Metabolic effects of dinoseb, diazinon and esfenvalerate in eyed eggs and alevins of Chinook salmon (Oncorhynchus tshawytscha) determined by 1H NMR metabolomics.

Pesticide pulses in the Sacramento River, California, originate from storm-water discharges and non-point source aquatic pollution that can last from a few days to weeks. The Sacramento River and its tributaries have historically supported the majority of California's Chinook salmon (Oncorhynchus tshawytscha) spawning grounds. Three pesticides currently used in the Sacramento Valley-- dinoseb, diazinon, and esfenvalerate-- were chosen to model the exposure of salmon embryos to storm-water discharges. Static-renewal (96 h) exposures to eyed eggs and alevins resulted in both toxicity and significant changes in metabolism assessed in whole-embryo extracts by (1)H nuclear magnetic resonance (NMR) spectroscopy based metabolomics and HPLC with UV detection (HPLC-UV). The 96-h LC(50) values of eyed eggs and alevins exposed to dinoseb were 335 and 70.6 ppb, respectively, and the corresponding values for diazinon were 545 and 29.5 ppm for eyed eggs and alevins, respectively. The 96-h LC(50) of eyed eggs exposed to esfenvalerate could not be determined due to lack of mortality at the highest exposure concentration, but in alevins was 16.7 ppb. All esfenvalerate exposed alevins developed some degree of lordosis or myoskeletal abnormality and did not respond to stimulus or exhibit normal swimming behavior. ATP concentrations measured by HPLC-UV decreased significantly in eyed eggs due to 250 ppb dinoseb and 10 and 100 ppb esfenvalerate (p < 0.05). Phosphocreatine, as measured by HPLC-UV, decreased significantly in eyed eggs due to 250 ppb dinoseb, 10 and 100 ppb esfenvalerate, and 100 ppm diazinon (p < 0.05). Principal components analyses of (1)H NMR metabolite fingerprints of eyed egg and alevin extracts revealed both dose-dependent and mechanism of action-specific metabolic effects induced by the pesticides. Furthermore, NMR based metabolomics proved to be more sensitive than HPLC-UV in identifying significant changes in sublethal metabolism of pesticide exposed alevins. In conclusion, we have demonstrated several benefits of a metabolomics approach for chemical risk assessment, when used in conjunction with a fish embryo assay, and have identified significant metabolic perturbations to the early life stages of Chinook salmon by currently used pesticides.

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.

Metabolic changes in Japanese medaka (Oryzias latipes) during embryogenesis and hypoxia as determined by in vivo 31P NMR.

In vivo (31)P nuclear magnetic resonance spectroscopy (NMR) was used to determine phosphometabolite changes in medaka (Oryzias latipes) during embryogenesis and hypoxia. NMR data were acquired using a flow-through NMR tube perfusion system designed to both deliver oxygenated water to embryos and accommodate a hypoxic challenge. Measurements of embryogenesis at 12- and 24-h intervals throughout 8 days of development (n = 3 per time point, 900 embryos per replicate) and during acute hypoxia (n = 6, 900 embryos at Iwamatsu stage 37 per replicate) were performed via NMR, and replicate samples (n = 4, 250 embryos each) were flash frozen for HPLC analysis. The hypoxic challenge experiment consisted of data acquisition with recirculating water (pre-hypoxic control period; 1 h), without recirculating water (hypoxic challenge; 1 h), then again with recirculating water (recovery period; 1.3 h). Concentrations of ATP, phosphocreatine (PCr), orthophosphate (P(i)), phosphomonoesters (PME), phosphodiesters (PDE), and intracellular pH (pH(i)) were determined by NMR, and ATP, ADP, AMP, GTP, GDP, and PCr were also determined via HPLC. During embryogenesis, [ATP] and [PCr] as determined by HPLC increased from 1-day post fertilization (DPF) levels of 0.93+/-0.08 and 2.48+/-0.21 micromol/mg (dry tissue), respectively, to 7.24+/-0.77 and 15.66+/-1.08 micromol/mg, respectively, by day 8. [ATP] and [PCr] measured by both NMR and HPLC fluctuated over 1-3 DPF, then increased significantly (p<0.05) over 3-8 DPF, while [PME] and [PDE] decreased (p<0.05) throughout embryogenesis. NMR and HPLC measurements revealed 1-3, 4-5, and 6-8 DPF as periods of embryogenesis significantly different from each other (p<0.05), and representing important transitions in metabolism and growth. During hypoxic challenge, [ATP] and [PCr] declined (p<0.05), [PME] and [PDE] decreased slightly, and [P(i)] increased (p<0.05). All phosphometabolites returned to pre-hypoxia concentrations during recovery. The pH(i) decreased (p<0.05) from 7.10+/-0.03 to 6.94+/-0.03 as a result of hypoxia, and failed to return to pre-hypoxic levels within the 1.3-h recovery phase. Results demonstrate the utility of in vivo (31)P NMR to detect significant alterations in phosphorylated nucleotides and phosphometabolites at specific developmental stages during medaka development and that late-stage medaka utilize PCr to generate ATP under hypoxic conditions.

Utilizing in vivo nuclear magnetic resonance spectroscopy to study sublethal stress in aquatic organisms.

In vivo nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for characterizing the sublethal actions of physical and chemical stressors in live, intact organisms. In particular, 31P NMR is ideal for observing perturbations to cellular energetics since critical metabolite concentrations, including phosphagens, ATP and inorganic phosphate (Pi), can be measured non-invasively and in real time. This technique's versatility is demonstrated not only in the diversity of organisms that can be studied, but also in its broad-ranging applicability to environmental research. Illustrative studies include the actions of copper in adult red abalone (Haliotis rufescens) and changes in energetically important metabolites in developing medaka embryos (Oryzias latipes). Advantages and disadvantages of in vivo NMR will be discussed.