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S Awasthi

Publications and source records attributed to S Awasthi.

At least 55 records · Page 3Linked to original sources
PubMed1999

Induction of glutathione S-transferase hGST 5.8 is an early response to oxidative stress in RPE cells.

PURPOSE: To delineate the role of the glutathione S-transferase (GST) isozyme hGST 5.8 in protection mechanisms against oxidative stress, the effect of low-level transient exposure of H2O2 to retinal pigmented epithelial (RPE) cells on hGST 5.8 and other enzymes involved in defense against oxidative stress was examined. METHODS: Cultured human RPE cells were exposed to 50 microM H2O2 for 20 minutes. Subsequently, the cells were washed and resuspended in the culture media. The cells were pelleted and lysed, and the levels of lipid peroxidation products including thiobarbituric acid-reactive substances (TBARS), glutathione (GSH), glutathione peroxidase (GPX), glucose 6-phosphate dehydrogenase, glutathione reductase, GST, catalase (CAT), and superoxide dismutase (SOD) were determined and compared with levels in control cells. Total GSTs were purified by GSH-affinity chromatography, and the isozymes were separated by isoelectric focusing, characterized, and quantitated. hGST 5.8 was quantitated by an immunologic method as well as by determining activity toward its preferred substrate, 4-hydroxynonenal (4-HNE). Kinetic constants of hGST 5.8 purified from H2O2-treated cells were also determined and compared with those of control cells. RESULTS: Exposure of RPE cells to 50 microM H2O2 for 20 minutes showed a significant increase in TBARS (1.8-fold) and gamma-glutamyl cysteine synthetase (gamma-GCS) activity (1.6-fold). A significant increase (1.2-fold) was also observed in GPX activity toward cumene hydroperoxide, but CAT and SOD activities remained unchanged. There was no significant increase in GST activity toward 1-chloro-2, 4-dinitrobenzene but GST activity toward 4-HNE was increased by 1.4- to 1.8-fold. The increase in GST activity toward 4-HNE was associated with a 2.8-fold increase in protein of the isozyme hGST 5.8, which uses 4-HNE as the preferred substrate. CONCLUSIONS: Results of these studies show that the induction of hGST 5.8, which is involved in the detoxification of the lipid peroxidation products 4-HNE and hydroperoxides, may be an early adaptive response of RPE cells exposed to low levels of transient oxidative stress. It is suggested that this isozyme may be crucial for protecting the RPE from low levels of chronic oxidative stress. Observed increases in GPX and gamma-GCS activities are consistent with this idea, because GPX activity is also expressed by hGST 5.8, and gamma-GCS is the rate-limiting enzyme in biosynthesis of GSH, the substrate for hGST 5.8.

Benzene Derivatives↗
PubMed1999

Effectiveness of BCG vaccination against tuberculous meningitis.

OBJECTIVE: To assess the protective effectiveness of BCG vaccination against tuberculous meningitis, while controlling for age, nutrition and socio-economic status, in children 1 month to 12 years of age. DESIGN: Case-control study. SETTING: Secondary care referral and teaching hospital. METHODS: Cases were those conforming to the definition of tuberculous meningitis and controls were patients admitted after every third consecutive case included in the study from September 1995 till the end of August 1997 and who did not suffer from any central nervous system disorder. RESULTS: Among the 192 cases and 70 controls, BCG scar was present in 57.8% and 75.7%, respectively. The crude odd's ratio (OR)for tuberculosis meningitis with a BCG scar was 0.44 (95% CI, .24-0.81; p = 0.008), while the adjusted OR was 0.53 (95% CI, 0.26-1.06; p value = 0.07) after controlling for weight, age, sex and place of residence. Higher weight for age and urban residence were associated with a decreased risk of tuberculous meningitis in the logistic model. CONCLUSIONS: BCG vaccination offers protection against tuberculous meningitis. Since improvement in weight for age was associated with a decreased risk of disease, further studies are needed to evaluate the association, if any, between nutritional status and vaccine efficacy.

Age Distribution↗
PubMed1998

Gender-related differences in susceptibility of A/J mouse to benzo[a]pyrene-induced pulmonary and forestomach tumorigenesis.

Benzo[a]pyrene (BP) is a suspected human carcinogen and is known to produce tumors in the lung and forestomach of mice. Glutathione (GSH) S-transferases (GST) play a major role in the detoxification of the ultimate carcinogen of BP, (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+)-anti-BPDE). Previous studies have shown gender-related differences in the expression of GST isoenzymes in mice. The present study was designed to test the hypothesis whether gender-related differences in the expression of GST isoenzymes can affect the susceptibility of mice to BP-induced lung and forestomach tumorigenesis. The expression of pi class isoenzyme mGSTP1-1, which is highly efficient in the detoxification of (+)-anti-BPDE, was approximately 3.0- and 1.5-fold higher in the liver and forestomach of male A/J mouse, respectively, as compared with the female. The levels of other major GST isoenzymes, mGSTA3-3 (alpha class), mGSTM1-1 (mu class) and mGSTA4-4 (alpha class), were also significantly higher in the liver of the male mouse as compared with the female. While pulmonary mGSTP1-1 expression did not differ significantly between male and female A/J mice, the expression of mGSTA3-3, mGSTM1-1 and mGSTA4-4 was significantly higher (1.4-4.0-fold) in the lung of the male A/J mouse as compared with the female. At lower concentrations of BP (0.5 mg BP/mouse), the tumor incidence/multiplicity was significantly higher in the lung as well as in the forestomach of female mice as compared with male mice. For example, while 30% of the female mice developed pulmonary tumors 26 weeks after the first 0.5 mg BP administration, none of the male mice had tumors in their lungs. At higher doses of BP (1.5 mg BP/mouse), however, this differential was either abolished or relatively less pronounced. Our results suggest that up to a certain threshold of BP exposure the levels of GST isoenzymes may be an important determinant of susceptibility to BP-induced tumorigenesis in mice.

Animals↗
PubMed1998

ATP-dependent transport of glutathione conjugate of 7beta, 8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles.

Glutathione (GSH) S-transferases (GSTs) have an important role in the detoxification of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9, 10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogen of benzo[a]pyrene. However, the fate and/or biological activity of the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is not known. We now report that (-)-anti-BPD-SG is a competitive inhibitor (Ki 19 microM) of Pi-class isoenzyme mGSTP1-1, which among murine hepatic GSTs is most efficient in the GSH conjugation of (+)-anti-BPDE. Thus the inhibition of mGSTP1-1 activity by (-)-anti-BPD-SG might interfere with the GST-catalysed GSH conjugation of (+)-anti-BPDE unless one or more mechanisms exist for the removal of the conjugate. The results of the present study indicate that (-)-anti-BPD-SG is transported across canalicular liver plasma membrane (cLPM) in an ATP-dependent manner. The ATP-dependent transport of (-)-anti-[3H]BPD-SG followed Michaelis-Menten kinetics (Km 46 microM). The ATP dependence of the (-)-anti-BPD-SG transport was confirmed by measuring the stimulation of ATP hydrolysis (ATPase activity) by the conjugate in the presence of cLPM protein, which also followed Michaelis-Menten kinetics. In contrast, a kinetic analysis of ATP-dependent uptake of the model conjugate S-[3H](2,4-dinitrophenyl)-glutathione ([3H]DNP-SG) revealed the presence of a high-affinity and a low-affinity transport system in mouse cLPM, with apparent Km values of 18 and 500 microM respectively. The ATP-dependent transport of (-)-anti-BPD-SG was inhibited competitively by DNP-SG (Ki 1.65 microM). Likewise, (-)-anti-BPD-SG was found to be a potent competitive inhibitor of the high-affinity component of DNP-SG transport (Ki 6.3 microM). Our results suggest that GST-catalysed conjugation of (+)-anti-BPDE with GSH, coupled with ATP-dependent transport of the resultant conjugate across cLPM, might be the ultimate detoxification pathway for this carcinogen.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1998

ATP-Dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity.

Dinitrophenyl S-glutathione (DNP-SG) ATPase is a 38 kDa membrane protein expressed in erythrocytes and other tissues. Although stimulation of ATP hydrolysis catalyzed by DNP-SG ATPase has been demonstrated in the presence of several structurally unrelated amphiphilic ions, structural and functional properties of this protein have not been well-defined. In the present study, we have developed an improved protocol for the purification of DNP-SG ATPase and investigated its kinetic and substrate-binding properties. The purification procedure was based on highly specific elution of the 38 kDa protein from DNP-SG affinity resin in the presence of ATP. The protein could not be eluted using either ADP or adenosine-5'-[beta,gamma-methylene]triphosphate (methylene-ATP), a nonhydrolyzable analogue of ATP. Doxorubicin (DOX), a weakly basic anthracycline chemotherapy agent, was found to be the preferred activator for stimulation of ATP hydrolysis by the enzyme. ATP binding to the enzyme was demonstrated using 8-azido-ATP photoaffinity labeling and binding of trinitrophenyl (TNP)-ATP, a fluorescent analogue of ATP. The photoaffinity labeling of DNP-SG ATPase (38 kDa) was saturable with respect to 8-azido ATP (Kd = 2 microM), indicating that the enzyme was capable of specific and saturable binding to ATP. DNP-SG binding was evident from the purification procedure itself and was also demonstrable by quenching of tryptophan fluorescence. Results of quenching of tryptophan fluorescence as well as radioactive isotope-binding studies indicated that DOX was bound to the purified protein as well.

Adenosine Triphosphatases↗
PubMed1998

ATP-Dependent human erythrocyte glutathione-conjugate transporter. II. Functional reconstitution of transport activity.

Purified dinitrophenyl S-glutathione (DNP-SG) ATPase was reconstituted into artificial liposomes prepared from soybean asolectin. Electron micrography confirmed the formation of unilamellar vesicles with an average radius of 0.25 micron. Intravesicular volume estimated by incorporation of radiolabled inulin into the vesicles was found to be 19.7 +/- 1.3 microL/mL reconstitution solution. Accumulation of the glutathione-conjugate of CDNB, DNP-SG, and of doxorubicin (DOX) in the proteoliposomes was increased in the presence of ATP as compared to equimolar ADP or adenosine 5'-[beta,gamma-methylene]triphosphate tetralithium. ATP-dependent transmembrane movement of DOX and DNP-SG into DNP-SG ATPase-reconstituted vesicles was saturable with respect to time, sensitive to the osmolarity of the assay medium, and temperature dependent. The energy of activation was found to be 12 and 15 kcal/mol for DNP-SG and DOX, respectively. Optimal temperature for transport was 37 degrees C. Saturable transport was demonstrated for DNP-SG (Vmax of 433 +/- 20 nmol/min/mg of protein, KmATP = 2.4 +/- 0. 3 mM and KmDNP-SG = 36 +/- 5 microM) as well as DOX (Vmax = 194 +/- 19 nmol/min/mg of protein, KmATP = 2.5 +/- 0.6 mM and KmDOX = 2.4 +/- 0.7 microM). The kinetic data for both DNP-SG and DOX transport were consistent with a random bi-bi sequential reaction mechanism. DOX was found to be a competitive inhibitor of DNP-SG transport with Kis of 1.2 +/- 0.2 microM and DNP-SG was found to be a competitive inhibitor of DOX transport with Kis of 13.3 +/- 2.6 microM.

Adenosine Triphosphatases↗
PubMed1998

Protein oxidation biomarkers in hyperoxic lung injury in rats: effects of U-74389.

Hyperoxia, used therapeutically in the treatment of respiratory insufficiencies, can cause lung injury, probably through the actions of reactive oxygen species. The present studies were designed to test the hypothesis that oxidation of specific proteins would provide useful biomarkers of the onset of tissue injury, and thereby provide clues as to the mechanisms responsible. We exposed adult male Sprague-Dawley rats to room air or to greater than 95% O2 for 60 h and examined proteins in pleural effusion and broncho-alveolar lavage (BAL) fluids, and in lung tissue homogenates and subfractions. Oxidation of protein thiols was assessed by derivatization with monobromobimane, separation by electrophoresis, and visualization of the fluorescent thioether derivatives. Derivatization with 2,4-dinitrophenylhydrazine (DNPH), electrophoresis, and western analysis was employed to assess a different class of oxidative modifications, frequently termed 'protein carbonyls'. In addition, we investigated the effects of the 21-aminosteroid U-74389G, 10 mg/kg, given intraperitoneally every 12 h, on biomarkers of protein oxidation and on manifestations of lung injury. Hyperoxia caused lung injury evidenced by pleural effusions, increases in BAL protein concentrations, and pulmonary edema; U-74389G attenuated the first two indices of lung injury, but did not alter edema. Protein thiol status of the fractions studied were not affected notably by hyperoxia, or by the aminosteroid. The formation of DNPH-reactive sites on a limited number of proteins by hyperoxia was observed, and some of these effects were attenuated in the animals given U-74389G. Histological examination of lung tissues showed accumulation of intra-alveolar protein exudates in hyperoxic rats, and a significant attenuation of this effect was observed in the animals treated with U-74389G. In conclusion, studies of shifts in protein thiol status that may be caused by hyperoxia will require increasingly specific methods of analysis, and characterization of the specific DNPH-reactive proteins formed in hyperoxia may provide critical insights into the mechanisms of lung injury. Administration of U-74389G offers some degree of protection against hyperoxia and attenuation of these biomarkers of oxidation, but the precise mechanisms by which this protection is effected will require additional study.

Animals↗
PubMed1998

Allylamine and beta-aminopropionitrile-induced vascular injury: enhanced expression of high-molecular-weight proteins.

In the present study we describe changes in aorta at the protein level associated with allylamine (AA) and beta-aminopropionitrile (beta APN) induced vascular toxicity in a rat model. This model represents a remarkable synergistic, necrotizing toxic effect of these combined toxins, and our rationale was to examine protein expression in order to shed light on the mechanisms underlying this synergism. Rats were given AA (100 mg/kg body weight/day) and beta APN (1 g/kg body weight/day) by gavage for 10 d; this protocol has been shown to result in smooth-muscle necrosis, but no visible connective tissue changes. Soluble and insoluble fractions from AA + beta APN- or from beta APN-treated aorta showed enhanced expression of three high-molecular-weight protein bands (ranges between approximately 120 and 95 kD). The time course of induction of proteins showed the appearance of AA + beta APN-induced specific proteins at d 3 of AA + beta APN treatment. Partial purification and characterization suggested that AA + beta APN specific proteins are likely to be collagen proteins (type I). Thus, the data presented in this article help in understanding the vascular toxicity induced by AA + beta APN or by beta APN, in that we have described an altered phenotypic expression of collagenous proteins indicative of selective medial vascular toxicity.

Administration, Oral↗
PubMed1998

Glutathione S-transferases of rabbit lung macrophages.

The catalytic activities of glutathione S-transferases (GSTs), particularly the alpha-class isozymes, can provide protection against oxidative stress through GSH-mediated metabolism of reactive products of lipid peroxidation. Lipid peroxidation products from oxidative metabolism in alveolar macrophages play an important role in mediating and regulating inflammatory response and injury in the lung. The rabbit has been used as an important animal model for studies of the role of alveolar macrophages in pulmonary pathology. Although rabbit lung macrophages display GST activity, the isozyme-specific expression of GSTs and the catalytic properties of these isozymes has not previously been defined. In present studies, we have purified the GST isozymes of rabbit alveolar macrophages obtained by bronchoalveolar lavage and performed immunologic and kinetic characterization of the purified isozymes. Results of our studies indicate the presence of three alpha-class isozymes (pI 10.2, 9.3, and 6.0) and one micro-class isozyme (pI 7.2). N-terminal sequence analysis of the micro-class isozyme indicated that it was distinct from the two previously described micro-class isozymes of rabbit. Kinetic studies indicated that two cationic alpha-class GSTs (pI 10.2 and 9.3) contribute the large majority of selenium independent GSH-peroxidase activity toward dilinoleoyl phosphatidylcholine hydroperoxide (kcat/Km values of 83.4 and 31.9 s-1 . M-1 . 10(3), respectively). A third alpha-class GST (pI 6.0) was shown to have highest catalytic activity toward conjugation of the 4-hydroxynonenal (4HNE) with GSH (kcat/Km = 1900 s-1 . M-1 . 10(3)). Structural and immunologic characterization of this GST isozyme indicated that it belongs to a subclass of the alpha-classGSTs selectively expressed in mesodermal origin cells that are exposed to high levels of oxidative stress and are characterized by high specific activity toward both lipid hydroperoxides and 4-HNE.

Aldehydes↗
PubMed1998

The role of glutathione S-transferases as a defense against reactive electrophiles in the blood vessel wall.

The glutathione transferases (GSTs) are a family of ubiquitous enzymes that catalyze the conjugation of reduced glutathione (GSH) with reactive electrophiles. Rat vascular tissue contains GST isoforms that represent a major cellular defense mechanism against atherogenic alpha,beta-unsaturated aldehydes (Misra et al., Toxicol. Appl. Pharmacol. 133, 27-33, 1995). In this study we examined the role of GSTs in providing protection to cultured neonatal vascular smooth muscle cells (VSMCs) from the alpha,beta-unsaturated carbonyl cardiovascular toxins, allylamine and its metabolite, acrolein. Confluent cultured cells were exposed to 2 to 10 microM allylamine (a cardiovascular toxin that is metabolized in vivo and in vitro by VSMCs to the reactive aldehyde, acrolein) or to acrolein (2-10 microM) for 48 h; dose-cytotoxicity curves were generated utilizing a tetrazolium-dependent cytotoxicity assay. Concommittant treatment with sulfasalazine, an established inhibitor of GST, was found to markedly increase allylamine- or acrolein-induced cytotoxicity, decreasing the LC50 by two- to threefold at 50 to 100 microM sulfasalazine. A clonogenic survival assay in VSMCs exposed to these compounds for 4 h confirmed lethal toxicity and enhanced toxicity following cotreatment with sulfasalazine. Isobologram analysis (which statistically defines the limits of additivity of two independent treatments) showed that the sulfasalazine effect on both allylamine and acrolein cytotoxicity was supraadditive, or synergistic. Sulfasalazine was not cytotoxic to VSMCs in the range of concentrations that augmented acrolein or allylamine cytoxicity; total GST activity was inhibited, however, in a dose-dependent manner in that range. GST purified by GSH-affinity chromatography from pelleted untreated cells gave specific activities and kinetic constants consistent with those previously reported for rat aorta total GSTs. The catalytic efficiency (Kcat/Vm) was found to be much greater for 4-hydroxy-2-nonenal than for 1-chloro-2,4-dinitrobenzene (0.058 vs 0.4 s-1 mM-1). Western blot of purified total GSTs using antibodies against rec-mGSTA4-4 revealed a single band at 25 kDa, confirming the presence of a GST isozyme immunologically similar to rat GST8-8, which is known to utilize alpha,beta-unsaturated carbonyls as preferred substrates. Our data indicate that GSTs are an important defense in the vascular media, protecting blood vessels against alpha,beta-unsaturated carbonyl cardiovascular toxins that are involved in initiating atherosclerotic lesions.

Acrolein↗
PubMed1998

Mechanisms of anticarcinogenic properties of curcumin: the effect of curcumin on glutathione linked detoxification enzymes in rat liver.

Curcumin, an antioxidant isolated from turmeric (curcuma longa), has been shown to attenuate chemical carcinogenesis in rodents. Previous studies have shown that curcumin causes an increase in glutathione S-transferase (GST) activity in rodent liver which may contribute to its anti-cancer and anti-inflammatory activities. Since the effects of curcumin on specific GST isozymes and other glutathione (GSH)-linked enzymes are incompletely defined, we have examined in the present studies the effect of curcumin on hepatic non-protein sulfhydryls and GSH-linked enzymes in male Sprague-Dawley rats. When rats were fed curcumin at doses from 1 to 500 mg kg-1 body weight daily for 14 days, the induction of hepatic GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was found to be biphasic, with maximal induction of about 1.5 fold at the 25 to 50 mg kg-1 body weight dosage. At higher doses, a decrease was observed in the activity and in the rats treated with 500 mg kg-1 curcumin this activity was below the levels observed in controls. In contrast, GST activity towards 4-hydroxynonenal (4-HNE) increased in a saturable, dose dependent manner. Western-blot analyses of liver cytosols revealed that curcumin caused a dose dependent induction of rGST 8-8, an isozyme which is known to display the highest activity towards 4-HNE, a highly toxic product of lipid peroxidation. Glutathione peroxidase (GPx) activity towards cumene hydroperoxide in liver homogenate was also found to be increased in a saturable manner with respect to curcumin dose. Our results suggest that induction of enzymes involved in the detoxification of the electrophilic products of lipid peroxidation may contribute to the anti-inflammatory and anti-cancer activities of curcumin.

Animals↗
PubMed1998

Cause-specific mortality in under fives in the urban slums of Lucknow, north India.

We recorded the causes of death in the preceding 3 years in a slum population of 24,196 children less than 5 years of age in Lucknow, North India. Of 1469 deaths recorded, 298 were stillbirths. For each death, a 'verbal autopsy' was conducted by interviewing the parents to ascertain the cause of death. 71.8 per cent of deaths were at home; 94.5 per cent of hospital deaths had no death certificate. Excluding stillbirths, 69.9 per cent of deaths were in the first year of life. Leading causes of death in the neonatal period were prematurity (38.5 per cent) and tetanus (36.4 per cent). Beyond the neonatal period, the leading causes of death were pneumonia (23.4 per cent), diarrhoeal disease (20.9 per cent), and malnutrition and/or anaemia (11.4 per cent). The existent mortality-recording system was under-reporting at least one third of the deaths. We conclude that stillbirths and neonatal mortality can be reduced by improved antenatal and natal care. In the postnatal period, since most of the mortality is due either to infective diseases or malnutrition, interventions to improve the nutritional status of preschool children can reduce this.

Age Distribution↗
PubMed1998

Family expenditure on sickness episodes of pre-school children in urban slums of Lucknow, north India.

This study investigated family expenditure on the sickness episodes of pre-school children in 32 urban slums of Lucknow, North India. Between July 1995 and January 1996, 396 children between the ages of 1.5 to 3.5 years were contacted. The main objective was to discover the direct medical cost of illness incurred by their families in the preceding months. Point prevalence of major morbidities, in the preceding month, were as follows: 17.2% respiratory and 6.3% diarrhoeal. A health care provider saw 31.4% [95% confidence interval (CI): 26.9-36.01] children. In > 95% of the cases, the health care provider was a self-employed, non-governmental dispenser (NGD). More than half of NGDs did not have a recognized medical degree. The mean family expenditure on sickness in 1 month was RS 12.27 [standard deviation (SD): RS 23.81], approximately 1% of their income (1 US $ = RS 32). Respiratory illness was the primary cause of financial burden to the community. The isolated symptoms of fever was the most expensive to treat, with diarrhoea and dysentery costs being similar. Immunizing a child saved money. We conclude that since the NGDs are the predominant health care providers in the urban slums, and since neither the nature of medicines dispensed by them nor their additional costs can be ascertained, steps must be taken to monitor their dispensing practices and to educate them about rational drug therapy.

Analysis of Variance↗
PubMed1997

Induction of glutathione S-transferase pi as a bioassay for the evaluation of potency of inhibitors of benzo(a)pyrene-induced cancer in a murine model.

There is a growing need for short-term and cost-effective bioassay to assess the efficacy of potential chemo-preventive agents. We report that the induction of glutathione (GSH) S-transferase pi (mGSTP1-1) by a chemo-preventive agent can be used as a reliable marker to assess its efficacy in retarding chemical carcinogenesis induced by benzo(a)pyrene (BP), which is a widespread environmental pollutant and believed to be a risk factor in human chemical carcinogenesis. This conclusion is based on 1) the relative contribution of mGSTP1-1 of the liver and forestomach of female A/J mice in the detoxification of the ultimate carcinogenic metabolite of BP, (+)-anti-7,8-dihydroxy-9, 10-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene [(+)-anti-BPDE]; and 2) a positive correlation between the induction of hepatic and forestomach mGSTP1-1 by 5 naturally occurring organosulfides (OSCs) from garlic (diallyl sulfide, diallyl disulfide, diallyl trisulfide, dipropyl sulfide and dipropyl disulfide) and their effectiveness in preventing BP-induced forestomach neoplasia in mice. In the liver, the combined contribution of other GSTs in the detoxification of (+)-anti-BPDE was far less than the contribution of mGSTP1-1 alone. Likewise, in the forestomach, the contribution of mGSTP1-1 far exceeded the combined contribution of other GSTs. Studies on the effects of OSCs against BP-induced forestomach neoplasia revealed a good correlation between their chemo-preventive efficacy and their ability to induce mGSTP1-1 expression in the liver (r = -0.89; p < 0.05) as well as in the forestomach (r = -0.97; p < 0.05). Our results suggest that the induction of mGSTP1-1 may be a reliable marker for evaluating the efficacy of potential inhibitors of BP-induced cancer in a murine model.

Allyl Compounds↗
PubMed1997

Mechanism of differential catalytic efficiency of two polymorphic forms of human glutathione S-transferase P1-1 in the glutathione conjugation of carcinogenic diol epoxide of chrysene.

The kinetics of the conjugation of glutathione (GSH) with anti-1, 2-dihydroxy-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-CDE), the activated form of the widespread environmental pollutant chrysene, catalyzed by two naturally occurring polymorphic forms of the pi class human GSH S-transferase (hGSTP1-1), has been investigated. The polymorphic forms of hGSTP1-1, which differ in their primary structure by a single amino acid in position 104, exhibited preference for the GSH conjugation of (+)-anti-CDE, which is a far more potent carcinogen than (-)-anti-CDE. When concentration of anti-CDE was varied (5-200 microM and the GSH concentration was kept constant at 2 mM, both hGSTP1-1(I104) and hGSTP1-1(V104) obeyed Michaelis-Menten kinetics. However, the Vmax of GSH conjugation of anti-CDE was approximately 5.3-fold higher for the V104 variant than for the I104 form. Calculation of catalytic efficiency (kcat/Km) thus resulted in a value for hGSTP1-1(V104), 28 mM-1 s-1, that was 7.0-fold higher than that for hGSTP1-1(I104), 4 mM-1 s-1. The mechanism of the differences in the kinetic properties of hGSTP1-1 isoforms toward anti-CDE was investigated by molecular modeling of the two proteins with GSH conjugation products in their active sites. These studies revealed that the enantioselectivity of hGSTP1-1 for (+)-anti-CDE and the differential catalytic efficiencies of the V104 and I104 forms of hGSTP1-1 in the GSH conjugation of (+)-anti-CDE were due to the differences in the active-site architecture of the two proteins. The results of the present study, for the first time, provide evidence for the toxicological relevance of GSTP1-1 polymorphism in humans and suggest that the population polymorphism of hGSTP1-1 variants with disparate enzyme activities may, at least in part, account for the differential susceptibility of individuals to environmental carcinogens such as anti-CDE and possibly other similar carcinogens.

Animals↗
PubMed1997

Active site architecture of polymorphic forms of human glutathione S-transferase P1-1 accounts for their enantioselectivity and disparate activity in the glutathione conjugation of 7beta,8alpha-dihydroxy-9alpha,10alpha-ox y-7,8,9,10-tetrahydrobenzo(a)pyrene.

In this study, we demonstrate that the active site architecture of the human glutathione (GSH) S-transferase Pi (GSTP1-1) accounts for its enantioselectivity in the GSH conjugation of 7beta,8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9, 10-tetrahydrobenzo(a) pyrene (anti-BPDE), the ultimate carcinogen of benzo(a)pyrene. Furthermore, we report that the two polymorphic forms of human GSTP1-1, differing in their primary structure by a single amino acid in position 104, have disparate activity toward (+)-anti-BPDE, which can also be rationalized in terms of their active site structures. When concentration of (+)-anti-BPDE, which among four BPDE isomers is the most potent carcinogen, was varied and GSH concentration was kept constant at 2 mM (saturating concentration), both forms of hGSTP1-1 [hGSTP1-1(V104) and hGSTP1-1(I104)] obeyed Michaelis-Menten kinetics. The V(max) of GSH conjugation of (+)-anti-BPDE was approximately 3.4-fold higher for hGSTP1-1(V104) than for hGSTP1-1(I104). Adherence to Michaelis-Menten kinetics was also observed for both isoforms when (-)-anti-BPDE, which is a weak carcinogen, was used as the variable substrate. However, (-)-anti-BPDE was a relatively poor substrate for both isoforms as compared with (+)-anti-BPDE. Moreover, there were no significant differences between hGSTP1-1(V104) and hGSTP1-1(I104) in either V(max) or K(m) for (-)-anti-BPDE. The mechanism of differences in kinetic properties and enantioselectivity of hGSTP1-1 variants toward anti-BPDE was investigated by modeling of the two proteins with conjugation product molecules in their active sites. Molecular modeling studies revealed that the differences in catalytic properties of hGSTP1-1 variants as well as the enantioselectivity of hGSTP1-1 in the GSH conjugation of anti-BPDE can be rationalized in terms of the architecture of their active sites. Our results suggest that the population polymorphism of hGSTP1-1 variants with disparate enzyme activities may, at least in part, account for the differential susceptibility of individuals to carcinogens such as anti-BPDE and possibly other similar carcinogens.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1997

Increased resistance to oxidative stress in transfected cultured cells overexpressing glutathione S-transferase mGSTA4-4.

Peroxidation of unsaturated fatty acids in membrane phospholipids is one of the multiple cytotoxic effects of oxidative stress. Lipid peroxidation is significant because a single initiating event triggers a chain reaction, thus amplifying the initial stimulus. Many oxidative stress-related pathologies have been linked to lipid peroxidation. Mouse glutathione S-transferase mGSTA4-4 exhibits high glutathione conjugating activity with toxic products of lipid peroxidation, e.g., 4-hydroxynon-2-enal. In addition, mGSTA4-4 has glutathione peroxidase activity toward phospholipid hydroperoxides. On the basis of these catalytic properties, we have previously proposed that the enzyme may be physiologically important in alleviating the cytotoxic effects of lipid peroxidation. We have now experimentally confirmed this hypothesis by transfecting HepG2 cells with mGSTA4 cDNA, and demonstrating a protective effect of expressed mGSTA4-4 protein on cells exposed during plating to H2O2, organic hydroperoxides, and phosphatidylcholine hydroperoxide. As compared to cells transfected with insert-free vector, a larger proportion of mGSTA4-transfected cells was able to attach to the culture dish, and continued to divide in the presence of the above compounds. In addition to alleviating the cytotoxic effects of oxidative stress, mGSTA4-4 may interfere with the subtoxic but cytostatic signals generated by a low-level pro-oxidant state.

Animals↗