Biomedical subjects

E Maser

Publications and source records attributed to E Maser.

At least 19 recordsLinked to original sources

PubMed2005

11Beta-hydroxysteroid dehydrogenase type 1: purification from human liver and characterization as carbonyl reductase of xenobiotics.

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion of 11-oxo glucocorticoids to their 11-hydroxy metabolites, thereby controlling access of glucocorticoid hormones to the glucocorticoid receptor. Interestingly, evidence is emerging that 11beta-HSD1 fulfills an additional role in the metabolism of xenobiotic carbonyl compounds. In our studies, 11beta-HSD1 was identified as a microsomal reductase that initiates the final detoxification of xenobiotics by reducing them to alcohols that are easier to conjugate and eliminate. With its pluripotent substrate specificities for glucocorticoids and xenobiotics, 11beta-HSD1 adds to an expanding list of those hydroxysteroid dehydrogenases which, on the one hand, are capable of catalyzing the carbonyl reduction of non-steroidal carbonyl compounds, and which, on the other hand, exhibit great specificity to their physiological steroid substrates. It is conceivable that large interferences must occur between endogenous steroid metabolism and the detoxification of xenobiotic compounds on the level of hydroxysteroid dehydrogenases.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗

PubMed2001

Two-dimensional gel electrophoresis: a powerful method to elucidate cellular responses to toxic compounds.

Humans are exposed to a variety of environmental toxicants and combinations thereof, and a large number of interacting factors contribute to an individual's risk for disease. Therefore, new strategies in toxicological research are needed for efficient screening of environmental hazards on complex living systems. The rapidly expanding field of proteomics relies heavily upon the use of two-dimensional gel electrophoresis (2-DE) of protein samples. 2-DE is a key separation technique in proteome analysis due to its advantage of simultaneous separation of thousands of proteins at a time, excellent reproducibility, and ability to exhibit post-translational modifications. Therefore, 2-D proteome analysis is becoming a popular method of choice to detect differentially expressed proteins between proteome profiles after exposure to toxicants. The goal of this study was to examine the response of pancreas carcinoma cells to increasing concentrations of the cytotoxic agent daunorubicin (DRC). The proteomic investigation revealed a number of proteins that were up-regulated by DRC treatment, some in a dose-dependent manner. However, these changes were not seen by reverse transcriptase-polymerase chain reaction. The determination of proteome changes following exposure to xenobiotics will aid our understanding of the mechanisms of their toxicity as well as providing the possibility for the establishment of biomarkers that can be used in risk assessment as well as for the identification of individual susceptibility factors.

Adenocarcinoma↗

PubMed2001

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni: biological significance, three-dimensional structure and gene regulation.

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) catalyses the oxidoreduction at carbon 3 of steroid hormones and is postulated to initiate the complete mineralisation of the steroid nucleus to CO(2) and H(2)O in Comamonas testosteroni. The enzyme was found to be functional towards a variety of steroid substrates, including the steroid antibiotic fusidic acid. The enzyme also catalyses the carbonyl reduction of non-steroidal aldehydes and ketones such as a novel insecticide. It is suggested that 3alpha-HSD/CR contributes to important defense strategies of C. testosteroni against natural and synthetic toxicants. The 3alpha-HSD/CR gene (hsdA) is 774 base pairs long and the deduced amino acid sequence comprises 258 residues with a calculated molecular mass of 26.4 kDa. A homology search revealed 3alpha-HSD/CR as a new member of the short-chain dehydrogenase/reductase (SDR) superfamily. Upon gel permeation chromatography the purified enzyme elutes as a 49.4 kDa protein indicating a dimeric nature of 3alpha-HSD/CR. The protein was crystallised and the structure solved by X-ray analysis. The crystal structure reveals one homodimer per asymmetric unit, thereby verifying its dimeric nature. Dimerisation takes place via an interface essentially built-up by helix alphaG and strand betaG of each subunit. So far, this type of intermolecular contact has exclusively been observed in homotetrameric SDRs, but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3alpha-HSD/CR by the presence of a predominantly alpha-helical subdomain, which is missing in all other SDRs of known structure. The promoter domain was localised within the 93 bp region upstream of hsdA and the transcriptional start site was identified at 28 bp upstream of the translation start site. Interestingly, hsdA expression was found to be under negative control by two repressor proteins, the genes of which were found in opposite direction downstream or overlapping with hsdA. Based on our results, we propose that induction of hsdA expression in C. testosteroni by steroids actually appears to be a de-repression by preventing the binding of repressor proteins to regulatory regions.

3-Hydroxysteroid Dehydrogenases↗

PubMed2001

A model on the regulation of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase expression in Comamonas testosteroni.

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds. The enzyme has recently been cloned and characterized by our group. A strong induction of enzyme activity is observed in the presence of steroids like testosterone. In the present investigation, two repressor proteins (Rep1 and Rep2) containing 78 and 420 amino acids, respectively, were found to regulate 3alpha-HSD/CR gene (hsdA) expression. Gel shift experiments showed that Rep2 binds to a 10 nucleotide sequence 9 bp upstream of the hsdA promoter. The deletion of this cis-regulating sequence significantly increases hsdA expression. About 1633 bp further upstream, a second ten nucleotide sequence, complementary to the first one, was found, which is also recognized by Rep2 and increases hsdA expression, if deleted. To purify the repressor proteins, the genes encoding each were cloned into His-tag expression vectors and overexpressed in Escherichia coli. Rep1 does not bind to DNA but may bind to 3alpha-HSD/CR mRNA as predicted by its secondary structure. Concluding from our data, induction of 3alpha-HSD/CR in C. testosteroni by steroids in fact appears to be a de-repression, where the steroidal 'inducer' prevents the binding of the two repressor proteins to the hsdA promoter and mRNA, respectively.

3-Hydroxysteroid Dehydrogenases↗

PubMed2001

Characterization of enzymes participating in carbonyl reduction of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human placenta.

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) has been identified as one of the strongest nitrosamine carcinogens in tobacco products in all species tested. Carbonyl reduction to 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronosylation is considered to be the main detoxification pathway in humans. In previous investigations, we have identified a microsomal NNK carbonyl reductase as being identical to 11beta-hydroxysteroid dehydrogenase 1, a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Recently, we provided evidence that carbonyl reduction of NNK does also take place in cytosol from mouse and human liver and lung. In human liver cytosol, carbonyl reductase, a SDR enzyme, and AKR1C1, AKR1C2 and AKR1C4 from the aldo-keto reductase (AKR) superfamily were demonstrated to be responsible for NNK reduction. Since NNK and/or its metabolites can diffuse through the placenta and reach fetal tissues, we now investigated NNK carbonyl reduction in the cytosolic fraction of human placenta in addition to that in microsomes. Concluding from the sensitivity to menadione, ethacrynic acid, rutin and quercitrin as specific inhibitors, mainly carbonyl reductase (EC 1.1.1.184) seems to perform this reaction in human placenta cytosol. The presence of carbonyl reductase was confirmed by RT-PCR. This is the first report to provide evidence that NNAL formation in placenta is mediated by carbonyl reductase.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗

PubMed2001

Human 11beta-hydroxysteroid dehydrogenase 1/carbonyl reductase: additional domains for membrane attachment?

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a membrane integrated glycoprotein, which physiologically performs the interconversion of active and inactive glucocorticoid hormones and which also participates in xenobiotic carbonyl compound detoxification. Since 11beta-HSD 1 is fixed to the endoplasmic reticulum (ER) with a N-terminal membrane spanning domain, the enzyme is very difficult to purify in an active state. Upon expression experiments in Escherichia coli, 11beta-HSD 1 turns out to be hardly soluble without detergents. This study describes attempts to increase the solubility of 11beta-HSD 1 via mutagenesis experiments by generating several truncated forms expressed in E. coli and the yeast Pichia pastoris. Furthermore, we investigated if the codon for methionine 31 in human 11beta-HSD 1 could serve as an alternative start codon, thereby leading to a soluble form of the enzyme, which lacks the membrane spanning segment. Our results show that deletion of the hydrophobic membrane spanning domain did not alter the solubility of the enzyme. In contrast, the enzyme remained bound to the ER membrane even without the N-terminal membrane anchor. However, activity could not be found, neither with the truncated protein expressed in E. coli nor with that expressed in P. pastoris. Hydrophobicity plots proved the hydrophobic nature of 11beta-HSD 1 and indicated the existence of additional membrane attachment sites within its primary structure.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMed2001

Expression and NNK reducing activities of carbonyl reductase and 11beta-hydroxysteroid dehydrogenase type 1 in human lung.

The tobacco specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), which is found in high amounts in tobacco products, is believed to play an important role in lung cancer induction in smokers. NNK requires metabolic activation by cytochrome P450 mediated alpha-hydroxylation to exhibit its carcinogenic properties. On the other hand, NNK is inactivated by carbonyl reduction to its alcohol-equivalent 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronidation and final excretion into urine or bile. Carbonyl reduction and alpha-hydroxylation are the predominant pathways in man, and it has been postulated that the extent of these competing pathways determines the individual susceptibility to lung cancer. Moreover, only a minor part of all habitual smokers develop lung cancer, suggesting the existence of susceptibility genes. Microsomal 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) (EC 1.1.1.146) and cytosolic carbonyl reductase (CR) (EC 1.1.1.184) have been shown to be mainly responsible for NNAL formation in liver and lung. In the present study, we performed comparative investigations of human lung tissue samples from several patients with respect to the expression and activity of 11beta-HSD 1 and carbonyl reductase. We observed varying levels in 11beta-HSD 1 and carbonyl reductase expression in these patients, as revealed by RT-PCR and ELISA. Also, the tissue samples showed a different activity and inhibitor profile for both enzymes. According to our results, variations in the expression and activity of NNK carbonyl reducing enzymes may constitute a major determinant in the overall NNK detoxification capacity and thus may be linked to the great differences observed in the individual susceptibility of tobacco-smoke related lung cancer.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗

PubMed2001

Regulation of the steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comamonas testosteroni.

The Comamonas testosteroni 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase gene (hsdA) codes for an adaptive enzyme in the degradation of steroid compounds. However, no information was available on the molecular regulation of steroid-inducible genes nor on the mechanism of steroid signaling in procaryotes. We, therefore, investigated the cis- and trans-acting elements of hsdA expression to infer the mechanism of its molecular regulation by steroids. The gene was localized on a 5.257-kilobase EcoRI fragment of C. testosteroni chromosomal DNA. The promoter was characterized, and the transcriptional start site was identified. Two palindromic operator domains were found upstream of hsdA. A new gene coding for a trans-acting negative regulator (repressor A, RepA) of hsdA expression was characterized. The specific interaction between RepA, testosterone, and the operator domain is demonstrated. From our results we conclude that hsdA is under negative transcriptional control by an adjacent gene product (RepA). Accordingly, induction of hsdA by steroids in fact is a derepression, where steroidal inducers bind to the repressor, thereby preventing its binding to the hsdA operator.

Alcohol Oxidoreductases↗

PubMed2000

The crystal structure of 3alpha -hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase/reductase family.

The crystal structure of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (3alpha-HSDH) as well as the structure of its binary complex with NAD(+) have been solved at 1.68-A and 1.95-A resolution, respectively. The enzyme is a member of the short chain dehydrogenase/reductase (SDR) family. Accordingly, the active center and the conformation of the bound nucleotide cofactor closely resemble those of other SDRs. The crystal structure reveals one homodimer per asymmetric unit representing the physiologically active unity. Dimerization takes place via an interface essentially built-up by helix alphaG and strand betaG of each subunit. So far this type of intermolecular contact has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3alpha-HSDH by the presence of a predominantly alpha-helical subdomain which is missing in all other SDRs of known structure.

3-Hydroxysteroid Dehydrogenases↗

PubMed2000

Human 11beta-hydroxysteroid dehydrogenase type 1 is enzymatically active in its nonglycosylated form.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a microsomal enzyme responsible for the reversible interconversion of active 11beta-hydroxyglucocorticoids into inactive 11-ketosteroids and by this mechanism regulates access of glucocorticoids to the glucocorticoid receptor. The enzyme has also been proven to participate in xenobiotic carbonyl compound detoxification. 11beta-HSD 1 is anchored within the membranes of the endoplasmic reticulum (ER) by its N-terminus, whereby its active site protrudes into the lumen of the ER. In the primary structure of 11beta-HSD 1 three Asn-X-Ser glycosylation motifs have been identified. However, the importance of N-linked glycosylation of 11beta-HSD 1 for catalytic activity has been controversely discussed. To clarify if glycosylation is essential for enzyme activity, we performed deglycosylation experiments of native 11beta-HSD 1 from human liver as well as site-directed mutagenesis to remove potential glycosylation sites upon overexpression in Pichia pastoris. The altered proteins were examined regarding their catalytic activity towards their physiological glucocorticoid substrates. The molecular size of the various 11beta-HSD 1 forms was analyzed by immunoblotting with a polyclonal antibody raised against 11beta-HSD 1 protein from human liver. By stepwise enzymatic deglycosylation of native 11beta-HSD 1 we could demonstrate that all potential glycosylation sites carry N-linked oligosaccharide residues under physiological conditions. Interestingly, complete deglycosylation did not affect enzyme activity, neither in the reductive (cortisone) nor in the oxidative (cortisol) direction. Upon overexpression in the yeast P. pastoris, 11beta-HSD 1 did not undergo glycosylation, but, in spite of this, yielded a fully active enzyme. Our results conclusively demonstrate that 11beta-HSD 1 does not need to be glycosylated to perform its physiological role as glucocorticoid oxidoreductase.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗

PubMed2000

Functional expression, purification, and characterization of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.

3alpha-Hydroxysteroid dehydrogenase (3alpha-HSD) catalyzes the oxidoreduction at carbon 3 of steroid hormones and is postulated to initiate the complete mineralization of the steroid nucleus to CO(2) and H(2)O in Comamonas testosteroni. By this activity, 3alpha-HSD provides the basis for C. testosteroni to grow on steroids as sole carbon and energy source. 3alpha-HSD was cloned and overexpressed in E. coli and purified to homogeneity by an affinity chromatography system as His-tagged protein. The recombinant enzyme was found to be functional as oxidoreductase toward a variety of steroid substrates, including androstanedione, 5alpha-dihydrotestosterone, androsterone, cholic acid, and the steroid antibiotic fusidic acid. The enzyme also catalyzes the carbonyl reduction of nonsteroidal aldehydes and ketones such as metyrapone, p-nitrobenzaldehyde and a novel insecticide (NKI 42255), and, based on this pluripotent substrate specificity, was named 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR). It is suggested that 3alpha-HSD/CR contributes to important defense strategies of C. testosteroni against natural and synthetic toxicants. Antibodies were generated in rabbits against the entire 3alpha-HSD/CR protein, and may now be used for evaluating the pattern of steroid induction in C. testosteroni on the protein level. Upon gel permeation chromatography the purified enzyme elutes as a 49.4 kDa protein revealing for the first time the dimeric nature of 3alpha-HSD/CR of C. testosteroni.

3-Hydroxysteroid Dehydrogenases↗

PubMed2000

Heterogeneity of 11beta-hydroxysteroid dehydrogenase type 1/microsomal carbonyl reductase (11beta-HSD/CR) in guinea pig tissues. Purification of the liver form suggests modification in the cosubstrate binding site.

11beta-hydroxysteroid dehydrogenase (11beta-HSD) and xenobiotic carbonyl reductase activities were determined in guinea pig tissue microsomes. The data indicate the presence of a NADP(H) dependent form, distinct from the known type I isozyme. Purification of 11beta-HSD-1 from liver microsomes resulted in two distinct peaks, resolved by dye-ligand chromatography, indicating differences in the cosubstrate binding site. Immunoblot analysis using anti 11beta-HSD-1 antibodies reveals the presence of similar structural determinants between the enzyme forms. Both have an apparent molecular mass of 32 kDa, suggesting protein modifications occurring in the type 1 isozyme which account for the differences in chromatographic behaviour.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗

PubMed2000

Molecular and structural aspects of xenobiotic carbonyl metabolizing enzymes. Role of reductases and dehydrogenases in xenobiotic phase I reactions.

The major metabolic pathways involved in synthesis and disposition of carbonyl and hydroxyl group containing compounds are presented, and structural and functional characteristics of the enzyme families involved are discussed. Alcohol and aldehyde dehydrogenases (ADH, ALDH) participate in oxidative pathways, whereas reductive routes are accomplished by members of the aldo-keto reductase (AKR), short-chain dehydrogenases/reductases (SDR) and quinone reductase (QR) superfamilies. A wealth of biochemical, genetic and structural data now establishes these families to constitute important phase I enzymes.

Animals↗

PubMed2000

Human 11beta-hydroxysteroid dehydrogenase 1/carbonyl reductase: recombinant expression in the yeast Pichia pastoris and Escherichia coli.

Detoxification of aldehydes and ketones generally proceeds via reduction to their corresponding alcohols, which are then conjugated and eliminated. We focused our interest on 11beta-hydroxysteroid-dehydrogenase type 1 (11beta-HSD 1), a pluripotent enzyme which physiologically performs the interconversion of active and inactive glucocorticoid hormones, and which also participates in xenobiotic carbonyl compound detoxification. 11beta-HSD 1 belongs to the protein superfamily of the short-chain dehydrogenases/reductases (SDR), and has been structurally and functionally characterized. 11beta-HSD 1 is a glycosylated membrane protein which is very difficult to purify in an active state. In addition, expression levels in humans differ in a wide range. In order to facilitate biochemical and molecular studies on the significance of human 11beta-HSD 1 in detoxification processes, we have successfully performed the overexpression of recombinant human 11beta-HSD 1 in the yeast Pichia pastoris and in Escherichia coli. Recombinant 11beta-HSD 1 from E. coli was purified to homogeneity and used to generate a polyclonal antibody. The enzyme had no enzymatic activity, possibly due to the lack of glycosylation and/or incorrect folding in E. coli. In contrast, 11beta-HSD 1 overexpressed in P. pastoris was enzymatically active towards its physiological glucocorticoid substrates as well as towards xenobiotic carbonyl compounds. In western blot experiments the antibody crossreacted with both recombinant 11beta-HSD 1 forms and with the native enzyme from mouse and human liver. In conclusion, recombinant 11beta-HSD 1 from P. pastoris serves as a valuable tool for future studies on carbonyl compound detoxification.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗

PubMed2000

Modulation of daunorubicin toxicity by liposomal encapsulation and use of specific inhibitors in vitro.

Anthracyclines serve as a valuable tool in chemotherapy, but their usefulness is often limited by the occurrence of resistance mechanisms in tumor cells. Resistance of tumor cells is a multifactorial event, where several mechanisms act concurrently, including drug efflux and enzymatic drug inactivation. Liposomal encapsulation of anthracyclines has been discussed as a successful regimen to overcome drug resistance. Our investigations were carried out on a daunorubicin (DRC) sensitive breast cancer cell line and two DRC resistant sublines generated thereof. In all three cell lines, the extent of DRC detoxification via carbonyl reduction to daunorubicinol (DRCOL) was determined. In addition, rutin, the most effective inhibitor of carbonyl reducing enzymes, was tested to affect DRCOL formation. DRC IC(50) values were determined in relation to several combinations of DRC administration, (a) liposomal encapsulated DRC, (b) addition of verapamil (inhibitor of drug efflux), (c) addition of rutin (inhibitor of DRC carbonyl reduction). We could show that DRC sensitive and resistant breast cancer cell lines are able to catalyze DRC detoxification via carbonyl reduction to DRCOL. Rutin was shown to inhibit this reaction, but could not serve as an enhancer of DRC toxicity in MTT tests. Verapamil was effective only in resistant cells due to the overexpression of P-glycoprotein 170. Liposomal encapsulation of DRC did not show the expected increase in DRC toxicity in the present tumor cell model.

Antibiotics, Antineoplastic↗

PubMed2000

Development of daunorubicin resistance in tumour cells by induction of carbonyl reduction.

A resistant descendant of the human stomach carcinoma cell line EPG85-257 was selected in the presence of increasing concentrations of daunorubicin (DRC). To avoid the expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP), cells were cultured in the presence of verapamil. The resulting cells were used to evaluate an induced carbonyl reduction as a new determinant in DRC resistance. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide) toxicity assay was performed to estimate sensitivity to DRC in both cell lines. IC50 values of DRC increased almost 8-fold in the resistant descendants compared to the parental cell line. P-gp transcripts were detectable in both cell lines at only very low levels, and no significant alterations between sensitive and resistant cells were observed. MRP mRNA expression was markedly higher compared to P-gp mRNA, but, as was the case with P-gp, MRP mRNA levels in sensitive and resistant cells showed no alteration. This was probably due to the effect of the presence of verapamil during cell selection. Another known drug resistance factor, the lung resistance-related protein (LRP), was not at all detectable. Interestingly, resistant cells possessed 6-fold higher levels of DRC carbonyl-reducing activity, leading to the less toxic 13-hydroxy metabolite daunorubicinol (DRCOL). The 6-fold higher DRCOL formation roughly parallels the 8-fold increase in DRC IC50 values during cell selection, and therefore may account for DRC resistance in these cells. The determination of specific carbonyl reducing enzymes, known to be involved in DRC detoxification, revealed that mRNA expression of carbonyl reductase (EC 1.1.1.184), aldose reductase (EC 1.1.1.21), and dihydrodiol dehydrogenase 2 (EC 1.3.1.20) increased in the resistant descendant. In contrast, the phase II-conjugating enzyme activities of glutathione S-transferases were significantly lower in resistant than in sensitive cells, whereas those of glucuronosyl transferase were not detectable in either cell line. Apparently, conjugating enzymes are not involved in DRC resistance in human stomach carcinoma cells. These studies indicate that DRC resistance in human stomach carcinoma cells may appear as a result of an induction of metabolic DRC inactivation via carbonyl reduction to the less active 13-hydroxy metabolite DRCOL.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Carbonyl reduction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by cytosolic enzymes in human liver and lung.

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen, independent of the route and type of administration. There are competing metabolic activation and detoxification pathways. NNK is activated by alpha-hydroxylation at either the methyl or methylene carbonyl adjacent to the N-nitroso group to yield intermediates that methylate and pyridyloxobutylate DNA. Detoxification of NNK in humans usually occurs via carbonyl reduction to its hydroxy product NNAL, which undergoes glucuronosylation and final excretion. In vitro studies on NNK metabolism have usually been performed with tissue homogenates, microsomal fractions and/or purified microsomal enzymes, but cytosolic metabolism of NNK has been ignored until today. The results of this study demonstrate that cytosolic fractions of human liver and lung also participate in NNK metabolism. We provide evidence that a substantial degree of NNK carbonyl reduction occurs by cytosolic enzymes and that these enzymes may contribute to NNK detoxification in human liver and lung. The relative contribution of cytosolic vs. microsomal NNK carbonyl reduction is nearly identical in liver, whereas it is more than 3-fold higher in lung microsomes compared to lung cytosol. The inhibition profile suggested that mainly carbonyl reductase (EC 1.1.1.184) was active in cytosol of both organs. The expression of carbonyl reductase mRNA in liver and lung was proven by reverse transcription-(RT)-PCR. In conclusion, the results of this study provide the first data on cytosolic enzymes participating in NNK detoxification in human liver and lung.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗

PubMed2000

Purification and characterization of oxidoreductases-catalyzing carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human liver cytosol.

1. Four enzymes were purified to homogeneity from human liver cytosol and were demonstrated to be responsible for carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). 2. Carbonyl reductase (EC 1.1.1.184), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, was compared with three isoenzymes of the aldo-keto reductase (AKR) superfamily in terms of enzyme kinetics, co-substrate dependence and inhibition pattern. 3. AKR1C1, 1C2 and 1C4, previously designated as dihydrodiol dehydrogenases (DD1, DD2 and DD4), showed lower K(m) (0.2, 0.3 and 0.8 mM respectively) than did carbonyl reductase (7 mM), whereas carbonyl reductase exhibited the highest enzyme efficiency (Vmax/K(m)) for NNK. Multiplication of enzyme efficiencies with the relative quantities of individual enzymes in cytosol resulted in a rough estimate of their contributions to total alcohol metabolite formation. These were approximately 60% for carbonyl reductase, 20% each for AKR1C1 and 1C2, and 1% for AKR1C4. 4. Except for AKR1C4, the enzymes had a strong preference for NADPH over NADH, and the highest activities were measured with an NADPH-regenerating system. Carbonyl reductase activity was extensively inhibited by menadione, rutin and quercitrin, whereas medroxyprogesterone acetate, phenolphthalein and flufenamic acid were potent inhibitors of AKR1C1, 1C2 and 1C4. 5. In conclusion, cytosolic members of the SDR and AKR superfamilies contribute to reductive NNK detoxification in human liver, the enzymes responsible being carbonyl reductase and aldoketo reductases of the AKRIC subfamily.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗