| Feeding experiments were conducted in a static system to determine the toxicityeffects of copper and cadmium and the protective role of dietary selenium andα-lipoic acid on abalone, Haliotis discus hannai Ino. Results of the studies arepresented as follows:1、The aim of this study was to study the effects of waterborne copper (Cu) onsurvival, antioxidant response, lipid peroxidation and metal accumulation in abaloneHaliotis discus hannai. Experimental animals (initial weight:7.49±0.01g) wereexposed to graded concentrations of waterborne Cu (0.02mg/L,0.04mg/L,0.06mg/L,0.08mg/L) for28days, respectively. Activities of the antioxidant enzymes(catalase, CAT; superoxide dismutase, SOD; glutathione peroxidases, GPx;glutathione S-transferase, GST), contents of the reduced glutathione (GSH), totalantioxidative competence (T-AOC) and malondiadehyde (MDA) in thehepatopancreas, and metal accumulation in hepatopancreas and muscles wereanalyzed after0,1,3,6,10,15,21,28days of metal exposure, respectively. Resultsshowed that0.04mg/L,0.06mg/L and0.08mg/L Cu caused100%death of abaloneon the21st,10th and6th day, respectively. However, no dead abalone was foundduring28-day waterborne Cu exposure at0.02mg/L Cu. Generally, activities of SOD,GST and T-AOC in hepatopancreas under all Cu concentrations followed a decreasingtrend as the exposure time prolonged. Activities of CAT, GPx and the content of GSHin all Cu exposure treatments were higher than those in the control. Contents of MDAin hepatopancreas in0.02mg/L Cu treatments significantly increased first and thendecreased to the control level. Contents of MDA in hepatopancreas in0.04mg/L, 0.06mg/L,0.08mg/L waterborne Cu were higher than that in the control. The metalsaccumulation in both hepatopancreas and muscles of abalone significantly increasedwith the increase of waterborne metals concentration and exposure time. These resultsindicated that waterborne Cu induced the oxidantive stress of abalone. Moreover, CAT,GPx and the content of GSH played more important role to resist Cu stress. Thereducing of anti-oxidant capacity and the rising of lipid peroxidation may lead to thedead of ablone H. discus hannai.2、A feeding experiment was conducted to determine the possible protective effects ofselenium (Se) against waterborne Cu toxicity on juvenile abalone Haliotis discushannai Ino. Purified diets supplemented with0.10mg/kg,1.31mg/kg and4.20mg/kgof Se, respectively, were fed to abalone (initial weight:3.17±0.01g) for two monthsunder waterborne Cu exposure (0.02mg/L) in a static system. A novelmetallothionein (MT) and metal regulation of transcription (MTF-1) was cloned fromabalone by homology cloning with degenerate primers and RACE techniques. Thefull length of HdhMT cDNA was551bp, consisting of a5’ untranslated region (UTR)of108bp, a3’ UTR of255bp, and an open reading frame (ORF) of188bp. Thededuced protein has69amino acid residues with a calculated molecular mass of6.996kDa and a predicted isoelectric point of5.16. The The full length of HdhMTF-1cDNA was2278bp, consisting of a5’ untranslated region (UTR) of25bp, a3’ UTRof626bp, and an open reading frame (ORF) of1566bp. The deduced protein has522amino acid residues with a calculated molecular mass of59.63kDa and a predictedisoelectric point of5.61. The results showed that there were no significant differencesin specific growth rate (SGR) and survival of abalone among different treatments. Thetreatment with1.31mg/kg dietary Se treatment significantly decreased the Cuconcentration in the serum and hepatopancreas compared with the control. Moreover,1.31mg/kg dietary Se had lowest Cu concentration in shell, muscle, mantle, gill,hepatopancreas and serum compared with the control and4.20mg/kg dietary Setreatments. Thioredoxin reductase (TrxR) activity and the content of the reducedglutathione (GSH) in hepatopancreas fed with Se supplements were significantlyincreased than those in the control. Se-dependent glutathione peroxidase (Se-GPx) activity fed with1.5mg/kg dietary Se treatment had no significant differencecompared with than in the control. However, Se-GPx activity with4.20mg/kg dietarySe treatment was significant decreased compared with other treatments. Themalondiadehyde (MDA) content and protein carbonyl content in hepatopancreas withdietary1.31mg/kg Se group were significantly decreased than that in the control.Genes (HdhMT and HdhMTF-1) were also detected in abalone fed with graded dietscontaining selenium using real-time PCR assays, respectively. Results indicated thatthe content of MT, the expression of HdhMT mRNA and HdhMTF-1mRNAincreased and reached the maximum at1.31mg/kg dietary selenium. But they weredown-regulated by dietary4.20mg/kg selenium. The present work indicated dietaryselenium could increase the antioxidant capacity and decreased the content of MDAand protein carbonyl content to some extent. Results showed that the MT content, theexpression of HdhMT mRNA and HdhMTF-1mRNA increased with dietary seleniumcontents. These data indicated that MT and the MT regulatory pathway by MTF-1mayplay important role to detoxify Cu toxicity in abalone H. discus hannai. Moreover,protection of1.31mg/kg Se had a better effect on abalone than4.20mg/kg Setreatment to against waterborne Cu in abalone.3、A feeding experiment was conducted to determine the possible protective effects ofα-lipoic acid (LA) against waterborne Cu toxicity on juvenile abalone Haliotis discushannai Ino. Purified diets supplemented with0g/kg,0.7g/kg and2.1g/kg of LA,respectively, were fed to abalone (initial weight:3.17±0.01g) for two months underwaterborne Cu exposure (0.02mg/L) in a static system. The results showed that thespecific growth rate (SGR) of abalone was increased with the dietary LA content underwaterborne Cu exposure, but no significant difference was found among all thetreatments. The treatment with0.7and2.1g/kg dietary LA treatment significantlydecreased the Cu concentration in the serum, hepatopancreas, gill and mantlecompared with the control. In muscle,2.1g/kg dietary LA treatment had significantlower Cu concentration compared with the control, and Cd concentration in0.7g/kgdietary LA treatment had no significant difference with that in the control. Activity ofsuperoxide dismutase (SOD), catalase (CAT), Se-dependent glutathione peroxidase (Se-GPx, glutathione S-transferase (GST) and the content of glutathione (GSH) inhepatopancreas were significant increased with dietary LA treatment in thisexperiment. The malondiadehyde (MDA) content in0.7g/kg and2.1g/kg dietary LAwere significant decreased compared with the control. Protein carbonyl content andDNA strand-breaks in dietary2.1g/kg LA were significant decreased compared withthe control and0.7g/kg dietary LA. The MT content in dietary0.7g/kg and2.1g/kgLA were significant higher than the control. The expression of HdhMT mRNA in0.7g/kg dietary LA was significantly higher than the control, and the expression ofMTF-1mRNA in0.7g/kg and2.1g/kg dietary LA was significantly higher than thecontrol. The present work indicated dietary LA could increase the antioxidant capacityand decreased the content of MDA, protein carbonyl content and DNA strand-breaks.Results showed that the MT content, the expression of HdhMT mRNA andHdhMTF-1mRNA increased with dietary selenium contents. These data indicatedthat MT and the MT regulatory pathway by MTF-1may play important role todetoxify Cu toxicity in abalone H. discus hannai.4、The aim of this study was to study the effects of waterborne cadmium (Cd) onsurvival, antioxidant response, lipid peroxidation and metal accumulation in abaloneHaliotis discus hannai. Experimental animals (initial weight:7.49±0.01g) wereexposed to graded concentrations of waterborne Cd (0.025mg/L,0.05mg/L,0.25mg/L,0.5mg/L) for28days, respectively. Activities of the antioxidant enzymes(catalase, CAT; superoxide dismutase, SOD; glutathione peroxidases, GPx;glutathione S-transferase, GST), contents of the reduced glutathione (GSH), totalantioxidative competence (T-AOC) and malondiadehyde (MDA) in thehepatopancreas, and metal accumulation in hepatopancreas and muscles wereanalyzed after0,1,3,6,10,15,21,28days of metal exposure, respectively. Resultsshowed that no dead abalone was found during28-day waterborne Cd exposure. Themetals accumulation in both hepatopancreas and muscles of abalone significantlyincreased with the increase of waterborne metals concentration and exposure time.Moreover, the hepatopancreas accumulated greatly higher levels of Cd than themuscles. Generally, activities of CAT, SOD and GST in hepatopancreas under all Cd concentrations followed the same trend as the exposure time prolonged. Theseactivities were firstly increased and then decreased to the control level and increasedagain. However, activity of GPx followed a decreasing trend. The level of T-AOC inhepatopancreas were firstly increased and then decreased to the control level andincreased again in0.05mg/L,0.25mg/L and0.5mg/L waterborne Cd. In the presentstudy, the first significant change for Cd exposure was found on day1for SOD, day3for GST, GSH and T-AOC, day6for CAT and GPx, respectively. Contents of MDA inhepatopancreas in all Cd treatments had no significant difference with that in thecontrol. These results indicated that waterborne Cd induced the oxidantive stress ofabalone. Moreover, SOD, CAT, GST and the content of GSH played more importantrole to resist Cd stress. The most sensitive indicators under Cd exposure was SOD,followed by GST、GSH and T-AOC in hepatopancreas of abalone. The anti-oxidantsystem in abalone may protect the animals from the lipid peroxidation.5、A feeding experiment was conducted to determine the possible protective effects ofselenium (Se) against waterborne Cd toxicity on juvenile abalone Haliotis discushannai Ino. Purified diets supplemented with0.10mg/kg,1.31mg/kg and4.20mg/kgof Se, respectively, were fed to abalone (initial weight:3.17±0.01g) for two monthsunder waterborne Cd exposure (0.35mg/L) in a static system. The results showed thatthere were no significant differences in specific growth rate (SGR) and survival ofabalone among different treatments, and SGR of4.20mg/kg dietary Se treatment hadmaximum value. The treatment with1.31and4.20mg/kg dietary Se treatmentsignificantly decreased the Cd concentration in the serum, hepatopancreas, gill andmantle compared with the control. In muscle,4.20mg/kg dietary Se had significantlower Cd concentration compared with the control and1.31mg/kg dietary Setreatments. Activity of thioredoxin reductase (TrxR), Se-dependent glutathioneperoxidase (Se-GPx), thioredoxin peroxidase (TPx) and the content of the reducedglutathione (GSH) in hepatopancreas with Se supplements were significantlyincreased than those in the control. The malondiadehyde (MDA) content, proteincarbonyl content and DNA strand-breaks in hepatopancreas with dietary1.31mg/kgand4.20mg/kg Se group were significantly decreased than that in the control. The MT content in hepatopancreas in1.31mg/kg and4.20mg/kg dietary Se wassignificant increased than that in the control. The present work indicated dietaryselenium could increase the antioxidant capacity and decreased the content of MDA,protein carbonyl content and DNA strand-breaks under waterborne Cd exposure.Moreover, MT may play important role to detoxify Cd toxicity in abalone H. discushannai.6、A feeding experiment was conducted to determine the possible protective effects ofα-lipoic acid (LA) against waterborne Cd toxicity on juvenile abalone Haliotis discushannai Ino. Purified diets supplemented with0mg/kg,0.7g/kg and2.1mg/kg of LA,respectively, were fed to abalone (initial weight:3.17±0.01g) for two months underwaterborne Cd exposure (0.35mg/L) in a static system. The results showed that thespecific growth rate (SGR) of abalone was increased with the dietary LA content underwaterborne Cu exposure, but no significant difference was found among all thetreatments. The treatment with0.7and2.1g/kg dietary LA treatment significantlydecreased the Cd concentration in the serum, hepatopancreas, gill and mantlecompared with the control. In muscle,2.1g/kg dietary Se treatment had significantlower Cd concentration compared with the control, and Cd concentration in0.7g/kgdietary Se treatment had no significant difference with that in the control. Activitycatalase (CAT), Se-dependent glutathione peroxidase (Se-GPx, glutathioneS-transferase (GST) and the content of glutathione (GSH) in hepatopancreas weresignificant increased with dietary LA treatment in this experiment. Themalondiadehyde (MDA) content in2.1g/kg dietary LA were significant decreasedcompared with the control and0.7g/kg. Protein carbonyl content and DNAstrand-breaks in dietary0.7g/kg and2.1g/kg LA were significant decreased comparedwith the control. The MT content and the expression of HdhMT and HdhMTF-1mRNA was significantly higher than the control.The present work indicated dietaryLA could increase the antioxidant capacity and decreased the content of MDA,protein carbonyl content and DNA strand-breaks. Results showed that the MT content,the expression of HdhMT mRNA and HdhMTF-1mRNA increased with dietary LAcontents. These data indicated that MT and the regulatory pathway by MTF-1played important role to detoxify Cd toxicity in abalone H. discus hannai. |
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