The metabolism of 2- 13C/ 15N-glycine and U- 13C-glucose was determined in four tissue blocks (adductor muscle, stomach and digestive gland, mantle, and gills) of the Eastern oyster ( Crassostrea virginica) using proton ( 1H) and carbon-13 ( 13C) nuclear magnetic resonance (NMR) spectroscopy. The oysters were treated in aerated seawater with three treatments (5.5 mM U- 13C-glucose, 2.7 mM 2- 13C/ 15N-glycine, and 5.5 mM U- 13C-glucose plus 2.7 mM 2- 13C/ 15N-glycine) and the relative mass balance and 13C fractional enrichments were determined in the four tissue blocks. In all tissues, glycine was metabolized by the glycine cycle forming serine exclusively in the mitochondria by the glycine cleavage system forming 2,3- 13C-serine. In muscle, a minor amount of serine-derived pyruvate entered the Krebs cycle as substantiated by detection of a trace of 2,3- 13C-aspartate. In all tissues, U- 13C-glucose formed glycogen by glycogen synthesis, alanine by glycolysis, and glutamate and aspartate through the Krebs cycle. Alanine was formed exclusively from glucose via alanine transaminase and not glycine via alanine-glyoxylate transaminase. Based on isotopomer analysis, pyruvate carboxylase and pyruvate dehydrogenase appeared to be equal points for pyruvate entry into the Krebs cycle. In the 5.5 mM U- 13C-glucose plus 2.7 mM 2- 13C/ 15N-glycine emergence treatment used to simulate 12 h of “low tide”, oysters accumulated more 13C-labeled metabolites, including both anaerobic glycolytic and aerobic Krebs cycle intermediates. The aerobic metabolites could be the biochemical result of the gaping behavior of mollusks during emergence. The change in tissue distribution and mass balance of 13C-labeled nutrients (U- 13C-glucose and 2- 13C/ 15N-glycine) provides the basis for a new quantitative fluxomic method for elucidating sub-lethal environmental effects in marine organisms called whole body mass balance phenotyping (WoMBaP ).
References
[1]
Smart, R.C.; Hodgeson, E. Molecular and Biochemical Toxicology, 4th ed. ed.; Wiley, John & Sons, Incorporated: Hoboken, NJ, USA, 2008; p. 948.
[2]
Klaassen, C.D. Casarett & Doull’s Toxicology: The Basic Science of Poisons, 7th ed. ed.; McGraw-Hill Professional: New York, NY, USA, 2007; p. 1280.
[3]
Lee, H.; Tikunov, A.; Stoskopf, M.K.; Macdonald, J.M. Applications of chemical shift imaging to marine sciences. Mar. Drugs 2010, 8, 2369–2383, doi:10.3390/md8082369.
[4]
Tikunov, A.P.; Winnike, J.H.; Tech, K.; Jeffries, R.E.; Semelka, C.T.; Martin, J.; McClelland, R.; Graves, L.M.; Macdonald, J.M. Fluxomics by NMR spectroscopy from cells to organisms focusing on liver. Curr. Metabolomics 2013, 1, 128–159, doi:10.2174/2213235X11301020003.
[5]
Tikunov, A.; Johnson, C.B.; Lee, H.; Stoskopf, M.K.; Macdonald, J.M. Metabolomic investigations of American oysters Using 1H-NMR spectroscopy. Mar. Drugs 2010, 8, 2578–2596, doi:10.3390/md8102578.
[6]
Fan, T.W.; Higashi, R.M.; Macdonald, J.M. Emergence and recovery response of phosphate metabolites and intracellular pH in intact Mytilus edulis as examined in situ by in vivo 31P-NMR.. Biochim. Biophys. Acta (BBA)—Mol. Cell Res. 1991, 1092, 39–47, doi:10.1016/0167-4889(91)90176-X.
[7]
Higashi, R.M.; Fan, T.W.-M.; Macdonald, J.M. Monitoring of metabolic responses of intact Haliotis (abalones) under salinity stress by 31P surface probe localized NMR. J. Exp. Zool. 1989, 249, 350–356, doi:10.1002/jez.1402490317.
[8]
Hines, A.; Oladiran, G.S.; Bignell, J.P.; Stentiford, G.D.; Viant, M.R. Direct sampling of organisms from the field and knowledge of their phenotype: Key recommendations for environmental metabolomics. Environ. Sci. Technol. 2007, 41, 3375–3381, doi:10.1021/es062745w.
[9]
Jones, O.A.H.; Spurgeon, D.J.; Svendsen, C.; Griffin, J.L. A metabolomics based approach to assessing the toxicity of the polyaromatic hydrocarbon pyrene to the earthworm Lumbricus rubellus. Chemosphere 2008, 71, 601–609, doi:10.1016/j.chemosphere.2007.08.056.
[10]
Tuffnail, W.; Mills, G.A.; Cary, P.; Greenwood, R. An environmental 1H NMR metabolomic study of the exposure of the marine mussel Mytilus edulis to atrazine, lindane, hypoxia and starvation. Metabolomics 2009, 5, 33–43, doi:10.1007/s11306-008-0143-1.
[11]
Viant, M.R.; Bundy, J.G.; Pincetich, C.A.; de Ropp, J.S.; Tjeerdema, R.S. NMR-derived developmental metabolic trajectories: An approach for visualizing the toxic actions of trichloroethylene during embryogenesis. Metabolomics 2005, 1, 149–158, doi:10.1007/s11306-005-4429-2.
[12]
Macdonald, J.M.; Schmidlin, O.; James, T.L. In vivo monitoring of hepatic glutathione in anesthetized rats by 13C NMR. Magn. Reson. Med. 2002, 48, 430–439, doi:10.1002/mrm.10244.
[13]
Jo, P.G.; Choi, Y.K.; Choi, C.Y. Cloning and mRNA expression of antioxidant enzymes in the Pacific oyster, Crassostrea gigas in response to cadmium exposure. Comp. Biochem. Physiol. Part C 2008, 147, 460–469.
[14]
Ivanina, A.V.; Sokolov, E.P.; Sokolova, I.M. Effects of cadmium on anaerobic energy metabolism and mRNA expression during air exposure and recovery of an intertidal mollusk Crassostrea virginica. Aquat. Toxicol. 2010, 99, 330–342, doi:10.1016/j.aquatox.2010.05.013.
Meng, J.; Zhu, Q.; Zhan, L.; Li, C.; Li, L.; She, Z.; Huang, B.; Zhang, G. Genome and transcriptome analyses provide insight into the euryhaline adaptation mechanism of Crassostrea gigas. PLoS One 2013, 8, e58563.
[17]
Cunningham, P.A.; Tripp, M.R. Accumulation, tissue distribution and elimination of 203HgCl2 and CH3 203HgCl in the tissues of the American oyster Crassostrea virginica. Mar. Biol. 1975, 31, 321–334, doi:10.1007/BF00392089.
[18]
Denton, G.R.W.; Burdon-Jones, C. Influence of temperature and salinity on the uptake, distribution and depuration of mercury, cadmium and lead by the black-lip oyster Saccostrea echinata. Mar. Biol. 1981, 64, 317–326.
[19]
Martincié, D.; Nürnberg, N.W.; Stoeppler, M.; Branica, M. Bioaccumulation of heavy metals by bivalves from Lim Fjord (North Adriatic Sea). Mar. Biol. 1984, 81, 177–188, doi:10.1007/BF00393116.
[20]
Bryan, G.W.; Langston, W.J. Bioavailability, accumulation and effects of heavy metals in sediments with special reference to United Kingdom estuaries: A review. Environ. Pollut. 1992, 76, 89–131, doi:10.1016/0269-7491(92)90099-V.
[21]
Kurochkin, I.O.; Ivanina, A.V.; Eilers, S.; Downs, C.A.; May, L.A.; Sokolova, I.M. Cadmium affects metabolic responses to prolonged anoxia and reoxygenation in eastern oysters (Crassostrea virginica). Am. J. Physiol. Regul. Integr. Comp. Physiol. 2009, 297, 1262–1272, doi:10.1152/ajpregu.00324.2009.
[22]
Paynter, K.T.; Karam, G.A.; Ellis, L.L.; Bishop, S.H. Subcellular distribution of aminotransferases, and pyruvate branch point enzymes in gill tissue from four bivalves. Comp. Biochem. Physiol. Part B: Comp. Biochem. 1985, 82, 129–132, doi:10.1016/0305-0491(85)90140-3.
[23]
Donald, K.M.; Hawkins, A.J.S.; Smerdon, G.R. Transcript analysis of the genes encoding aminopeptidase N and alanine aminotransferase, two enzymes involved in protein turnover, in the pacific oyster, Crassotrea gigas. Comp. Biochem. Physiol. 2001, 128, 459–467, doi:10.1016/S1096-4959(00)00345-6.
[24]
Katz, J.; Lee, W.-N.; Wals, P.A.; Bergner, E.A. Studies of glycogen synthesis and the Krebs cycle by mass isotopomer analysis with [U-13C]Glucose in rats. J. Biol. Chem. 1989, 264, 12994–13001.
Greenway, S.C.; Storey, K.B. Seasonal change and prolonged anoxia affect the kinetic properties of phosphofructokinase and pyruvate kinase in oysters. J. Comp. Physiol. 2000, 170, 285–293, doi:10.1007/s003600000098.
[27]
Le Moulla, G.; Bacca, H.; Huvet, A.; Moal, J.; Pouvreau, S.; van Wormhoudt, A. Transcriptional regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase in the adductor muscle of the oyster Crassostrea gigas during prolonged hypoxia. J. Exp. Zool. 2007, 307, 371–382.
[28]
Hammen, C.S. Metabolism of the oyster, Crassostrea virginica. Am. Zool. 1969, 9, 309–318.
[29]
Lannig, G.; Eilers, S.; P？rtner, H.O.; Sokolova, I.M.; Bock, C. Impact of ocean acidification on energy metabolism of oyster, Crassostrea gigas-changes in metabolic pathways and thermal response. Mar. Drugs 2010, 8, 2318–2339, doi:10.3390/md8082318.
[30]
Dickinson, G.H.; Ivanina, A.V.; Matoo, O.B.; P？rtner, H.O.; Lannig, G.; Bock, C.; Beniash, E.; Sokolova, I.M. Interactive effects of salinity and elevated CO2 levels on juvenile eastern oysters, Crassostrea virginica. J. Exp. Biol. 2012, 215, 29–43, doi:10.1242/jeb.061481.
[31]
Sussarellu, R.; Fabioux, C.; le Moullac, G.; Fleury, E.; Moraga, D. Transcriptomic response of the Pacific oyster Crassostrea gigas to hypoxia. Mar. Genomics 2010, 3, 133–143, doi:10.1016/j.margen.2010.08.005.
[32]
Macey, B.M.; Achilihu, I.O.; Burnett, K.G.; Burnett, L.E. Effects of hypercapnic hypoxia on inactivation and elimination of Vibrio campbellii in the Eastern oyster, Crassostrea virginica. Appl. Environ. Microbiol. 2008, 74, 6077–6084, doi:10.1128/AEM.00317-08.
[33]
Chapman, R.W.; Mancia, A.; Beal, M.; Veloso, A.; Rathburn, C.; Blair, A.; Holland, A.F.; Warr, G.W.; Didinato, G.; Sokolova, I.M.; et al. The transcriptomic responses of the eastern oyster, Crassostrea virginica, to environmental conditions. Mol. Ecol. 2011, 20, 1431–1449, doi:10.1111/j.1365-294X.2011.05018.x.
[34]
David, E.; Tanguy, A.; Pichavant, K.; Moraga, D. Response of the Pacific oyster Crassostrea gigas to hypoxia exposure under experimental conditions. FEBS J. 2005, 272, 5635–5652, doi:10.1111/j.1742-4658.2005.04960.x.
[35]
Dewar, B.J.; Keshari, K.; Jeffries, R.; Dzeja, P.; Graves, L.M.; Macdonald, J.M. Metabolic assessment of a novel chronic myelogenous leukemic cell line and an imatinib resistant subline by 1H NMR spectroscopy. Metabolomics 2010, 6, 439–450, doi:10.1007/s11306-010-0204-0.
[36]
Fan, T.W.M.; Colmer, T.D.; Lane, A.N.; Higashi, R.M. Determination of metabolites by 1H NMR and GC: Analysis for organic osmolytes in crude tissue extracts. Anal. Biochem. 1993, 214, 260–271, doi:10.1006/abio.1993.1486.
[37]
Fan, T.W.M.; Higashi, R.M.; Lane, A.N.; Jardetzky, O. Combined use of 1H-NMR and GC-MS for metabolite monitoring and in vivo 1H-NMR assignments. Biochim. Biophys. Acta 1986, 882, 154–167, doi:10.1016/0304-4165(86)90150-9.
[38]
Szyperski, T. 13C-NMR, MS and metabolic flux balancing in biotechnology research. Q. Rev. Biophys. 1998, 31, 41–106, doi:10.1017/S0033583598003412.
