Toxic actions of dinoseb in medaka (Oryzias latipes) embryos as determined by in vivo 31P NMR, HPLC-UV and 1H NMR metabolomics.

Changes in metabolism of Japanese medaka (Oryzias latipes) embryos exposed to dinoseb (2-sec-butyl-4,6-dinitrophenol), a substituted dinitrophenol herbicide, were determined by in vivo (31)P NMR, high-pressure liquid chromatography (HPLC)-UV, and (1)H NMR metabolomics. ATP and phosphocreatine (PCr) metabolism were characterized within intact embryos by in vivo (31)P NMR; concentrations of ATP, GTP, ADP, GDP, AMP and PCr were determined by HPLC-UV; and changes in numerous polar metabolites were characterized by (1)H NMR-based metabolomics. Rangefinding exposures determined two sublethal doses of dinoseb, 50 and 75 ppb, in which embryos survived from 1-day post fertilization (DPF) through the duration of embryogenesis. In vivo (31)P NMR data were acquired from 900 embryos in 0, 50, and 75 ppb dinoseb at 14, 62, and 110 h (n = 6 groups) after initiation of exposure. After 110 h, embryos were observed for normal development and hatching success, then either preserved in 10% formalin for growth analysis or flash frozen and extracted for HPLC-UV and (1)H NMR analysis. Dinoseb exposure at both concentrations resulted in significant declines in [ATP] and [PCr] at 110 h as measured by in vivo (31)P NMR (p < 0.01), HPLC-UV (p < 0.001) and NMR-based metabolomics. Reduced eye growth and diminished heart rate occurred in a concentration-dependent fashion. Metabolic effects measured by in vivo (31)P NMR showed a significant increase in orthophosphate levels (P(i); p < 0.05), and significant decreases in [ATP], [PCr] and the PCr/P(i) ratio (p < 0.05). Metabolomics revealed a dose-response relationship between dinoseb and endogenous metabolite changes, with both dinoseb concentrations producing significantly different metabolic profiles from controls (p < 0.05). Metabolic changes included decreased concentrations of ATP, PCr, alanine and tyrosine, and increased concentrations of lactate with medaka embryotoxicity. This study demonstrated that medaka embryos respond to dinoseb with significant changes in metabolism, reduced growth and heart rates, and increased abnormal development and post-exposure mortality. All three analytical methods confirmed similar trends, and utilization of PCr to compensate for ATP loss was found to be a consistent indicator of sublethal stress-one that could be used to quantify stress associated with medaka embryotoxicity.

[1]  山本 時男.  Medaka (killifish) : biology and strains , 1975 .

[2]  D. Monty,et al.  Toxicological effects of cadmium chloride on the canine testis following various routes of administration , 1977 .

[3]  M. Viant,et al.  Metabolic changes in Japanese medaka (Oryzias latipes) during embryogenesis and hypoxia as determined by in vivo 31P NMR. , 2005, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[4]  J. E. Richards,et al.  Dinoseb presence in agricultural subsurface drainage from potato fields in northwestern New Brunswick, Canada , 1989, Bulletin of environmental contamination and toxicology.

[5]  D. Woodward.  Toxicity of the Herbicides Dinoseb and Picloram to Cutthroat (Salmo clarki) and Lake Trout (Salvelinus namaycush) , 1976 .

[6]  A. Suresh,et al.  Shifts in protein metabolism in some organs of freshwater fish, Cyprinus carpio under mercury stress. , 1991, Biochemistry international.

[7]  E Holmes,et al.  Metabonomic characterization of genetic variations in toxicological and metabolic responses using probabilistic neural networks. , 2001, Chemical research in toxicology.

[8]  B. Roelofsen,et al.  31P-NMR determination of phosphomonoesters in relation to phospholipid biosynthesis in testis of the rat at different ages. , 1991, Biochimica et biophysica acta.

[9]  F. Podo.  Tumour phospholipid metabolism , 1999, NMR in biomedicine.

[10]  Ronald S. Tjeerdema,et al.  NMR-derived developmental metabolic trajectories: an approach for visualizing the toxic actions of trichloroethylene during embryogenesis , 2005, Metabolomics.

[11]  M. Viant,et al.  Utilizing in vivo nuclear magnetic resonance spectroscopy to study sublethal stress in aquatic organisms. , 2002, Marine environmental research.

[12]  H. Blum,et al.  Reversible injury: creatinine kinase recovery restores bioenergetics and function. , 1996, The Journal of surgical research.

[13]  K. S. Rao,et al.  Tissue specific alteration of aminotransferases and total ATPases in the fish (Tilapia mossambica) under methyl parathion impact , 1984 .

[14]  S. Shivaji,et al.  31P Nuclear Magnetic Resonance Studies on the Phosphorus-Containing Metabolites of the Developing Embryos of a Freshwater Catfish, Clarias batrachus (L.) , 1999, Marine Biotechnology.

[15]  Hinton,et al.  Thiobencarb-induced embryotoxicity in medaka (Oryzias latipes): stage-specific toxicity and the protective role of chorion. , 2000, Aquatic toxicology.

[16]  T. Iwamatsu.  Stages of normal development in the medaka Oryzias latipes , 1994, Mechanisms of Development.

[17]  D. E. Atkinson.  The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. , 1968, Biochemistry.

[18]  Mark R Viant,et al.  NMR-based metabolomics: a powerful approach for characterizing the effects of environmental stressors on organism health. , 2003, Environmental science & technology.

[19]  A. Ally,et al.  Rapid determination of creatine, phosphocreatine, purine bases and nucleotides (ATP, ADP, AMP, GTP, GDP) in heart biopsies by gradient ion-pair reversed-phase liquid chromatography. , 1992, Journal of chromatography.

[20]  C. Franklin,et al.  Metabolic recovery in herring larvae following strenuous activity , 1996 .

[21]  M. Viant.  Improved methods for the acquisition and interpretation of NMR metabolomic data. , 2003, Biochemical and biophysical research communications.

[22]  J. Ruíz-Cabello,et al.  Phospholipid metabolites as indicators of cancer cell function , 1992, NMR in biomedicine.

[23]  A. Van der Linden,et al.  Effects of sublethal copper exposure on muscle energy metabolism of common carp, measured by 31P‐nuclear magnetic resonance spectroscopy , 1997 .

[24]  M. Mayes,et al.  Acute toxicity tests with Daphnia magna straus and Pimephales promelas rafinesque in support of national pollutant discharge elimination permit requirements , 1986 .

[25]  J. Klaunig,et al.  Preliminary studies on the usefulness of medaka, Oryzias latipes, embryos in carcinogenicity testing. , 1984, National Cancer Institute monograph.

[26]  B. Herman,et al.  Blebbing, free Ca2+ and mitochondrial membrane potential preceding cell death in hepatocytes , 1987, Nature.

[27]  David E. Hinton,et al.  Age-dependent changes in toxicity of N-nitroso compounds to Japanese medaka (Oryzias latipes) embryos , 1990 .

[28]  K. Nicolay,et al.  Phosphorylation state of red and white muscle in tilapia during graded hypoxia: an in vivo31P-NMR study. , 1999, American journal of physiology. Regulatory, integrative and comparative physiology.

[29]  Franca Podo,et al.  Magnetic resonance spectroscopy in biology and medicine : functional and pathological tissue characterization , 1992 .

[30]  E. Faustman,et al.  Developmental toxicity of four model alkylating agents on japanese medaka fish (Oryzias latipes) embryos , 1987 .

[31]  T. Fan.  Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures , 1996 .

[32]  J. Joly,et al.  Morphogenesis of the optic tectum in the medaka (Oryzias latipes): A morphological and molecular study, with special emphasis on cell proliferation , 1999, The Journal of comparative neurology.

[33]  M. Denison,et al.  Dioxinlike properties of a trichloroethylene combustion-generated aerosol. , 1996, Environmental health perspectives.

[34]  M. Viant,et al.  Correlation between heat-shock protein induction and reduced metabolic condition in juvenile steelhead trout (Oncorhynchus mykiss) chronically exposed to elevated temperature , 2003, Fish Physiology and Biochemistry.

[35]  John C. Lindon,et al.  Pattern recognition methods and applications in biomedical magnetic resonance , 2001 .

[36]  C. Palmeira,et al.  Interactions of herbicides 2,4-D and dinoseb with liver mitochondrial bioenergetics. , 1994, Toxicology and applied pharmacology.

[37]  Hinton,et al.  The role of development and duration of exposure to the embryotoxicity of diazinon. , 2000, Aquatic toxicology.

[38]  Sunia A Trauger,et al.  Metabonomic assessment of toxicity of 4‐fluoroaniline, 3,5‐difluoroaniline and 2‐fluoro‐4‐methylaniline to the earthworm Eisenia veneta (rosa): Identification of new endogenous biomarkers , 2002, Environmental toxicology and chemistry.