Computational analysis of human medium-chain dehydrogenases/reductases revealing substrate- and coenzyme-binding characteristics.

The medium-chain dehydrogenase/reductase (MDR) superfamily has more than 600,000 members in UniProt as of March 2023. As the family has been growing, the proportion of functionally characterized proteins has been falling behind. The aim of this project was to investigate the binding pockets of nine different MDR protein families based on sequence conservation patterns and three-dimensional structures of members within the respective families. A search and analysis methodology was developed. Using this, a total of 2000 eukaryotic MDR sequences belonging to nine different families were identified. The pairwise sequence identities within each of the families were 80-90 % for the mammalian sequences, like the levels observed for alcohol dehydrogenase, another MDR family. Twenty conserved residues were identified in the coenzyme part of the binding site by matching structural and conservation data of all nine protein families. The conserved residues in the substrate part of the binding pocket varied between the nine MDR families, implying divergent functions for the different families. Studying each family separately made it possible to identify multiple conserved residues that are expected to be important for substrate binding or catalysis of the enzymatic reaction. By combining structural data with the conservation of the amino acid residues in each protein, important residues in the binding pocket were identified for each of the nine MDRs. The obtained results add new positions of interest for the binding and activity of the enzyme family as well as fit well to earlier published data. Three distinct types of binding pockets were identified, containing no, one, or two tyrosine residues.

Computational studies of human class V alcohol dehydrogenase - the odd sibling.

BACKGROUND: All known attempts to isolate and characterize mammalian class V alcohol dehydrogenase (class V ADH), a member of the large ADH protein family, at the protein level have failed. This indicates that the class V ADH protein is not stable in a non-cellular environment, which is in contrast to all other human ADH enzymes. In this report we present evidence, supported with results from computational analyses performed in combination with earlier in vitro studies, why this ADH behaves in an atypical way. RESULTS: Using a combination of structural calculations and sequence analyses, we were able to identify local structural differences between human class V ADH and other human ADHs, including an elongated ß-strands and a labile α-helix at the subunit interface region of each chain that probably disturb it. Several amino acid residues are strictly conserved in class I-IV, but altered in class V ADH. This includes a for class V ADH unique and conserved Lys51, a position directly involved in the catalytic mechanism in other ADHs, and nine other class V ADH-specific residues. CONCLUSIONS: In this study we show that there are pronounced structural changes in class V ADH as compared to other ADH enzymes. Furthermore, there is an evolutionary pressure among the mammalian class V ADHs, which for most proteins indicate that they fulfill a physiological function. We assume that class V ADH is expressed, but unable to form active dimers in a non-cellular environment, and is an atypical mammalian ADH. This is compatible with previous experimental characterization and present structural modelling. It can be considered the odd sibling of the ADH protein family and so far seems to be a pseudoenzyme with another hitherto unknown physiological function.

The mammalian alcohol dehydrogenase genome shows several gene duplications and gene losses resulting in a large set of different enzymes including pseudoenzymes.

Mammalian alcohol dehydrogenase (ADH) is a protein family divided into six classes and the number of known family members is increasing rapidly. Several primate genomes are completely analyzed for the ADH region, where higher primates (human and hominoids) have seven genes of classes ADH1-ADH5. Within the group of non-hominoids apes there have been further duplications and species with more than the typical three isozymic forms for ADH1 are present. In contrast there are few completely analyzed ADH genomes in the non-primate group of mammals, where an additional class has been identified, ADH6, that has been lost during the evolution of primates. In this study 85 mammalian genomes with at least one ADH gene have been compiled. In total more than 500 ADH amino acid sequences were analyzed for patterns that distinguish the different classes. For ADH1-ADH4 intensive investigations have been performed both at the functional and at structural levels. However, a corresponding functional protein to the ADH5 gene, which is found in most ADH genomes, has never been detected. The same is true for ADH6, which is only present in non-primates. The entire mammalian ADH family shows a broad spectrum of gene duplications and gene losses where the numbers differ from six genes (most non-primate mammals) up to ten genes (vole). Included in these sets are examples of pseudogenes and pseudoenzymes.

Analysis of mammalian alcohol dehydrogenase 5 (ADH5): characterisation of rat ADH5 with comparisons to the corresponding human variant.

Alcohol dehydrogenase 5 (ADH5) is a member of the mammalian alcohol dehydrogenase family of yet undefined functions. ADH5 was first identified at the DNA level in human and deer mouse. A rat alcohol dehydrogenase structure of similar type has been isolated at the cDNA level using human ADH5 as a screening probe, where the rat cDNA structure displayed several atypical properties. mRNA for rat ADH5 was found in multiple tissues, especially in the kidney. In vitro translation experiments indicated that rat ADH5 is expressed as efficiently as ADH1 and furthermore, rat ADH5 was readily expressed in COS cells fused to Green Fluorescent Protein. However, no soluble ADH5 protein could be heterologously expressed in Escherichia coli cells with expression systems successfully used for other mammalian ADHs, including fused to glutathione-S-transferase. Molecular modelling of the enzyme indicated that the protein does not fold in a productive way, which can be the explanation why no stable and active ADH5 has been isolated. These results indicate that ADH5, while readily expressed at the mRNA level, does not behave similarly to other mammalian ADHs investigated. The results, in vitro and in silico, suggest an unstable ADH5 structure, which can explain for why no active and stable protein can be isolated. Further possibilities are conceivable: the ADH5 protein may have to interact with a stabiliser, or the gene is actually a pseudogene.

Mammalian alcohol dehydrogenases--a comparative investigation at gene and protein levels.

Mammalian alcohol dehydrogenase (ADH) can be divided into six classes, ADH1-ADH6, according to primary structure and function, where the classes are further subdivided into isozymes and allelic forms. With the increasing amount of available genomic data a general pattern is possible to trace within the mammalian ADH gene and protein families. The transcriptional order for the ADH genes in all mammalian genomes is the same (ADH4-ADH1-ADH6-ADH5-ADH2-ADH3), but the cluster is found on different chromosomes in different species. However, in primates only ADH1-ADH5 are present, where the loss of ADH6 may have occurred simultaneously as the split into ADH1 isoforms. ADH3, also denoted glutathione-dependent formaldehyde dehydrogenase and S-nitrosoglutathione reductase, is identified as the last gene in the ADH transcriptional order, but several pseudogenes for ADH3 have been traced at other chromosomes. The flanking genes outside the ADH genome are similar or identical for all species showing that a larger DNA region has been duplicated and further evolved. However, the only entirely completed ADH genomes are those from primates and rodents. The latest identified ADH forms, ADH5 (class V) and ADH6 (class VI), are truly different classes and both are very diverged in contrast to ADH3, which is the most conserved class of all ADHs. ADH5 and ADH6 have been identified at the gene and transcriptional levels only, and their functions are still an enigma.

Enrichment of ligands with molecular dockings and subsequent characterization for human alcohol dehydrogenase 3.

Alcohol dehydrogenase 3 (ADH3) has been assigned a role in nitric oxide homeostasis due to its function as an S-nitrosoglutathione reductase. As altered S-nitrosoglutathione levels are often associated with disease, compounds that modulate ADH3 activity might be of therapeutic interest. We performed a virtual screening with molecular dockings of more than 40,000 compounds into the active site of human ADH3. A novel knowledge-based scoring method was used to rank compounds, and several compounds that were not known to interact with ADH3 were tested in vitro. Two of these showed substrate activity (9-decen-1-ol and dodecyltetraglycol), where calculated binding scoring energies correlated well with the logarithm of the k (cat)/K (m) values for the substrates. Two compounds showed inhibition capacity (deoxycholic acid and doxorubicin), and with these data three different lines for specific inhibitors for ADH3 are suggested: fatty acids, glutathione analogs, and cholic acids.

Medium-chain fatty acids and glutathione derivatives as inhibitors of S-nitrosoglutathione reduction mediated by alcohol dehydrogenase 3.

Alcohol dehydrogenase 3 (ADH3) has emerged as an important regulator of protein S-nitrosation in its function as S-nitrosoglutathione (GSNO) reductase. GSNO depletion is associated with various disease conditions, emphasizing the potential value of a specific ADH3 inhibitor. The present study investigated inhibition of ADH3-mediated GSNO reduction by various substrate analogues, including medium-chain fatty acids and glutathione derivatives. The observed inhibition type was non-competitive. Similar to the Michaelis constants for the corresponding omega-hydroxy fatty acids, the inhibition constants for fatty acids were in the micromolar range and showed a clear dependency on chain length with optimal inhibitory capacity for eleven and twelve carbons. The most efficient inhibitors found were undecanoic acid, dodecanoic acid and dodecanedioic acid, with no significant difference in inhibition constant. All glutathione-derived inhibitors displayed inhibition constants in the millimolar range, at least three orders of magnitudes higher than the Michaelis constants of the high-affinity substrates GSNO and S-hydroxymethylglutathione. The experimental results as well as docking simulations with GSNO and S-methylglutathione suggest that for ADH3 ligands with a glutathione scaffold, in contrast to fatty acids, a zinc-binding moiety is imperative for correct orientation and stabilization of the hydrophilic glutathione scaffold within a predominantly hydrophobic active site.

The Janus face of alcohol dehydrogenase 3.

Many carbonyl metabolizing enzymes are equally involved in xenobiotic and endogenous metabolism, but few have been investigated in terms of substrate competition and interference between different cellular pathways. Mammalian alcohol dehydrogenase 3 (ADH3) represents the key enzyme in the formaldehyde detoxification pathway by oxidation of S-hydroxymethylglutathione [HMGSH; the glutathione (GSH) adduct of formaldehyde]. In addition, several studies have established ADH3 as S-nitrosoglutathione (GSNO) reductase in endogenous NO homeostasis during the last decade. GSNO depletion associates with various diseases including asthma, and evidence for a causal relationship between ADH3 and asthma pathology has been put forward. In a recent study, we showed that ADH3-mediated alcohol oxidation, including HMGSH oxidation, is accelerated in presence of GSNO which is concurrently reduced under immediate cofactor recycling [C.A. Staab, J. Alander, M. Brandt, J. Lengqvist, R. Morgenstern, R.C. Grafström, J.-O. Höög, Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors, Biochem. J. 413 (2008) 493-504]. Thus, considering the usually low cytosolic free NADH/NAD(+) ratio, formaldehyde may trigger and promote GSNO reduction by enzyme-bound cofactor recycling. These findings provided evidence for formaldehyde-induced, ADH3-mediated GSNO depletion with potential direct implications for asthma. Furthermore, analysis of product formation as a function of GSH concentrations suggested that, under conditions of oxidative stress, GSNO reduction can lead to the formation of glutathione sulfinamide and its hydrolysis product glutathione sulfinic acid, both potent inhibitors of glutathione transferase activity.

Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors.

GSNO (S-nitrosoglutathione) is emerging as a key regulator in NO signalling as it is in equilibrium with S-nitrosated proteins. Accordingly, it is of great interest to investigate GSNO metabolism in terms of competitive pathways and redox state. The present study explored ADH3 (alcohol dehydrogenase 3) in its dual function as GSNOR (GSNO reductase) and glutathione-dependent formaldehyde dehydrogenase. The glutathione adduct of formaldehyde, HMGSH (S-hydroxymethylglutathione), was oxidized with a k(cat)/K(m) value approx. 10 times the k(cat)/K(m) value of GSNO reduction, as determined by fluorescence spectroscopy. HMGSH oxidation in vitro was greatly accelerated in the presence of GSNO, which was concurrently reduced under cofactor recycling. Hence, considering the high cytosolic NAD(+)/NADH ratio, formaldehyde probably triggers ADH3-mediated GSNO reduction by enzyme-bound cofactor recycling and might result in a decrease in cellular S-NO (S-nitrosothiol) content in vivo. Formaldehyde exposure affected S-NO content in cultured cells with a trend towards decreased levels at concentrations of 1-5 mM, in agreement with the proposed mechanism. Product formation after GSNO reduction to the intermediate semimercaptal responded to GSH/GSNO ratios; ratios up to 2-fold allowed the spontaneous rearrangement to glutathione sulfinamide, whereas 5-fold excess of GSH favoured the interception of the intermediate to form glutathione disulfide. The sulfinamide and its hydrolysis product, glutathione sulfinic acid, inhibited GST (glutathione transferase) activity. Taken together, the findings of the present study provide indirect evidence for formaldehyde as a physiological trigger of GSNO depletion and show that GSNO reduction can result in the formation of GST inhibitors, which, however, is prevented under normal cellular redox conditions.

Bioinformatics processing of protein and transcript profiles of normal and transformed cell lines indicates functional impairment of transcriptional regulators in buccal carcinoma.

Normal and two transformed buccal keratinocyte lines were cultured under a standardized condition to explore mechanisms of carcinogenesis and tumor marker expression at transcript and protein levels. An approach combining three bioinformatic programs allowed coupling of abundant proteins and large-scale transcript data to low-abundance transcriptional regulators. The analysis identified previously proposed and suggested novel protein biomarkers, gene ontology categories, molecular networks, and functionally impaired key regulator genes for buccal/oral carcinoma.

The application of normal, SV40 T-antigen-immortalised and tumour-derived oral keratinocytes, under serum-free conditions, to the study of the probability of cancer progression as a result of environmental exposure to chemicals.

In vitro models are currently not considered to be suitable replacements for animals in experiments to assess the multiple factors that underlie the development of cancer as a result of environmental exposure to chemicals. An evaluation was conducted on the potential use of normal keratinocytes, the SV40 T-antigen-immortalised keratinocyte cell line, SVpgC2a, and the carcinoma cell line, SqCC/Y1, alone and in combination, and under standardised serum-free culture conditions, to study oral cancer progression. In addition, features considered to be central to cancer development as a result of environmental exposure to chemicals, were analysed. Genomic expression, and enzymatic and functional data from the cell lines reflected many aspects of the transition of normal tissue epithelium, via dysplasia, to full malignancy. The composite cell line model develops aberrances in proliferation, terminal differentiation and apoptosis, in a similar manner to oral cancer progression in vivo. Transcript and protein profiling links aberrations in multiple gene ontologies, molecular networks and tumour biomarker genes (some proposed previously, and some new) in oral carcinoma development. Typical specific changes include the loss of tumour-suppressor p53 function and of sensitivity to retinoids. Environmental agents associated with the aetiology of oral cancer differ in their requirements for metabolic activation, and cause toxic effects to cells in both the normal and the transformed states. The results suggest that the model might be useful for studies on the sensitivity of cells to chemicals at different stages of cancer progression, including many aspects of the integrated roles of cytotoxicity and genotoxicity. Overall, the properties of the SVpgC2a and SqCC/Y1 cell lines, relative to normal epithelial cells in monolayer or organotypic culture, support their potential applicability to mechanistic studies on cancer risk factors, including, in particular, the definition of critical toxicity effects and dose-effect relationships.

Functionality of allelic variations in human alcohol dehydrogenase gene family: assessment of a functional window for protection against alcoholism.

Alcohol dehydrogenase (ADH) catalyses the rate-determining reaction in ethanol metabolism. Genetic association studies of diverse ethnic groups have firmly demonstrated that the allelic variant ADH1B*2 significantly protects against alcoholism but that ADH1C*1, which is in linkage with ADH1B*2, produces a negligible protection. The influence of other potential candidate genes/alleles within the human ADH family, ADH1B*3 and ADH2, remains unclear or controversial. To address this question, functionalities of ADH1B3 and ADH2 were assessed at a physiological level of coenzyme and substrate range. Ethanol-oxidizing activities of recombinant ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2 and ADH2 were determined at pH 7.5 in the presence of 0.5 mm NAD with 2-50 mm ethanol. The activity differences between ADH1B2 and ADH1B1 were taken as a threshold for effective protection against alcoholism and those between ADH1C1 and ADH1C2 as a threshold for null protection. Over 2-50 mm ethanol, the activities of ADH1B3 were found 2.9-23-fold lower than those of ADH1B2, largely attributed to the Km effect (ADH1B2, 1.8 mm; ADH1B3, 61 mm). Strikingly, the ADH1B3 activity was only 84% that of ADH1B1 at a low ethanol concentration, 2 mm, but increased 10-fold at 50 mm. Corrected for relative expression levels of the enzyme in liver, the hepatic ADH2 activities were estimated to be 18-97% those of ADH1B1 over 2-50 mm ethanol and were 28-140% of the activity differences between ADH1C1 and ADH1C2. The assessment based on the proposed functional window for the human ADH gene family indicates that ADH1B*3 may show some degree of protection against alcoholism and that the ADH2 functional variants appear to be negligible for this protection.

Enzymatic mechanism of low-activity mouse alcohol dehydrogenase 2.

ADH2 is a member of one of the six classes of mammalian alcohol dehydrogenases, which catalyze the reversible oxidation of alcohols using NAD(+) as a cofactor. Within the ADH2 class, the rodent enzymes form a subgroup that exhibits low catalytic activity with all substrates that were examined, as compared to other groups, such as human ADH2. The low activity can be ascribed to the rigid nature of the proline residue at position 47 as the activity can be increased by approximately 100-fold by substituting Pro47 with either His (as found in human ADH2), Ala, or Gln. Mouse ADH2 follows an ordered bi-bi mechanism, and hydride transfer is rate-limiting for oxidation of benzyl alcohols catalyzed by the mutated and wild-type enzymes. Structural studies suggest that the mouse enzyme with His47 has a more closed active site, as compared to the enzyme with Pro47, and hydride transfer can be more efficient. Oxidation of benzyl alcohol catalyzed by all forms of the enzyme is strongly pH dependent, with pK values in the range of 8.1-9.3 for turnover numbers and catalytic efficiency. These pK values probably correspond to the ionization of the zinc-bound water or alcohol. The pK values are not lowered by the Pro47 to His substitution, suggesting that His47 does not act as a catalytic base in the deprotonation of the zinc ligand.

Modelling of normal and premalignant oral tissue by using the immortalised cell line, SVpgC2a: a review of the value of the model.

Normal oral keratinocytes (NOKs), and a Simian virus 40 T-antigen-immortalised oral keratinocyte line termed SVpgC2a, were cultured in an effort to model the human oral epithelium in vitro, including normal and dysplastic tissue. Monolayer and organotypic cultures of NOKs and SVpgC2a were successfully established in a standardised serum-free medium with high levels of amino acids, by using regular tissue culture plastic for monolayers and collagen gels containing oral fibroblasts as the base for generating tissue equivalents. NOKs express many characteristics of normal tissue, including those associated with terminal squamous differentiation. After > 150 passages, SVpgC2a cells retained an immortal, nontumourigenic phenotype that, relative to NOKs, was associated with aberrant morphology, enhanced proliferation, deficiency in terminal differentiation, proneness to apoptosis, and variably altered expression of structural epithelial markers. Transcript and protein profiling, as well as activity assays, demonstrated the expression of multiple xenobiotic-metabolising enzymes in SVpgC2a cells, some of which were higher in comparison to NOKs. A generally preserved, or even activated, ability for xenobiotic metabolism in long-term cultures of SVpgC2a cells indicated that this cell line could be useful in safety testing protocols--for example, in the development of consumer products in the oral health care field. However, SVpgC2a cells displayed some features reminiscent of a severe oral dysplasia, implying that this cell line could also to some extent serve as a model of a premalignant oral epithelium.

Reduction of S-nitrosoglutathione by human alcohol dehydrogenase 3 is an irreversible reaction as analysed by electrospray mass spectrometry.

Human alcohol dehydrogenase 3/glutathione-dependent formaldehyde dehydrogenase was shown to rapidly and irreversibly catalyse the reductive breakdown of S-nitrosoglutathione. The steady-state kinetics of S-nitrosoglutathione reduction was studied for the wild-type and two mutated forms of human alcohol dehydrogenase 3, mutations that have previously been shown to affect the oxidative efficiency for the substrate S-hydroxymethylglutathione. Wild-type enzyme readily reduces S-nitrosoglutathione with a kcat/Km approximately twice the kcat/Km for S-hydroxymethylglutathione oxidation, resulting in the highest catalytic efficiency yet identified for a human alcohol dehydrogenase. In a similar manner as for S-hydroxymethylglutathione oxidation, the catalytic efficiency of S-nitrosoglutathione reduction was significantly decreased by replacement of Arg115 by Ser or Lys, supporting similar substrate binding. NADH was by far a better coenzyme than NADPH, something that previously has been suggested to prevent reductive reactions catalysed by alcohol dehydrogenases through the low cytolsolic NADH/NAD+ ratio. However, the major products of S-nitrosoglutathione reduction were identified by electrospray tandem mass spectrometry as glutathione sulfinamide and oxidized glutathione neither of which, in their purified form, served as substrate or inhibitor for the enzyme. Hence, the reaction products are not substrates for alcohol dehydrogenase 3 and the overall reaction is therefore irreversible. We propose that alcohol dehydrogenase 3 catalysed S-nitrosoglutathione reduction is of physiological relevance in the metabolism of NO in humans.

The mammalian alcohol dehydrogenases interact in several metabolic pathways.

Mammalian alcohol dehydrogenases (ADHs), including ADH1-ADH5/6, interact extensively in the oxidation and reduction of alcohols and aldehydes. ADH1 and ADH2 are involved in several metabolic pathways besides the oxidation of ethanol and have also been shown to be involved in drug transformations. The ADH2 enzymes show further complexity among the species, e.g. in enzymatic characteristics where the rodent forms essentially lack ethanol-oxidizing capacity. ADH3 (glutathione-dependent formaldehyde dehydrogenase) has been shown to catalyze the reductive breakdown of S-nitrosoglutathione, indicating involvement in nitric oxide metabolism. Mass spectrometry identified the major enzymatic product as glutathione sulfinamide. This reductive breakdown directly interferes with the formaldehyde scavenging that has been proposed to be the physiological action of ADH3. The human ADH5 and rodent ADH6 seem to be the corresponding enzymes due to their similar behavior. None of these latter ADHs have so far been assigned to any function. They can be expressed as recombinant proteins but no enzymatic activity has been detected.

Species variations in cutaneous alcohol dehydrogenases and aldehyde dehydrogenases may impact on toxicological assessments of alcohols and aldehydes.

Alcohol dehydrogenase (ADH; EC. 1.1.1.1) and aldehyde dehydrogenase (ALDH; EC 1.2.1.3) play important roles in the metabolism of both endogenous and exogenous alcohols and aldehydes. The expression and localisation patterns of ADH (1-3) and ALDH (1-3) were investigated in the skin and liver of the mouse (BALB/c and CBA/ca), rat (F344) and guinea-pig (Dunkin-Hartley), using Western blot analysis and immunohistochemistry with class-specific antisera. ALDH2 expression and localisation was also determined in human skin, while ethanol oxidation, catalysed by ADH, was investigated in the mouse, guinea-pig and human skin cytosol. Western blot analysis revealed that ADH1, ADH3, ALDH1 and ALDH2 were expressed, constitutively, in the skin and liver of the mouse, rat and guinea-pig. ADH2 was not detected in the skin of any rodent species/strain, but was present in all rodent livers. ALDH3 was expressed, constitutively, in the skin of both strains of mouse and rat, but was not detected in guinea-pig skin and was absent in all livers. Immunohistochemistry showed similar patterns of expression for ADH and ALDH in both strains of mouse, rat, guinea-pig and human skin sections, with localisation predominantly in the epidermis, sebaceous glands and hair follicles. ADH activity (apparent V(max), nmoles/mg protein/min) was higher in liver (6.02-16.67) compared to skin (0.32-1.21) and lower in human skin (0.32-0.41) compared to mouse skin (1.07-1.21). The ADH inhibitor 4-methyl pyrazole (4-MP) reduced ethanol oxidation in the skin and liver in a concentration dependent manner: activity was reduced to approximately 30-40% and approximately 2-10% of the control activity, in the skin and liver, respectively, using 1 mM 4-MP. The class-specific expression of ADH and ALDH enzymes, in the skin and liver and their variation between species, may have toxicological significance, with respect to the metabolism of endogenous and xenobiotic alcohols and aldehydes.

Oxidation of celecoxib by polymorphic cytochrome P450 2C9 and alcohol dehydrogenase.

AIMS: Celecoxib is a novel selective cyclooxygenase-2 inhibitor, which is subject to extensive hepatic metabolism. The aims of the present in vitro investigation were 1) to compare the rate of celecoxib hydroxylation by different genetic variants of cytochrome P450 2C9 (CYP2C9), and 2) to identify the enzyme(s) involved in the formation of the major metabolite carboxycelecoxib. METHODS: Hydroxycelecoxib formation was studied in human liver microsomes from 35 genotyped livers, as well as in yeast microsomes with recombinant expression of different P450 variants. Carboxycelecoxib formation was studied in liver microsomes incubated in the absence or presence of liver cytosol. The metabolites were identified and quantified by h.p.l.c. In addition, hydroxycelecoxib oxidation by different variants of recombinant human alcohol dehydrogenase (ADH1-3) was analysed by spectrophotometric monitoring of NADH generation from NAD+. RESULTS: The intrinsic clearance of celecoxib hydroxylation was significantly lower for yeast-expressed CYP2C9.3 (0.14 ml min-1 nmol-1 enzyme) compared with CYP2C9.1 (0.44 ml min-1 nmol-1 enzyme). In human liver microsomes, a significant 2-fold decrease in the rate of hydroxycelecoxib formation was evident in CYP2C9*1/*3 samples compared with CYP2C9*1/*1 samples. There was also a marked reduction (up to 5.3 times) of hydroxycelecoxib formation in a liver sample genotyped as CYP2C9*3/*3. However, the CYP2C9*2 samples did not differ significantly from CYP2C9*1 in any of the systems studied. Inhibition experiments with sulphaphenazole (SPZ) or triacetyloleandomycin indicated that celecoxib hydroxylation in human liver microsomes was mainly dependent on CYP2C9 and not CYP3A4. The further oxidation of hydroxycelecoxib to carboxycelecoxib was completely dependent on liver cytosol and NAD+. Additional experiments showed that ADH1 and ADH2 catalysed this reaction in vitro with apparent K m values of 42 micro m and 10 micro m, respectively, whereas ADH3 showed no activity. CONCLUSIONS: The results confirm that CYP2C9 is the major enzyme for celecoxib hydroxylation in vitro and further indicate that the CYP2C9*3 allelic variant is associated with markedly slower metabolism. Furthermore, it was shown for the first time that carboxycelecoxib formation is dependent on cytosolic alcohol dehydrogenase, presumably ADH1 and/or ADH2.