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.

Cadmium chloride inhibits lactate gluconeogenesis in mouse renal proximal tubules: An in vitro metabolomic approach with (13)C NMR.

Using isolated mouse renal proximal tubules incubated with lactate as substrate, we have found that the addition of 1-50 μM cadmium chloride (CdCl2) caused a concentration-dependent decrease in lactate utilization, in glucose production and in the cellular level of ATP, coenzyme A, acetyl-coenzyme A and glutathione (reduced and oxidized forms). Combining enzymatic and (13)C NMR measurements in a cellular metabolomic approach, we have shown that, in the presence of 10 μM CdCl2, fluxes through the key-enzymes of gluconeogenesis, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were greatly depressed by cadmium. This was accompanied by a reduction in fluxes through the enzymes of the tricarboxylic acid cycle.

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.

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.

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.

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.

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.

Cadmium chloride inhibits lactate gluconeogenesis in isolated human renal proximal tubules: a cellular metabolomic approach with 13C-NMR.

As part of a study on cadmium nephrotoxicity, we studied the effect of cadmium chloride (CdCl2) in isolated human renal proximal tubules metabolizing the physiological substrate lactate. Dose-effect experiments showed that 10-500 μM CdCl2 reduced lactate removal, glucose production and the cellular levels of ATP, coenzyme A, acetyl-coenzyme A and of reduced glutathione in a dose-dependent manner. After incubation with 5 mM L: -[1-(13)C]-, or L: -[2-(13)C]-, or L: -[3-(13)C] lactate or 5 mM L: -lactate plus 25 mM NaH(13)CO3 as substrates, substrate utilization and product formation were measured by both enzymatic and carbon 13 NMR methods.

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.

Ifosfamide metabolite chloroacetaldehyde inhibits cell proliferation and glucose metabolism without decreasing cellular ATP content in human breast cancer cells MCF-7.

Chloroacetaldehyde (CAA), a product of hepatic metabolism of the widely used anticancer drug ifosfamide (IFO), has been reported to decrease cancer cell proliferation. The basis of this effect is not completely known but has been attributed to a drop of cellular ATP content. Given the importance of glucose metabolism and of the 'Warburg effect' in cancer cells, we examined in the present study the ability of CAA to inhibit cancer cell proliferation by altering the glycolytic pathway.

Uranyl nitrate inhibits lactate gluconeogenesis in isolated human and mouse renal proximal tubules: a 13C-NMR study.

As part of a study on uranium nephrotoxicity, we investigated the effect of uranyl nitrate in isolated human and mouse kidney cortex tubules metabolizing the physiological substrate lactate. In the millimolar range, uranyl nitrate reduced lactate removal and gluconeogenesis and the cellular ATP level in a dose-dependent fashion. After incubation in phosphate-free Krebs-Henseleit medium with 5 mM L-[1-13C]-, or L-[2-13C]-, or L-[3-13C]lactate, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods.

Targets of chloroacetaldehyde-induced nephrotoxicity.

Chloroacetaldehyde, one of the main products of hepatic ifosfamide metabolism, contributes to its nephrotoxicity. However, the pathophysiology of this toxicity is not fully understood. The present work examined the time and dose effects of clinically relevant concentrations of chloroacetaldehyde (25-75microM) on precision-cut rat renal cortical slices metabolizing a physiological concentration of lactate.

Study of a new indirect method based on absolute norms of variation to detect autologous blood transfusion.

The aim of this study was to investigate an indirect method based on a determination of absolute norms of variation in biological markers that could be used to identify autologous blood transfusion within the framework of the fight against doping. The selection of markers was made from experimental variations obtained during different phases including an increase in training volume at sea level, high altitude training, blood withdrawal and autologous blood reinfusion. The global statistical method was then developed in order to fix absolute norms of variation for each selected marker.

Mechanisms of the ifosfamide-induced inhibition of endocytosis in the rat proximal kidney tubule.

The Fanconi syndrome is a common side effect of the chemotherapeutic agent ifosfamide. Current evidences suggest that chloroacetaldehyde (CAA), one of the main metabolites of ifosfamide activation, contributes to its nephrotoxicity. However, the pathophysiology of CAA-induced Fanconi syndrome is not fully understood.

In vivo mesna and amifostine do not prevent chloroacetaldehyde nephrotoxicity in vitro.

Chloroacetaldehyde (CAA) is the putative metabolite responsible for ifosfamide-induced nephrotoxicity. Whereas evidence suggests that sodium 2-mercaptoethanesulfonate (mesna) and amifostine protect renal cells against CAA toxicity in vitro, their efficacy in clinical studies is controversial. To better understand the discrepancy between in vivo and in vitro results, we combined the in vivo intraperitoneal administration of either saline or mesna (100 mg/kg) or amifostine (200 mg/kg) in rats and the in vitro study of CAA toxicity to both proximal tubules and precision-cut renal cortical slices.

[Renal and urinary tract disease national research program].

The National Institute of Health and Medical Research (Inserm), the Society of Nephrology, and the French Kidney Foundation recognized the need to create a National Research Program for kidney and urinary tract diseases. They organized a conference gathering 80 researchers to discuss the state-of-the art and evaluate the strengths and weaknesses of kidney and urinary tract disease research in France, and to identify research priorities. From these priorities emerged 11 of common interest: 1) conducting epidemiologic studies; 2) conducting large multicenter cohorts of well-phenotyped patients with blood, urine and biopsy biobanks; 3) developing large scale approach: transcriptomics, proteomics, metabolomics; 4) developing human and animal functional imaging techniques; 5) strengthening the expertise in renal pathology and electrophysiology; 6) developing animal models of kidney injury; 7) identifying nontraumatic diagnostic and prognostic biomarkers; 8) increasing research on the fetal programming of adult kidney diseases; 9) encouraging translational research from bench to bedside and to population; 10) creating centers grouping basic and clinical research workforces with critical mass and adequate logistic support; 11) integrating and developing european research programs.

Glutamine gluconeogenesis in the small intestine of 72 h-fasted adult rats is undetectable.

Recent reports have indicated that 48-72 h of fasting, Type 1 diabetes and high-protein feeding induce gluconeogenesis in the small intestine of adult rats in vivo. Since this would (i) represent a dramatic revision of the prevailing view that only the liver and the kidneys are gluconeogenic and (ii) have major consequences in the metabolism, nutrition and diabetes fields, we have thoroughly re-examined this question in the situation reported to induce the highest rate of gluconeogenesis. For this, metabolically viable small intestinal segments from 72 h-fasted adult rats were incubated with [3-13C]glutamine as substrate.

Intrinsic gluconeogenesis is enhanced in renal proximal tubules of Zucker diabetic fatty rats.

Recent studies indicate that renal gluconeogenesis is substantially stimulated in patients with type 2 diabetes, but the mechanism that is responsible for such stimulation remains unknown. Therefore, this study tested the hypothesis that renal gluconeogenesis is intrinsically elevated in the Zucker diabetic fatty rat, which is considered to be an excellent model of type 2 diabetes. For this, isolated renal proximal tubules from diabetic rats and from their lean nondiabetic littermates were incubated in the presence of physiologic gluconeogenic precursors.

Identification of novel targets of cephaloridine in rabbit renal proximal tubules synthesizing glutamine from alanine.

Cephaloridine, which accumulates in the renal proximal tubule, is a model compound used for studying the toxicity of antibiotics towards this nephron segment. Several studies have demonstrated that cephaloridine alters renal intermediary and energy metabolism, but the mechanism by which this compound interferes with renal metabolic pathways remains incompletely understood. In an attempt to improve our knowledge in this field, we have studied the influence of cephaloridine on the synthesis of glutamine, which represents a key metabolic process involving several important enzymatic steps in the rabbit kidney.

Characteristics of glutamine metabolism in human precision-cut kidney slices: a 13C-NMR study.

The metabolism of glutamine, a physiological substrate of the human kidney, plays a major role in systemic acid-base homoeostasis. Not only because of the limited availability of human renal tissue but also in part due to the lack of adequate cellular models, the mechanisms regulating the renal metabolism of this amino acid in humans have been poorly characterized. Therefore given the renewed interest in their use, human precision-cut renal cortical slices were incubated in Krebs-Henseleit medium (118 mM NaCl, 4.

Metabolic viability and pharmaco-toxicological reactivity of cryopreserved human precision-cut renal cortical slices.

We have tested the suitability of cryopreserved human precision-cut renal cortical slices for metabolic and pharmaco-toxicological studies. The viability of these slices and their pharmaco-toxicological reactivity were assessed using intracellular ATP and protein contents, lactate dehydrogenase (LDH) leakage, lactate and glutamine metabolism and the ammoniagenic effect of valproate. Despite a decrease in ATP and protein contents when compared with those of fresh slices, cryopreserved slices did not show any LDH leakage and retained the capacity to metabolize glutamine and lactate.

Complexity of glutamine metabolism in kidney tubules from fed and fasted rats.

Glutamine is an important renal glucose precursor and energy provider. In order to advance our understanding of the underlying metabolic processes, we studied the metabolism of variously labelled [13C]glutamine and [14C]glutamine molecules and the effects of fasting in isolated rat renal proximal tubules. Absolute fluxes through the enzymes involved, including enzymes of four different cycles operating concomitantly, were assessed by combining mainly the 13C NMR data with an appropriate model of glutamine metabolism.

Inhibition of glutamine synthetase in the mouse kidney: a novel mechanism of adaptation to metabolic acidosis.

As part of a study on the regulation of renal ammoniagenesis in the mouse kidney, we investigated the effect of chronic metabolic acidosis on glutamine synthesis by isolated mouse renal proximal tubules. The results obtained reveal that, in tubules from control mice, glutamine synthesis occurred at high rates from glutamate and proline and, to a lesser extent, from ornithine, alanine, and aspartate. A 48 h, metabolic acidosis caused a marked inhibition of glutamine synthesis from near-physiological concentrations of both alanine and proline that were avidly metabolized by the tubules; metabolic acidosis also greatly stimulated glutamine utilization and metabolism.

Effect of starvation on glutamine ammoniagenesis and gluconeogenesis in isolated mouse kidney tubules.

It has been shown recently that glutamine is taken up by the mouse kidney in vivo. However, knowledge about the fate of this amino acid and the regulation of its metabolism in the mouse kidney remains poor. Given the physiological and pathophysiological importance of renal glutamine metabolism and the increasing use of genetically modified mice in biological research, we have conducted a study to characterize glutamine metabolism in the mouse kidney.

The anaplerotic substrate alanine stimulates acetate incorporation into glutamate and glutamine in rabbit kidney tubules. A (13)C NMR study.

Although acetate, the main circulating volatile fatty acid in humans and animals, is metabolized at high rates by the renal tissue, little is known about the precise fate of its carbons and about the regulation of its renal metabolism. Therefore, we studied the metabolism of variously labeled [(13)C]acetate and [(14)C]acetate molecules and its regulation by alanine, which is also readily metabolized by the kidney, in isolated rabbit renal proximal tubules. With acetate as the sole substrate, 72% of the C-1 and 49% of the C-2 of acetate were released as CO(2); with acetate plus alanine, the corresponding values were decreased to 49 and 25%.