Protocols and Applications of Cellular Metabolomics in Safety Studies Using Precision-Cut Tissue Slices and Carbon 13 NMR.

Numerous xenobiotics are toxic to human and animal cells by interacting with their metabolism, but the precise metabolic step affected and the biochemical mechanism behind such a toxicity remain often unknown. In an attempt to reduce the ignorance in this field, we have developed a new approach called cellular metabolomics. This approach, developed in vitro, provides a panoramic view not only of the pathways involved in the metabolism of physiological substrates of any normal or pathological human or animal cell but also of the beneficial and adverse effects of xenobiotics on these metabolic pathways. Unlike many cell lines, precision-cut tissue slices, for which there is a renewed interest, remain metabolically differentiated for at least 24-48 h and allow to study the effect of xenobiotics during short-term and long-term incubations. Cellular metabolomics (or metabolic flux analysis), which combines enzymatic and carbon 13 NMR measurements with mathematical modeling of metabolic pathways, is illustrated in this brief chapter for studying the effect of insulin on glucose metabolism in rat liver precision-cut slices and of valproate on glutamine metabolism in human renal cortical precision-cut slices. The use of very small amounts of test compounds allows to predict their toxic effect and eventually their beneficial effects very early in the research and development processes. Cellular metabolomics is complementary to other omics approaches, but, unlike them, provides functional, mechanistic, and dynamic pieces of information by measuring enzymatic fluxes.

Use of precision-cut renal cortical slices in nephrotoxicity studies.

1.Unlike cell lines and primary cells in culture, precision-cut tissue slices remain metabolically differentiated for at least 24-48 h and allow to study the effect of xenobiotics during short-term and long-term incubations. 2.In this article, we illustrate the use of such an experimental model to study the nephrotoxic effects of (i) chloroacetaldehyde, a metabolite of the anticancer drug ifosfamide, (ii) of cobalt chloride, a potential leakage product of the cobalt-containing nanoparticles, and (iii) of valproate, a widely used antiepileptic drug. 3.Since all the latter test compounds, like many toxic compounds, negatively interact with cellular metabolic pathways, we also illustrate our biochemical toxicology approach in which we used not only enzymatic but also carbon 13 NMR measurements and mathematical modelling of metabolic pathways. 4.This original approach, which can be applied to any tissue, allows to predict the nephrotoxic effects of milligram amounts of test compounds very early during the research and development processes of drugs and chemicals. This approach, combined with the use of cells that retain their in vivo metabolic properties and, therefore, are predictive, reduces the risk, the time and cost of such processes.

Brain slices from glutaminase-deficient mice metabolize less glutamine: a cellular metabolomic study with carbon 13 NMR.

In the brain, glutaminase is considered to have a key role in the provision of glutamate, a major excitatory neurotransmitter. Brain slices obtained from wild-type (control) and glutaminase-deficient (GLS1+/-) mice were incubated without glucose and with 5 or 1 mmol/L [3-(13)C]glutamine as substrate. At the end of the incubation, substrate removal and product formation were measured by both enzymatic and carbon 13 nuclear magnetic resonance ((13)C-NMR) techniques. Slices from GLS1+/- mice consumed less [3-(13)C]glutamine and accumulated less [3-(13)C]glutamate. They also produced less (13)CO(2) but accumulated amounts of (13)C-aspartate and (13)C-gamma-aminobutyric acid (GABA) that were similar to those found with brain slices from control mice. The newly formed glutamine observed in slices from control mice remained unchanged in slices from GLS1+/- mice. As expected, flux through glutaminase in slices from GLS1+/- mice was found diminished. Fluxes through all enzymes of the tricarboxylic acid cycle were also reduced in brain slices from GLS1+/- mice except through malate dehydrogenase with 5 mmol/L [3-(13)C]glutamine. The latter diminutions are consistent with the decreases in the production of (13)CO(2) also observed in the slices from these mice. It is concluded that the genetic approach used in this study confirms the key role of glutaminase for the provision of glutamate.

Effects of valproate on glutamate metabolism in rat brain slices: a (13)C NMR study.

Sodium valproate is a drug widely used for the treatment of epilepsy and mood disorders. We studied the effect of valproate on cerebral energy metabolism by incubating rat brain slices with 5 mM [3-(13)C]glutamate in the absence and the presence of 1 mM valproate. Substrate removal and product formation were measured by enzymatic and carbon 13 NMR methods. Fluxes through the enzymatic steps involved were calculated with an original mathematical model. We demonstrate that, in the presence of valproate, glutamate consumption and aspartate accumulation and labeling were inhibited, whereas GABA accumulation and labeling were increased. Consistent with these observations, this drug inhibited the unidirectional flux from glutamate to α-ketoglutarate and fluxes through several enzymes (gamma aminobutyric acid aminotransferase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, malic enzyme, pyruvate dehydrogenase, pyruvate carboxylase and citrate synthase). By contrast, glutamic acid decarboxylase flux was increased. With 2 mM glutamate+1 mM valproate and with 5 mM glutamate+2 mM valproate, GABA and aspartate labelings were similarly altered. On the basis of the effects of valproate, it is concluded that our cellular model and our cellular metabolomic approach appear suitable to study the beneficial and adverse interactions of neurotropic compounds with the cerebral metabolic pathways.

Rat brain slices oxidize glucose at high rates: a (13)C NMR study.

Since glucose is the main cerebral substrate, we have characterized the metabolism of various (13)C glucose isotopomers in rat brain slices. For this, we have used our cellular metabolomic approach that combines enzymatic and carbon 13 NMR techniques with mathematical models of metabolic pathways. We identified the fate and the pathways of the conversion of glucose carbons into various products (pyruvate, lactate, alanine, aspartate, glutamate, GABA, glutamine and CO(2)) and determined absolute fluxes through pathways of glucose metabolism. After 60 min of incubation, lactate and CO(2) were the main end-products of the metabolism of glucose which was avidly metabolized by the slices. Lactate was also used at high rates by the slices and mainly converted into CO(2). High values of flux through pyruvate carboxylase, which were similar with glucose and lactate as substrate, were observed. The addition of glutamine, but not of acetate, stimulated pyruvate carboxylation, the conversion of glutamate into succinate and fluxes through succinate dehydrogenase, malic enzyme, glutamine synthetase and aspartate aminotransferase. It is concluded that, unlike brain cells in culture, and consistent with high fluxes through PDH and enzymes of the tricarboxylic acid cycle, rat brain slices oxidized both glucose and lactate at high rates.

Metabolic fate of a high concentration of glutamine and glutamate in rat brain slices: a ¹³C NMR study.

This study was performed to analyze the metabolic fate of a high concentration (5 mM) of glutamine and glutamate in rat brain slices and the participation of these amino acids in the glutamine-glutamate cycle. For this, brain slices were incubated for 60 min with [3-¹³C]glutamine or [3-¹³C]glutamate. Tissue plus medium extracts were analyzed by enzymatic and ¹³C NMR measurements and fluxes through pathways of glutamine and glutamate metabolism were calculated. We demonstrate that both substrates were utilized and oxidized at high rates by rat brain slices and served as precursors of neurotransmitters, tricarboxylic acid (TCA) cycle intermediates and alanine. In order to determine the participation of glutamine synthetase in the appearance of new glutamine molecules with glutamine as substrate, brain slices were incubated with [3-¹³C]glutamine in the presence of methionine sulfoximine, a specific inhibitor of glutamine synthetase. Our results indicate that 36.5% of the new glutamine appeared was glutamine synthetase-dependent and 63.5% was formed from endogenous substrates. Flux through glutamic acid decarboxylase was higher with glutamine than with glutamate as substrate whereas fluxes from α-ketoglutarate to glutamate and through glutamine synthetase, malic enzyme, pyruvate dehydrogenase, pyruvate carboxylase and citrate synthase were in the same range with both substrates.

Protocols and applications of cellular metabolomics in safety studies using precision-cut tissue slices and carbon 13 NMR.

Numerous xenobiotics are toxic to human and animal cells by interacting with their metabolism, but the precise metabolic step affected and the biochemical mechanism behind such a toxicity often remain unknown. In an attempt to reduce the ignorance in this field, we have developed a new approach called cellular metabolomics. This approach, developed in vitro, provides a panoramic view not only of the pathways involved in the metabolism of physiologic substrates of any normal or pathologic human or animal cell but also of the beneficial and adverse effects of xenobiotics on these metabolic pathways. Unlike many cell lines, precision-cut tissue slices, for which there is a renewed interest, remain metabolically differentiated for at least 24-48 h and allow to study the effect of xenobiotics during short-term and long-term incubations. Cellular metabolomics (or cellular metabonomics), which combines enzymatic and carbon 13 NMR measurements with mathematical modeling of metabolic pathways, is illustrated in this brief chapter for studying the effect of insulin on glucose metabolism in rat liver precision-cut slices, and of valproate on glutamine metabolism in human renal cortical precision-cut slices. The use of very small amounts of test compounds allows to predict their toxic effect and eventually their beneficial effects very early in the research and development processes. Cellular metabolomics is complementary to other omics approaches, but, unlike them, provides functional and dynamic pieces of information by measuring enzymatic fluxes.