Studies on the mechanism of haloacetonitriles toxicity: inhibition of rat hepatic glutathione S-transferases in vitro

Acetonitrile (AN) and seven of its halogenated derivatives known to be water disinfectant by-products were evaluated for their action on hepatic cytosolic glutathione S-transferase (GST) activity using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Increasing concentrations of acetonitrile, monofluoroacetonitrile (MFAN), monochloroacetonitrile (MCAN), and monobromoacetonitrile (MBAN) up to 10 mM failed to produced 50% inhibition of the activity of GST enzyme. However, dichloroacetonitrile (DCAN), trichloroacetonitrile (TCAN), dibromoacetonitrile (DBAN), and monoiodoacetonitrile (MIAN) were potent inhibitors with 150 values of 2.49, 0.34, 0.82, and 4.44 mM, respectively. At concentrations equivalent to their 150, MIAN, DCAN, and DBAN decrease both apparent Km and Vmax of the enzyme activity toward glutathione (GSH) to 20-50% of control. TCAN significantly increases both apparent Km and Vmax for GSH to 650 and 120% of control values, respectively. The inhibitory effect of haloacetonitriles (HAN) on hepatic GST activity toward CDNB was found to be a mixed type. The inhibitory effect of DCAN, DBAN, and TCAN on the hepatic GST activity was found to be reversible and the activity was completely recovered after dialysis of the inhibited enzyme. MIAN, however, inhibited GST activity in an irreversible manner. Haloacetonitriles' induced inhibition of hepatic GST activity in vitro is consistent with that observed in vivo. The data presented in this study show that haloacetonitriles induced reversible inhibition of hepatic GST activities, and this effect may lead to decreased detoxification of other electrophilic chemicals.     

The cyclopentenone product of lipid peroxidation, 15-A(2t)-isoprostane (8-isoprostaglandin A(2)), is efficiently conjugated with glutathione by human and rat glutathione transferase A4-4

Glutathione transferases (GSTs) are a large family of enzymes that can be divided into different classes based on structure. There has been considerable interest in the ability of GSTs to conjugate and inactivate endogenously derived reactive lipid peroxidation products that contain alpha,beta-unsaturated carbonyl moieties such as 4-hydroxyalkenals. One enzyme with prominent activity toward these substrates is human GST A4-4. Recently, we described a novel series of compounds termed A(2)/J(2)-isoprostanes (IsoPs) that are formed endogenously in humans from the free radical-initiated peroxidation of arachidonic acid. These compounds contain alpha,beta-unsaturated carbonyl groups and have structures similar to cyclooxygenase-derived PGA(2) and PGJ(2). Because of their chemical reactivity, these compounds may mediate tissue injury associated with oxidant stress. Herein, we report that the A-ring IsoP 15-A(2t)-IsoP (8-iso-PGA(2)) is efficiently conjugated to glutathione (GSH) by human GST A4-4 with a k(cat)/K(m) value of >200 s(-)(1) mM(-)(1). The k(cat)/K(m) value for conjugation of 15-A(2t)-IsoP by the homologous rat GST A4-4 is >2000 s(-)(1) mM(-)(1). Similar high enzyme activities were observed when PGA(2) was used as a substrate. In contrast, the human GSTs A1-1, M1-1, M2-2, P1-1, and T1-1 and rat GST T2-2 did not significantly metabolize 15-A(2t)-IsoP. These studies have therefore defined a potentially important route by which cyclopentenone IsoPs are metabolized that may serve as a mechanism for the inactivation of these highly reactive compounds.     

Organ distribution and kinetics of Glutathione transferase from African river prawn, Macrobrachium vollenhovenii (Herklots)

The distribution of glutathione transferase (GST) in the major organs of African river prawn (Macrobrachium vollenhovenii) was studied. All the organs studied had GST activity. The specific activity of the extract from the hepatopancreas was highest while that from the muscle lowest. Purified GST from the hepatopancreas which could conjugate glutathione (GSH) with only 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBDCl) among some electrophilic substrates tested, had a K(m)(NBDCl) of 2.2+/-0.12 mmol l(-1) while the K(m)CDNB was 2.03+/-0.29 mmol l(-1). Chloride ion, a product of the enzymatic reaction readily inhibited the conjugation of CDNB with GSH with an I50 of 0.12 mmol l(-1), whereas chloride ion up to 0.6 mol l(-1) had no inhibitory effect on the conjugation of GSH with NBDCl. However, nitrite inhibited the two reactions but the K(i) for the conjugation of NBDCl was lower than the K(i) for the conjugation of CDNB. The enzyme had an optimum temperature of 40 degrees C and an activation energy of 35.1 kJ/mol. The overall results show that M. vollenhovenii GST (mvGST) uses different mechanisms for different electrophilic substrates. The high K(m) of mvGST for the electrophilic substrates may be a special physiological adaptation for effective xenobiotic detoxication.     

Mouse liver glutathione S-transferase isoenzyme activity toward aflatoxin B1-8,9-epoxide and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide

As part of the studies of the biochemical basis for species differences in biotransformation of the carcinogen aflatoxin B1 (AFB1) and its modulation by phenolic antioxidants, we have investigated the role of mouse liver glutathione S-transferase (GST) isoenzymes in the conjugation of AFB1-8,9-epoxide. Isoenzymes of GST were purified to electrophoretic homogeneity from Swiss-Webster mouse liver cytosol by affinity chromatography and chromatofocusing. The isoenzyme fractions were characterized in terms of activity toward surrogate substrates and immunologic cross-reactivity with antisera to rat GSTs. The major isoenzymes were identified as SW 4-4, SW 3-3, and SW 1-1. The specific activity of SW 4-4 toward AFB1-8,9-epoxide was at least 50- and 150-fold greater than that of SW 3-3 and SW 1-1, respectively. Relatively high activity toward another epoxide carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, was observed with both SW 4-4 and SW 3-3. SW 1-1 had the highest activity toward 1-chloro-2,4-dinitrobenzene (CDNB) whereas SW 4-4 had relatively low CDNB activity. Following pretreatment with 0.75% butylated hydroxyanisole in the diet, the fraction of total GST contributed by SW 1-1 appeared to increase dramatically, whereas in control mice SW 3-3 constituted the predominant isoenzyme. The high GST activity of mouse liver cytosol toward AFB1-8,9-epoxide is apparently due to an isoenzyme that contributes little to the overall cytosolic CDNB activity.     

Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and mouse

To determine the mechanisms which mediate species- and treatment-related differences in susceptibility to aflatoxin B1 (AFB), we conducted a comparative study of the effects of dietary butylated hydroxyanisole (BHA) on the hepatic in vivo DNA binding and in vitro biotransformation of AFB in the rat and mouse. Mice are resistant to the hepatocarcinogenic effects of AFB, and BHA pretreatment has been shown to inhibit the carcinogenic effects of AFB in the highly susceptible rat. Rats and mice were fed a control diet or an identical diet containing 0.75% BHA for 10 days. On the 11th day, one-half of the control and BHA animals were administered [3H]AFB (0.25 mg/kg in dimethyl sulfoxide) via intraperitoneal injection. Animals were killed 2 hr later and covalent binding of AFB to hepatic DNA was determined. The remaining animals were killed for preparation of hepatic subcellular fractions used in in vitro assays. BHA treatment resulted in a decrease in in vivo hepatic AFB-DNA adduct formation in mice to 68% of control, but, in rats, treatment decreased AFB-DNA binding to 18% of control. Furthermore, hepatic AFB-DNA binding in control mice was only 1.2% of that measured in control rats. The rate of in vitro activation of AFB to the epoxide was 3.4-fold greater in control mice relative to control rats. BHA pretreatment increased the activation of AFB in mice 3.3-fold, but had no effect on oxidative metabolism in rats. Control mice had 52 times greater glutathione S-transferase (GST) activity toward the AFB-epoxide, but only 2.6 times greater GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB), compared to that of control rats. In mice, BHA did not significantly increase GST activity toward the AFB-epoxide, but increased GST activity toward CDNB 3.1-fold. In rats, BHA increased GST activity toward the AFB-epoxide and CDNB by 3.2- and 2.1-fold, respectively. Epoxide hydrolase activity toward p-nitrostyrene oxide in mice was only 52% of the activity in rats. BHA increased epoxide hydrolase activity 3.8- and 2.5-fold in mice and rats, respectively. These data indicate that mice have high levels of an AFB-epoxide-specific GST activity relative to that of the rat. The rate of formation of the AFB-epoxide and the activity of epoxide hydrolase appear to be relatively unimportant under conditions of high GST activity, whereas elevated GST activity, and thus inactivation of the AFB-epoxide, appears to be the critical component in species- and BHA-induced differences in AFB-DNA adduct formation and, presumably, AFB hepatocarcinogenicity.     

Detoxification of 1-chloro-2,4-dinitrobenzene in MCF7 breast cancer cells expressing glutathione S-transferase P1-1 and/or multidrug resistance protein 1.

We examined the roles of glutathione S-transferase (GST) P1-1 and the glutathione S-conjugate (GS-X) transporter, multidrug resistance protein 1 (MRP1), singly or in combination, in the detoxification of 1-chloro-2,4-dinitrobenzene (CDNB). Derivatives of MCF7 breast carcinoma cells expressing GST P1-1 and MRP1 alone or in combination were developed. Detoxification was measured in cells as formation of the glutathione conjugate of CDNB, S-(2,4-dinitrophenyl)-glutathione (DNP-SG), efflux of DNP-SG, and ultimately protection from CDNB cytotoxicity. MRP1 expression in the absence of GST P1-1 confers a three- to fourfold resistance to CDNB, which is associated with a >10-fold increase in the maximum rate of DNP-SG efflux. DNP-SG efflux in MRP1-expressing MCF7 cells was ATP-dependent and exhibited an apparent Km for DNP-SG of 95 microM. MRP1 expression alone, however, had no effect on DNP-SG formation. Combined expression of GST P1-1 and MRP1 increased the rates of DNP-SG formation when cells were exposed to 10 microM CDNB. Moreover, combined expression of GSTP1-1 with MRP1 moderately augmented MRP1-mediated resistance to CDNB but only during short term (10 min) exposures to CDNB where IC50 values were in the 8-10 microM range. In contrast, expression of GST P1-1 in the absence of MRP1 slightly sensitized cells to the toxicity of CDNB (10 min exposures), despite increasing rates of DNP-SG formation. The sensitization to CDNB in cells expressing GST P1-1 alone was associated with increased intracellular accumulation of DNP-SG, indicating that DNP-SG may contribute to CDNB toxicity. The potential toxicity of DNP-SG is also suggested by the finding that inhibition of DNP-SG formation by prior glutathione depletion confers resistance to CDNB cytotoxicity in MRP1-poor MCF7 cells. Altogether, our results demonstrate that glutathione conjugation and MRP1-mediated conjugate efflux can operate together to confer resistance to CDNB. The data indicate that MRP1-mediated conjugate efflux is required for cytoprotection from CDNB because its conjugate (DNP-SG), when present at high intracellular levels, may also be toxic to cells.     

In vitro interaction of dithiocarb with rat liver glutathione S-transferases

The in vitro interaction of dithiocarb (DTC) with rat liver glutathione S-transferase was studied, using reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. The inhibition of the GST activity by DTC was dose dependent, but not linear. The different GST isoenzymes were inhibited to different degrees. Kinetic studies revealed uncompetitive inhibition towards GSH for GST AA, and an intermediate kinetic pattern between uncompetitive and noncompetitive inhibition for the other GST isoenzymes. With respect to CDNB, mixed type inhibition was found for most GST isoenzymes, and nearly uncompetitive inhibition for GST AA and M. Titration of remaining GSH in appropriate incubation mixtures with DTC revealed no GST catalyzed conjugation of DTC with GSH. It is concluded that DTC interact with GST by direct binding to these proteins. This binding could have a protective function against DTC.     

Amine metabolism: a novel path to coronary artery vasospasm.

We hypothesized that allylamine (AA) induces subendocardial necrosis in mammals via coronary artery (CA) vasospasm. Additionally, AA toxicity is likely dependent on the enzyme semicarbazide-sensitive amine oxidase (SSAO), which is highly expressed in the aorta of rats and humans. We tested whether AA or acrolein (1, 10, 100, and 1000 microM), a highly reactive product of AA metabolism by SSAO, could contract CA or thoracic aorta (TA) in vitro and if the AA effects involved SSAO. AA or acrolein produced a similar pattern of responses in both CA and TA rings at 100 and 1000 microM, including (1) increased basal tension, (2) enhanced agonist-induced contraction (hypercontractility or vasospasm), (3) remarkable, agonist-induced slow wave vasomotion (vasospasm), and (4) irreversible reduction in vessel contractility after 1 mM exposure. Endothelium-dependent acetylcholine-induced relaxation was not altered during vasospasm in either vessel. Pretreatment with the SSAO inhibitor semicarbazide (1 mM; 10 min) prevented or significantly reduced the majority of AA's effects in both CA and TA rings and inhibited 100% of the SSAO activity present in rat TA and human CA and TA. We propose a two-step model for AA induction of CA vasospasm and resultant myocardial necrosis: (1) metabolism of AA to acrolein by coronary arterial SSAO activity and (2) acrolein induction of CA vasospasm independent of endothelial injury-a novel path.     

Gender dependent effects of cigarette smoke on hepatic and pulmonary xenobiotic metabolizing enzymes in rats

The adult male and female rats were exposed to cigarette smoke (CS) 5 times a day, with 1 h intervals, for 3 days in a chamber where smoke and fresh air lead alternatively and were killed 16 h after the last treatments and hepatic and pulmonary monooxygenase (MO) activities (aniline-4-hydroxylase, AH; aminopyrine-N-demethylase, AMND; 7-ethoxyresorufin-O-deethylase, EROD; p-nitroanisole-O-demethylase, p-NAOD), lipid peroxidation (LP) and reduced glutathione (GSH) levels and glutathione-S-transferases (GSTs) activities toward several substrates (1-chloro-2,4-dinitrobenzene, CDNB; 1,2-dichloro-4-nitrobenzene, DCNB; ethacrynic acid, EAA; 1,2-epoxy-3-(p-nitrophenoxy)-propane, ENPP) were determined. CS significantly increased hepatic AMND, EROD and p-NAOD activities whereas it unaltered AH activity in both genders as compared with controls. In the lung, EROD and p-NAOD activities were also significantly increased by CS in both genders. Pulmonary AH activity, however, significantly increased in males but remained unchanged in females. Pulmonary AMND activity significantly increased in females but remained unaltered in males. A significant decrease was noted in the LP level of males, while that of females was unaltered by CS in the liver. Pulmonary GSH and LP, and hepatic GSH levels were significantly increased by CS in both genders. In males, GST activities toward CDNB and DCNB did not alter, whereas GST activities toward EAA and ENPP significantly increased and decreased, respectively, in the liver. In females, CS significantly increased hepatic GST activity toward DCNB but it was ineffective on the other hepatic GST activities. All pulmonary GST activities of males were significantly depressed by CS. In females, however, CS significantly increased pulmonary GST activities toward CDNB and DCNB but was ineffective on GST activities toward EAA and ENPP. These results suggest that gender related differences exist in the modulations of hepatic GST, and pulmonary MO and GST activities but not in those of hepatic MO activities, by CS in rats.     

Subcellular distribution of glutathione S-transferase in Chinese fetal liver

Subcellular fractions were isolated from Chinese fetal liver at 4-8 months of age for the determination of glutathione S-transferase (GST). Using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, GST activity was found to be 66 +/- 34 nmol/(min.mg protein), mainly in the cytosol. The GST activities were detected principally in microsomes and their values were 66 +/- 31 and 144 +/- 83 nmol/(min.mg protein), respectively, when assayed with p-nitrobenzyl chloride (PNB) and ethacrynic acid (EA) as substrates. There were no age and sex-related differences in GST activities for any of the substrates studied during fetal development. The Km values of GST for CDNB, PNB and EA were 1112, 1039 and 205 mumol/L, respectively. The conjugation of GST may play an important role in fetal hepatic metabolism of toxic electrophiles.     

Induction of glutathione S-transferase activity and protein expression in brown bullhead (Ameiurus nebulosus) liver by ethoxyquin.

The inducibility of hepatic cytosolic glutathione S-transferases (GSTs) was examined in brown bullheads, a freshwater fish that is highly susceptible to hepatic neoplasia following exposure to carcinogen-contaminated sediments. Juvenile bullheads were fed a semi-purified antioxidant-free diet supplemented with ethoxyquin (0.5% w/w dissolved in 3% corn oil), a prototypical rodent GST-inducing agent, twice daily for 14 days. Control bullheads received the antioxidant-free diet supplemented with corn oil (3% w/w). A significant increase (1.6-fold, p < or = 0.01) in hepatic cytosolic GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) was observed in the ethoxyquin-treated bullheads relative to control fish. A trend toward increased GST-NBC activity was observed in the ethoxyquin-treated fish (1.2-fold, p = 0.06), whereas no treatment-related effects were observed on GST activities toward ethacrynic acid (ECA). In contrast, GST activity toward (+/-)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) was repressed in affinity-purified cytosolic fractions prepared from ethoxyquin-treated bullheads relative to control bullheads. Silver staining and densitometric analysis of isoelectric-focused, affinity-purified GST proteins revealed increased expression of two basic GST-like isoforms in ethoxyquin-treated fish. In summary, exposure to ethoxyquin increases brown bullhead GST-CDNB catalytic activity and hepatic cationic GST protein expression. However, the increase in overall GST-CDNB activity by ethoxyquin is associated with repression of GST-BPDE activity, suggesting differential effects on hepatic bullhead GST isoforms by ethoxyquin. The potential repression of bullhead GST isoforms that conjugate the carcinogenic metabolites of PAH metabolism under conditions of environmental chemical exposure could be a contributing factor in the sensitivity of bullheads to pollutant-associated neoplasia.     

Identification of theta-class glutathione S-transferase in liver cytosol of the marmoset monkey

The presence of theta-class glutathione S-transferase (GST) in marmoset monkey liver cytosol was investigated. An anti-peptide antibody targeted against the C-terminus of rGSTT1 reacted with a single band in marmoset liver cytosol that corresponded to a molecular weight of 28 kDa. The intensity of the immunoreactive band was not affected by treatment of marmoset monkeys with 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbitone, rifampicin or clofibric acid. Similarly, activity towards methyl chloride (MC) was unaffected by these treatments. However, GST activity towards 1,2-epoxy- 3-(p-nitrophenoxy)-propane (EPNP) was increased in marmosets treated with phenobarbitone (2.6-fold) and rifampicin (2.6-fold), activity towards dichloromethane (DCM) was increased by 50% after treatment of marmosets with clofibric acid, and activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was raised slightly (30–42% increases) after treatment with phenobarbitone, rifampicin or clofibric acid. Compared with humans, marmoset liver cytosol GST activity towards DCM was 18-fold higher, activity towards MC was 7 times higher and activity towards CDNB was 4 times higher. Further, EPNP activity was clearly detectable in marmoset liver cytosol samples, but was undetectable in human samples. Immunoreactive marmoset GST was partially purified by affinity chromatography using hexylglutathione-Sepharose and Orange A resin. The interaction of immunoreactive marmoset GST was similar to that found previously for rat and human GSTT1, suggesting that this protein is also a theta class GST. However, unlike rat GSTT1, the marmoset enzyme was not the major catalyst of EPNP conjugation. Instead, immunoreactivity was closely associated with activity towards MC. In conclusion, these results provide evidence for the presence of theta-class GST in the marmoset monkey orthologous to rGSTT1 and hGSTT1. Received: 18 October 1999 / Accepted: 8 December 1999     

Soluble glutathione S-transferase isoenzymes in rat brain

The soluble glutathione S-transferase (GST) isoenzymes in rat brain were investigated using 1-chloro-2,4-dinitrobenzene (CDNB) as the second substrate. The percentages of the different CDNB-GST isoenzymes found were: anionic GST: 3.7%, GST D + E: 35.3%, GST C: 27.9%, GST B: 0.5%, GST A: 13.9% and GST AA: 18.6%. The percentages of isoenzymes are quite different from those measured in liver, testis and prostate. An important detoxication role of GST in brain is suggested.     

Circadian variation of hepatic glutathione S-transferase activities in the mouse

1. The circadian variation in glutathione S-transferase (GST) activity was studied in the hepatic cytosolic fraction of the male and female mouse. A circadian variation in GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was observed in the male, the activity being higher in the light phase (07:00-19:00 h) than in the dark phase (19:00-07:00 h) during a day under normal lighting conditions. 2. The circadian variation was only existed from June to October. The difference between the lowest activity (at 01:00 h) and the highest activity (at 13:00 h) was maximum in August. 3. In both the normal and reversed light/dark cycle (lights on 07:00 and 19:00 h, respectively), reduced glutathione (GSH) content was lowest in the middle of the light period and highest in the middle of the dark period and GST activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) exhibited opposite peaks and troughs. GST activities toward CDNB and 1,2-dichloro-4-nitrobenzene (DCNB) during the normal lighting schedule was higher at 13:00 h than at 01:00 h, but no differences were observed under reversed lighting conditions. 4. A circadian variation in GST activity for CDNB and DCNB was also observed in the female in a similar manner to the male, but the variation in the activity for EPNP was not observed in the female. 5. Thus, the circadian variation of hepatic GST activities in mouse were dependent on the enzyme substrates used, and seemed to be reflected by the difference in each isozyme levels. The daily change in the hepatic GSH levels is also thought involved, at least in part, in the regulation of GST activity.     

Mouse strain differences in glutathione S-transferase activity and aflatoxin B1 biotransformation

Previous studies have suggested that mice are resistant to the carcinogenic effects of aflatoxin B1 (AFB1) and that this resistance is largely the result of expression of an isoenzyme of glutathione S-transferase (GST) with high activity toward AFB1-8,9-epoxide. Significant interstrain differences in cytosolic GST activities toward a variety of substrates have been reported in mice. If such differences exist for the conjugation of AFB1-8,9-epoxide, then there may be significant mouse strain differences in susceptibility to AFB1-induced hepatocarcinogenicity. The hepatic microsomal and cytosolic biotransformation of AFB1 was studied in 8 different strains of mice fed a purified diet. GST-mediated conjugation of AFB1-8,9-epoxide with glutathione and GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA) and cumene hydroperoxide (CHP) were determined with cytosolic fractions from 8-10 pooled livers. Specific activities of cytochrome-P-450-mediated oxidation of AFB1 to aflatoxin Q1 (AFQ1), aflatoxin M1 (AFM1), and aflatoxin P1 (AFP1), as well as the reactive intermediate AFB1-8,9-epoxide, were determined with hepatic microsomal fractions from each mouse strain. No striking differences in specific activity between mouse strains were observed for any of the P-450- or GST-mediated enzymatic pathways measured, although some statistically significant differences were found. GST specific activities toward AFB1-8,9-epoxide, CDNB, DCNB, ECA and CHP ranged from 1.5-2.1, 2,830-5,370, 81-144, 38-69 and 32-73 nmol/mg protein/min, respectively. The rate of formation of AFB1-8,9-epoxide ranged from 208 to 465 pmol/mg protein/min. The specific activities of AFQ1,AFM1, and AFP1 formation by microsomes ranged from 36-70, 161-326, and 252-426 pmol/mg protein/min, respectively. Mice fed a standard rodent chow diet showed evidence of microsomal and cytosolic enzyme induction when compared to mice fed a purified diet. The lack of substantial differences in enzyme specific activities between mouse strains suggests that interstrain variations in the hepatocarcinogenic effects of AFB1 in mice should not be large.     

Xenobiotic metabolizing and antioxidant enzymes in normal and neoplastic human breast tissue.

An investigation was made of ethoxyresorufin O-deethylase (EROD) activity, a cytochrome P450 (CYP) dependent enzyme mainly catalyzed by CYP1A1, glutathione S-transferase (GST) activity toward the substrates 1-chloro-2,4- dinitrobenzene (CDNB) and ethacrynic acid (EAA), reduced glutathione (GSH) levels, and antioxidant enzyme (AOE) activity namely catalase (CAT) and selenium- dependent glutathione peroxidase (Se-GPx) in tumor and surrounding tumor-free (normal) tissues in female breast cancer patients. Wide interindividual variations were found in the enzyme activities in both tumor and normal breast tissues. No significant differences were noted between mean EROD and CAT activities in tumor and normal breast tissues. The mean activities of CDNB GST, EAA GST and Se-GPx and GSH levels in tumor tissue were significantly higher than those in normal breast tissue. These results show that CYP, GST and AOE behave differentially in breast tumors.     

The inhibition of human glutathione S-transferases activity by plant polyphenolic compounds ellagic acid and curcumin.

Glutathione S-transferases (GSTs) are multifunctional detoxification proteins that protect the cell from electrophilic compounds. Overexpression of GSTs in cancer results in resistance to chemotherapeutic agents and inhibition of the over expressed GST has been suggested as an approach to combat GST-induced resistance. The inhibition of human recombinant GSTs by natural plant products was investigated in this study. Using 1-chloro-2,4 dinitrobenzene (CDNB) as a substrate, ellagic acid and curcumin were shown to inhibit GSTs A1-1, A2-2, M1-1, M2-2 and P1-1 with IC(50) values ranging from 0.04 to 5 microM whilst genistein, kaempferol and quercetin inhibited GSTs M1-1 and M2-2 only. The predominant mode of inhibition with respect to the G and H-sites were mixed inhibition and uncompetitive to a lesser extent. The K(i) (K(i)(')) values for ellagic acid and curcumin with respect to GSH and CDNB were in the range 0.04-6 microM showing the inhibitory potency of these polyphenolic compounds. Ellagic acid and curcumin also showed time- and concentration-dependent inactivation of GSTs M1-1, M2-2 and P1-1 with curcumin being a more potent inactivator than ellagic acid. These results facilitate the understanding of the interaction of human GSTs with plant polyphenolic compounds with regards to their role as chemomodulators in cases of GST-overexpression in malignancies.     

Several glutathione S-transferase isozymes that protect against oxidative injury are expressed in human liver mitochondria

The mitochondrial environment is rich in reactive oxygen species (ROS) that may ultimately peroxidize membrane proteins and generate unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE). We had previously demonstrated the presence of hGSTA4-4, an efficient catalyst of 4HNE detoxification, in human liver mitochondria to the exclusion of the cytosol. In the present study, GSH-affinity chromatography was used in conjunction with biochemical and proteomic analysis to determine the presence of additional cytosolic glutathione S-transferases (GSTs) in human hepatic mitochondria. HPLC-subunit analysis of GSH affinity-purified liver mitochondrial proteins indicated the presence of several potential mitochondrial GST isoforms. Electrospray ionization-mass spectrometry analysis of eluted mitochondrial GST subunits yielded molecular masses similar to those of hGSTP1, hGSTA1 and hGSTA2. Octagonal matrix-assisted laser desorption/ionization time of flight mass spectrometry and proteomics analysis using MS-FIT confirmed the presence of these three GST subunits in mitochondria, and HPLC analysis indicated that the relative contents of the mitochondrial GST subunits were hGSTA1>hGSTA2>hGSTP1. The mitochondrial localization of the alpha and pi class GST subunits was consistent with immunoblotting analysis of purified mitochondrial GST. Enzymatic studies using GSH-purified mitochondrial GST fractions demonstrated the presence of significant GST activity using the nonspecific GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), as well as 4HNE, delta(5)-androstene-3,17-dione (ADI), and cumene hydroperoxide (CuOOH). Interestingly, the specific mitochondrial GST activities toward 4HNE, a highly toxic alpha,beta-unsaturated aldehyde produced during the breakdown of membrane lipids, exceeded that observed in liver cytosol. These observations are suggestive of a role of GST in protecting against mitochondrial injury during the secondary phase of oxidative stress, or modulation of 4HNE-mediated mitochondrial signaling pathways. However, other properties of mitochondrial GST, such as conjugation of environmental chemicals and binding of lipophilic non-substrate xenobiotics and endogenous compounds, remain to be investigated.     

Inhibition of glutathione S-transferases by thonningianin A, isolated from the African medicinal herb, Thonningia sanguinea, in vitro.

There is evidence that increased expression of glutathione S-transferase (EC: 2.5.1.18, GST) is involved in resistance of tumor cells against chemotherapeutic agents. In this study we investigated the inhibitory effects of thonningianin A (Th A), a novel antioxidant isolated from the medicinal herb, Thonningia sanguinea on uncharacterized rat liver GST and human GST P1-1. Using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, rat liver cytosolic GST activity was inhibited by Th A in a concentration dependent manner with 50% inhibition concentration (IC50) of 1.1 microM. When Th A was compared with known potent GST inhibitors the order of inhibition was tannic acid>cibacron blue>hematin>Th A>ethacrynic acid with CDNB as substrate. Th A also exhibited non-competitive inhibition towards both CDNB and glutathione. Furthermore, using 1,2-dichloro-4-nitrobenzene, ethacrynic acid and 1,2-epoxy-3-(p-nitrophenoxy) propane as substrates Th A at 1.0 microM inhibited cytosolic GST by 2%, 12% and 36% respectively. Human GST P1-1 was also inhibited by Th A with an IC50 of 3.6 microM. While Th A showed competitive inhibition towards CDNB it exhibited non-competitive inhibition towards GSH of the human GST P1-1. These results suggest that Th A represents a new potent GST in vitro inhibitor.     

Hepatic mixed function oxygenase activity and glutathione S-transferase activity in mice following ethanol consumption and withdrawal

The effect of an 8 day liquid diet containing 7% v/v ethanol and the effect of ethanol withdrawal on several drug metabolizing enzyme activities, cytochrome P-450 content and glutathione S-transferase activity (GST) has been studied in male C57/BL mice. After treatment, hepatic microsomal activities toward benzphetamine (BNZ), biphenyl (BPH) and dimethylnitrosamine (DMN) and cytosolic GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) were determined. Ethanol treatment caused a differential time dependent increase in the metabolism of the 4 xenobiotics. Increased BPH-4-hydroxylase activity correlated most closely with that of the increased concentration of hepatic P-450. That is, both values were increased (5.8-fold) over controls after 8 days of ethanol treatment. Ethanol withdrawal (24 h) resulted in a 46% reduction in the P-450 content and a 26% reduction in BPH-4-hydroxylase activity compared to the elevated values at day 8. By 48 h, the values were no different from controls. DNA-N-demethylase, BNZ-N-demethylase and GST activities all increased after 4 days of ethanol treatment and remained the same at 8 days. However, ethanol withdrawal resulted in differential time dependent changes in the activities towards BNZ, DMN, and CDNB. While DMN-N-demethylase activity returned to control activity within 24 h, BNZ-N-demethylase activity did not change for the first 24 h of withdrawal, but returned to control activity by 48 h. GST activity had not decreased by 48 h of withdrawal. These data suggest that ethanol induces several cytochrome P-450 isozymes that have a time difference in induction by ethanol and reduction following ethanol withdrawal. Furthermore, ethanol induction of GSTs occurs quickly (4 days) and remains elevated at least 48 h after ethanol withdrawal.