• Biomedical Science Letters
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• v.7 no.4
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• pp.191-196
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• 2001

To evaluate an effect of cyclohexanone (CHO) treatment on the serum levels of glutathione S-transferase (GST) activity in acute liver damaged animals, acute liver damage was induced in rats with pretreatment of 50% $CCl_4$ in olive oil (0.1 ml/100 g body wt) intraperitoneally 14 times every other day. To liver damaged rats, CHO (1.56 g/kg body wt, i. p.) was injected once and then rats were sacrificed at 4 hours after injection of CHO. Increasing rate of GST activity to the control in serum was higher in CHO-treated rats pretreated with CCL$_4$ than the $CCl_4$-pretreated those. All the more, the injection of CHO to the liver damaged rats led to more enhanced liver damage on the basis of liver functional findings, i. e., serum levels of alanine aminotransferase (ALT) activity, liver weight per body weight, and malondialdehyde content. The changing pattern of serum ALT activity was similar with that of GST activity, whereas that of liver in both enzymes differed more or less from each other; the liver GST activity in CHO-treated rats pretreated with $CCl_4$ being more increased tendency than that of $CCl_4$-pretreated rats. Concomitantly the injection of CHO showed a increasing tendency of liver GST activity compared with the control. Furthermore, CHO injection to the liver damaged rats showed somewhat higher Vmax in the kinetics of liver GST enzymes. In conclusion, injection of CHO to the liver damaged animals led to more increased activity of serum GST, and it may be chiefly caused by the alteration of membrane permeability.

• Bulletin of the Korean Chemical Society
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• v.24 no.8
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• pp.1188-1192
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• 2003

In order to study the role of residue in the active site of glutathione S-transferase (GST), Tyr 108 residue in human GST P1-1 was replaced with alanine, phenylalanine and tryptophan by site-directed mutagenesis to obtain mutants Y108A, Y108F and Y108W. These three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitutions of Tyr108 significantly affected $K_m^{CDNB}$ and $K_m^{ETA}$, whereas scarcely affected $K_m^{GSH}$. The substitutions of Tyr108 also significantly affected $I_{50}$ of ETA, an electrophilic substrate-like compound. The effect of these substitutions on kinetic parameters and the response to inhibition suggests that tyrosine 108 in hGST P1-1 contributes to the binding of the electrophilic substrate and a major determinant in the binding of CDNB is the aromatic ring of Tyr108, not its hydroxyl group.

• BMB Reports
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• v.31 no.4
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• pp.399-404
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• 1998

In order to gain further insight on the relationship between structure and function of glutathione S-transferase (GST), the six active-site mutants, R13T, K44T, Q51A, Q64A, S65A, and D98A, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The active-site mutants showed marked differences in substrate specificity. The substitution of Gln51 with threonine resulted in a drastic decrease in the specific activities to <10% of the wild-type value. The substitution of Arg13 with threonine resulted in more decreased specific activity toward cumene hydroperoxide and in the $I_{50}$ values of S-(2,4-dinitrophenyl) glutathione and benanstatin A. These results suggest that the substitution of Arg13 with threonine changes the conformation of the active site to increase the affinity for the product or electrophilic substrate. Lys44 seems to be in the vicinity of the H-site of hGST P1-1 or may contribute to some extents to the electrophile binding.

• Bulletin of the Korean Chemical Society
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• v.28 no.5
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• pp.772-776
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• 2007

In order to study the role of residue in the active site of glutathione S-transferase (GST), Arg13 residue in human GST P1-1 was replaced with alanine, lysine and leucine by site-directed mutagenesis to obtain mutants R13A, R13K and R13L. These three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. Mutation of Arg13 into Ala caused a substantial reduction of the specific activity by 10-fold. Km GSH, Km DCNB and Km EPNP values of R13A were approximately 2-3 fold larger than those of the wild type. Mutation of Arg13 into Ala also significantly affected I50 values of S-methyl-GSH that compete with GSH and ethacrynic acid, an electrophilic substrate-like compound. These results appeared that the substitution of Arg13 with Ala resulted in significant structural change of the active site. Mutation of Arg13 into Leu reduced the catalytic activity by approximately 2-fold, whereas substitution by Lys scarcely affected the activity, indicating the significance of a positively charged residue at position 13. Therefore, arginine 13 participates in catalytic activity as mainly involved in the construction of the proper electrostatic field and conformation of the active site in human GST P1-1.

• Bulletin of the Korean Chemical Society
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• v.22 no.1
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• pp.77-83
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• 2001

To gain further insight into the relationship between structure and function of glutathione S-transferase (GST), the four cysteine mutants, C14S, C47S, C101S and C169S, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized glutathione (GSH). The catalytic activities of the four mutant enzymes were characterized with five different substrates as well as by their binding to four different inhibitors. Cys14 seems to participate in the catalytic reaction of GST by stabilizing the conformation of the active-site loop, not in the GSH binding directly. The substitution of Cys47 with serine significantly reduces the affinity of GSH binding, although it does not prevent GSH binding. On the other hand, the substitution of Cys101 with serine appears to change the binding affinity of electrophilic substrate by inducing a conformational change of the $\alpha-helix$ D. Cys169 seems to be important for maintaining the stable conformation of the enzyme. In addition, all four cysteine residues are not needed for the steroid isomerase activity of human glutathione S-transferase P1-1.

• Journal of Food Hygiene and Safety
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• v.6 no.1
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• pp.13-26
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• 1991

This study was performed for searching for non-hepatectomy medium-term bioassay model by using newborn female rats. Newborn female Sprague-Dawley rats (1 day old) were given an intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DENA). After three weeks, all rats were weaned and divided into three groups. Group 1 were fed on diets containing 0.01% 2-acetylaminofluorene (2-AAF) as a promoter for three weeks. Group 2 were given 0.05% phenobarbital (PB) in drinking water as a promoter for 8 weeks. Group 3 was control group. The autopsy was carried out at 4 weeks and 8 weeks after weaning. Preneoplastic lesions were indentified with immunohistochemical staining for glutathione S-transferase placental form (GST-P). In liver weight to body weight ratios, group 2 showed significant difference from group 1 (p<0.001) at 4 weeks after weaning. Group 1 and group 2 showed significant difference from group 3 at 8 weeks after weaning (p<0.0I, p<0.001), respectively. In quantitative analysis for GST-P positive lesion area by using Image Analyzer, group 1 and group 2 represented significant difference in comparison with group 3 at early 4 weeks after weaning (p

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.178-185
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• 2008

Avian influenza virus (AIV) is recognized as key to the emergence of pandemic influenza for humans; there are growing concerns that AIV H9N2 may become more efficient to transmit to humans in the near future, since the infection of poultry with AIV H9N2 has been common in recent years. In this study, we aimed to produce antisera recognizing the HA and NA proteins of AIV H9N2. Initially, coding sequences corresponding to the N-terminal regions of the HA and NA proteins of the Korean AIV H9N2 (A/Ck/Kr/MS96/96) isolated from a domestic chicken were amplified from the genomic RNA. Following cloning of the amplified cDNA fragments into pGEX4T-1 vector, two GST-fusion proteins (GST-HAln and GST-NAn) were expressed in E. coli BL21 and purified with glutathione sepharose columns; the recombinant GST-HAln and GST-NAn proteins were both used as immunogens in rabbits. The antigenicity of the rabbit antisera was analyzed by immunoblotting of the cell lysates prepared from AIV H9N2-infected MDCK cells. Overall, the recombinant HAln and NAn proteins fused to the C-terminus of GST and the rabbit antisera raised against the corresponding recombinant proteins would provide a valuable reagent for AIV diagnosis and basic research.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.4
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• pp.1611-1616
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• 2014

Spirogyra neglecta, a freshwater green alga, is a local food in the northern and northeastern parts of Thailand. This investigation explored the anticarcinogenicity of S neglecta and its possible cancer chemopreventive mechanisms in rats divided into 14 groups. Groups 1 and 10 served as positive and negative control groups, respectively. Groups 1-9 were intraperitoneally injected with diethylnitrosamine (DEN) once a week for 3 weeks. Groups 10-14 received normal saline instead. One week after the last DEN injection, groups 2-5 were administered for 9 consecutive weeks various doses of S neglecta extract (SNE) and dried S neglecta (SND), mixed with basal diet. Groups 6-9 and 11-14 similarly were administered various doses of SNE and SND starting from the first week of the experiment. Administration of SNE and SND was not associated with formation of glutathione-Stransferase placental form (GST-P) positive foci in rat liver. SNE and SND during initiation phase significantly reduced the number of GST-P positive foci in rats injected with DEN. The number of GST-P also diminished in groups treated with SNE and SND after injection with DEN, except for the low dose extract group. SNE showed stronger anticarcinogenic potency than SND. Furthermore, SNE also decreased the number of Ki-67 positive cells. However, the numbers of TUNEL-positive cells in the liver of the SNE-treated groups were not statistically different from the controls. The GST activity in 50 mg/kg bw of SNE and 1% of SND groups was significantly increased as compared to the positive control. In conclusion, Spirogyra neglecta (Hassall) K$\ddot{u}$tzing showed cancer chemopreventive properties at the early stages of diethylnitrosamine-induced hepatocarcinogenesis in rats. Possible inhibitory mechanisms include enhancement of the activities of some detoxifying enzymes and/or suppression of precancerous cells.

• Toxicological Research
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• v.27 no.4
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• pp.247-251
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• 2011

To clarify whether inhibitory effect of estrogen on liver tumor is associated with cell proliferation, we investigated its role in diethylnitrosamine (DEN)-induced rat preneoplastic lesions, with time sequenced manners. F344 male rats (n = 90) were divided into three groups at 5 weeks of age. The mini-osmotic pumps providing a continuous infusion of DEN was implanted into the abdominal cavity of each animal in group 1, 2 and 3 at 6 weeks of age. To see the effect of estrogen, pellet containing 1 or 10 ${\mu}g$ of estradiol-3-benzoate (EB) was implanted subcutaneously in the animals of groups 2 or 3, respectively, one week prior to DEN treatment. Ten animals of each group were euthanized at 10, 14 and 18 weeks after DEN treatment. Liver tissues at each time point were fixed in 10% phosphate-buffered formalin and were processed and embedded in paraffin and 5 ${\mu}g$ sections mounted on a silanized slide. Glutathione S-transferase placental form (GST-P) positive foci and 5-bromo-2-deoxyuridine (BrdU) labeling cells were detected at each time point. Area of GST-P positive foci in DEN+EB 1 or 10 ${\mu}g$ group was significantly decreased compared to DEN alone at 14 weeks (p < 0.01 or p < 0.05, respectively) an at 18 weeks (p < 0.05 or p < 0.01, respectively). BrdU index in DEN+EB 1 or 10 ${\mu}g$ groups was significantly decreased compared to DEN alone at 14 weeks and at 18 weeks (p < 0.01). Taken together, we conclude that EB treatment decrease the DEN-induced liver preneoplastic lesions and this may be associated with decrease of cellular proliferation.

• Journal of Nutrition and Health
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• v.38 no.1
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• pp.20-29
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• 2005

This study is conducted to determine the effects of dietary levels of corn and tuna oils on the formation of preneoplastic lesions in die-thylnitrosamine (DEN) induced rat hepatocarcinogenesis. Weanling male Sprague-Dawley rats were fed 2.5, 5, 15, 25% (w/w) corn or tuna oils. Hepatocellular carcinogenesis was induced by DEN (200 mg/kg body weight) and two-thirds partial hepactectomy was carried out 3 weeks later and were sacrificed 8 weeks after DEN initiation. Tuna oil group showed smaller area of placental glutathione S-transferase (GST-P) positive foci than com oil group. Com oil group of 25% (w/w) showed the widest area of GST -P positive foci, and tuna oil group showed significantly smaller area of GST-P positive foci than com oil in 25% (w/w) level but had no differences between oil levels. Thio-barbituric acid reactive substances (TBARS) content was the highest in 25% (w/w) level of tuna oil group fed long chain and highly polyunsaturated fatty acids. Also serum ${\gamma}$ -glutamyltranspeptidase (GGT) activities in 25% level of tuna oil group were significantly higher than by other levels. As oil contents increased, glucose 6-phosphatase (G6Pase) seems to decrease in com oil groups but remained the same in tuna oil groups. Glutathione reductase (GR) activities were significantly higher in tuna oil group, and the higher the level of tuna oil, the higher GR activities. But Cu/Zn superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities didn't seem to be influenced by levels and kind of dietary fats. Therefore, as oil levels increased, com oil rich in n-6 fatty acids promoted carcinogenesis but tuna oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) of n-3 fatty acids suppressed. Although lipid peroxidation products were elevated in 25% (w/w) tuna oil group, GST-P positive foci didn't increase. Therefore pre-neoplastic lesions might be reduced through mediation of a lipid peroxidation process in tuna oil. As fat contents of tuna oil increased, elevated GR activities may give a rise to produce more reduced glutathione in order to protect against free radical attack, and high G6Pase activities remained the same and they contributed to membrane stability. So tuna oil diet seems to protect hepatocarcinogenesis.