Glutathione S-transferase Isoforms Research Articles

Constitutive mRNA Expression of Various Glutathione S-Transferase Isoforms in Different Tissues of Mice

Glutathione S-transferase (Gst) enzymes are instrumental in protecting cellular macromolecules against electrophiles and products of oxidative stress. Of interest primarily to pharmacologists and toxicologists is the ability of these enzymes to metabolize cancer chemotherapeutic drugs, insecticides, herbicides, and carcinogens. Thus, constitutive expression of Gsts might determine a tissue's ability to handle certain forms of chemical stress. In the present study, the constitutive mRNA expression of 19 different Gst enzymes was investigated in 14 different tissues in mice. The information obtained from the present study could be distilled into a few generalized principles: in all tissues examined, multiple isoforms of Gst were constitutively expressed; several isoforms, such as Gstk1, Gstm1, Gstm4, Gstm6, and Gstt1, were expressed in most of the tissues studied; at least five Gst isoforms were highly expressed in the gonads, about three in heart, and at least one in brain (Gstm5). Gender differences in the expression of various Gst isoforms were pronounced. With a few exceptions, most of the Gst isoforms expressed in kidney showed higher expression in females than males; the same trend was observed for heart and gonads. At least eight Gst isoforms showed very high expression in stomach. This was a unique finding in the current study because drug-metabolizing enzymes that are highly expressed in the gastrointestinal (GI) tract tend to have the highest expression in small intestine with low or no expression in the stomach. In summary, most Gst isoforms are most highly expressed in the GI tract and liver, which strongly suggests an important role of many Gst isoforms in detoxification of ingested xenobiotics.

Functional Promiscuity Correlates with Conformational Heterogeneity in A-class Glutathione S-Transferases

The structurally related glutathione S-transferase isoforms GSTA1-1 and GSTA4-4 differ greatly in their relative catalytic promiscuity. GSTA1-1 is a highly promiscuous detoxification enzyme. In contrast, GSTA4-4 exhibits selectivity for congeners of the lipid peroxidation product 4-hydroxynonenal. The contribution of protein dynamics to promiscuity has not been studied. Therefore, hydrogen/deuterium exchange mass spectrometry (H/DX) and fluorescence lifetime distribution analysis were performed with glutathione S-transferases A1-1 and A4-4. Differences in local dynamics of the C-terminal helix were evident as expected on the basis of previous studies. However, H/DX demonstrated significantly greater solvent accessibility throughout most of the GSTA1-1 sequence compared with GSTA4-4. A Phe-111/Tyr-217 aromatic-aromatic interaction in A4-4, which is not present in A1-1, was hypothesized to increase core packing. "Swap" mutants that eliminate this interaction from A4-4 or incorporate it into A1-1 yield H/DX behavior that is intermediate between the wild type templates. In addition, the single Trp-21 residue of each isoform was exploited to probe the conformational heterogeneity at the intrasubunit domain-domain interface. Excited state fluorescence lifetime distribution analysis indicates that this core residue is more conformationally heterogeneous in GSTA1-1 than in GSTA4-4, and this correlates with greater stability toward urea denaturation for GSTA4-4. The fluorescence distribution and urea sensitivity of the mutant proteins were intermediate between the wild type templates. The results suggest that the differences in protein dynamics of these homologs are global. The results suggest also the possible importance of extensive conformational plasticity to achieve high levels of functional promiscuity, possibly at the cost of stability.

The GSTP1 Gene Is a Susceptibility Gene for Childhood Asthma and the GSTM1 Gene Is a Modifier of the GSTP1 Gene

Background: Bronchial asthma is a chronic airway disorder characterized by bronchial inflammation. Oxidative stress is a key component of inflammation. Glutathione S-transferase P1 (GSTP1), the abundant isoform of glutathione S-transferases (GSTs) in lung epithelium, plays a key role in cellular protection against oxidative stress. Several studies have shown that the GSTP1 geneis involved in the pathogenesis of asthma and a gene-gene interaction may occur within the GST gene superfamily. Methods: We screened single-nucleotide polymorphisms (SNPs) at the GSTP1 locus and performed an association study in the Japanese population using two independent case-control groups (group 1: 391 pediatric patients with asthma, 462 adult patients with asthma, and 639 controls, and group 2: 115 pediatric patients with asthma and 184 controls). The effect of GSTM1 null/present genotype on the association between GSTP1 Ile105Val and asthma was also investigated. Results: We identified 20 SNPs at this locus and found this region consisted of one linkage disequilibrium block represented by four SNPs (tag SNPs). The association between the Ile105Val polymorphism in the GSTP1 gene and childhood asthma was significant in both groups (p = 0.047 in group 1, and p = 0.021 in group 2). This association was only significant in patients with GSTM1-positive genotype in both groups (group 1: GSTM1 present p = 0.013 and GSTM1 null p = 0.925, and group 2: GSTM1 present p = 0.015 and GSTM1 null p = 0.362). Conclusions: These findings suggest that the GSTP1 gene is a childhood asthma susceptible gene, and the GSTM1 gene is a modifier gene of GSTP1 for the risk of childhood asthma in the Japanese population.

Sequence, biochemical characteristics and expression of a novel Sigma-class of glutathione S-transferase from the intertidal copepod, Tigriopus japonicus with a possible role in antioxidant defense

Glutathione S-transferases (GSTs) play a major role in detoxification of xenobiotics and antioxidant defense. Here we report full-length cDNA sequence of a novel Sigma-class of GST (GST-S) from the intertidal copepod Tigriopus japonicus. The full sequence was of 1136 bp in length containing an open reading frame (ORF) of 651 bp that encoded 217 amino acid residues. The recombinant Tigriopus GST-S was highly expressed in transformed Escherichia coli. Kinetic properties and effects of pH, temperature and chemical inhibitors on Tigriopus GST-S were also studied. The expression of GST-S was studied using real-time RT-PCR in response to exposure to two oxidative stresses-inducing agents, viz., hydrogen peroxide (H 2O 2) and heavy metals (copper, manganese). It was observed that H 2O 2 (2 mM) exposure down-regulated its expression at the initial stage but there was recovery and up-regulation shortly afterwards. In case of heavy metal exposure there was concentration-dependent increase in Tigriopus GST-S gene expression up to 24 h. These results suggest that Tigriopus GST-S expression is modulated by prooxidant chemicals and it may play a role against oxidative stress. A majority of other GST isoforms is known to play an important role in antioxidant defense. This study provides a preliminary insight into the possible antioxidant role for Sigma-class of GST in T. japonicus.

Relationship between glutathione-S-transferase P1 Ile105Val polymorphism and docetaxel neurosensory toxicity

2527 Background: Glutathione-S-transferases (GST) regulate the cellular response to oxidative stress and various anticancer agents. We recently underlined the importance of oxidative stress in the side effects of taxanes [Alexandre et al,J Natl Cancer Inst 2006;98:236–44], and investigated the relationship between the GST isoforms M1, T1 and P1 genes polymorphisms and docetaxel-induced peripheral neuropathy. Methods: We retrospectively analysed GST polymorphisms in a cohort of cancer patients (pts) treated with docetaxel and entered in a clinical trial database. A clinical neurologic evaluation (according to the NCI-CTC v2.0) was performed at baseline and at each treatment cycle. The GST M1 (null genotype), GST T1 (null genotype), and GST P1 (Ile105Val and Ala114Val) polymorphisms were determined using PCR, followed by either sequencing or RFLP techniques. The relationship between GST polymorphisms and grade = 2 neurosenrory toxicity (NST) as primary endpoint was studied, using the Chi2-square (with Yates correction) and the Fischer’s two-tailed exact test. Results: Fifty-eight pts were included : F/M: 29/29; median age: 61 years (range: 47–75). Primary tumor: 27.6% breast, 27.6% prostate, 24.1% lung and 20.7% other cancers. Pts received docetaxel 75–100 mg/m2 given as single-agent. A total of 261 cycles were administered (median/pt: 4, range 2–12). Ten pts developed grade = 2 NST. Twenty-seven pts (47%) were homozygous for the GST P1 105Ile allele, and 31 pts were either homozygous or heterozygous for the GST P1 105Val allele. Grade = 2 NST was significantly more common in pts with GST P1 105Ile/105Ile genotype (8/27 pts, 30%) compared with patients with 105Ile/105Val or 105Val/105Val GSTP1 genotype (2/31 pts, 6,5%; P = 0.047). Pts who were genotyped as GST P1 105Ile/105Ile had a higher risk of developing a grade = 2 NST than did those with other GSTP1 genotypes (OR 6.11; 95% CI, 1.17–31.94; P < 0.05). Conclusions: We found a significant correlation between GST P1 105Ile homozygous genotype and the development of a docetaxel-induced peripheral neuropathy. No significant financial relationships to disclose.

Transfection of HepG2 cells with hGSTA4 provides protection against 4-hydroxynonenal-mediated oxidative injury

4-Hydroxynonenal (4-HNE) is a mutagenic α,β-unsaturated aldehyde produced during oxidative injury that is conjugated by several glutathione S-transferase (GST) isoforms. The alpha class human GSTA4-4 enzyme (hGSTA4-4) has a particularly high catalytic efficiency toward 4-HNE conjugation. However, hGST4-4 expression is low in most human cells and there are other aldehyde metabolizing enzymes that detoxify 4-HNE. In the current study, we determined the effect of over-expression of hGSTA4 mRNA on the sensitivity of HepG2 cells to 4-HNE injury. HepG2 cells transfected with an hGSTA4 vector construct exhibited high steady-state hGSTA4 mRNA, high GST–4-HNE catalytic activities, but lower basal glutathione (GSH) concentrations relative to insert-free vector (control) cells. Exposure to 4-HNE elicited an increase in GSH concentrations in the control and hGSTA4 cells, although the dose-response of GSH induction differed among the two cell types. Specifically, hGSTA4 cells had significantly higher GSH concentrations when exposed to 5–15 μM 4-HNE, but not at 20 μM 4-HNE, suggesting extensive GSH utilization at high concentrations of 4-HNE. The hGSTA4 cells exhibited a significant growth advantage relative to control cells in the absence of 4-HNE, and a trend towards increased growth at low dose exposures to 4-HNE. However, the hGSTA4 cells did not exhibit a growth advantage relative to control cells at higher 4-HNE exposures associated with increased GSH utilization. As expected, the hGSTA4 cells showed resistance to 4-HNE stimulated lipid peroxidation at all 4-HNE doses. In summary, our data indicates that over-expression of hGSTA4 at levels conferring high GST–4-HNE conjugating activity confers a partial growth advantage to HepG2 cells and protects against 4-HNE oxidative injury. However, the loss of proliferative capacity of hGSTA4 cells challenged with levels of 4-HNE associated with severe oxidative stress indicates a role of other aldehyde metabolizing enzymes, and/or GSH–electrophile transporter proteins, in providing full cellular protection against 4-HNE toxicity.

Expression of genes for redox-dependent glutathione S-transferase isoforms GSTP1-1 and GSTA4-4 in tumor cell during the development doxorubicin resistance

Expression of genes for redox-dependent glutathione S-transferase isoforms GSTP1-1 and GSTA4-4 in tumor cells K562, MCF-7, and SKOV-3 was studied during the development of resistance to doxorubicin. It was found that the development of resistance was accompanied by predominant increase in the expression of hGSTP1 gene in MCF-7 cells, and hGSTA4 gene in resistant K562/DOX and SKVLB cells.

Changes of antioxidants and GSH-associated enzymes in isoproturon-treated maize

The recommended field dose (RFD) of isoproturon induced significant accumulations of H2O2 in the leaves of 10-d-old maize seedlings throughout the following 20 d; the accumulation increased with time and also with herbicide dose. Meanwhile, low doses significantly increased ascorbic acid, glutathione and thiols while high doses caused diminutions. Superoxide dismutase (SOD; EC 1.15.1.1) activity was significantly enhanced up to the 12th d whereas ascorbate peroxidase (APX; EC 1.11.1.7) activity was significantly reduced after the fourth d onwards. Catalase (CAT; EC 1.11.1.6) and guaiacol peroxidase (GPX; EC 1.11.1.7) activities were similarly increased during the first 4 d but decreased from the 12th and the eighth d, respectively. Low doses increased SOD and GPX activities but high doses led to diminutions whereas CAT and APX were reduced by all doses. The activities of γ-glutamyl-cysteine synthethase (γ-GCS; EC 6.3.2.2) and glutathione synthethase (GSS; EC 6.3.2.3) were enhanced for 4 d; high doses caused general reductions. Isoproturon significantly reduced activities of glutathione S-transferase (GST; EC 2.5.1.18) isoforms [GST(CDNB), GST(ALA), or GST(MET)] after the fourth d, however, it had no effect on GST(ATR). Similar reductions in activities of glutathione peroxidase (GSPX; EC 1.15.1.1) and glutathione reductase (GR; EC 1.6.4.2) were detected up to the 16th and the 12th d, respectively. The activities of GST isoforms, GSPX and GR were reduced by high doses. These changes seemed to be related and might point to an oxidative stress state that exacerbated with prolonged time and/or increased isoproturon dose.

Effect of herbicides on glutathione S-transferases in the earthworm, Eisenia fetida

Earthworms have been studied as a readily available, easily maintainable and cheap test species for assessing chemical pollution, and may be an alternative to in vivo rodent bioassays. The current investigation aims to characterize detoxification enzymes in Eisenia fetida and stress response against two herbicides with different modes of action, namely, fenoxaprop and metolachlor. Herbicides were applied to soil containing earthworms. Animals were then collected, sacrificed and shock-frozen. Extracted protein was analyzed for glutathione S-transferase (GST) activity using CDNB (1-chloro-2,4-dinitrobenzene), DCNB (1,2-dichloro-4-nitrobenzene), pNBC (p-nitrobenzylchloride), PNOBC (p-nitrobenz-o-ylchloride) and selected herbicides. GST isoenzymes were partially purified by affinity chromatography and molecular weights were estimated by SDS-PAGE. In E. fetida protein extracts, GST activity towards model compounds ranked as CDNB>DCNB>PNBOC>PNBC. Fluorodifen was not conjugated at all, but fenoxaprop and metolachlor were conjugated at low rates. Furthermore, the GST isoenzyme pattern changed during the incubation with herbicides, either due to stress or as a defense reaction. After incubation with monochlorobimane, a strong fluorescence of the intestinal tract and the intersegments was observed, indicating organ-specific GST induction. According to the author's knowledge, here, for the first time, evidence is presented that E. fetida GST are also capable of conjugating a wider range of xenobiotic substrates. Different forms of GST were observed and changes in GST isoforms due to the herbicide treatment were also noticed. GST conjugation rates varied between different herbicides used in this experiment. It might be assumed that herbicides may well be detoxified by earthworms, to a certain extent, but that they are also potent stress factors influencing the detoxification system of the animal. High doses or long exposure might lead to deleterious effects on earthworms and limit their survival rate. The use of the animals as bioindicators for herbicides and herbicide residues seems very promising, but is surely influenced by the lack of detoxification for some compounds. Conjugation of several xenobiotics with model substances and herbicides is proven in the earthworm E. fetida. However, E. fetida has only limited capabilities of detoxifying herbicidal compounds. Different isoforms of GST were involved and altered in their activity after treatment. The accumulation of GS-conjugates and their determination via fluorescence microscopy is a quick and secure, additional marker for exposure that should be further developed to complement existing biotests. The described methods and endpoints might help to understand the complex reaction of earthworms towards herbicides and lead to an adapted test methodology.

Glutathione S-transferase cytosolic isoform in the pink-shrimp, Farfantepenaeus brasiliensis, from Conceição Lagoon, Santa Catarina Island, SC, Brazil

Contaminant input into the environment can affect the biochemical responses of exposed organisms. Activity of conjugation enzymes, such as glutathione S-transferase (GST) has been proposed as a biomarker of susceptibility to the presence of potentially damaging xenobiotics. The aim of the present study was to evaluate the activity of total and Pi-class GST in the hepatopancreas of pink-shrimp Farfantepenaeus brasiliensis. The shrimp were caught at a single site at Conceição Lagoon and transplanted to another site (Canto da Lagoa — CA) in the same lagoon, and to the laboratory (LAB), where they were kept for 15 days. This experiment was carried out in winter 2003 and in summer 2004. Shrimp were sampled before the exposure, at a zero time (t0), and after 15-days at CA site and LAB. The activity of total GST and Pi-isoform were analyzed using CDNB and ethacrynic acid (EA) as substrates, respectively. Shrimp caught in winter showed higher levels of Pi-class GST than those caught in summer, for both t0 and CA groups. Moreover, differences in the activity of this GST isoform between groups were observed only in winter, where the animals from t0 and CA groups presented higher activity when compared to those kept in the laboratory. This difference could be associated either to changes in the contaminant input or to other water quality parameters in this ecosystem. The elevated GST Pi activity observed in the shrimp of both t0 and CA groups sampled in winter could be related to salinity, since it was higher in winter than in summer. We could also suggest a possible association of this finding to a seasonal metabolic variation in this penaeid. If that is the case, one should take into account these alterations when considering the use of this GST isoform as biomarker in F. brasiliensis in environmental monitoring programs.

Gene expression profiles of drug‐metabolizing enzymes and transporters with an overexpression of hepatocyte growth factor

It is important to elucidate the precise mechanism of drug metabolism during hepatic regeneration. Although cytochromes P450 (CYPs) are well known to be down-regulated in growth-stimulated cells, the overall gene expression profile of drug metabolizing enzymes are still not fully understood during hepatic regeneration. In this study, we investigated the gene expression profiles of such enzymes with an overexpression of hepatocyte growth factor (HGF). Gene expression profiles were obtained using the Affymetrix MOE430A GeneChip oligonucleotide microarray by comparing HGF transgenic mice and wild-type mice. HGF produced a general decrease in mice with the expression of CYP isoforms such as Cyp1a2, Cyp2b10, Cyp2c, Cyp2d9, Cyp3a11, Cyp4a10, and Cyp7a1. Some isoforms of alcohol dehydrogenase, aldehyde dehydrogenase, and carboxylesterase also decreased. In the phase II enzymes, some isoforms of glutathione S-transferase and UDP-glucuronosyl transferase showed a reduced expression, although the sulfotransferase did not. In phase III transporters, some organic anion transporter and organic cation transporters were down-regulated. Among the nuclear receptors that are known to regulate the drug-metabolizing enzymes, small heterodimer partner and constitutive androstane receptor were down-regulated with an HGF overexpression. The protein level and enzymatic activity of Cyp2c decreased with an HGF overexpression. We furthermore investigated the inducibility of Cyp2b10 with xenobiotic inducers. Although the basal expression of Cyp2b10 was repressed, the inducibility was not abolished with the HGF overexpression. HGF down-regulated not only CYPs but also some drug-metabolizing enzymes, transporters, and nuclear receptors. We thus have to take in our mind the low basal expression of drug metabolizing enzymes, when treating patients with a regenerative liver state.

Characterization of hepatic glutathione S-transferases in coho salmon ( Oncorhynchus kisutch)

The glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes which protect against chemical injury. In contrast to mammals, GST expression in fish has not been extensively characterized, especially in the context of detoxifying waterborne pollutants. In the Northwestern United States, coho salmon ( Oncorhynchus kisutch) are an important species of Pacific salmon with complex life histories that can include exposure to a variety of compounds including GST substrates. In the present study we characterized the expression of coho hepatic GST to better understand the ability of coho to detoxify chemicals of environmental relevance. Western blotting of coho hepatic GST revealed the presence of multiple GST-like proteins of approximately 24–26 kDa. Reverse phase HPLC subunit analysis of GSH affinity-purified hepatic GST demonstrated six major and at least two minor potential GST isoforms which were characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI MS–MS) and Fourier transform-ion cyclotron resonance (FT-ICR) MS analyses. The major hepatic coho GST isoforms consisted of a pi and a rho-class GST, whereas GSTs representing the alpha and mu classes constituted minor isoforms. Catalytic studies demonstrated that coho cytosolic GSTs were active towards the prototypical GST substrate 1-chloro-2,4-dinitrobenzene, as well as towards ethacrynic acid and nitrobutyl chloride. However, there was no observable cytosolic GST activity towards the pesticides methyl parathion or atrazine, or products of oxidative stress, such as cumene hydroperoxide and 4-hydroxynonenal. Interestingly, coho hepatic cytosolic fractions had a limited ability to bind bilirubin, reflecting a potential role in the sequestering of metabolic by-products. In summary, coho salmon exhibit a complex hepatic GST isoform expression profile consisting of several GST classes, but may have a limited a capacity to conjugate substrates of toxicological significance such as pesticides and endogenous compounds associated with cellular oxidative stress.

Expression of xenobiotic and steroid hormone metabolizing enzymes in human breast carcinomas

The potential to metabolize endogenous and exogenous substances may influence breast cancer development and tumor growth. Therefore, the authors investigated the protein expression of Glutathione S-transferase (GST) isoforms and cytochrome P450 (CYP) known to be involved in the metabolism of steroid hormones and endogenous as well as exogenous carcinogens in breast cancer tissue to obtain new information on their possible role in tumor progression. Expression of GST pi, mu, alpha and CYP1A1/2, 1A2, 3A4/5, 1B1, 2E1 was assessed by immunohistochemistry for primary breast carcinomas of 393 patients from the German GENICA breast cancer collection. The percentages of positive tumors were 50.1 and 44.5% for GST mu and CYP2E1, and ranged from 13 to 24.7% for CYP1A2, GST pi, CYP1A1/2, CYP3A4/5, CYP1B1. GST alpha was expressed in 1.8% of tumors. The authors observed the following associations between strong protein expression and histopathological characteristics: GST expression was associated with a better tumor differentiation (GST mu, p = 0.018) and with reduced lymph node metastasis (GST pi, p = 0.02). In addition, GST mu expression was associated with a positive estrogen receptor and progesterone receptor status (p < 0.001). CYP3A4/5 expression was associated with a positive nodal status (p = 0.018). Expression of CYP1B1 was associated with poor tumor differentiation (p = 0.049). Our results demonstrate that the majority of breast carcinomas expressed xenobiotic and drug metabolizing enzymes. They particularly suggest that GST mu and pi expression may indicate a better prognosis and that strong CYP3A4/5 and CYP1B1 expression may be key features of nonfavourable prognosis.

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

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

4-hydroxynonenal detoxification by mitochondrial glutathione S-transferase is compromised by short-term ethanol consumption in rats.

4-Hydroxynonenal (HNE), a toxic lipid peroxidation product, has been implicated in mitochondrial damage in rat liver by ethanol consumption. The present study assessed the effects of short-term in vivo ethanol exposure on HNE detoxification by mitochondrial glutathione S-transferase (GST). Male Sprague Dawley rats were administered 5 doses of ethanol (4 g/kg) at 12 hr intervals by gavage. Pair-fed rats that received isocaloric dextrose instead of ethanol served as controls. Mitochondrial and submitochondrial fractions were prepared from the livers. Mitochondrial contents of HNE and HNE-glutathione conjugate were measured by high-performance liquid chromatography. GST isoforms were identified by Western blots in submitochondrial fractions. Whereas there was an 80% increase in mitochondrial HNE content after ethanol consumption, there was a 42% decrease in the content of HNE-glutathione conjugate, compared with controls (p< 0.05). After ethanol exposure, the GST activities toward HNE in intact mitochondria and in the membranous fraction were decreased by 37% and 45% (p< 0.05), respectively, whereas that in the aqueous fraction was unchanged. Kinetic analysis of HNE conjugation by the membrane-associated GST showed that ethanol decreased the V(max) nearly by half (p< 0.05), whereas it did not affect the K(m). HNE conjugation by the aqueous GST demonstrated a higher K(m) than that of the membrane-associated GST, although its kinetics were not significantly altered by ethanol. Immunochemical analysis with Western blots demonstrated that both the membranous and the aqueous fractions of mitochondria contain GST-alpha and GST-mu isoforms, whereas GST-pi was absent. HNE detoxification by mitochondrial GST is compromised by short-term ethanol consumption, which may contribute to elevated mitochondrial HNE content and hence its toxicity in the ethanol-exposed liver.

Low detoxification capacity in the ileal pouch mucosa of patients with ulcerative colitis

An ileal pouch-anal anastomosis has become the most widely accepted procedure for surgical treatment of patients with ulcerative colitis (UC). The primary function of the ileum within the pouch changes from absorption to storage. Malignancies have been described in the pouch mucosa. The detoxifying glutathione S-transferase (GST) enzymes are involved in the mucosal protection against toxins and carcinogens. Levels of GSTs are much higher in the ileum as compared with the colon. The adaptation of the ileal pouch mucosa into a more colon-like phenotype possibly influences the activity and levels of GST. This study compares the detoxification capacity of GST of the afferent ileal limb mucosa with the ileal pouch mucosa of patients with UC. Biopsies from normal-appearing mucosa from the ileal pouch and the ileal afferent limb were obtained from 18 patients with UC. GST isoforms were quantified by immunoblotting. GST activity was measured spectrophotometrically, and glutathione and cysteine levels were determined by high-performance liquid chromatography. The GST activity and GSTA1+A2 levels were significantly lower in the pouch compared with the afferent ileal limb of patients with UC, whereas the GSTP1 levels were higher in the pouch. No differences were observed in the levels of GSTM1, GSTT1, glutathione, or cysteine. The lower GST detoxification activity in the pouch mucosa of patients with UC may result in higher levels of toxins and carcinogens and thus partly contribute to the risk of developing malignancies in the pouch.

In Vitro Metabolism of (Nitrooxy)butyl Ester Nitric Oxide-Releasing Compounds: Comparison with Glyceryl Trinitrate

We investigated the in vitro metabolism of two (nitrooxy)butyl ester nitric oxide (NO) donor derivatives of flurbiprofen and ferulic acid, [1,1'-biphenyl]-4-acetic acid-2-fluoro-alpha-methyl-4-(nitrooxy)butyl ester (HCT 1026) and 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 4-(nitrooxy)butyl ester (NCX 2057), respectively, in rat blood plasma and liver subcellular fractions compared with (nitrooxy)butyl alcohol (NOBA) and glyceryl trinitrate (GTN). HCT 1026 and NCX 2057 undergo rapid ubiquitous carboxyl ester hydrolysis to their respective parent compounds and NOBA. The nitrate moiety of this latter is subsequently metabolized to inorganic nitrogen oxides (NOx), predominantly in liver cytosol by glutathione S-transferase (GST) and to a lesser extent in liver mitochondria. If, however, in liver cytosol, the carboxyl ester hydrolysis is prevented by an esterase inhibitor, the metabolism at the nitrate moiety level does not occur. In blood plasma, HCT 1026 and NCX 2057 are not metabolized to NOx, whereas a slow but sustained NO generation in deoxygenated whole blood as detected by electron paramagnetic resonance indicates the involvement of erythrocytes in the bioactivation of these compounds. Differently from NOBA, GTN is also metabolized in blood plasma and more quickly metabolized by different GST isoforms in liver cytosol. The cytosolic GST-mediated denitration of these organic nitrates in liver limits their interaction with other intracellular compartments to possible generation of NO and/or their subsequent availability and bioactivation in the systemic circulation and extrahepatic tissues. We show the possibility of modulating the activity of hepatic cytosolic enzymes involved in the metabolism of (nitrooxy)butyl ester compounds, thus increasing the therapeutic potential of this class of compounds.

Inhibition of human glutathione transferases by multidrug resistance chemomodulators in vitro

Reversal of the drug-resistance phenotype in cancer cells usually involves the use of a chemomodulator that inhibits the function of a resistance-related protein. The aim of this study was to investigate the effects of MDR chemomodulators on human recombinant glutathione S-transferase (GSTs) activity. IC50 values for 15 MDR chemomodulators were determined using 1-chloro-dinitrobenzene (CDNB), cumene hydroproxide (CuOOH) and anticancer drugs as substrates. GSTs A1, P1 and M1 were inhibited by O6-benzylguanine (IC50s around 30 μM), GST P1-1 by sulphinpyrazone (IC50 = 66 μM), GST A1-1 by sulphasalazine, and camptothecin (34 and 74 μM respectively), and GST M1-1 by sulphasalazine, camptothecin and indomethacin (0.3, 29 and 30 μM respectively) using CDNB as a substrate. When ethacrynic acid (for GST P1-1), CuOOH (for A1-1) and 1,3-bis (2-chloroethyl)-1-nitrosourea (for GST M1-1) were used as substrates, these compounds did not significantly inhibit the GST isoforms. However, progesterone was a potent inhibitor of GST P1-1 (IC50 = 1.4 μM) with ethacrynic acid as substrate. These results suggest that the target of chemomodulators in vivo could be a specific resistance-related protein.

Increased sensitivity of Hep G2 cells toward the cytotoxicity of cisplatin by the treatment of piper betel leaf extract

Piper betel leaves (PBL) are used in Chinese folk medicine for the treatment of various disorders. PBL has the biological capabilities of de-toxication, anti-oxidation and anti-mutation. In this study we first examined the effect of PBL extract on the activity of Glutathione S-transferase (GST) isoforms, and found that it inhibited total GST and the alpha class of GST (GSTA), but not the pi class of GST (GSTP), and the mu class of GST (GSTM), activity in Hep G2 cells. RT-PCR results verified a reduction in the expression of GSTA1. Next, we examined whether PBL extract could increase the sensitivity of Hep G2 cells to anti-cancer drugs. The data showed that the cytotoxicity of cisplatin was significantly enhanced by the presence of PBL extract, accompanied by a reduction in the expression of multidrug resistance protein 2 (MRP2). These effects of PBL extract were compared to its major constitute, eugenol. Although eugenol decreased MRP2 level more effectively than PBL extract, it exhibited less sensitizing effect. In conclusion, we demonstrated that PBL extract was able to increase the sensitivity of Hep G2 cells to cisplatin via at least two mechanisms, reducing the expression of MRP2 and inhibiting the activity of total GST and the expression of GSTA. The data of this study support an application of PBL as an additive to reduce drug resistance.

Differential protein expression profiles in anterior gills of Eriocheir sinensis during acclimation to cadmium

Using a proteomic approach, we characterized different protein expression profiles in anterior gills of the Chinese mitten crab, Eriocheir sinensis, after cadmium (Cd) exposure. Two experimental conditions were tested: (i) an acute exposure (i.e. 500 μg Cd l −1 for 3 days) for which physiological, biochemical and ultrastructural damage have been observed previously; (ii) a chronic exposure (i.e. 50 μg Cd l −1 for 30 days) resulting in physiological acclimation, i.e. increased resistance to a subsequent acute exposure. Two-dimensional gel electrophoresis (2-DE) revealed six protein spots differentially expressed after acute, and 31 after chronic Cd exposure. From these spots, 15 protein species were identified using MS/MS micro-sequencing and MS BLAST database searches. Alpha tubulin, glutathione S-transferase and crustacean calcium-binding protein 23 were down-regulated after an acute exposure, whereas another glutathione S-transferase isoform was up-regulated. Furthermore, analyses revealed the over-expression of protein disulfide isomerase, thioredoxin peroxidase, glutathione S-transferase, a proteasome subunit and cathepsin D after chronic exposure. Under the same condition, ATP synthase beta, alpha tubulin, arginine kinase, glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase were down-regulated. These results demonstrate that acute and chronic exposure to waterborne Cd induced different responses at the protein expression level. Protein identification supports the idea that Cd mainly exerts its toxicity through oxidative stress induction and sulfhydryl-group binding. As a result, analyses showed the up-regulation of several antioxidant enzymes and chaperonins during acclimation process. The gill proteolytic capacity seems also to be increased. On the other hand, the clearly decreased abundance of several enzymes involved in energy transfer suggests that chronic metal exposure induced an important metabolic reshuffling.