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Biomedical subjects

E Maser

Publications and source records attributed to E Maser.

At least 55 records · Page 3Linked to original sources
PubMed1996

Induction of daunorubicin carbonyl reducing enzymes by daunorubicin in sensitive and resistant pancreas carcinoma cells.

Daunorubicin (DRC) and other anthracyclines are valuable cytotoxic agents in the clinical treatment of certain malignancies. However, as is the case with virtually all anticancer drugs, tumor cell resistance to these agents is one of the major obstacles to successful chemotherapy. In addition to an increased energy-dependent efflux of chemotherapeutic agents, enzymatic drug-inactivating mechanisms also contribute to multidrug resistance of tumor cells. In the case of DRC, carbonyl reduction leads to 13-hydroxydaunorubicinol (DRCOL), the major metabolite of DRC with a significantly lower antineoplastic potency compared to the parent drug. In the present study, we compared two pancreas carcinoma cell lines (a DRC-sensitive parental line and its DRC-resistant subline) with respect to their capacity of DRC inactivation via carbonyl reduction. In addition, we cultured the two cell lines in the presence of increasing sublethal concentrations of DRC. Evidence is presented that DRC treatment itself leads to a concentration-dependent induction of DRC carbonyl reduction in subcellular fractions of both the sensitive and resistant pancreas carcinoma cells, resulting, surprisingly, in different susceptibilities to DRC. The principal difference between the two cell lines becomes most apparent at high-dose DRC supplementation (1 microgram/mL), at which DRC resistant cells exhibited higher inducibility of DRC-inactivating enzymes, whereas respective sensitive cells already showed an impairment of cellular viability. The use of the diagnostic model substrates metyrapone and p-nitrobenzaldehyde reveals that this adaptive enhancement of DRC inactivation can be attributed to the induction of DRC carbonyl reductases different from known aldehyde and carbonyl reductases. In conclusion, these findings suggest that inactivation of anthracyclines by carbonyl reduction is inducible by the substrate itself, a fact that might be considered as one of the enzymatic mechanisms that contribute to the acquired resistance to these drugs.

Alcohol Oxidoreductases↗
PubMed1996

Antibiotic resistance and enhanced insecticide catabolism as consequences of steroid induction in the gram-negative bacterium Comamonas testosteroni.

The effects of steroid induction on antibiotic resistance against the fungal steroid fusidic acid (ramycin; 16-(acetyloxy)-3 alpha,11 alpha-dihydroxy-29-dammara-17(20), 24-dien-21-oic-acid) as well as on carbonyl reduction and degradation of the novel anti-insect agent NKI 42255 (2-(1-imidazolyl)-1-(4-methoxyphenyl)-2-methyl-1-propanone) were studied in the Gram-negative soil bacterium Comamonas testosteroni strain ATCC 11996. Cells grown with testosterone as inducing agent showed a 5-6-fold elevation of antibiotic resistance against the fungal steroid fusidic acid. Furthermore, testosterone induction caused a faster uptake and different metabolism of the anti-insect agent NKI 42255 compared to control cultures, revealing carbonyl reduction of the substrate keto group as an initial degradation step in induced cells. It is concluded that the formerly described steroid inducible hydroxysteroid dehydrogenases/carbonyl reductases present in Comamonas testosteroni contribute to these altered phenotypes, thus establishing steroid-inducible catabolic pathways as important defense processes against natural and synthetic toxicants in certain bacteria, which are present in the intestinal microflora of mammalian species as well as in soil samples.

Anti-Bacterial Agents↗
PubMed1996

Biotransformation and detoxification of insecticidal metyrapone analogues by carbonyl reduction in the human liver.

1. The carbonyl reduction of insecticidal metyrapone analogues to their hydroxyl metabolites by human liver microsomes and cytosol was examined. Metabolite quantification was performed by means of hplc determination and inhibition experiments, using specific carbonyl reductase inhibitors, were conducted. 2. The cytotoxicity of the ketones and their hydroxy metabolites was assessed with the MTT test, using Chang liver cells. 3. It was found that the alcohol derivatives are the major metabolite, both in microsomes and cytosol. The microsomal reductive metabolism, considered to be mediated by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) (EC 1.1.1.146), was more extensive than the cytosolic carbonyl reduction. In each case, this metabolism was inhibited significantly by equimolar concentrations of the microsomal 11 beta-HSD inhibitor glycyrrhetinic acid and the cytosolic carbonyl reductase inhibitor quercitrin, respectively. 4. The parent ketones were more cytotoxic than their alcohol metabolites. 5. These results demonstrate that the metyrapone analogues are extensively metabolized by human liver microsomes, presumably by 11 beta-HSD, to the less cytotoxic and readily excretable alcohols. 6. Since the metyrapone analogues can inhibit ecdysone 20-monooxygenase (EC 1.14.99.22), our results indicate potential application of these compounds as insecticides, which would be safer for humans, due to their reductive detoxification, mainly by the hepatic microsomal 11 beta-HSD, to the less toxic hydroxy metabolites.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1995

Cloning and primary structure of murine 11 beta-hydroxysteroid dehydrogenase/microsomal carbonyl reductase.

Screening of a mouse liver lambda gt 11 cDNA library with a rat liver 11 beta-hydroxysteroid dehydrogenase cDNA (11 beta-HSDr1A) and subsequent screening with an isolated mouse probe, resulted in the isolation and structure determination of a mouse cDNA encoding an amino acid sequence which is very similar to human and rat 11 beta-hydroxysteroid dehydrogenases (78% and 86% similar, respectively), and also to other known vertebrate 11 beta-hydroxysteroid dehydrogenase structures. Open-reading-frame analysis and the deduced amino acid sequence predict a protein with a molecular mass of 32.3 kDa which belongs to the superfamily of the short-chain dehydrogenase proteins. The amino acid sequence contains two potential glycosylation sites. These data are in agreement with information on the glycoprotein character of the native enzyme. This kind of post-translational modification seems to be a determining factor concerning the equilibrium of the catalyzed 11 beta-dehydrogenation/11-oxo reduction step [Obeid, J., Curnow, K. M., Aisenberg, J. & White, P.C. (1993) Mol. Endocrinol. 7, 154-160; Agarwal, A.K., Tusie-Luna, M.T., Monder, C. & White, P.C. (1990) Mol. Endocrinol. 4, 1827-1832]. After in vitro transcription/translation of the mouse cDNA, immunoprecipitation with anti-(microsomal carbonyl reductase) serum and N-terminal sequence analysis of the purified protein confirms the identity of microsomal 11 beta-hydroxysteroid dehydrogenase with the previously described, microsomal-bound xenobiotic carbonyl reductase [Maser, E. & Bannenberg, G. (1994) Biochem. Pharmacol. 47, 1805-1812], and points to an involvement of the 11 beta-HSD1A isoform in the reductive phase-I metabolism of xenobiotic compounds, besides its endocrinological functions. The alignment and comparison to other hydroxysteroid dehydrogenase forms of the same protein superfamily allows the identification of important residues in the 11 beta-HSD primary structure.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1995

Comparison of 11 beta-hydroxysteroid dehydrogenase in spontaneously hypertensive and Wistar-Kyoto rats.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) modulates glucocorticoid interactions with mineralocorticoid and glucocorticoid receptors in vivo, by converting 11 beta-hydroxyglucocorticoids to their inactive 11-ketone derivatives. Defective 11 beta-oxidation of glucocorticoids has been associated with hypertension. The objective of this study was to investigate whether 11 beta-HSD contributes to the occurrence of hypertension in spontaneously hypertensive rats (SHRs). The liver and kidney microsomal oxidations of corticosterone (the physiological glucocorticoid in rats) in organs from juvenile (3 weeks old) and adult (3 months old) SHR and Wistar-Kyoto (WKY) rats, with NAD and NADP, show no differences between rat strains. For cortisol, with NADP, adult SHRs show (1.3-3 times; P < 0.05) lower kidney microsomal oxidation rates. The liver microsomal reduction of cortisone shows remarkable interstrain differences; with NADH, reduction is conducted only by adult WKY rats, whereas with NADPH, juvenile animals show similar reduction rates, but at adulthood, only WKYs reduce cortisone. Using Western blot analysis with antibodies against 11 beta-HSD1, positive signals are obtained only for liver microsomes, appearing somewhat lower in SHRs for juvenile but not adult animals. Urinary corticosterone/11-dehydrocorticosterone ratios (measured in adult animals) are not different between rat strains, but are elevated after administration of corticosterone in both strains (although significant only in SHRs). The data provide no indications for exaggerated stimulation of renal corticosteroid receptors, due to modified 11 beta-HSD, in SHRs. However, the experiments suggest the existence of multiple 11 beta-HSDs, in addition to 11 beta-HSD1 and 11 beta-HSD2, some of which may be modified in SHR, but the nature and physiological role of these 11 beta-HSDs is unclear.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1994

11 beta-hydroxysteroid dehydrogenase mediates reductive metabolism of xenobiotic carbonyl compounds.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) is considered to confer mineralocorticoid specificity on the non-selective Type I adrenocorticoid receptor by converting active 11-hydroxyglucocorticoids to receptor-inactive 11-oxo metabolites, in mineralocorticoid target tissues like the kidney. However, 11 beta-HSD is also present in the liver, where it may regulate steroid exposure to the glucocorticoid Type II receptor. Because of the much higher activities compared to that in kidney, liver 11 beta-HSD possibly has additional functions besides the metabolism of glucocorticoids. In the present investigation we have isolated 11 beta-HSD from mouse liver microsomes and demonstrate that the homogeneously purified enzyme is also capable of catalyzing the reductive metabolism of xenobiotic carbonyl compounds such as metyrapone, p-nitroacetophenone and p-nitrobenzaldehyde. Enzyme kinetic studies revealed that, in addition to NADP+, mouse liver 11 beta-HSD also accepts NAD+ as cosubstrate for glucocorticoid 11 beta-dehydrogenation. NADH as cosubstrate for 11-oxoreduction plays only a minor role compared to that with NADPH, a fact which is also true for xenobiotic carbonyl reduction. Inhibition experiments revealed strong sensitivity of xenobiotic carbonyl reduction to glucocorticoids. The competitive nature of this inhibition suggests that both glucocorticoids and xenobiotic carbonyl substances bind to the same catalytically active site of 11 beta-HSD. High enzyme activities were also found in microsomal fractions of the ovary and adrenal gland but, although expressing considerable glucocorticoid 11-dehydrogenation activity (one third that of liver), almost no carbonyl reduction was detectable in kidney microsomes. Immunoblot analysis with polyclonal antibodies directed against the liver 11 beta-HSD did not yield an immunological crossreaction in the same tissues. In conclusion, corresponding to the cytosolic aldo-keto reductases, microsomal 11 beta-HSD of liver may be considered to play a role in the phase I biotransformation of pharmacologically relevant carbonyl substances as well as protecting organisms against toxic carbonyl compounds by converting them to less lipophilic and more soluble and conjugatable metabolites. Discrepancies in bioactivity together with the lack of response to anti-liver 11 beta-HSD antibodies strongly indicate the existence of distinct forms of 11 beta-HSD to be present in kidney, adrenal gland and ovary. The ability of xenobiotic carbonyl reduction might be another distinguishing feature among the various 11 beta-HSD isozymes.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1994

The purification of 11 beta-hydroxysteroid dehydrogenase from mouse liver microsomes.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) is a microsomal enzyme complex responsible for the interconversion of active 11-hydroxy glucocorticoids to inactive 11-oxo metabolites. It has long been controversially discussed whether 11-dehydrogenation and 11-oxoreduction are catalysed by a single bidirectional enzyme or if the 11 beta-HSD system comprises 2 kinetically distinct microsomal enzyme activities, 11-dehydrogenase and 11-oxoreductase. However, 11-oxoreduction of homogeneously purified 11 beta-HSD could not be demonstrated under in vitro conditions until today. We have purified 11 beta-HSD from mouse liver microsomes to homogeneity by a purification method which affords a gentle membrane protein solubilization as well as providing a favourable detergent surrounding during the various chromatographic steps. Following 11-dehydrogenation of corticosterone and 11-oxoreduction of dehydrocorticosterone simultaneously throughout the entire purification procedure we could demonstrate that 11 beta-HSD retains both oxidative and reductive activities in almost the same ratio, which is also true for the homogeneously purified enzyme. Deducing from the coincidentally increasing specific activities of 11-dehydrogenation and 11-oxoreduction the conclusion can be drawn that both activities reside within the same protein. Furthermore, in addition to NADP(H) also NAD(H) can serve as cosubstrate, which is mainly true for the oxidative direction. In conclusion, our results provide evidence that the oxidative and reductive behaviour of 11 beta-HSD can be explained by the concept of a unique, reversible oxidoreductase thus disproving the two enzyme theory.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1994

Ontogenic pattern of carbonyl reductase activity of 11 beta-hydroxysteroid dehydrogenase in mouse liver and kidney.

1. The ontogenic pattern of xenobiotic carbonyl reducing activity and glucocorticoid 11 beta-oxidoreducing activity of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) in mouse liver and kidney was examined. In addition, the expression of this enzyme was investigated by means of immunoblot analysis in the same tissues. 2. In liver, the foetus shows low or no enzyme activities. After birth both activities increase dramatically with age and remain then on a high plateau until the time of sexual maturity (4 weeks). After maturity, the enzyme activities decline to intermediate values. The developmental pattern of immunological expression of the liver enzyme corresponds well with that of the enzyme activity. 3. Considerable activities of xenobiotic carbonyl reduction and glucocorticoid 11 beta-oxidoreduction are also present after birth in all developmental stages of the kidney. However, no immunological crossreaction was found in any stages with the antibody against the liver 11 beta-HSD suggesting the presence of a structurally different isozyme form in the kidney. 4. The dramatic increase of both activities during the peri- and postnatal developmental periods suggest a potentially biological significance of the liver 11 beta-HSD isozyme in early animal life. 5. Besides being involved in 11 beta-glucocorticoid metabolism in particular the liver enzyme seems to play an additional role as xenobiotic carbonyl reductase.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1992

A novel membrane associated carbonyl reducing enzyme is present in smooth endoplasmic reticulum of mouse liver.

Evidence supporting the existence of two distinct carbonyl (metyrapone) reducing enzymes which differ in subcellular localization and immunological homology has been provided. A soluble enzyme, designated as carbonyl reductase (EC 1.1.1.184) is located in the cytosol. The distribution of the second, membrane associated, MPON-reductase shows an excellent linear correlation to NADPH-cytochrome c reductase and, on the other hand, is reciprocal to the RNA/protein ratio of submicrosomal preparations. This indicates that the membrane associated MPON-reductase is exclusively located in the smooth endoplasmic reticulum. Using antibodies against the purified membrane associated enzyme the extent of immunological crossreaction corresponds well to the specific activities of MPON-reductase in the granular fractions, thus further confirming the localization of this enzyme within this organelle. The absence of antigenic crossreaction to cytosolic MPON-reductase indicates differences also in terms of the immunological relationship between the two enzymes.

Alcohol Oxidoreductases↗
PubMed1992

Characterization of carbonyl reducing activity in continuous cell lines of human and rodent origin.

Carbonyl reduction was investigated in the continuous cell lines V79, NCI-H322 and C2REV7 by using the ketone compound metyrapone as a substrate. Metyrapone reducing enzymes were characterized by evaluating the cosubstrate requirement and by testing the sensitivity of this reaction to specific inhibitors. All cell lines were found to produce metyrapol at a linear rate over a time course of at least 48 hr, when tested in cultured monolayers. In general, cytosolic metyrapone reduction exceeds microsomal activity several-fold in all three cell lines. Quercitrin turned out to be the strongest inhibitor in all fractions, except in NCI-H322 microsomes where it had no effect. Consequently, carbonyl reductase is suspected to be responsible for metyrapone reduction in the cytosol and microsomes of V79 and C2REV7 cells as well as in the cytosol of NCI-H322 cells. Simultaneous sensitivity towards quercitrin, dicoumarol, indomethacin and 5 alpha-dihydrotestosterone in some cases points to the existence of different isozymes of carbonyl reductase. In NCI-H322 microsomes only dicoumarol and indomethacin decrease metyrapol formation, thus pointing to an isozyme of NAD(P)H:quinone-oxidoreductase. Concerning cosubstrate requirements metyrapone reducing enzymes show a strong preference for NADPH, thus confirming the involvement of carbonyl reductase in this reaction. In conclusion, carbonyl reduction of metyrapone in continuous cell lines is mediated by carbonyl reductases due to the common sensitivity towards the diagnostic inhibitor quercitrin and due to the strong preference for NADPH as cosubstrate. According to its maintenance in permanent cell lines carbonyl reductase seems to be an essential and constitutive enzyme, which probably fills an important role in normal cell physiology.

Alcohol Oxidoreductases↗
PubMed1992

Functional and immunological relationships between metyrapone reductase from mouse liver microsomes and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni.

3 Alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from Pseudomonas testosteroni was shown to reduce the xenobiotic carbonyl compound metyrapone (MPON). Reversely, MPON reductase purified from mouse liver microsomes and previously characterized as aldehyde reductase, was competitively inhibited by 3 alpha-HSD steroid substrates. For MPON reduction both enzymes can use either NADH or NADPH as co-substrate. Immunoblot analysis after native and SDS gel electrophoresis of 3 alpha-HSD gave a specific crossreaction with the antibodies against the microsomal mouse liver MPON reductase pointing to structural homologies between these enzymes. In conclusion, there seem to exist structural as well as functional relationships between a mammalian liver aldehyde reductase and prokaryotic 3 alpha-HSD. Moreover, based on the molecular weights and the co-substrate specificities microsomal mouse liver MPON reductase and Pseudomonas 3 alpha-HSD seem to be members of the short-chain alcohol dehydrogenase family.

Alcohol Oxidoreductases↗
PubMed1992

High carbonyl reductase activity in adrenal gland and ovary emphasizes its role in carbonyl compound detoxication.

Carbonyl reduction has been studied in liver, kidney, adrenal gland and ovary of female Wistar and Sprague-Dawley rats as well as of female NMRI mice by using metyrapone as a substrate and by means of direct HPLC analysis of the reduced alcohol metabolite metyrapol. Carbonyl reducing activities were found in all tissues examined so far, with that in rat ovary and adrenal gland cytosol exceeding the liver cytosolic specific activity severalfold: 15-fold and 12-fold in the Wistar strain; 12-fold and 7-fold in the Sprague-Dawley strain, respectively. In general, Wistar rat enzyme activities were about four times higher than those of Sprague-Dawley rats in all fractions, which indicates an interesting genetic difference between the two rat strains. Due to the sensitivity towards the diagnostic inhibitor quercitrin, carbonyl reductase (EC 1.1.1.184) seems to be mainly responsible for metyrapone reduction in rat and mouse adrenal gland and ovary cytosol. However, sensitivity towards dicoumarol in microsomal fractions of mouse tissues points to the involvement of further carbonyl reducing enzymes. Western blot experiments revealed immunological differences between metyrapone reductase from liver microsomes and respective enzymes of all other tissues. In conclusion, the difference in tissue and intracellular distribution suggests that several enzymes are involved in carbonyl reduction of metyrapone and the intracellular multiplicity of the enzymes may have some relation to their significance in carbonyl compound detoxification. These results support the hypothesis that carbonyl reductases, besides their participation in the metabolism of physiologically occurring substances, provide the enzymatic basis for the detoxification of xenobiotic carbonyl compounds in adrenal gland and ovary which have escaped their metabolic conversion by the liver.

Adrenal Glands↗
PubMed1992

Homologies between enzymes involved in steroid and xenobiotic carbonyl reduction in vertebrates, invertebrates and procaryonts.

Evidence is reported for the existence of a structurally and functionally related and probably evolutionarily conserved class of membrane-bound liver carbonyl reductases/hydroxysteroid dehydrogenases involved in steroid and xenobiotic carbonyl metabolism. Carbonyl reduction was investigated in liver microsomes of 8 vertebrate species, as well as in insect larvae total homogenate and in purified 3 alpha-hydroxysteroid dehydrogenase preparations of the procaryont Pseudomonas testosteroni, using the ketone compound 2-methyl-1,2 di-(3-pyridyl)-1-propanone (metyrapone) as substrate. The enzyme activities involved in the metyrapone metabolism were screened for their sensitivity to several steroids as inhibitors. In all fractions tested, steroids of the adrostane or pregnane class strongly inhibited xenobiotic carbonyl reduction, whereas only in the insect and procaryotic species could ecdysteroids inhibit this reaction. Immunoblot analysis with antibodies against the respective microsomal mouse liver metyrapone reductase revealed strong crossrections in all fractions tested, even in those of the insect and the procaryont. A similar crossreaction pattern was achieved when the same fractions were incubated with antibodies against 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. The mutual immunoreactivity of the antibody species against proteins from vertebrate liver microsomes, insects and procaryonts suggests the existence of structural homologies within these carbonyl reducing enzymes. This is further confirmed by limited proteolysis of purified microsomal mouse liver carbonyl reductase and subsequent analysis of the peptide fragments with antibodies specifically purified by immunoreactivity against this respective crossreactive antigen. These immunoblot experiments revealed a 22 kDa peptide fragment which was commonly recognized by all antibodies and which might represent a conserved domain of the enzyme.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

Carbonyl reduction of metyrapone in human liver.

Carbonyl reduction was investigated in cytosolic and microsomal fractions of human liver using the ketone metyrapone as a substrate. The cytosolic enzyme has a stronger preference for NADPH over NADH than the microsomal enzyme: the former shows only 14% of the NADPH-supported activity while the latter exhibits 36% activity with NADH. Barbitone and quercitrin, the classic inhibitors of carbonyl reductases, do not affect metyrapone reduction in either fraction. Dicumarol and indomethacin, the specific inhibitors of NAD(P)H: quinone-oxidoreductase and dihydrodiol dehydrogenase, respectively, only slightly decreased metyrapol formation. In contrast, 5 alpha-dihydrotestosterone, the active form of the androgen steroid testosterone, inhibited metyrapone reduction very strongly in the microsomal fractions and is postulated to be the physiological substrate of the enzyme. This resembles the situation in mouse liver [E. Maser and K. J. Netter, Biochem Pharmacol 38: 3049-3054, 1989] where microsomal metyrapone reductase was inhibited by steroids and the purified enzyme was demonstrated to mediate androsterone oxidation. Immunoblot analysis revealed antigenic cross-reaction of antibodies against the 34 kDa metyrapone reductase from mouse liver microsomes with the homologous protein in human liver microsomes pointing to structural homologies between the respective enzymes of the two species. These results--together with previous findings, which have shown that there exist functional as well as structural relationships between microsomal mouse liver metyrapone reductase and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni [E. Maser, U. Oppermann and K. J. Netter, Eur J Pharmacol 183:1366, 1990]--suggest that metyrapone reduction in human liver microsomes might be catalysed by a microsomal hydroxysteroid dehydrogenase.

Alcohol Oxidoreductases↗