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Exposure to traffic-related pollutants, including diesel exhaust, is associated with increased risk of cardiopulmonary disease and mortality; however, the precise biochemical pathways underlying these effects are not known. To investigate biological response mechanisms underlying exposure to traffic related pollutants, we used an integrated molecular response approach that included high-resolution metabolomic profiling and peripheral blood gene expression to identify biological responses to diesel exhaust exposure. Plasma samples were collected from 73 non-smoking males employed in the US trucking industry between February 2009 and October 2010, and analyzed using untargeted high-resolution metabolomics to characterize metabolite associations with shift- and week-averaged levels of elemental carbon (EC), organic carbon (OC) and particulate matter with diameter ≤ 2.5 μm (PM2.5). Metabolic associations with EC, OC and PM2.5 were evaluated for biochemical processes known to be associated with disease risk. Annotated metabolites associated with exposure were then tested for relationships with the peripheral blood transcriptome using multivariate selection and network correlation. Week-averaged EC and OC levels, which were averaged across multiple shifts during the workweek, resulted in the greatest exposure-associated metabolic alterations compared to shift-averaged exposure levels. Metabolic changes associated with EC exposure suggest increased lipid peroxidation products, biomarkers of oxidative stress, thrombotic signaling lipids, and metabolites associated with endothelial dysfunction from altered nitric oxide metabolism, while OC exposures were associated with antioxidants, oxidative stress biomarkers and critical intermediates in nitric oxide production. Correlation with whole blood RNA gene expression provided additional evidence of changes in processes related to endothelial function, immune response, inflammation, and oxidative stress. We did not detect metabolic associations with PM2.5. This study provides an integrated molecular assessment of human exposure to traffic-related air pollutants that includes diesel exhaust. Metabolite and transcriptomic changes associated with exposure to EC and OC are consistent with increased risk of cardiovascular diseases and the adverse health effects of traffic-related air pollution.

Background: Chronic exposure to certain metals plays a role in disease development. Integrating untargeted metabolomics with urinary metallome data may contribute to better understanding the pathophysiology of diseases and complex molecular interactions related to environmental metal exposures. To discover novel associations between urinary metal biomarkers and metabolism networks, we conducted an integrative metallome-metabolome analysis using a panel of urinary metals and untargeted blood metabolomic data from the Strong Heart Family Study (SHFS). Methods: The SHFS is a prospective family-based cohort study comprised of American Indian men and women recruited in 2001–2003. This nested case-control analysis of 145 participants of which 50 developed incident diabetes at follow up in 2006–2009, included participants with urinary metal and untargeted metabolomic data. Concentrations of 8 creatinine-adjusted urine metals/metalloids [antimony (Sb), cadmium (Cd), lead (Pb), molybdenum (Mo), selenium (Se), tungsten (W), uranium (U) and zinc (Zn)], and 4 arsenic species [inorganic arsenic (iAs), monomethylarsonate (MMA), dimethylarsinate (DMA), and arsenobetaine (AsB)] were measured. Global metabolomics was performed on plasma samples using high-resolution Orbitrap mass spectrometry. We performed an integrative network analysis using xMWAS and a metabolic pathway analysis using Mummichog. Results: 8,810 metabolic features and 12 metal species were included in the integrative network analysis. Most metal species were associated with distinct subsets of metabolites, forming single-metal-multiple-metabolite clusters (|r|>0.28, p-value 0.17, p-value

BACKGROUND: Air pollution exposure has been linked with diminished fertility. Identifying the metabolic changes induced by periconception air pollution exposure among women could enhance our understanding of the potential biological pathways underlying air pollution’s reproductive toxicity. OBJECTIVE: To identify serum metabolites associated with periconception air pollution exposure and evaluate the extent to which these metabolites mediate the association between air pollution and live birth. METHODS: We included 200 women undergoing a fresh assisted reproductive technology (ART) cycle at Massachusetts General Hospital Fertility Center (2005–2015). A serum sample was collected during stimulation, and untargeted metabolic profiling was conducted using liquid chromatography with ultra-high-resolution mass spectrometry. Exposure to nitrogen dioxide (NO(2)), ozone (O(3)), fine particulate matter

Electronic cigarettes (e-cig) are an increasingly popular alternative to traditional smoking but have been in use for too short of a period of time to fully understand health risks. Furthermore, associated health risks are difficult to evaluate because of a large range of flavoring agents and their combinations for use with e-cig. Many flavoring agents are generally regarded as safe but have limited studies for effects on lung. Vanillin, an aromatic aldehyde, is one of the most commonly used flavoring agents in e-cig. Vanillin is electrophilic and can be redox active, with chemical properties expected to interact within biologic systems. Because accumulating lung metabolomics studies have identified metabolic disruptions associated with idiopathic pulmonary fibrosis, asthma and acute respiratory distress syndrome, we used human bronchial epithelial cells (BEAS-2B) with high-resolution metabolomics analysis to determine whether these disease-associated pathways are impacted by vanillin over the range used in e-cig. A metabolome-wide association study showed that vanillin perturbed specific energy, amino acid, antioxidant and sphingolipid pathways previously associated with human disease. Analysis of a small publicly available human dataset showed associations with several of the same pathways. Because vanillin is a common and high-abundance flavorant in e-cig, these results show that vanillin has potential to be mechanistically important in lung diseases and warrants in vivo toxicity testing in the context of e-cig use.

Glucocorticoid resistance (GR) is associated with exposure to chronic stress and an increased risk of metabolic and inflammatory disorders in both animal and human populations. Studies on ethnic disparities highlight the African-American (AA) population as having a high propensity to both GR and chronic stress exposure. Glucocorticoids and inflammation play a very important role in pregnancy outcome and fetal development. To date, however, the metabolites and metabolic pathways associated with GR during pregnancy have not been identified, obscuring the mechanisms by which adverse health consequences arise, and thus impeding targeted therapeutic intervention. The objective of this study was to perform untargeted high-resolution metabolomics (HRM) profiling on 273 pregnant AA women, to identify metabolites and metabolic pathways associated with GR during the first trimester of pregnancy and to evaluate their cross-sectional association with birth outcomes and psychosocial variables related to chronic stress exposure. For this study, GR was determined by the concentration of dexamethasone required for 50% inhibition (Dex IC50) of the cytokine tumor-necrosis factor alpha (TNF-alpha) release in vitro in response to a standard dose of lipopolysaccharide. The results for Metabolome-Wide Association Studies (MWAS) and pathway enrichment analysis for serum metabolic associations with Dex IC50, showed energy (nicotinamide and TCA cycle), amino acid, and glycosphingolipid metabolism as top altered pathways. Bioinformatic analysis showed that GR, as indicated by elevated Dex IC50 in the pregnant women, was associated with increased inflammatory metabolites, oxidative stress related metabolites, increased demand for functional amino acids to support growth and development, and disruption in energy-related metabolites. If confirmed in future studies, targeting these physiologically significant metabolites and metabolic pathways may lead to future assessment and intervention strategies to prevent inflammatory and metabolic complications observed in pregnant populations.

Intestinal homeostasis is regulated in-part by reactive oxygen species (ROS) that are generated in the colonic mucosa following contact with certain lactobacilli. Mechanistically, ROS can modulate protein function through the oxidation of cysteine residues within proteins. Recent advances in cysteine labeling by the Isotope Coded Affinity Tags (ICATs) technique has facilitated the identification of cysteine thiol modifications in response to stimuli. Here, we used ICATs to map the redox protein network oxidized upon initial contact of the colonic mucosa with Lactobacillus rhamnosus GG (LGG). We detected significant LGG-specific redox changes in over 450 proteins, many of which are implicated to function in cellular processes such as endosomal trafficking, epithelial cell junctions, barrier integrity, and cytoskeleton maintenance and formation. We particularly noted the LGG-specific oxidation of Rac1, which is a pleiotropic regulator of many cellular processes. Together, these data reveal new insights into lactobacilli-induced and redox-dependent networks involved in intestinal homeostasis.

Background: High-resolution metabolomics (HRM) is emerging as a sensitive tool for measuring environmental exposures and biological responses. The aim of this analysis is to assess the ability of high-resolution metabolomics (HRM) to reflect internal exposures to complex traffic-related air pollution mixtures. Methods: We used untargeted HRM profiling to characterize plasma and saliva collected from participants in the Dorm Room Inhalation to Vehicle Emission (DRIVE) study to identify metabolic pathways associated with traffic emission exposures. We measured a suite of traffic-related pollutants at multiple ambient and indoor sites at varying distances from a major highway artery for 12 weeks in 2014. In parallel, 54 students living in dormitories near (20 m) or far (1.4 km) from the highway contributed plasma and saliva samples. Untargeted HRM profiling was completed for both plasma and saliva samples; metabolite and metabolic pathway alternations were evaluated using a metabolome-wide association study (MWAS) framework with pathway analyses. Results: Weekly levels of traffic pollutants were significantly higher at the near dorm when compared to the far dorm (p

Human disease occurs due to gene-environment (G × E) interactions. Oxidants cause reversible and irreversible changes to living systems, and these can be beneficial (oxidative eustress) or harmful (oxidative distress). The present article focuses on the network of developmental, defense, and repair programs that maintain health in the presence of an oxidizing environment. We consider these programs as genetically encoded exposure memory systems that evolved to allow individuals to adapt to environment during life span, thereby improving fitness for survival and reproduction. Importantly, adaptations to improve resistance to one stress can limit adaptability to other types of stress, resulting in a cumulative decline in tolerance to environmental stresses. Omics tools are now widely available to help examine the cumulative decline in network flexibility due to G × E interactions. One can thus envisage ways to monitor cumulative impacts of oxidative stress to support new antioxidant strategies to improve healthy longevity.

Background Toxicant-associated steatohepatitis has been described in adults but less is known regarding the role of toxicants in liver disease of children. Perfluoroalkyl substances (PFAS) cause hepatic steatosis in rodents, but few previous studies have examined PFAS effects on severity of liver injury in children. Objectives We aimed to examine the relationship of PFAS to histologic severity of nonalcoholic fatty liver disease (NAFLD) in children. Methods Seventy-four children with physician-diagnosed NAFLD were recruited from Children’s Healthcare of Atlanta between 2007 and 2015. Biopsy-based liver histological features were scored for steatosis, lobular and portal inflammation, ballooning, and fibrosis. Plasma concentrations of perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonic acid (PFHxS), and untargeted plasma metabolomic profiling, were determined using liquid chromatography with high-resolution mass spectrometry. A metabolome-wide association study coupled with pathway enrichment analysis was performed to evaluate metabolic dysregulation associated with PFAS. A structural integrated analysis was applied to identify latent clusters of children with more severe form of NAFLD based on their PFAS levels and metabolite pattern. Results Patients were 7–19 years old, mostly boys (71%), Hispanic (51%), and obese (85%). The odds of having nonalcoholic steatohepatitis (NASH), compared to children with steatosis alone, was significantly increased with each interquartile range (IQR) increase of PFOS (OR: 3.32, 95% CI: 1.40–7.87) and PFHxS (OR: 4.18, 95% CI: 1.64–10.7). Each IQR increase of PFHxS was associated with increased odds for liver fibrosis (OR: 4.44, 95% CI: 1.34–14.8), lobular inflammation (OR: 2.87, 95% CI: 1.12–7.31), and higher NAFLD activity score (β coefficient 0.46; 95% CI: 0.03, 0.89). A novel integrative analysis identified a cluster of children with NASH, characterized by increased PFAS levels and altered metabolite patterns including higher plasma levels of phosphoethanolamine, tyrosine, phenylalanine, aspartate and creatine, and decreased plasma levels of betaine. Conclusions Ηigher PFAS exposure was associated with more severe disease in children with NAFLD. PFAS may be an important toxicant contributing to NAFLD progression; however larger, longitudinal studies are warranted to confirm these findings.

Non-invasive measures of the response of individual patients to cancer therapeutics is an emerging strategy in precision medicine. Platelets offer a potential dynamic marker for metabolism and bioenergetic responses in individual patients since they have active glycolysis and mitochondrial oxidative phosphorylation and can be easily isolated from a small blood sample. We have recently shown how the bioenergetic-metabolite interactome can be defined in platelets isolated from human subjects by measuring metabolites and bioenergetics in the same sample. In the present study, we used a model system to assess test the hypothesis that this interactome is modified by xenobiotics using exposure to the anti-cancer drug doxorubicin (Dox) in individual donors. We found that unsupervised analysis of the metabolome showed clear differentiation between the control and Dox treated group. Dox treatment resulted in a concentration-dependent decrease in bioenergetic parameters with maximal respiration being most sensitive and this was associated with significant changes in over 166 features. A metabolome-wide association study of Dox was also conducted, and Dox was found to have associations with metabolites in the glycolytic and TCA cycle pathways. Lastly, network analysis showed the impact of Dox on the bioenergetic-metabolite interactome and revealed profound changes in the regulation of reserve capacity. Taken together, these data support the conclusion that platelets are a suitable platform to predict and monitor therapeutic efficacy as well as anticipate susceptibility to toxicity in the context of precision medicine., Graphical abstract Image 1, Highlights • Chemotherapeutic drug doxorubicin dose-dependently inhibited bioenergetic parameters in platelets. • Doxorubicin induced metabolite alterations in multiple pathways that modulate cellular bioenergetics. • The interactome revealed novel interactions between bioenergetic parameters and metabolites in platelets. • The integration of platelet bioenergetics with the metabolome can be utilized for precision medicine strategies.

Background: Maternal exposure to traffic-related air pollution during pregnancy has been shown to increase the risk of adverse birth outcomes and neurodevelopmental disorders. By utilizing high-resolution metabolomics (HRM), we investigated perturbations of the maternal serum metabolome in response to traffic-related air pollution to identify biological mechanisms. Methods: We retrieved stored mid-pregnancy serum samples from 160 mothers who lived in the Central Valley of California known for high air particulate levels. We estimated prenatal traffic-related air pollution exposure (carbon monoxide, nitric oxides, and particulate matter

The exposome is a complement to the genome that includes non-genetic causes of disease. Multiple definitions are available, with salient points being global inclusion of exposures and behaviors, and cumulative integration of associated biologic responses. As such, the concept is both refreshingly simple and dauntingly complex. This article reviews high-resolution metabolomics (HRM) as an affordable approach to routinely analyze samples for a broad spectrum of environmental chemicals and biologic responses. HRM has been successfully used in multiple exposome research paradigms and is suitable to implement in a prototype universal exposure surveillance system. Development of such a structure for systematic monitoring of environmental exposures is an important step toward sequencing the exposome because it builds upon successes of exposure science, naturally connects external exposure to body burden and partitions the exposome into workable components. Practical results would be repositories of quantitative data on chemicals according to geography and biology. This would support new opportunities for environmental health analysis and predictive modeling. Complementary approaches to hasten development of exposome theory and associated biologic response networks could include experimental studies with model systems, analysis of archival samples from longitudinal studies with outcome data and study of relatively short-lived animals, such as household pets (dogs and cats) and non-human primates (common marmoset). International investment and cooperation to sequence the human exposome will advance scientific knowledge and also provide an important foundation to control adverse environmental exposures to sustain healthy living spaces and improve prediction and management of disease. Keywords: Mass spectrometry, Biomonitoring, Analytical chemistry, Metabolomics, Environmental surveillance

Metazoan genomes encode exposure memory systems to enhance survival and reproductive potential by providing mechanisms for an individual to adjust during lifespan to environmental resources and challenges. These systems are inherently redox networks, arising during evolution of complex systems with O2 as a major determinant of bioenergetics, metabolic and structural organization, defense, and reproduction. The network structure decreases flexibility from conception onward due to differentiation and cumulative responses to environment (exposome). The redox theory of aging is that aging is a decline in plasticity of genome–exposome interaction that occurs as a consequence of execution of differentiation and exposure memory systems. This includes compromised mitochondrial and bioenergetic flexibility, impaired food utilization and metabolic homeostasis, decreased barrier and defense capabilities and loss of reproductive fidelity and fecundity. This theory accounts for hallmarks of aging, including failure to maintain oxidative or xenobiotic defenses, mitochondrial integrity, proteostasis, barrier structures, DNA repair, telomeres, immune function, metabolic regulation and regenerative capacity., Graphical abstract, Highlights • A redox interface connects an organism and its environment. • Genetically encoded exposure memory systems evolved along with multicellularity in an O2-rich environment. • Exposure memory allows an individual to adapt to resources and threats during lifespan. • Aging is an irreversible decline in adaptability due to execution of exposure memory systems.

A combination of the herbicide paraquat (PQ) and fungicide maneb (MB) has been linked to Parkinson's disease. Previous studies show that this involves an additive toxicity with at least two different mechanisms. However, detailed understanding of mixtures is often difficult to elucidate because of the multiple ways by which toxic agents can interact. In the present study, we used a combination of transcriptomics and metabolomics to investigate mechanisms of toxicity of PQ and MB in a neuroblastoma cell line. Conditions were studied with concentrations of PQ and MB that each individually caused 20% cell death and together caused 50% cell death. Transcriptomic and metabolomic samples were collected at time points prior to significant cell death. Statistical and bioinformatic methods were applied to the resulting 30,869 transcripts and 1358 metabolites. Results showed that MB significantly changed more transcripts and metabolites than PQ, and combined PQ + MB impacted more than MB alone. Transcriptome–metabolome-wide association study (TMWAS) showed that significantly changed transcripts and metabolites mapped to two network substructures, one associating with significant effects of MB and the other included features significantly associated with PQ + MB. The latter contained 4 clusters of genes and associated metabolites, with one containing genes for two cation transporters and a cation transporter regulatory protein also recognized as a pro-apoptotic protein. Other clusters included stress response genes and transporters linked to cytoprotective mechanisms. MB also had a significant network structure linked to cell proliferation. Together, the results show that the toxicologic mechanism of the combined neurotoxicity of PQ and MB involves network level interactions and that TMWAS provides an effective approach to investigate such complex mechanisms.

Due to its short lifespan, ease of use and age-related pathologies that mirror those observed in humans, the common marmoset (Callithrix jacchus) is poised to become a standard nonhuman primate model of aging. Blood and extracellular fluid possess two major thiol-dependent redox nodes involving cysteine (Cys), cystine (CySS), glutathione (GSH) and glutathione disulfide (GSSG). Alteration in these plasma redox nodes significantly affects cellular physiology, and oxidation of the plasma Cys/CySS redox potential (EhCySS) is associated with aging and disease risk in humans. The purpose of this study was to determine age-related changes in plasma redox metabolites and corresponding redox potentials (Eh) to further validate the marmoset as a nonhuman primate model of aging. We measured plasma thiol redox states in marmosets and used existing human data with multivariate adaptive regression splines (MARS) to model the relationships between age and redox metabolites. A classification accuracy of 70.2% and an AUC of 0.703 were achieved using the MARS model built from the marmoset redox data to classify the human samples as young or old. These results show that common marmosets provide a useful model for thiol redox biology of aging., Graphical abstract, Highlights • Characterization of the Common Marmoset as a model for aging research. • Plasma thiol redox measurements in marmosets ranging in age from 2–16 years. • Similar to humans, marmosets exhibit age-related alterations in plasma thiol redox metabolites. • Marmoset redox data can be used to classify humans as young or old.

Adoption of metabolic screening into routine health care has the potential to support Personalized Medicine; however, before informed decisions can be made on the basis of metabolic phenotyping, heterogeneity and confounding factors will have to be understood. This can only be accomplished through a data-driven approach, particularly when considering dietary and environmental influences. Identification of how phenotypic and lifestyle traits contribute to definition of an individual’s metabolic phenotype is required for meaningful interpretation of metabolic associations. In this chapter, we discuss the feasibility of metabolomics as a tool for population screening and for defining the human metabolic phenotype. Inherent factors contributing to the metabolic phenotype, including circadian rhythm, gender, age, and disease status, are reviewed. Because of the all-encompassing influence of human–environment interactions, we also discuss how environmental factors affect the metabolic phenotype and the use of metabolomics for broad-spectrum chemical biomonitoring. We close with a case study identifying biological responses to insecticide exposure in a healthy population.

Most current studies of reactive oxygen species (ROS) production report globally averaged measurements within the cell; however, ROS can be produced in distinct subcellular locations and have local effects in their immediate vicinity. A microfluidic platform for high-throughput single-cell imaging allows mitochondrial ROS production to be monitored as varying in both space and time. Using this systems biology approach, single-cell variability can be viewed within a population. We discuss single-cell monitoring of contributors to mitochondrial redox state-mitochondrial hydrogen peroxide or superoxide-through the use of a small molecule probe or targeted fluorescent reporter protein. Jurkat T lymphoma cells were stimulated with antimycin A and imaged in an arrayed microfluidic device over time. Differences in single-cell responses were observed as a function of both inhibitor concentration and type of ROS measurement used.

Drug-induced liver injury (DILI) is common and nearly all classes of medications can cause liver disease. Most cases of DILI are benign, and improve after drug withdrawal. It is important to recognize and remove the offending agent as quickly as possible to prevent the progression to chronic liver disease and/or acute liver failure. There are no definite risk factors for DILI, but pre-existing liver disease and genetic susceptibility may predispose certain individuals. Although most patients have clinical symptoms that are identical to other liver diseases, some patients may present with symptoms of systemic hypersensitivity. Treatment of drug and herbal-induced liver injury consists of rapid drug discontinuation and supportive care targeted to alleviate unwanted symptoms.

Publisher Summary Redox-signaling mechanisms function in cell regulation and growth control. Agents altering glutathione (GSH) concentration affect the transcription of detoxification enzymes, cell proliferation, and apoptosis. GSH, GSH S-conjugates, and its disulfide form (GSSG), or the redox state of the GSH/GSSG couple provide a mechanistic control or signal for functional changes. A loss or oxidation of GSH occurs in association with differentiation both in vitro and in vivo and during apoptosis. In contrast, increases in GSH and/or a reduction of the GSH/GSSG pool is associated with growth stimulation by nutrients and growth factors. Thus, the balance of GSH and GSSG may not only reflect oxidative stress but may reflect changes in redox signaling and control. The most widely used indicator of the redox state of the GSH pool is the ratio of GSH to GSSG. The cellular balance of GSH and GSSG provides a dynamic indicator of oxidative stress. Although GSH is synthesized continuously and has various fates in cells, these rates are generally slow relative to the turnover by the oxidation-reduction cycle. The activity of the GSSG reductase is dependent on GSSG concentration. During acute oxidative stress, GSH concentration decreases and the associated increase in GSSG concentration results in an increased turnover of the GSH/GSSG cycle.

Publisher Summary This chapter focuses on the absorption and tissue distribution of Glutathione (GSH) distribution in natural products. GSH is present in most plant and animal tissues from which the human diet is derived. Much of this GSH is bioavailable because cells of the gastrointestinal tract have Na+-dependent and Na+-independent uptake mechanisms, and net absorption of GSH from the lumen into the vascular circulation occurs in the small intestine. In addition, GSH can function as an antioxidant in the ingesta, can maintain ascorbate in a reduced and functional form, can directly react with and inactivate toxic electrophiles in the diet, and can be broken down to yield cysteine. Studies have shown that dietary GSH enhances metabolic clearance of dietary peroxidized lipids and decreases their net absorption and that consumption of foods high in GSH content is associated with about a 50% reduction in risk of oral and pharyngeal cancer. Glutathione is readily oxidized by food processing and preparation, and many foods contain reactive electrophilic compounds that can remove GSH even after food is ingested. Although knowledge of GSH contents of foods remains incomplete, studies have provided insight into the range of values and provide some useful generalizations.

Publisher Summary Suspensions of intact, fully functional cells from adult mammalian tissues have widespread utility in biochemical research. This chapter presents the method for the preparation of cells from the rat kidney involving perfusion with a medium containing ethyleneglycol-bis(aminoethyl ether)tetraacetic acid (EGTA) to weaken cell adhesions by the removal of Ca 2+ , followed by perfusion with a medium containing collagenase to hydrolyze the connective material. The resultant preparation contains mainly spherical cells of uniform size, which are predominantly derived from the tubular epithelium. The preparation procedure is rapid and the cells retain viability in vitro for at least 4 hr. The cells are comparable to intact kidney tissue with regard to amino acid transport, glutathione synthesis, drug metabolism, and respiratory characteristics. The preparation is, therefore, suitable for a wide variety of metabolic studies. Because it is of great importance to ensure the metabolic integrity of isolated cells, the chapter also summarizes the characteristics of this preparation, methods for the assessment of cell viability, and incubation conditions found most suitable for short-term experiments.