Biotransformation of fluorinated drugs and xenobiotics by the model fungus Cunninghamella elegans.

Some species of the genus Cunninghamella (C. elegans, C. echinulata and C. blaskesleeana) produce the same phase I and phase II metabolites when incubated with xenobiotics as mammals, and thus are considered microbial models of mammalian metabolism. This had made these fungi attractive for metabolism studies with drugs, pesticides and environmental pollutants. As a substantial proportion of pharmaceuticals and agrochemicals are fluorinated, their biotransformation has been studied in Cunninghamella fungi and C. elegans in particular. This article details the methods employed for cultivating the fungi in planktonic and biofilm cultures, and extraction and analysis of fluorinated metabolites. Furthermore, protocols for the heterologous expression of Cunninghamella cytochromes P450 (CYPs), which are the enzymes associated with phase I metabolism, are described.

Substrate promiscuity of xenobiotic-transforming hydrolases from stream biofilms impacted by treated wastewater.

Organic contaminants enter aquatic ecosystems from various sources, including wastewater treatment plant effluent. Freshwater biofilms play a major role in the removal of organic contaminants from receiving water bodies, but knowledge of the molecular mechanisms driving contaminant biotransformations in complex stream biofilm (periphyton) communities remains limited. Previously, we demonstrated that biofilms in experimental flume systems grown at higher ratios of treated wastewater (WW) to stream water displayed an increased biotransformation potential for a number of organic contaminants. We identified a positive correlation between WW percentage and biofilm biotransformation rates for the widely-used insect repellent, N,N-diethyl-meta-toluamide (DEET) and a number of other wastewater-borne contaminants with hydrolyzable moieties. Here, we conducted deep shotgun sequencing of flume biofilms and identified a positive correlation between WW percentage and metagenomic read abundances of DEET hydrolase (DH) homologs. To test the causality of this association, we constructed a targeted metagenomic library of DH homologs from flume biofilms. We screened our complete metagenomic library for activity with four different substrates, including DEET, and a subset thereof with 183 WW-related organic compounds. The majority of active hydrolases in the metagenomic library preferred aliphatic and aromatic ester substrates while, remarkably, only a single reference enzyme was capable of DEET hydrolysis. Of the 626 total enzyme-substrate combinations tested, approximately 5% were active enzyme-substrate pairs. Metagenomic DH family homologs revealed a broad substrate promiscuity spanning 22 different compounds when summed across all enzymes tested. We biochemically characterized the most promiscuous and active enzymes identified based on metagenomic analysis from uncultivated Rhodospirillaceae and Planctomycetaceae. In addition to characterizing new DH family enzymes, we exemplified a framework for linking metagenome-guided hypothesis generation with experimental validation. Overall, this study expands the scope of known enzymatic contaminant biotransformations for metagenomic hydrolases from WW-receiving stream biofilm communities.

Metagenomic landscape of sediments of river Ganga reveals microbial diversity, potential plastic and xenobiotic degradation enzymes.

The Ganga is the largest river in India, serves as a lifeline for agriculture, drinking water, and religious rites. However, it became highly polluted due to the influx of industrial wastes and untreated sewages, leading to the decline of aquatic biodiversity. This study investigated the microbial diversity and plastic-xenobiotic degrading enzymes of six sediment metagenomes of river Ganga at Prayagraj (RDG, TSG, SDG) and Devprayag (KRG, BNG, BRG). The water quality parameters, higher values of BOD (1.8-3.7 ppm), COD (23-29.2 ppm) and organic carbon (0.18-0.51%) were recorded at Prayagraj. Comparative analysis of microbial community structure between Prayagraj and Devprayag revealed significant differences between Bacteroidetes and Firmicutes, which emerging as the predominant bacterial phyla across six sediment samples. Notably, their prevalence was highest in the BRG samples. Furthermore, 25 OTUs at genus level were consistent across all six samples. Alpha diversity exhibited minimal variation among samples, while beta diversity indicated an inverse relationship between species richness and diversity. Co-occurrence network analysis established that genera from the same and different groups of phyla show positive co-relations with each other. Thirteen plastic degrading enzymes, including Laccase, Alkane-1 monooxygenase and Alkane monooxygenase, were identified from six sediment metagenomes of river Ganga, which can degrade non-biodegradable plastic viz. Polyethylene, Polystyrene and Low-density Polyethelene. Further, 18 xenobiotic degradation enzymes were identified for the degradation of Bisphenol, Xylene, Toluene, Polycyclic aromatic hydrocarbon, Styrene, Atrazene and Dioxin etc. This is the first report on the identification of non-biodegradable plastic degrading enzymes from sediment metagenomes of river Ganga, India. The findings of this study would help in pollution abatement and sustainable management of riverine ecosystem.

Interaction with B-type lamin reveals the function of Drosophila Keap1 xenobiotic response factor in nuclear architecture.

BACKGROUND: The Keap1-Nrf2 pathway serves as a central regulator that mediates transcriptional responses to xenobiotic and oxidative stimuli. Recent studies have shown that Keap1 and Nrf2 can regulate transcripts beyond antioxidant and detoxifying genes, yet the underlying mechanisms remain unclear. Our research has uncovered that Drosophila Keap1 (dKeap1) and Nrf2 (CncC) proteins can control high-order chromatin structure, including heterochromatin. METHODS AND RESULTS: In this study, we identified the molecular interaction between dKeap1 and lamin Dm0, the Drosophila B-type lamin responsible for the architecture of nuclear lamina and chromatin. Ectopic expression of dKeap1 led to an ectopic localization of lamin to the intra-nuclear area, corelated with the spreading of the heterochromatin marker H3K9me2 into euchromatin regions. Additionally, mis-regulated dKeap1 disrupted the morphology of the nuclear lamina. Knocking down of dKeap1 partially rescued the lethality induced by lamin overexpression, suggesting their genetic interaction during development. CONCLUSIONS: The discovered dKeap1-lamin interaction suggests a novel role for the Keap1 oxidative/xenobiotic response factor in regulating chromatin architecture.

Detection and Imaging of Exposure-Related Metabolites and Xenobiotics in Hard Tissues by Laser Sampling and Mass Spectrometry.

The utility of two novel laser-based methods, laser ablation electrospray ionization (LAESI) and laser desorption ionization (LDI) from silicon nanopost array (NAPA), is explored via local analysis and mass spectrometry imaging (MSI) of hard tissues (tooth and hair) for the detection and mapping of organic components. Complex mass spectra are recorded in local analysis mode from tooth dentin and scalp hair samples. Nicotine and its metabolites (cotinine, hydroxycotinine, norcotinine, and nicotine) are detected by LAESI-MS in the teeth of rats exposed to tobacco smoke. The intensities of the detected metabolite peaks are proportional to the degree of exposure. Incorporating ion mobility separation in the LAESI-MS analysis of scalp hair enables the detection of cotinine in smoker hair along with other common molecular species, including endogenous steroid hormones and some lipids. Single hair strands are imaged by MALDI-MSI and NAPA-LDI-MSI to explore longitudinal variations in the level of small molecules. Comparing spectra integrated from NAPA-LDI-MSI and MALDI-MSI images reveals that the two techniques provide complementary information. There were 105 and 82 sample-related peaks for MALDI and NAPA, respectively, with an overlap of only 16 peaks, indicating a high degree of complementarity. Enhanced molecular coverage and spatial resolution offered by LAESI-MS and NAPA-LDI-MSI can reveal the distributions of known and potential biomarkers in hard tissues, facilitating exposome research.

The response of Anisakis simplex (s. s.) to anthelmintics - Specific changes in xenobiotic metabolic processes.

Anisakiasis is a parasitic disease transmitted through the consumption of raw or undercooked fish and cephalopods that are infected with larvae of Anisakis simplex (sensu stricto) or Anisakis pegreffii. The purpose of this study was to investigate how A. simplex (s. s.) responds to the influence of anthelmintics such as ivermectin (IVM) and pyrantel (PYR). In vitro experiments were conducted using larvae at two developmental stages of A. simplex (s. s.) (L3 and L4) obtained from Baltic herring (Clupea harengus membras). Larvae were cultured with different concentrations of IVM or PYR (1.56, 3.125, and 6.25 µg/mL) for various durations (3, 6, 9, and 12 h) under anaerobic conditions (37 °C, 5% CO2). The gene expression of actin, ABC transporter, antioxidant enzymes, γ-aminobutyric acid receptors, and nicotinic acetylcholine receptors, as well as the oxidative status were analyzed. The results showed that A. simplex (s. s.) L3 stage had lower mobility when cultured with PYR compared to IVM. The analysis of relative gene expression revealed significant differences in the mRNA level of ABC transporters after treatment with IVM and PYR, compared to the control group. Similar patterns were observed in the gene expression of antioxidant enzymes in response to both drugs. Furthermore, the total antioxidant capacity (TAC) and glutathione S-transferase (GST) activity were higher in the treatment groups than in the control group. These findings suggest a relationship between the expression of the studied genes, including those related to oxidative metabolism, and the effectiveness of the tested drugs.

MetSim: Integrated Programmatic Access and Pathway Management for Xenobiotic Metabolism Simulators.

Xenobiotic metabolism is a key consideration in evaluating the hazards and risks posed by environmental chemicals. A number of software tools exist that are capable of simulating metabolites, but each reports its predictions in a different format and with varying levels of detail. This makes comparing the performance and coverage of the tools a practical challenge. To address this shortcoming, we developed a metabolic simulation framework called MetSim, which comprises three main components. A graph-based schema was developed to allow metabolism information to be harmonized. The schema was implemented in MongoDB to store and retrieve metabolic graphs for subsequent analysis. MetSim currently includes an application programming interface for four metabolic simulators: BioTransformer, the OECD Toolbox, EPA's chemical transformation simulator (CTS), and tissue metabolism simulator (TIMES). Lastly, MetSim provides functions to help evaluate simulator performance for specific data sets. In this study, a set of 112 drugs with 432 reported metabolites were compiled, and predictions were made using the 4 simulators. Fifty-nine of the 112 drugs were taken from the Small Molecule Pathway Database, with the remainder sourced from the literature. The human models within BioTransformer and CTS (Phase I only) and the rat models within TIMES and the OECD Toolbox (Phase I only) were used to make predictions for the chemicals in the data set. The recall and precision (recall, precision) ranked in order of highest recall for each individual tool were CTS (0.54, 0.017), BioTransformer (0.50, 0.008), Toolbox in vitro (0.40, 0.144), TIMES in vivo (0.40, 0.133), Toolbox in vivo (0.40, 0.118), and TIMES in vitro (0.39, 0.128). Combining all of the model predictions together increased the overall recall (0.73, 0.008). MetSim enabled insights into the performance and coverage of in silico metabolic simulators to be more efficiently derived, which in turn should aid future efforts to evaluate other data sets.

Physiologically based kinetic (PBK) modeling as a new approach methodology (NAM) for predicting systemic levels of gut microbial metabolites.

There is a clear need to develop new approach methodologies (NAMs) that combine in vitro and in silico testing to reduce and replace animal use in chemical risk assessment. Physiologically based kinetic (PBK) models are gaining popularity as NAMs in toxico/pharmacokinetics, but their coverage of complex metabolic pathways occurring in the gut are incomplete. Chemical modification of xenobiotics by the gut microbiome plays a critical role in the host response, for example, by prolonging exposure to harmful metabolites, but there is not a comprehensive approach to quantify this impact on human health. There are examples of PBK models that have implemented gut microbial biotransformation of xenobiotics with the gut as a dedicated metabolic compartment. However, the integration of microbial metabolism and parameterization of PBK models is not standardized and has only been applied to a few chemical transformations. A challenge in this area is the measurement of microbial metabolic kinetics, for which different fermentation approaches are used. Without a standardized method to measure gut microbial metabolism ex vivo/in vitro, the kinetic constants obtained will lead to conflicting conclusions drawn from model predictions. Nevertheless, there are specific cases where PBK models accurately predict systemic concentrations of gut microbial metabolites, offering potential solutions to the challenges outlined above. This review focuses on models that integrate gut microbial bioconversions and use ex vivo/in vitro methods to quantify metabolic constants that accurately represent in vivo conditions.

Molecular and functional characterization of heat-shock protein 70 in Aphis gossypii under thermal and xenobiotic stresses.

Aphis gossypii, a globally distributed and economically significant pest of several crops, is known to infest a wide range of host plants. Heat shock proteins (Hsps), acting as molecular chaperones, are essential for the insect's environmental stress responses. The present study investigated the molecular characteristics and expression patterns of AgHsp70, a heat shock protein gene, in Aphis gossypii. Our phylogenetic analysis revealed that AgHsp70 shared high similarity with homologs from other insects, suggesting a conserved function across species. The developmental expression profiles of AgHsp70 in A. gossypii showed that the highest transcript levels were observed in the fourth instar nymphs, while the lowest levels were detected in the third instar nymphs. Heat stress and exposure to four different xenobiotics (2-tridecanone, tannic acid, gossypol, and flupyradifurone (4-[(2,2-difluoroethyl)amino]-2(5H)-furanone)) significantly up-regulated AgHsp70 expression. Knockdown of AgHsp70 using RNAi obviously increased the susceptibility of cotton aphids to 2-tridecanone, gossypol and flupyradifurone. Dual-luciferase reporter assays revealed that gossypol and flupyradifurone significantly enhanced the promoter activity of AgHsp70 at a concentration of 10 mg/L. Furthermore, we identified the transcription factor heat shock factor (HSF) as a regulator of AgHsp70, as silencing AgHSF reduced AgHsp70 expression. Our results shed light on the role of AgHsp70 in xenobiotic adaptation and thermo-tolerance.

Comparing the oxidative functions of neutrophil myeloperoxidase and cytochrome P450 enzymes in drug metabolism.

Drug metabolism is an essential process that chemically alters xenobiotic substrates to activate or terminate drug activity. Myeloperoxidase (MPO) is a neutrophil-derived haem-containing enzyme that is involved in killing invading pathogens, although consequentially, this same oxidative activity can produce metabolites that damage host tissue and play a role in various human pathologies. Cytochrome P450s (CYPs) are a superfamily of haem-containing enzymes that are significantly involved in the metabolism of drugs by functioning as monooxygenases and can be induced or inhibited, resulting in significant drug-drug interactions that lead to unanticipated adverse drug reactions. In this review, the functions of drug metabolism of MPO and CYPs are explored, along with their involvement and association for common enzymatic pathways by certain xenobiotics. MPO and CYPs metabolize numerous xenobiotics, although few reported studies have made a direct comparison between both enzymes. Additionally, we employed molecular docking to compare the active site and haem prosthetic group of MPO and CYPs, supporting their similar catalytic activities. Furthermore, we performed LCMS analysis and observed a shared hydroxylated mefenamic acid metabolite produced in both enzymatic systems. A proper understanding of the enzymology and mechanisms of action of MPO and CYPs is of significant importance when enhancing the beneficial functions of drugs in health and diminishing their damaging effects on diseases. Therefore, awareness of drugs and xenobiotic substrates involved in MPO and CYPs metabolism pathways will add to the knowledge base to foresee and prevent potential drug interactions and adverse events.

Transcriptomic profile of human iPSC-derived podocyte-like cells exposed to a panel of xenobiotics.

Podocytes play a critical role in the formation and maintenance of the glomerular filtration barrier and injury to these cells can lead to a breakdown of the glomerular barrier causing permanent damage leading to progressive chronic kidney disease. Matured podocytes have little proliferative potential, which makes them critical cells from a health perspective, but also challenging cells to maintain in vitro. Differentiating podocyte-like cells from induced pluripotent stem cells (iPSC) provides a novel and continuous source of cells. Here, we investigated the effect of a 24-h exposure to eight compounds, including the known glomerular toxins doxorubicin and pamidronate, on transcriptomic alterations in iPSC derived podocytes. Doxorubicin (50 nM), pamidronate (50 µM), sodium arsenite (10 µM), and cyclosporine A (15 µM) had a strong impact on the transcriptome, gentamicin (450 µg/ml), lead chloride (15 µM) and valproic acid (500 µM) had a mild impact and busulfan (50 µM) exhibited no impact. Gene alterations and pathways analysis provided mechanistic insight for example, doxorubicin exposure affected the p53 pathway and dedifferentiation, pamidronate activated several pathways including HIF1alpha and sodium arsenite up-regulated oxidative stress and metal responses. The results demonstrate the applicability of iPSC derived podocytes for toxicological and mechanistic investigations.

Can xenobiotics support the growth of Mn(II)-oxidizing bacteria (MnOB)? A case of phenol-utilizing bacteria Pseudomonas sp. AN-1.

Biogenic manganese oxides (BioMnOx) produced by Mn(II)-oxidizing bacteria (MnOB) have garnered considerable attention for their exceptional adsorption and oxidation capabilities. However, previous studies have predominantly focused on the role of BioMnOx, neglecting substantial investigation into MnOB themselves. Meanwhile, whether the xenobiotics could support the growth of MnOB as the sole carbon source remains uncertain. In this study, we isolated a strain termed Pseudomonas sp. AN-1, capable of utilizing phenol as the sole carbon source. The degradation of phenol took precedence over the accumulation of BioMnOx. In the presence of 100 mg L-1 phenol and 100 µM Mn(II), phenol was entirely degraded within 20 h, while Mn(II) was completely oxidized within 30 h. However, at the higher phenol concentration (500 mg L-1), phenol degradation reduced to 32% and Mn(II) oxidation did not appear to occur. TOC determination confirmed the ability of strain AN-1 to mineralize phenol. Based on the genomic and proteomics studies, the Mn(II) oxidation and phenol mineralization mechanism of strain AN-1 was further confirmed. Proteome analysis revealed down-regulation of proteins associated with Mn(II) oxidation, including MnxG and McoA, with increasing phenol concentration. Notably, this study observed for the first time that the expression of Mn(II) oxidation proteins is modulated by the concentration of carbon sources. This work provides new insight into the interaction between xenobiotics and MnOB, thus revealing the complexity of biogeochemical cycles of Mn and C.

CF2-II Alternative Splicing Isoform Regulates the Expression of Xenobiotic Tolerance-Related Cytochrome P450 CYP6CY22 in Aphis gossypii Glover.

The expression of P450 genes is regulated by trans-regulatory factors or cis-regulatory elements and influences how endogenous or xenobiotic substances are metabolized in an organism's tissues. In this study, we showed that overexpression of the cytochrome P450 gene, CYP6CY22, led to resistance to cyantraniliprole in Aphis gossypii. The expression of CYP6CY22 increased in the midgut and remaining carcass of the CyR strain, and after repressing the expression of CYP6CY22, the mortality of cotton aphids increased 2.08-fold after exposure to cyantraniliprole. Drosophila ectopically expressing CYP6CY22 exhibited tolerance to cyantraniliprole and cross-tolerance to xanthotoxin, quercetin, 2-tridecanone, tannic acid, and nicotine. Moreover, transcription factor CF2-II (XM_027994540.2) is transcribed only as the splicing variant isoform CF2-II-AS, which was found to be 504 nucleotides shorter than CF2-II in A. gossypii. RNAi and yeast one-hybrid (Y1H) results indicated that CF2-II-AS positively regulates CYP6CY22 and binds to cis-acting element p (-851/-842) of CYP6CY22 to regulate its overexpression. The above results indicated that CYP6CY22 was regulated by the splicing isoform CF2-II-AS, which will help us further understand the mechanism of transcriptional adaption of cross-tolerance between synthetic insecticides and plant secondary metabolites mediated by P450s.

A serum-induced gene signature in hepatocytes is associated with pediatric nonalcoholic fatty liver disease.

OBJECTIVE: Pediatric nonalcoholic fatty liver disease (NAFLD) is a growing problem, but its underlying mechanisms are poorly understood. We used transcriptomic reporter cell assays to investigate differences in transcriptional signatures induced in hepatocyte reporter cells by the sera of children with and without NAFLD. METHODS: We studied serum samples from 45 children with NAFLD and 28 children without NAFLD. The sera were used to induce gene expression in cultured HepaRG cells and RNA-sequencing was used to determine gene expression. Computational techniques were used to compare gene expression patterns. RESULTS: Sera from children with NAFLD induced the expression of 195 genes that were significantly differentially expressed in hepatocytes compared to controls with obesity. NAFLD was associated with increased expression of genes promoting inflammation, collagen synthesis, and extracellular matrix remodeling. Additionally, there was lower expression of genes involved in endobiotic and xenobiotic metabolism, and downregulation of peroxisome function, oxidative phosphorylation, and xenobiotic, bile acid, and fatty acid metabolism. A 13-gene signature, including upregulation of TREM1 and MMP1 and downregulation of CYP2C9, was consistently associated with all diagnostic categories of pediatric NAFLD. CONCLUSION: The extracellular milieu of sera from children with NAFLD induced specific gene profiles distinguishable by a hepatocyte reporter system. Circulating factors may contribute to inflammation and extracellular matrix remodeling and impair xenobiotic and endobiotic metabolism in pediatric NAFLD.

An update of skin permeability data based on a systematic review of recent research.

The cutaneous absorption parameters of xenobiotics are crucial for the development of drugs and cosmetics, as well as for assessing environmental and occupational chemical risks. Despite the great variability in the design of experimental conditions due to uncertain international guidelines, datasets like HuskinDB have been created to report skin absorption endpoints. This review updates available skin permeability data by rigorously compiling research published between 2012 and 2021. Inclusion and exclusion criteria have been selected to build the most harmonized and reusable dataset possible. The Generative Topographic Mapping method was applied to the present dataset and compared to HuskinDB to monitor the progress in skin permeability research and locate chemotypes of particular concern. The open-source dataset (SkinPiX) includes steady-state flux, maximum flux, lag time and permeability coefficient results for the substances tested, as well as relevant information on experimental parameters that can impact the data. It can be used to extract subsets of data for comparisons and to build predictive models.

Costs of molecular adaptation to the chemical exposome: a focus on xenobiotic metabolism pathways.

Organisms adapt to their environment through different pathways. In vertebrates, xenobiotics are detected, metabolized and eliminated through the inducible xenobiotic-metabolizing pathways (XMP) which can also generate reactive toxic intermediates. In this review, we will discuss the impacts of the chemical exposome complexity on the balance between detoxication and side effects. There is a large discrepancy between the limited number of proteins involved in these pathways (few dozens) and the diversity and complexity of the chemical exposome (tens of thousands of chemicals). Several XMP proteins have a low specificity which allows them to bind and/or metabolize a large number of chemicals. This leads to undesired consequences, such as cross-inhibition, inefficient metabolism, release of toxic intermediates, etc. Furthermore, several XMP proteins have endogenous functions that may be disrupted upon exposure to exogenous chemicals. The gut microbiome produces a very large number of metabolites that enter the body and are part of the chemical exposome. It can metabolize xenobiotics and either eliminate them or lead to toxic derivatives. The complex interactions between chemicals of different origins will be illustrated by the diverse roles of the aryl hydrocarbon receptor which binds and transduces the signals of a large number of xenobiotics, microbiome metabolites, dietary chemicals and endogenous compounds. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

New insights into xenobiotic tolerance of Antarctic bacteria: transcriptomic analysis of Pseudomonas sp. TNT3 during 2,4,6-trinitrotoluene biotransformation.

The xenobiotic 2,4,6-trinitrotoluene (TNT) is a highly persistent environmental contaminant, whose biotransformation by microorganisms has attracted renewed attention. In previous research, we reported the discovery of Pseudomonas sp. TNT3, the first described Antarctic bacterium with the ability to biotransform TNT. Furthermore, through genomic analysis, we identified distinctive features in this isolate associated with the biotransformation of TNT and other xenobiotics. However, the metabolic pathways and genes active during TNT exposure in this bacterium remained unexplored. In the present transcriptomic study, we used RNA-sequencing to investigate gene expression changes in Pseudomonas sp. TNT3 exposed to 100 mg/L of TNT. The results showed differential expression of 194 genes (54 upregulated and 140 downregulated), mostly encoding hypothetical proteins. The most highly upregulated gene (> 1000-fold) encoded an azoreductase enzyme not previously described. Other significantly upregulated genes were associated with (nitro)aromatics detoxification, oxidative, thiol-specific, and nitrosative stress responses, and (nitro)aromatic xenobiotic tolerance via efflux pumps. Most of the downregulated genes were involved in the electron transport chain, pyrroloquinoline quinone (PQQ)-related alcohol oxidation, and motility. These findings highlight a complex cellular response to TNT exposure, with the azoreductase enzyme likely playing a crucial role in TNT biotransformation. Our study provides new insights into the molecular mechanisms of TNT biotransformation and aids in developing effective TNT bioremediation strategies. To the best of our knowledge, this report is the first transcriptomic response analysis of an Antarctic bacterium during TNT biotransformation.

Development and evaluation of a comprehensive workflow for suspect screening of exposome-related xenobiotics and phase II metabolites in diverse human biofluids.

Suspect and non-target screening (SNTS) methods are being promoted in order to decode the human exposome since a wide chemical space can be analysed in a diversity of human biofluids. However, SNTS approaches in the exposomics field are infra-studied in comparison to environmental or food monitoring studies. In this work, a comprehensive suspect screening workflow was developed to annotate exposome-related xenobiotics and phase II metabolites in diverse human biofluids. Precisely, human urine, breast milk, saliva and ovarian follicular fluid were employed as samples and analysed by means of ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometry (UHPLC-HRMS/MS). To automate the workflow, the "peak rating" parameter implemented in Compound Discoverer 3.3.2 was optimized to avoid time-consuming manual revision of chromatographic peaks. In addition, the presence of endogenous molecules that might interfere with the annotation of xenobiotics was carefully studied as the employment of inclusion and exclusion suspect lists. To evaluate the workflow, limits of identification (LOIs) and type I and II errors (i.e., false positives and negatives, respectively) were calculated in both standard solutions and spiked biofluids using 161 xenobiotics and 22 metabolites. For 80.3 % of the suspects, LOIs below 15 ng/mL were achieved. In terms of type I errors, only two cases were identified in standards and spiked samples. Regarding type II errors, the 7.7 % errors accounted in standards increased to 17.4 % in real samples. Lastly, the use of an inclusion list for endogens was favoured since it avoided 18.7 % of potential type I errors, while the exclusion list caused 7.2 % of type II errors despite making the annotation workflow less time-consuming.

Transcriptomics and Metabolomics Reveal Tomato Consumption Alters Hepatic Xenobiotic Metabolism and Induces Steroidal Alkaloid Metabolite Accumulation in Mice.

SCOPE: Tomato consumption is associated with many health benefits including lowered risk for developing certain cancers. It is hypothesized that tomato phytochemicals are transported to the liver and other tissues where they alter gene expression in ways that lead to favorable health outcomes. However, the effects of tomato consumption on mammalian liver gene expression and chemical profile are not well defined. METHODS AND RESULTS: The study hypothesizes that tomato consumption would alter mouse liver transcriptomes and metabolomes compared to a control diet. C57BL/6J mice (n = 11-12/group) are fed a macronutrient matched diet containing either 10% red tomato, 10% tangerine tomato, or no tomato powder for 6 weeks after weaning. RNA-Seq followed by gene set enrichment analyses indicates that tomato type and consumption, in general, altered expression of phase I and II xenobiotic metabolism genes. Untargeted metabolomics experiments reveal distinct clustering between control and tomato fed animals. Nineteen molecular formulas (representing 75 chemical features) are identified or tentatively identified as steroidal alkaloids and isomers of their phase I and II metabolites; many of which are reported for the first time in mammals. CONCLUSION: These data together suggest tomato consumption may impart benefits partly through enhancing detoxification potential.

Redox Regulation of Xenobiotics by Reactive Sulfur and Supersulfide Species.

Significance: Routine exposure to xenobiotics is unavoidable during our lifetimes. Certain xenobiotics are hazardous to human health, and are metabolized in the body to render them less toxic. During this process, several detoxification enzymes cooperatively metabolize xenobiotics. Glutathione (GSH) conjugation plays an important role in the metabolism of electrophilic xenobiotics. Recent Advances: Recent advances in reactive sulfur and supersulfide (RSS) analyses showed that persulfides and polysulfides bound to low-molecular-weight thiols, such as GSH, and to protein thiols are abundant in both eukaryotes and prokaryotes. The highly nucleophilic nature of hydropersulfides and hydropolysulfides contributes to cell protection against oxidative stress and electrophilic stress. Critical Issues: In contrast to GSH conjugation to electrophiles that is aided by glutathione S-transferase (GST), persulfides and polysulfides can directly form conjugates with electrophiles without the catalytic actions of GST. The polysulfur bonds in the conjugates are further reduced by perthioanions and polythioanions derived from RSS to form sulfhydrated metabolites that are no longer electrophilic but rather nucleophilic, and differ from metabolites that are formed via GSH conjugation. Future Directions: In view of the abundance of RSS in cells and tissues, metabolism of xenobiotics that is mediated by RSS warrants additional investigations, such as studies of the impact of microbiota-derived RSS on xenobiotic metabolism. Metabolites formed from reactions between electrophiles and RSS may be potential biomarkers for monitoring exposure to electrophiles and for studying their metabolism by RSS. Antioxid. Redox Signal. 40, 679-690.