Shizukaol C alleviates trimethylamine oxide-induced inflammation through activating Keap1-Nrf2-GSTpi pathway in vascular smooth muscle cell.

BACKGROUND: Cardiovascular disease is one of the main causes of global mortality, and there is an urgent need for effective treatment strategies. Gut microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) promotes the development of cardiovascular diseases, and shizukaol C, a natural sesquiterpene isolated from Chloranthus multistachys with various biological activities, might exhibit beneficial role in preventing TMAO-induced vascular inflammation. PURPOSE: The purpose of this study was to investigate the anti-inflammatory effects and the underlying mechanisms of shizukaol C on TMAO-induced vascular inflammation. METHODS: The effect and underlying mechanism of shizukaol C on TMAO-induced adhesion molecules expression, bone marrow-derived macrophages (BMDM) adhesion to VSMC were evaluated by western blot, cell adhesion assay, co-immunoprecipitation, immunofluorescence assay, and quantitative Real-Time PCR, respectively. To verify the role of shizukaol C in vivo, TMAO-induced vascular inflammation model were established using guidewire-induced injury on mice carotid artery. Changes in the intima area and the expression of GSTpi, VCAM-1, CD68 were examined using haematoxylin-eosin staining, and immunofluorescence assay. RESULTS: Our data demonstrated that shizukaol C significantly suppressed TMAO-induced adhesion molecule expression and the bone marrow-derived macrophages (BMDM) adhesion in vascular smooth muscle cells (VSMC). Mechanically, shizukaol C inhibited TMAO-induced c-Jun N-terminal kinase (JNK)-nuclear factor-kappa B (NF-κB)/p65 activation, and the JNK inhibition was dependent on the shizukaol C-mediated glutathione-S-transferase pi (GSTpi) expression. By further molecular docking and protein-binding analysis, we demonstrated that shizukaol C directly binds to Keap1 to induce Nrf2 nuclear translocation and upregulated GSTpi expression. Consistently, our in vivo experiment showed that shizukaol C elevated the expression level of GSTpi in carotid arteries and alleviates TMAO-induced vascular inflammation. CONCLUSION: Shizukaol C exerts anti-inflammatory effects in TMAO-treated VSMC by targeting Keap1 and activating Nrf2-GSTpi signaling and resultantly inhibits the downstream JNK-NF-κB/p65 activation and VSMC adhesion, and alleviates TMAO-induced vascular inflammation in vivo, suggesting that shizukaol C may be a potential drug for treating TMAO-induced vascular diseases.

Evaluation of GSTP1, GSTA4 and AChE Gene Methylation in Bovine Lymphocytes Cultured In Vitro with Miconazole Alone and in Combination with Mospilan 20SP.

5-methylcytosine (5mC) is one of the most important epigenetic modifications. Its increased occurrence in regulatory sequences of genes, such as promoters and enhancers, is associated with the inhibition of their expression. Methylation patterns are not stable but are sensitive to factors such as the environment, diet, and age. In the present study, we investigated the effects of fungicide miconazole, both alone and in combination with the insecticide Mospilan 20SP, on the methylation status of bovine GSTP1, GSTA4, and AChE genes in bovine lymphocytes cultured in vitro. The methylation-specific PCR technique was used for the objectives of this study. We found that miconazole alone at concentrations of 1.25, 2.5, 5, 10, 25, and 50 µg/mL after 24 h exposure probably did not induce changes in methylation for all three genes analysed. The same results were found for the combination of pesticides at 24 h exposure and the following concentrations for each of them: 0.625, 1.25, 2.5, 5, and 12.5 µg/mL. Thus, we can conclude that the fungicide miconazole alone, as well as in combination with the insecticide Mospilan 20SP, was unlikely to cause changes to the methylation of bovine GSTP1, GSTA4, and AChE genes.

Identification of predictive markers of the therapeutic effect of eribulin chemotherapy for locally advanced or metastatic breast cancer.

BACKGROUND: The recently developed reagent, eribulin mesylate (eribulin), is a microtubule dynamics inhibitor with a mechanism of action that differs from those of taxanes and vinca alkaloids. This drug is considered to be a promising chemotherapeutic agent for the treatment of locally advanced or metastatic breast cancer (MBC). In this study, we investigated if variables such as tumor expression of ß-tubulin class III, glutathione S-transferase pi (GSTP) 1 or transducin-like enhancer of split (TLE) 3 might act as predictive factors on the therapeutic effect of eribulin chemotherapy. METHODS: The subjects included 52 patients with MBC who underwent chemotherapy with eribulin. The expression levels of Estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor (HER) 2, Ki67, ß-tubulin class III, GSTP-1 and TLE-3 were evaluated using immunostaining employing needle biopsy specimens. RESULTS: Patients with TLE3-negative tumors displayed significantly poorer outcomes regarding progression-free survival than patients with TLE3-positive tumors when prognosis within the group of patients with triple-negative breast cancer (TNBC) lesions was analyzed (p = 0.011, log-rank). In contrast, no such difference in prognosis was found in a comparison of TLE-3 positive/negative patients in the group of all patients (p = 0.433, log-rank) or of patients with non-TNBC lesions (p = 0.659, log-rank). Based on a univariate analysis of 22 TNBC cases, a better progression-free survival correlated significantly with a positive TLE3 expression in the tumor (p = 0.025). A multivariate logistic regression analysis including 22 patients with TNBC also showed that a positive TLE3 expression significantly correlated with a better progression-free survival (p = 0.037). CONCLUSIONS: Our findings suggest that TLE3 is a useful marker for predicting the therapeutic effect of eribulin chemotherapy for TNBC.

Design and Synthesis of Irreversible Analogues of Bardoxolone Methyl for the Identification of Pharmacologically Relevant Targets and Interaction Sites.

Semisynthetic triterpenoids such as bardoxolone methyl (methyl-2-cyano 3,12-dioxooleano-1,9-dien-28-oate; CDDO-Me) (4) are potent inducers of antioxidant and anti-inflammatory signaling pathways, including those regulated by the transcription factor Nrf2. However, the reversible nature of the interaction between triterpenoids and thiols has hindered attempts to identify pharmacologically relevant targets and characterize the sites of interaction. Here, we report a shortened synthesis and SAR profiling of 4, enabling the design of analogues that react irreversibly with model thiols, as well as the model protein glutathione S-transferase P1, in vitro. We show that one of these analogues, CDDO-epoxide (13), is comparable to 4 in terms of cytotoxicity and potency toward Nrf2 in rat hepatoma cells and stably modifies specific cysteine residues (namely, Cys-257, -273, -288, -434, -489, and -613) within Keap1, the major repressor of Nrf2, both in vitro and in living cells. Supported by molecular modeling, these data demonstrate the value of 13 for identifying site(s) of interaction with pharmacologically relevant targets and informing the continuing development of triterpenoids as novel drug candidates.

Cytogenetic damage in Turkish coke oven workers exposed to polycyclic aromatic hydrocarbons: Association with CYP1A1, CYP1B1, EPHX1, GSTM1, GSTT1, and GSTP1 gene polymorphisms.

The aim of this study was to determine the frequencies of chromosomal aberrations (CA) and cytochalasin-blocked micronuclei (CBMN) in peripheral blood lymphocytes from Turkish coke oven workers and the influence of CYP1A1, CYP1B1, EPHX1, GSTM1, GSTT1, and GSTP1 gene polymorphisms on these biomarkers. Cytogenetic analysis showed that occupational exposure significantly increased the CA and CBMN frequencies. Gene polymorphisms, on the other hand, did not affect CA or CBMN in either exposed or control subjects. However, due to the limited sample size, our findings need to be verified in future studies with a larger sample.

Biomarkers and bioaccumulation of clam Ruditapes philippinarum in response to combined cadmium and benzo[α]pyrene exposure.

Biochemical and molecular biomarkers (the contents of metallothionein (MT), glutathione (GSH), the activities of aryl hydrocarbon hydroxylase (AHH), glutathione S-transferase (GST) and superoxide dismutase (SOD) and the mRNA expressions of GST-pi and Cu, Zn-SOD) were evaluated in clams Ruditapes philippinarum exposed to cadmium (Cd, 15 µg/L) and benzo[α]pyrene (BaP, 0.01 µg/L) individually and in combination (15 µg/L Cd+0.01 µg/L BaP) for 21 days. The accumulation of Cd, BaP and the biomarkers measured in the gills and digestive glands of the clam showed significant increase in combination treatment and it was significantly higher than the Cd or BaP treatment (P>0.05). The contents of MT increased in Cd and Cd+BaP treatment, while AHH activities were increased in Bap and Cd+BaP treatment (P>0.05). GSH levels enhanced in Cd group and declined significantly in Cd+BaP treatment (P>0.05). The activities of GST, SOD, and mRNA expressions of GST-pi, Cu, Zn-SOD increased remarkably in the clams exposed to combined pollutants. In this study, a significant interaction was observed for Cd and BaP accumulation in the clam and the current findings demonstrate the differences in antioxidant response of the biomarkers in clam to single contaminant and the mixtures.

Ligustrazine derivate DLJ14 reduces multidrug resistance of K562/A02 cells by modulating GSTπ activity.

Multidrug resistance (MDR) of tumor cells is a major obstacle in chemotherapeutic cancer treatment. Over-expression of glutathione S-transferase π (GSTπ) is one of the mechanisms contributing to MDR. In this study, we investigated the reversal of MDR by DLJ14, a ligustrazine derivate, in adriamycin (Adr) resistant human myelogenous leukemia (K562/A02) cells by modulating the expression of GSTπ and the activity of GST-related enzymes. In the MTT test, DLJ14 showed a weak inhibition on proliferation of both K562/A02 and K562 cells, while verapamil at the same concentration showed a much stronger inhibition. The sensitivity of K562/A02 cells to cytotoxic killing by Adr was enhanced by incubation with DLJ14 as a result of the increased intracellular accumulation of Adr. The accumulation of Adr induced by DLJ14 may due to down regulation of GST-related enzyme activity. Western blot analysis and RT-PCR showed that DLJ14 was able to inhibit the protein expression and mRNA expression of GSTπ in K562/A02 cells. Moreover, DLJ14 increased the expression of cellular c-Jun NH(2)-terminal kinase (JNK) in K562/A02 cells exposure to Adr. This is consistent with the inhibition of GSTπ. These results demonstrate that DLJ14 may be an attractive new agent for the chemosensitization of cancer cells.

Soy phytoestrogens modify DNA methylation of GSTP1, RASSF1A, EPH2 and BRCA1 promoter in prostate cancer cells.

BACKGROUND: The aim of this study was to determine the effects of soy phytoestrogens on the methylation of promoter genes in prostate tumors. The incidence of prostate cancer in Asia is thirty percent lower than in Western countries. Since soy phytoestrogens represent a large portion of the Asian diet, evidence suggests their protective effect against prostate cancer. MATERIALS AND METHODS: In three human prostate cancer cell lines, methylation-specific-PCR was used to determine the effect of soy isoflavones (genistein and daidzein), compared to known demethylating agent 5-azacytidine as control in the promoter regions of glutathione S-transferase P1 (GSTP1), Ras association domain family 1 (RASSF1A), ephrin B2 (EPHB2) and breast cancer 1 (BRCA1) genes. In parallel, immunohistochemistry was used to assess the effects of genistein, daidzein and 5-azacytidine treatment on the corresponding protein expression. RESULTS: All studied promoters, with the exception of that for BRCA1, were strongly methylated without treatment. After treatment by phytoestrogens, demethylation of GSTP1 and EPHB2 promoter regions was observed and an increase in their protein expression was demonstrated by immunohistochemistry. CONCLUSION: Epigenetic modifications of DNA, such as the promoter CpG island demethylation of tumor suppressor genes, might be related to the protective effect of soy on prostate cancer.

Rofecoxib augments anticancer effects by reversing intrinsic multidrug resistance gene expression in BGC-823 gastric cancer cells.

OBJECTIVE: To investigate combined chemotherapeutic effects of rofecoxib in combination with 5-fluorouracil (5-FU), cisplatin (DDP) and etoposide (VP-16) in vitro, and to explore the potential mechanisms in modulating multidrug resistance (MDR) expression. METHODS: The BGC-823 gastric cancer cell line was incubated for 48 h with 0.1 micromol/L rofecoxib, 5-FU, DDP and VP-16 (1 microg/mL, 10 microg/mL and 100 microg/mL) alone, and combined with rofecoxib, respectively. Methyl-thiazolyl-tetrazolium and the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-yriphosphate nick-end labeling assays were performed to calculate inhibitory rates and apoptotic index. Middle effects principles (CI values) were used to determine the interaction between rofecoxib and chemotherapeutic agents. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were employed to determine expression of MDR1, multidrug resistance-associated protein 1 (MRP1), glutathione S-tranferase-pi (GST-pi) mRNA and protein in gastric cancer cells administered by rofecoxib, respectively. RESULTS: Both anticancer drugs such as 5-FU, DDP and VP-16 and rofecoxib inhibited the cells' proliferation and induced apoptosis in a dose-dependent manner, and a more significant inhibition was achieved when the cells were co-treated with anticancer drugs and rofecoxib. There was a synergetic role when different concentrations of chemotherapeutic agents were combined with rofecoxib (all CI < 1, P < 0.01 or 0.05). RT-PCR analyses of MDR gene families in BGC-823 gastric cancer cells revealed a strong expression in MRP1 and GST-pi mRNA, but MDR1 mRNA was undetectable. After administration with different concentrations of rofecoxib (0.1, 1.0, 10 micromol/L), significant downregulation of MRP1 and GST-pi mRNA was observed (MRP1: from 0.984 +/- 0.093-0.513 +/- 0.098; GST-pi: from 1.078 +/- 0.201-0.472 +/- 0.084, P < 0.01 or 0.05). In addition, MRP1 and GST-pi protein expression induced by rofecoxib were also reduced (P < 0.01 or 0.05). CONCLUSION: Rofecoxib, a specific cyclooxygenase-2 inhibitor, plays a chemotherapeutic sensitizer role in various anticancer agents on the BGC-823 gastric cancer cell line, which could be partly explained by its ability to reverse the intrinsic MRP1 and GST-piin vitro.

Suppressive effect of sulindac on branch duct-intraductal papillary mucinous neoplasms.

BACKGROUND: When considering surgery for branch duct-intraductal papillary mucinous neoplasms (BD-IPMNs) with suspected malignancy, it should be recognized that these lesions are frequently multifocal and are usually found in elderly patients with potential comorbidities that could affect the outcome of surgery. Clinical trials of chemoprevention have been conducted for a wide variety of malignancies. METHODS: Twenty-two BD-IPMN patients participated in the trial at our institution from June 2004 to January 2007. Ten of the 22 patients who rejected surgical therapy although their lesions or clinical symptoms met the criteria for surgical resection of the International Association of Pancreatology guidelines were assigned to the treatment group. Sulindac (150 mg twice daily) and omeprazole (20 mg once daily) were administered to these patients for 18 months. The remaining 12 patients comprised the control group. Branch duct diameter and mural nodule heights were monitored by either magnetic resonance cholangiopancreatography (MRCP) or computed tomography (CT) and by endoscopic ultrasonography (EUS). RESULTS: Both branch duct diameter and mural nodule height of BD-IPMNs in the treatment group were significantly reduced, while those in the control group were unchanged. Immunohistochemical staining for cyclooxygenase-1 and -2 was negative in hyperplasia, adenoma and carcinoma portions of resected pancreatic specimens but was clearly positive for glutathione-S-transferase pi (GST-pi), suggesting that GST-pi is a putative target molecule for sulindac. CONCLUSIONS: Although a larger scale randomized controlled study is needed in future, the present results suggest the promise of chemoprevention of carcinoma derived from BD-IPMNs by sulindac.

Effects of atrazine on hepatic metabolism and endocrine homeostasis in rainbow trout (Oncorhynchus mykiss).

The herbicide atrazine (ATZ) is one of the most widely used pesticides in the world and is now under scrutiny for its alleged capacity to disrupt the endocrine system. Exhibiting negligible interaction with the estrogen receptor (ER), ATZ's mode of action remains to be elucidated. ATZ may act as an inducer of the enzyme aromatase, which converts androgens to estrogens, although other mechanisms should also be taken into consideration such as impairment of hepatic metabolism. Therefore we administered juvenile rainbow trout (Oncorhynchus mykiss) a dose of either 2 or 200 microg ATZ/kg, or of carrier control phosphate buffered saline (PBS) and we measured plasma concentrations of testosterone (T), 17beta-estradiol (E2) and vitellogenin (Vtg) 6 days after exposure. Simultaneously we analyzed hepatic gene expression of cytochrome P450 (CYP) 1A and pi-class glutathione S-transferase (GST-P), and catalase (CAT) activity. Although sex steroid levels showed no significant alterations, we found a dose-dependent increase in Vtg and a concomitant decrease in CYP1A. There was no effect of ATZ on GST-P mRNA levels but GST-P was positively correlated with CYP1A. Also, CYP1A was negatively correlated with liver CAT and E2, and varied with T concentrations in a hormetic manner. The results showed that ATZ can alter hepatic metabolism, induce estrogenic effects and oxidative stress in vivo, and that these effects are linked.

Glutathione transferase P1: an endogenous inhibitor of allergic responses in a mouse model of asthma.

RATIONALE: Although epidemiological studies have linked asthma susceptibility and severity to polymorphisms in human glutathione transferase Pi (GSTP) 1, there is no direct evidence for a functional involvement of GSTP1 in processes that are pathognomic of asthma. OBJECTIVES: To examine the role of GSTP1 in modulating the development of allergic airways disease. METHODS: Allergic airways disease was induced in wild-type (WT) and Gstp-null mice employing both acute and chronic models. Eosinophilia, goblet cells, and remodeling were quantified by histological assessment; respiratory function was determined using invasive methods. ELISA was used to evaluate Th2 cytokines, eotaxin, and phospho-c-Jun. Gstp1/2 expression was quantified by reverse transcriptase-polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: Compared with allergic WT mice, eosinophilia, goblet cell hyperplasia, airway remodeling, lung resistance, and IL-5 were enhanced in allergic Gstp-null mice. However, the protective efficacy of GSTP1 was mouse-strain dependent, and associated with inherent variation in expression of Gstp1. Although elevated levels of phospho-c-Jun were detected in Gstp-null mice, treatment of WT mice with a GSTP/c-Jun N-terminal kinase (JNK) inhibitory peptide enhanced phospho-c-Jun and significantly attenuated allergic responses. CONCLUSIONS: GSTP1 attenuates the severity of allergic airways disease. However, the efficacy of GSTP1 correlated with mouse strain-dependent variation in Gstp1 expression. Although GSTP1 attenuated c-Jun phosphorylation, treatment with a GSTP/JNK inhibitory peptide revealed an inverse relationship between c-Jun phosphorylation and allergic responses, indicating that the mechanism by which GSTP attenuates allergic responses is not dependent on the JNK/c-Jun axis. Our data, together with epidemiological evidence, suggest variation in expression and/or function of this protein is an important determinant in asthma pathophysiology.

Expression of MRP1 and GSTP1-1 modulate the acute cellular response to treatment with the chemopreventive isothiocyanate, sulforaphane.

A major component of the anticarcinogenic activity of the dietary chemopreventive agent sulforaphane (SFN) is attributed to its ability to induce expression of phase II detoxification genes containing the antioxidant response element (ARE) within their promoters. Because SFN is a reactive electrophile--readily forming conjugates with glutathione (GSH)--we asked whether expression of glutathione S-transferase (GST) P1-1 and the GSH conjugate efflux pump, multidrug resistance or resistance-associated protein (MRP) 1, would significantly modify the cellular response to SFN exposure. This was investigated using GST- and MRP1-poor parental MCF7 cells and transgenic derivatives expressing GSTP1-1 and/or MRP1. Compared with parental cells, expression of GSTP1-1 alone enhanced the rate of intracellular accumulation of SFN and its glutathione conjugate, SFN-SG--an effect that was associated with increased ARE-containing reporter gene induction. Expression of MRP1 greatly reduced SFN/SFN-SG accumulation and resulted in significant attenuation of SFN-mediated induction of ARE-containing reporter and endogenous gene expression. Coexpression of GSTP1-1 with MRP1 further reduced the level of induction. Depletion of GSH prior to SFN treatment or the substitution of tert-butylhydroquinone for SFN abolished the effects of MRP1/GSTP1-1 on ARE-containing gene induction-indicating that these effects are GSH dependent. Lastly, analysis of NF-E2-related factor 2 (Nrf2)--a transcription factor operating via binding to the ARE--showed that the increased levels of Nrf2 following SFN treatment were considerably less sustained in MRP1-expressing, especially those coexpressing GSTP1-1, than in MRP1-poor cells. These results suggest that the regulating effects of MRP1 and GSTP1-1 expression on SFN-dependent induction of phase II genes are ultimately mediated by altering nuclear Nrf2 levels.

Modulation of gene methylation by genistein or lycopene in breast cancer cells.

Dietary agents with chemopreventive potential, including soy-derived genistein and tomato-derived lycopene, have been shown to alter gene expression in ways that can either promote or potentially inhibit the carcinogenic processes. To begin to explore the mechanisms by which these agents may be acting we have examined the DNA methylation modulating capacity of genistein or lycopene for several genes relevant to breast cancer in the breast cancer cell lines MCF-7 and MDA-MB-468, as well as in immortalized but noncancer fibrocystic MCF10A breast cells. We find using methylation specific PCR (MSP) that a low, nontoxic concentration of genistein (3.125 microM, resupplemented every 48 hr for 1 week) or a single dose of lycopene (2 microM) partially demethylates the promoter of the GSTP1 tumor suppressor gene in MDA-MB-468 cells. RT-PCR studies confirm a lack of GSTP1 expression in untreated MDA-MB-468, with restoration of GSTP1 expression after genistein or lycopene treatment. The RARbeta2 gene however, was not demethylated by genistein or lycopene in either of these breast cancer cell lines. But, lycopene (2 microM, once per week for 2 weeks) did induce demethylation of RARbeta2 and the HIN-1 genes in the noncancer MCF10A fibrocystic breast cells. These data show for the first time that the tomato carotenoid lycopene has direct DNA demethylating activity. In summary, both genistein and lycopene, at very low, dietarily relevant concentrations can potentially mitigate tumorigenic processes via promoter methylation modulation of gene expression.

Quantitative analysis of liver GST-P foci promoted by a chemical mixture of hexachlorobenzene and PCB 126: implication of size-dependent cellular growth kinetics.

The objectives of this study were twofold: (1) evaluating the carcinogenic potential of the mixture of two persistent environmental pollutants, hexachlorobenzene (HCB) and 3,3',4,4',5-pentachlorobiphenyl (PCB 126), in an initiation-promotion bioassay involving the development of pi glutathione S-transferase (GST-P) liver foci, and (2) analyzing the GST-P foci data using a biologically-based computer model (i.e., clonal growth model) with an emphasis on the effect of focal size on the growth kinetics of initiated cells. The 8-week bioassay involved a series of treatments of initiator, two-thirds partial hepatectomy, and daily oral gavage of the mixture of two doses in male F344 rats. The mixture treatment significantly increased liver GST-P foci development, indicating carcinogenic potential of this mixture. Our clonal growth model was developed to simulate the appearance and development of initiated GST-P cells in the liver over time. In the model, the initiated cells were partitioned into two subpopulations with the same division rate but different death rates. Each subpopulation was further categorized into single cells, mini- (2-11 cells), medium- (12-399 cells), and large-foci (>399 cells) with different growth kinetics. Our modeling suggested that the growth of GST-P foci is size-dependent; in general, the larger the foci, the higher the rate constants of division and death. In addition, the modeling implied that the two doses promoted foci development in different manners even though the experimental foci data appeared to be similar between the two doses. This study further illustrated how clonal growth modeling may facilitate our understanding in chemical carcinogenic process.

Epigenetic changes in the rat livers induced by pyrazinamide treatment.

Drug-induced liver injury, including drug-induced hepatotoxicity during the treatment of tuberculosis infection, is a major health problem with increasingly significant challenges to modern hepatology. Therefore, the assessment and monitoring of the hepatotoxicity of antituberculosis drugs for prevention of liver injury are great concerns during disease treatment. The recently emerged data showing the ability of toxicants, including pharmaceutical agents, to alter cellular epigenetic status, open a unique opportunity for early detection of drug hepatotoxicity. Here we report that treatment of male Wistar rats with antituberculosis drug pyrazinamide at doses of 250, 500 or 1000 mg/kg/day body weight for 45 days leads to an early and sustained decrease in cytosine DNA methylation, progressive hypomethylation of long interspersed nucleotide elements (LINE-1), and aberrant promoter hypermethylation of placental form glutathione-S-transferase (GSTP) and p16(INK4A) genes in livers of pyrazinamide-treated rats, while serum levels of bilirubin and activity of aminotransferases changed modestly. The early occurrence of these epigenetic alterations and their association with progression of liver injury specific pathological changes indicate that alterations in DNA methylation may be useful predictive markers for the assessment of drug hepatotoxicity.

Modulation of human glutathione s-transferases by polyphenon e intervention.

PURPOSE: Green tea consumption has been associated with decreased risk of certain types of cancers in humans. Induction of detoxification enzymes has been suggested as one of the biochemical mechanisms responsible for the cancer-preventive effect of green tea. We conducted this clinical study to determine the effect of repeated green tea polyphenol administration on a major group of detoxification enzymes, glutathione S-transferases (GST). METHODS: A total of 42 healthy volunteers underwent a 4-week washout period by refraining from tea or tea-related products. At the end of the washout period, a fasting blood sample was collected, and plasma and lymphocytes were isolated for assessment of GST activity and level. Following the baseline evaluation, study participants underwent 4 weeks of green tea polyphenol intervention in the form of a standardized Polyphenon E preparation at a dose that contains 800 mg epigallocatechin gallate (EGCG) once a day. Polyphenon E was taken on an empty stomach to optimize the oral bioavailability of EGCG. Upon completion of the intervention, samples were collected for postintervention GST assessment. RESULTS: Four weeks of Polyphenon E intervention enhanced the GST activity in blood lymphocytes from 30.7 +/- 12.2 to 35.1 +/- 14.3 nmol/min/mg protein, P = 0.058. Analysis based on baseline activity showed that a statistically significant increase (80%, P = 0.004) in GST activity was observed in individuals with baseline activity in the lowest tertile, whereas a statistically significant decrease (20%, P = 0.02) in GST activity was observed in the highest tertile. In addition, Polyphenon E intervention significantly increased the GST-pi level in blood lymphocytes from 2,252.9 +/- 734.2 to 2,634.4 +/- 1,138.3 ng/mg protein, P = 0.035. Analysis based on baseline level showed that this increase was only significant (P = 0.003) in individuals with baseline level in the lowest tertile, with a mean increase of 80%. Repeated Polyphenon E administration had minimal effects on lymphocyte GST-mu and plasma GST-alpha levels. There was a small but statistically significant decrease (8%, P = 0.003) in plasma GST-alpha levels in the highest tertile. CONCLUSIONS: We conclude that 4 weeks of Polyphenon E administration resulted in differential effects on GST activity and level based on baseline enzyme activity/level, with GST activity and GST-pi level increased significantly in individuals with low baseline enzyme activity/level. This suggests that green tea polyphenol intervention may enhance the detoxification of carcinogens in individuals with low baseline detoxification capacity.

In vitro drug response and molecular markers associated with drug resistance in malignant gliomas.

PURPOSE: Drug resistance in malignant gliomas contributes to poor clinical outcomes. We determined the in vitro drug response profiles for 478 biopsy specimens from patients with the following malignant glial histologies: astrocytoma (n = 71), anaplastic astrocytoma (n = 39), glioblastoma multiforme (n = 259), oligodendroglioma (n = 40), and glioma (n = 69). EXPERIMENTAL DESIGN: Samples were tested for drug resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, dacarbazine, paclitaxel, vincristine, and irinotecan. Biomarkers associated with drug resistance were detected by immunohistochemistry, including multidrug resistance gene-1, glutathione S-transferase pi (GSTP1), O(6)-methylguanine-DNA methyltransferase (MGMT), and mutant p53. RESULTS: In vitro drug resistance in malignant gliomas was independent of prior therapy. High-grade glioblastomas showed a lower level of extreme drug resistance than low-grade astrocytomas to cisplatin (11% versus 27%), temozolomide (14% versus 27%), irinotecan (33% versus 53%), and BCNU (29% versus 38%). A substantial percentage of brain tumors overexpressed biomarkers associated with drug resistance, including MGMT (67%), GSTP1 (49%), and mutant p53 (41%). MGMT and GSTP1 overexpression was independently associated with in vitro resistance to BCNU, whereas coexpression of these two markers was associated with the greatest degree of BCNU resistance. CONCLUSIONS: Assessment of in vitro drug response and profiles of relevant tumor-associated biomarkers may assist the clinician in stratifying patient treatment regimens.

Dietary selenium intake and genetic polymorphisms of the GSTP1 and p53 genes on the risk of esophageal squamous cell carcinoma.

Few studies have assessed potential effect modifications by polymorphisms of susceptibility genes on the association between selenium intake and esophageal squamous cell carcinoma (ESCC). We studied the joint effects of dietary selenium and the GSTP1 and p53 polymorphisms on ESCC risk in a population-based case-control study with 218 ESCC cases and 415 controls in Taixing City, China. Dietary selenium intake was estimated from a food frequency questionnaire with 97 food items. GSTP1 and p53 polymorphisms were detected by RFLP-PCR assays. Logistic regression analyses were done to estimate odds ratios (OR) and 95% confidence intervals (95% CI). Reduced ESCC risk was observed among individuals in the highest quartile of dietary selenium intake (adjusted OR, 0.31; 95% CI, 0.13-0.70) with a dose-dependent gradient (P(trend) = 0.01). The p53 Pro/Pro genotype was associated with increased risk of ESCC compared with the Arg/Arg genotype (adjusted OR, 2.02; 95% CI, 1.19-3.42). When combined with selenium consumption, an obvious increased risk was observed among individuals with the p53 Pro/Pro or GSTP1 Ile/Ile genotype with adjusted ORs of 3.19 (95% CI, 1.74-5.84) and 1.90 (95% CI, 1.03-3.51), respectively. Among smokers and alcohol drinkers, elevation of ESCC risk was more prominent among p53 Pro/Pro individuals who consumed a low level of dietary selenium (adjusted OR, 3.59; 95% CI, 1.49-8.66 for smokers and 6.19; 95% CI, 1.83-20.9 for drinkers). Our study suggests that the effect of dietary selenium on the risk of ESCC may be modulated by tobacco smoking, alcohol drinking, and p53 Pro/Pro and GSTP1 Ile/Ile genotypes.

Transcriptional and post-transcriptional regulation of glutathione S-transferase P1 expression during butyric acid-induced differentiation of K562 cells.

Over-expression of glutathione S-transferase P1 is related to chemotherapeutic drug resistance as well as to differentiation of human erythroleukemia cells. In opposition to previously described differentiating inducers which enhance the GST-resistance phenotype, time- and concentration-dependent activation of both erythroid and megakaryocytic differentiation pathways by butyric acid progressively diminished GSTP1 mRNA expression. GSTP1 mRNA expression decreased by 25% (p<0.01) and 64% (p<0.01) in 1mM and 2mM butyric acid-differentiated K562 cells, respectively. These results were associated to both a reduction of GATA-1 binding activity to the GSTP1 promoter and to a posttranscriptional destabilization of GSTP1 mRNA in a concentration dependent manner. Indeed, GSTP1 mRNA half-life decreased from 43.8 to 36.2 h and 12.6 h in 1mM- and 2mM-treated cells, respectively.