The Effect of Silver Nanoparticles on the Digestive System, Gonad Morphology, and Physiology of Butterfly Splitfin (Ameca splendens).

The aim of this study was to determine the effects of silver nanoparticles (AgNPs) on the morphology and enzymatic activity of butterfly splitfin (Ameca splendens). Individuals of both sexes, aged about five months, were exposed to AgNPs at concentrations of 0 (control group), 0.01, 0.1, and 1.0 mg/dm3 for 42 days. On the last day of the experiment, the fish were euthanized, subjected to standard histological processing (anterior intestine, liver, and gonads), and analysed for digestive enzyme activity in the anterior intestine and oxidative stress markers in the liver. Fish in the AgNP 0.01 and 0.1 groups had the lowest anterior intestinal fold and enterocyte height. However, there were no statistically significant changes in the digestive enzyme activity in the anterior intestine. Analysis of enzymatic activity in the liver showed an increase in superoxide dismutase activity in fish in the AgNP 0.1 group. Histological analyses showed that AgNPs inhibited meiotic divisions at prophase I in a non-linear manner in ovaries and testes. In the AgNP 0.1 and 1.0 groups, the area occupied by spermatocytes was lower compared to the other groups. These results indicate that exposure to AgNPs may lead to disturbances in morphology and enzymatic activity in the liver and intestine and may lead to disruption of reproduction in populations.

Chronic hypoxia and hyperoxia alter tissue-specific fatty acid profile and FD6D and elongase gene expression levels in rainbow trout (Oncorhynchus mykiss).

Commercially important trout species, especially rainbow trout, are under great threat due to several negative factors affecting oxygen levels in water such as global warming and eutrophication. In our study, rainbow trout (Oncorhynchus mykiss) was exposed to chronic (for 28 days) hypoxia (4.0 ± 0.5 mg/L) and hyperoxia (12 ± 1.2 mg/L) in order to evaluate the alteration of fatty acid profiles in muscle, liver and gill tissues. In addition, delta-6-desaturase and elongase gene expression profiles were measured in liver, kidney and gill tissues. The amount of saturated fatty acids increased by oxygen applications in the liver, while it decreased in the muscle and gill tissues compared to normoxia (p < 0.05). Monounsaturated fatty acids levels increased in muscle and gill (p < 0.05). Although n-3 polyunsaturated fatty acid (PUFA) decreased in muscle tissue, n-6 PUFA increased (p < 0.05). The n-3/n-6 ratio decreased in muscle tissue in response to the both exposures (p < 0.05) as well as eicosapentaenoic acid/docosahexaenoic acid ratio (p < 0.05). Hypoxia exposure generally increased delta-6-desaturase and elongase mRNA levels in all tissues (p < 0.05). However, gene expression profiles were variable in fish exposed to hyperoxia. As a result of oxygen exposures, the lipid profile of muscle tissue, which stores dense fat, was negatively affected more than that of liver and gill tissues. We determined that the change in expression levels was tissue specific.

Inhibitory effects of sulfenimides on human and bovine carbonic anhydrase enzymes.

A series of sulfenimide derivatives (1a-i) were investigated as inhibitors of human (hCA-I, hCA-II) and bovine (bCA) carbonic anhydrase enzymes. The compounds were synthesised by the reaction of substituted thiophenols with phthalimide by means of an effective, simple and eco-friendly method and the structures were confirmed by IR, 1H NMR, 13C NMR, MS and elemental analysis. All derivatives except for the methyl derivative (1b) exhibited effective inhibitory action at low micromolar concentrations on human isoforms, but only four derivatives (1e, 1f, 1h, 1i) inhibited the bovine enzyme. The bromo derivative (1f) was found to be strongest inhibitor of all three enzymes with KI values of 0.023, 0.044 and 20.57 µM for hCA-I, hCA-II and bCA, respectively. Results of our study will make valuable contributions to carbonic anhydrase inhibition studies for further investigations since inhibitors of this enzyme are important molecules for medicinal chemistry.

Effects of hypoxia and hyperoxia on growth parameters and transcription levels of growth, immune system and stress related genes in rainbow trout.

Hypoxia and hyperoxia are disparate stressors which can have destructive influences on fish growth and physiology. It is yet to be determined if hypoxia and hyperoxia have a cumulative effect in aquatic ecosystems that affect biological parameters in fish, and to understand if this is associated with gene expression. Here we address whether growth performance and expressions of growth, immune system and stress related genes were affected by hypoxia and hyperoxia in fish. Rainbow trout was chosen as the study organism due to its excellent service as biomonitor. After an acclimatization period, fish were exposed to hypoxia (4.0 ± 0.5 ppm O2), normoxia (7.5 ± 0.5 ppm O2) and hyperoxia (12 ± 1.2 ppm O2) for 28 days. At 6 h, 12 h, 24 h, 48 h, 72 h and 28 days, samples were collected. Hypoxia and hyperoxia negatively affected weight gain (WG), specific growth rate (SGR), survival rate (SR) and feed conversion ratio (FCR). The best WG, SGR, SR and FCR values occurred in fish exposed to normoxia, whereas hypoxia was most suppressive on growth and hyperoxia showed intermediate suppression of these parameters. Gene expression analyses were performed in liver and results revealed that long term exposure caused reduced growth hormone-I (GH-I) and insulin like growth factor I-II (IGF I-II) levels in both hypoxia and hyperoxia-treated fish. Heat shock protein (HSP70) levels increased in both hypoxia and hyperoxia treatment, and both exposures caused elevation of leptin (LEP) expression in long-term exposure. Overall data indicate that both hypoxia and hyperoxia cause stress in rainbow trout and negatively affects growth parameters.

Dietary inclusion of royal jelly modulates gene expression and activity of oxidative stress enzymes in zebrafish.

Here we investigated the effects of different levels of royal jelly in zebrafish (Danio rerio) diets [0.0% (D1); 0.1% (D2); 0.4% (D3); 1.6% (D4) vs 6.4% (D5)] on the activity and expression profiles of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione S-transferase. Muscle, liver and kidney tissue samples were obtained from fish fed during 8 weeks. In these tissues, enzyme activity was determined by means of spectrophotometer and gene expression by quantitative real-time PCR. mRNA levels of the enzymes were elevated in almost all diet groups compared to the control (D1). It was determined that enzyme activities were also increased in general by supplementation of royal jelly although some decreases were also observed. However, the significant correlation between gene expression and enzyme activity was not observed in all tissues. It was concluded that main regulation occurs with post-translational modifications although effects at transcriptomic level demonstrated a snap variation.

Goose fat, a promising nutrient for fish feeding, activates antioxidant enzymes in rainbow trout, Oncorhynchus mykiss.

The objective of this experiment was to test effects of different dietary lipids in rainbow trout feeding on the activity and expression of antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione S-transferase (GST). Four iso-nitrogenous and iso-lipidic casein-gelatin based experimental diets were formulated. The sources of dietary lipids were cod liver oil (CO, rich in polyunsaturated fatty acids), goose fat (GF, rich in saturated fatty acids and monounsaturated fatty acids), soybean oil (SO, rich in linoleic acid), and a blend of CO, GF and SO. Dietary treatments had no significant effect on growth performance and survival was not affected. SOD, GPx and GST enzymes had the maximum activity in GF diet. However qPCR data showed that SOD and GPx mRNA levels were minimum in GF group. Overall data showed that rainbow trout liver enzymes were activated upon GF diet probably activating the enzyme structure itself without stimulating gene expression.

Influence of pesticides on the pH regulatory enzyme, carbonic anhydrase, from European Seabass liver and bovine erythrocytes.

The objective of this study was to assess the inhibitory effects of six commonly used pesticides, cyhalothrin, cypermethrin, dichlorvos, methamidophos, chlorpyrifos and methylparathion, on the pH regulatory enzyme carbonic anhydrase (CA) of Dicentrarchus labrax (European Seabass) liver (dCA) and bovine erythrocytes (bCA). Results of the study showed that the pesticides displayed quite variable inhibition profiles with K(I) values ranging from 0.376 to 26.164µM against dCA, and from 1.174 to 53.281µM against bCA. Methylparathion was the most effective inhibitor for both enzymes. Overall data show that all of the tested pesticides inhibit both dCA and bCA at low concentrations indicating that indiscriminate use of these pesticides might cause disruption of acid base regulation resulting in animal deaths. Our results also point out that susceptibility to these pesticides varies among CAs from different organisms.

Chronic toxicity of pesticides to the mRNA expression levels of metallothioneins and cytochrome P450 1A genes in rainbow trout.

The hazardous effects of pesticides on various metabolic pathways are a great problem for environmental health and should be well determined. In the present study, the authors treated rainbow trout with 0.6 µg/L deltamethrin for 28 days and 1.6 mg/L 2,2-dichlorovinyl dimethyl phosphate for 21 days. After this time period, the authors observed alterations in mRNA expression levels of MT-A, MT-B and CYP-1A. Chronic exposure to low levels of pesticides may have a more significant effect on fish populations than acute poisoning. While both pesticides caused a significant increase on mRNA levels of MT-A and CYP-1A, MT-B mRNA levels were increased significantly only upon deltamethin administration. The significant increase in mRNA levels of the corresponding genes may be considered as a defence mechanism in addition to the antioxidants against oxidative stress, as well as a detoxification mechanism against adverse effects of pesticides.

Impact of deltamethrin exposure on mRNA expression levels of metallothionein A, B and cytochrome P450 1A in rainbow trout muscles.

Metallothioneins (MT) are widely utilized to identify specific responses to heavy metal pollution. In addition, there is evidence demonstrating that in vertebrates MT synthesis is stimulated by different endogenous and exogenous agents in particular compounds leading to production of ROS. Also, cytochrome P450 1A can enhance the generation of ROS. On this basis, MT and CYP 1A induction can be considered as biomarkers of oxidative stress. In the current study, we examined the influences of pesticide administration on the expression of MT-A, MT-B and CYP 1A. For this purpose, we produced muscle metallothionein-A, metallothionein-B and cytochrome P450 1A cDNAs and used quantitative RT-PCR to assay mRNAs in rainbow trout exposed to acute and long-term deltamethrin administration. We observed that deltamethrin exposure significantly (p<0.05) increased the expression levels of Cyp1A, MT-A and MT-B in a time dependent manner. Results of our study contributes to the identification of inducers of such biomarkers in addition to well known agents.

Influence of cobalt and zinc exposure on mRNA expression profiles of metallothionein and cytocrome P450 in rainbow trout.

The present research aims to evaluate the effects of cobalt and zinc exposure of rainbow trout (Oncorhynchus mykiss) on metallothioneins and cytocrome P450. Mature rainbow trouts were exposed to 10 mg/L CoCl(2).6H(2)O and 1 mg/L ZnSO(4).7H(2)O. After 6, 12, 24, and 48 h of treatment, expressions of muscle MT-A, MT-B, and CYP P4501A1 mRNAs were measured by means of quantitative real-time polymerase chain reaction. During the exposure experiments, no mortalities occurred. We observed that expression levels of all genes increased with exposure time. Since the organism has not learned how to completely dispose of heavy metals and tends to bioaccumulate them, our results indicate that cobalt and zinc exposure may result in accumulation of the non-eliminated metals which may lead to fish death.

IGF and GH mRNA levels are suppressed upon exposure to micromolar concentrations of cobalt and zinc in rainbow trout white muscle.

The objective of this study was to assess the effects of cobalt and zinc exposure of rainbow trout (Oncorhynchus mykiss) on insulin like growth factors (IGF) and growth hormone (GH). Mature rainbow trouts were exposed to 0.42, 2.1, 4.2, 21 and 42µmol/L Co(2+) (added as CoCl(2)·6H(2)O) and 0.34, 1.7, 3.4, 17 and 34µmol/L Zn(2+) (added as ZnSO(4)i·7H(2)O). After 6, 12, 24 and 48h of treatment, expressions of white muscle IGF-I, IGF-II and GH mRNAs were measured by means of quantitative Real Time PCR. During the exposure experiments, no mortalities occurred. The most effective metal concentrations, which caused significant alterations, were determined to be 42µmol/L Co(2+) (10mg CoCl(2)·6H(2)O/L) and 3.4µmol/L Zn(+2) (1mg ZnSO(4)·7H(2)O/L). The following results were obtained for these concentrations. Expression of IGF-I did not change at 6h in zinc treatment while the decrease (p<0.05) was observed at 12h and 24h, and this decrease became stronger at 48h. Cobalt exposure caused a decrease in IGF-I mRNA level at 6h, 12h, 24h and 48h (p<0.05). Both zinc and cobalt exposure resulted in significant decreases in GH expression at 6h. Exposure of trout to Zn resulted in a decrease in expression of IGF-II starting from 6h whereas the significant decrease started at 6h in cobalt exposure and this decrease elevated at 24h. The results indicate that micromolar cobalt and zinc exposure causes significant attenuation in the expressions of these three genes' time dependently. Our findings show that IGF-I is the most resistant and GH is the most sensitive component against cobalt and zinc exposure. We conclude that IGF/GH axis might be strongly affected by the short term exposure to low micromolar concentrations of zinc and cobalt due to alterations of these genes.

Acute and long-term genotoxicity of deltamethrin to insulin-like growth factors and growth hormone in rainbow trout.

We report here the acute and long-term influences of deltamethrin on the expression of IGF-I, IGF-II and GH-I in rainbow trout muscles. We treated rainbow trouts with different concentrations of deltamethrin (0.25 microg/L, 1 microg/L and 2.5 microg/L) and observed the alterations in mRNA expression levels of IGF-I, IGF-II and GH-I at different time intervals (at 6th, 12th, 24th, 48th, 72nd hours and 30th day). The mRNA levels significantly decreased with increasing deltamethrin concentrations for acute administration. Interestingly, a significant recovery in GH-I expression was seen after the 72nd hour up to 30th day while no significant differences were observed for IGF-I and IGF-II between the same time intervals. Here we demonstrate that deltamethrin exposure decreases the expression of IGF-I, IGF-II and GH-I in rainbow trout which might cause undesirable outcomes not only in growth, but also in development and reproduction.

The effect of peptide absorption on PepT1 gene expression and digestive system hormones in rainbow trout (Oncorhynchus mykiss).

The present study evaluates the effect of protein source (dipeptides, free amino acids, and intact protein) on development and growth of Salmonid fish alevin. Specifically, we follow the expression of oligopeptide transporter protein PepT1 in the intestine of rainbow trout (Oncorhynchus mykiss). Fish were fed exogenously one of four diets: three formulated (lysyl-glycine dipeptide supplemented diet - PP, free lysine and glycine supplemented diet - AA, control diet with no lysine - CON) or commercial starter (Aller Futura - AF). Fish increased mean body weight 8 fold with PP- and AA-supplemented diets resulting in significantly higher weight gain than fish fed CON. Statistical analysis revealed a significant increase in relative PepT1 expression of fish fed experimental diets. Immunohistochemical staining with PepT1 antibody showed the presence of the transporter protein in the brush border membrane of the proximal intestinal enterocytes of fish from all experimental groups. Leptin immunoreactivity occurred not only in the gastric glands but also in proximal intestine and pyloric caeca of fish fed PP, AA and AF diets. Leptin immunoreactivity was also observed in hepatocyte cytoplasm and pancreatic acinar cells. Gastrin/CCK immunoreactive cells were present in the proximal intestine and pyloric caeca.