Induction Of Glutathione S-transferase Research Articles

The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance.

The glutathione S-transferases (GST) represent a major group of detoxification enzymes. All eukaryotic species possess multiple cytosolic and membrane-bound GST isoenzymes, each of which displays distinct catalytic as well as noncatalytic binding properties: the cytosolic enzymes are encoded by at least five distantly related gene families (designated class alpha, mu, pi, sigma, and theta GST), whereas the membrane-bound enzymes, microsomal GST and leukotriene C4 synthetase, are encoded by single genes and both have arisen separately from the soluble GST. Evidence suggests that the level of expression of GST is a crucial factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals. In this article the biochemical functions of GST are described to show how individual isoenzymes contribute to resistance to carcinogens, antitumor drugs, environmental pollutants, and products of oxidative stress. A description of the mechanisms of transcriptional and posttranscriptional regulation of GST isoenzymes is provided to allow identification of factors that may modulate resistance to specific noxious chemicals. The most abundant mammalian GST are the class alpha, mu, and pi enzymes and their regulation has been studied in detail. The biological control of these families is complex as they exhibit sex-, age-, tissue-, species-, and tumor-specific patterns of expression. In addition, GST are regulated by a structurally diverse range of xenobiotics and, to date, at least 100 chemicals have been identified that induce GST; a significant number of these chemical inducers occur naturally and, as they are found as nonnutrient components in vegetables and citrus fruits, it is apparent that humans are likely to be exposed regularly to such compounds. Many inducers, but not all, effect transcriptional activation of GST genes through either the antioxidant-responsive element (ARE), the xenobiotic-responsive element (XRE), the GST P enhancer 1(GPE), or the glucocorticoid-responsive element (GRE). Barbiturates may transcriptionally activate GST through a Barbie box element. The involvement of the Ah-receptor, Maf, Nrl, Jun, Fos, and NF-kappa B in GST induction is discussed. Many of the compounds that induce GST are themselves substrates for these enzymes, or are metabolized (by cytochrome P-450 monooxygenases) to compounds that can serve as GST substrates, suggesting that GST induction represents part of an adaptive response mechanism to chemical stress caused by electrophiles. It also appears probable that GST are regulated in vivo by reactive oxygen species (ROS), because not only are some of the most potent inducers capable of generating free radicals by redox-cycling, but H2O2 has been shown to induce GST in plant and mammalian cells: induction of GST by ROS would appear to represent an adaptive response as these enzymes detoxify some of the toxic carbonyl-, peroxide-, and epoxide-containing metabolites produced within the cell by oxidative stress. Class alpha, mu, and pi GST isoenzymes are overexpressed in rat hepatic preneoplastic nodules and the increased levels of these enzymes are believed to contribute to the multidrug-resistant phenotype observed in these lesions. The majority of human tumors and human tumor cell lines express significant amounts of class pi GST. Cell lines selected in vitro for resistance to anticancer drugs frequently overexpress class pi GST, although overexpression of class alpha and mu isoenzymes is also often observed. The mechanisms responsible for overexpression of GST include transcriptional activation, stabilization of either mRNA or protein, and gene amplification. In humans, marked interindividual differences exist in the expression of class alpha, mu, and theta GST. The molecular basis for the variation in class alpha GST is not known. (ABSTRACT TRUNCATED)

Ellagic acid induces transcription of the rat glutathione S-transferase-Ya gene.

Induction of glutathione S-transferase (GST) enzymes can increase detoxification of carcinogens and reduce carcinogen-induced mutagenesis and tumorigenesis. To determine if the anticarcinogen ellagic acid induces cellular enzymes which detoxify carcinogens, we examined the effect of ellagic acid on the expression of glutathione S-transferase-Ya. Rats fed ellagic acid demonstrated significant increases in total hepatic GST activity, hepatic GST-Ya activity and hepatic GST-Ya mRNA. To determine if the observed increase in GST-Ya mRNA was due to ellagic acid inducing transcription of the GST-Ya gene, transfection studies were performed with plasmid constructs containing various portions of the 5' regulatory region of the rat GST-Ya gene. The transfection studies demonstrated that ellagic acid increased GST-Ya mRNA by inducing transcription of the GST-Ya gene and demonstrated that this induction is mediated through the antioxidant responsive element of the GST-Ya gene.

Induction of CYP1A and glutathione S-transferase activities by 2,3,7,8-tetrachlorodibenzo-p-dioxin in human hepatocyte cultures.

Induction of CYP1A and glutathione S-transferase activities with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in human hepatocytes in primary culture to investigate the variability of inducibility and the potency of TCDD. Determining induction of 7-ethoxyresorufin O-deethylase activity, preferentially catalyzed by CYP1A isozymes, we obtained concentration-response diagrams in TCDD-treated hepatocyte cultures from transplant donors and patients undergoing hepatic surgery. At a concentration of 10(-10) M TCDD approximately half-maximal induction of CYP1A was observed. Northern analysis of CYP1A gene expression showed a similar concentration-response relationship. In comparison with rat hepatocytes, human hepatocytes were about 10-fold less sensitive towards the CYP1A-inducing effect of TCDD. No pronounced interindividual differences in the inducing potency of TCDD (concentration which leads to half-maximal induction) were obvious in the six human individuals studied, whereas the efficacy of CYP1A induction was highly variable. In addition, inducibility of glutathione S-transferase (GST) activity also revealed a considerable degree of interindividual variation, i.e. a complete lack of induction in three out of six hepatocyte preparations and a highly variable efficacy of GST induction among responders which was not related to CYP1A inducibility.

Effect of fish oil on the development of AOM-induced glutathione S-transferase placental form positive hepatocellular foci in male F344 rats.

Omega-3 fatty acids, which are contained in fish oils and certain vegetable oils in contrast to corn oil or safflower oil rich in omega-6 fatty acids (linoteic acid), have been reported to reduce the carcinogenesis in several organs. In this study, the modifying effect of menhaden fish oil was investigated on the occurrence of azoxymethane (AOM)-induced glutathione S-transferase placental form (GST-P) positive hepatocellular foci, recognized preneoplastic lesions in the liver, in male F344 rats. Starting at five weeks of age, groups of animals were fed ad libitum a semipurified diet containing 5% corn oil (low fat). At seven weeks of age, all animals except the vehicle-treated groups were injected subcutaneously with AOM (15 mg/kg body wt, 1x/wk for 2 wks). Four days after the second injection, groups of animals were fed the diets containing 4% menhaden oil + 1% corn oil (low fish oil diet), 22.5% menhaden oil + 1% corn oil (high fish oil diet), and 5% corn oil. Thirty-four weeks after AOM injections, all animals were necropsied. Livers were sectioned and performed immunohistochemical staining of GST-P for quantitative analysis of enzyme altered foci of the liver. The results demonstrate that the density and the unit area of AOM-induced enzyme altered foci in the liver were significantly lower in the high fish oil group (0.60 +/- 0.08/cm2, 3.0 +/- 0.4 x 10(-4)) than in the 5% corn oil group (2.71 +/- 0.33/cm2, 16.6 +/- 2.6 x 10(-4)) and the low fish oil group (1.66 +/- 0.33/cm2, 11.1 +/- 1.9 x 10(-4)).(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-dependent inhibition of glutathione S-transferase placental form-positive hepatocellular foci induction in the rat by methyl propyl disulfide and propylene sulfide from garlic and onions.

Two organosulfur compounds, methyl propyl disulfide (MPD) and propylene sulfide (PS) from garlic and onions, were studied for their modifying effects on hepatocarcinogenesis in the F344 rats. Modifying potential was scored by comparing the number and area per cm2 of induced glutathione S-transferase placental form (GST-P)-positive foci in the liver. MPD and PS significantly reduced both these parameters of GST-P-positive foci in a dose-dependent manner. To investigate possible mechanisms of inhibition, ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SAT) activities were measured. In MPD and PS-high dose-treated liver tissue there was a tendency for their decrease, albeit non-significant, which suggested that the inhibitory effect might have been caused by decreased cell proliferation associated with decreased polyamine biosynthesis. In evaluating relationships between diet and cancer, it is thus necessary to consider various effects in assessing possible protective roles of garlic and onions.

Microsomal Glutathione S-Transferase (GST) Isozymes in Aspergillus ochraceus TS: Induction by 3-Methylcholanthrene

Induction of microsomal glutathione S-transferase isozymes in Aspergillus ochraceus TS is reported. Among the inducers of mammalian drug-metabolising enzymes 3-methylcholanthrene, benzo(a)pyrene and polychlorinated biphenyl enhanced the microsomal GST activity quite considerably. On the other hand phenobarbital, β-naphthoflavone and trans-stilbene oxide were almost without any effect. To our knowledge this seems to be the first report on induction of microsomal GST in lower eukaryotes. A significant increase (7.9 fold) in GST activity was observed with the cells induced by 3-methylcholanthrene. Moreover, three new GST isozymes were found to be present in 3-methylcholanthrene induced microsomes. The isozymes are basic in nature. The GST isozymes were found to be catalytically distinct from each other.

Regulation of aflatoxin B1-metabolizing aldehyde reductase and glutathione S-transferase by chemoprotectors.

Ingestion of aflatoxin B1 (AFB1) represents a major risk factor in the aetiology of human hepatocellular carcinoma. In the rat, the harmful effects of AFB1 can be prevented by the administration of certain drugs which induce hepatic detoxification enzymes. We have previously shown that treatment of rats with the chemoprotector ethoxyquin (EQ) results in a marked increase in expression of the Alpha-class glutathione S-transferase (GST) Yc2 subunit which has high activity towards AFB1-8,9-epoxide [Hayes, Judah, McLellan, Kerr, Peacock and Neal (1991) Biochem. J. 279, 385-398]. To allow an assessment of whether the increased expression of GST Yc2 represents a general adaptive resistance mechanism to chemical stress, that is invoked by both chemoprotectors and carcinogens, we have examined the effects of EQ, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), phenobarbital (PB), AFB1, 3-methylcholanthrene (3-MC) and clofibrate on the AFB1-glutathione-conjugating activity and the GST subunit levels in rat liver. In addition, the effect of these drugs on the hepatic levels of an aldehyde reductase (AFB1-AR) that metabolizes the cytotoxic dialdehydic form of AFB1 has been studied as this enzyme also appears to be important in chemoprotection. Administration of the antioxidants EQ, BHA or BHT, as well as PB, led to a marked increase in levels of the GST Yc2 subunit in rat liver, and this increase coincided with a substantial rise in the GST activity towards AFB1-8,9-epoxide; neither AFB1, 3-MC nor clofibrate caused induction of Yc2 or any of the GST subunits examined. Among the xenobiotics studied, EQ was found to be the most effective inducing agent for the Yc2 subunit as well as Yc1, Yb1 and Yf. However, PB was equally as effective as EQ in increasing levels of the Ya-type subunits, although it was not found to be as potent an inducer of the other GST subunits, including Yc2. In addition to induction of GST, EQ caused a substantial increase in the hepatic content of AFB1-AR. Both BHA and BHT were also able to induce this enzyme but, by contrast, PB was found to be a poor inducer of AFB1-AR. AFB1, 3-MC and clofibrate were unable to serve as inducers of this reductase. The presence of Alpha-class GST, including the Yc2 subunit, was examined in various rat tissues. Constitutive expression of Yc2 was found in the epididymis at levels comparable with that observed in the liver from EQ-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative studies on the effect of butylated hydroxyanisole on glutathione and glutathione S-transferases in the tissues of male and female CD-1 mice

1. Male CD-1 mice had about 1.6-fold higher glutathione (GSH), 2-fold higher glutathione S-transferase (GST) activity and 2.8-fold higher GST protein in their livers as compared to the female mice. 2. When mice were fed a diet containing 0.75% BHA for 2 weeks, a 1.8-fold increase was observed in GSH levels of female mice liver as opposed to only 1.2-fold increase in male mice. BHA caused 10-fold increase in GST activity and protein in livers of female mice as compared to only about 3–4-fold increase in livers of males. Differential induction of GSH and GST in males and females was also observed in other tissue besides liver but was not as remarkable. 3. Sex-related differences were also observed in the induction of the α- and μ- and π-classes ofGSTs by BHA; most noticeable being GST π, which was induced to about 10-fold in female liver as opposed to only 3.4-fold in male liver.

INDUCTION OF HEPATIC GLUTATHIONE S-TRANSFERASE BY MUSK XYLENE IN MICE

The inducing effects of musk xylene, a non-mutagenic carcinogen, on hepatic glutathione S-transferase (GST) in mice were investigated. Musk xylene posessed a potency of inducing all isozymes of mouse GSTs, whereas the effects were different between male and female mice. Induction of both GST MI (α class) and GST MII (π class) by musk xylene was observed only ip female. GST MIII (μ class) was induced up to about 5-fold by musk xylene in both sexes. The induction of MIII was higher than those of the other isozymes. There were strain differences in the induction of MII and MIII by musk xylene in female mice. The induction observed in C57BL/6N female mice was higher than that in DBA/2N female mice. These results indicate that musk xylene possesses multifunctional effects on GST induction in male and female mice.

Perturbation of glutathione status and generation of oxidative stress in mouse skin following application of contact allergenic sesquiterpene lactones and isothiocyanates.

1. The sensitizing or non-sensitizing status of selected sesquiterpene lactones and isothiocyanates was confirmed in mouse by open epicutaneous application. 2. Glutathione status of mouse skin was determined 12 h after lactone/isothiocyanate application; glutathione S-transferase activity also was determined 12 h after lactone application. 3. NAD(P)H utilization by rat liver microsomal preparations exposed to the sesquiterpene lactones and isothiocyanates was measured. 4. A correlation was observed between sensitizing status and the ability to perturb glutathione status, to induce glutathione S-transferase activity, and to stimulate NAD(P)H utilization. 5. It was concluded that sensitizing sesquiterpene lactones and isothiocyanates could induce oxidative stress in mouse skin, possibly as a result of their reductive metabolism.

Effect of restricted caloric intake on the development of the azoxymethane-induced glutathione S-transferase placental form positive hepatocellular foci in male F344 rats

The modifying effect of 30% caloric restriction on the occurrence of azoxymethane (AOM)-induced glutathione S-transferase placental form (GST-P) positive hepatocellular foci was investigated in male F344 rats. Starting at 5 weeks of age, groups of animals were fed ad libitum a high-fat (23.5%) semipurified diet. At 7 weeks of age, all animals except the vehicle-treated groups were s.c. injected with AOM (15 mg/kg body wt., once weekly for 2 weeks). Four days after the second injection, groups of animals were continued on high-fat diet and fed ad libitum (ad libitum group) whereas other groups were restricted no 70% of total calories (calorie-restricted group) consumed by the ad libitum group, but received the same amounts of fiber, vitamins and minerals. Thirty-two weeks after AOM injections, all animals were necropsied and livers were sectioned and stained for GST-P by a immunohistochemical technique for quantitative analysis of enzyme altered foci of the liver. Comparing AOM treated groups. The density and the unit area of enzyme altered foci were significantly lower in the calorie-restricted group (3.84 ± 1.55/cm 2, 7.96 ± 5.43%) than in the ad libitum group (10.14 ± 3.62/cm 2, 28.11 ± 12.33%). The size of foci was also reduced in the calorie-restricted group (17.15 × 10 −3 mm 2 vs. 32.36 × 10 −3 mm 2). The incidence and density of hepatocellular foci in rats fed calorie restricted diet were significantly lower than those in rats fed ad libitum, comparing vehicle-treated groups. These results indicate that calorie restriction inhibited the occurrence of both of spontaneous and AOM induced GST-P positive foci in rats.

A possible modulatory influence of melatonin on representative phase I and II drug metabolizing enzymes in 9,10-dimethyl-1,2-benzanthracene induced rat mammary tumorigenesis.

The oncosuppressive effect of melatonin on 9,10-dimethyl-1,2-benzanthracene (DMBA) induced rat mammary tumorigenesis led us to assess its possible modulatory influence on representative hepatic and mammary drug metabolizing enzymes in DMBA treated female Holtzman rats, reared in short and long photoperiods. Melatonin treated rats in either photoperiod showed a significant induction in hepatic and mammary levels of glutathione (GSH) and cytosolic activities of glutathione S-transferase (GST) when compared with the corresponding controls, along with a significant drop in hepatic microsomal contents of cytochromes b5 and P450. This induction of GSH and GST, and depletion of cytochromes b5 and P450 by melatonin may possibly be related to its anticarcinogenic potential in this tumor model.

Liver medium-term bioassay in rats for screening of carcinogens and modifying factors in hepatocarcinogenesis

The present report describes a study of the hepatocarcinogenic potential of a second large assay series of 94 compounds carried out using the rapid bioassay system (DEN-PH model) developed in this laboratory and based on the two-step concept of hepatocarcinogenesis. Male F344 rats were initially given a single dose of diethylnitrosamine (DEN, 200 mg/kg body weight ip) and, starting 2 wk later, were treated with test compounds for 6 wk and then killed, all rats being subjected to a two-thirds partial hepatectomy at wk 3. Carcinogenic potential was scored by comparing the numbers (no./cm 2) and areas (mm 2/cm 2) of induced glutathione S-transferase placental form (GST-P) positive foci in the livers of groups of about 15 rats with those of corresponding control groups given DEN alone. Positive was scored for a significant increase ( P < 0.05) in quantitative values of GST-P positive foci, negative for no change or a decrease. Results for the 94 compounds were also compared with previously published data from Salmonella/microsome (Ames) tests and long-term carcinogenicity studies in rats and mice. Of the known liver carcinogens, 14 out of 14 (100%) mutagenic (Ames test) compounds and 10 out of 12 (83%) non-mutagenic compounds gave positive results in our DEN-PH system (mean 92%). Two hepatocarcinogenic peroxisome proliferators did not enhance the development of GST-P positive foci. Carcinogens other than hepatocarcinogens gave fewer positive results (five out of 17, 29%). One of the 13 compounds reported as non-carcinogenic, malathion, gave positive results in the DEN-PH assay, suggesting that this compound is a weak hepatocarcinogen or tumour promoter for hepatocarcinogenesis based on the two-stage hypothesis for carcinogenesis. The present study also provided information regarding the inhibitory potential of nine compounds. The practical usefulness and benefits of the DEN-PH protocol for the rapid screening of carcinogenic agents are discussed.

Species and sex differences of aflatoxin B 1-induced glutathione S-transferase placental form in single hepatocytes

Species and sex differences of aflatoxin B 1 (AFB 1)-induced glutathione S-transferase placental form (GST-P) positive single hepatocytes have been investigated 48 h after an intraperitoneal injection of AFB 1 to young male and female Fischer rats (2 mg AFB 1/kg body wt) and male Syrian golden hamsters (6 mg AFB 1/kg body wt). The presence of GST-P positive hepatocytes was examined by the immunohistochemical method. Male rats formed three times as many AFB 1-induced GST-P positive hepatocytes as females. Pretreatment of both male and female rats with an inhibitor of GSH synthesis, buthionine sulfoximine (BSO) (4 mmol/kg body wt), 2 h and 4 h before AFB 1 injection increased AFB 1-induced GST-P positive hepatocytes by about 120% above the controls. Male hamsters formed several-fold less AFB 1-induced GST-P positive hepatocytes than male rats. Pretreatment with BSO did not increase AFB 1-induced GST-P positive hepatocytes in hamsters even though it produced an increase in hepatic necrosis. It appears that GSH and GSH S-transferases play an important role in modulating hepatic AFB 1-DNA binding and AFB 1-induced GST-P positive hepatocytes in rats and hamsters.

Lead-induced lipid peroxidation and antioxidant defense components of developing chick embryos

Administration of lead (1.25 and 2.5 μmol/kg egg weight) to 14-day-old chick embryos enhanced the level of lipid peroxides (LPO) in tissues of liver, brain, and heart. Accumulation of LPO was maximum at 9 h after treatment with lead and returned to normal level by 72 h. Further, we have studied the levels of glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), superoxida dismutase (SOD), and catalase. AT 9 h posttreatment, the hepatic GR was reduced significantly with the induction of GST and considerable depletion of GSH. However, in brain and heart, both GR and GST activities were unaltered with significant reduction of GSH. Further, an increase of non-Se-dependent GPx and SOD activities were observed in liver, brain, and heart. Similarly, at 72 h, although the GPx activity was found decreased in liver and brain, the GST, catalase, and SOD activities were significantly increased in all the three tissues alike, suggesting tissue-specific changes of antioxidant defense components in response to lead treatment. Our results suggest that the elevated levels of GST, SOD, and catalase at 72 h were successful in bringing LPO levels back to normal.

Effects of indole-3-carbinol on biotransformation enzymes in the rat: in vivo changes in liver and small intestinal mucosa in comparison with primary hepatocyte cultures

Groups of male Wistar rats were fed semi-synthetic diets containing 0, 200 or 500 mg indole-3-carbinol (I3C)/kg for 2, 7, 14 or 28 days. After 2 days, P-450 activities were already induced, but the isoenzyme pattern induced was different in the liver and the small intestine. Hepatic P4501 A1, P4501 A2 and P4502 B1 apoprotein levels were dose-relatedly enhanced, whereas in the small intestine induced levels of P4502 B1 and P4501 A1 were detected but P4501 A2 was not induced. Pentoxy- and ethoxyresorufin dealkylation (PROD and EROD) were dose-relatedly enhanced in the liver (5- and 7-fold, respectively, in the higher dose group) as well as in the small intestine (8- and 13-fold, respectively, at 500 mg I3C/kg diet). Testosterone 16α- and 16β-hydroxylation in the small intestine were enhanced (6-9-fold) from day 2 onwards, but in the liver these activities were only slightly enhanced from day 7 onwards. Thus, the major forms induced in the liver appear to be P4501 A1, P4501 A2, P4502 B1 and, to a lesser extent, P4503 A, whereas in the small intestine all of the effects that were found are associated with only one cytochrome P-450, P4502 B1. After 2 days I3C (500 mg/kg) induced glutathione S-transferase in the liver (1.3-fold) and small intestine (1.5-fold). Hepatic glucuronyl transferase (GT1) was induced (about 1.6-fold) after 7, 14 and 28 days. DT-diaphorase was induced in the liver (2.7-fold) and small intestine (1.5-fold) after 14 days of exposure to 500 mg I3C/kg diet. Treatment of rat hepatocytes with indole-3-acetonitrile and 3,3′-diindolylmethane, but not I3C and indole-3-carboxaldehyde, enhanced EROD activity and halved testosterone 16α- and 2α-hydroxylation. All four indoles slightly induced glutathione S-transferase in cultured hepatocytes. Thus, the in vitro studies suggest that the in vivo effects of I3C have to be attributed to indole-condensation products, such as 3,3′-diindolylmethane, but not to I3C itself.

Activity of glutathione-S-transferase in rat liver and kidneys after administration of lead or cadmium.

The influence of an acute dose of lead nitrate or cadmium chloride on the activity of glutathione-S-transferase (GST) was investigated in rats. CdCl2 (10 mumol/kg) did not influence the enzyme activity in either organ. In contrast Pb(NO3)2 (100 mumol/kg) caused a significant increase of GST activity in both organs. SDS polyacrylamide gel electrophoresis (SDS-PAGE) showed that in liver the activity enhancement is due mainly to the induction of the isoenzyme GST 7-7, while in the kidneys the activity of all the isoenzymes is increased.

Changes in levels of hepatic biotransformation enzymes and haemoglobin levels in female plaice ( Pleuronectes platessa) after oral administration of a technical polychlorinated biphenyl mixture (Clophen A40)

Female plaice ( Pleuronectes platessa) were orally dosed with a gelatin capsule containing a solution of the technical PCB mixture Clophen A40 in sunflower oil. They were compared to plaice injected with a gelatin capsule containing only the sunflower oil at 10 and 16 days after injection. Even at 16 days after injection, the increase in concentrations of individual CB congeners in muscle was proportional to their contribution in Clophen A40. Biochemical effects are related to increases in concentrations of well-separable CB congeners in muscle, which increased by factors between 1.6 and 64 compared to the reference group of fish. Of both sampling points, total cytochrome P-450 levels were higher than the control groups, but surprisingly ethoxyresorufin- O-deethylase (EROD) activities did not differ between the groups. Enzyme-linked immunosorbent assay (ELISA) showed increased concentrations of the inducible cytochrome P-450IA1 in PCB-treated fish. The apparent lack of EROD induction may be due to competitive substrate inhibition by certain CB congeners present in the sample. The activity of glutathione- S-transferase (GST) (with CDNB as model substrate) was significantly elevated by PCB-treatment at day 16, but not at day 10. A longer time interval between injection with PCBs and induction of GST compared to P-450 monooxygenase activities has been reported earlier and may indicate that in fish both groups of enzymes are regulated individually and not as an [Arylhydrocarbon] gene battery as appears to be the case in mammals. Haemoglobin concentrations and MCHC were decreased in fish treated with Clophen A40. Haematocrit values did not differ between groups of fish.

Modulation of glutathione S-transferases and glutathione peroxidase by the anticarcinogen butylated hydroxyanisole in murine extrahepatic organs.

Induction of glutathione S-transferases (GST) by the anticarcinogen butylated hydroxyanisole (BHA) has been examined in lung, kidney and small intestine of male and female BALB/c mice. BHA produced maximal induction of GST in the gut and although it increased GST levels in the kidney, it had little effect on pulmonary GST. Dietary BHA induced Alpha (Ya and Yk), Mu (Yb) and Pi (Yf) class GST subunits at least 10-fold in the small intestine but, by contrast, selenium-dependent glutathione peroxidase activity was reduced by approximately 4-fold in this organ following BHA treatment. In the kidney, all of the GST subunits, apart from Yk in males, showed modest levels of induction by BHA. However, a pronounced sex difference in the expression of renal alpha class subunits in both control and BHA-treated mice was observed, with female mice expressing approximately 4-fold greater levels of Ya and Yk than male mice. All renal GST were localized primarily in the proximal tubules. Dietary BHA was found to have the least inductive effect in the lung, where the GST were localized solely in the bronchi. The pulmonary Mu class GST subunits were induced approximately 2-fold by BHA; the expression of other GST was marginally increased by this inducer. Alpha class GST was also subject to sexual differentiation in the lung with female mice possessing higher levels of Yc and Yk than males. The Ya-type subunit was not detected in the lung nor was it induced by BHA.

Ethoxyquin-induced resistance to aflatoxin B1 in the rat is associated with the expression of a novel Alpha-class glutathione S-transferase subunit, Yc2, which possesses high catalytic activity for aflatoxin B1-8,9-epoxide

A purification scheme has been devised for two ethoxyquin-inducible Alpha-class glutathione S-transferases (GSTs) which possess at least 25-fold greater activity towards aflatoxin B1 (AFB1)-8,9-epoxide than that exhibited by the GSTs (i.e. F, L, B and AA) that have been described previously. These two enzymes are both heterodimers and both contain a subunit of Mr 25,800. This subunit has been isolated from both of the GST isoenzymes and, after cleavage with CNBr, it has been subjected to automated amino acid sequencing. The primary structure of the Mr 25,800 subunit revealed that it forms part of a subfamily of Alpha-class GSTs which possess closest identity (about 92%) with the Yc subunit of apparent Mr 27,500, which is encoded by the recombinant cDNA clone pGTB42 [Telakowski-Hopkins, Rodkey, Bennett, Lu & Pickett (1985) J. Biol. Chem. 260, 5820-5825]. As these two GSTs possess less than 70% sequence identity with the Ya1 and Ya2 subunits, both of Mr 25,500, the constitutively expressed Yc subunit of Mr 27,500 has been renamed Yc1 and the ethoxyquin-inducible GST of Mr 25,800 has been designated Yc2. Using this nomenclature, the two GSTs with high activity for AFB1-8,9-epoxide are Ya1Yc2 and Yc1Yc2. Although evidence suggests that induction of Yc2 is responsible for the high detoxification capacity of livers from ethoxyquin-treated rats for AFB1-8,9-epoxide, resistance towards AFB1 may be multifactorial in this instance as dietary ethoxyquin also induces the Ya1, Ya2 and Yc1 subunits about 2.2-, 10.9- and 2.7-fold respectively. Besides the induction of GST by ethoxyquin, activity towards AFB1-8,9-epoxide is also elevated in the livers of neonatal rats and in livers that contain preneoplastic nodules. Western blotting experiments show that Yc2 is not present in hepatic cytosol from adult rats fed on normal diets but is expressed in neonatal rat livers and in the livers of adult rats that contain preneoplastic nodules that have arisen as a consequence of consuming diets contaminated with AFB1.