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  • Direct comparison of the nature of mouse and human GST T1-1 and the implications on dichloromethane carcinogenicity. 11884241

    Dichloromethane (DCM) is a hepatic and pulmonary carcinogen in mice exposed to high doses by inhalation. It has been shown previously that the incidence of liver and lung tumors does not increase in rats or hamsters exposed to the dihaloalkane under conditions similar to those that produced tumors in mice. The biological consequences of DCM exposure to humans is therefore uncertain. The carcinogenic effects of DCM in the mouse are caused by the interaction with DNA of a glutathione (GSH) conjugate that is produced by the class theta glutathione S-transferase T1-1 (GST T1-1). The species specificity is thought to be due to the greater amount of transferase activity in mouse target organs and specific nuclear localization of GST T1-1 in target cells. This paper directly compares the relative capacity and locality of DCM activation in mouse and human tissues. The results show that mouse GST T1-1 is more efficient in catalyzing the conjugation of DCM with GSH than the orthologous human enzyme. In addition, the mouse expresses higher levels of the transferase than humans in hepatic tissue. Histochemical analysis confirmed the presence of GST T1-1 in the nucleus of mouse liver cells. However, in human liver GST T1-1 was detected in bile duct epithelial cells and hepatocyte nuclei but was also present in the cytoplasm. Taking this information into account, it is unlikely that humans have a sufficiently high capacity to activate DCM for this compound to be considered to represent a carcinogenic risk.
    Tipo de documento:
    Referencia
    Referencia del producto:
    ABS1653
    Nombre del producto:
    Anti-GSTT1 Antibody

  • Determining estrogenic steroids in Taipei waters and removal in drinking water treatment using high-flow solid-phase extraction and liquid chromatography/tandem mass spec ... 17428520

    River water and wastewater treatment plant (WWTP) effluents from metropolitan Taipei, Taiwan were tested for the presence of the pollutants estrone (E1), estriol (E3), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2) using a new methodology that involves high-flow solid-phase extraction and liquid chromatography/tandem mass spectrometry. The method was also used to investigate the removal of the analytes by conventional drinking water treatment processes. Without adjusting the pH, we extracted 1-L samples with PolarPlus C18 Speedisks under a flow rate exceeding 100 mL/min, in which six samples could be done simultaneously using an extraction station. The adsorbent was washed with 40% methanol/60% water and then eluted by 50% methanol/50% dichloromethane. The eluate was concentrated until almost dry and was reconstituted by 20 μL of methanol. Quantitation was done by LC-MS/MS-negative electrospray ionization in the selected reaction monitoring mode with isotope-dilution techniques. The mobile phase was 10 mM N-methylmorpholine aqueous solution/acetonitrile with gradient elution. Mean recoveries of spiked Milli-Q water were 65–79% and precisions were within 2–20% of the tested concentrations (5.0–200 ng/L). The method was validated with spiked upstream river water; precisions were most within 10% of the tested concentrations (10–100 ng/L) with most RSDs < 10%. LODs of the environmental matrixes were 0.78–7.65 ng/L. A pre-filtration step before solid-phase extraction may significantly influence the measurement of E1 and EE2 concentrations; disk overloading by water matrix may also impact analyte recoveries along with ion suppression. In the Taipei water study, the four steroid estrogens were detected in river samples (ca. 15 ng/L for E2 and EE2 and 35−45 ng/L for E1 and E3). Average levels of 19–26 ng/L for E1, E2, and EE2 were detected in most wastewater effluents, while only a single effluent sample contained E3. The higher level in the river was likely caused by the discharge of untreated human and farming waste into the water. In the drinking water treatment simulations, coagulation removed 20–50% of the estrogens. An increased dose of aluminum sulfate did not improve the performance. Despite the reactive phenolic moiety in the analytes, the steroids were decreased only 20–44% of the initial concentrations in pre- or post-chlorination. Rapid filtration, with crushed anthracite playing a major role, took out more than 84% of the estrogens. Except for E3, the whole procedure successfully removed most of the estrogens even if the initial concentration reached levels as high as 500 ng/L.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo

  • Evidence that human class Theta glutathione S-transferase T1-1 can catalyse the activation of dichloromethane, a liver and lung carcinogen in the mouse. Comparison of the ... 9307035

    The cDNA encoding human glutathione S-transferase (GST) T1 has been expressed as two recombinant forms in Escherichia coli that could be purified by affinity chromatography on either IgG-Sepharose or nickel-agarose; one form of the transferase was synthesized from the pALP 1 expression vector as a Staphylococcus aureus protein A fusion, whereas the other form was synthesized from the pET-20b expression vector as a C-terminal polyhistidine-tagged recombinant. The yields of the two purified recombinant proteins from E. coli cultures were approx. 15 mg/l for the protein A fusion and 25 mg/l for the C-terminal polyhistidine-tagged GST T1-1. The purified recombinant proteins were catalytically active, although the protein A fusion was typically only 5-30% as active as the histidine-tagged GST. Both recombinant forms could catalyse the conjugation of glutathione with the model substrates 1,2-epoxy-3-(4'-nitrophenoxy)propane,4-nitrobenzyl chloride and 4-nitrophenethyl bromide but were inactive towards 1-chloro-2,4-dinitrobenzene, ethacrynic acid and 1-menaphthyl sulphate. Recombinant human GST T1-1 was found to exhibit glutathione peroxidase activity and could catalyse the reduction of cumene hydroperoxide. In addition, recombinant human GST T1-1 was found to conjugate glutathione with dichloromethane, a pulmonary and hepatic carcinogen in the mouse. Immunoblotting with antibodies raised against different transferase isoenzymes showed that GST T1-1 is expressed in a large number of human organs in a tissue-specific fashion that differs from the pattern of expression of classes Alpha, Mu and Pi GST. Most significantly, GST T1-1 was found in only low levels in human pulmonary soluble extract of cells, suggesting that in man the lung has little capacity to activate the volatile dichloromethane.
    Tipo de documento:
    Referencia
    Referencia del producto:
    ABS1653
    Nombre del producto:
    Anti-GSTT1 Antibody

  • Physiological and pathophysiological applications of sensitive ELISA methods for urinary deoxycorticosterone and corticosterone in rodents. 19577584

    Deoxycorticosterone (DOC: a weak mineralocorticoid) is the precursor to corticosterone (B: the major glucocorticoid in rodents) and aldosterone (the major mineralocorticoid). The genes Cyp11b1 and Cyp11b2 that encode the enzymes responsible for DOC to B (11beta-hydroxylase) and DOC to aldosterone (aldosterone synthase) conversions are located on the same chromosome. The aim of this study was to develop sensitive and specific ELISA methods to quantify urinary DOC and B concentrations to assess the physiological and genetic control of the Cyp11b1/b2 locus. Antibodies raised in rabbits against DOC and B and horse radish peroxidase-goat anti-rabbit IgG enzyme tracer were used to develop the assays. Urine samples collected from mice held in metabolic cages were extracted with dichloromethane and reconstituted in assay buffer. The assays were validated for specificity, sensitivity, parallelism, accuracy and imprecision. Cross-reactivities with major interfering steroids were minimal: DOC assay (progesterone=0.735% and corticosterone=0.045%), and for B assay (aldosterone=0.14%, 11-dehydro-B=0.006%, cortisol=0.016% and DOC=0.04%) and minimum detection limit for DOC ELISA was 2.2 pg/mL (6.6 pmol/L), and for B ELISA was 6.2 pg/mL (17.9 pmol/L). The validity of urinary DOC and B ELISAs was confirmed by the excellent correlation between the results obtained before and after solvent extraction and HPLC (DOC ELISA: Y=1.092X-0.054, R(2)=0.988; B ELISA: Y=1.047X-0.226, R(2)=0.996). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. The methods were used in a series of metabolic cage studies which demonstrated that (i) females produce more DOC and corticosterone than males; (ii) DOC and corticosterone respond to ACTH treatment but not dietary sodium restriction; (iii) DOC:B ratios in Cyp11b1 null mice were >200-fold greater than wild type.
    Tipo de documento:
    Referencia
    Referencia del producto:
    12-348
    Nombre del producto:
    Goat Anti-Rabbit IgG Antibody, HRP-conjugate

  • Formulating erythropoietin-loaded sustained-release PLGA microspheres without protein aggregation. 18620011

    We report a simple method to microencapsulate erythropoietin (EPO, a protein easily denatured and antigenized by contact with water-organic solvent interfaces) into poly(lactic-co-glycolic acid) (PLGA) microspheres with minimal aggregation. This formulation process involved an aqueous-aqueous emulsion formed at reduced temperature. EPO was first dissolved in water together with dextran (MW=70,000) and polyethylene glycol (MW=8000), followed by a freezing process during which dextran separated out as the dispersed phase with EPO partitioned in preferentially. The frozen sample was then lyophilized to powder and washed with dichloromethane to remove the PEG continuous phase. Once loaded in the polysaccharide particles, 1-4 microm in diameter, EPO gained resistance to organic solvents and was encapsulated into PLGA microspheres without significant aggregation (2%). EPO released from the composite PLGA microspheres in a sustained-release manner with minimal burst (20% at the first day) and incomplete (20%) release. Single injection of these EPO-loaded PLGA microspheres to mice resulted in a red blood cell elevation equivalent to twelve injections of the solution formulation. An ELISA assay suggested that the mice injected with the EPO-loaded PLGA microspheres did not develop anti-EPO IgG more than those given solution form EPO.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AP188P
    Nombre del producto:
    Mouse Anti-Rabbit IgG Antibody, HRP conjugate, Species Adsorbed

  • Protection conferred by selenium deficiency against aflatoxin B1 in the rat is associated with the hepatic expression of an aldo-keto reductase and a glutathione S-transf ... 9331086

    Fischer 344 rats fed on a diet that is deficient in selenium are more resistant to the hepatocarcinogen aflatoxin B1 (AFB1) than those fed on a selenium-sufficient diet. Hepatic cytosol from either selenium-deficient Fischer 344 rats or Hooded Lister rats possesses a marked increase in both reductase activity toward AFB1-dialdehyde and glutathione S-transferase (GST) activity toward AFB(1)-8,9-epoxide than hepatic cytosol from selenium-sufficient rats. The elevation in hepatic AFB1-aldehyde reductase (AFAR) activity in selenium-deficient animals is accompanied by an increase of 11- and 15-fold in the levels of AFAR protein in liver cytosol from Fischer 344 and Hooded Lister rats, respectively. The amount of AFAR protein in selenium-sufficient and -deficient Fischer rats was modulated by treatment with N-acetylcysteine; this antioxidant reduced basal expression of AFAR but did not modulate the relative overexpression of AFAR during selenium deficiency. The enhanced capacity to conjugate glutathione with AFB(1)-8,9-epoxide in selenium-deficient livers from Fischer 344 and Hooded Lister rats is associated with a 5- and 7-fold increase, respectively, in the hepatic levels of the AFB1-metabolizing alpha-class GSTA5 subunit. The elevated levels of AFAR and GSTA5 protein in the selenium-deficient animals coincided with increases in the steady-state levels of their mRNAs. In selenium-deficient Fischer 344 rats, AFAR and GSTA5 were both found to be expressed throughout the centrilobular and midzonal areas of the liver lobule but were essentially absent from periportal hepatocytes. The effect of selenium insufficiency is pleiotropic, and it was also noted that the theta-class GSTT1 is overexpressed 3- and 10-fold in livers of selenium-deficient Hooded Lister and Fischer 344 rats. Inasmuch as GSTT1 is responsible for the metabolic activation of dihaloalkanes, selenium deficiency may increase the susceptibility of rats to mutagens such as dichloromethane.
    Tipo de documento:
    Referencia
    Referencia del producto:
    ABS1653
    Nombre del producto:
    Anti-GSTT1 Antibody

  • Inhibition by aminoacyl-chloromethane protease inhibitors of superoxide anion production by phorbol-ester-stimulated human neutrophils. The labeled target is a membrane p ... 2171934

    In a previous paper, we described the kinetic characteristics of the inhibition exerted by the protease inhibitors tosylphenylalanyl and tosyllysyl chloromethanes on superoxide production by human polymorphonuclear leukocytes when stimulated by phorbol esters [E. C. Conseiller & F. Lederer (1989) Eur. J. Biochem. 183, 107-114]. The results suggested the existence of a specific target which was affinity labeled by the inhibitors. The target appeared to be neither a protease, nor intracellular enzymes which can be inhibited in vitro by the chloromethanes (protein kinase C, hexokinase and enzymes of the hexose monophosphate shunt). In the present work, using the cell-free reconstitution assay for superoxide production, we substantiate the hypothesis that the chloromethanes, target is on the plasma membrane. We have radiolabeled the membranes of cells inactivated before or after phorbol ester stimulation, using either [3H]KBH4 reduction after reaction with unlabeled inactivator, or tritiated tosylphenylalanyl chloromethane. In all cases, besides a certain background of non-specific labeling, a radioactive band of Mr 15,000 can be observed upon SDS/PAGE of radiolabeled membranes. We suggest that it is the chemical modification of this protein which is responsible for inactivation of superoxide production. Its identity and its role in the oxidative burst remain to be determined.
    Tipo de documento:
    Referencia
    Referencia del producto:
    20-221
    Nombre del producto:
    NAD Cofactor