Identification of Carassius auratus gibelio liver cell proteins interacting with the GABAA receptor γ2 subunit using a yeast two-hybrid system.

The γ-aminobutyric acid type A (GABAA) receptor is an important pentameric inhibitory neurotransmitter receptor, and the γ2 subunit of this receptor plays a key role in potentiation of the GABAA response. We previously detected that the expression of GABAA receptor in the livers of Carassius auratus gibelio significantly increased after medication (avermectin and difloxacin treatment). In order to better understand the mechanism of action of the GABAA receptor γ2 subunit in the livers of C. auratus gibelio, we constructed a C. auratus gibelio liver cDNA library (the titer value of 1.2 × 106 cfu/mL) and identified the proteins that interact with the GABAA receptor γ2 subunit by using a yeast two-hybrid assay. The yeast two-hybrid screening yielded seven positive clones, namely, prelid3b, cdc42, sgk1, spg21, proteasome, chia.5, and AP-3 complex subunit beta-1, all of which have been annotated by the NCBI database. The functions of these proteins are complex; therefore, additional studies are required to determine the specific interactions of these proteins with the GABAA receptor γ2 subunit in the liver of C. auratus gibelio. Although the interactions identified by the yeast two-hybrid system should be considered as preliminary results, the findings of this study may provide further direction and a foundation for future research focusing on the mechanisms of the GABAA receptor γ2 subunit in C. auratus gibelio livers.

Molecular characterization, expression and functional analysis of NOD1, NOD2 and NLRC3 in Nile tilapia (Oreochromis niloticus).

The nucleotide-binding oligomerization domain proteins NOD1, NOD2 and NLRC3 are cytoplasmic pattern recognition receptors (PRRs) of the Nod-like receptor (NLR) family. In the present study, the Nile tilapia (Oreochromis niloticus) NOD1 (ntNOD1), NOD2 (ntNOD2) and NLRC3 (ntNLRC3) genes were cloned and characterized. The full-length ntNOD1, ntNOD2 and ntNLRC3 genes were 3924, 3886 and 4574 bp, encoding 941, 986 and 1130 amino acids, respectively. The three Nod-like receptors have a NACHT domain and a C-terminal leucine-rich repeat (LRR) domain. In addition, ntNOD1 and ntNOD2 have a N-terminal CARD domain (ntNOD2 has two). Phylogenetic analysis showed that the three NLRs are highly conserved. Tissue expression analysis of the three receptors revealed that the highest mRNA and protein levels of ntNOD1, ntNOD2 and ntNLRC3 were in the spleen. The expression patterns of NLRs during embryonic development showed that the expression levels of ntNOD2 and ntNLRC3 significantly increased from 2 to 8 days post-fertilization (dpf). The expression levels of ntNOD1 significantly increased from 2 to 6 dpf, decreased at 7 dpf and then increased at 8 dpf. Upon stimulation with an intraperitoneal injection of Streptococcus agalactiae, expression levels of the ntNOD1, ntNOD2 and ntNLRC3 mRNA and protein were clearly altered in the blood, spleen, kidney, intestine and gill. Furthermore, after cotransfection with an NF-κB reporter plasmid, NF-κB activation in ntNOD1-overexpressing 293T cells significantly increased compared with that in control cells, before or after i-EDPA-stimulation. By contrast, compared with control, ntNOD2 and ntNLRC3 had no effect on NF-κB activation in 293T cells, when their potential ligands were not stimulated. However, after MDP-stimulation, ntNOD2 and ntNLRC3 overexpression increased NF-κB activation in 293T cells. NOD1 and NLRC3 were uniformly distributed throughout the cytoplasm in 293T cells, whereas NOD2 was distributed throughout the cytoplasm and nucleus. Our results indicate that the three Nod-like receptors are functionally conserved and may play pivotal roles in defense against pathogens such as Streptococcus agalactiae.

Toxicity and accumulation of zinc pyrithione in the liver and kidneys of Carassius auratus gibelio: association with P-glycoprotein expression.

Zinc pyrithione (ZPT) is a broad-spectrum antibacterial and antifungal agent; therefore, it is widely used in industry and civilian life. It is discharged into the aquatic environment with industrial and civilian waste water. Carassius sp. is one of the most widely distributed and farmed fish in China. The effects of aquatic ZPT on Carassius sp. remain unknown. In this study, we determined the acute toxicity of ZPT on Carassius sp. The results showed that the median lethal concentration (LC50 96 h) of ZPT on Carassius sp. cultivated in freshwater or water with 1.5 or 3 ‰ salinity was 0.163, 0.126, and 0.113 mg/L, respectively. ZPT has a higher affinity to the liver than the kidney, with a prolonged tissue residual time. P-glycoprotein (P-gp), an ATP-binding cassette transporter, was found to be induced in the liver and kidney tissues of these Carassius spp. after ZPT treatment, based on the determination of its mRNA and protein levels by quantitative real-time reverse transcription polymerase chain reaction and immunohistochemistry, respectively. The ZPT accumulation and magnitude of P-gp induction were also affected by the salinity of the cultivation water. These results suggest that aquatic ZPT is potentially toxic to Carassius sp. We speculate that P-gp induction may play a protective role for Carassius sp. Our findings provide a basis for assessing the potential risk of ZPT to aquatic animals including Carassius sp.

Analysis of Saprolegnia parasitica Transcriptome following Treatment with Copper Sulfate.

BACKGROUND: Massive infection caused by oomycete fungus Saprolegnia parasitica is detrimental to freshwater fish. Recently, we showed that copper sulfate demonstrated good efficacy for controlling S. parasitica infection in grass carp. In this study, we investigated the mechanism of inhibition of S. parasitica growth by copper sulfate by analyzing the transcriptome of copper sulfate-treated S. parasitica. To examine the mechanism of copper sulfate inhibiting S. parasitica, we utilized RNA-seq technology to compare differential gene expression in S. parasitica treated with or without copper sulfate. RESULTS: The total mapped rates of the reads with the reference genome were 90.50% in the control group and 73.50% in the experimental group. In the control group, annotated splice junctions, partial novel splice junctions and complete novel splice junctions were about 83%, 3% and 14%, respectively. In the treatment group, the corresponding values were about 75%, 6% and 19%. Following copper sulfate treatment, a total 310 genes were markedly upregulated and 556 genes were markedly downregulated in S. parasitica. Material metabolism related GO terms including cofactor binding (33 genes), 1,3-beta-D-glucan synthase complex (4 genes), carboxylic acid metabolic process (40 genes) were the most significantly enriched. KEGG pathway analysis also determined that the metabolism-related biological pathways were significantly enriched, including the metabolic pathways (98 genes), biosynthesis of secondary metabolites pathways (42 genes), fatty acid metabolism (13 genes), phenylalanine metabolism (7 genes), starch and sucrose metabolism pathway (12 genes). The qRT-PCR results were largely consistent with the RNA-Seq results. CONCLUSION: Our results indicate that copper sulfate inhibits S. parasitica growth by affecting multiple biological functions, including protein synthesis, energy biogenesis, and metabolism.

Inhibition of dioscin on Saprolegnia in vitro.

As one of the most serious pathogens in the freshwater aquatic environment, Saprolegnia can induce a high mortality rate during the fish egg incubation period. This study investigated the anti-Saprolegnia activity of a total of 108 plants on Saprolegnia parasitica in vitro and Dioscorea collettii was selected for further studies. By loading on an open silica gel column and eluting with petroleum ether-ethyl acetate-methanol, dioscin (C45H72O16) was isolated from D. collettii. Saprolegnia parasitica growth was inhibited significantly when dioscin concentration was more than 2.0 mg L(-1). When compared with formalin and hydrogen peroxide, dioscin showed a higher inhibitory effect. As potential inhibition mechanisms, dioscin could cause the S. parasitica mycelium morphologic damage, dense folds, or disheveled protuberances observed by field emission scanning electron microscopy and the influx of Propidium iodide. The structural changes in the treated mycelium were indicative of an efficient anti-Saprolegnia activity of dioscin. The oxidative stress results showed that dioscin also accumulated reactive oxygen species excessively and increased total antioxidant and superoxide dismutase activity. These situations could render S. parasitica more vulnerable to oxidative damage. Additionally, when dioscin concentration was less than 2.0 mg L(-1), the survival rate of embryos was more than 70%. Therefore, the use of dioscin could be a viable way of preventing and controlling saprolegniasis.

Chitosan Influences the Expression of P-gp and Metabolism of Norfloxacin in Grass Carp.

The aim of this study was to investigate the relationship between the administration of chitosan (CTS), expression of permeability glycoprotein (P-gp), and the metabolism of norfloxacin (NOR) in Grass Carp Ctenopharyngodon idella. Fish were administrated with a single dose of either NOR, CTS, 1:5 NOR-CTS or 1:10 NOR-CTS. The P-gp expression was analyzed by immunohistochemistry and real time-PCR. The concentration of NOR was determined using HPLC. The mRNA and protein expression of P-gp in the fish intestine was significantly enhanced following a single dosage of 40 mg/kg NOR, and peak expression occurred at 3 h after drug administration (P < 0.05). A single dosage of both 1:5 NOR-CTS and 1:10 NOR-CTS reduced the intestinal P-gp expression to levels significantly lower than that from NOR alone (P < 0.05), but significantly higher than that from the control (P < 0.05). Interestingly, CTS alone also led to a slight decrease in P-gp expression. In addition, pharmacokinetic assays revealed a marked increase in area under the curve (AUC) of NOR with 1:5 and 1:10 NOR-CTS, by approximately 1.5-fold and threefold, respectively. Finally, the relative bioavailability of NOR after a single oral dosage of 1:5 and 1:10 NOR-CTS was enhanced to 148.02% and 304.98%, respectively. In this study, we demonstrated that the transmembrane glycoprotein P-gp regulates NOR metabolism in the intestine of Grass Carp, suggesting that NOR may be a direct substrate of P-gp. More importantly, we showed that CTS can inhibit P-gp expression in a dose-dependent manner and improve the relative bioavailability of NOR in this species.

Tissue accumulation and toxicity of isothiazolinone in Ctenopharyngodon idellus (grass carp): association with P-glycoprotein expression and location within tissues.

Isothiazolinone is widely used as a broad-spectrum fungicide in various industries, such as oil, paper, pesticide, dyes, tanning and cosmetics. There is an increasing concern over protection of the aquatic environment due to its large-scale use. The acute toxicity (LC50) of isothiazolinone in Ctenopharyngodon idellus was investigated. The residual time and accumulation in tissues, P-glycoprotein mRNA level and localization of P-glycoprotein in the liver and kidney were also analyzed. The LC50 (48 h) values of isothiazolinone to C. idellus were 0.53±0.17 mg/L and 0.41±0.08 mg/L at 15 °C and 25 °C, respectively. The LC50 values decreased as the temperature increased. The accumulation of isothiazolinone in livers and kidneys in the high temperature group (25 °C) was significantly greater than that of the low temperature group (15 °C). Prolonged tissue residual time of isothiazolinone was seen in all the groups. There were significant differences in P-glycoprotein mRNA expression between isothiazolinone-treated groups and control samples (P<0.05-0.01). Temperature affected accumulation and toxicity of isothiazolinone.

Inhibition of CYP450 1A and 3A by berberine in crucian carp Carassius auratus gibelio.

Berberine has long been considered as an antibiotic candidate in aquaculture. However, studies regarding its effects on drug-metabolizing enzymes in fish are still limited. In the present study, the effects of berberine on cytochrome P4501A (CYP1A) and CYP3A in crucian carp were investigated. Injection of different concentrations of berberine (0, 5, 25, 50, and 100mg/kg) inhibited the CYP1A mRNA expression, thereby inhibiting further the catalytic activity of CYP1A-related ethoxyresorufin-O-deethylase (EROD). Furthermore, both CYP1A expression and EROD activity were further inhibited with increasing berberine concentrations. In addition, the CYP3A expressions at both the mRNA and the protein levels were downregulated by higher berberine concentrations. The catalytic activity of CYP3A-related erythromycin N-demethylase (ERND) was also inhibited by berberine at a dose of no less than 25mg/kg. Moreover, at the berberine concentration exceeding 25mg/kg, the inhibition of CYP3A expression and ERND activity increased with increasing berberine concentrations. In vitro experiments were also performed. When berberine was pre-incubated with the crucian carp liver microsomes, it competitively inhibited the corresponding EROD activity with the IC(50) of 11.7 µM. However, the ERND activity was slightly inhibited by berberine with the IC(50) of 206.4 µM. These results suggest that, in crucian carp, berberine may be a potent inhibitor to CYP1A, whereas the CYP3A inhibition needs a higher concentration of berberine.

Effects of cytochrome P450 1A substrate (difloxacin) on enzyme gene expression and pharmacokinetics in crucian carp (hybridized Prussian carp).

Cytochrome P450s (CYPs) play a prominent role in drug metabolism and biotransformation which are distributed in liver of aquatic animals. However, limited information is available about CYP genes involved in drug metabolism in fish. In the present study, we explore CYP1A characterization for DIF metabolism. Firstly, we cloned and characterized the full-length cDNA sequence of a CYP1A gene from crucian carp (hybridized Prussian carp), the predicted protein sequence for CYP1A comprise 496 amino acids. The heme-binding region of the CYP1A, encompassing the amino acid sequence GLGKRRCIG, which is identical to the same region of other homologues. Secondly, we studied the difloxacin (DIF) kinetics and the effects of DIF on their corresponding CYP1A mRNA levels in liver of crucian carp. CYP1A1 mRNA expression was analyzed by real-time PCR, and DIF concentration was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). Results showed that the concentration of DIF in liver reached its peak (67.70 mg kg(-1)) at 0.5h, while the CYP1A1 gene expression was at the lowest point. CYP1A mRNA was down-regulated by 6.5 mg ml(-1) DIF in the liver of crucian carp. Thus, our work confirmed that DIF is both the substrate and inhibitor of CYP1A. The information provided a model for the potential utility of gene expression analysis and drug metabolization in fish.

Effects of fish cytochromes P450 inducers and inhibitors on difloxacin N-demethylation in kidney of Chinese idle (Ctenopharyngodon idellus).

Cytochromes P450 (CYPs) play key roles in drug metabolism which are widely distributed in kidney in aquatic organisms. CYP(s) mainly catalyzed the N-demethylation reaction of difloxacin (DIF) biotransformation to sarafloxacin (SAR). However, limited information is available about CYP investigation in fish. In order to supply useful information on CYP(s) characterization for DIF N-demethylation, the present study assessed the effects of fish potent CYP inducers and inhibitors on DIF N-demethylation and the inductive and inhibitive enzyme kinetics in kidney of Chinese idle (Ctenopharyngodon idellus) by reversed-phase high-performance liquid chromatography (RP-HPLC). Results demonstrated that the amounts of SAR formation pretreated by ß-naphthoflavone (BNF) increased by 1.1-fold and α-naphthoflavone (ANF) inhibited SAR formation level by 0.6-fold at the third day. Enzymatic parameters V(max) and Cl(int) of DIF N-demethylase were increased by 0.56- and 0.38-fold due to ß-naphthoflavone (BNF) pretreatment. DIF N-demethylation inhibition by varying ANF concentrations represented a mixed-type inhibition with the value of the inhibition constants (K(i)) 12.9mg/kg. BNF and ANF are the separate typical inducer and inhibitor for CYP1A in fish. Thus, we suggest that CYP1A may be responsible for DIF N-demethylation in kidney. This study provides instructive information to ensure treatment success in fisheries medication with two or more drugs.

Effects of fish CYP inducers on difloxacin N-demethylation in kidney cell of Chinese idle (Ctenopharyngodon idellus).

A drug-drug interaction occurs when the effect of one drug is altered by the presence of another drug which is generally associated with the induction of cytochrome P450s (CYPs) activity. Thus, unexpected treatment failures often happen resulting from inappropriate coadministration in fisheries. However, little information is available about CYP induction in fish. The reaction of difloxacin (DIF) biotransformation to sarafloxacin (SAR) belongs to N-demethylation catalyzed mainly by CYP(s). In order to supply useful information on CYP induction, the present study assessed the effects of fish-specific CYP inducers on DIF N-demethylation and enzyme kinetics in kidney cell of Chinese idle (CIK; grass carp (Ctenopharyngodon idellus)) by RP-HPLC. Results demonstrated that the amounts of SAR formation and enzymatic parameters Clint and Vmax were significantly increased due to beta-naphthoflavone (BNF) pretreatment. Therefore, we suggest that CYP1A may be involved in DIF N-demethylation in CIK. This study provides instructive information to ensure treatment success via avoiding CYP induction in fisheries.

Effects of mammalian CYP3A inducers on CYP3A-related enzyme activities in grass carp (Ctenopharyngodon idellus): Possible implications for the establishment of a fish CYP3A induction model.

Unexpected drug-drug interactions in fish are generally associated with the induction of CYP3A activity and may lead to the formation of drug residues and thus threaten the safety of fishery products. However, little information is available about CYP3A induction in fish. In the present study, we determined the in vivo and in vitro effects of typical mammalian CYP3A inducers (rifampicin, phenobarbital and dexamethasone) on CYP3A-related enzyme activities in a freshwater teleost, the grass carp (Ctenopharyngodon idellus). Our results showed that the response to rifampicin was similar for grass carp liver cell line (GCL), liver microsomes and the primary hepatocytes of grass carp, as indicated by the activity of aminopyrine N-demethylase (APND). When erythromycin N-demethylase (ERND) and 6beta-testosterone hydroxylase (6beta-TOH) were taken into consideration, the GCL displayed a greater capacity for conducting CYP3A metabolism and induction than the C. idellus kidney cell line (CIK). Using erythromycin and testosterone as substrates, we demonstrated that CYP3A catalysis exhibited non-Michaelis-Menten kinetics in GCL cells, and that V(max)/K(m) values were significantly increased due to rifampicin-treatment. Overall, this study may have implications for the use of GCL as a CYP3A induction model to identify physiological changes in fish as well as the similarities or differences between fish and mammals.