Pi GST Research Articles

The effect of cloudy apple juice on hepatic and mammary gland phase I and II enzymes induced by DMBA in female Sprague-Dawley rats

Apples abundant in phenolic compounds show a variety of biological activities that may contribute to health beneficial effects against cardiovascular diseases, diabetes, obesity and cancer. We investigated the effect of cloudy apple juice (CAJ) on the hepatic and mammary gland carcinogen metabolizing enzymes, DNA damage and liver injury, altered by 7,12-dimethylbenz[a]anthracene (DMBA). Sprague-Dawley female rats were gavaged with CAJ (10 ml/kg b.w.) for 28 consecutive days. DMBA was administered i.p. on the 27th and the 28th days. In the liver, feeding with CAJ decreased the activities of CYP1A1 and 1A2 and increased phase II enzymes. The activities of all enzymes tested were enhanced in the animals treated with DMBA alone and in combination with CAJ. The most significant changes in the level of the hepatic enzymes tested were observed for GST alpha and NQO1. In mammary gland CAJ induced an increase in the level of GST mu and GST pi, while DMBA and CAJ combined administration elevated GST pi only. This may be beneficial as GST pi is involved in the DMBA detoxification. Additionally, pretreatment with CAJ reduced the level of most of the blood biochemical liver and kidney markers elevated as a result of DMBA treatment. These findings indicate that CAJ may interfere with enzyme system involved in carcinogen metabolism. However, this effect seems to be dependent on tissue and carcinogen and is moderately effective in the case of DMBA. Moreover, CAJ can also provide some protection against the liver and kidney damage.

In vitro and in vivo toxicological evaluation of extract and fractions from Baccharis trimera with anti-inflammatory activity

Ethnopharmacological relevanceBaccharis trimera (Less) DC. (Asteraceae), popularly known in Brazil as “carqueja”, have been used in folk medicine to treat gastrointestinal, hepatic and renal diseases, and inflammatory processes as rheumatism. Aim of the studyTo evaluate the in vitro and in vivo toxicological effects of anti-inflammatory Baccharis trimera aqueous extract and fractions. Materials and methodsAqueous extract of Baccharis trimera (AEBt) was produced by infusion in boiling water. After lyophylization AEBt was extracted with 80% ethanol, originating the ethanolic supernatant fraction (EFBt) and the aqueous sediment fraction (AFBt). Anti-inflammatory properties of AEBt, EFBt or AFBt (3, 30 or 300μg/kg b.w.) were evaluated by the carrageenan-induced mouse paw edema using indomethacin (10mg/kg) as positive control. The growth of rat hepatoma cells (HTC) and human embryo kidney epithelial cells (HEK) was determined by protein staining assay. Cytotoxicity was assayed by the tetrazolium salt (MTT) reduction. Cyclosporin was used as reference cytotoxic drug for spleen cells and doxorubicin for HTC and HEK cells. For in vivo toxicological evaluation SW male mice were daily and oral (gavage) treated with extract/fractions at 4.2mg/kg or 42mg/kg during 15 days. After treatment liver or kidney cells were submitted to comet assay to determine the DNA damage index, and the glutathione S-transferase activity was assayed towards ETHA (class Pi) and CDNB (several classes). Mutagenicity was evaluated by the Ames test using Salmonella typhimurium strains TA97, TA98, TA100, and TA102. ResultsThe anti-inflammatory effects of EFBt were higher than those of AEBt or AFBt. Mice treatment (3–300μg/kg) with AFBt reduced the paw edema (3h) at lower levels (29.2–37.3%; P<0.01), than those observed for AEBt (44.7–54.2%; P<0.001), EFBt (49.3–58.2%; P<0.001) or indomethacin (64.6%, P<0.001, 10mg/kg). The growth of kidney cells (HEK) was inhibited by AEBt (IC50 182.6μg/ml), EFBt (IC50 78.1μg/ml) and AFBt (IC50 86.2μg/ml), with lower effects on HTC hepatic cell (IC50 308.8μg/ml, 396.5μg/ml and 167.9μg/ml, respectively). As evaluated by MTT test, AFBt exhibited cytotoxicity for HEK cells (IC50 372.5μg/ml), but none for HTC ones; by the way, AFBt stimulated spleen cells (EC50 2.2μg/ml) while cyclosporine, a cytotoxic reference drug inhibited them with IC50 of 0.42μg/ml; the IC50 for doxorubicin for HEK and HTC cells was 0.28μg/ml and 14.4μg/ml, respectively, at 96h. No mutagenic potential was observed. Mice treatment with AEBt or AFBt at 42mg/kg for 15 days altered the kidney relative weight, but not at 4.2mg/kg. Baccharis trimera did not change liver, spleen or popliteal lymph node relative weight. DNA damage index of kidney cells was observed on mice treated with AEBt/AFBt, but not on animals treated with EFBt, while DNA lesions were detected on liver cells only after AFBt treatment. The general activities of hepatic GST and Pi GST were reduced by EFBt and AFBt treatment, respectively. ConclusionsBaccharis trimera did not show mutagenicity, inhibited the GST activity, a hepatic detoxification enzyme, and induced in vivo (genotoxicity) and in vitro toxicological effects to kidney cells.

Class Pi Glutathione Transferase Unfolds via a Dimeric and Not Monomeric Intermediate: Functional Implications for an Unstable Monomer

Cytosolic class pi glutathione transferase P1-1 (GSTP1-1) is associated with drug resistance and proliferative pathways because of its catalytic detoxification properties and ability to bind and regulate protein kinases. The native wild-type protein is homodimeric, and whereas the dimeric structure is required for catalytic functionality, a monomeric and not dimeric form of class pi GST is reported to mediate its interaction with and inhibit the activity of the pro-apoptotic enzyme c-Jun N-terminal kinase (JNK) [Adler, V., et al. (1999) EMBO J. 18, 1321-1334]. Thus, the existence of a stable monomeric form of wild-type class pi GST appears to have physiological relevance. However, there are conflicting accounts of the subunit's intrinsic stability since it has been reported to be either unstable [Dirr, H., and Reinemer, P. (1991) Biochem. Biophys. Res. Commun. 180, 294-300] or stable [Aceto, A., et al. (1992) Biochem. J. 285, 241-245]. In this study, the conformational stability of GSTP1-1 was re-examined by equilibrium folding and unfolding kinetics experiments. The data do not demonstrate the existence of a stable monomer but that unfolding of hGSTP1-1 proceeds via an inactive, nativelike dimeric intermediate in which the highly dynamic helix 2 is unfolded. Furthermore, molecular modeling results indicate that a dimeric GSTP1-1 can bind JNK. According to the available evidence with regard to the stability of the monomeric and dimeric forms of GSTP1-1 and the modality of the GST-JNK interaction, formation of a complex between GSTP1-1 and JNK most likely involves the dimeric form of the GST and not its monomer as is commonly reported.

Activation of the antioxidant enzyme 1-CYS peroxiredoxin requires glutathionylation mediated by heterodimerization with pi GST.

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, can protect cells against membrane oxidation through glutathione (GSH)-dependent reduction of phospholipid hydroperoxides to corresponding alcohols. However, purified native or recombinant enzyme in vitro generally lacks GSH peroxidase (GPx) activity because of oxidation of its single conserved cysteine. Reduction of the resultant oxidized cysteine is difficult because of its protected location within the homodimer formed by the oxidized protein monomers. Partial purification of 1-cysPrx from bovine lung revealed the presence of pi GST in an active preparation, while purification to homogeneity yielded enzyme that inactivated with time. We show that heterodimerization of 1-cysPrx with GSH-saturated pi GST results in glutathionylation of the oxidized cysteine in 1-cysPrx followed by subsequent spontaneous reduction of the mixed disulfide and restoration of enzymatic activity. Maximum activation of 1-cysPrx occurred with a 1:1 molar ratio of GSH-saturated pi GST and a 2:1 molar ratio of GSH to 1-cysPrx. Liposome-mediated delivery of oxidized recombinant enzyme into NCI-H441 cells that lack 1-cysPrx but express pi GST resulted in 1-cysPrx activation, whereas activation in MCF7 cells required co-delivery of pi GST. Our data indicate a physiological mechanism for glutathionylation of the oxidized catalytic cysteine of 1-cysPrx by its heterodimerization with pi GST followed by its GSH-mediated reduction and enzyme activation.

Purification and Characterisation of Hepatic Glutathione Transferases from a Herbivorous Marsupial, the Brushtail Possum ( Trichosurus vulpecula)

A single glutathione transferase isoenzyme was purified from hepatic cytosol of the brushtail possum and shown to represent 3.6 ± 0.3% of the total cytosolic protein. Characterisation of the enzyme, termed Possum GST 1–1, indicated that it possessed similar catalytic activity and structural homology with isoenzymes belonging to the alpha class of glutathione transferases. This homodimeric GST exhibited a single band with an apparent molecular mass of 25.4 kDa on sodium dodecyl sulphate-polyacrylamide gels and an apparent pI of 9.8. Inhibition studies demonstrated that Possum GST 1–1 displays binding affinity for a range of inhibitors similar to that shown by alpha class GSTs purified from other mammals. Immunoblot analysis demonstrated immuno-cross reactivity between Possum GST 1–1 and antisera raised against human alpha GST, while this GST did not cross-react with antisera raised against human mu and pi GST. N-terminal sequencing of purified Possum GST 1–1 revealed that the N-terminus of the protein is chemically blocked. Sequence analysis of three internal peptide sequences demonstrated homology with mammalian alpha GSTs. Of particular interest is the significant substrate specificity that Possum GST 1–1 displays with both organic and inorganic hydroperoxides. It is proposed that this substrate specificity is an evolutionary adaptation to a diet high in potentially toxic plant allelochemicals.

Cross-linking of human placenta pi class glutathione S-transferase dimer by chlorambucil.

Interaction of chlorambucil and the glutathione-depleted human placenta pi class glutathione S-transferase (pi GST) results in the formation of a complex between the drug and the protein at physiological pH. This complex is not formed in the presence of glutathione or S-hexylglutathione. Molecular mass measurement of the reaction product using matrix-assisted laser desorption mass spectrometry indicates that one molecule of chlorambucil cross-links two subunits of the homodimeric protein. A combination of enzymic proteolysis, high performance liquid chromatography, and mass spectrometry reveals that chlorambucil alkylation occurs at cysteine 47 of one subunit and cysteine 101 of the second subunit. This result supports the idea that conformational changes occur in glutathione-depleted pi GST, which allow the bifunctional tether of chlorambucil to cross-link the two subunits of the protein.

Appearance of oval cells in the liver of rats after long-term exposure to ethanol

Epidemiological studies show an increased risk of developing liver cancer among alcoholics. There is some agreement that ethanol itself is not carcinogenic, but it may enhance the tumorigenic process by inducing drug-metabolizing enzymes, suppression of the immune system or by affecting DNA repair enzymes. Precisely how ethanol predisposes or promotes the development of hepatoma is unknown. Hepatocarcinogenesis induced by a choline-deficient, ethionine-supplemented (CDE) diet produces extensive alteration of the liver architecture with the emergence and rapid proliferation of oval cells. This study examines whether chronic alcohol consumption induces the proliferation of oval cells. Oval cells induced in rats maintained on a 5% ethanol liquid diet (ELD) for up to 24 months, or fed a CDE diet for up to 4 weeks, are compared using a panel of liver-specific markers. In CDE-treated rats, oval cells staining positively for alpha-fetoprotein (AFP), pi-class glutathione S-transferase (pi GST), and the embryonic form of pyruvate kinase (M2-PK) are observed after 1 week. Similar cells are seen in ELD-treated rats after 2 months. Their numbers increase with time, and incorporation of [3H]thymidine confirms they are a dividing population. Acute damage induced by partial hepatectomy and CCI4 poisoning did not induce the appearance of oval cells. We conclude that chronic ethanol consumption induces oval cell proliferation. We suggest that, in addition to other proposed mechanisms, an alteration in cellular composition of the liver be considered as an explanation for the increased incidence of liver cancer among alcoholics. (Hepatology 1996 Jan;23(1):145-54)

Appearance of oval cells in the liver of rats after long-term exposure to ethanol.

Epidemiological studies show an increased risk of developing liver cancer among alcoholics. There is some agreement that ethanol itself is not carcinogenic, but it may enhance the tumorigenic process by inducing drug-metabolizing enzymes, suppression of the immune system or by affecting DNA repair enzymes. Precisely how ethanol predisposes or promotes the development of hepatoma is unknown. Hepatocarcinogenesis induced by a choline-deficient, ethionine-supplemented (CDE) diet produces extensive alteration of the liver architecture with the emergence and rapid proliferation of oval cells. This study examines whether chronic alcohol consumption induces the proliferation of oval cells. Oval cells induced in rats maintained on a 5% ethanol liquid diet (ELD) for up to 24 months, or fed a CDE diet for up to 4 weeks, are compared using a panel of liver-specific markers. In CDE-treated rats, oval cells staining positively for alpha-fetoprotein (AFP), pi-class glutathione S-transferase (pi GST), and the embryonic form of pyruvate kinase (M2-PK) are observed after 1 week. Similar cells are seen in ELD-treated rats after 2 months. Their numbers increase with time, and incorporation of [3H]thymidine confirms they are a dividing population. Acute damage induced by partial hepatectomy and CCI4 poisoning did not induce the appearance of oval cells. We conclude that chronic ethanol consumption induces oval cell proliferation. We suggest that, in addition to other proposed mechanisms, an alteration in cellular composition of the liver be considered as an explanation for the increased incidence of liver cancer among alcoholics.

The expression of glutathione transferase isoenzymes in human malignant lymphoma biopsies.

Glutathione transferase (GST) activity as well as the expression of different classes of GST isoenzymes were studied in 14 lymphoma biopsies. The GST activity measured with 1-chloro-2,4-dinitrobenzene as a substrate, varied almost 9-fold. The expression of GSTs classes Pi, Alpha and Mu was studied by immunoblotting using antibodies against human GSTs. All lymphoma samples displayed high levels of class Pi GST. Class Alpha and Mu GSTs expression varied from not detectable to high. The observations were confirmed by quantitation of the three classes of GST with an ELISA technique. Nine of the patients were treated with bifunctional alkylating agents. A correlation between a clinical complete response to chemotherapy and low expression of GST Alpha was noted (p < 0.02).

Conjugation of microsome generated and synthetic aflatoxin B 1-8,9 epoxide and styrene oxide to glutathione by purified glutathione S-transferases from hamster and mouse livers

Glutathione (GSH) conjugation of microsome-mediated and synthetic aflatoxin B 1 (AFB 1)-epoxide and styrene oxide has been studied with purified glutathione transferases (GSTs) from mouse and hamster liver cytosols. In hamster, with microsomally activated epoxide, the alpha group of GSTs show about 10-fold more activity than the mu group. With the synthetic AFB 1 epoxide, the mu enzymes designated H 3B and C show considerable activity although less than alpha, whereas H 3A and D demonstrate similar ranges of activity as the alpha group. The pi class of GST could not be assayed due to its absence in the hamster liver. The mouse liver cytosols show 3.6-fold greater activity than hamster cytosol in microsome mediated assay system. The mouse alpha and mu enzymes have similar levels of activity in the microsome mediated system; this activity could not be determined with the pi GST due to shortage of this enzyme. The alpha group has 2- and 5-fold higher activity than mu and pi group of GSTs, respectively, with the synthetic epoxide of AFB 1. With styrene oxide, the purified GSTs from hamster liver show total loss of activity whereas in the mouse alpha, mu and pi classes of GSTs have similar range of activity as the cytosol. The role of alpha and mu isozymes of GST in rendering these animals resistant to hepatocarcinogenecity is suggested.

Gender-related differences in expression of murine glutathione S-transferases and their induction by butylated hydroxyanisole

The basal levels of mu and pi class glutathione S-transferases RNA were 18-fold higher in the male mouse liver as compared with the female. When 0.75% (w/w) BHA was included in the diet it altered the RNA levels of alpha, mu, pi GST classes and mGSTA4-4 in a tissue and sex specific manner. The most marked induction of RNA was seen for the mu class GSTs of female liver, lung and kidney (52, 10 and 8-fold, respectively), and of male liver and kidney (25 and 3.5-fold, respectively), the pi class GSTs of female liver, lung, and kidney (11, 10, and 5-fold, respectively), and mGSTA4-4 of female liver (4-fold). The effect of BHA on the induction of the mu and pi class GST RNA was 2-9 fold greater in female as compared with male tissues. The degree of induction of GST RNA did not correlate directly with changes in GST protein indicating that post-transcriptional events regulating GST expression may be affected by BHA particularly for GST mu and mGSTA4-4.

Glutathione S-transferase in bone marrow metastases of disseminated neuroblastoma.

Antisera to each of the three main cytosolic forms of glutathione S-transferase (GST; alpha, mu, and pi) has been used to characterise GST expression by metastatic neuroblastoma in bone marrow trephine biopsies taken from 15 patients at presentation and from five of this group at relapse. There was no correlation between expression of extra-nuclear alpha or mu GST and outcome, and no consistent pattern at relapse. Seven of eight expressing nuclear pi GST at presentation died of resistant disease. Three of five cases with no detectable nuclear pi class GST remain alive and disease free. The results provide no encouragement for further investigation of alpha or mu GST in this disease but larger studies of uniformly treated patients may show whether nuclear pi GST expression at presentation indicates likely relapse.

Isoenzyme-specific quantitative immunoassays for cytosolic glutathione transferases and measurement of the enzymes in blood plasma from cancer patients and in tumor cell lines

Enzyme-linked immunoassays (ELISAs) based on the double-antibody sandwich technique have been developed for the quantitative analysis of the major human cytosolic class Pi, Mu and Alpha glutathione transferases (GSTs). The procedures were optimized with respect to antibody concentration for coating of plates as well as other parameters in order to achieve high sensitivity and accuracy. No cross-reactivity was detected between members of the three different classes of GSTs or among the Mu class GSTs M2-2, M3-3 and M4-4 with the ELISA for GST M1-1. The ELISAs have been applied to establish the cytosolic GST profiles of 10 cell lines and to monitor the plasma GST levels in cancer patients. The results revealed that the class Pi GST was the dominant isoenzyme in six (LS 174T, HCT-8, Hu 549 Pat, K-562, U-937 and Hu 549) out of nine tumor cell lines and immortalized hepatocytes (Chang Liver). The isoenzymes A1-1 and M1-1 were determined to be the major GST components in Hep G2 and HeLa cells, respectively. In a clinical study, the majority of the patients with urinary bladder cancer were found to have increased plasma levels of both GST A1-1 and GST P1-1 (10/15), while patients with renal cancer frequently showed increases only in GST P1-1 (5/8). The results demonstrate that the ELISAs are suitable for analyzing GST phenotypes in both normal and tumor cells and in monitoring plasma levels of GSTs in cancer patients.

EXPRESSION OF GLUTATHIONE-S-TRANSFERASE (GST) SUBTYPES IN RESECTED LUNG-TUMORS - RELATIONSHIP TO HISTOLOGICAL TYPE AND PROLIFERATION INDEXES

Samples from 19 resected lung tumours have been analysed for the levels of GST alpha, mu and pi mRNA and protein. No significant survival disadvantage was found for patients with tumours containing any of the GST subtypes at the levels of either protein or mRNA. Both GST mu and GST pi mRNAs were expressed at significantly higher levels in squamous carcinomas than adenocarcinomas but GST alpha mRNA was not. The significant increases in expression of mu and pi GST in squamous relative to adenocarcinomas of the lung is not related to differences in the proliferation rates between these cancers.

Squid glutathione S-transferase. Relationships with other glutathione S-transferases and S-crystallins of cephalopods.

Glutathione S-transferase (GST, EC 2.5.1.18) was purified from the digestive gland of the squid Ommastrephes sloani pacificus. It had high enzymatic activity for the 1-chloro-2,4-dinitrobenzene substrate and was composed of a major and a minor polypeptide band, both with molecular masses near 25 kDa on SDS-polyacrylamide gels. GST cDNA clones were derived from the digestive gland mRNA. The deduced GSTs of the longest cDNAs (pGST5 and pGST11) containing the entire coding sequence have a molecular mass near 23 kDa. Sequence comparisons showed that the squid GST is 42-44% identical to both squid and octopus S-crystallins (the major proteins of the lens), 32-34% identical to class pi and 29-32% identical to class alpha GSTs of vertebrates, and 19-23% identical to other GSTs of vertebrates and insects. Northern blot hybridization revealed that GST mRNAs were much more abundant in the digestive gland than in the testis, mantle, or lens. Analysis of a squid GST gene indicated that it has an exon-intron structure similar to that of the vertebrate class pi GST gene. An apparently novel repetitive element was identified in the 5'-flanking sequence of the squid GST gene. Our results suggest that multiple duplications of an ancestral GST gene gave rise to a family of enzymatically inactive crystallins specialized for lens refraction and one (or two) active GST enzyme expressed preferentially, but not exclusively, in the digestive gland in squids. This differs from the innovation of refractive function from a metabolic enzyme by increased expression in the lens with minimal or no gene duplication, as occurred among the enzyme-crystallins of vertebrates.

What role do glutathione S-transferases play in the cellular response to ionizing radiation?

The glutathione S-transferases (GST's) are cytosolic dimeric proteins that are composed of three family members, alpha, pi, and mu, and a fourth microsomal member. These four family members are primarily involved in cellular detoxification of xenobiotics and hydroperoxides. Recently, a strong correlation has been found between the overexpression of GST's and resistance to chemotherapeutic drugs. In comparison to chemotherapy, little is known about the role of GST's in the cellular response to ionizing radiation. To determine which GST's may be involved in this response, we have identified Chinese hamster ovary cell lines that possess different levels of alpha and pi GST isozyme activity. The survival of these cell lines to ionizing radiation was similiar to that of wild-type Chinese hamster ovary-KI cells from which they were derived. Although differences in GST levels did not affect ionizing radiation sensitivity per se, we found that ionizing radiation decreased the amount of cytosolic pi GST without affecting alpha GST levels. Taken together, these data suggest that GST's are involved in the cellular response against oxidative stress generated by ionizing radiation.

Distribution of glutathione S-transferase isoenzymes in primary epithelial tumors of the ovary

Rahilly M, Al Nafussi A, Harrison DJ. Distribution of glutathione S-transferase isoenzymes in primary epithelial tumors of the ovary. Int J Gynecol Cancer 1991; 1: 268–274. The glutathione S-transferases play a central role in the detoxification of electrophilic xenobiotics, including cytotoxic drugs and carcinogens, by conjugation with reduced glutathione. There are four major classes of human GST: pi, alpha, mu and microsomal GST. GST pi has been implicated in the progression of carcinogenesis in both animal and human studies. This study examined constitutive GST isoenzyme expression in 60 primary epithelial tumors of the ovary, including benign, borderline and frankly malignant categories. GST pi was the predominant isoenzyme, being strongly expressed in all tumor types. The intensity of GST pi staining was independent of grade in all categories of carcinoma. Thus GST pi has no potential role as a marker of differentiation or tumor progression in epithelial ovarian tumors. GST alpha, which localizes to steroid-producing cells in normal ovary, was useful in demonstrating enzymatically active stromal cells in ovarian carcinomas with functioning stroma. GSTs, particularly the alpha and microsomal isoenzymes, have been implicated in both intrinsic and acquired resistance to cancer chemotherapy. Alpha, mu and microsomal GSTs displayed a heterogeneous staining pattern in all tumor types. This heterogeneity of expression may contribute to intrinsic drug resistance in ovarian carcinomas.

Mutation of an evolutionarily conserved tyrosine residue in the active site of a human class Alpha glutathione transferase

Human class Alpha glutathione transferase (GST) A1—1 has been subjected to site-directed mutagenesis of a Tyr residue conserved in all classes of cytosolic GSTs. The change of Tyr 8→Phe lowers the specific activities with three substrates to 2–8% of the values for the wild-type enzyme. The changes in the kinetic parameters k cat/ K max, V max and S 0.5 show that the decreased activities are partly due to a reduced affinity for glutathione. The effect is reflected in lowered k cat values, suggesting that the hydroxyl group of Tyr 8 is involved in the activation of glutathione. The proposal of such a role for the Tyr residue has support from the 3D structure of a pig lung class Pi GST [Reinemer et al. (1991) EMBO J. 10, 1997–2005]. Thus, Tyr 8 appears to be the first active site residue established as participating in the chemical mechanism of a GST.

Drug-metabolizing enzyme expression in human normal, peritumoral and tumoral colorectal tissue samples

In the present study, we investigated Phase I (cytochrome P450; DT-diaphorase, DTD) and Phase II (epoxide hydrolase, EH; glutathione-S-transferases, GSTs) enzymes in normal colon from patients without colorectal adenocarcinoma and in peritumoral and tumoral tissues from patients with colorectal adenocarcinoma. No significant changes in levels of cytochrome P450IIIA4 (the only P450 detectable in this tissue), EH, GSTs and DTD activity were found between normal and peritumoral tissues. In tumoral tissue, compared with peritumoral tissues, we observed significant decreases in cytochrome P450IIIA4 (-50%, P less than 0.002) and EH (-60%, P less than 0.03), no change in DTD activity and significant increases in GST pi (+40%, P less than 0.03) and total GST activity (+30%, P less than 0.01). The numerous changes observed in tumoral tissues suggest that variations in drug-metabolizing enzyme expression in colorectal adenomatous polyps could represent pretumoral markers. Moreover, a better understanding of the expression of these enzymes in tumoral tissues would help us to choose the most appropriate colon tumor cell lines for the testing of new anti-cancer drugs.

Effects of inducers of drug metabolism on basic hepatic forms of mouse glutathione transferase.

The cytosolic glutathione transferases (GSTs) with basic pI values have been studied in mouse liver after treatment with 2,3-t-butylhydroxyanisole (BHA), cafestol palmitate (CAF), phenobarbital (PB), 3-methylcholanthrene (3-MC) and trans-stilbene oxide (t-SBO). The cytosolic GST activity was induced by all compounds except for 3-MC. Three forms of GST were isolated by means of affinity chromatography and f.p.l.c. The examination of protein profiles and enzymic activities with specific substrates showed that the three GSTs correspond to those found in control animals, i.e. GSTs MI, MII and MIII. The class Mu GST MIII accounted for the major effect of induction, whereas the class Alpha GST MI and the class Pi GST MII were unchanged or somewhat down-regulated. The greatest induction was obtained with BHA, PB and CAF. The activities of other glutathione-dependent enzymes were also studied. An increase in glutathione reductase and thioltransferase activities was observed after BHA, PB or CAF treatment; glyoxalase I and Se-dependent glutathione peroxidase were depressed in comparison with the control group in all cases studied.