PubMed Health⌕ Search

Biomedical subjects

W H Peters

Publications and source records attributed to W H Peters.

At least 19 recordsLinked to original sources
PubMed1994

Monochlorobimane does not selectively label glutathione in peripheral blood mononuclear cells.

Monochlorobimane (MCB) has been used by several investigators as a fluorescent label for quantifying glutathione (GSH) levels in human peripheral blood mononuclear cells (PBMC). This paper describes a biochemical evaluation of this approach. PBMC were incubated with MCB (10-100 microM) and the fluorescence in extracellular medium and cell lysates was measured. Nonlinear curves were obtained in both cases and no "plateau" was reached. The majority of the fluorescence was in the medium. Gel permeation (Sephadex G-25) of the lysate indicated a linear increase in protein-bimane adduct formation, reaching about 50% of the intracellular fluorescence after 1 h. Fractionation of the deproteinized samples with Sephadex G-10 showed that only about one-third of the "low-molecular-weight" fluorescence could be ascribed to GSH-bimane, in either the lysate or the medium. Furthermore, about 40% of the free GSH in lysates appeared unbound even after 1 h of incubation. These data are in line with our observation of an extremely low activity in PBMCs of glutathione S-transferase under the conditions employed. Our findings indicate that many variables influence the cellular fluorescence, including the presence of alternative metabolic pathways for MCB and the rapid excretion of GSH-bimane out of the cell. This lack of specificity limits the value of MCB as a GSH probe for PBMC and confirms earlier suggestions that a careful biochemical evaluation is a prerequisite for its application to any particular cell type.

Adult↗
PubMed1992

Induction of rat hepatic and intestinal glutathione S-transferases by dietary butylated hydroxyanisole.

To obtain insight into the protection mechanism of butylated hydroxyanisole (BHA), a widely used food preservative with anticarcinogenic properties, we investigated the effects of dietary BHA on rat hepatic and intestinal glutathione S-transferase (GST) enzyme activity, and GST isozyme levels. In the proximal small intestine and liver, BHA supplementation significantly increased GST enzyme activity as compared with controls (2.3- and 1.7-fold, respectively, P less than 0.05). GST class alpha and mu contents were significantly higher only in the small intestine (1.6-2.1-fold and 1.3-1.5-fold, respectively, P less than 0.05), whereas GST class pi was significantly induced in liver (4.6-fold, P less than 0.05).

Animals↗
PubMed1992

Biochemical characterization of resistance to mitoxantrone and adriamycin in Caco-2 human colon adenocarcinoma cells: a possible role for glutathione S-transferases.

Cytotoxicity of Adriamycin on human colon adenocarcinoma cell lines was investigated. Concentrations of Adriamycin producing 50% inhibition were very similar in HT29, Sw480, Sw620, and Sw1116 cells, whereas Caco-2 cells were relatively insensitive. As compared to the Sw1116 cell line, Caco-2 cells were also insensitive to mitoxantrone. Sensitivity to cisplatin, 5-fluorouracil, or ethacrynic acid was comparable in both cell lines. To find the mechanism for this mitoxantrone and Adriamycin resistance, several potential Adriamycin-detoxifying systems were characterized and quantified in both Sw1116 and Caco-2 cells. No dramatic differences in glutathione content and expression of both selenium dependent- and independent glutathione peroxidase, UDP-glucuronyltransferase, and cytochrome P-450 were found. However, highly significant differences in glutathione S-transferase activity were present, the expression of both class pi and class alpha glutathione S-transferases being much higher in the Caco-2 cell line. In addition, a slightly higher content of P-170 glycoprotein was present in the Caco-2 cells. These findings suggest that glutathione S-transferases, and to a lesser extent the P-170 glycoprotein, may be involved in mitoxantrone and Adriamycin resistance of Caco-2 colon carcinoma cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Expression of drug-metabolizing enzymes and P-170 glycoprotein in colorectal carcinoma and normal mucosa.

Resistance to chemotherapy is a significant problem in the treatment of colorectal carcinomas. To obtain insight into the mechanism of drug resistance, the expression of P-170 glycoprotein and biotransformation enzymes that are potentially able to contribute to drug resistance were investigated in paired samples of normal mucosa and tumors from 24 patients with colorectal cancer. In the tumors, glutathione S-transferase (GST) enzyme activity and content of GST-pi and P-170 glycoprotein were increased significantly compared with normal mucosa (P less than 0.03, P less than 0.003, and P less than 0.02, respectively). In contrast, GST-alpha and -mu, present in minor amounts compared with GST-pi, were downregulated in the tumor. Cytochrome P-450(4,5,6) and UDP-glucuronyltransferase (towards 4-nitrophenol and bilirubin) levels were significantly lower in the tumors (P less than 0.0001 and P less than 0.0002, respectively). Because decreased expression of cytochrome P-450 and increased levels of GST-pi and the P-170 glycoprotein have been implicated in (multi)drug resistance, these findings strongly suggest that in colorectal tumors the inherent resistance is multifactorial. Research to overcome this resistance should therefore be directed toward a combined treatment that eliminates all of these different mechanisms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Epidermal growth factor receptor levels are lower in carcinomatous than in normal colorectal tissue.

A series of 24 paired samples of colorectal carcinoma and the respective normal colorectal mucosa were analysed for Epidermal Growth Factor Receptor (EGFR) content by means of a standardised ligand binding assay. We, for the first time, found that EGFR levels are statistically significantly higher in normal colorectal mucosa biopsy samples than they are in colorectal carcinoma biopsy samples, the median EGFR levels being 77.5 fmol mg-1 of membrane protein (range 35-239), against 46 fmol mg-1 of membrane protein (range 22-81), respectively, P less than 0.001. In addition, we found that there are significant regional differences in EGFR expression in the normal human colon mucosa. The EGFR levels were significantly higher in samples from the proximal part of the colon than they were in samples from the distal part, the median EGFR levels being 124 fmol mg-1 of membrane protein (range 70-239) vs 55 fmol mg-1 membrane protein (range 35-156), P less than 0.05. The EGFR levels of the colorectal carcinoma samples did not show any regional variation.

Aged↗
PubMed1992

Cyclosporin metabolism by human gastrointestinal mucosal microsomes.

The in vitro metabolism of the immunosuppressant cyclosporin (CsA) by human gastrointestinal mucosal microsomes has been studied. Macroscopically normal intestinal (n = 4) and liver (n = 2) tissue was obtained from kidney transplant donors, and microsomes prepared. Intestinal metabolism was most extensive with duodenal protein (15% conversion to metabolites M1/M17 after 2 h incubation at 37 degrees C; metabolite measurement by h.p.l.c). Western blotting confirmed the presence of P-4503A (enzyme subfamily responsible for CsA metabolism) in duodenum and ileum tissue, but not in colon tissue. The results of this study indicate that the gut wall may play a role in the first-pass metabolism of CsA, and could therefore be a contributory factor to the highly variable oral bioavailability of CsA.

Biological Availability↗
PubMed1991

Combined in vitro modulation of adriamycin resistance.

In a P-glycoprotein-negative cell line, GLC4-Adr90, a 75-fold acquired Adriamycin (Adr) resistance coincided with a reduced cellular Adr level, an increased detoxifying capacity (glutathione (GSH) and glutathione S-transferase (GST) elevated), and a reduced topoisomerase-II (topo-II) activity compared with the parent cell line GLC4. The effect on Adr resistance of buthionine sulfoximine (BSO, GSH synthesis inhibitor), was studied alone or in combination with verapamil (drug-efflux inhibitor), docosahexaenoic acid (membrane lipid domain affector), ethacrynic acid (GST inhibitor), aphidicolin (DNA-polymerase-alpha inhibitor) or novobiocin (NOV, topo-II inhibitor). Cytotoxicity was tested using a microculture tetrazolium assay. In GLC4-Adr90, BSO and NOV increased Adr-induced cytotoxicity 12.9-fold and 1.8-fold respectively. The combination of BSO plus NOV showed an additive effect, decreasing the Adr resistance factor from 75 to 2.7. Combination of modulators of Adr resistance directed at different resistance mechanisms appears promising in vitro.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Genetic deficiency of human class mu glutathione S-transferase isoenzymes in relation to the urinary excretion of the mercapturic acids of Z- and E-1,3-dichloropropene.

Mononuclear lymphocytes were isolated from the blood of 12 individuals, who had been exposed to the vapour of the soil fumigant 1,3-dichloropropene (DCP). Western blot experiments were performed on the crude lymphocyte homogenates, using a monoclonal antibody against human hepatic glutathione S-transferase (GST) isoenzyme mu, to determine the presence or absence of mu-class isoenzymes mu and/or psi. Nine of the individuals were found to be positive for mu and/or psi, the remaining three individuals being negative. In addition, all individuals showed a positive staining on immunoblot of a protein of somewhat lower molecular mass than the hepatic standard. This protein was bound by the S-hexylglutathione affinity column, and presumably constitutes a new mu-class isoenzyme, which is not subject to genetic polymorphism. Determination of the specific activities of individual human GST isoenzymes towards Z-(cis-) and E-(trans-)-DCP demonstrated that mu-class isoenzymes show a considerably higher specific activity with Z-DCP than alpha-class or pi-class isoenzymes. In addition, mu-class isoenzymes were found to be 2- to 3-fold more active with Z-DCP than with E-DCP. Their activity towards E-DCP was similar to the specific activity of alpha-class isoenzymes. Genetic polymorphism for mu-class isoenzymes could thus be a determinant in the extent of excretion of mercapturic acids from Z- and E-DCP. The urinary excretion of Z- and E-DCP mercapturic acids and the respiratory exposure to Z- and E-DCP were determined for nine and eight phenotyped individuals, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcysteine↗
PubMed1991

Biotransformation enzymes in human intestine: critical low levels in the colon?

Biotransformation or drug-metabolising enzymes have an important function in the detoxication of ingested toxic, carcinogenic, or tumour promoting compounds. Enzyme activity and isoenzyme composition of three biotransformation systems: glutathione S-transferase, uridine diphosphate-glucuronosyltransferase, and cytochrome P-450 were studied in normal small and large intestinal mucosa from three kidney donors. The activity of most drug-metabolising enzymes decreases slightly from proximal to distal small intestine, whereas in the mucosa of the large intestine a sharp fall in activity was observed. The isoenzyme composition for each of the three biotransformation systems changed from the small to the large intestine. Class Alpha glutathione S-transferases were not expressed in the colon, in contrast to the small intestine where both Alpha and Pi class isoenzymes are present. In addition, with monoclonal antibodies fewer protein bands for UDP-glucuronosyltransferases and cytochrome P-450 were detected in the colon. In the small intestine both isoforms P-450(4) and P-450(5) were present, whereas in the colon only reduced amounts of cytochrome P-450(4) could be visualised. For UDP-glucuronosyltransferase, 53 and 54 kDa proteins could be detected in the small intestine, but in the colon there was only weak staining of the 54 kDa band. In the normal human colon enzymes are less active and there are fewer isoenzymes present in the mucosa than in the small intestine. This implies a lower level of the detoxifying potential in the colon, which might be important in regard to the high rates of carcinogenesis in the colon.

Adolescent↗
PubMed1990

Quantification of human hepatic glutathione S-transferases.

Human hepatic glutathione S-transferase (GST) subunits were characterized and quantified with the aid of a recently developed h.p.l.c. method. In 20 hepatic tissue specimens the absolute amounts of the basic Class Alpha subunits B1 and B2, the near-neutral Class Mu subunits mu and psi and the acidic subunit pi were determined. The average total amount of GST was 37 micrograms/mg of cytosolic protein, with the Class Alpha GST being the predominant class (84% of total GSTs), and pi as the sole representative of the Class Pi GSTs present in the lowest concentration (4% of total GSTs). Large interindividual differences were observed for all subunits, with variations up to 27-fold, depending on the subunit. For the Class Alpha GST-subunits B1 and B2, a biphasic ratio was observed. The genetic polymorphism of the subunits mu and psi was confirmed by h.p.l.c. analysis, and correlated with the enzymic glutathione conjugation of trans-stilbene oxide and with Western blotting of cytosols, using a monoclonal anti-(Class Mu GST) antibody. Of the 20 livers examined, ten contained only mu, whereas the occurrence of psi alone, and the combination of mu and psi, were found in only one liver each.

Chromatography, High Pressure Liquid↗
PubMed1990

A monoclonal antibody against pig gastric H+/K(+)-ATPase, which binds to the cytosolic E1.K+ form.

Monoclonal antibodies were raised against a purified membrane fraction from hog gastric mucosa containing H+/K(+)-ATPase. The properties of one of these monoclonal antibodies (5B6) were further evaluated. On immunoblot it recognized the 95 kDa peptide of the H+/K(+)-ATPase-rich membrane fraction. The K(+)-ATPase activity was inhibited by 65% under standard assay conditions (pH 7.0). At pH 6.0 and 8.0 this enzyme activity was inhibited by 40% and 100%, respectively. The maximal inhibition in inside-out vesicles was also 65% at pH 7.0. The inhibition was uncompetitive with respect to K+ and noncompetitive with respect to ATP. Mg2(+)-ATPase activity and K(+)-dependent p-nitrophenylphosphatase activity were not influenced. The monoclonal antibody lowered the steady-state phosphorylation level at pH 6.0, 7.0 and 8.0 by 30%, 40% and 60% respectively. The rate of the K(+)-stimulated dephosphorylation step was not inhibited. These findings demonstrate that 5-B6 recognizes the E1.K+ dephosphoenzyme at the cytosolic side.

4-Nitrophenylphosphatase↗
PubMed1990

Immunodetection with a monoclonal antibody of glutathione S-transferase mu in patients with and without carcinomas.

Several monoclonal antibodies against human liver glutathione S-transferase mu were developed. One of these monoclonal antibodies, called GST-3H4 was further characterized and used in this study. In hepatic tissue, after immunoblotting, GST-3H4 strains a 27 kDa protein with a pI value of 6.2. GST-3H4 recognizes other human class-mu glutathione S-transferases, but does not detect acidic or basic glutathione S-transferases. By immunodetection with this monoclonal antibody, glutathione S-transferase mu can be demonstrated in human breast, stomach, liver, small and large intestine, mononuclear blood cells, kidney and placenta. A 100% correlation is found in the distribution of glutathione S-transferase mu when different tissues or mononuclear blood cells from the same individuals are investigated. In 62.5% of the mononuclear blood cells from controls, glutathione S-transferase mu is present. In patients with polyposis coli, breast cancer or colon cancer a similar distribution is found. Therefore no important role for glutathione S-transferase mu deficiencies in the aetiology of these diseases is suggested.

Animals↗
PubMed1990

Expression of glutathione S-transferases in normal gastric mucosa and in gastric tumors.

Glutathione S-transferases from both normal gastric mucosa and its matched gastric tumors from 10 different patients were investigated. The transferases were purified and subsequently the isoenzyme composition was studied. Glutathione S-transferase (GST)-pi was present in all specimens in large amounts. Class alpha GSTs were present in 9 out of 10 normal specimens and in six tumors. In malignant tissue, expression of GST-pi was increased at the expense of class alpha GST. In six patients, the ratio GST-pi/GST-alpha was higher in tumorous versus normal tissue. On a Western blot, using a monoclonal antibody, GST-mu was shown to be present in both normal and malignant tissue from four patients, the other six patients completely missed the enzyme in their gastric tissue. When present, GST-mu amounts to only a few per cent of total GST protein. GST-pi was quantified by densitometric analysis of Western blots, treated with a monoclonal antibody against GST-pi. Both total GST enzyme activity as well as the absolute amounts of GST-pi protein were significantly higher in the tumors, as compared to its matched normal mucosa. The importance of this overexpression of GST-pi was previously unknown. However, the frequent occurrence of this phenomenon in many refractory tumors, and as shown now also in gastric cancers, suggests a role for GST-pi in the mechanism of anti-cancer drug resistance.

Adult↗
PubMed1989

Time-dependent activity and expression of glutathione S-transferases in the human colon adenocarcinoma cell line Caco-2.

The human colon carcinoma cell line Caco-2 was examined for glutathione S-transferase (GST) composition and activity. Freshly seeded cells and cells until 4 days after confluency contain only the placental (Pi) form of glutathione transferase. Cells in culture for longer periods start to express class-Alpha GST isoenzymes. Confluent cells in culture for 20 days or longer contain up to 90% class-Alpha GST. Class-Mu GSTs are not detectable. GST activity gradually increases from 564 +/- 28 to 5381 +/- 165 nmol/min per mg of protein at day 0 and 32 after confluency respectively. With regard to GST composition, Caco-2 cells in culture for longer periods most resemble small-intestinal cells, whereas short-time cultures have characteristics of colonic cells. This cell line is very well suited for the study of both the in vitro properties and the expression of class Alpha and Pi GSTs.

Adenocarcinoma↗
PubMed1989

Cytochromes P-450 in the intestinal mucosa of man.

With monoclonal antibodies against cytochrome P-450(5) and P-450(4,5,6), 52 and 54 kDa bands are visualized in microsomes from proximal as well as distal human small intestine. These bands most probably correspond to cytochrome P-450(5) and P-450(4), respectively. This and several other cytochrome P-450 related proteins are present in hepatic microsomes from the same patient. In both hepatic and intestinal microsomes from this patient cytochrome P-450(8) is hardly detectable. In contrast to the small intestine and liver, large intestinal tissue from several other patients does not contain cytochrome P-450(5). Here the 54 kDa isoenzyme, possibly cytochrome P-450(4), is most prominent. Earlier we found a higher content of total cytochrome P-450 in the proximal as compared to the distal small intestine. A similar distribution is now found with regard to aldrin epoxidase activity.

Cytochrome P-450 Enzyme System↗
PubMed1989

Human intestinal glutathione S-transferases.

Cytosolic glutathione S-transferases were purified from the epithelial cells of human small and large intestine. These preparations were characterized with regard to specific activities, subunit and isoenzyme composition. Isoenzyme composition and specific activity showed little variation from proximal to distal small intestine. Specific activities of hepatic and intestinal enzymes from the same patient were comparable. Hepatic enzymes were mainly composed of 25 kDa subunits. Transferases from small intestine contained 24 and 25 kDa subunits, in variable amounts. Colon enzymes were composed of 24 kDa subunits. In most preparations, however, minor amounts of 27 and 27.5 kDa subunits were detectable. Separation into isoforms by isoelectric focusing revealed striking differences: glutathione S-transferases from liver were mainly basic or neutral, enzymes from small intestine were basic, neutral and acidic, whereas large intestine contained acidic isoforms only. The intestinal acidic transferase most probably was identical with glutathione S-transferase Pi, isolated from human placenta. In the hepatic preparation, this isoform was hardly detectable. The specific activity of glutathione S-transferase showed a sharp fall from small to large intestine. In proximal and distal colon, activity seemed to be about equal. In the ascending colon there might be a relationship between specific activity of glutathione S-transferases and age of the patient, activity decreasing with increasing age.

Adult↗