Differential expression of S1P receptor subtypes in human bladder transitional cell carcinoma.

PURPOSE: Sphingosine 1 phosphate (S1P), S1P receptors (S1PRs) and their signaling pathways play an important role in the fate of cancer cells. The expression pattern of S1PR subtypes (S1PR1-S1PR5) may alter in cancer development stages, depending on the origin and the pathologic features of tumors. The present study aimed to examine the relationship between plasma S1P levels and the expression of S1PR subtypes in bladder tumors. METHODS/PATIENTS: These changes were evaluated in terms of the pathologic grades and stages of human bladder cancer samples. For this, tumor biopsies from 41 new bladder cancer patients as well as 26 normal-looking bladder tissues were collected and processed for immunohistochemistry (IHC) and quantitative real-time RT-PCR of S1PR subtypes. Plasma S1P level was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The results show that tissue S1PR1, S1PR2 and S1PR3 are over-expressed in all tumors regardless of their pathological grade (~ 3, ~ 6 and ~ 104 folds, respectively). These results were corroborated by IHC data showing accumulation of S1PR subtypes 1 and 2 in the tissues. Plasma S1P in the plasma samples from patients was in the range of control samples (Controls; 256 ± 47; patients, 270 ± 41). CONCLUSIONS: Overexpression of S1PR1, S1PR2 and S1PR3 in bladder tumor biopsies which were corroborated with the pathological grades and stages may suggest that S1PR profile in tumor biopsies is a promising marker in the diagnosis of bladder carcinoma.

Inhibition of breast tumor growth and abnormal angiogenesis in mice treated with endothelial cells and their progenitor mesenchymal stem cells derived from bone marrow.

Incorporation of endothelial cells or their progenitor cells into newly sprouting blood vessels can contribute to tissue vascularization after ischemic injury. However, the interaction of the stem cells-derived endothelial cells with angiogenesis within tumors is not well understood. The aim of this study was to examine the efficiency of endothelial-like cells derived from MSCs in controlling breast tumor growth associated with abnormal angiogenesis. For this purpose, Balb/c mouse model of breast carcinoma was developed and subjected to intra tumor (I.T)/intra venous (I.V) therapy with undifferentiated MSCs or endothelial cells derived from them. The homing of the stem cells was approved by measuring different markers as well as tracing green fluorescence protein (GFP)-labeled MSCs in the tumors. Tumor growth was measured following cell therapy using a digital caliper. At the end of treatment period (30 days) the angiogenesis markers; VEGFR2 expression as well as micro-vessel density (MVD) using CD31 were estimated in tumor tissues. Stem cell transplantation to mice bearing breast tumors resulted in tumor growth suppression in all experimental groups. The endothelial markers; CD31 and VEGFR2 were down regulated following I.T delivery of the endothelial cells. Accordingly, angiogenesis was suppressed following I.T administration of endothelial cells which was associated with increased focal necrosis in the tumors. In conclusion, data show that endothelial cells directly injected into tumors is more efficient compared to undifferentiated MSCs in controlling tumor-associated angiogenesis and tumor growth.

Effect of Curcumin on Aspergillus parasiticus Growth and Expression of Major Genes Involved in the Early and Late Stages of Aflatoxin Biosynthesis.

BACKGROUND: The effect of curcumin as a natural safe compound with different biological activities was examined on fungal growth and aflatoxin production in Aspergillus parasiticus NRRL 2999. METHODS: The fungus was cultured in presence of serial two-fold concentrations of curcumin (125-2000 µg/ml) in yeast extract sucrose broth for 3 days at 28°C. Mycelia dry weight was determined as an index of fungal growth, while aflatoxin production was assessed by high performance liquid chromatography (HPLC). The expression of ver-1, nor-1, pksA, omtA and aflR genes in aflatoxin biosynthetic pathway was evaluated by real time PCR. RESULTS: Curcumin strongly inhibited aflatoxin B(1) production in the range of 26.6 to 94.9% by serial two-fold concentrations from 125 to 2000 µg/ml. Fungal growth was also inhibited by the compound in the range of 34.0 to 60.8%. Analysis of the expression of aflatoxin pathway genes by real time PCR showed that curcumin inhibited the expression of ver-1, nor-1, pksA, omtA and aflR genes at concentrations of 250 and 1000 µg/ml. In concentration of 1000 µg/ml, gene expression was reduced by 31.3%, 44.6%, 57.1% 110.9% and 286.7% accordingly. Reduction in the expression of aflatoxin biosynthesis genes was significant only for aflR. In ferric reducing ability of plasma (FRAP) assay, curcumin showed strong antioxidant activity at all concentrations tested. CONCLUSION: Curcumin may be employed successfully as a good candidate in controlling of toxigenic fungal growth on food and feed and subsequent contamination with aflatoxins in practice.

A novel aflatoxin-binding Bacillus probiotic: Performance, serum biochemistry, and immunological parameters in Japanese quail.

Two experiments were performed to screen bacilli isolated from quails for their aflatoxin removal potential and to assess the efficiency of their amelioration of experimental aflatoxicosis. Nonhemolytic bacilli were selected for in vitro aflatoxin B1 (AFB1) removal and conventional probiotic tests. The isolate with the highest scores was selected for assessment in field experiments and was identified as Berevibacillus laterosporus (Bl). In the second experiment, 125 male Japanese quails (21 d old) were divided into 5 groups with 5 replications to compare the toxin removal efficiency of Bl with that of a commercial toxin binder, improved Millbond-TX (IMTX). The experimental groups were as follows: Control (without any feed additive or AFB1); AFB1 (2.5 mg/kg); AFB1+Bl (2.5 mg/kg+10(8) cfu/mL); AFB1+IMTX (2.5 mg/kg+2.5 g/kg); and Bl (10(8) cfu/mL). The greatest BW gain and slaughter and carcass weights were found in the Bl group and the lowest values were observed in the AFB1 group (P<0.05). Feeding AFB1 alone to the chicks resulted in a significant decrease in serum albumin, total protein, and glucose and cholesterol levels but a significant increase in serum uric acid, urea, creatinin and phosphorus (P<0.05). Treatment of birds on AFB1 with Bl restored these to their original levels (P<0.05). AFB1+Bl-fed birds had serum aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and alkaline phosphatase enzyme activity similar to control birds (P<0.05). Antibody titer against Newcastle disease virus was found to be lowest in the AFB1 group but highest in the Bl group (P<0.05). Antibody production against sheep red blood cells was lower in the AFB1 group compared with the AFB1+Bl group (P<0.05). Berevibacillus laterosporus supplementation of the AFB1 diet restored the skin response to 2,4-dinitro 1-chlorobenzene to levels comparable with control birds (P<0.05). It can be concluded that selected indigenous Bl is a promising probiotic with AFB1 removal potential.

Inhibitory effects of dietary caraway essential oils on 1,2-dimethylhydrazine-induced colon carcinogenesis is mediated by liver xenobiotic metabolizing enzymes.

The effects of dietary essential oils prepared from caraway seeds on colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in rats has been studied. The number of aberrant crypt foci (ACF) and aberrant crypt (AC) induced by DMH were found to be significantly inhibited in colon of rats treated with essential oils in diet (0.01 and 0.1%). To find out the mechanism(s) by which the essential oils reduced colon premalignancies, plasma, liver, and colon tissues were collected and analyzed for parameters related to oxidative stress and xenobiotic metabolizing enzymes. Lack of influence of caraway extracts on hepatic lipid peroxidation products, superoxide dismutase (SOD), catalase (CAT) and ferric reducing ability of plasma (FRAP) may suggest that the oils do not interfere with these factors. However, it was clearly shown that DMH-related changes in hepatic and colonic cytochrome P4501A1 (CYP1A1) and glutathione S-transferae (GST) activities were recovered in liver but not in colon tissue in animals treated with caraway oil preparations. In conclusion, histopathological and biochemical data clearly showed that inhibition of colon premalignant lesions induced by DMH is mediated by interference of caraway oil components in the activities of the main hepatic xenobiotic metabolizing enzymes.

Fabrication and kinetic studies of a novel silver nanoparticles-glucose oxidase bioconjugate.

In this study, a new effective, pH and thermally stable glucose oxidase (GOX)-silver nanoparticles (AgNPs) bioconjugate was designed. AgNPs were synthesized based on the reduction of silver nitrate (AgNO(3)) by sodium borohydride (NaBH(4)) using two simple procedures. Periodic acid was used for oxidation of the GOX and emission of Lucifer yellow (LyCH) was monitored by spectrofluorometer for evaluation of the oxidation properties of the GOX. The oxidized GOX (Ox-GOX) was immobilized on AgNPs by its sugar moieties via 6-aminohexanoic acid (6AHA) as linker. A sample of the synthesized bioconjugate was loaded on 7.5% non-denaturing polyacrylamide gel electrophoresis (PAGE) to confirm its structural and physical stability. The results from enzymatic activity assay showed that the bioconjugate, GOX and Ox-GOX were similar in stability and activity in acidic and basic pH (optimum pH=7.0-8.0). Based on the results from thermal stability assay, it was found that the activity of the bioconjugate was found to be higher at lower temperatures. The V(max) of the bioconjugate, GOX, and Ox-GOX was estimated as 28.6, 6.2, and 6 IU microg(-1) enzyme and the K(m) was calculated as 2.7, 9, and 9.5 mM, respectively. It was found that the immobilization method improves the activity and stability of the GOX in different pH and temperatures. As a conclusion, the proposed method opens up the way to the development of a new bioconjugate with potential use in sensing, and many find potential applications in clinical diagnostics, medicine, and industries.

Sodium selenite improves the in vitro follicular development by reducing the reactive oxygen species level and increasing the total antioxidant capacity and glutathione peroxide activity.

BACKGROUND: The aim of this study was to investigate the effect of sodium selenite (SS) on reactive oxygen species (ROS) production, total antioxidant capacity (TAC) and glutathione peroxide (GPx) activity of cultured pre-antral follicles derived from vitrified and non-vitrified ovarian tissue. METHODS: Immature mouse ovaries were vitrified, and mechanically isolated pre-antral follicles from vitrified and non-vitrified samples were cultured in TCM 199 medium supplemented with different concentrations (0, 5 and 10 ng/ml) of SS. Follicular, oocyte and embryo development was assessed. In parallel, ROS, TAC and GPx levels were analyzed after 0, 12, 24, 48, 72 and 96 h of culture. RESULTS: Development rates of follicles, oocytes and embryos were significantly higher in SS-supplemented groups (P < 0.005). ROS production was increased, and TAC levels and GPx activities were decreased after 24 h of culture of pre-antral follicles in vitrified and non-vitrified groups, whereas in the presence of SS, ROS production was decreased and TAC levels and selenium-dependent GPx-specific activities were increased after 96 h of culture. Vitrified and non-vitrified samples responded in a similar manner. CONCLUSION: SS caused an increase in follicular TAC level and GPx activity and a decrease in ROS level, thus improving the in vitro development of follicles.

Hepatoprotective effects of gamma-irradiated caraway essential oils in experimental sepsis.

Irradiation is an important method of processing herbal drugs, while our understanding of the effects of gamma-irradiation on pharmacological properties of seed products such as caraway essential oils is however still very limited. In this study, caraway seeds were irradiated at dose levels of 0, 10 and 25kGy. After extracting the essential oils, the effects of fresh and gamma-irradiated caraway oils (100mg/kg b.w) on preventing septic-related oxidative liver injury induced by cecal ligation and puncture (CLP) model were investigated by measuring oxidative stress parameters in the liver. CLP operation caused a marked increase in myeloperoxidase (MPO) activity which was readily reversed in rats treated with fresh and irradiated caraway oils. Likewise, thiobarbituric acid reactive substances (TBARS) level in the liver was compensated in rats treated with the fresh and irradiated caraway oils. Moreover, liver GSH which was initially depleted due to CLP was recovered by essential oil treatments. The protective role of oils was further confirmed by showing that liver function tests (ALT/AST) as well as histopathological changes following CLP operation were recovered in rats treated with oils from either fresh or irradiated caraway seeds. These data may suggest that gamma-irradiation to caraway seeds at 10 and 25kGy has no influence on the antioxidative properties of caraway essential oils.

The effects of different concentrations of sodium selenite on the in vitro maturation of preantral follicles in serum-free and serum supplemented media.

PURPOSE: This study was to investigate the effect of sodium selenite (SS) on in vitro maturation of mouse preantral follicles. METHODS: The isolated preantral follicles were cultured in TCM 199 medium supplemented with different concentrations (0, 5, 10, 15 ng/ml) of SS and 3 mg/ml bovine serum albumin (BSA) or 5% Fetal Bovine Serum (FBS). The ovulation was induced by addition of 1.5 IU/ml human chorionic gonadotropin. The size and development of follicles and oocytes were assessed by calibrated eyepiece. RESULTS: The survival rates of follicles in FBS supplemented groups containing 5 and 10 ng/ml SS (88.23%, 90.83%) were higher than other groups (P < 0.05 and P < 0.001 respectively). The mean diameter of follicles (199.84 +/- 15.58 microm) and the percentage of MII oocyte (33.08%) were higher in FBS supplemented group containing 10 ng/ml SS (P < 0.001). CONCLUSION: The sodium selenite and FBS improve the in vitro growth and maturation of mouse preantral follicles.

Functional hepatocyte-like cells derived from human bone marrow mesenchymal stem cells on a novel 3-dimensional biocompatible nanofibrous scaffold.

AIM: To supporting growth and functional differentiation of adult stem cells into hepatocytes in a well-controlled manner, we performed differentiation of human bone marrow mesenchymal stem cells (hBMSCs) to hepatocytes-like cells on a constructed 3-dimensional (3D) nanofibrous biocompatible scaffold. METHODS: After characterization of the hBMSCs isolated from human bone marrow, the performance of the cells seeded and their proliferation on the scaffold was evaluated by scanning electron microscopy (SEM) and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Different approaches such as immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), and biochemical assays were used to estimate the ability of hBMSC-derived cells to express hepatocyte-specific markers. RESULTS: Scanning electron micrographs and MTT analysis revealed the cells were able to expand and remained biologically and metabolically active for 21 days. Immunocytochemical analysis of albumin and alfa-fetoprotein showing the accumulation of these markers in differentiated cells was confirmed by RT-PCR. Additional markers such as cytochrome P450 3A4, cytokeratin-18, and cytokeratin-19 detected by RT-PCR showed progressive expression during 3 weeks of differentiation on 3D scaffold. The hepatocyte-like cells displayed several characteristics of metabolic functions as judged by production of albumin, urea, transferrin, serum glutamic pyruvic transaminase (SGPT), and serum oxaloacetate aminotransferase (SGOT). Levels of above-mentioned markers, except SGOT in differentiated cells on scaffold, were found to be significantly greater than in the 2D culture system (p<0.05). CONCLUSION: Overall data suggest that the engineered nanofibrous scaffold is a conductive matrix for functional hBMSC-derived hepatocyte-like cells and is promising for maintenance of hepatocytes suitable for implantation.

Efficient replacing of fetal bovine serum with human platelet releasate during propagation and differentiation of human bone-marrow-derived mesenchymal stem cells to functional hepatocyte-like cells.

OBJECTIVES: The aim of this study was to find out substitution effect of fetal bovine serum (FBS) with human platelet releasate (HPR) as a major growth factor source during expansion and differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) into hepatocytes. METHODS: Propagation and differentiation potential of hBMSCs into hepatocyte-like cells in a medium fortified with HPR instead of FBS were investigated with morphological, cytochemical and molecular experiments. RESULTS: Multiplex analysis showed that HPR was more efficient than FBS in supporting hBMSC outgrowth. The proliferation rate of MSC in presence of HPR (derived from 10(9) platelets/ml) was about threefold greater than that of FBS (P < 0.001). Despite the differences in MTT value, hBMSCs-driven HPR or FBS did not differ in terms of gross morphology, immunophenotype and osteogenic differentiation potential. Hepatic differentiation of hBMSCs was successfully performed in the media enriched with HPR. Immunoreactivity of cells with monoclonal antibodies against for albumin and alpha-fetoprotein (AFP) was even more positive in hepatocytes differentiated in presence of HPR as compared to that of FBS. The gene expression of albumin, AFP and cytokeratin-18 at mRNA levels in differentiated cells attest to supporting role of HPR in hepatic differentiation media. These findings were further confirmed with greater urea production (approximately twofold) in the culture media of cells differentiated under HPR compared to that in FBS (P < 0.001). CONCLUSION: Human platelet releasate is an efficient and safe substitute for FBS in culture media used for expansion and differentiation of hBMSCs to hepatocyte.

Relationship between genetic polymorphism of glutathione S-transferase-p1 and p53 protein accumulation in Iranian esophageal squamous cell carcinoma patients.

BACKGROUND: It has been reported that the activity of glutathione S-transferase (GST) is over-expressed in plasma and esophagus biopsies in Iranian patients suffering from esophageal squamous cell carcinoma (SCC). The aim of this study was to find out the frequency of GST-P genotypes in these patients. Moreover, the association of GST-P genotypes with p53 protein accumulation in esophageal epithelium was investigated. MATERIALS AND METHODS: DNA isolated from paraffin-embedded tissue biopsies from patients suffering from esophageal SCC (n = 56) were collected. polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using Alw261 enzyme was applied to determine GST-P genotypes (Ile 105 Val). All the samples were also subjected to immunohistochemistry (IHC) for p53. RESULTS: The frequency of GST-P genotypes in Iranian esophagus SCC patients for Ile/Ile, Ile/Val and Val/Val was 73.2, 21.5 and 5.3%. There was no association between GST-P polymorphism and p53 accumulation in esophageal epithelial cells. CONCLUSIONS: The frequency of GST-P polymorphism was not associated with p53 protein accumulation in esophagus epithelium. The frequency of polymorphic variants of GST-P, Ile/Ile, Ile/Val and Val/Val in SCC patients may suggest that Ile to Val substitution in GST-P gene dose not represent susceptibility to SCC in high-risk Iranian population.

Comparison of glutathione S-transferase-Pi expression at mRNA levels in oesophageal mucosa using RT-PCR-ELISA in individuals with reflux diseases, adenocarcinoma and squamous cell carcinoma.

OBJECTIVES: To develop a RT-PCR technique coupled with Enzyme Linked Immunosobant Assay (ELISA) i.e. RT-PCR-ELISA for measurement of class-Pi glutathione S-transferase (GST-P)-specific mRNA in esophageal diseases. METHODS: In this study, 66 esophageal tissue biopsies diagnosed as non-erosive reflux disease (NERD), gastroesophageal reflux disease (GERD), adenocarcinoma (ADC), and squamous cell carcinoma (SCC) were used. Standardization of the RT-PCR-ELISA was carried out using specific GST-Pi and beta-actin primers, biotin labeled probe, DIG-labeling RT-PCR and anti-DIG-HRP conjugate. RESULTS: The results of RT-PCR-ELISA based on OD ratio of GST-Pi mRNA/beta-actin showed that there was no significant difference in GST-Pi expression in normal, NERD and GERD samples. Overexpression of GST-Pi in malignant tissues (ADC and SCC) was distinguishable. The OD ratio of GST-Pi mRNA expression to beta-actin mRNA was 1.17+/-0.13 and 1.3+/-0.13 in ADC and SCC samples, respectively, which is significantly higher (P<0.05) than matching control (0.78+/-0.06 and 0.85+/-0.07). CONCLUSIONS: RT-PCR-ELISA showed that GST-Pi expression was not altered in GERD and NERD esophagus, whereas, in ADC and SCC samples, it was significantly higher (P<0.05) as compared to inflamed and normal tissues.

Suppressive effects of caraway (Carum carvi) extracts on 2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin-dependent gene expression of cytochrome P450 1A1 in the rat H4IIE cells.

Cytochrome P450 1A1 (CYP1A1) is among the cytochrome P450 classes known to convert xenobiotics and endogenous compounds to toxic and/or carcinogenic metabolites. Suppression of CYP1A1 over expression by certain compounds is implicated in prevention of cancer caused by chemical carcinogens. Chemopreventive agents containing high levels of flavonoids and steroids-like compounds are known to suppress CYP1A1. This study was carried out for assessment of the genomic and proteomic effects of caraway (Carum carvi) extracts containing high levels of both flavonoids and steroid-like substances on ethoxy resorufin dealkylation (EROD) activity and CYP1A1 at mRNA levels. Rat hepatoma cells co-treated with a CYP1A1 inducer i.e. TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) and different preparations of caraway extracts at concentrations of 0, 0.13, 1.3, and 13 microM in culture medium. After incubation (37 degrees C and 7% CO2 for 20 h), changes in EROD specific activity recorded and compared in cells under different treatments. The results show that caraway seed extract prepared in three different organic solvents suppressed the enzyme activity in hepatoma cells in a dose-dependent manner. The extracts added above 0.13 microM could significantly inhibit EROD activity and higher levels of each extract (1.3 and 13 microM) caused approximately 10-fold suppression in the enzyme activity. Accordingly, data obtained from the RT-PCR (TaqMan) clearly showed the suppressive effects of plant extract on CYP1A1-related mRNA expression. These data clearly show that substances in caraway seeds extractable in organic solvents can potentially reverse the TCDD-dependent induction in cytochrome P450 1A1.

Production of novel recombinant single-domain antibodies against tandem repeat region of MUC1 mucin.

Recently, the existence of "heavy-chain" antibody in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to peptides. In addition, there was not any report of production of single-domain antibodies in two-humped camels (Camelus bactrianus). In the present study, these questions are addressed. We showed the feasibility of immunizing old world camels, cloning the repertoire of the variable domain of their heavy-chain antibodies, panning and selection, leading to the successful identification of minimum-sized antigen binders. Antigen-specific fragments of the heavy-chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable, highly soluble, and react specifically and with high affinity to the antigens. In this study, we immunized two camels (Camelus dromedarius and Camelus bactrianus) with homogenized cancerous tissues, synthetic peptide, and human milk fat globule membrane (HMFG), and generated two V(HH) libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the tandem repeat region of MUC1. The camels' single-domain V(HH) harbor the original, intact antigen binding site and reacted specifically and with high affinity to the tandem repeat region of MUC1. Indeed soluble, specific antigen binders and good affinities (in the range of 0.2 x 10(9) M(-1) to 0.6 x 10(9) M(-1)) were identified from these libraries. This is the first example of the isolation of camel anti-peptide V(HH) domains.

Measurement of glutathione S-transferase and its class-pi in plasma and tissue biopsies obtained after laparoscopy and endoscopy from subjects with esophagus and gastric cancer.

OBJECTIVES: To develop an indirect enzyme-linked immunosorbent assay (ELISA) for measuring class-pi glutathione S-transferase (GST) in plasma, and tissue biopsies obtained from upper gastrointestinal cancer (UGI Ca) patients. METHODS: GST activity and GST-pi concentration were detected in normal human squamous esophageal epithelium, normal gastric cardia and their corresponding malignant tumor biopsies. RESULTS: Plasma GST was significantly higher (p < 0.05) in UGI Ca patients as compared to those obtained from normal individuals. Plasma GST-pi concentration in normal subjects was 6.6 +/- 1.9 ng/mg protein, whereas it was higher in UGI Ca patients (esophageal, 10.0 +/- 1.8; gastric, 10.7 +/- 1.7 ng/mL, p

Acetaminophen-glutathione conjugate formation in a coupled cytochrome P-450-glutathione S-transferase assay system mediated by subcellular preparations from adult and weanling rat tissues.

Previous studies from this laboratory indicated that glutathione (GSH) conjugate formation with acetaminophen (APAP) is remarkably induced in liver of weanling rats in response to a single overdose of the drug administered intraperitoneally (ip). Increased APAP-GSH conjugation has been attributed to inducible glutathione S-transferases (GSTs) in dividing hepatocytes. In order to verify this finding, an in vitro reconstitution assay containing liver microsomes (source of cytochrome P-450) and cytosolic fractions (source of GST) from livers and kidneys of adult and weanling rats has been established. In vitro incubation of the reaction mixture was followed by solvent extraction, enzymatic digestion and HPLC analysis of the conjugate. Under controlled conditions, in vitro, the rate of APAP-GSH conjugation reflected the GST activity of cytosolic sample added to incubation system. The activity of cytosolic GST in catalyzing this reaction was measured using cytosols prepared from various tissue sources, particularly from animals pretreated with dietary butylated hydroxylanisole (BHA). The extent of APAP-GSH conjugate formation mediated by cytosols varied in this order: BHA-treated adult liver>BHA-treated weanling liver>control adult liver>control weanling liver>BHA-adult kidney>control adult kidney>BHA weanling kidney>control weanling kidney. In contrast to findings obtained from in vivo experiments, the rate of GST-dependent APAP conjugate formation with GSH in vitro is not induced in the presence of exogenous drug.

Effects of neem leaf extract on production of aflatoxins and activities of fatty acid synthetase, isocitrate dehydrogenase and glutathione S-transferase in Aspergillus parasiticus.

The relationship between the activities of 3 cytosolic enzymes with aflatoxin biosynthesis in Aspergillus parasiticus cultured under different conditions has been investigated in order to find out the role of each enzyme in aflatoxin biosynthesis. Basically the activity of isocitrate dehydrogenase (IDH) was higher in non-toxigenic strains as compared to its counterpart toxigenic fungi (p < 0.05). In contrast, the activities of fatty acid synthase (FAS) as well as glutathione S-transferase (GST) were higher (P < 0.05) in toxigenic strains than that of the non-toxigenic fungi. Aflatoxin production was inhibited in fungi grown in presence of various concentrations of neem leaf extract. Aflatoxin was at its lowest level (>90% inhibition) when the concentration of neem extract was adjusted to 50% (v/v). No significant changes in FAS and IDH activities were observed when aflatoxin synthesis was under restraints by neem (Azadirachta indica) leaf extract. During a certain period of time of culture growth, when aflatoxin production reached to its maximum level, the activity of FAS was slightly induced in the toxigenic strains fed with a low concentration (1.56% v/v) of the neem leaf extract. At the time (96 h) when aflatoxin concentration reached to its maximum levels, the activity of GST in the toxigenic fungi was significantly higher (i.e., 7-11 folds) than that of non-toxigenic strains. The difference was highest in mycelial samples collected after 120 h. However unlike FAS and IDH, GST was readily inhibited (approximately 67%) in mycelia fed with 1.56% v/v of the neem extract. The inhibition reached to maximum of 80% in samples exposed to 6.25-12.5% of the extract. These results further substantiate previous finding that there is a positive correlation between GST activity and aflatoxin production in fungi.

Unusual profile and high prevalence of p53 mutations in esophageal squamous cell carcinomas from northern Iran.

Over 15,000 human tumor p53 mutations have been recorded in the scientific literature, including over 700 mutations in esophageal tumors. There are no data on p53 mutations in esophageal cancer patients from Iran yet; however, this country experiences one of the highest cancer mortality rates in the world for esophageal squamous cell carcinomas (ESCCs). The causes of this high cancer burden in Iran remain obscure and do not appear to be related to tobacco and alcohol consumption, the two major risk factors identified in Europe and North America. Because molecular analysis of tumors can provide clues to endogenous or environmental factors contributing to high cancer risk, we examined 74 Iranian ESCCs for the presence of mutations in exons 5-8 of the p53 gene by PCR and direct sequencing. Forty-eight of the 74 tumors (65%) had one or more p53 gene point mutations, including 5 patients with two or more mutations and one with a tandem mutation in codon 242. Surprisingly, over one-third of the 54 mutations we identified were transitions at CpG sites (20 of a total of 54 mutations, or 37%), a class of mutation that is significantly less common (16% of mutations) in the compilation of ESCC mutations from other countries (chi2 statistic, P < 0.0002), whereas transversions, which the literature shows to be common in ESCCs from non-Iranian patients, were infrequent in the tumors we examined here. Elevated levels of cyclooxygenase-2 and inducible nitric oxide synthase were observed in 74 and 91%, respectively, of tumors from Tehran as determined by immunohistochemistry, and high COX-2 expression correlated significantly with the presence of a p53 mutation in the tumor. Mediators of the inflammatory response in esophageal mucosa, perhaps in conjunction with specific dietary or cultural practices in Iran, may contribute importantly to the p53 mutation load in Iranian ESCC patients.

Kinetic studies of aflatoxin B1-glutathione conjugate formation in liver and kidneys of adult and weanling rats.

Aflatoxin B1(AFB1)-glutathione(GSH) conjugation is the major pathway for the detoxification of aflatoxin metabolites. This reaction is catalysed by GSH S-transferase (GST) and play a major role in modulation of AFB1 adduct formation to nuclear DNA. Changes recorded in hepatic GST activity during development of rats can alter the balance between AFB1-GSH conjugation and AFB1-DNA adduct formation. Measurment of cytosolic GST using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate showed that the enzyme activity is initially lower in weanling tissues as compared to that of adults. But nevertheless hepatic and renal cytosolic GST activity is increased significantly in growing rats pretreated with AFB1. Kinetic studies of AFB1-GSH conjugate formation in kidneys and livers of the two-age groups of rats treated with a single i.p. dose of AFB1 (400 microg/kg b.w.) revealed that at the end of 24 h of AFB1 administration the rate of the conjugate formation in kidneys of immature rats was approximately twice of that measured in adults. Age-related differences in the GST activity as well as AFB1-GSH conjugation was more pronounced in kidneys. The conjugate formation in kidneys of growing rats during 6-24 h following AFB1 administration shows that urinary excretion of aflatoxin metabolites is relatively rapid in growing rats. The major portion of the AFB1-GSH is formed in liver but contribution of the renal tissue to the formation of detoxification metabolites can not be ruled out. These data demonstrate that aflatoxin metabolites are eliminated more efficiently from kidneys of a growing rat. AFB1-induced GST induction in renal tissues of growing animals during 24 h of the carcinogen administration could be considered as an important mechanism for GSH conjugate formation and aflatoxin detoxification. Therefore GST induction in response to hepatotoxic drugs can confer resistance to young animals being exposed for the first time to such drugs. It is also worthmentioning that the GST activity measured before AFB1 administration does not reflect the rate of AFB1 detoxification via GSH conjugation.