Glutathione metabolism modeling: a mechanism for liver drug-robustness and a new biomarker strategy.

BACKGROUND: Glutathione metabolism can determine an individual's ability to detoxify drugs. To increase understanding of the dynamics of cellular glutathione homeostasis, we have developed an experiment-based mathematical model of the kinetics of the glutathione network. This model was used to simulate perturbations observed when human liver derived THLE cells, transfected with human cytochrome P452E1 (THLE-2E1 cells), were exposed to paracetamol (acetaminophen). METHODS: Human liver derived cells containing extra human cytochrome P4502E1 were treated with paracetamol at various levels of methionine and in the presence and absence of an inhibitor of glutamyl-cysteine synthetase (GCS). GCS activity was also measured in extracts. Intracellular and extracellular concentrations of substances involved in glutathione metabolism were measured as was damage to mitochondria and proteins. A bottom up mathematical model was made of the metabolic pathways around and including glutathione. RESULTS: Our initial model described some, but not all the metabolite-concentration and flux data obtained when THLE-2E1 cells were exposed to paracetamol at concentrations high enough to affect glutathione metabolism. We hypothesized that the lack of correspondence could be due to upregulation of expression of glutamyl cysteine synthetase, one of the enzymes controlling glutathione synthesis, and confirmed this experimentally. A modified model which incorporated this adaptive response adequately described the observed changes in the glutathione pathway. Use of the adaptive model to analyze the functioning of the glutathione network revealed that a threshold input concentration of methionine may be required for effective detoxification of reactive metabolites by glutathione conjugation. The analysis also provided evidence that 5-oxoproline and ophthalmic acid are more useful biomarkers of glutathione status when analyzed together than when analyzed in isolation, especially in a new, model-assisted integrated biomarker strategy. CONCLUSION: A robust mathematical model of the dynamics of cellular changes in glutathione homeostasis in cells has been developed and tested in vitro. GENERAL SIGNIFICANCE: Mathematical models of the glutathione pathway that help examine mechanisms of cellular protection against xenobiotic toxicity and the monitoring thereof, can now be made.

Systems biology tools for toxicology.

An important goal of toxicology is to understand and predict the adverse effects of drugs and other xenobiotics. For pharmaceuticals, such effects often emerge unexpectedly in man even when absent from trials in vitro and in animals. Although drugs and xenobiotics act on molecules, it is their perturbation of intracellular networks that matters. The tremendous complexity of these networks makes it difficult to understand the effects of xenobiotics on their ability to function. Because systems biology integrates data concerning molecules and their interactions into an understanding of network behaviour, it should be able to assist toxicology in this respect. This review identifies how in silico systems biology tools, such as kinetic modelling, and metabolic control, robustness and flux analyse, may indeed help understanding network-mediated toxicity. It also shows how these approaches function by implementing them vis-à-vis the glutathione network, which is important for the detoxification of reactive drug metabolites. The tools enable the appreciation of the steady state concept for the detoxification network and make it possible to simulate and then understand effects of perturbations of the macromolecules in the pathway that are counterintuitive. We review how a glutathione model has been used to explain the impact of perturbation of the pathway at various molecular sites, as would be the effect of single-nucleotide polymorphisms. We focus on how the mutations impact the levels of glutathione and of two candidate biomarkers of hepatic glutathione status. We conclude this review by sketching how the various systems biology tools may help in the various phases of drug development in the pharmaceutical industry.

Multiscale modelling approach combining a kinetic model of glutathione metabolism with PBPK models of paracetamol and the potential glutathione-depletion biomarkers ophthalmic acid and 5-oxoproline in humans and rats.

A key role of the antioxidant glutathione is detoxification of chemically reactive electrophilic drug metabolites within the liver. Therefore glutathione depletion can have severe toxic consequences. Ophthalmic acid and 5-oxoproline are metabolites involved in glutathione metabolism, which can be measured readily in the blood and urine and have been proposed as candidate biomarkers of hepatic glutathione content. However, currently it is unclear whether their concentrations in plasma exhibit a robust correlation with hepatic glutathione content. To explore this important question, we have developed a novel approach which combines a physiologically based pharmacokinetic (PBPK) model of metabolism and disposition of paracetamol (acetaminophen) with a previously developed mathematical systems model of hepatic glutathione homeostasis. Paracetamol is metabolised to reactive intermediates which deplete glutathione and cause toxicity when given at high doses. Our model correctly predicted that hepatic glutathione depletion following paracetamol administration resulted in elevated concentrations of 5-oxoproline and ophthalmic acid in blood and of 5-oxoproline in urine. However, we also found from the model that concentrations of both of the compounds were likely to be influenced by prolonged administration of paracetamol and by the concentrations of intracellular metabolites such as methionine. We conclude that care must be taken when extrapolating from concentrations of these biomarkers to hepatic glutathione status.

A mathematical modelling approach to assessing the reliability of biomarkers of glutathione metabolism.

One of the main pathways for the detoxification of reactive metabolites in the liver involves glutathione conjugation. Metabolic profiling studies have shown paradoxical responses in glutathione-related biochemical pathways. One of these is the increase in 5-oxoproline and ophthalmic acid concentrations with increased dosage of paracetamol. Experimental studies have thus far failed to resolve these paradoxes and the robustness of how these proposed biomarkers correlate with liver glutathione levels has been questioned. To better understand how these biomarkers behave in the glutathione system a kinetic model of this pathway was made. By using metabolic control analysis and by simulating biomarker levels under a variety of conditions, we found that 5-oxoproline and ophthalmic acid concentrations may not only depend on the glutathione but also on the methionine status of the cell. We show that neither of the two potential biomarkers are reliable on their own since they need additional information about the methionine status of the system to relate them uniquely to intracellular glutathione concentration. However, when both biomarkers are measured simultaneously a direct inference of the glutathione concentration can be made, irrespective of the methionine concentration in the system.

A Systems Biology Approach to Deciphering the Etiology of Steatosis Employing Patient-Derived Dermal Fibroblasts and iPS Cells.

Non-alcoholic fatty liver disease comprises a broad spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis. As a result of increases in the prevalences of obesity, insulin resistance, and hyperlipidemia, the number of people with hepatic steatosis continues to increase. Differences in susceptibility to steatohepatitis and its progression to cirrhosis have been attributed to a complex interplay of genetic and external factors all addressing the intracellular network. Increase in sugar or refined carbohydrate consumption results in an increase of insulin and insulin resistance that can lead to the accumulation of fat in the liver. Here we demonstrate how a multidisciplinary approach encompassing cellular reprogramming, transcriptomics, proteomics, metabolomics, modeling, network reconstruction, and data management can be employed to unveil the mechanisms underlying the progression of steatosis. Proteomics revealed reduced AKT/mTOR signaling in fibroblasts derived from steatosis patients and further establishes that the insulin-resistant phenotype is present not only in insulin-metabolizing central organs, e.g., the liver, but is also manifested in skin fibroblasts. Transcriptome data enabled the generation of a regulatory network based on the transcription factor SREBF1, linked to a metabolic network of glycerolipid, and fatty acid biosynthesis including the downstream transcriptional targets of SREBF1 which include LIPIN1 (LPIN) and low density lipoprotein receptor. Glutathione metabolism was among the pathways enriched in steatosis patients in comparison to healthy controls. By using a model of the glutathione pathway we predict a significant increase in the flux through glutathione synthesis as both gamma-glutamylcysteine synthetase and glutathione synthetase have an increased flux. We anticipate that a larger cohort of patients and matched controls will confirm our preliminary findings presented here.

HPLC-MS/MS methods for the quantitative analysis of 5-oxoproline (pyroglutamate) in rat plasma and hepatic cell line culture medium.

5-Oxoproline (5-OP; pyroglutamate) is an intermediate in the biosynthesis of the endogenous tripeptide glutathione and has been seen to be elevated in the biofluids and tissues of rats following the administration of glutathione-depleting hepatotoxic xenobiotics such as acetaminophen (paracetamol), bromobenzene and ethionine. As 5-OP is a potential biomarker for hepatotoxicity HPLC-MS/MS methods have been developed for its quantification in in vitro cell culture media and rat plasma. For the cell culture media the lower limit of quantification (LLOQ), defined as the lowest concentration on the calibration curve, was 10 ng/ml. Minimal carry over was observed for cell culture media between injections (less than 5% at all concentrations examined), precision and accuracy were generally better than 20% for within and between day analyses. For rat plasma a LLOQ of 50 ng/ml was obtained. Carry over for plasma was less than 5% for all concentrations, within and between batch accuracy and precision were generally better than 20%. The methods were linear for both sample types from the LLOQ up to 1 µg/ml. For samples obtained from rats subjected to chronic administration of the hepatotoxin methapyrilene, concentrations of 5-OP were not observed to increase significantly at any time point compared to controls. 5-OP was also determined in the culture media of human liver epithelial (THLE) cells transfected with cytochrome P450 2E1 (THLE-2E1). Following exposure of THLE-2E1 cells to acetaminophen, large increases in the concentrations of 5-OP were observed, which correlated with reduced cellular glutathione content and with cell toxicity. These results show that LC-MS/MS can be used to perform rapid, sensitive, and quantitative determination of 5-OP in vivo and in vitro and will enable additional investigations into the utility of 5-OP as a biomarker of liver drug-induced liver injury.

HPLC-MS/MS methods for the quantitative analysis of ophthalmic acid in rodent plasma and hepatic cell line culture medium.

Ophthalmic acid (OA), an endogenous tripeptide analogue of glutathione, has been suggested as a potential biomarker for paracetamol/acetaminophen hepatotoxicity. Here HPLC-MS/MS methods have been developed for the precise, sensitive and specific detection and quantification of OA in in vitro cell culture medium and plasma. For the cell culture medium the LLOQ was found to be 1 ng/ml, with less than 1% between sample carry over at all concentrations and precision below 15% for within day and below 9% for between day analyses. For rat plasma the presence of endogenous OA resulted in the LLOQ being 25 ng/ml (defined as the lowest concentration on the calibration curve where the base peak was less than 20% of the LLOQ). For the plasma assay the percentage carry over was less than 1% for all concentrations and within and between batch precision was below 21%. The methods were linear for both sample types from the LLOQ up to 5 µg/ml. The method was successfully applied to the determination of OA in samples obtained following the chronic administration of the rat hepatotoxin methapyrilene, where plasma OA concentrations were observed to show a weak negative correlation with those of established liver injury biomarkers such as aspartate aminotransferase (AST).