Predicting changes in renal metabolism after compound exposure with a genome-scale metabolic model.

The kidneys are metabolically active organs with importance in several physiological tasks such as the secretion of soluble wastes into the urine and synthesizing glucose and oxidizing fatty acids for energy in fasting (non-fed) conditions. Once damaged, the metabolic capability of the kidneys becomes altered. Here, we define metabolic tasks in a computational modeling framework to capture kidney function in an update to the iRno network reconstruction of rat metabolism using literature-based evidence. To demonstrate the utility of iRno for predicting kidney function, we exposed primary rat renal proximal tubule epithelial cells to four compounds with varying levels of nephrotoxicity (acetaminophen, gentamicin, 2,3,7,8-tetrachlorodibenzodioxin, and trichloroethylene) for six and twenty-four hours, and collected transcriptomics and metabolomics data to measure the metabolic effects of compound exposure. For the transcriptomics data, we observed changes in fatty acid metabolism and amino acid metabolism, as well as changes in existing markers of kidney function such as Clu (clusterin). The iRno metabolic network reconstruction was used to predict alterations in these same pathways after integrating transcriptomics data and was able to distinguish between select compound-specific effects on the proximal tubule epithelial cells. Genome-scale metabolic network reconstructions with coupled omics data can be used to predict changes in metabolism as a step towards identifying novel metabolic biomarkers of kidney function and dysfunction.

Mechanistic identification of biofluid metabolite changes as markers of acetaminophen-induced liver toxicity in rats.

Acetaminophen (APAP) is the most commonly used analgesic and antipyretic drug in the world. Yet, it poses a major risk of liver injury when taken in excess of the therapeutic dose. Current clinical markers do not detect the early onset of liver injury associated with excess APAP-information that is vital to reverse injury progression through available therapeutic interventions. Hence, several studies have used transcriptomics, proteomics, and metabolomics technologies, both independently and in combination, in an attempt to discover potential early markers of liver injury. However, the casual relationship between these observations and their relation to the APAP mechanism of liver toxicity are not clearly understood. Here, we used Sprague-Dawley rats orally gavaged with a single dose of 2 g/kg of APAP to collect tissue samples from the liver and kidney for transcriptomic analysis and plasma and urine samples for metabolomic analysis. We developed and used a multi-tissue, metabolism-based modeling approach to integrate these data, characterize the effect of excess APAP levels on liver metabolism, and identify a panel of plasma and urine metabolites that are associated with APAP-induced liver toxicity. Our analyses, which indicated that pathways involved in nucleotide-, lipid-, and amino acid-related metabolism in the liver were most strongly affected within 10 h following APAP treatment, identified a list of potential metabolites in these pathways that could serve as plausible markers of APAP-induced liver injury. Our approach identifies toxicant-induced changes in endogenous metabolism, is applicable to other toxicants based on transcriptomic data, and provides a mechanistic framework for interpreting metabolite alterations.

Systems-level computational modeling demonstrates fuel selection switching in high capacity running and low capacity running rats.

High capacity and low capacity running rats, HCR and LCR respectively, have been bred to represent two extremes of running endurance and have recently demonstrated disparities in fuel usage during transient aerobic exercise. HCR rats can maintain fatty acid (FA) utilization throughout the course of transient aerobic exercise whereas LCR rats rely predominantly on glucose utilization. We hypothesized that the difference between HCR and LCR fuel utilization could be explained by a difference in mitochondrial density. To test this hypothesis and to investigate mechanisms of fuel selection, we used a constraint-based kinetic analysis of whole-body metabolism to analyze transient exercise data from these rats. Our model analysis used a thermodynamically constrained kinetic framework that accounts for glycolysis, the TCA cycle, and mitochondrial FA transport and oxidation. The model can effectively match the observed relative rates of oxidation of glucose versus FA, as a function of ATP demand. In searching for the minimal differences required to explain metabolic function in HCR versus LCR rats, it was determined that the whole-body metabolic phenotype of LCR, compared to the HCR, could be explained by a ~50% reduction in total mitochondrial activity with an additional 5-fold reduction in mitochondrial FA transport activity. Finally, we postulate that over sustained periods of exercise that LCR can partly overcome the initial deficit in FA catabolic activity by upregulating FA transport and/or oxidation processes.

Mitochondrial structure and function are not different between nonfailing donor and end-stage failing human hearts.

During human heart failure, the balance of cardiac energy use switches from predominantly fatty acids (FAs) to glucose. We hypothesized that this substrate shift was the result of mitochondrial degeneration; therefore, we examined mitochondrial oxidation and ultrastructure in the failing human heart by using respirometry, transmission electron microscopy, and gene expression studies of demographically matched donor and failing human heart left ventricular (LV) tissues. Surprisingly, respiratory capacities for failing LV isolated mitochondria (n = 9) were not significantly diminished compared with donor LV isolated mitochondria (n = 7) for glycolysis (pyruvate + malate)- or FA (palmitoylcarnitine)-derived substrates, and mitochondrial densities, assessed via citrate synthase activity, were consistent between groups. Transmission electron microscopy images also showed no ultrastructural remodeling for failing vs. donor mitochondria; however, the fraction of lipid droplets (LDs) in direct contact with a mitochondrion was reduced, and the average distance between an LD and its nearest neighboring mitochondrion was increased. Analysis of FA processing gene expression between donor and failing LVs revealed 0.64-fold reduced transcript levels for the mitochondrial-LD tether, perilipin 5, in the failing myocardium (P = 0.003). Thus, reduced FA use in heart failure may result from improper delivery, potentially via decreased perilipin 5 expression and mitochondrial-LD tethering, and not from intrinsic mitochondrial dysfunction.-Holzem, K. M., Vinnakota, K. C., Ravikumar, V. K., Madden, E. J., Ewald, G. A., Dikranian, K., Beard, D. A., Efimov, I. R. Mitochondrial structure and function are not different between nonfailing donor and end-stage failing human hearts.

Catalytic Coupling of Oxidative Phosphorylation, ATP Demand, and Reactive Oxygen Species Generation.

Competing models of mitochondrial energy metabolism in the heart are highly disputed. In addition, the mechanisms of reactive oxygen species (ROS) production and scavenging are not well understood. To deepen our understanding of these processes, a computer model was developed to integrate the biophysical processes of oxidative phosphorylation and ROS generation. The model was calibrated with experimental data obtained from isolated rat heart mitochondria subjected to physiological conditions and workloads. Model simulations show that changes in the quinone pool redox state are responsible for the apparent inorganic phosphate activation of complex III. Model simulations predict that complex III is responsible for more ROS production during physiological working conditions relative to complex I. However, this relationship is reversed under pathological conditions. Finally, model analysis reveals how a highly reduced quinone pool caused by elevated levels of succinate is likely responsible for the burst of ROS seen during reperfusion after ischemia.

Open-Loop Control of Oxidative Phosphorylation in Skeletal and Cardiac Muscle Mitochondria by Ca(2.).

In cardiac muscle, mitochondrial ATP synthesis is driven by demand for ATP through feedback from the products of ATP hydrolysis. However, in skeletal muscle at higher workloads there is an apparent contribution of open-loop stimulation of ATP synthesis. Open-loop control is defined as modulation of flux through a biochemical pathway by a moiety, which is not a reactant or a product of the biochemical reactions in the pathway. The role of calcium, which is known to stimulate the activity of mitochondrial dehydrogenases, as an open-loop controller, was investigated in isolated cardiac and skeletal muscle mitochondria. The kinetics of NADH synthesis and respiration, feedback from ATP hydrolysis products, and stimulation by calcium were characterized in isolated mitochondria to test the hypothesis that calcium has a stimulatory role in skeletal muscle mitochondria not apparent in cardiac mitochondria. A range of respiratory states were obtained in cardiac and skeletal muscle mitochondria utilizing physiologically relevant concentrations of pyruvate and malate, and flux of respiration, NAD(P)H fluorescence, and rhodamine 123 fluorescence were measured over a range of extra mitochondrial calcium concentrations. We found that under these conditions calcium stimulates NADH synthesis in skeletal muscle mitochondria but not in cardiac mitochondria.

Feedback Regulation and Time Hierarchy of Oxidative Phosphorylation in Cardiac Mitochondria.

To determine how oxidative ATP synthesis is regulated in the heart, the responses of cardiac mitochondria oxidizing pyruvate to alterations in [ATP], [ADP], and inorganic phosphate ([Pi]) were characterized over a range of steady-state levels of extramitochondrial [ATP], [ADP], and [Pi]. Evolution of the steady states of the measured variables with the flux of respiration shows that: (1) a higher phosphorylation potential is achieved by mitochondria at higher [Pi] for a given flux of respiration; (2) the time hierarchy of oxidative phosphorylation is given by phosphorylation subsystem, electron transport chain, and substrate dehydrogenation subsystems listed in increasing order of their response times; (3) the matrix ATP hydrolysis mass action ratio [ADP] × [Pi]/[ATP] provides feedback to the substrate dehydrogenation flux over the entire range of respiratory flux examined in this study; and finally, (4) contrary to previous models of regulation of oxidative phosphorylation, [Pi] does not modulate the activity of complex III.

Influence of metabolic dysfunction on cardiac mechanics in decompensated hypertrophy and heart failure.

Alterations in energetic state of the myocardium are associated with decompensated heart failure in humans and in animal models. However, the functional consequences of the observed changes in energetic state on mechanical function are not known. The primary aim of the study was to quantify mechanical/energetic coupling in the heart and to determine if energetic dysfunction can contribute to mechanical failure. A secondary aim was to apply a quantitative systems pharmacology analysis to investigate the effects of drugs that target cross-bridge cycling kinetics in heart failure-associated energetic dysfunction. Herein, a model of metabolite- and calcium-dependent myocardial mechanics was developed from calcium concentration and tension time courses in rat cardiac muscle obtained at different lengths and stimulation frequencies. The muscle dynamics model accounting for the effect of metabolites was integrated into a model of the cardiac ventricles to simulate pressure-volume dynamics in the heart. This cardiac model was integrated into a simple model of the circulation to investigate the effects of metabolic state on whole-body function. Simulations predict that reductions in metabolite pools observed in canine models of heart failure can cause systolic dysfunction, blood volume expansion, venous congestion, and ventricular dilation. Simulations also predict that myosin-activating drugs may partially counteract the effects of energetic state on cross-bridge mechanics in heart failure while increasing myocardial oxygen consumption. Our model analysis demonstrates how metabolic changes observed in heart failure are alone sufficient to cause systolic dysfunction and whole-body heart failure symptoms.

Determination of the catalytic mechanism for mitochondrial malate dehydrogenase.

The kinetics of malate dehydrogenase (MDH) catalyzed oxidation/reduction of L-malate/oxaloacetate is pH-dependent due to the proton generated/taken up during the reaction. Previous kinetic studies on the mitochondrial MDH did not yield a consensus kinetic model that explains both substrate and pH dependency of the initial velocity. In this study, we propose, to our knowledge, a new kinetic mechanism to explain kinetic data acquired over a range of pH and substrate concentrations. Progress curves in the forward and reverse reaction directions were obtained under a variety of reactant concentrations to identify associated kinetic parameters. Experiments were conducted at physiologically relevant ionic strength of 0.17 M, pH ranging between 6.5 and 9.0, and at 25 °C. The developed model was built on the prior observation of proton uptake upon binding of NADH to MDH, and that the MDH-catalyzed oxidation of NADH may follow an ordered bi-bi mechanism with NADH/NAD binding to the enzyme first, followed by the binding of oxaloacetate/L-malate. This basic mechanism was expanded to account for additional ionic states to explain the pH dependency of the kinetic behavior, resulting in what we believe to be the first kinetic model explaining both substrate and pH dependency of the reaction velocity.

Characterization of the kinetics of cardiac cytosolic malate dehydrogenase and comparative analysis of cytosolic and mitochondrial isoforms.

Because the mitochondrial inner membrane is impermeable to pyridine nucleotides, transport of reducing equivalents between the mitochondrial matrix and the cytoplasm relies on shuttle mechanisms, including the malate-aspartate shuttle and the glycerol-3-phosphate shuttle. These shuttles are needed for reducing equivalents generated by metabolic reactions in the cytosol to be oxidized via aerobic metabolism. Two isoenzymes of malate dehydrogenase (MDH) operate as components of the malate-aspartate shuttle, in which a reducing equivalent is transported via malate, which when oxidized to oxaloacetate, transfers an electron pair to reduce NAD to NADH. Several competing mechanisms have been proposed for the MDH-catalyzed reaction. This study aims to identify the pH-dependent kinetic mechanism for cytoplasmic MDH (cMDH) catalyzed oxidation/reduction of MAL/OAA. Experiments were conducted assaying the forward and reverse directions with products initially present, varying pH between 6.5 and 9.0. By fitting time-course data to various mechanisms, it is determined that an ordered bi-bi mechanism with coenzyme binding first followed by the binding of substrate is able to explain the kinetic data. The proposed mechanism is similar to, but not identical to, the mechanism recently determined for the mitochondrial isoform, mMDH. cMDH and mMDH mechanisms are also shown to both be reduced versions of a common, more complex mechanism that can explain the kinetic data for both isoforms. Comparing the simulated activity (ratio of initial velocity to the enzyme concentration) under physiological conditions, the mitochondrial MDH (mMDH) activity is predicted to be higher than cMDH activity under mitochondrial matrix conditions while the cMDH activity is higher than mMDH activity under cytoplasmic conditions, suggesting that the functions of the isoforms are kinetically tuned to their individual physiological roles.

Analysis of the kinetics and bistability of ubiquinol:cytochrome c oxidoreductase.

Ubiquinol:cytochrome c oxidoreductase, bc1 complex, is the enzyme in the respiratory chain of mitochondria responsible for the transfer reducing potential from ubiquinol to cytochrome c coupled to the movement of charge against the electrostatic potential across the mitochondrial inner membrane. The complex is also implicated in the generation of reactive oxygen species under certain conditions and is thus a contributor to cellular oxidative stress. Here, a biophysically detailed, thermodynamically consistent model of the bc1 complex for mammalian mitochondria is developed. The model incorporates the major redox centers near the Qo- and Qi-site of the enzyme, includes the pH-dependent redox reactions, accounts for the effect of the proton-motive force of the reaction rate, and simulates superoxide production at the Qo-site. The model consists of six distinct states characterized by the mobile electron distribution in the enzyme. Within each state, substates that correspond to various electron localizations exist in a rapid equilibrium distribution. The steady-state equation for the six-state system is parameterized using five independent data sets and validated in comparison to additional experimental data. Model analysis suggests that the pH-dependence on turnover is primarily due to the pKa values of cytochrome bH and Rieske iron sulfur protein. A previously proposed kinetic scheme at the Qi-site where ubiquinone binds to only the reduced enzyme and ubiquinol binds to only the oxidized enzyme is shown to be thermodynamically infeasible. Moreover, the model is able to reproduce the bistability phenomenon where at a given overall flux through the enzyme, different rates of superoxide production are attained when the enzyme is differentially reduced.

Stimulatory effects of calcium on respiration and NAD(P)H synthesis in intact rat heart mitochondria utilizing physiological substrates cannot explain respiratory control in vivo.

Mitochondrial TCA cycle dehydrogenase enzymes have been shown to be stimulated by Ca(2+) under various substrate and ADP incubation conditions in an attempt to determine and understand the role of Ca(2+) in maintaining energy homeostasis in working hearts. In this study, we tested the hypothesis that, at physiological temperature and 1 mM extramitochondrial free magnesium, Ca(2+) can stimulate the overall mitochondrial NAD(P)H generation flux in rat heart mitochondria utilizing pyruvate and malate as substrates at both subsaturating and saturating concentrations. In both cases, we found that, in the physiological regime of mitochondrial oxygen consumption observed in the intact animal and in the physiological range of cytosolic Ca(2+) concentration averaged per beat, Ca(2+) had no observable stimulatory effect. A modest apparent stimulatory effect (22-27%) was observable at supraphysiological maximal ADP-stimulated respiration at 2.5 mM initial phosphate. The stimulatory effects observed over the physiological Ca(2+) range are not sufficient to make a significant contribution to the control of oxidative phosphorylation in the heart in vivo.

Identification of the catalytic mechanism and estimation of kinetic parameters for fumarase.

The enzyme fumarase catalyzes the reversible hydration of fumarate to malate. The reaction catalyzed by fumarase is critical for cellular energetics as a part of the tricarboxylic acid cycle, which produces reducing equivalents to drive oxidative ATP synthesis. A catalytic mechanism for the fumarase reaction that can account for the kinetic behavior of the enzyme observed in both isotope exchange studies and initial velocity studies has not yet been identified. In the present study, we develop an 11-state kinetic model of the enzyme based on the current consensus on its catalytic mechanism and design a series of experiments to estimate the model parameters and identify the major flux routes through the mechanism. The 11-state mechanism accounts for competitive binding of inhibitors and activation by different anions, including phosphate and fumarate. The model is identified from experimental time courses of the hydration of fumarate to malate obtained over a wide range of buffer and substrate concentrations. Further, the 11-state model is found to effectively reduce to a five-state model by lumping certain successive steps together to yield a mathematically less complex representation that is able to match the data. Analysis suggests the primary reaction route of the catalytic mechanism, with fumarate binding to the free unprotonated enzyme and a proton addition prior to malate release in the fumarate hydration reaction. In the reverse direction (malate dehydration), malate binds the protonated form of the enzyme, and a proton is generated before fumarate is released from the active site.

Centralizing neurofibromatosis experimental tool knowledge with the NF Research Tools Database.

Experimental tools and resources, such as animal models, cell lines, antibodies, genetic reagents and biobanks, are key ingredients in biomedical research. Investigators face multiple challenges when trying to understand the availability, applicability and accessibility of these tools. A major challenge is keeping up with current information about the numerous tools available for a particular research problem. A variety of disease-agnostic projects such as the Mouse Genome Informatics database and the Resource Identification Initiative curate a number of types of research tools. Here, we describe our efforts to build upon these resources to develop a disease-specific research tool resource for the neurofibromatosis (NF) research community. This resource, the NF Research Tools Database, is an open-access database that enables the exploration and discovery of information about NF type 1-relevant animal models, cell lines, antibodies, genetic reagents and biobanks. Users can search and explore tools, obtain detailed information about each tool as well as read and contribute their observations about the performance, reliability and characteristics of tools in the database. NF researchers will be able to use the NF Research Tools Database to promote, discover, share, reuse and characterize research tools, with the goal of advancing NF research. Database URL: https://tools.nf.synapse.org/.

Kinetic analysis and design of experiments to identify the catalytic mechanism of the monocarboxylate transporter isoforms 4 and 1.

Transport of lactate, pyruvate, and other monocarboxylates across the sarcolemma of skeletal and cardiac myocytes occurs via passive diffusion and by monocarboxylate transporter (MCT) mediated transport. The flux of lactate and protons through the MCT plays an important role in muscle energy metabolism during rest and exercise and in pH regulation during exercise. The MCT isoforms 1 and 4 are the major isoforms of this transporter in skeletal and cardiac muscle. The current consensus on the mechanism of these transporters, based on experimental measurements of labeled lactate fluxes, is that monocarboxylate-proton symport occurs via a rapid-equilibrium ordered mechanism with proton binding followed by monocarboxylate binding. This study tests ordered and random mechanisms by fitting experimental measurements of tracer exchange fluxes from MCT1 and MCT4 isoforms to theoretical predictions derived using relationships between one-way fluxes and thermodynamic forces. Analysis shows that: 1), the available kinetic data are insufficient to distinguish between a rapid-equilibrium ordered and a rapid-equilibrium random-binding model for MCT4; 2), MCT1 has a higher affinity to lactate than does MCT4; 3), the theoretical conditions for the so-called trans-acceleration phenomenon (e.g., increased tracer efflux from a vesicle caused by increased substrate concentration outside the vesicle) do not necessarily require the rate constant for the lactate and proton bound transporter to reorient across the membrane to be higher than that for the unbound transporter; and finally, 4), based on model analysis, additional experiments are proposed to be able to distinguish between ordered and random-binding mechanisms.

Identifying functional metabolic shifts in heart failure with the integration of omics data and a heart-specific, genome-scale model.

In diseased states, the heart can shift to use different carbon substrates, measured through changes in uptake of metabolites by imaging methods or blood metabolomics. However, it is not known whether these measured changes are a result of transcriptional changes or external factors. Here, we explore transcriptional changes in late-stage heart failure using publicly available data integrated with a model of heart metabolism. First, we present a heart-specific genome-scale metabolic network reconstruction (GENRE), iCardio. Next, we demonstrate the utility of iCardio in interpreting heart failure gene expression data by identifying tasks inferred from differential expression (TIDEs), which represent metabolic functions associated with changes in gene expression. We identify decreased gene expression for nitric oxide (NO) and N-acetylneuraminic acid (Neu5Ac) synthesis as common metabolic markers of heart failure. The methods presented here for constructing a tissue-specific model and identifying TIDEs can be extended to multiple tissues and diseases of interest.

Common phenotype of resting mouse extensor digitorum longus and soleus muscles: equal ATPase and glycolytic flux during transient anoxia.

Rates of ATPase and glycolysis are several times faster in actively contracting mouse extensor digitorum longus muscle (EDL) than soleus (SOL), but we find these rates are not distinguishable at rest. We used a transient anoxic perturbation of steady state energy balance to decrease phosphocreatine (PCr) reversibly and to measure the rates of ATPase and of lactate production without muscle activation or contraction. The rate of glycolytic ATP synthesis is less than the ATPase rate, accounting for the continual PCr decrease during anoxia in both muscles. We fitted a mathematical model validated with properties of enzymes and solutes measured in vitro and appropriate for the transient perturbation of these muscles to experimental data to test whether the model accounts for the results. Simulations showed equal rates of ATPase and lactate production in both muscles. ATPase controls glycolytic flux by feedback from its products. Adenylate kinase function is critical because a rise in [AMP] is necessary to activate glycogen phosphorylase. ATPase is the primary source of H+ production. The sum of contributions of the 13 reactions of the glycogenolytic and glycolytic network to total proton load is negligible. The stoichiometry of lactate and H+ production is near unity. These results identify a default state of energy metabolism for resting muscle in which there is no difference in the metabolic phenotype of EDL and SOL. Therefore, additional control mechanisms, involving higher ATPase flux and [Ca2+], must exist to explain the well-known difference in glycolytic rates in fast-twitch and slow-twitch muscles in actively contracting muscle.

Analysis of the diffusion of Ras2 in Saccharomyces cerevisiae using fluorescence recovery after photobleaching.

Binding, lateral diffusion and exchange are fundamental dynamic processes involved in protein association with cellular membranes. In this study, we developed numerical simulations of lateral diffusion and exchange of fluorophores in membranes with arbitrary bleach geometry and exchange of the membrane-localized fluorophore with the cytosol during fluorescence recovery after photobleaching (FRAP) experiments. The model simulations were used to design FRAP experiments with varying bleach region sizes on plasma membrane-localized wild-type GFP-Ras2 with a dual lipid anchor and mutant GFP-Ras2C318S with a single lipid anchor in live yeast cells to investigate diffusional mobility and the presence of any exchange processes operating in the time scale of our experiments. Model parameters estimated using data from FRAP experiments with a 1 microm x 1 microm bleach region-of-interest (ROI) and a 0.5 microm x 0.5 microm bleach ROI showed that GFP-Ras2, single or dual lipid modified, diffuses as single species with no evidence of exchange with a cytoplasmic pool. This is the first report of Ras2 mobility in the yeast plasma membrane. The methods developed in this study are generally applicable for studying diffusion and exchange of membrane-associated fluorophores using FRAP on commercial confocal laser scanning microscopes.

Genome-Scale Model-Based Identification of Metabolite Indicators for Early Detection of Kidney Toxicity.

Identifying early indicators of toxicant-induced organ damage is critical to provide effective treatment. To discover such indicators and the underlying mechanisms of toxicity, we used gentamicin as an exemplar kidney toxicant and performed systematic perturbation studies in Sprague Dawley rats. We obtained high-throughput data 7 and 13 h after administration of a single dose of gentamicin (0.5 g/kg) and identified global changes in genes in the liver and kidneys, metabolites in the plasma and urine, and absolute fluxes in central carbon metabolism. We used these measured changes in genes in the liver and kidney as constraints to a rat multitissue genome-scale metabolic network model to investigate the mechanism of gentamicin-induced kidney toxicity and identify metabolites associated with changes in tissue gene expression. Our experimental analysis revealed that gentamicin-induced metabolic perturbations could be detected as early as 7 h postexposure. Our integrated systems-level analyses suggest that changes in kidney gene expression drive most of the significant metabolite alterations in the urine. The analyses thus allowed us to identify several significantly enriched injury-specific pathways in the kidney underlying gentamicin-induced toxicity, as well as metabolites in these pathways that could serve as potential early indicators of kidney damage.

Genome-Scale Characterization of Toxicity-Induced Metabolic Alterations in Primary Hepatocytes.

Context-specific GEnome-scale metabolic Network REconstructions (GENREs) provide a means to understand cellular metabolism at a deeper level of physiological detail. Here, we use transcriptomics data from chemically-exposed rat hepatocytes to constrain a GENRE of rat hepatocyte metabolism and predict biomarkers of liver toxicity using the Transcriptionally Inferred Metabolic Biomarker Response algorithm. We profiled alterations in cellular hepatocyte metabolism following in vitro exposure to four toxicants (acetaminophen, carbon tetrachloride, 2,3,7,8-tetrachlorodibenzodioxin, and trichloroethylene) for six hour. TIMBR predictions were compared with paired fresh and spent media metabolomics data from the same exposure conditions. Agreement between computational model predictions and experimental data led to the identification of specific metabolites and thus metabolic pathways associated with toxicant exposure. Here, we identified changes in the TCA metabolites citrate and alpha-ketoglutarate along with changes in carbohydrate metabolism and interruptions in ATP production and the TCA Cycle. Where predictions and experimental data disagreed, we identified testable hypotheses to reconcile differences between the model predictions and experimental data. The presented pipeline for using paired transcriptomics and metabolomics data provides a framework for interrogating multiple omics datasets to generate mechanistic insight of metabolic changes associated with toxicological responses.