Edmund Maser

The SDR (Short-Chain Dehydrogenase/Reductase and Related Enzymes) Nomenclature Initiative

Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with presently over 46,000 members. In phylogenetic comparisons, members of this superfamily show early divergence where the majority have only low pairwise sequence identity, although sharing common structural properties. The SDR enzymes are present in virtually all genomes investigated, and in humans over 70 SDR genes have been identified. In humans, these enzymes are involved in the metabolism of a large variety of compounds, including steroid hormones, prostaglandins, retinoids, lipids and xenobiotics. It is now clear that SDRs represent one of the oldest protein families and contribute to essential functions and interactions of all forms of life. As this field continues to grow rapidly, a systematic nomenclature is essential for future annotation and reference purposes. A functional subdivision of the SDR superfamily into at least 200 SDR families based upon hidden Markov models forms a suitable foundation for such a nomenclature system, which we present in this paper using human SDRs as examples.

Approaching clinical proteomics: current state and future fields of application in fluid proteomics

Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter‐ and intra‐assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.

Carbonyl Reductase 1 Is a Predominant Doxorubicin Reductase in the Human Liver

A first step in the enzymatic disposition of the antineoplastic drug doxorubicin (DOX) is the reduction to doxorubicinol (DOX-OL). Because DOX-OL is less antineoplastic but more cardiotoxic than the parent compound, the individual rate of this reaction may affect the antitumor effect and the risk of DOX-induced heart failure. Using purified enzymes and human tissues we determined enzymes generating DOX-OL and interindividual differences in their activities. Human tissues express at least two DOX-reducing enzymes. High-clearance organs (kidney, liver, and the gastrointestinal tract) express an enzyme with an apparent Km of ∼140 μM. Of six enzymes found to reduce DOX, Km values in this range are exhibited by carbonyl reductase 1 (CBR1) and aldo-keto reductase (AKR) 1C3. CBR1 is expressed in these three organs at higher levels than AKR1C3, whereas AKR1C3 has higher catalytic efficiency. However, inhibition constants for DOX reduction with 4-amino-1-tert-butyl-3-(2-hydroxyphenyl)pyrazolo[3,4-d]pyrimidine (an inhibitor that can discriminate between CBR1 and AKR1C3) were identical for CBR1 and human liver cytosol, but not for AKR1C3. These results suggest that CBR1 is a predominant hepatic DOX reductase. In cytosols from 80 human livers, the expression level of CBR1 and the activity of DOX reduction varied >70- and 22-fold, respectively, but showed no association with CBR1 gene variants found in these samples. Instead, the interindividual differences in CBR1 expression and activity may be mediated by environmental factors acting via recently identified xenobiotic response elements in the CBR1 promoter. The variability in the CBR1 expression may affect outcomes of therapies with DOX, as well as with other CBR1 substrates.

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.

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.

Cytotoxic potency of H2O2 in cell cultures: Impact of cell concentration and exposure time

Using C6 glioma cells in this study we investigated in detail how exposure time and cell concentration affect the cytotoxic potency of H(2)O(2) in vitro. Median cytotoxic concentrations (EC(50)) decreased from 500 to 30 μM with increasing incubation time from 1 to 24h. Twenty-four hours proved to be sufficient to determine incipient cytotoxic concentrations of H(2)O(2). The incipient EC(50) values were linearly related to the cell concentration. A cell concentration-independent median cytotoxic cell dose (ED(50)) of 430 nmol/mg cell protein or 860 nmol/10(7) cells was derived. Median cytotoxic H(2)O(2) concentrations were completely eliminated from the culture medium at a rate proportional to both the H(2)O(2) and the cell concentrations. In contrast to EC(50) values the corresponding areas under the concentration versus time curve (AUC) were independent of the cell concentration and amounted to 1800 μM×min. With decreasing cell concentration the H(2)O(2) elimination decelerates and, thus, exposure to H(2)O(2) applied as a bolus approaches a continuous exposure to a steady H(2)O(2) concentration. Taken together, our results indicate that the cytotoxic potency of H(2)O(2) administered to cultured cells as a bolus is characterized by the AUC, which depends on its initial concentration, the ability of the cells to eliminate H(2)O(2), and the cell concentration. We recommend expressing the toxic potency of H(2)O(2) in vitro by the incipient toxic cell dose (e.g., nmol H(2)O(2)/mg cell protein or nmol H(2)O(2)/10(7) cells), in particular for comparative purposes.

Purification and characterization of AKR1B10 from human liver: role in carbonyl reduction of xenobiotics

Members of the aldo-keto reductase (AKR) superfamily have a broad substrate specificity in catalyzing the reduction of carbonyl group-containing xenobiotics. In the present investigation, a member of the aldose reductase subfamily, AKR1B10, was purified from human liver cytosol. This is the first time AKR1B10 has been purified in its native form. AKR1B10 showed a molecular mass of 35 kDa upon gel filtration and SDS-polyacrylamide gel electrophoresis. Kinetic parameters for the NADPH-dependent reduction of the antiemetic 5-HT3 receptor antagonist dolasetron, the antitumor drugs daunorubicin and oracin, and the carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) to the corresponding alcohols have been determined by HPLC. Km values ranged between 0.06 mM for dolasetron and 1.1 mM for daunorubicin. Enzymatic efficiencies calculated as kcat/Km were more than 100 mM–1 min–1 for dolasetron and 1.3, 0.43, and 0.47 mM–1 min–1 for daunorubicin, oracin, and NNK, respectively. Thus, AKR1B10 is one of the most significant reductases in the activation of dolasetron. In addition to its reducing activity, AKR1B10 catalyzed the NADP+-dependent oxidation of the secondary alcohol (S)-1-indanol to 1-indanone with high enzymatic efficiency (kcat/Km = 112 mM–1 min–1). The gene encoding AKR1B10 was cloned from a human liver cDNA library and the recombinant enzyme was purified. Kinetic studies revealed lower activity of the recombinant compared with the native form. Immunoblot studies indicated large interindividual variations in the expression of AKR1B10 in human liver. Since carbonyl reduction of xenobiotics often leads to their inactivation, AKR1B10 may play a role in the occurrence of chemoresistance of tumors toward carbonyl group-bearing cytostatic drugs.

11β-hydroxysteroid dehydrogenase type 1 is an important regulator at the interface of obesity and inflammation.

Systemic glucocorticoid excess, as exemplified by the Cushing syndrome, leads to obesity and all further symptoms of the metabolic syndrome. The current obesity epidemic, however, is not characterized by increased plasma cortisol concentrations, but instead comes along with chronic low-grade inflammation in adipose tissue and concomitant increased levels of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1, gene HSD11B1), a parameter known to cause obesity in a mouse model. 11beta-HSD1 represents an intracellular amplifier of active glucocorticoid, thus enhances the associated effects on the inflammatory response as well as on nutrient and energy metabolism, and may therefore cause and exacerbate obesity by local increase of glucocorticoid concentrations. Obtained by extensive literature and database searching, the present review includes comprehensive lists of primary glucocorticoid-sensitive genes and gene products as well as of the thus far known regulators of HSD11B1 expression with implication in inflammation and metabolic disease. Collectively, the data clearly show that, in addition to amplifying active glucocorticoid and thus profoundly modulating inflammation and nutrient metabolism, 11beta-HSD1 is subject to tight control of multiple additional immunomodulatory and metabolic regulators. Hence, 11beta-HSD1 acts at the interface of inflammation and obesity and represents an efficient integrator and effector of local inflammatory and metabolic state.

11β-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.

Molecular Cloning, Overexpression, and Characterization of Steroid-inducible 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase from Comamonas testosteroni A NOVEL MEMBER OF THE SHORT-CHAIN DEHYDROGENASE/REDUCTASE SUPERFAMILY

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonas testosteroni, a bacterium that is able to grow on steroids as the sole carbon source, catalyzes the oxidoreduction at position 3 of a variety of C19–27 steroids and the carbonyl reduction of a variety of nonsteroidal aldehydes and ketones. The gene of this steroid-inducible 3α-HSD/CR was cloned by screening a C. testosteroni gene bank with a homologous DNA probe that was obtained by polymerase chain reaction with two degenerative primers based on the N-terminal sequence of the purified enzyme. The 3α-HSD/CR gene 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 that amino acid sequences highly conserved in the short-chain dehydrogenase/reductase (SDR) superfamily are present in 3α-HSD/CR. Two consensus sequences of the SDR superfamily were found, an N-terminal Gly-X-X-X-Gly-X-Gly cofactor-binding motif and a Tyr-X-X-X-Lys segment (residues 155–159 in the 3α-HSD/CR sequence) essential for catalytic activity of SDR proteins. 3α-HSD/CR was overexpressed and purified to homogeneity, and its activity was determined for steroid and nonsteroidal carbonyl substrates. These results suggest that inducible 3α-HSD/CR from C. testosteroni is a novel member of the SDR superfamily.