GST Pi Research Articles

Acetaminophen Metabolism Does Not Contribute to Gender Difference in Its Hepatotoxicity in Mouse

Gender is an important factor in pharmacokinetics and pharmacodynamics. In the current study, gender difference in acetaminophen (APAP)-induced hepatotoxicity has been examined. Male and female mice were injected with a toxic dose of APAP (500 mg/kg, ip). Female mice were resistant to the hepatotoxic effects of APAP, depicted by serum alanine aminotransferase and sorbital dehydrogenase activities and histological analysis. Basal hepatic reduced glutathione (GSH) levels were lower in females than in males, suggesting that basal GSH level may not be a factor in determining the gender difference of APAP hepatotoxicity. APAP metabolism was slower in females than males, revealed by lower levels of glucuronidation and sulfation and higher amounts of free APAP in the livers of female mice. Lower basal Cyp1a2 mRNA levels and lower expression of Cyp1a2 and Cyp3a11 mRNAs after APAP dosing were also observed in females compared with males. However, there was no gender difference in N-acetyl-p-benzoquinone imine covalent binding 2 h after APAP administration, suggesting similar APAP bioactivation between genders. Moreover, liver Gst pi mRNA levels were significantly lower in females than males. This finding is consistent with a previous report, which showed that Gst pi knockout mice are protected from APAP-induced liver toxicity. In conclusion, gender difference of APAP-induced hepatotoxicity is not likely due to APAP metabolism. Perhaps, it is in part due to gender-dependent Gst pi expression. However, the mechanism underlying the association between reduction in Gst pi expression and hepatoprotective effect against APAP toxicity remains to be further explored.

Direct Evidence for Complex Formation Between 1‐Cys Peroxiredoxin and Glutathione S‐Transferase pi

A heterodimer complex forms between 1-Cys peroxiredoxin (1-Cys Prx) with a C-terminal His-tag and glutathione S-transferase pi (GST pi) upon incubation at pH 8.0 in buffer containing 20% 1,6-hexanediol, followed by dialysis against buffer containing glutathione (GSH) but lacking 1,6-hexanediol. The heterodimer can be purified by chromatography on nickel-nitriloacetic acid agarose in the presence of GSH. N-terminal sequencing shows the two proteins are present in equimolar amounts. In the heterodimer, 1-Cys Prx is fully active, while the GST pi activity is ~25% of that of the GST pi homodimer. In contrast, the 1-Cys Prx homodimer is inactive in the presence of free GSH. The yield of heterodimer is decreased in the absence of 1,6-hexanediol or of GSH. Rapid glutathionylation of 1-Cys Prx in the heterodimer is detected by immunoblotting. Subsequently, a disulfide-linked dimer is observed on SDS-PAGE and the free cysteine content is decreased by 2/heterodimer. Based on mutagenesis, for Prx, neither Cys-47 nor Ser-32 is required for heterodimer formation, but Cys-47 is essential for Prx activation. For GST pi, Cys-47 and Tyr-7 (at the GSH-binding site) are needed for heterodimer formation. We conclude that reactivation of oxidized 1-Cys Prx by GST pi occurs by heterodimerization of 1-Cys Prx and GST pi harboring bound GSH, followed by glutathionylation of 1-Cys Prx and then formation of an inter-subunit disulfide. Finally, the GSH-mediated reduction of the disulfide regenerates the active site sulfhydryl of 1-Cys Prx. (Supp. By NIH R01-CA66561, F31-GM75387 & P01-HL79063.)

Direct Evidence for the Formation of a Complex between 1-Cysteine Peroxiredoxin and Glutathione S-Transferase π with Activity Changes in Both Enzymes

Glutathione S-transferase pi (GST pi) has been shown to reactivate oxidized 1-cysteine peroxiredoxin (1-Cys Prx, Prx VI, Prdx6, and AOP2). We now demonstrate that a heterodimer complex is formed between 1-Cys Prx with a C-terminal His6 tag and GST pi upon incubation of the two proteins at pH 8.0 in buffer containing 20% 1,6-hexanediol to dissociate the homodimers, followed by dialysis against buffer containing 2.5 mM glutathione (GSH) but lacking 1,6-hexanediol. The heterodimer can be purified by chromatography on nickel-nitriloacetic acid agarose in the presence of GSH. N-Terminal sequencing showed that equimolar amounts of the two proteins are present in the isolated complex. In the heterodimer, 1-Cys Prx is fully active toward either H2O2 or phospholipid hydroperoxide, while the GST pi activity is approximately 25% of that of the GST pi homodimer. In contrast, the 1-Cys Prx homodimer lacks peroxidase activity even in the presence of free GSH. The heterodimer is also formed in the presence of S-methylglutathione, but no 1-Cys Prx activity is found under these conditions. The yield of heterodimer is decreased in the absence of 1,6-hexanediol or GSH. Rapid glutathionylation of 1-Cys Prx in the heterodimer is detected by immunoblotting. Subsequently, a disulfide-linked dimer is observed on SDS-PAGE, and the free cysteine content is decreased by 2 per heterodimer. The involvement of particular binding sites in heterodimer formation was tested by site-directed mutagenesis of the two proteins. For 1-Cys Prx, neither Cys47 nor Ser32 is required for heterodimer formation but Cys47 is essential for 1-Cys Prx activation. For GST pi, Cys47 and Tyr7 (at or near the GSH-binding site) are needed for heterodimer formation but three other cysteines are not. We conclude that reactivation of oxidized 1-Cys Prx by GST pi occurs by heterodimerization of 1-Cys Prx and GST pi harboring bound GSH, followed by glutathionylation of 1-Cys Prx and then formation of an intersubunit disulfide. Finally, the GSH-mediated reduction of the disulfide regenerates the reduced active-site sulfhydryl of 1-Cys Prx.

Inhibition of GSTpi Activity Increases the Sensitivity of MCL Cell Lines to Cisplatin, Cytarabin and Bortezomib, but Not Doxorubicin.

Preliminary data suggest drug resistance plays a major role in mantle cell lymphoma (MCL) and is linked to the aberrant expression of molecules such as Glutathion S-Transferase pi (GST pi) that catalyzes the nuclear conjugation of a broad variety of reactive electrophiles to the glutathion. We investigated in vitro the effect of the inhibition of GSTpi activity on the chemosensitivity of MCL cell lines by inhibiting the nuclear transport of GSTpi with Agaricus bisporus leptine (ABL).Methods. Four MCL cell lines (Granta, NCEB, REC and UPN1) were analyzed for GSTP1 transcript expression by RT-PCR analysis and for GSTP1 genetic Ile105Val polymorphism. The effect of ABL on the 4 MCL cell lines viability was examined using cells pre-treated with ABL (40μg/ml) for 10 h followed by treatment with various concentrations of doxorubicin (DOX), cisplatin (CDDP), cytarabin and bortezomib for 48 h. The cell viability was estimated by MTT assay repeated three times.Results. All the 4 cell lines expressed GSTPI transcript. Two of them (UPN1 and REC) were heterozygous for Ile105Val polymorphism, whereas NCEB and GRANTA were homozygous GSTP1-105Ile which has been correlated with an enzyme of high activity. DOX, CDDP, cytarabin and bortezomib have cytotoxic effects compared to non-treated cells, without any difference between the 4 MCL cell lines when considering their genetic polymorphism status. Co-administration of ABL caused a significant increasis of the cytotoxicity of CDDP and bortezomib in all cell lines (Student test: p between 0.0004 to 0.02 for CDDP; and p between 0.013 and 0.05 for velcade, depending on the cell lines). Co-administration of ABL caused an increasis of the cytotoxicity of cytarabin in 3 cell lines, the 4th being actually tested. No influence of ABL was detected on the cytotoxic effect of DOX. The analysis of the nuclear and the cytoplasmic localizations of GSTpi is currently realized by immunohistochemistry.Conclusion. These results suggest that inhibition of the nuclear transfer of GSTpi increases in vitro the MCL sensitivity to CDDP, cytarabin and velcade but not DOX.

Activity and expression of glutathione S-transferase pi in patients with amyotrophic lateral sclerosis

Glutathione S-transferase (GST, EC 2.5.1.18) is an enzyme responsible for inactivation of a large variety of toxic, electrophilic compounds and organic peroxides. GST activity and GST pi expression were studied in patients with amyotrophic lateral sclerosis (ALS). Studies were conducted on cerebrospinal fluid (CSF), blood serum and peripheral blood mononuclear cells (PBMC) obtained from 40 ALS patients. CSF from 30 subjects without neurological diseases and blood from 40 healthy blood donors were used as controls. GST activity assayed with 1-chloro-2,4-dinitrobenzene (substrate for transferase activity) and cumene peroxide (substrate for peroxidase activity) was significantly decreased in PBMC of ALS patients, as well as the GST pi expression on both mRNA and protein level. The mean peroxidase activity was however significantly increased in CSF and serum of ALS patients with the specificity of 80% and 73%, and the sensitivity of 78% and 85%, respectively. It can thus be concluded that the protective barrier formed by GST is originally affected in peripheral blood of ALS patients, and may increase their vulnerability to toxic effects of electrophilic compounds and organic peroxides. Studies on a larger group are needed to answer the question whether GSH-Px determination may be implicated in the diagnosis of ALS.

A Study of Glutathione S-transferase pi Expression in Central Nervous System of Subjects with Amyotrophic Lateral Sclerosis Using RNA Extraction from Formalin-Fixed, Paraffin-Embedded Material

The expression of glutathione S-transferase pi (GST pi), an enzyme responsible for inactivation of a large variety of toxic compounds was studied in spinal cord, motor and sensory brain cortex obtained from patients who died in the course of amyotrophic lateral sclerosis (ALS). The studies were performed on formalin-fixed, paraffin-embedded (FFPE) and freshly frozen tissues. The method of RNA isolation from FFPE was modified. A significant decrease of GST pi-mRNA expression was found in cervical spinal cord and motor brain cortex of ALS subjects comparing to analogue control tissues (P<0.01), as well as in motor cortex of ALS subjects comparing to their sensory cortex (P<0.05). In spinal cords the decrease in GST pi-mRNA expression was accompanied by a decrease of GST pi protein level. Results indicated lowered GST pi expression on both mRNA and protein levels in the regions of nervous system affected by ALS. The non-properly inactivated by GST toxic electrophiles and organic peroxides may thus contribute to motor neurons damage.

Flipping the Epigenetic Switch

Just as genetic instability is a hallmark of malignant cells, so too is epigenetic instability.1,2 A key difference is that, in contrast to permanent genetic alterations, epigenetic changes need not be permanent in either tumor cells or normal cells. Thus, when a cell adopts a new gene expression pattern as a result of epigenetic changes, that cell and its progeny reserve the capability to switch back to previous gene expression patterns. This phenotypic plasticity would provide epigenetic metastable or unstable cancer cells the ability to alter gene expression and appropriately adapt to its environment, as it takes its destructive journey from primary lesion to metastatic colony. For example, it is reasonable to propose that cancer cells would likely directly modulate the expression of genes involved in cell adhesion and motility or the transcription factors that control their expression during cancer progression. Early in cancer development these genes remain active in the primary tumor, however, as natural selection occurs, these genes need be silenced so that the tumor has the necessary phenotypic characteristics to metastasize. Following cancer cell colonization at a distant site, the malignant tumor may require reacquisition of those gene products that suppress the properties of motility and invasion, for example. Regulation of the expression of this complement of genes, through DNA methylation and related epigenetic mechanisms, provides a unique switch mechanism in tumor cells that is not as drastic as mutation. Interestingly, some tumor suppressor genes, such as p53, are typically mutated and deleted, and rarely, if ever, are silenced in association with DNA methylation.3 In contrast, some genes with tumor suppressor function, rarely, if ever, are mutated, and often are silenced by aberrant DNA methylation. Examples of this latter class of genes include MGMT, maspin, GST pi, and as is further reported here, ERβ in prostate cancer.4–10

Glutathione S-transferase pi as a target for tricyclic antidepressants in human brain.

GST pi, the main glutathione S-transferase isoform present in the human brain, was isolated from various regions of the brain and the in vitro effect of tricyclic antidepressants on its activity was studied. The results indicated that amitripyline and doxepin--derivatives of dibenzcycloheptadiene, as well as imipramine and clomipramine--derivatives of dibenzazepine, inhibit the activity of GST pi from frontal and parietal cortex, hippocampus and brain stem. All these tricyclics are noncompetitive inhibitors of the enzyme with respect to reduced glutathione and noncompetitive (amitripyline, doxepin) or uncompetitive (imipramine, clomipramine) with respect to the electrophilic substrate. Their inhibitory effect is reversible and it depends on the chemical structure of the tricyclic antidepressants rather than on the brain localization of the enzyme. We conclude that the interaction between GST pi and the drugs may reduce their availability in the brain and thus affect their therapeutic activity. On the other hand, tricyclic antidepressants may decrease the efficiency of the enzymatic barrier formed by GST and increase the exposure of brain to toxic electrophiles. Reactive electrophiles not inactivated by GST may contribute in adverse effects caused by these drugs.

Glutathione S-transferase pi as a target for tricyclic antidepressants in human brain.

GST pi, the main glutathione S-transferase isoform present in the human brain, was isolated from various regions of the brain and the in vitro effect of tricyclic antidepressants on its activity was studied. The results indicated that amitripyline and doxepin--derivatives of dibenzcycloheptadiene, as well as imipramine and clomipramine--derivatives of dibenzazepine, inhibit the activity of GST pi from frontal and parietal cortex, hippocampus and brain stem. All these tricyclics are noncompetitive inhibitors of the enzyme with respect to reduced glutathione and noncompetitive (amitripyline, doxepin) or uncompetitive (imipramine, clomipramine) with respect to the electrophilic substrate. Their inhibitory effect is reversible and it depends on the chemical structure of the tricyclic antidepressants rather than on the brain localization of the enzyme. We conclude that the interaction between GST pi and the drugs may reduce their availability in the brain and thus affect their therapeutic activity. On the other hand, tricyclic antidepressants may decrease the efficiency of the enzymatic barrier formed by GST and increase the exposure of brain to toxic electrophiles. Reactive electrophiles not inactivated by GST may contribute in adverse effects caused by these drugs.

Identification of the Ligand Binding Sites and Novel Drug Target Molecules by Photoaffinity Labeling

AbstractFor Abstract see ChemInform Abstract in Full Text.

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase pi.

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase pi (GST pi): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST pi with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST pi at pH 7.0 and 25 degrees C as assayed using mBBr as substrate, with a lesser effect on the enzyme's use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST pi with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.

Antioxidant enzymatic defences in human follicular fluid: characterization and age-dependent changes.

The aim of this work was to study the antioxidant enzymatic defences in human follicular fluid and investigate their possible changes during reproductive ageing. To this end, we tested the specific activities and protein expression of enzymes involved in reactive oxygen species (ROS) scavenging and in detoxification of ROS byproducts in follicular fluid from young (range 27-32 years, n = 12) and older (range 39-45 years, n = 12) women participating in an IVF programme. Results show that all the tested enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione transferase, glutathione reductase] were significantly expressed in human follicular fluid. However, when the two age groups were compared, we found that follicular fluid from older women exhibited a reduced level of glutathione transferase and catalase activities and a higher level of SOD activity. Immunoblot analysis revealed that ageing was associated with decreased protein expression of GST Pi isoform and did not affect SOD and catalase protein expression. Taken together, these findings indicate that reproductive ageing is accompanied by a change in the antioxidant enzymatic pattern that could impair ROS scavenging efficiency in the follicular environment.

Identification of the ligand binding sites and novel drug target molecules by photoaffinity labeling

Photoaffinity labeling is a useful and reliable method for 1) the identification of the ligand-target receptor and 2) the structural investigation of its binding site. Using photoaffinity labeling techniques, the binding sites of four typical calcium antagonists, 1,4-dihydropyridines, benzothiazepines, phenylalkylamines, and benzothiazines, were successfully identified within the primary structure of the skeletal muscle calcium channels. The results confirm pharmacological observations of the four antagonists, which had been proposed to interact allosterically with each other. Secondarily we demonstrated that human glutathione S-transferase class pi (GST pi) is specifically photolabeled by the antidiabetic agent sulfonylurea glibenclamide (GB) and it also inhibits the enzyme activities of glutathione conjugation by GB in a competitive manner for glutathione. These results indicate that GST pi is another target molecule of sulfonylurea since a subunit of ATP-sensitive potassium channels is well known to be a sulfonylurea receptor. This review focuses on photoaffinity labeling techniques as a useful tool for drug discovery and development.

Protein expression profiling of glutathione S-transferase pi null mice as a strategy to identify potential markers of resistance to paracetamol-induced toxicity in the liver.

GST pi (GSTP) is a member of the glutathione S-transferase (EC 2.5.1.18; GST) family of enzymes that catalyse the conjugation of electrophilic species with reduced glutathione and thus play an important role in the detoxification of electrophilic metabolites. Deletion of GSTP in mice has previously been shown to lead to enhanced susceptibility to chemical-induced skin carcinoma, consistent with its known metabolic functions. A decreased susceptibility to paracetamol hepatotoxicity has also been observed, which has not been fully explained. One possibility is that deletion of the GSTP gene locus results in compensatory changes in other proteins involved in defence against chemical stress. We have therefore used complementary protein expression profiling techniques to perform a systematic comparison of the protein expression profiles of livers from GSTP null and wild-type mice. Analysis of liver proteins by two-dimensional electrophoresis confirmed the absence of GSTP in null mice whereas GSTP represented 3-5% of soluble protein in livers from wild-type animals. There was a high degree of quantitative and qualitative similarity in other liver proteins between GSTP null and wild-type mice. There was no evidence that the absence of GSTP in null animals resulted in enhanced expression of other GST isoforms in the null mice (GST alpha, 1.48%, GST mu, 1.68% of resolved proteins) compared with the wild-type animals (GST alpha, 1.50%, GST mu, 1.40%). In contrast, some members of the thiol specific antioxidant family of proteins, notably antioxidant protein 2 and thioredoxin peroxidases, were expressed at a higher level in the GSTP null mouse livers. These changes presumably reflect the recently described role of GSTP in cell signalling and may underlie the protection against paracetamol toxicity seen in these animals.

Enhanced immunocytochemical expression of antioxidant enzymes in rat submandibular gland after normobaric oxygenation

In order to clarify the role of antioxidant enzymes in the male rat submandibular gland against short-term normobaric oxygenation, we performed immunocytochemical staining of manganese-containing superoxide dismutase (Mn-SOD), copper- and zinc-containing SOD (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase, and glutathione S-transferases (GST alpha, GST mu, and GST pi) between days 1 and 7 after normobaric oxygenation. Ultrastructural alterations and immunoreactivities for malondialdehyde (MDA), a lipid peroxidation-related molecule, of the acinar and ductal cells after the oxygenation were also investigated. Immunoreactivity for MDA was exhibited in the acinar cells throughout the experiment. On the other hand, immunoreactivity for the SODs, CAT, and GSTs was not altered, when compared to that of controls, but was significantly elevated in the granular, striated, and excretory ductal cells. Since an increase of lipid peroxidation as indicated by enhanced immunoreactivity for MDA was detected in the acinar and intercalated ductal cells, the results indicate that the enhanced antioxidant enzymes in the granular, striated, and excretory ductal cells play a crucial role in the self-defense system of the male rat submandibular gland against normobaric oxygenation.

Cloning and expression of a novel Mu class murine glutathione transferase isoenzyme.

The present study describes the cDNA cloning, expression and characterization of a novel Mu class murine glutathione transferase (GST) isoenzyme. Screening of a cDNA library from the small intestine of a female A/J mouse using consensus probes derived from Mu class murine GST genes (mGSTM1-mGSTM5) resulted in the isolation of a full-length cDNA clone of a previously unknown Mu class GST gene (designated as mGSTM7). The choice of tissue was based on our previous identification in female A/J mouse small intestine of a potentially novel Mu class GST isoenzyme. The deduced amino acid sequence of mGSTM7, which comprises of 218 amino acid residues, exhibited about 67-78% identity with other Mu class murine GSTs. Recombinant mGSTM7-7 cross-reacted with anti-(GST Mu) antibodies, but not with anti-(GST Alpha) or anti-(GST Pi) antibodies. The pI and the reverse-phase-HPLC elution profile of recombinant mGSTM7-7 were different from those of other Mu class murine GSTs. The substrate specificity of mGSTM7-7 was also different compared with other Mu class murine GSTs. Interestingly, mGSTM7 had a higher identity with the human Mu class isoenzyme hGSTM4 (87% identity and 94% similarity in the amino acid sequence) than with any of the known mouse Mu class GSTs. Specific activities of recombinant mGSTM7-7 and human GSTM4-4 were comparable towards several substrates. For example, similar to hGSTM4-4, recombinant mGSTM7-7 was poorly active in catalysing the GSH conjugation of 1-chloro-2,4-dinitrobenzene and ethacrynic acid, and lacked activity towards 1,2-dichloro-4-nitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane. These results suggested that hGSTM4-4 might be the human counterpart of mouse GSTM7-7. Reverse transcription-PCR analysis using mGSTM7-specific primers revealed that mGSTM7 is widely expressed in tissues of female A/J mice, including liver, forestomach, lung, kidney, colon and spleen.

Enhancement of the chemoprotective enzymes glucuronosyl transferase and glutathione transferase in specific organs of the rat by the coffee components kahweol and cafestol.

The coffee components kahweol and cafestol (K/C) have been reported to protect the colon and other organs of the rat against the formation of DNA adducts by 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and aflatoxin B1. PhIP is a cooked-food mutagen to which significant human exposure and a role in colon cancer etiology are attributed, and, interestingly, such cancers appear to develop at a lower rate in consumers of coffees with high amounts of K/C. Earlier studies in rodent liver have shown that a key role in the chemopreventive effect of K/C is likely to be due to the potential of these compounds to induce the detoxification of xenobiotics by glutathione transferase (GST) and to enhance the synthesis of the corresponding co-factor glutathione. However, mutagens like PhIP may also be detoxified by UDP-glucuronosyl transferase (UDPGT) for which data are lacking regarding a potential effect of K/C. Therefore, in the present study, we investigated the effect of K/C on UDPGT and, concomitantly, we studied overall GST and the pattern of individual GST classes, particularly GST-theta;, which was not included in earlier experiments. In addition, we analyzed the organ-dependence of these potentially chemopreventive effects. K/C was fed to male F344 rats at 0.122% in the chow for 10 days. Enzyme activities in liver, kidney, lung, colon, salivary gland, pancreas, testis, heart and spleen were quantified using five characteristic substrates and the hepatic protein pattern of GST classes alpha, mu, and pi was studied with affinity chromatography/HPLC. Our study showed that K/C is not only capable of increasing overall GST and GST classes alpha, mu, and pi but also of enhancing UDGPT and GST-theta. All investigated K/C effects were strongest in liver and kidney, and some response was seen in lung and colon but none in the other organs. In summary, our results show that K/C treatment leads to a wide spectrum of increases in phase II detoxification enzymes. Notably, these effects occurred preferentially in the well perfused organs liver and kidney, which may thus not only contribute to local protection but also to anti-carcinogenesis in distant, less stimulated organs such as the colon.

Identification of six mRNA sequences of genes related to multixenobiotic resistance (MXR) and biotransformation in Mytilus edulis

Tissue-specific gene expression of the MXR (multixenobiotic resistance) and biotrans- formation-related gene homologs for the multidrug resistance protein 1 (Mdr 1), the multidrug resis- tance protein (Mrp), the major vault protein (Mvp, i.e. the lung resistance protein, Lrp), glutathione S-transferase (Gst pi), heat-shock protein 70 gene (Hsp70), and cytochrome p450 (Cyp4A) was analysed in the blue mussel Mytilus edulis. Additionally, Topoisomerase II (TopoII) and actin were investigated as indicators for mitotic activity and as internal standards. We identified highly con- served regions in the corresponding genes of several other species, and synthesised degenerate olignucleotide primers designed to amplify these regions from M. edulis mRNA. Resulting RT-PCR products were cloned into a T-tailed vector and DNA-sequenced. The results were evaluated by com- puter-based sequence alignment with the known gene sequences of blueprint species. PCR amplified fragments were used as probes for Northern blot hybridisation to identify transcript sizes. Specific oligonucleotide primers were designed from the M. edulis sequences for each gene and used for semi-quantitative multiplex RT-PCR to investigate tissue-specific gene expression. We have cloned and DNA-sequenced segments of the M. edulis P-gp, Mvp(Lrp), Gst pi, Hsp70, TopoII and actin genes. Semi-quantitative multiplex RT-PCR rapidly identified characteristic differential gene- expression patterns in a range of mussel tissues, and is therefore a promising tool for comprehensive studies of gene regulation in marine invertebrates in adaptation to various physiological conditions, including responses to natural and anthropogenic stressors.

Sensitization to the cytotoxicity of melphalan by ethacrynic acid and hyperthermia in drug-sensitive and multidrug-resistant Chinese hamster ovary cells.

The ability of physical and pharmacological modulators to increase the cytotoxicity of melphalan was investigated in Chinese hamster ovary cells using a clonogenic cell survival assay. Hyperthermia has potential for use in cancer treatment, particularly as an adjuvant to chemotherapy or radiotherapy. Ethacrynic acid is a glutathione S-transferase inhibitor and also undergoes conjugation with glutathione. Interactions between hyperthermia (41-43 degrees C), ethacrynic acid and melphalan were evaluated in multidrug-resistant (CH(R)C5) cells with overexpression of P-glycoprotein (33.69-fold), and in drug-sensitive (AuxB1) cells. GST alpha was expressed at a higher level (3.65-fold) in CH(R)C5 cells than in sensitive cells, whereas levels of isoforms pi and mu were the same. GST pi was the most highly expressed isoform in the two cell populations. Ethacrynic acid was cytotoxic at elevated temperatures, while it caused little or no cytotoxicity at 37 degrees C. This effect occurred in drug-resistant and drug-sensitive cells, and attributes thermosensitizing properties to ethacrynic acid. Ethacrynic acid (20 microM) alone did not alter the cytotoxicity of melphalan at 37 degrees C. Hyperthermia potentiated drug cytotoxicity in cells, both with and without ethacrynic acid treatment. Ethacrynic acid could be useful in cancer treatment by acting as a thermosensitizer when combined with heat and by enhancing the cytotoxicity of melphalan at elevated temperatures. A major advantage arising from the use of regional hyperthermia is the ability to target drug cytotoxicity to the tumor volume. A useful finding is that ethacrynic acid, heat and/or melphalan are also effective against multidrug-resistant cells with overexpression of P-glycoprotein.

Glutathione transferase activities in renal carcinomas and adjacent normal renal tissues: factors influencing renal carcinogenesis induced by xenobiotics.

In general, the biological activation of nephrocarcinogenic chlorinated hydrocarbons proceeds via conjugation with glutathione. It has mostly been assumed that the main site of initial conjugation is the liver, followed by a mandatory transfer of intermediates to the kidney. It was therefore of interest to study the enzyme activities of subgroups of glutathione transferases (GSTs) in renal cancers and the surrounding normal renal tissues of the same individuals (n = 21). For genotyping the individuals with respect to known polymorphic GST isozymes the following substrates with differential specificity were used: 1-chloro-2,4-dinitrobenzene for overall GST activity (except GST theta); 7-chloro-4-nitrobenzo-2-oxa- 1,3-diazole for GST alpha; 1,2-dichloro-4-nitro-benzene for GST mu; ethacrynic acid and 4-vinylpyridine for GST pi; and methyl chloride for GST theta. In general, the normal tissues were able to metabolize the test substrates. A general decrease in individual GST enzyme activities was apparent in the course of cancerization, and in some (exceptional) cases individual activities, expressed in the normal renal tissue, were lost in the tumour tissue. The GST enzyme activities in tumours were independent of tumour stage, or the age and gender of the patients. There was little influence of known polymorphisms of GSTM1, GSTM3 and GSTP1 upon the activities towards the test substrates, whereas the influence of GSTT1 polymorphism on the activity towads methyl chloride was straightforward. In general, the present findings support the concept that the initial GST-dependent bioactivation step of nephrocarcinogenic chlorinated hydrocarbons may take place in the kidney itself. This should be a consideration in toxicokinetic modelling.