Abstract
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.
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Warburg, O. (1923) Versuche an uberiebendem Carcirnomgewebe. Biochem. Z. 142, 317–333.
Berry, M. N., and Friend, D. S. (1969) High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J. Cell. Biol. 43, 506–520.
Krumdieck, C. L., dos Santos, J. E., and Ho, K. J. (1980) A new instrument for the rapid preparation of tissue slices. Anal. Biochem. 104, 118–123.
Hirsch, G. H. (1976) Differential effects of nephrotoxic agents on renal transport and metabolism by use of in vitro techniques. Environ. Health Perspect. 15, 89–99.
Bach, P., and Lock, E. (1985) The use of renal tissue slices, perfusion and infusion techniques to assess nephrotoxicity related changes. In: (Bach, P. H., and Lock, E. A., eds.) Nephrotoxicity Assessment and Pathogenesis. Monographs of Applied Toxicology. Vol. 1. New-York: Wiley, pp. 505–518.
Berndt, W. O. (1987) Renal slices and perfusion. In: (Bach, P. H., and Lock, E. A., eds.) Nephrotoxicity: The Experimental and Clinical Situation. Martin Nijhoff Publishers, Boston, MA, USA pp. 301–316.
Bach, P. H., Vickers, A. E. M., Fisher, R., et al. (1996) The use of tissue slices for pharmacotoxicology studies. Altern. Lab. Anim. 24, 893–923.
Parrish, A. R., Gandolfi, A. J., and Brendel, K. (1995) Precision-cut tissue slices: applications in pharmacology and toxicology. Life Sci. 57, 1887–1901.
Lerche-Langrand, C., and Toutain, H. J. (2000) Precision-cut liver slices: characteristics and use for in vitro pharmaco-toxicology. Toxicology 153, 221–253.
Vickers, A. E., and Fisher, R. L. (2004) Organ slices for the evaluation of human drug toxicity. Chem. Biol. Interact. 150, 87–96.
Vickers, A. E., and Fisher, R. L. (2005) Precision-cut organ slices to investigate target organ injury. Expert Opin. Drug Metab. Toxicol. 1, 687–699.
De Graaf, I. A. M., Groothuis, G. M. M., and Olinga, P. (2007) Precision-cut tissue slices as a tool to predict metabolism of novel drugs. Expert Opin. Drug Metab. Toxicol. 3, 879–898.
Nicholson, J. K., Lindon, J. C., and Holmes, E. (1999) “Metabonomics”: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica 29, 1181–1189.
Lamprecht, W., and Trautchold, I. (1974) Adenosine-5′-triphosphate. Determination with hexokinase and glucose-6-phosphate dehydrogenase. In: (Bergmeyer, H., ed.) Methods of Enzymatic Analysis. Vol. 4. New York: Academic Press, pp. 2101–2110.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
Bergmeyer, H. U., and Bernt, E. (1974) Lactate dehydrogenase: UV assay with pyruvate and NADH. In: (Bergmeyer, H., ed.) Methods of Enzymatic Analysis. Vol. 2. New-York: Academic Press, pp. 574–579.
Baverel, G., Bonnard, M., D’Armagnac de Castanet, E., and Pellet, M. (1978) Lactate and pyruvate metabolism in isolated renal tubules of normal dogs. Kidney Int. 14, 567–575.
Baverel, G., and Lund, P. (1979) A role for bicarbonate in the regulation of mammalian glutamine metabolism. Biochem. J. 184, 599–606.
Shaka, A. J., Keeler, J., Frenkiel, T., and Freeman, R. (1983) An improved sequence for broadband decoupling: Waltz 16. J. Magn. Reson. 52, 335–338.
Howarth, O. W., and Lilley, D. M. J. (1978) Carbon-13-NMR of peptides and proteins. Prog. NMR Spectrosc. 12, 1–40.
Canioni, P., Alger, J. R., and Shulman, R. G. (1983) Natural abundance Carbon-13 nuclear magnetic resonance spectroscopy of liver and adipose tissue of the living rat. Biochemistry 22, 4974–4980.
Martin, G., Chauvin M.F., Dugelay S., and Baverel G. (1994) Non-steady state model applicable to NMR studies for calculating flux rates in glycolysis, gluconeogenesis, and citric acid cycle. J. Biol. Chem. 269, 26034–26039.
Martin G., Chauvin, M. F., and Baverel, G. (1997) Model applicable to NMR studies for calculating flux rates in five cycles involved in glutamate metabolism. J. Biol. Chem. 272, 4717–4728.
Dugelay, S., Chauvin, M. F., and Megnin-Chanet, F., et al. (1999) Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study. Biochem. J. 342, 555–566.
Conjard, A., Dugelay, S., Chauvin, M. F., Durozard, D., Baverel, G., and Martin, G. (2002) The anaplerotic substrate alanine stimulates acetate incorporation into glutamate and glutamine in rabbit kidney tubules. A 13C NMR study. J. Biol. Chem. 277, 29444–29454.
Vincent, N., Martin, G., and Baverel, G. (1992) Glycine, a new regulator of glutamine metabolism in isolated rat-liver cells. Biochim. Biophys. Acta. 1175, 13–20.
Vercoutere, B., Durozard, D., Baverel, G., and Martin, G. (2004) Complexity of glutamine metabolism in kidney tubules from fed and fasted rats. Biochem. J. 378, 485–495.
Simon, D., and Penry, J. K. (1975) Sodium di-N-propylacetate (DPA) in the treatment of epilepsy. A review. Epilepsia 16, 549–573.
Coulter, D. L., and Allen, R. J. (1980) Secondary hyperammonaemia: a possible mechanism for valproate encephalopathy. Lancet 1, 1310–1311.
Powell-Jackson, P. R., Tredger, J. M., and Williams, R. (1984) Hepatotoxicity to sodium valproate: a review. Gut 25, 673–681.
Warter, J. M., Marescaux, C., Chabrier, G., Rumbach, L., Micheletti, B., and Imler, M. (1984) Renal glutamine metabolism in man during treatment with sodium valproate. Rev. Neurol. (Paris) 140, 370–371.
Ferrier, B., Martin, M., and Baverel, G. (1988) Valproate-induced stimulation of renal ammonia production and excretion in the rat. J. Clin. Chem. Clin. Biochem. 26, 65–67.
Elhamri, M., Ferrier, B., Martin, M., and Baverel, G. (1993) Effect of valproate, sodium 2-propyl-4-pentenoate and sodium 2-propyl-2-pentenoate on renal substrate uptake and ammoniagenesis in the rat. J. Pharmacol. Exp. Ther. 266, 89–96.
Vittorelli, A., Gauthier, C., Michoudet, C., Martin, G., and Baverel, G. (2005) Characteristics of glutamine metabolism in human precision-cut kidney slices: a 13C-NMR study. Biochem. J. 387, 825–834.
Durozard, D, and Baverel, G. (1987) Gas chromatographic method for the measurement of sodium valproate utilization by kidney tubules. J. Chromatogr. 414, 460–464.
Durozard, D., Martin, G., and Baverel, G. (1991) Valproate-induced alterations of coenzyme A and coenzyme A ester concentrations in human kidney tubules metabolizing glutamine. Contrib. Nephrol. 92, 103–108.
Acknowledgments
The authors would like to thank Claudie Pinteur, Rémi Nazaret, and Lara Koneckny for their technical assistance as well as Claire Morel for secretarial assistance. This work was supported by grants from the European Commission [project numbers: BIO4-CT97-2145 (Euroslice) and STREP 032731 (CellNanoTox)] and from INSERM.
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Baverel, G. et al. (2011). Protocols and Applications of Cellular Metabolomics in Safety Studies Using Precision-Cut Tissue Slices and Carbon 13 NMR. In: Gautier, JC. (eds) Drug Safety Evaluation. Methods in Molecular Biology, vol 691. Humana Press. https://doi.org/10.1007/978-1-60761-849-2_12
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DOI: https://doi.org/10.1007/978-1-60761-849-2_12
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