Study On The Effects Of ABCG Transporter On Florfenicol Metabolism In Portunus Trituberculatus

ATP-binding cassette transporter(ABC)sub-family G is a member of ATP-binding cassette(ABC)transporters family,which is an integral membrane protein.They catalyze the transportation of diverse substrates,including exogenous compounds,sugars,metabolic products,ions,amino acids,lipids,using the energy from ATP hydrolysis.According to the present utilization of florfenicol(FLR)in China,we used the Portunus trituberculatus,which is a commercially important aquaculture species.The inhibitor of ABCG transporter-Fumitremorgin C(FTC)was compared to study the distribution and elimination of FLR in major tissues of P.trituberculatus.We cloned an ABC transporter from Portunus trituberculatus(designated ptABCG),along with a study of the FLR on ABCG gene expression.Meanwhile,the activities of metabolia enzymes and the expression of metabolize-related genes were determined in hepatopancreas and haemolymph,and the antioxidative capacity and immune parameters were also determined to study the effect of FLR on hepatopancreas and haemolymph in P.trituberculatus.The results obtained are as follows.1.The study of transport activity of ABCG to florfenicol in P.trituberculatus tissuesWe characterized the distribution and elimination of FLR in major tissues of P.trituberculatus,in comparison with and without of a potent and selective inhibitor-FTC.Results showed that florfenicol concentrations in the FTC groups were higher than without inhibitor groups.In elimination stage,the elimination rate of inhibitor groups was slower than without inhibitor groups,and in FTC+FLR goups,the elimination half-life(T1/2)in hemolymph?hepatopancreas?gill and muscle was 0.85?1.25?1.91?1.35 h,while in FLR groups,the T1/2 was 0.80?1.00?1.72?1.73 h.So we conferred that ABCG transporter promoted the FLR metabolism in Portunus trituberculatus.From the perspective of the transport activity of ABCG to FLR,after 40 mg/kg IV FLR injection,the ABCG activity in hemolymph,hepatopancreas,gill,and muscle were peak at 3,3,3,and 6 h,respectively.The transport activity was consistent with the FLR accumulations in different tissues,which further proves the transport function of ABCG to FLR in P.trituberculatus.2.Effects of FLR on relative expression of ptABCG in different tissues of Portunus trituberculatusFor further study the role of ABCG transporter in the process of FLR metabolism,using reverse transcription PCR and RACE cloning get the length of the Portunus trituberculatus ABCG cDNA sequence(GenBank login number: KY242401).The full-length was 2473 bp,including 25 bp of 5'non coding regin(untranslated regions,UTR),a 701 bp 3'noncodong regin and a 1747 bp open reading frame(ORF)which encoded a polypeptide of 578 amino acids.Bioinformatics analysis indicated that ptABCG shares many similarities with the ABC transmembrane transporter,including two conserved regions: a highly conserved nucleotide binding domains(NDB)and a transmembrane domain(TMD).TMD has inside-out and outside-in transmembrane helicals,thus can judge the cDNA sequence is ABCG gene sequences of Portunus trituberculatus.RT-PCR method was used to analyze the effects of FLR on relative expression of ptABCG in different tissues of Portunus trituberculatus.Result showed the ptABCG relative expression in the FTC+FLR groups were restrained,significantly lower than FLR groups(P<0.05).With the inhibitor weakened,the expression levels increased rapidly and respectively peaked at 12,6,and 12 h in hepatopancreas,gills,and muscle,then decreased gradurally to the control level.This results suggest that ABCG transporter palys a certain role in FLR metabolism.3.Effects of FLR on metabolic detoxification system in Portunus trituberculatusThe purpose of this study was to investigate the impact of FLR on metabolic detoxification system.Detoxification enzyme activities of phase I(Aminopyrine N-demethylase,APND),phase II(glutathione-S-transferase,GST)and related CYP2,GST gene expression and phase ? ABCG gene expression were determined,and results showed that all the detoxification enzyme activities and gene expressions were affected significantly in a time-dose dependent manner,while control groups have no obvious change.APND activity and CYP2 mRNA expression were decreased in hepatopancreas and hemolymph at 1,3,and 6 h after FLR injection.In hepatopancreas,compared to the control group,GST and GST mRNA were induced by FLR in a dose dependent manner.In hemolymph,different doses FLR have different effects on GST mRNA expression.Low and middle dosages(20 and 40 mg/kg)induced GST mRNA expression,while high dosage(80 mg/kg)suppressed it.The ABCG mRNA in the hepatopancreas,gills and muscles of P.trituberculatus were significantly up-regulated.These results suggested that the ptABCG transposter might play an important role in the physiological changes related to metabolism and cell detoxification that occur when accumulated a large number of drugs in body.4.Effects of FLR on physical health parameters of Portunus trituberculatusFlorfenicol(FLR)is a commonly used antibiotic for the treatment of bacterial diseases in Portunus trituberculatus.The aim of this study was to investigate the effects of FLR on immune response and antioxidant status of P.trituberculatus.Results showed antibacterial activities and phenol oxidase(PO)activity in plasma were decreased significantly compared to control during the experiment(P<0.05).There were no significant differences in bacteriolytic activity between the treatments and control groups(P>0.05).It showed that at 6 and 12 h after FLR injection,FLR affected antioxidant ststus of Total antioxidative capacity(T-AOC)?SuPeroxide Dismutases(SOD)and Glutathione(GSH)in hepatopancreas,and the effects appearred at 3 h in plasma.The antioxidant status in low and middle dosage(20 and 40 mg/kg)groups recovered to the control level,while in high dosage(80 mg/kg),FLR suppressed all of them during the whole experiment.FLR has significant effects on immune and antioxidant parameters of Portunus trituberculatus,therefore,when using antibiotics to prevention and control of diseases,we should comprehensively consider the effects of drugs dose to physical health.