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Biomedical subjects

M L Clapper

Publications and source records attributed to M L Clapper.

33 records · Page 2Linked to original sources
PubMed1995

Susceptibility to hepatocellular carcinoma is associated with genetic variation in the enzymatic detoxification of aflatoxin B1.

Aflatoxin B1 (AFB1) has been postulated to be a hepatocarcinogen in humans, possibly by causing p53 mutations at codon 249. AFB1 is metabolized via the phase I and II detoxification pathways; hence, genetic variation at those loci may predict susceptibility to the effects of AFB1. To test this hypothesis, genetic variation in two AFB1 detoxification genes, epoxide hydrolase (EPHX) and glutathione S-transferase M1 (GSTM1), was contrasted with the presence of serum AFB1-albumin adducts, the presence of hepatocellular carcinoma (HCC), and with p53 codon 249 mutations. Mutant alleles at both loci were significantly overrepresented in individuals with serum AFB1-albumin adducts in a cross-sectional study. Mutant alleles of EPHX were significantly overrepresented in persons with HCC, also in a case-control study. The relationship of EPHX to HCC varied by hepatitis B surface antigen status and indicated that a synergistic effect may exist. p53 codon 249 mutations were observed only among HCC patients with one or both high-risk genotypes. These results indicate that individuals with mutant genotypes at EPHX and GSTM1 may be at greater risk of developing AFB1 adducts, p53 mutations, and HCC when exposed to AFB1. Hepatitis B carriers with the high-risk genotypes may be an even greater risk than carriers with low-risk genotypes. These findings support the existence of genetic susceptibility in humans to the environmental carcinogen AFB1 and indicate that there is a synergistic increase in risk of HCC with the combination of hepatitis B virus infection and susceptible genotype.

Aflatoxin B1↗
PubMed1995

Chemopreventive activity of Oltipraz against N-nitrosobis(2-oxopropyl)amine (BOP)-induced ductal pancreatic carcinoma development and effects on survival of Syrian golden hamsters.

The synthetic dithiolethione Oltipraz has marked cancer chemopreventive and phase II enzyme inducing activity in various animal carcinogenesis models, but has not been examined in any animal models of ductal pancreatic cancer relevant to the human disease. The chemopreventive potential of Oltipraz on pancreatic tumor incidence and multiplicity was examined in the N-nitrosobis(2-oxopropyl)-amine (BOP)-induced ductal pancreatic adenocarcinoma model in Syrian hamsters. Animals were maintained on control semipurified diets or semipurified diets containing 300 and 600 mg/kg Oltipraz beginning 2 weeks prior to BOP initiation and throughout the 26 week study. Oltipraz at 300 mg/kg had no effect on the incidence or multiplicity of preneoplastic, neoplastic or metastatic lesions, while at 600 mg/kg dietary Oltipraz the incidence of pancreatic adenocarcinomas was reduced significantly (P < or = 0.05) compared to BOP-treated controls. Dietary Oltipraz at both doses had a significant influence on reducing mortality and morbidity in tumor-bearing animals with metastatic disease. At 26 weeks, total hepatic glutathione-S transferase (GST) activity and GST mu activity were elevated significantly in Oltipraz-treated animals, while total pancreatic GST activity was reduced, albeit not significantly. Serum lipase activity, a marker for pancreatic damage, exhibited a progressive decline in BOP-treated animals administered Oltipraz compared to BOP-treated controls at 12 weeks of the study; by week 26, lipase activity was comparable in all groups and reduced compared to activity at week 12. Positive nuclear immunostaining for the p53 tumor suppressor protein, a hallmark of human pancreatic cancer and a transient response to DNA damage, was observed in only a small percentage of BOP-induced pancreatic lesions and was not influenced Oltipraz administration. Further chemoprevention and pharmacologic studies of Oltipraz in relevant animal models of ductal pancreatic cancer could provide a foundation for future studies in human populations at potential risk for pancreatic cancer.

Animals↗
PubMed1994

Coordinate induction of glutathione S-transferase alpha, mu, and pi expression in murine liver after a single administration of oltipraz.

The antischistosomal agent oltipraz displays a unique ability to inhibit chemically induced carcinogenesis in a variety of animal models. Its apparent lack of carcinogen specificity and low toxicity make it an attractive candidate for further development as a chemopreventive agent. The mechanism by which oltipraz affords cellular protection is thought to involve the modulation of phase II detoxication enzymes. The present study examines the regulation of each class of glutathione S-transferase (EC 2.5.1.18) in mice after a single oral administration of oltipraz. Glutathione S-transferase activity in the liver increased in a dose-dependent manner after drug exposure. Oltipraz administration (1 g/kg, by gavage) elevated glutathione S-transferase activity to a maximum (4.5-fold) on day 4 after treatment. Western blot analyses demonstrated the induction of all three classes of glutathione S-transferase (alpha, mu, and pi) by oltipraz. Our murine studies suggest that the chemopreventive activity of oltipraz may be due in part to its ability to elevate glutathione S-transferase-mu activity. Consistent with this possibility, associations between the glutathione S-transferase-mu-null phenotype and increased risk for lung, larynx, and bladder cancer have been recently demonstrated in humans. Coordinate elevations in enzymatic activity were preceded by significant elevations in glutathione S-transferase alpha, mu, and pi RNA on day 2 after treatment. Although nuclear run-on assays confirmed the transcriptional induction of all three classes, the maintenance of elevations in enzymatic activity after RNA levels returned to base-line suggests that additional mechanisms are required to regulate glutathione S-transferase expression. Preclinical findings are presented that characterize the response of each class of glutathione S-transferase to oltipraz exposure and support the use of these enzymes as intermediate markers of the chemopreventive activity of oltipraz.

Animals↗
PubMed1993

SR2508 (etanidazole) pharmacokinetics and biochemical effects in tumor and normal tissues of scid mice bearing HT-29 human colon adenocarcinoma.

Several lines of evidence implicate glutathione (GSH) depletion and/or GSH transferase inhibition in the sensitizing action of nitroimidazoles to alkylating agents. To characterize this interaction, scid mice bearing subcutaneously implanted HT-29 colon tumor (0.75 to 1.25 cm diameter) were treated with SR2508 (2 g/kg, i.p.). At intervals following treatment, samples of blood, liver, spleen, kidney and central non-necrotic tumor core and tumor periphery were obtained and analyzed for SR2508 content by high-pressure liquid chromatography. Tissues were assayed spectrophotometrically for GSH and GSH transferase. SR2508 plasma pharmacokinetics in this model were similar to those described previously (t 1/2 beta = 5.83 hr). The volume of distribution of 0.32 L/kg suggests minimal tissue binding. In tumor periphery and core samples SR2508 levels peaked at 1 hr, and declined exponentially in parallel with plasma. During the terminal phase core SR2508 levels were 10-fold and tumor periphery levels 4.3-fold those of concurrent plasma concentrations. Consistent with these data, tumor GSH levels in both periphery and core fell below 30% of control at 4 hr, and remained depressed > 12 hr. Delayed recovery of GSH content of tumor tissue may explain in part the selectivity of SR2508 for tumor (oxic or hypoxic). GSH transferase activity in tumor was inhibited both at the center and periphery to 75 and 71% of control, respectively, and it appeared that recovery occurred more slowly in the hypoxic core. The mild degree of inhibition observed does not support an important role for inhibition of GSH transferase in sensitization by SR2508 in this tumor. The pronounced selective depletion of GSH in tumor supports the further development of SR2508 in the reversal of alkylating agent resistance.

Adenocarcinoma↗
PubMed1993

Time course of glutathione S-transferase elevation in Walker mammary carcinoma cells following chlorambucil exposure.

Resistance of Walker 256 rat mammary carcinoma cells to chlorambucil has been shown to be accompanied by a specific increase in the A2-2 subunit of glutathione S-transferase (GST) (Buller et al., Mol Pharmacol 31: 575-578, 1987). Analysis of the time course of GST activity following chlorambucil exposure revealed a 7.5- and 3-fold elevation on day 7 post-treatment in Walker-sensitive (WS) and Walker-resistant (WR) cells, respectively. Flow activated cell sorting (FACS) analyses using antibodies specific for rat liver cytosolic GST supported these results and demonstrated the heterogeneous response of WS cells to chlorambucil exposure. The range of GST levels in drug-treated cells was very broad as compared to that of untreated cells. Transcripts for each class of GST (alpha, mu and pi) were quantified for days 1-9 post-treatment from densitometric scans of RNA slot blots. Elevations in GST alpha RNA preceded increases in GST activity (day 7) in both WS and WR cells. Because fluctuations in GSTA1-1 transcripts were not observed, it was concluded that the increased expression of the alpha class must be attributed to increases in GSTA2-2 transcripts. Amplification of the GST genes in drug-treated cells was not present. These results support the role of GSTA2-2 in the detoxification of chlorambucil. The time course of the cellular response to chlorambucil suggests that the elevation of GSTA2-2 transcripts following alkylating agent exposure may represent only one component of a series of events which collectively confer protection and lead to the establishment of drug resistance.

Animals↗
PubMed1991

Phase I study of thiotepa in combination with the glutathione transferase inhibitor ethacrynic acid.

The glutathione transferases comprise a family of isoenzymes, one or more of which are involved in the conjugation of alkylating agents to glutathione (GSH). Increased GSH transferase activity has been shown to underlie acquired resistance to several alkylating agents. Ethacrynic acid inhibits the isoenzymes of GSH transferase with 50% inhibitory concentration values ranging from 0.3 to 6.0 microM and has been shown to restore sensitivity to alkylating agents in drug-resistant animal tumor models. We entered 27 previously treated patients with advanced cancer on a study of ethacrynic acid (25 to 75 mg/m2 p.o. every 6 h for 3 doses) and thiotepa (30 to 55 mg/m2 i.v. 1 h after the second dose of ethacrynic acid). The major toxicity of ethacrynic acid was diuresis, which was observed at every dose level; in addition, severe metabolic abnormalities occurred at 75 mg/m2. At 50 mg/m2, the diuretic effects were manageable. Myelosuppression was the most important effect of the combination. Two of seven courses of ethacrynic acid, 50 mg/m2, and thiotepa, 55 mg/m2, were associated with grade 3 or 4 neutropenia and/or thrombocytopenia. Nausea/vomiting greater than or equal to grade 2 was observed in 16% of courses. GSH transferase activity was assayed spectrophotometrically in the peripheral mononuclear cells of all patients. At each dose level, activity decreased following ethacrynic acid administration, with recovery by 6 h. Administration of ethacrynic acid, 50 mg/m2, resulted in a mean nadir of transferase activity of 37% of control. The pharmacokinetics of thiotepa and its principal metabolite TEPA were studied in 23 patients. The plasma disappearance of thiotepa fit a two-compartment open model with a terminal half-life of approximately 2 h. Plasma TEPA levels peaked at a mean of 2.16 h following thiotepa administration. The harmonic mean terminal half-life of TEPA was 10.4 h, and the TEPA area under the curve (AUC) did not increase with increasing thiotepa dose. The AUC of thiotepa was approximately twice, and the clearance about one-half, of the values obtained in a previous study of single agent thiotepa. The AUC of TEPA was lower than that previously observed. The data suggest that ethacrynic acid inhibits enzymes involved in the metabolic disposition of thiotepa, including its oxidative desulfuration to TEPA. The severity of the platelet toxicity was correlated with the AUC of thiotepa, but not with that of TEPA. This combination of thiotepa and ethacrynic acid will be tested further in Phase II trials.

Adult↗
PubMed1991

Glutathione S-transferases in normal and malignant human colon tissue.

This study focuses on the GST composition of a tissue intrinsically resistant to chemotherapy, the human colon. GSTs were purified from matched pairs of colon tissue (normal and tumor) using glutathione affinity chromatography. The mean GST activity of colon tumors was 1.5-fold higher than that of normal tissue, with tumors of the sigmoid colon showing the greatest increase (2.3-fold). Two-dimensional gel electrophoresis and Western blot analysis of purified enzymes demonstrated the presence of all three GST classes (alpha, mu and pi) in colon, with GST pi being both the predominant isozyme in normal and malignant tissues. The level of alpha class subunits was the same in normal and tumor tissues, while the mu class subunits were decreased in tumors. A protein copurifying with GSTs from both normal and tumor tissue did not crossreact with GST antibodies, but instead reacted with a polyclonal antibody to glyoxylase I. This enzyme existed as a dimer in its native state. Upon boiling, monomeric subunits were produced with a molecular mass of 22.6 kDa and an isoelectric point more acidic than GST pi. Increased amounts of glyoxylase I were also found in tumor vs. normal colon. The apparent elevated levels of these glutathione-associated detoxifying enzymes in colon tumors may contribute to their intrinsic drug resistance.

Adult↗
PubMed1991

Contribution of patient history to the glutathione S-transferase activity of human lung, breast and colon tissue.

Overexpression of the glutathione S-transferases (GSTs) and their involvement in the detoxification of anticancer agents has prompted numerous investigations of the enzyme activity of human tumor tissue. This study represents an in-depth evaluation of the contribution of patient history and pathological status to the GST activity of various human tissues. GST activity was elevated significantly in tumors of the lung, breast and colon as compared to unmatched and matched normal tissue from the same organ. The GST activity of primary breast tumors varied significantly with the stage of the tumor. Breast tumors previously treated with both radiation and chemotherapy had significantly lower levels of GST activity than untreated tumors. Neither progesterone nor estrogen receptor content was associated with the GST activity in primary breast tumors. Colon metastases possessed higher levels of GST activity than primary colon tumors but enzyme activity was independent of the Duke's classification of the tumor. Only tumors of the left colon had levels of GST activity that were higher than those of adjacent normal mucosa. No relationship was evident between either age or sex and the GST activity of any of the tissues examined. GST activity levels may reflect the site-specific ability of tissues to provide cellular protection against xenobiotics.

Biomarkers, Tumor↗
PubMed1989

Identification of a glutathione S-transferase associated with microsomes of tumor cells resistant to nitrogen mustards.

Walker 256 rat mammary carcinoma cells resistant to chlorambucil (WR) exhibited an approximate 4-fold increase in glutathione S-transferase (GST) activity using 1-chloro-2,4-dinitrobenzene as compared to the sensitive parent cell line (WS). WR cells maintained without biannual exposure to chlorambucil (WRr) reverted to the sensitive phenotype and possessed GST levels equivalent to WS. Mitochondria, microsomes and cytosol were isolated from WS, WR and WRr cell lines and analyzed for their GST composition. GST activity in each subcellular compartment of resistant cells was increased over the sensitive cells. Antibodies raised against total rat liver cytosolic GST crossreacted in resistant cells with two microsomal proteins (25.7 kD and 29 kD). The 29 kD protein was not detected in microsomal fractions from either WS or WRr and this protein was found to be dissimilar from cytosolic GST subunits in its isoelectric point (pI 6.7) and migration on two-dimensional polyacrylamide gels. In addition, the 29 kD microsome-associated GST from WR cells was immunologically distinct from a 14 kD GST subunit previously identified in rat liver microsomes. These data implicate the induction of a specific microsomal GST subunit in WR cells following drug selection and suggest its potential involvement in the establishment of cellular resistance to chlorambucil.

Animals↗
PubMed1987

Glutathione S-transferases in human prostate.

A number of human prostatic tissue biopsies have been analyzed for glutathione S-transferase activity, using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Samples from nine patients (age range 61-90) with benign prostatic hypertrophy who had received no prior chemotherapy had a mean glutathione S-transferase activity of 137 +/- 44 nmol/min per mg with a range of 97-237. A qualitative comparison of the glutathione S-transferase of normal prostate and benign prostatic hypertrophy samples was carried out. Approximately 260-fold purification was achieved using glutathione-Sepharose affinity chromatography, with glutathione S-transferase accounting for approximately 0.19-0.33% of the total protein. Substrate specificity determinations suggested similar, but not identical, glutathione S-transferase subunits in normal prostate and benign prostatic hypertrophy. One- and two-dimensional electrophoresis (isoelectric focusing and 12.5% SDS-polyacrylamide gel electrophoresis) identified at least seven stained polypeptides in the purified glutathione S-transferase preparations. These ranged in Mr from approximately 24,000 to 28,500 and in pI from near neutral to basic. Western blot analysis using polyclonal antibodies raised against rat liver glutathione S-transferase suggested crossreactivity with five of the human isoenzymes in both normal prostate and benign prostatic hypertrophy. One of the glutathione S-transferases, present in both normal prostate and benign prostatic hypertrophy, had an Mr of approx. 24,000 and a near-neutral pI and crossreacted immunologically with a polyclonal antibody raised against human placental glutathione S-transferase (Yf, subunit 7 or pi). These data suggest that four glutathione S-transferases are expressed in human prostate, with subunits from each of the major classes alpha, mu and pi. These are characterized as Ya, Yb, Yb' and Yf (analogous alternative nomenclature subunits 1, 3, 4 and 7).

Aged↗
PubMed1987

Glutathione S-transferases in nitrogen mustard-resistant and -sensitive cell lines.

Tumor cell resistance to alkylating agents was studied by examining Walker 256 rat mammary carcinoma cells differentially sensitive to nitrogen mustards. A resistant subpopulation (WR) was selected by exposure to chlorambucil. WR cells showed approximately a 15-fold resistance to the cytotoxic effects of nitrogen mustards and elevated glutathione S-transferase (GST) activity when compared to the sensitive parent cell line (WS). To extend these findings, the GSTs from WR and WS were purified by affinity chromatography on S-hexylglutathione coupled to epoxy-activated agarose. Substrate specificity experiments using purified GSTs demonstrated different profiles of enzyme activity for WR and WS and suggested differential isoenzyme expression in these two cell lines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that the major GST present in both WR and WS was a 26,000-Da subunit that was immunologically distinct from the rat liver GSTs. This GST subunit cross-reacted with antibodies against anionic human placental GST. In addition, three GST forms common to rat liver (29,500, 28,500 and 27,500 molecular weight) were also identified. Overexpression of the 29,500-Da protein was observed in WR cells. These data suggest that differential expression of GST subunits may contribute to the nitrogen mustard-resistant phenotype.

Alkylating Agents↗
PubMed1986

Cardiovascular defects in rat embryos cultured on serum from rats chronically exposed to phenytoin.

Headfold-stage rat embryos were cultured for 48 hours on serum from rats chronically exposed to phenytoin for periods as long as from conception until 11 months of age. Serum from phenytoin-exposed rats caused approximately 50% of the cultured rat embryos to develop cardiovascular defects as compared to 12% for controls. These morphological abnormalities included hemorrhaging of blood vessels within the embryo, pericardial edema, and absence of yolk sac circulation. Neither serum glucose nor phosphate levels nor serum osmolality were appreciably affected by phenytoin treatment. However, serum protein concentration was reduced in rats exposed to phenytoin as compared to controls. An absence of the serum protein hemopexin was associated with the reduction in serum protein levels but did not appear to be responsible for the observed cardiovascular defects.

Animals↗
PubMed1984

Human serum teratogenicity studied by rat embryo culture: epilepsy, anticonvulsant drugs, and nutrition.

Epileptic women have a greater risk for spontaneous abortions and children with birth defects than do nonepileptics. In a unique approach to identifying causes of these problems, we have cultured whole rat embryos for 48 h on blood sera from epileptics. In the first part of the study, three embryos were cultured on each serum sample from 128 different epileptics being treated with either single anticonvulsants or no drug to compare the teratogenicity of these drugs. Sera from subjects receiving either phenobarbital or no drug had comparable frequencies of cultured embryo abnormalities, which were lower than those from subjects taking phenytoin, valproic acid, or carbamazepine. In the second phase of the study, attempts to identify causes for serum teratogenicity led to the finding that the abnormalities and reduced embryo growth produced by many serum samples could be completely overcome by adding vitamins and/or amino acids to the serum. Of 53 samples tested, 32 (60%) were corrected by supplementation (17 of 23 phenytoin, seven of nine phenobarbital, six of 12 carbamazepine, none of six valproic acid, and two of three no drug). Although the results of this study provided a general assessment of drug teratogenicity that agreed with other studies and emphasized the role of nutrition in fetal defects, the importance of individual differences in causes of teratogenicity was also noted.

Amino Acids↗
PubMedDate not supplied

Identification of a teratogenic drug-protein complex in sera of phenytoin-treated monkeys.

Whole rat embryos were cultured for 48 h on sera drawn from monkeys before and 10 h after phenytoin gavage (275 mg/kg body weight). Sera from treated monkeys caused exencephaly, anophthalmia, microcephaly, and incomplete ventral curvature when used as culture media, whereas sera drawn from the same monkeys before treatment supported normal embryonic development. To identify the cause of serum teratogenicity, isolated constituents of teratogenic sera were added to nonteratogenic sera for testing by embryo culture. Serum extracts containing free phenytoin and its free metabolites were not teratogenic. Teratogenicity was found associated with serum proteins. Using polyacrylamide gel electrophoresis (PAGE) and an antibody that recognized phenytoin and its metabolites, we were able to demonstrate that phenytoin was bound to a protein of 80,000 daltons. Addition of this same antibody to teratogenic sera from dosed monkeys improved the development of cultured embryos and provided additional support for this complex as the proximal teratogen. Use of the antibody to follow the uptake and distribution of phenytoin in cultured embryos suggested that only the phenytoin-protein complex (and not phenytoin itself) was able to pass through the yolk sac and reach the tissues of the embryo proper. These results suggested that a drug-protein complex may serve to transport drugs from their site of activation in the maternal liver to the developing embryo.

Abnormalities, Drug-Induced↗