Metabolite profiling of the post-ovulatory oocytes of the common carp, Cyprinus carpio: A 1H NMR-based metabolomics approach.

A metabolomics study was conducted to investigate the molecular bases of oocyte over-ripening in common carp, Cyprinus carpio from a metabolic point of view. The ovulation was induced in fish brooders by intramuscular injection of pituitary extract and oocytes were collected four times post-ovulation with 30 min intervals. A set of 32 metabolites were identified on the NMR spectra of the oocytes, which mainly included energy-linked metabolites, amino acids, methylated metabolites and citric acid cycle (TCA) intermediates. PCA and PLS-DA models clearly separated the post ovulations times, indicating the effects of post-ovulation time on oocyte metabolome content. Based on the loading plot outputs, 15 metabolites including tryptophan, cysteine, AMP, tyrosine, valine, creatine phosphate (PCr), ATP, leucine, inosine, malate, acetate, TMAO, glucose, fumarate and lysine had more effects on the separation of post ovulation times. According to the results of metabolite profiling, the concentrations of glutamine, alanine, tryptophan, lysine and cysteine mostly significantly (P < 0.01) increased at 90 and 120 min post-ovulation. The concentrations of PCr, ATP, inosine and guanosine were relatively stable until 60 min post-ovulation, while significantly (P < 0.01) decreased at 90 and 120 min post ovulation. The TCA metabolites succinate, malate and fumarate significantly (P < 0.01) elevated at 90 and 120 min post-ovulation. AMP concentrations remained relatively unchanged until 30 min and then progressively decreased with time post ovulation (P < 0.01). The concentrations of lactate showed significant elevations at 90 and 120 min post ovulation (P < 0.01). In conclusion, the energetic potentials of the oocytes reduced with time post ovulation. There were apparent elevations in the concentrations of free amino acids, which may be associated with the onset of proteolytic activities in the post ovulatory oocytes. In addition, we found some changes in the apoptotic-related metabolites, which may support the results of previous studies regarding the oxidative stress and following apoptosis in post ovulatory oocytes of fish.

Metabolite profiling, histological and oxidative stress responses in the grey mullet, Mugil cephalus exposed to the environmentally relevant concentrations of the heavy metal, Pb (NO3)2.

In this study, a metabolomics approach was applied to investigate the metabolic responses of grey mullet, Mugil cephalus to toxicity induced by heavy metal, Pb (NO3)2. In addition, the study was followed by assessing the peroxidation index and histology of liver as supplementary data. Pb (NO3)2 exposure affected the plasma metabolome, especially four group metabolites including amino acids, methylated metabolites, energetic metabolites and citric acid intermediates. Pb (NO3)2 in medium and high concentrations (15 and 25 µg/l) increased the levels of plasma amino acids compared to control (P < 0.01). In contrast, Pb (NO3)2 decreased the plasma levels of methylated metabolites (P < 0.01). The ketogenic metabolites and glycerol levels significantly elevated in fish exposed to 25 µg/l Pb (NO3)2 (P < 0.01). The plasma glucose levels increased in treatment, 5 µg/l Pb (NO3)2 and after a decline in treatment 15 µg/l Pb (NO3)2 elevated again in treatment 25 µg/l Pb (NO3)2 (P < 0.01).The plasma levels of lactate significantly increased in fish exposed to 5 and 15 µg/l Pb (NO3)2 and then declined to initial levels in treatment, 25 µg/l Pb (NO3)2 (P < 0.01). The plasma levels of TCA cycle intermediates significantly elevated in treatments 15 and 25 µg/l Pb (NO3)2 (P < 0.01). As a biomarker of oxidative stress, the plasma levels of malondialdehyde (MDA) showed significant increases in Pb (NO3)2 exposed fish (P < 0.01). During exposure period, wide ranges of liver tissue damages were also observed in Pb (NO3)2 exposed fish. In conclusion, exposure to Pb (NO3)2 affected the metabolome content of blood in grey mullet, mainly through inducing the biochemical pathways related to the metabolism of the amino acids, energetic metabolites and methylated metabolites. Our results may help to understand the effects of heavy metals on fish hematology from a molecular point of view.

Immunostimulating effects of Ginkgo biloba extract against toxicity induced by organophosphate pesticide, diazinon in rainbow trout, Oncorhynchus mykiss: innate immunity components and immune-related genes.

The immunostimulating and therapeutic properties of Ginkgo biloba (GB) have always been the focus of traditional medicine over thousands of years. During last decade, special attentions were paid to use of GB in aquaculture to enhance fish health and survival. In the present study, we investigated for the first time the immunogenic effects of dietary GB against oxidative and toxicity induced by organophosphate pesticide, diazinon. In non-diazinon-exposed fish, the plasma total immunoglobulin, lysozyme activity, and peroxidase activity significantly elevated after 60-day experiment in fish supplemented with 1 and 2 g GB/kg diet (p < 0.05). The respiratory burst activity and complement activity significantly increased only in groups supplemented with 0.5 g GB/kg diet (p < 0.05). Furthermore, the peroxidase activity, total immunoglobulin, and lysozyme activity significantly declined in groups supplemented with 4 g GB/kg diet during feeding trial (p < 0.05). There were no significant differences in expression of interleukin 1 beta (IL-1ß) and transforming growth factor beta 1 (TGF-ß1) genes in kidney between control group (non-GB-supplemented fish) and GB-supplemented fish (p > 0.05). In diazinon-exposed fish, all immunity components significantly decreased during exposure in control and those fed 0.5 and 4 g GB/kg diet (p < 0.05). In fish fed 1 and 2 g GB/kg diet, no alternations were found in immunity components during exposure period (p > 0.05). In addition, diazinon induced the expression of IL-1ß and TGF-ß1 genes in control and fish fed 0.5 and 4 g GB/kg diet (p < 0.05). No significant changes were observed in expression of IL-1ß and TGF-ß1 genes in fish supplemented with 1 and 2 g GB/kg (p > 0.05). In conclusion, the results of the present study suggest an immunogenic role for dietary GB at optimum dietary levels (1-2 g GB/kg diet) against toxicity induced by diazinon. Nevertheless, GB at high dietary levels (4 g GB/kg diet) showed immunosuppressive effects, which makes it necessary to optimize its levels in diet.

NMR-based metabolomic study on the toxicological effects of pesticide, diazinon on adaptation to sea water by endangered Persian sturgeon, Acipenser persicus fingerlings.

NMR-based metabolomics was applied to explore metabolic impacts of diazinon on sea water adaptation of Persian sturgeon fingerlings, Acipenser persicus. Fingerlings were exposed to sub-lethal concentrations of diazinon in freshwater (FW) for 96 h (short-term trial) and 12 days (long-term trial) and then exposed in brackish water (BW) (12 mg L-1 salinity) for 24 h. After 96 h and 12 days exposure in FW, identified metabolites (amino acids, osmolytes, energy metabolites) showed different change-patterns compared to control group (P < 0.05) as follow: (A) short-term trial: higher plasma levels of glucose, lactate (in all diazinon-exposed fish), acetate and acetoacetate (in 0.9 mg L-1diazinon treatment); lower levels of creatine (in all diazinon-exposed fish), trimethylamine-N-oxide, choline, taurine, betaine, N,N-dimethylglycine and almost all amino acids in fish exposed to high concentrations of diazinon (0.54 and 0.9 mg L-1 diazinon). (B) Long-term trial: higher plasma levels of lipid oxidation metabolites and almost all amino acids in fish exposed to 0.54 and 0.9 mg L-1 diazinon; lower levels of creatine, trimethylamine-N-oxide, N,N-dimethylglycine, betaine, choline (in all diazinon-exposed fish), glucose (in 0.54 and 0.9 mg L-1diazinon treatments) and taurine (in 0.9 mg L-1 diazinon treatment). When fish were exposed in BW for 24 h, the plasma levels of osmolytes decreased significantly in almost all experimental groups of short-term and long-term trial (P < 0.05). In short-term trial, the plasma levels of glucose in all groups and lactate in 0.18 and 0.54 mg L-1 diazinon treatments increased after salinity challenge (P < 0.05). However, a significant decrease was observed in lactate levels in 0.9 mg L-1 diazinon treatment (P < 0.05). Also, the plasma levels of amino acids decreased mostly in fish of control group than exposed fish (P < 0.05). The plasma glycerol concentration showed a significant decrease only in fish of 0.54 mg L-1 diazinon treatment (P < 0.05). In long term trial, the energetic metabolites (acetate, acetoacetate, glycerol) showed significant increases mostly in fish exposed to high concentrations of diazinon (P < 0.05). Phosphocreatine was detected only in groups exposed to 0.54 and 0.9 mg L-1 diazinon. Some amino acids decreased in control and diazinon-exposed groups while glycine (in control and 0.18 mg L-1 diazinon treatment), glutamine and alanine (in 0.9 mg L-1 diazinon treatment) elevated significantly after 24 h acclimation in BW (P < 0.05). Our results may help to understand the effects of pesticides on fish osmoregulation from a metabolic approach.

Effects of diazinon on adaptation to sea-water by the endangered Persian sturgeon, Acipenser persicus, fingerlings.

To replenish the depleting populations of sturgeon fishes especially Persian sturgeon, Acipenser persicus in the Caspian Sea, millions of Persian sturgeon fingerlings are farmed through artificial propagation and released into the Iranian river estuaries annually. Fish osmoregulation is a vital physiological process that can be affected during the release. Many Iranian river estuaries are under the influence of pesticides originating from farming activities that may affect osmoregulation. In this study, Persian sturgeon fingerlings were exposed to sublethal concentrations (0, 0.18, 0.54, 0.9mgL(-)(1)) of diazinon for 96h (short-term trial) and 12 days (long-term trial) in fresh water (FW) and then fish were exposed in brackish water (BW) for 24h. After 96h and 12 days of exposure in FW, the lower levels of plasma triidothyronine (T3), thyroxine (T4), Na(+), Cl(-), K(+), gill Na(+)/K(+)- ATPase activity and number of chloride cells were observed in exposed fish (0.54 and 0.9mgL(-)(1) diazinon) compared to control group and 0.18mgL(-)(1) diazinon treatment. Also, higher levels of plasma cortisol (except 0.18mgL(-)(1) diazinon treatment in long-term trial) were observed in diazinon exposed fish compared to control group. However, gill Na(+)/K(+)-ATPase activity and the number of chloride cells were higher in fingerlings exposed to diazinon compared than control. When fish were exposed in BW for 24h, the following changes occurred: (a) in short-term trial: increases in cortisol and Cl(-) levels (0.54mgL(-)(1) diazinon ), Na(+) (0.9mgL(-)(1) diazinon ) and gill Na(+)/K(+)-ATPase activity (0.18mgL(-)(1) diazinon ). In control group, cortisol, T4, Na(+), gill Na(+)/K(+)-ATPase activity and the number of chloride cells increased significantly. (b) In long-term trial: increases in K(+) levels in fish exposed to 0.9mgL(-)(1) diazinon, Na+ in all diazinon concentrations and decreases in chloride cells number in fish exposed to 0.18mgL(-)(1) diazinon. In control group, significant increases were observed in cortisol, T3, Na(+) and chloride cells number. Finally, gill showed many histopathological damages during exposure in FW and BW. Our results suggest that the contamination of river estuaries with diazinon may alter the osmoregulation ability of released Persian sturgeon fingerlings, which could lead to a failure in their restocking program in the Caspian Sea.