Study Of Biomarker Response Of Metallothionein And Heat Shock Proteins In Macrobrachium Rosenbergii In Exposure To Heavy Metals & Thermal Stress

1.1 Background and Experimental SetupThough various biomarkers have been proposed for assessment of toxicity but metallothioneins and Hsp70 family have been given prime importance in this regards and hence these were extensively studied in various organisms.However,literature also shows some controversial data,especially in case of Hsp70 family,regarding their validity as biomarkers for assessment of environmental toxicity.Furthermore,most of studies had been conducted by using high doses of individual heavy metals and less attention was paid to fairly environmentaly relavent doses individually as well as in combinations.Therefore,we have used indvidual as well as combined dose concentrations within limits of standards set by EPA(environmental protection agency). The maximum contamination limits(MCL) for Cd,Cu,and Zn is 5,1300 and 1000μg/l respectively.We have used two concentrations of each of Cd(2.5 & 5μg/l),Cu(250 & 500μg/l) and Zn(250 & 500μg/l) for 0,1,2,4 and 8 hours exposure in addition to combined doses as Cd+Cu(2.5+250μg/l),Cd+Zn(2.5+250μg/l),Cu+Zn(250+250μg/l), Cd+Cu+Zn(2.5+250+250μg/l),Cd+Cu+Zn with heat shock(2.5+250+250μg/l+28℃) and heat shock of 35℃for 2 hours of exposure.The sampling was done at room temperature(25℃).1.2 Metallothionein of M.rosenbergiiWe isolated and characterized a novel MT cDNA contaning 433 bp with 189bp in ORF, 95bp in 5'UTR and 149bp in 3'UTR,from the hepatopancreas of Macrobrachium rosenbergii(GenBank accession,EU871044).It encodes 61 amino acid residues.A comparision of putative Mar-MT-I peptide showed 6 to 93%sequence identity(with Daphnia pulex,EU307302 and Palaemonetes pugio,AY935987 respectively) with an average 66%with MT of other crustaceans.The MT-I of Macrobrachium rosenbergii, like Palaemonetes pugio(AY935987) has 17 cysteines residues instead of 18(which exist in majority) and 4 additional amino acids DCKG(aspartic acid,cysteine,lysine and glycine) at position 16 to 19.Furthermore,16 amino acid residues existing at different positions in Mar-MT-I have not been found in any other species at the same position except Palaemonetes pugio.1.3 Mar-MT-I as BiomarkerThe biomarker response of Mar-MT-I gene in the hepatopancreas reveals that its expression profile pattern is same under different exposure levels of cadmium,copper and zinc but at the same time unique for each of these metals tested.However,variations occurs in mRNA levels with respect to concentration of dose exposure and the mRNA expression profile pattern resulted in response to low dose is achieved in less time in case of high dose exposure of cadmium and copper.The zinc has reductional effect.The reason for such reduction is probably this MT-I isoform is copper induced and responses to copper and cadmium very well but not to zinc,and the zinc reduces its induction to switch on the induction of another isoform(may be called as zinc induced,MT-Ⅱ). Further,MT-I also expresses normally in ovary,hapatopancreas,gut and thorasic ganglia (level wise from higher to lower,respectively) of M.rosenbergii.However,no expression was found in gills and muscles under normal conditions,and in the gills under heavy metal toxicity too.The quick and remarkable changes in the basal level of MT-I in the hapatopancreas of M.rosenbergii provides an insight into cadmium,copper or zinc pollution.However,this ability of MT-I isoform is functional very well in exposure to single contaminant(either cadmium,copper or zinc) in the environment;and the response is quite different in case of different mixtures of these contaminants.In case of synergistic effects we noted that the presence of Cd(2.5μg/l) with Cu or Zn(each 250μg/l) reduces the induction of MT-I.While the combination of Cu+Zn(250+250μg/l), Cd+Cu+Zn(2.5+250+250μg/l) and Cd+Cu+Zn(2.5+250+250μg/l) along with 28℃heat shock showed nonsignificant effect on basal levels of MT-I mRNA.However,MT-I mRNA level was drastically lowered to almost none in case of higher thermal stress (35℃) for 2 hours of exposure.This reveals a homeostatic response that under high temperature the expression of normally other genes is switched off along with the switching on of heat shock response.However,at this stage we noted a signicicant increase in the heat shock proteins,Hsc70.All such responses of MT-I were found in the hepatopancreas while in the gills no induction was observed in response to any concentration of these metals tested upto 8 hours exposure.To date,according to our knowledge its first report about the lack of an important MT isoform in the gills of crustacean,Macrobrachium rosenbergii.1.4 Hsp70 family as BiomarkerAmong the 70kDa heat shock protein family,the inducible member Hsp70 did not express in both of the tissues,the hepatopancreas and gills in exposure to heavy metals. The failure of its expression is a new finding,but contrary to earlier studies.On the other hand the expression of the constitutive member Hsc70 in the hepatopancreas showed an over all about 20%fluctuation in the basal levels in exposure to cadmium(2.5 & 5μg/l), revealing that up to 5μg/l of cadmium is not too much toxic.While the effects of low and high dose concentrations of copper were quite different from each other.The low dose (250μg/l) significantly increased the expression about 3 folds at 1^st hour while high dose (500μg/l) about two folds at 4^th hour of exposure.This shows that up to 500μg/l of copper is toxic too.On the other hand,the both dose concentrations(250μg/l and 500μg/l) of zinc showed a gradual increase,but not in a time and dose dependent manner.This shows both these doses of zinc are less toxic as compared to that of copper.In case of combined heavy metals Cd with either Cu or Zn or combination of thase three metals plus mild heat shock has significant reductional effect while thermal stress(35℃) alone has significant increasing effect on Hsc70 gene expression.The heavy metals effect on Hsc70 gene expression in the gills revealed 2.5μg/l of cadmium has significant reductional effect for long exposure(8 hr),while 5μg/l of cadmium has insignificant effect for the same exposure duration(as in case of 2.5μg/l). In case of copper exposure,a dose concentration of 250μg/l showed significant increasing effect at 1^st hour while 500μg/l at 4^th hour.However,250μg/l and 500μg/l of zinc showed a significant reductional effect at 4^th and 2^nd hours of exposure,respectively. In combined heavy metals,all the combinatios(Cd+Zn,Cd+Cu,Cu+Zn and Cd+Cu+Zn) a showed significant reductional effect;while the later combination with 3℃thermal stress showed a mild(insignificant) reduction in the expression level.However,thermal stress of 35℃showed significant increasing effect.1.5 Relationship between Mar-MT and Hsc70Our study showed that there is well defined relationship between the expression levels of Hsc70 and Mar-MT-I genes.The point where the expression level of Hsc70 is low,the MT is relatively higher than its basal level and vice versa.However,in case of thermal stress the level of MT-I always goes down,while that of Hsc70 up.Therefore,their application(as biomarker set) in the same package yields better assessment of heavy metal pollution instead of either one as alone.Furthermore,the expression level of Hsc70 is lower in the tissues where MTs are expressed e.g in the hepatopancreas is comperatively lower than the gills.