The Research Of Effects Caused By Fishery Drugs Based On Type A Of Gamma-aminobutyric Acid Receptor

Gamma-aminobutyric acid (GABA) is an important inhibitoryneurotransmitter which mainly distributs in the central nervous system (CNS). In thebody, GABA is synthesized from glutamate irreversibly and metabolized to succinicsemialdehyde by glutamic acid decarboxylase (GAD) and GABA transaminase(GABA-T), respectively. And GAD has two isoforms, namely GAD65and GAD67.Gamma-aminobutyric acid receptors (GABA receptor, GABAR) refer to thesites which can identify and combine with GABA on synaptic membranes. Aftercombined with GABA, ion permeability of cell membrane changed, and that would leadto neural inhibited consequently. According to sensitivity difference to agonists andinhibitors, GABARs have three subtypes, namely GABAAR, GABABR and GABACR.GABAAreceptors are ligand-operated chloride channels that are assembled from fivesubunits in a heteropentameric manner, which belong to the cys-loop ligand-gated ionchannel family, and122is the predominant GABAAreceptor subtype which has twocopies of1and2in each subunit.To date, informations about distributions and quantities of GABAAreceptorsin aquatic animals and effects of fishery drugs on expressions of GABAAreceptors arestill limited. For carassius auratus gibelio is the one of widely bred freshwater fishspecies in China, C. auratus gibelio was employed in this study. Then by using reversetranscription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR(qPCR), and high performance liquid chromatography (HPLC), the tissue specificdistributions and quantitative detections of GABAAR were executed firstly. Andresearches of effects caused by avermectin (AVM) and difloxacin (DIF) based on type Aof gamma-aminobutyric acid receptors were conducted secondly.Section Ⅰ: Distributions and quantitative detections of GABAR and the enzymesrelate to GABA in C.auratus gibelioFirstly, the tissue specific distributions of GABAAR2a (AR2a) andGABAAR2b (AR2b) were study with RT-PCR technique in C. auratus gibelio.Results showed that GABAAR2a and GABAAR2b genes expressed in both brain andperipheral organs such as liver, kidney, heart, intestine, swim bladder, gill, muscle andfin. In addition, GAD65and GAD67, and GABA-T genes also expressed in these tissuesmentioned above. Secondly, the mRNA expressions of AR2a, AR2b, GAD, and GABA-T ofdifferent tissues and the expression differences of AR2a and AR2b between twodevelopmental stages were quantified in C. auratus gibelio. Results showed that themajorities of AR2a, AR2b, GAD, and GABA-T were all mainly synthesized in brain.However, a considerable amount of GABA-T was secreted from the peripheral tissues,especially in the liver. Moreover, the expression of AR2a and AR2b genes in differenttissues varied with body weight changed.Section Ⅱ: researches of effects caused by Avermectin (AVM) based on type A ofGABAR in C.auratus gibelioAvermectin (AVM) is a widely used drug for parasite prevention andcontrolling in aquaculture. In order to research the effects caused by AVM based on typeA of GABAR in fish, the acute toxicity of AVM to C. auratus gibelio and the tissueresiduals of AVM in C. auratus gibelio were detected in this section. Results showedthat the median lethal concentrations (LC50) of AVM at24h,48h and96h to C.auratus gibelio(60.04±5.02g body weight) were0.127,0.071and0.039mg/Lrespectively, So safe concentration (SC) of AVM was0.0039mg/L. And the existencesof AVM in the brain of C. auratus gibelio were detected under the median lethalconcentrations of AVM at24h,48h and96h and its safety concentration (0.0039mg/L). The brain residual of AVM under24h LC50at24h was0.292±0.005μg/g whichwas the highest one. Furthermore, the AVM contents in brain of C. auratus gibelio havepositive correlations with the initial drug concentrations. The results showed that AVMcould penetrate through blood brain barrier into brain of C. auratus gibelio underdifferent drug concentrations.At the same time, the results showed that the expressions of AR2a andAR2b genes in cerebrum, midbrain, medulla oblongata and cerebellum of CNS weremost significant decreased (P<0.01) at different concentrations and different time pointsafter AVM used in C. auratus gibelio, and cerebellum was the tissue in which theexpressions of AR2a and AR2b genes were lowestly down-regulated. But in theperipheral tissues, the expressions of AR2a gene were increased significantly (P<0.01)in liver and kidney, while it’s expressions in muscle were significant decreased (P<0.05).Meanwhile, the expressions of AR2b gene in liver, kidney and muscle were mostsignificantly down-regulated except that in kidney under96h LC50at24h, and thechanges of AR2a and AR2b genes happened in liver, kidney and muscle have nocorrelation with the concentrations of AVM. The results mentioned above showed thatexpressions of AR2gene have decline trend after AVM processed in C. auratusgibelio.What’s more, there also showed that the expressions of GAD and GABA-T genes were affected by AVM in C. auratus gibelio. To GAD, the expression of GAD65and GAD67genes were most significantly down-regulated in the tissues of CNSdetected in this paper, and medulla oblongata was the organ which the expressions ofGAD65and GAD67genes were most significantly down-regulated. However, theexpressions of GAD65and GAD67genes in liver, kidney and muscle were mostsignificantly down-regulated except happened at24h with96h LC50at which theexpressions of GAD65and GAD67genes were increased significantly and thedown-regulations happened in muscle were most obvious. To GABA-T, the expressionsof GABA-T genes in erebrum, midbrain, cerebellum and medulla oblongata of CNS inC. auratus gibelio were all decreased significantly (P<0.05). However, the expressionsof GABA-T genes in liver, kidney and muscle were decreased significantly (P<0.05)except happened in kidney at24h with96h LC50which was significant up-regulated.All the results mentioned above indicated that the synthesis and metabolism rates ofGABA were decreased.Section Ⅲ: researches of effects caused by Fluoroquinolones (FQs) based on type Aof GABAR in C.auratus gibelioIn order to research the effects caused by FQs based on type A of GABAR infish, the acute toxicity of DIF (one kind of FQs) to C. auratus gibelio and the tissueresiduals of DIF in C. auratus gibelio were detected firstly in this section. The resultsshowed that the median lethal dose (LD50) of DIF at96h to C. auratus gibelio with60.04±5.02g body weight was2840mg/kg, and the drug could be judged as a kind oflow toxic substances to C. auratus gibelio. And the detections of DIF in the brain atdifferent time points with two drug doses indicated that DIF could penetrate throughblood brain barrier into the brain of C. auratus gibelio. In addition, theconcentration-time curve in brain was the gentlest one in the four organs with20mg/kg.Combining with the result there has a highest residual content (0.392±0.007μg/g) ofDIF in brain at960thh, it would inferred that brain could used as the target tissue fordrug residue analysis.The results showed that the expressions of AR2a genes in cerebrum,midbrain, cerebellum and medulla oblongata of CNS were all most significantlydecreased (P<0.05) in C. auratus gibelio with96h LD50and20mg/kg of DIF. But forAR2b, the expressions in telencephalon, mesencephalon and cerebella wereup-regulated except that the expression changed in medulla oblongata. Cerebrum is thesenior sports center in fish, so it may be speculated that the increasing expression ofAR2b gene could lead to nerve excitability in C. auratus gibelio. In the peripheraltissues, the expressions of AR2a and AR2b genes in liver, kidney and muscle were allmost significant increased especially that in muscle. All results showed that the expression of AR2a genes showed increasing trends in C. auratus gibelio after the useof DIF.And to GAD, the expressions of GAD65and GAD67genes in telencephalon,mesencephalon, cerebella and medulla oblongata of CNS were increased or mostsignificantly increased with96h LD50of DIF, but all declined with20mg/kg of DIF. Soit could be speculated that in brain of C. auratus gibelio, the synthesis of GABA wasincreased after the use of DIF with96h LD50, while the situation was reversed with20mg/kg. To GABA-T, the expressions of GABA-T genes both in CNS and in theperipheral tissues detected in this paper were all most significantly increased with96hLD50. This indicated that the metabolisms of GABA in those organs were enhanced afterthe use of DIF with96h LD50.In conclusion, although AR2a, AR2b GAD, and GABA-T genes were allfound distributing in CNS (telencephalon, mesencephalon, cerebella, medulla oblongata)and in peripheral organs (liver, kidneys, heart, intestine, swim bladder, gill, muscle andfin), but the vast majority of them were secreted in the former. The researches of effectscaused by fishery drugs based on type A of GABAR showed that, in vast majority ofcases, the expressions of the receptors were down-regulated which was caused by AVMand DIF in C.auratus gibelio. And the expressions of GAD and GABA were alsoaffected by AVM and DIF, but it was still hard to judge the content changes of GABAwithin body.