hydatidosisis term used to refer to infection with the parasite larva in humans and echinococcosis restricted to infection with the adult stage in carnivorous animals ( 1 ) . recently , the genetics , structure and function of the human cytosolic gst enzyme with emphasis on their roles in the cellular metabolism has been defined ( 2 ) . gst activity was detected in most mammalian tissues , especially in the liver which plays a key role in detoxification . there are different classes of gst isozymes that diverge in their specificity to xenobiotic or endogenous substrates ( 3 ) . gst enzymes involved in the cellular detoxification of a broad range of chemical substrates ( 4 ) . apart from reaction from their endogenous metabolism , gsts of helminth parasite may protect against exogenous xenobiotics as a result of immune effectors mechanisms from the host ( 5 ) . significantly higher activity has been found in intestinal cestodes and digeneas , compared with parasitic nematodes ( 6 ) . gsts activity assay has been demonstrated in the cytosol of protoscolices from sheep hydatid cysts ( 7 ) . diagnosis of hydatid disease is done by a combination of clinical signs , imaging techniques , cyst fluid examination , serological tests and molecular techniques ( 9 ) . the prevalence of liver hydatid cyst was reported 4.7% in people of peru country using recombinant antigen , epc1 glutathione s - transferase [ repc1-gst ] , in western blot technique ( 10 ) . however , the hydatid cyst diagnosis technique is under developing due to specificity and sensitivity problems . in the present study , gst enzyme activity of hydatid cyst protoscolices ( parasite ) , healthy and cystic liver tissues were compared and mentioned gst enzyme importance for diagnostic biomarker in hydatid cyst disease . ten samples of parasites were obtained from 10 liver infected with hydatid cysts of sheep slaughtered at a localabattoir ( karaj , iran ) . parasites samples were washed 3 times with pbs buffer , ph 7.2 , freeze - thawed 3 - 6 times in liquid nitrogen and and water bath 37c respectively and sonicated in a 150w ultrasonic disintegrator,10 sec on and 5 sec off on ice until no intact psc were visible microscopically ( approximately 15 min ) . then resulted suspension was centrifuged ( 10000 g for 30 min at 4c ) and supernatant was stored at -20c ( 11 ) . sheep livers ( 10 health liver samples and 10 infected samples ) were obtained at a local abattoir and washed 3 times with pbs buffer ph 7.2 . then they were homogenized with 3 volumes of homogenizing buffer , pbs ph 6.5 , in a glass homogenizer , so the suspension were centrifuged ( 10000 g for 30 minat 4c ) and supernatant stored at -20c ( 12 ) . the protein concentration in the extract solutions of protoscolices and sheep liver tissues were estimated by the method of bradford using bovine serum albumin as the standard ( 12 ) . gsts activity was assayed spectro photometrically at 25c with reduced glutathione ( gsh ) and 1-chloro-2 , 4-dinitrobenzene ( cdnb ) as substrates . protosolices and liver extract samples were removed from -20c freezer and allowed to thaw on ice . cdnb 100 mm from 4c and gsh 100 mm from -20c freezer were removed and allowed to thaw at room temperature , when thawed , incubated at 30c in water bath . for each assay was prepared one ml of assay cocktail ( 980lpbs ph 6.5 , 10l of 100 mm cdnb and 10 l of 100 mm gsh ) , then removed 100 l of cocktail and its remaining placed 900 l of it into 1.5 ml cuvette . to zero spectrophotometer , was used 1 ml of distilled water and to the blank cuveet added 100l pbs to 900 l of cocktail and measured absorbance at 340 nm , every1 minute , for 3 min . to the test cuvette was added 100 l of sample to 900 l cocktail , mixed and measured absorbance at 340 nm as above ( 13 ) . sds - page and coomassie blue staining were used to separate and stain the protein components of samples respectively . molecular weights of sample proteins were compared with respect to the protein marker ( 12 ) . to detect the molecular weight , rf(ratio factor ) of ladder bands was calculated , standard curve was prepared in excel software and finally proteins mw were determined . independent t - test was performed to compare the mean values of protein concentration and enzyme activity between healthy and cystic liver tissues or parasite and liver tissues . ten samples of parasites were obtained from 10 liver infected with hydatid cysts of sheep slaughtered at a localabattoir ( karaj , iran ) . parasites samples were washed 3 times with pbs buffer , ph 7.2 , freeze - thawed 3 - 6 times in liquid nitrogen and and water bath 37c respectively and sonicated in a 150w ultrasonic disintegrator,10 sec on and 5 sec off on ice until no intact psc were visible microscopically ( approximately 15 min ) . then resulted suspension was centrifuged ( 10000 g for 30 min at 4c ) and supernatant was stored at -20c ( 11 ) . sheep livers ( 10 health liver samples and 10 infected samples ) were obtained at a local abattoir and washed 3 times with pbs buffer ph 7.2 . then they were homogenized with 3 volumes of homogenizing buffer , pbs ph 6.5 , in a glass homogenizer , so the suspension were centrifuged ( 10000 g for 30 minat 4c ) and supernatant stored at -20c ( 12 ) . the protein concentration in the extract solutions of protoscolices and sheep liver tissues were estimated by the method of bradford using bovine serum albumin as the standard ( 12 ) . gsts activity was assayed spectro photometrically at 25c with reduced glutathione ( gsh ) and 1-chloro-2 , 4-dinitrobenzene ( cdnb ) as substrates . protosolices and liver extract samples were removed from -20c freezer and allowed to thaw on ice . cdnb 100 mm from 4c and gsh 100 mm from -20c freezer were removed and allowed to thaw at room temperature , when thawed , incubated at 30c in water bath . for each assay was prepared one ml of assay cocktail ( 980lpbs ph 6.5 , 10l of 100 mm cdnb and 10 l of 100 mm gsh ) , then removed 100 l of cocktail and its remaining placed 900 l of it into 1.5 ml cuvette . to zero spectrophotometer , was used 1 ml of distilled water and to the blank cuveet added 100l pbs to 900 l of cocktail and measured absorbance at 340 nm , every1 minute , for 3 min . to the test cuvette was added 100 l of sample to 900 l cocktail , mixed and measured absorbance at 340 nm as above ( 13 ) . sds - page and coomassie blue staining were used to separate and stain the protein components of samples respectively . molecular weights of sample proteins were compared with respect to the protein marker ( 12 ) . to detect the molecular weight , rf(ratio factor ) of ladder bands was calculated , standard curve was prepared in excel software and finally proteins mw were determined . independent t - test was performed to compare the mean values of protein concentration and enzyme activity between healthy and cystic liver tissues or parasite and liver tissues . the mean values of protein concentrations and enzyme activities for parasite , healthy and infected liver samples are presented in table 1 . protein concentration of healthy liver was higher than infected liver ( p < 0.05 ) . significant higher gst specific activities in cystic liver samples was observed as compared with healthy liver ( p < 0.05 ) . statistical t - test showed gst enzyme activity of parasite was lower than healthy liver tissues ( p < 0.05 ) . extract samples of protoscolices , healthy and cystic liver tissues were analyzed by sds - page electrophoresis and the results are shown in fig . 1 . sds - page gel shows gst protein bands with 24kda in parasite and 25kda in healthy liver samples . similar cross protein bands is observing in parasite and liver sample but has been not recorded by databse . the mean values of protein amounts and gsts activity for parasite , healthy and cystic liver samples molecular weight of proteins ( kda ) from the extracts of parasite and liver tissues in sds - page . lanes 1 - 3 are healthy liver samples . lanes 4 - 6 are cystic liver samples . lanes 7 - 9 are parasite samples ( protein marker , peqgold i , lot - no . 64072 ) identified proteins of parasite according to molecular weight by using protein database(http://web.expasy.org ) identified proteins of sheep liver tissue according to molecular weight by using protein database(http://web.expasy.org ) the mean values of protein concentrations and enzyme activities for parasite , healthy and infected liver samples are presented in table 1 . protein concentration of healthy liver was higher than infected liver ( p < 0.05 ) . significant higher gst specific activities in cystic liver samples was observed as compared with healthy liver ( p < 0.05 ) . statistical t - test showed gst enzyme activity of parasite was lower than healthy liver tissues ( p < 0.05 ) . extract samples of protoscolices , healthy and cystic liver tissues were analyzed by sds - page electrophoresis and the results are shown in fig . 1 . sds - page gel shows gst protein bands with 24kda in parasite and 25kda in healthy liver samples . similar cross protein bands is observing in parasite and liver sample but has been not recorded by databse . the mean values of protein amounts and gsts activity for parasite , healthy and cystic liver samples molecular weight of proteins ( kda ) from the extracts of parasite and liver tissues in sds - page . lanes 7 - 9 are parasite samples ( protein marker , peqgold i , lot - no . 64072 ) identified proteins of parasite according to molecular weight by using protein database(http://web.expasy.org ) identified proteins of sheep liver tissue according to molecular weight by using protein database(http://web.expasy.org ) function of hepatocytesmay be disturbed in the presence of infections ( 15 ) . in our study the reduction of protein synthesis as a result of hydatid cyst causes to decrease protein concentration . gsts can make up to 10% of cytosolic protein in some mammalian organs ( 16 ) . hepatic cells contain high levels of gst enzyme which has been found to be an indicator of hepatocyte injury in transplantation , toxicity and infections ( 17 ) . the hydatid cyst infection stimulates oxidative stress and toxin production in hepatic cells . from the point of biochemical defense view gst enzyme be able to neutralization of these toxins , therefore we expect to increase activity level of this enzyme . increase of liver protein and gst in infected mice indicates the occurrence of oxidative stress in hepatocytes due to infection ( 18 ) . serum gsta is a more sensitive marker than transaminases ( alanine aminotransferase , asparatate aminotransferase and alkaline transferase ) for monitoring and as an early analyst of hepatic damage ( 19 ) . therefore gst activity difference between healthy and infected host liver tissue could be concerned for hydatid cyst diagnosis . however , other infections cause to hepatocyte damage , thus gst elevation must be evaluated with other clinical and paraclinical parameters of hydatid cyst disease . mammalian cytosolic gsts are dimeric , with both subunits being from the same class of gsts , although not necessarily identical . gst enzyme molecular weight of parasite is reported 24 - 27 kda ( 13 ) . in this research protein bands with 24kda and 25kda were found in parasite and liver tissues respectively . gst activity in cystic liver tissue could be concerned as a biomarker for hydatid cyst diagnosis with other parameters of hydatidosis .
abstractbackgroundthe aim of this study was to detect the glutathione s - transferase(gst ) enzyme activity of healthy / cystic liver as a diagnostic biomarker for hydatidosis . in order to compare with liver tissue , 
 the level of the gsts enzyme activity of parasite was also determined.methodsparasites were collected from sheep liver tissue with hydatid cysts at a local abattoir and washed with pbs buffer . 
 collected parasites and liver tissues were sonicated or homogenized respectively . 
 extract solution samples were centrifuged and stored at - 20c . 
 gst enzyme activities were measured in the extract of parasite and liver tissue samples ( healthy and infected livers ) . 
 protein amounts and protein bands were detected using bradford and sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) methods respectively . to determine significant difference between two groups , two - sample t - test was performed.resultsgst specific activities of healthy / infected livers and parasites were estimated 304 , 1297 and 146 u / ml / mg respectively . 
 significant higher gst specific activities in cystic liver than healthy liver was observed ( p < 0.05 ) . 
 t - test analysis showed gst activity of parasite was lower than healthy liver tissue . 
 sds - page showed gst protein bands with 24 kda in parasite samples and25 kda in liver tissues.conclusiongst activity incystic liver tissue could be concerned as a biomarker for hydatid cyst diagnosis with other hydatid disease parameters .