and metabolomics isomer separations.

Co-Occurrence and Combinatory Effects of Alternaria Mycotoxins and other Xenobiotics of Food Origin: Current Scenario and Future Perspectives.

Mycotoxins are low-molecular weight compounds produced by diverse genera of molds that may contaminate food and feed threatening the health of humans and animals. Recent findings underline the importance of studying the combined occurrence of multiple mycotoxins and the relevance of assessing the toxicity their simultaneous exposure may cause in living organisms. In this context, for the first time, this work has critically reviewed the most relevant data concerning the occurrence and toxicity of mycotoxins produced by Alternaria spp., which are among the most important emerging risks to be assessed in food safety, alone or in combination with other mycotoxins and bioactive food constituents. According to the literature covered, multiple Alternaria mycotoxins may often occur simultaneously in contaminated food, along with several other mycotoxins and food bioactives inherently present in the studied matrices. Although the toxicity of combinations naturally found in food has been rarely assessed experimentally, the data collected so far, clearly point out that chemical mixtures may differ in their toxicity compared to the effect of toxins tested individually. The data presented here may provide a solid foothold to better support the risk assessment of Alternaria mycotoxins highlighting the actual role of chemical mixtures on influencing their toxicity.

A Generic Liquid Chromatography-Tandem Mass Spectrometry Exposome Method for the Determination of Xenoestrogens in Biological Matrices.

We are constantly exposed to a variety of environmental contaminants and hormones, including those mimicking endogenous estrogens. These highly heterogeneous molecules are collectively referred to as xenoestrogens and hold the potential to affect and alter the delicate hormonal balance of the human body. To monitor exposure and investigate potential health implications, comprehensive analytical methods covering all major xenoestrogen classes are needed but not available to date. Herein, we describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of multiple classes of endogenous as well as exogenous estrogens in human urine, serum, and breast milk to enable proper exposure and risk assessment. In total, 75 analytes were included, whereof a majority was successfully in-house validated in the three matrices. Extraction recoveries of validated analytes ranged from 71% to 110% and limits of quantification from 0.015 to 5 µg/L, 0.03 to 14 µg/L, and 0.03 to 4.6 µg/L in urine, serum, and breast milk, respectively. The applicability of the novel method was demonstrated in proof-of-principle experiments by analyzing urine from Austrian individuals and breast milk from Austrian and Nigerian individuals. Thereby, we proved the methods' feasibility to identify and quantify different classes of xenoestrogens simultaneously. The results illustrate the general importance of multiclass exposure assessment in the context of the exposome paradigm. Specifically, they highlight the need for estimating total estrogenic burden rather than single analyte or chemical class measurements and its potential impact in endocrine disruption and hormone related diseases including cancers.

Residues of xenobiotics in the environment and phytotoxic activity in Armenia.

The article presents the results of a dynamic study (spring, summer, and fall) of the residues of certain xenobiotics such as organochlorine pesticides (OCPs), synthetic surfactants (SSs) in surface water, soil, sludge, snow and phytotoxic activity in Ararat and Lori marzes of Armenia (2016-2017). A comparative analysis of the environmental status showed that all pollutants studied such as γ-hexachlorocyclohexane (γ-HCH), 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (4,4'-DDT), and p-p'-dichlorodiphenyldichloroethylene (4,4'-DDE) were identified in Ararat marz with the average concentrations and detection rates higher than in Lori marz. The prominent contaminant was γ-HCH. The findings indicated the concentrations of OCPs below of regulatory concern. In Ararat marz an increase in the number of sterile pollen grains of certain wild plants was revealed (14.8-26.0%), compared with Lori marz with the levels within the contingent standard of 5-11%. The phytotoxic activity of soil samples from the Ararat marz studied on Avena sativa seedlings was significantly higher in the summer-fall period. These data correlated with monitoring findings showed an increase in the levels of γ-HCH, 4,4'-DDT, SS in the soil and sludge sampled in summer-fall in Ararat marz. This difference in the environmental status can be considered as the result of active agriculture in Ararat marz, whose share of contribution to a relevant branch of the economy of Armenia is two times higher than in Lori marz, 15.4% and 7.9%, respectively. Mentioned rates of agricultural production may be one of the reasons for the environmental deterioration in Ararat marz. The detection of 4,4-DDT, γ-HCH residues testifies the circulation of these formulations in the environment indicating their use in recent past and confirming their persistence. Although since the 70s of the last century the application of OCPs in the Republic of Armenia has been prohibited. The current situation may be explained by low awareness of farmers on different issues related to the safe management of pesticides. To ensure safe working conditions and raise awareness among the farmers we have developed "Recommendations on Safety requirements when working with pesticides" that are approved by the State Service for Food Safety at the Ministry of Agriculture of RA and included in the reference booklets for farmers as guiding information. Our study shows the understanding of associations between the deterioration of the environmental status and share of agriculture contribution to the economy that provides the evidence for future research programs.

Development and validation of an analytical procedure to detect spatio-temporal differences in antidepressant use through a wastewater-based approach.

Wastewater-based epidemiology applies the analysis of human metabolic excretion products of xenobiotics in wastewater to estimate the community-wide use of these compounds. A new bioanalytical method was developed, optimised and validated for the analysis of a broad range of antidepressants and their metabolites at trace concentrations in influent wastewater. The assay was based on solid-phase extraction and liquid chromatography coupled to tandem mass spectrometry. For most compounds, Oasis® HLB cartridges were used for sample preparation. Oasis® MCX cartridges were used for extraction of normirtazapine, moclobemide, sertraline, and melitracen in particular. The Kinetex XBC18 column with a gradient elution resulted in appropriate separation for the analytes under investigation. Validation was done according to the European Medicines Agency guidelines on bioanalytical method validation. For 27 compounds, the performance criteria met the requirements for method validation. For these analytes, the lower limit of quantification (LLOQ) ranged between 1 and 25 ng/L. Furthermore, all targeted biomarkers showed high in-sample stability during 24 h, with the exception of mianserin. The validated assay was applied to influent wastewater samples collected from four wastewater treatment plants in Belgium. Among these four locations, a total of 18 out of 27 biomarkers for antidepressant use were present in the samples in concentrations above the LLOQ. Additionally, the proposed methodology proved capable of analysing high resolution spatio-temporal trends. Mann-Kendall trend analyses showed that antidepressant use is stable throughout the week, except for trazodone which increased throughout the week.

The metagenomic landscape of xenobiotics biodegradation in mangrove sediments.

Metagenomics is a powerful approach to study microorganisms present in any given environment and their potential to maintain and improve ecosystem health without the need of cultivating these microorganisms in the laboratory. In this study, we combined a cultivation-independent metagenomics approach with functional assays to identify the detoxification potential of microbial genes evaluating their potential to contribute to xenobiotics resistance in oil-impacted mangrove sediments. A metagenomic fosmid library containing 12,960 clones from highly contaminated mangrove sediment was used in this study. For assessment of metal resistance, clones were grown in culture medium with increasing concentrations of mercury. The analyses metagenomic library sequences revealed the presence of genes related to heavy metals and antibiotics resistance in the oil-impacted mangrove microbiome. The taxonomic profiling of these sequences suggests that at the genus level, Geobacter was the most abundant genus in our dataset. A functional screening assessment of the metagenomic library successfully detected 24 potential heavy metal tolerant clones, six of which were capable of growing with increased concentrations of mercury. The genetic characterization of selected clones allowed the detection of genes related to detoxification processes, such as chromate transport protein ChrA, haloacid dehalogenase-like hydrolase, lipopolysaccharide transport system, and 3-oxoacyl-[acyl-carrier-protein] reductase. Clones were capable of growing in medium containing increased concentrations of metals and antibiotics, but none manifested strong mercury removal from culture medium characteristic of mercuric reductase activity. These results suggest that resistance to xenobiotic stress varies greatly and that additional studies to elucidate the potential of metal biotransformation need to be carried out with the goal of improving bioremediation application.

The influence of progesterone on bovine uterine fluid energy, nucleotide, vitamin, cofactor, peptide, and xenobiotic composition during the conceptus elongation-initiation window.

Conceptus elongation coincides with one of the periods of greatest pregnancy loss in cattle and is characterized by rapid trophectoderm expansion, commencing ~ Day 13 of pregnancy, i.e. before maternal pregnancy recognition. The process has yet to be recapitulated in vitro and does not occur in the absence of uterine gland secretions in vivo. Moreover, conceptus elongation rates are positively correlated to systemic progesterone in maternal circulation. It is, therefore, a maternally-driven and progesterone-correlated developmental phenomenon. This study aimed to comprehensively characterize the biochemical composition of the uterine luminal fluid on Days 12-14 - the elongation-initiation window - in heifers with normal vs. high progesterone, to identify molecules potentially involved in conceptus elongation initiation. Specifically, nucleotide, vitamin, cofactor, xenobiotic, peptide, and energy metabolite profiles of uterine luminal fluid were examined. A total of 59 metabolites were identified, of which 6 and 3 displayed a respective progesterone and day effect, whereas 16 exhibited a day by progesterone interaction, of which 8 were nucleotide metabolites. Corresponding pathway enrichment analysis revealed that pyridoxal, ascorbate, tricarboxylic acid, purine, and pyrimidine metabolism are of likely importance to to conceptus elongation initiation. Moreover, progesterone reduced total metabolite abundance on Day 12 and may alter the uterine microbiome.

Uric acid analogue as a possible xenobiotic marker of uric acid transporter Urat1 in rats.

The inhibitor of uric acid reabsorptive transporter URAT1 in kidney is drawing attention as a drug target for hyperuricemia. However, it is difficult to evaluate efficacy of URAT1 inhibitors in vivo using laboratory animals due to species difference in uric acid metabolism. In the present study, the usefulness of exogenously administering uric acid analogues resistant to uricase was investigated for in vivo evaluation of transport activity of rUrat1 in rats. Uptake of examined four uric acid analogues by rUrat1-expressing Xenopus oocytes was significantly higher than that by water-injected oocytes. In metabolism studies, disappearance of these compounds was negligible, while uric acid was significantly decreased. When oxypurinol was administered to rats, fractional excretion (FE) was 0.4, suggesting reabsorption of oxypurinol. Moreover, FE of oxypurinol was tended to be increased, but not statistically different, by co-administration of a uricosuric agent FYU-981, while plasma concentration of oxypurinol was not affected. These results suggested that oxypurinol is a potential uric acid analogue, although it was not suitable as a probe of uric acid in in vivo study. Our findings may contribute to discovery and development of novel uricosuric agent targeting URAT1.

Ultrasound assisted dispersive liquid-liquid microextraction for fast and accurate analysis of chloramphenicol in honey.

Honey is a food produced from honey bee widely used for the sweetening power and for its biological properties. In order to prevent the infection of the hive, different xenobiotics (antibiotics, pesticide) were frequently employed. One of these substances is the chloramphenicol, that given its chemical stability could often found in food. Chloramphenicol have several side effects in humans after their ingestion and for this reason their intake must be avoid. The aim of this study, was developed an ultrasound-assisted dispersive liquid-liquid microextraction method coupled with UHPLC MS/MS determination, for fast and accurate analysis of chloramphenicol in honey. The parameters affecting on extraction efficiency were carefully optimized using an experimental design in order to maximized the recovery reducing matrix effects. After the optimization the method was validated and successfully applied to 66 honey samples.

EPA's non-targeted analysis collaborative trial (ENTACT): genesis, design, and initial findings.

In August 2015, the US Environmental Protection Agency (EPA) convened a workshop entitled "Advancing non-targeted analyses of xenobiotic chemicals in environmental and biological media." The purpose of the workshop was to bring together the foremost experts in non-targeted analysis (NTA) to discuss the state-of-the-science for generating, interpreting, and exchanging NTA measurement data. During the workshop, participants discussed potential designs for a collaborative project that would use EPA resources, including the ToxCast library of chemical substances, the DSSTox database, and the CompTox Chemicals Dashboard, to evaluate cutting-edge NTA methods. That discussion was the genesis of EPA's Non-Targeted Analysis Collaborative Trial (ENTACT). Nearly 30 laboratories have enrolled in ENTACT and used a variety of chromatography, mass spectrometry, and data processing approaches to characterize ten synthetic chemical mixtures, three standardized media (human serum, house dust, and silicone band) extracts, and thousands of individual substances. Initial results show that nearly all participants have detected and reported more compounds in the mixtures than were intentionally added, with large inter-lab variability in the number of reported compounds. A comparison of gas and liquid chromatography results shows that the majority (45.3%) of correctly identified compounds were detected by only one method and 15.4% of compounds were not identified. Finally, a limited set of true positive identifications indicates substantial differences in observable chemical space when employing disparate separation and ionization techniques as part of NTA workflows. This article describes the genesis of ENTACT, all study methods and materials, and an analysis of results submitted to date. Graphical abstract ᅟ.

Xenobiotic biotransformation, oxidative stress and obesogenic molecular biomarker responses in Tilapia guineensis from Eleyele Lake, Nigeria.

Despite the important ecosystem and hydrological roles of coastal and inland waters, there are no established biomonitoring protocols for evaluating environmental, wildlife and human health for these coastlines in Nigeria. In the present study, contaminants tissue burden and effects at molecular and physiological levels, were investigated in Tilapia guineensis from a man-made lake (Eleyele Lake) that is used for municipal domestic water supply and compared to a reference site (Igboho Lake). Gene expression of phase I and II biotransformation systems, oxidative stress and obesogen responses were analyzed using real-time PCR, and these results were related to general health index (condition factor: CF) and muscle burden of trace metals, aliphatic and polycyclic aromatic hydrocarbons (PAHs). We observed a significant increase in phase I and II biotransformation systems, oxidative stress and obesogen responses in male and female fish from Eleyele lake compared with the reference site. Overall, our data showed significant relationships between biological responses and tissue concentrations of metals and PAHs for the Eleyele lake compared with the reference site. Given that a positive influence on genes and pathways associated with metabolic status has been previously associated with peroxisome proliferator activated receptors (PPARs), xenobiotic compounds that activate PPARs may produce changes in energy and metabolic processes, leading to obesity. The high CF (>1 = good health condition) observed, coupled with the high muscle burden of inorganic and organic contaminants in fish from Eleyele lake, suggest a potential obesogenic effect in these fishes. These findings represent co-relational evidence that the Eleyele lake is contaminated and consequently affecting biological and physiological integrity of organisms inhabiting the lake. These findings also suggest potential health risks for humans, since the lake is extensively used for domestic water supply and fisheries.