Search PubMed⌕ Search

Biomedical subjects

B Ketterer

Publications and source records attributed to B Ketterer.

At least 55 records · Page 3Linked to original sources
PubMed1993

Preferential increase of glutathione S-transferase class alpha transcripts in cultured human hepatocytes by phenobarbital, 3-methylcholanthrene, and dithiolethiones.

In rodents, a diversity of compounds are able to protect against acute and chronic toxicities of various xenobiotics including carcinogens, at least in part through induction of drug-metabolizing enzymes including glutathione S-transferase (GST) enzymes. We have posed the question as to whether or not these compounds also induce GSTs in human liver. Primary human hepatocyte cultures were exposed to phenobarbital, 3-methylcholanthrene, and two dithiolethiones [1,2-dithiole-3-thione and its 5-(2-pyrazinyl)-4-methyl derivative, oltipraz], and steady-state mRNA levels of GST classes alpha, mu, and pi were determined by Northern blot analysis. After 3 daily treatments, the two dithiolethiones were the most potent inducers; phenobarbital was also effective but to a lesser extent and 3-methylcholanthrene increased GST mRNA in only 2 of the 6 samples, although it stimulated cytochrome P-450 1A2 mRNA in all cell preparations. Whatever the compound only GSTA1 and/or A2 transcripts were induced. GST M1 mRNAs were not responsive or only slightly responsive, and GST P1 mRNAs, which were mostly undetectable in control cells, were not affected by treatment with any of the four chemicals. Large individual variations were observed in the level of induction of GST A1 and/or A2 mRNAs, and no sex difference could be demonstrated. These results clearly indicate that phenobarbital, 3-methylcholanthrene, and dithiolethiones are able to markedly increase mRNA levels of GST in human hepatocytes and that the GST alpha class is preferentially involved.

Cells, Cultured↗
PubMed1993

Quantitation of tissue- and sex-specific induction of rat GSH transferase subunits by dietary 1,2-dithiole-3-thiones.

Male and female Sprague--Dawley rats were maintained for 5 days on control diet or diet containing 0.075% (w/w) 1,2-dithiole-3-thione (D3T) or 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz). The content of GSH transferase (GST) subunits 1a, 1b, 2, 3, 4, 6, 7, 8, 10 and 11 in the soluble fraction of liver, kidney, small intestine, stomach and lung of control and D3T-fed animals was determined. Liver and kidney were similarly analysed for the oltipraz-fed animals. Significant induction of GST subunits by D3T was seen in all tissues, the most substantial being subunit 7 in male liver (approximately 50-fold) and subunits 1b, 3 and 10 in male and female small intestine (5- to 16-fold). Generally subunits 7, 10, 1b and 3 were most affected, while subunits 6 and 8 were hardly inducible. Oltipraz caused qualitatively similar inductions in the liver and kidney. The effect of GSH transferase induction on the hepatic capacity for GSH conjugation of aflatoxin B1 exo-8,9-oxide is assessed.

Animals↗
PubMed1992

Stereoselective conjugation of 2-bromocarboxylic acids and their urea derivatives by rat liver glutathione transferase 12-12 and some other isoforms.

Glutathione (GSH) conjugation of the separate enantiomers of five 2-bromocarboxylic acids and some of their urea derivatives by rat liver GSH transferases (GSTs) was studied. The liver cytosolic fraction conjugated all compounds, except for (R)-2-bromoisovaleric acid, with a variable degree of stereoselectivity. A GST pool, prepared by S-hexyl-GSH affinity chromatography, conjugated the urea derivatives at a somewhat higher rate but had very little activity towards the carboxylic acids, indicating that much activity towards the latter substrates was due to transferases not bound by the affinity column. Therefore, the activity was studied of some pure GSTs that are bound only slightly by the affinity column towards the separate enantiomers of 2-bromovaleric acid (BV), its urea derivative and 2-bromo-3-phenylpropionic acid (BPP). No activity was detected with transferases 5-5 and 8-8. Transferase 1-1 was active towards all compounds with high activity towards the urea derivatives. Transferase 12-12 showed high, stereospecific activity towards the R enantiomers of BV, its urea derivative and BPP.

Animals↗
PubMed1992

Overexpression of the multidrug resistance gene product in adult rat hepatocytes during primary culture.

Expression of P-glycoprotein (P-gp), the product of multidrug resistance gene(s), was investigated in primary cultures of normal adult rat hepatocytes. Levels of P-gp mRNAs determined by Northern blotting and of P-gp measured by immunoblotting increased in parallel with time in culture. As in normal liver, P-gp was found to be localized on the membrane of bile canaliculus-like structures. This increased expression of P-gp was associated with decreased intracellular retention of doxorubicin, which could be restored by compounds such as verapamil and cyclosporin; doxorubicin (and also vincristine) was more cytotoxic to early than to late cultures. As in preneoplastic and neoplastic liver, overexpression of P-gp in cultured hepatocytes was associated with differential changes in drug-metabolizing enzymes, including increased glutathione S-transferase 7-7. Functional P-gp over-expression was observed in the absence of xenobiotic exposure or cell division; it could be linked to cellular stress occurring during cell isolation and plating. Increased expression of P-gp was blocked by actinomycin D, indicating its dependence on increased transcription, while cycloheximide led to a superinduction suggesting negative regulation by a protein factor.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Expression of glutathione S-transferase during rat liver development.

The ontogeny of rat liver glutathione S-transferase (EC 2.5.1.18) (GSTs) during foetal and postnatal development was investigated. The GSTs are dimers, the subunits of which belong to three multigene families, Alpha (subunits 1, 2, 8 and 10), Mu (subunits 3, 4, 6, 9 and 11) and Pi (subunit 7) [Mannervik, Alin, Guthenberg, Jennsson, Tahir, Warholm & Jörnvall (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7202-7206; Kispert, Meyer, Lalor, Coles & Ketterer (1989) Biochem. J. 260, 789-793]. There is considerable structural homology within each gene family, with the result that whereas reverse-phase h.p.l.c. successfully differentiates individual subunits, immunocytochemical and Northern-blotting analyses may only differentiate families. Enzymic activity, h.p.l.c. and Northern blotting indicated that expression of GST increased from very low levels at 12 days of foetal growth to substantial amounts at day 21. At birth, GST concentrations underwent a dramatic decline and remained low until 5-10 days post partum, after which they increased to adult levels. During the period under study, GST subunits underwent differential expression. The Mu family had a lower level of expression than the Alpha family, and, within the Alpha family, subunit 1 was more dominant in the adult than the foetus. Subunit 2 is the major form in the foetus. Most noteworthy were subunits 7 and 10, which were prominent in the foetus, but present at low levels post partum. Immunocytochemical analysis of the 17-day foetal and newborn rat livers showed marked differences in the distribution of GSTs in hepatocytes. In the 17-day foetal liver Pi greater than Alpha greater than Mu whereas in the newborns Alpha greater than Mu much greater than Pi. Erythropoietic cells were not stained for any of the three GST families. Steady-state mRNA concentrations in the foetus correlated with the relative transcription of the Alpha, Mu and Pi class genes. However, in those genes expressed post partum, namely the Alpha and Mu class, low transcriptional activity was associated with high concentrations of mRNA. This suggests that there is a switch from transcriptional control to post-transcriptional control at birth. GST 7-7 appears to be regulated predominantly by transcription throughout the period of liver development under observation.

Aging↗
PubMed1992

Chlorambucil-monoglutathionyl conjugate is sequestered by human alpha class glutathione S-transferases.

The spontaneous reaction of 110 microM chlorambucil (4-[p-[bis(2-chloroethyl)amino]phenyl]-butanoic acid; CHB) with 5 mM GSH at 37 degrees C in physiological phosphate-buffered saline for 35 min gave primarily the monoglutathionyl derivative, 4-[p-[N-2-chloroethyl,N-2-S-glutathionylethyl]amino]phenyl]-butano ic acid; CHBSG) and the diglutathionyl derivative, 4-[p-[bis(2-S-glutathionylethyl]amino]phenyl]-butanoic acid (CHBSG2) with small amounts of the hydroxy-derivatives: 4-[p-[N-2-chloroethyl,N-2-hydroxy-ethyl]amino] phenyl-butanoic acid (CHBOH) and 4-[p-[N-2-S-glutathionylethyl-2-hydroxyethyl]amino]phenyl]-butanoi c acid (CHBSGOH). The inclusion of approximately physiological amounts of human glutathione S-transferases (GSTs) A1-1, A2-2, P1-1, M1a-1a M3-3 or P1-1 (for nomenclature see Mannervik et al., 1992, Biochem. J., 282, 305) had little or no catalytic effect on these reactions as determined by loss of CHB. However, GTSs A1-1 and A2-2 were associated with a significant increase of CHBSG at the expense of CHBSG2 + CHBSGOH suggesting that these GTs sequestered CHBSG at the active site. This interpretation was supported by inhibition studies which showed that CHBSG was a pure competitive inhibitor of the activity of GSTs A1-1 and A2-2 towards 1-chloro-2,4-dinitrobenzene with Ki's of 1.3 and 1.2 microM respectively. GSH transferases P1-1 and M1a-1a were inhibited by CHBSG above 10 microM. Incubation of 2 microM CHB, a concentration which may be of more significance for chemotherapy, in the presence or absence of GST A1-2 (20-50 microM) showed catalysis of GSH monoconjugation equivalent to 18% of the spontaneous rate. However, the dominant effect again was the sequestration of CHBSG which reached 74.3 +/- 1.5 (SEM)% of the total reactants at 60 min compared to 28.9 +/- 0.3(SEM)% in controls. CHBSG, although possessing a potential electrophilic centre, showed no detectable alkylation of plasmid DNA but indirect evidence was obtained that it alkylated other cellular macromolecules. It is concluded that the contribution of GSTs to catalysis of CHB detoxication will depend on factors not previously considered, namely the relative molarities of CHB, CHBSG and GSTs, and the cellular capacity to excrete CHBSG to relieve product inhibition.

Chlorambucil↗
PubMed1992

Effects of ethanol, dexamethasone and RU 486 on expression of cytochromes P450 2B, 2E, 3A and glutathione transferase pi in a rat hepatoma cell line (Fao).

The aim of our study was to investigate the suitability of Fao cells, derived from the Reuber H35 rat hepatoma as a tool for studying regulation of drug-metabolizing enzymes and drug metabolism. Fao cells express P450 2B, 2E, 3A and GST pi and were used to study the effects different inducers on these enzymes. Ethanol considerably increased the amounts of P450 2E and, to a lesser extent, P450 2B and GST pi mRNA and protein. Dexamethasone decreased the amounts of P450 2B, 3A and GST pi mRNAs, but had no appreciable effect per se upon the protein concentration of these enzymes. However, it antagonized the induction of P450 2E, 2B and GST pi by ethanol, even at the protein level. RU 486 decreased P450 2B protein and P450 2E mRNA and protein levels without effecting P450 3A and GST pi expression. RU 486 did not antagonize the dexamethasone effects, suggesting that at least some of these effects are not mediated by the glucocorticoid receptor. These data indicate that these cells constitute a suitable tool for studying the regulation of drug-metabolizing enzyme expression and drug metabolism.

Animals↗
PubMed1992

Elucidation of catalytic specificities of human cytochrome P450 and glutathione S-transferase enzymes and relevance to molecular epidemiology.

A number of different approaches have been used to determine the catalytic selectivity of individual human enzymes toward procarcinogens. Studies with cytochrome P450 (P450) enzymes and glutathione S-transferases are summarized here, and recent work with pyrrolizidine alkaloids, aflatoxins, 4,4'-methylenebis(2-chloroaniline), and ethyl carbamate is discussed. In some cases a single enzyme can catalyze both activation and detoxication reactions of a chemical. The purification and characterization of human lung P4501A1 and the development of a noninvasive assay for human P4502E1 are also described.

Aflatoxin B1↗
PubMed1992

The human glutathione S-transferase supergene family, its polymorphism, and its effects on susceptibility to lung cancer.

Cytosolic glutathione S-transferases (GSTs) are a supergene family of dimeric enzymes capable of detoxifying a number of carcinogenic electrophiles. Of the numerous components of tobacco smoke, the polycyclic aromatic hydrocarbons appear to be the principal compounds that yield substrates for these enzymes, GSTM1-1 being effective with those PAH derivatives so far studied; however, the gene locus for GSTM1 is polymorphic, containing two well-characterized expressing genes and a null allele. Use of cDNA for GSTM1-1 or appropriate fragments of genomic clones as probes in Southern blots indicated that the null allele is due to the absence of GSTM1. In preliminary experiments, described here, with lung tissue from smokers, levels of 32P-postlabeled nuclease P1-enhanced DNA adducts were inversely correlated with levels of antigen cross-reacting with antibody to GSTM1-1, suggesting that initiation depends on the expression of GSTM1-1. Since similar quantities of DNA adducts and GSTM1-1 activity have been shown to occur in bronchial and peripheral lung, however, the development of malignancy, which is usually in the bronchial region, presumably depends on additional factors that bring about promotion and progression, which are not necessarily affected by GSTM1 expression. Two epidemiological studies have been carried out in which a possible correlation between the absence of GSTM1 and lung cancer incidence is considered. In the first, involving a U.S. population sample, smokers with and without lung cancer were phenotyped, and a highly significant correlation between the absence of GSTM1-1 activity and adenocarcinoma of the lung was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

A novel glutathione transferase (13-13) isolated from the matrix of rat liver mitochondria having structural similarity to class theta enzymes.

A rat liver mitochondrial-matrix fraction was prepared and shown to have 1-chloro-2,4-dinitrobenzene(CDNB)-metabolizing glutathione transferase (GST) activity. Further fractionation by sequential gel filtration, isoelectric focusing or chromatofocusing and hydroxyapatite chromatography yielded three GSTs of pI 9.3, 8.9 and 7.5, none of which bound to a GSH-agarose affinity matrix. Most of the activity was associated with the pI-9.3 form, which was selected for further study. Its activity was tested with the following potential substrates in addition to CDNB: 1,2-dichloro-4-nitrobenzene, p-nitrobenzyl chloride, trans-4-phenylbut-3-en-2-one, 1,2-epoxy-3-(p-nitrophenoxy)propane, ethacrynic acid, menaphthyl sulphate, cumene hydroperoxide, linoleic acid hydroperoxide and 4-hydroxynon-2-enal. Appreciable activity was obtained only with CDNB and ethacrynic acid (82 and 26 mumol/min per mg of protein respectively). The apparent Km for GSH, using 1 mM-CDNB, was 1.9 mM. The enzyme is a dimer of subunit Mr 26,500. It has a free N-terminus, which has enabled the first 33 amino acids to be sequenced. This portion of primary structure has a sequence in common with members of the Theta class of GSTs (eg. 36% identity with subunit 12) and also a sequence which might function as a mitochondrial import signal. It is novel and has been named 'GST 13-13'.

Amino Acid Sequence↗
PubMed1991

Glutathione transferase pi its minimal promoter and downstream cis-acting element.

Fragments of the human glutathione S-transferase pi gene and 15 kb of its 5' flanking region have been fused to the chloramphenicol acetyl transferase (CAT) reporter gene. Transfection into a number of human cell lines (Hela, HepG2, MCF7 and EJ) has demonstrated that the AP1 binding site, located between nucleotides -58 and -65 (Cowell et al. 1988. Biochem. J. 255, 79-83), is essential for basal level promoter activity. We have also identified a positive cis-acting DNA element between nucleotides +8 and +72 which seems to be part of the promoter. No other regulatory activity was identified within the 17 kb analyzed.

Animals↗
PubMed1991

Theta, a new class of glutathione transferases purified from rat and man.

Glutathione transferases (GSTs) of a novel class, which it is proposed to term Theta, were purified from rat and human liver. Two, named GST 5-5 and GST 12-12, were obtained from the rat, and one, named GST theta, was from the human. Unlike other mammalian GSTs they lack activity towards 1-chloro-2,4-dinitrobenzene and are not retained by GSH affinity matrices. Only GST 5-5 retains full activity during purification, and its activities towards the substrates 1,2-epoxy-3-(p-nitrophenoxy)propane, p-nitrobenzyl chloride, p-nitrophenethyl bromide, cumene hydroperoxide, dichloromethane and DNA hydroperoxide are 185, 86, 67, 42, 11 and 0.03 mumol/min per mg of protein respectively. Earlier preparations of GST 5-5 or GST E were probably a mixture of GST 5-5 and GST 12-12, which was largely inactive, and may also have been contaminated by less than 1% with another GSH peroxidase of far greater activity. Partial analysis of primary structure shows that subunits 5, 12 and theta are related to each other, particularly at the N-terminus, where 25 of 27 residues are identical, but have little relationship to the Alpha, Mu and Pi classes of mammalian GSTs. They do, however, show some relatedness to subunit I of Drosophila melanogaster [Toung, Hsieh & Tu (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 31-35] and the dichloromethane dehalogenase of Methylobacterium DM4 [La Roche & Leisinger (1990) J. Bacteriol, 172, 164-171].

Amino Acid Sequence↗