Presence Of Alcohol Dehydrogenase Research Articles

Potential of Microorganisms to Decrease the "Beany" Off-Flavor: A Review.

Vegetable proteins are in high demand due to current issues surrounding meat consumption and changes in eating habits, but they are still not accepted by consumers due to their strong bitterness, astringent taste, and "beany" off-flavor. This review aimed to give an overview of the "beany" off-flavor and the potential of microorganisms to decrease it. Twenty-six volatile compounds were identified from the literature as contributing to the "beany" off-flavor, and their formation pathways were identified in a legume matrix, pea. Biotechnological ways to improve the flavor by reducing these volatile compounds were then looked over. As aldehydes and ketones are the main type of compounds directly linked to the "beany" off-flavor, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) were focused on. By converting aldehyde and ketones into alcohols or carboxylic acids, these two enzymes have the potential to decrease the off-flavor. The presence of the two enzymes in a selection of microorganisms (Lactobacillus acidophilus, Limosilactobacillus fermentum, Lactiplantibacillus plantarum, Streptococcus thermophilus, Saccharomyces cerevisiae, and Gluconobacter suboxydans) was done with a catabolism and a bioinformatical study. Finally, the correlation between the presence of the enzyme and the efficacy to improve the flavor was investigated by comparison with the literature. The presence of ADH and/or ALDH in the strain metabolism seems linked to an odor improvement. Especially, a constitutive enzyme (ADH or ALDH) in the catabolism should give better results, showing that some fermentative types are more inclined to better the flavor. Obligatory fermentative strains, with a constitutive ADH, or acetic acid bacteria, with constitutive ADH and ALDH, show the best results and should be favored to reduce the amount of compounds involved in the "beany" off-flavor and diminish that off-flavor in legume proteins.

Alcohol intoxication resistance and alcohol dehydrogenase levels differ between the honeybee castes

Various animal models are used in the study of alcoholism, with the honeybee (Apis mellifera L.) among them. Here, we tested the hypothesis that foragers show higher intoxication resistance to alcohol than nurses, an issue thus far not investigated. To this end, we measured the latency to full sedation when exposed to alcohol in foragers, nurses and reverted nurses. In addition, we measured alcohol dehydrogenase (ADH) levels in these worker castes. Caste status was confirmed by comparison of the size of their hypopharyngeal glands. We detected high intoxication resistance to alcohol and presence of ADH in foragers. In nurses, we detected significantly lower intoxication resistance to alcohol and no ADH. These between-caste differences cannot be explained by the age difference between castes as in reverted nurses, characterized by similar age to foragers, we detected an intermediate intoxication resistance to alcohol and no ADH. Our results suggest possible natural exposure to alcohol in different castes of workers. As such, we further develop the honeybee as a model in alcoholism-related research and open new research avenues.

Alcohol dehydrogenase of Candida albicans triggers differentiation of THP-1 cells into macrophages

Candida albicans proteins located on the cell wall and in the cytoplasm have gained great attention because they are not only involved in cellular metabolism and the maintenance of integrity but also interact with host immune systems. Previous research has reported that enolase from C. albicans exhibits high immunogenicity and effectively protects mice against disseminated candidiasis. In this study, alcohol dehydrogenase (ADH) of C. albicans was cloned and purified for the first time, and this study focused on evaluating its effects on the differentiation of the human monocytic cell line THP-1. The morphological features of THP-1 cells exposed to ADH were similar to those of phorbol-12-myristate acetate-differentiated (PMA-differentiated) macrophages. Functionally, ADH enhanced the adhesion, phagocytosis, and killing capacities of THP-1 cells. A flow cytometric assay demonstrated that ADH-induced THP-1 cells significantly increased CD86 and CD11b expression. The production of IL-1β, IL-6, and TNF-α by cells increased in the presence of ADH. As expected, after pretreatment with a MEK inhibitor (U0126), ADH-induced THP-1 cells exhibited unaltered morphological features, eliminated ERK1/2 phosphorylation, prevented CD86/CD11b upregulation and inhibited pro-inflammatory cytokine increase. Collectively, these results suggest that ADH enables THP-1 cells to differentiate into macrophages via the ERK pathway, and it may play an important role in the immune response against fungal invasion.

Estimation of bisphenol A—Human toxicity by 3D cell culture arrays, high throughput alternatives to animal tests

Bisphenol A (BPA) has been widely used for manufacturing polycarbonate plastics and epoxy resins and has been extensively tested in animals to predict human toxicity. In order to reduce the use of animals for toxicity assessment and provide further accurate information on BPA toxicity in humans, we encapsulated Hep3B human hepatoma cells in alginate and cultured them in three dimensions (3D) on a micropillar chip coupled to a panel of metabolic enzymes on a microwell chip. As a result, we were able to assess the toxicity of BPA under various metabolic enzyme conditions using a high-throughput and micro assay; sample volumes were nearly 2,000 times less than that required for a 96-well plate. We applied a total of 28 different enzymes to each chip, including 10 cytochrome P450s (CYP450s), 10 UDP-glycosyltransferases (UGTs), 3 sulfotransferases (SULTs), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase 2 (ALDH2). Phase I enzyme mixtures, phase II enzyme mixtures, and a combination of phase I and phase II enzymes were also applied to the chip. BPA toxicity was higher in samples containing CYP2E1 than controls, which contained no enzymes (IC50, 184±16μM and 270±25.8μM, respectively, p<0.01). However, BPA-induced toxicity was alleviated in the presence of ADH (IC50, 337±17.9μM), ALDH2 (335±13.9μM), and SULT1E1 (318±17.7μM) (p<0.05). CYP2E1-mediated cytotoxicity was confirmed by quantifying unmetabolized BPA using HPLC/FD. Therefore, we suggest the present micropillar/microwell chip platform as an effective alternative to animal testing for estimating BPA toxicity via human metabolic systems.

Alcohol Dehydrogenase- and Rat Liver Cytosol-Dependent Bioactivation of 1-Chloro-2-hydroxy-3-butene to 1-Chloro-3-buten-2-one, a Bifunctional Alkylating Agent

1,3-Butadiene (BD) is an air pollutant whose toxicity and carcinogenicity have been considered primarily mediated by its reactive metabolites, 3,4-epoxy-1-butene and 1,2,3,4-diepoxybutane, formed in liver and extrahepatic tissues by cytochromes P450s. A possible alternative metabolic pathway in bone marrow and immune cells is the conversion of BD to the chlorinated allylic alcohol 1-chloro-2-hydroxy-3-butene (CHB) by myeloperoxidase in the presence of hydrogen peroxide and chloride ion. In the present study, we investigated the in vitro bioactivation of CHB by alcohol dehydrogenases (ADH) under in vitro physiological conditions (pH 7.4, 37 °C). The results provide clear evidence for CHB being converted to 1-chloro-3-buten-2-one (CBO) by purified horse liver ADH and rat liver cytosol. CBO readily reacted with glutathione (GSH) under assay conditions to form three products: two CBO-mono-GSH conjugates [1-chloro-4-(S-glutathionyl)butan-2-one (3) and 1-(S-glutathionyl)-3-buten-2-one (4)] and one CBO-di-GSH conjugate [1,4-bis(S-glutathionyl)butan-2-one (5)]. CHB bioactivation and the ratios of the three GSH conjugates formed were dependent upon incubation time, GSH and CHB concentrations, and the presence of ADH or rat liver cytosol. The ADH enzymatic reaction followed Michaelis-Menten kinetics with a K(m) at 3.5 mM and a k(cat) at 0.033 s(-1). After CBO was incubated with freshly isolated mouse erythrocytes, globin dimers were detected using SDS-PAGE and silver staining, providing evidence that CBO can act as a protein cross-linking agent. Collectively, the results provide clear evidence for CHB bioactivation by ADH and rat liver cytosol to yield CBO. The bifunctional alkylating ability of CBO suggests that it may play a role in BD toxicity and/or carcinogenicity.

Sequential injection system for the enzymatic determination of ethanol in alcoholic beverages with in-line dilution

A sequential injection system for the enzymatic determination of ethanol in wines with spectrophotometric detection was developed. The determination was based on the enzymatic reaction involving ethanol and NAD+ in the presence of alcohol dehydrogenase. A gas-diffusion unit was introduced in the system to perform an in-line dilution. It was possible to operate the system with a reagent consumption of 1.3 mg of NAD+, 0.45 U of ADH, and 32 μL of sample, per assay with a determination rate of 21 h−1.The results obtained from the analysis of wine samples were statistically equivalent to those obtained by the reference method, with good repeatability; the relative standard deviation (n = 10) was <1% with a LOQ of 0.3% (v/v).

Natural ethanol availability directed the evolution of short‐chain alcohol dehydrogenase in animals

The presence of alcohol dehydrogenase (ADH) in animals has been assumed to be a consequence of chronic exposure to ethanol. Therefore, in this work we investigate if natural ethanol availability directed the evolution of type II or short‐chain ADHs evolution. In animals, 15‐hydroxyProstaglandin dehydrogenase (PGDH) is the closest homologous to type II ADHs. In contrast, bona fide type II ADHs and ADH‐like proteins appear to be restricted to Diptera (flies). Thus, it can be assumed that the origin of PGDH predates the origin of type II ADHs in Diptera. Adh locus in Drosophila has undergone at least five duplication events, and all them occurred within the Diptera. Because point mutations in Adh can abolish ADH activity in this fly, it can be considered the unique fly enzyme involved in ethanol metabolism.The natural breeding substrates of Drosophila are fermenting fruits. The Drosophila genus is among the most diverse of the Diptera, encompassing &gt;2,000 species. The efficient exploitation of such niches is the reasons for their successful worldwide spreading. Thus, induction of type II Adh by ethanol exposure, a positive correlation between ADH activity and ethanol resistance, ethanol utilization as a nutrient, and the fact that flies and type II Adh diversification occurred in concert with angiosperm diversification, strongly suggest that type II ADHs were recruited to allow larval flies to exploit new restricted niches with high ethanol content. Supported by DGAPA‐UNAM grant IN208510.

Ethanol determination by an amperometric bienzyme sensor based on a Clark-type transducer

The aim of this work is to develop a method for ethanol determination in some alcoholic beverages, by using an alcohol dehydrogenase/peroxidase-based amperometric enzyme electrode. The enzymes alcohol dehydrogenase and horseradish peroxidase, as well as the coenzyme nicotinamide adenine dinucleotide (NAD+) were immobilized on a nylon (Biodyne A) membrane. The enzyme-membrane was consecutively attached to the polyethylene membrane of a Clark oxygen electrode, which functioned as a transducer. Ethanol is oxidized by NAD+ in the presence of alcohol dehydrogenase; the NADH produced is aerobically oxidized by horseradish peroxidase. The rate of molecular oxygen consumption, which is directly proportional to the alcohol concentration in the sample, is amperometrically monitored with the oxygen electrode-based biosensor.The analytical characteristics of the biosensor (linear range, sensitivity, selectivity, response time, stability) were investigated. The value of the current intensity was monitored as a function of time, for different ethanol concentrations. The obtained calibration graph was linear within the range 10–80mM. The values of the ethanol content obtained for the analysed beverages ranged between 4.56% (v/v) for Belheimer beer and 41.83% for Stalinskaya vodka.

Enantioselective Synthesis of (R)- and (S)-1-2H-1-Octanol and Their Corresponding Amines

Both enantiomers of 1-2H-1-octanol were obtained by the enzymatic reduction of deuterated octanal in the presence of alcohol dehydrogenases (ADH) (ADH-T or ADH-LB) as the biocatalyst in good yield, purity, and enantiomeric excess (>95%). The cofactor nicotinamide adenine dinucleotide phosphate was regenerated by the addition of isopropanol. To simplify the synthetic route, the direct reduction of octanal using the same enzymes and the same cofactor but adding deuterated isopropanol was evaluated. This provided a one-step procedure from a commercially available starting compound to both enantiomers of 1-2H-1-octanol in good yields (>80%) and good enantiomeric excess (∼97%). The (S)-alcohols were converted to their corresponding (R)-amines, which showed ee's around 90%.

Natural alcohol exposure: Is ethanol the main substrate for alcohol dehydrogenases in animals?

Alcohol dehydrogenase (ADH) activity is widely distributed in all phyla. In animals, three non-homologous NAD(P) +-dependent ADH protein families are reported. These arose independently throughout evolution and possess different structures and mechanisms of reaction: type I (medium-chain) ADHs are zinc-containing enzymes and comprise the most studied group in vertebrates; type II (short-chain) ADHs lack metal cofactor and have been extensively studied in Drosophila; and type III ADHs are iron-dependent/-activated enzymes that were initially identified only in microorganisms. The presence of these different ADHs in animals has been assumed to be a consequence of chronic exposure to ethanol. By far the most common natural source of ethanol is fermentation of fruit sugars by yeast, and available data support that this fruit trait evolved in concert with the characteristics of their frugivorous seed dispersers. Therefore, if the presence of ADHs in animals evolved as an adaptive response to dietary ethanol exposure, then it can be expected that the enzymogenesis of these enzymes began after the appearance of angiosperms with fleshy fruits, because substrate availability must precede enzyme selection. In this work, available evidence supporting this possibility is discussed. Phylogenetic analyses reveal that type II ADHs suffered several duplications, all of these restricted to flies (order Diptera). Induction of type II Adh by ethanol exposure, a positive correlation between ADH activity and ethanol resistance, and the fact that flies and type II Adh diversification occurred in concert with angiosperm diversification, strongly suggest that type II ADHs were recruited to allow larval flies to exploit new restricted niches with high ethanol content. In contrast, phyletic distribution of types I and III ADHs in animals showed that these appeared before angiosperms and land plants, independently of ethanol availability. Because these enzymes are not induced by ethanol exposure and possess a high affinity and/or catalytic efficiency for non-ethanol endogenous substrates, it can be concluded that the participation of types I and III ADHs in ethanol metabolism can be considered as incidental, and not adaptive.

NAD+/NADH-Sensitive Quantum Dots: Applications To Probe NAD+-Dependent Enzymes and To Sense the RDX Explosive

Phenyl boronic acid-functionalized CdSe/ZnS quantum dots (QDs) were synthesized. The modified particles bind nicotinamide adenine dinucleotide (NAD(+)) or 1,4-dihydronicotinamide adenine dinucleotide (NADH). The NAD(+)-functionalized QDs are effectively quenched by an electron transfer process, while the NADH-modified QDs are inefficiently quenched by the reduced cofactor. These properties enable the implementation of the QDs for the fluorescence analysis of ethanol in the presence of alcohol dehydrogenase. The NADH-functionalized QDs were used for the optical analysis of the 1,3,5-trinitrotriazine, RDX explosive, with a detection limit that corresponded to 1 x 10(-10) M. We demonstrate cooperative optical and catalytic functions of the core-shell components of the QDs in the analysis of RDX.

Amplification of electrocatalytic oxidation of NADH based on cysteine nanolayers

A biosensor for the determination of alcohol based on cysteine (Cys) nanolayers has been constructed. The utilization of the redox-inactive, self-assembled monolayer (SAM) modified gold ultra micro-electrode (UME) for NADH electroanalysis is introduced as a new modified gold Cys-UME. This Cys-UME prevents fouling and was found to be stable and ill-defined under subsequent potential sweeps, and different NADH concentrations. NADH oxidation gave a linear correlation between the peak current and NADH concentration (1–5 mM). Also, excellent electro-catalytic activity (enhancing the oxidation peak current) was observed towards NADH oxidation, with activation overpotential of about 250 mV lower than that of the bare electrode. The use of different alcohol concentrations in the presence of alcohol dehydrogenase (ADH) gave an increase in the NADH oxidation peak current, indicating that the electrochemical NADH oxidation at mild potentials could lead to an enzymatically active NAD+.

Electrocatalytic reduction of NAD+ at glassy carbon electrode modified with single-walled carbon nanotubes and Ru(III) complexes

A simple procedure was developed to prepare a glassy carbon electrode modified with carbon nanotubes and Ruthenium (III) complexes. First, 25 μl of dimethyl sulfoxide–carbon nanotubes solutions (0.4 mg/ml) was cast on the surface of the glassy carbon electrode and dried in air to form a carbon nanotube film at the electrode surface. Then, the glassy carbon/carbon nanotube-modified electrode was immersed into a Ruthenium (III) complex solution (direct deposition) for a short period of time (10–20 s for multiwalled carbon nanotubes and 20–40 s for single-walled carbon nanotubes). The cyclic voltammograms of the modified electrode in aqueous solution shows a pair of well-defined, stable, and nearly reversible redox couple, Ru(III)/Ru(II), with surface-confined characteristics. The attractive mechanical and electrical characteristics of carbon nanostructures and unique properties and reactivity of Ru complexes are combined. The transfer coefficient (α), heterogeneous electron transfer rate constants (ks), and surface concentrations (Γ) for the glassy carbon/single-walled carbon nanotubes/Ru(III) complex-, glassy carbon/multiwalled carbon nanotubes/Ru(III) complex-, and glassy carbon/Ru(III) complex-modified electrodes were calculated using the cyclic voltammetry technique. The modified electrodes showed excellent catalytic activity, fast response time, and high sensitivity toward the reduction of nicotinamide adenine dinucleotide in phosphate buffer solutions at a pH range of 4–8. The catalytic cathodic current depends on the nicotinamide adenine dinucleotide concentration. In the presence of alcohol dehydrogenase, the modified electrode exhibited a response to addition of acetaldehyde. Therefore, the main product of nicotinamide adenine dinucleotide electroreduction at the Ru(III) complex/carbon nanotube-modified electrode was the enzymatically active NADH. The purposed sensor can be used for acetaldehyde determination.

Assaying uridine diphosphoglucose dehydrogenase in the presence of alcohol dehydrogenase

Assaying uridine diphosphoglucose dehydrogenase in the presence of alcohol dehydrogenase

4-Hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one Induces Mitochondrial Dysfunction and Apoptosis upon Its Intracellular Oxidation

We investigated the mechanism of cell death induced by a furoquinolinone derivative, namely, 4-hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one (HOFQ), in the dark. Mitochondrial depolarization was found to be a causative event in HOFQ-induced apoptosis that was blunted either by replacing the 4-hydroxymethyl group with a methyl one, or by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase (ADH). In vitro enzymatic assay demonstrated that HOFQ is a substrate of ADH. In isolated mitochondria HOFQ was without effect, whereas in the presence of ADH and NAD(+) it caused the opening of the permeability transition pore, indicating that HOFQ-oxidized products affect mitochondrial function directly. Finally, an analogue bearing the formyl group at the C-4 position mimicked all the effects exerted by HOFQ. In conclusion, these results suggest that the direct action on mitochondria of HOFQ-oxidized products are responsible for their cytotoxicity, which might be exacerbated, but hardly determined, by photodynamic action and/or binding to DNA.

Liquid chromatographic determination of ethyl alcohol in body fluids

A high-performance liquid chromatographic technique for ethyl alcohol determination in body fluids is proposed. Ethyl alcohol is quantitatively converted into acetaldehyde-phenylhydrazone by oxidation in the presence of alcohol dehydrogenase, nicotinamide–adenine dinucleotide and phenylhydrazine. The derivative is suitable for reversed-phase liquid chromatography and ultraviolet detection at 276 nm. The limits of linearity, detection and quantification as well as accuracy and reproducibility were investigated in water, serum and whole blood. Analytical responses were linear within the 0.008 to 5 g/l range, and the limit of quantification was 0.02 g/l both in aqueous standard and in biological matrix assays. Mean analytical recovery of ethyl alcohol in blood serum averaged 98.2±4.2%, imprecision (CV%) at 0.80 g/l was 2.2%, and the limit of quantification was 0.02 g/l. Serum concentrations of persons that avoided alcoholic beverages for a week were less than the limit of quantification. Ethyl alcohol concentrations in serum and whole blood compared well with those obtained by headspace gas chromatography. This simple and reliable procedure, which was also used for a urine assay, could be suitable for validation of the screening procedures used to monitor ethanol abuse.

Enzyme-Amplified Microtiter Plate Assay for Ethanol: Its Application to the Detection of Apparent Ethanol in Peanuts

A colorimetric microtiter plate assay amplified by aldehyde dehydrogenase, in the presence of alcohol dehydrogenase, nicotinamide adenine dinucleotide, diaphorase, and p-iodonitrotetrazolium violet was developed for detection of protein-bound apparent ethanol (i.e., ethanol and other primary alcohols) in peanuts of differing maturity and curing (stackpole) stages. Data showed that at each curing stage mature peanuts had a lower level of protein-bound ethanol than immature peanuts and that at each maturity stage the level of protein-bound ethanol decreased during curing. This change in the level of protein-bound ethanol suggests that peanut maturity and curing have an effect on the binding of ethanol to peanut proteins. The implication of this is that the extent of flavor binding might contribute to the consequent difference in flavors between mature/cured and immature/cured peanuts.

Anaerobic regulation of the adhE gene, encoding the fermentative alcohol dehydrogenase of Escherichia coli

The regulation of the adhE gene, which encodes the trifunctional fermentative acetaldehyde-alcohol dehydrogenase of Escherichia coli, was investigated by the construction of gene fusions and by two-dimensional protein gel electrophoresis. Both operon and protein fusions of adhE to lacZ were induced 10- to 20-fold by anaerobic conditions, and both fusions were repressed by nitrate, demonstrating that regulation is at the level of transcription. Nitrate repression of phi (adhE-lacZ) expression, as well as of alcohol dehydrogenase enzyme activity, was partly relieved by a mutation in narL. Mutations in rpoN or fnr had no effect on the expression of adhE. Two-dimensional protein gels demonstrated that increases in the amount of adhE protein correlated with increases in enzyme activity, demonstrating that induction was due to synthesis of new protein, not to activation of preexisting protein. When oxidized sugar derivatives such as gluconate or glucuronate were used as carbon sources, the anaerobic expression of phi (adhE-lacZ) was greatly reduced, whereas when sugar alcohols such as sorbitol were used, the expression was increased compared with expression when glucose was the carbon source. This observation suggested that induction of phi (adhE-lacZ) might depend on the level of reduced NADH, which should be highest with sorbitol-grown cells and lowest with glucuronate-grown cells. When phi (adhE-lacZ) was present in a strain deleted for the adhE structural gene, anaerobic expression of phi (adhE-lacZ) was approximately 10-fold higher than in an adhE+ strain. Since the presence of alcohol dehydrogenase would serve to decrease NADH levels, this finding again implies that the adhE gene is regulated by the concentration of reduced NAD. Introduction of a pgi (phosphoglucose isomerase) mutation reduced the anaerobic induction of phi(adhE-lacZ) when the cells were grown on glucose, but had little effect on fructose-grown cells. Pyruvate did not overcome the pgi effect, but glycerol 3-phosphate did, which is again consistent with the possibility that adhE expression responds to the level of reduced NAD rather than to a glycolytic intermediate.

A Modification and Validation of Two Urine Ethanol Procedures for Use with the Monarch® 2000 Chemistry System

A modification of two commercially available enzymatic ethanol urine assays for use with the Monarch 2000 Chemistry System (Instrumentation Laboratory) is described. Both the Syva EMIT st Urine Ethyl Alcohol Assay and the Sigma Diagnostics Alcohol in Urine Assay, which utilize the reduction of nicotinamide adenine dinucleotide (NAD) to NADH associated with the oxidation of ethanol in the presence of alcohol dehydrogenase (ADH), were adapted to spectrophotometrically determine ethanol concentration. Precision was evaluated over a 3-day period. Within-day (n = 9) and total (n = 27) coefficients of variation (CV) were less than 7% for the controls greater than or equal to 200 mg/L (20 mg/dL). Enzymatic assay results utilizing the Monarch procedure were compared to a gas chromatographic (GC) reference method (n = 100 samples). Regression analysis of assay data with each reagent compared to the reference method resulted in correlation coefficients r = 0.972 (Syva) and 0.948 (Sigma). Both methods exhibited nonlinear results and therefore quantitative applications cannot be made. No false positive or negative results were encountered with either reagent, indicating that the assay is acceptable as a positive/negative screen for urine ethanol for a threshold less than or equal to 20 mg/dL.

Mutagenicity of methylazoxymethanol acetate in the presence of alcohol dehydrogenase, aldehyde dehydrogenase, and rat liver microsomes inSalmonella typhimurium his G46

Methylazoxymethanol (MAM) is the short-lived toxic and carcinogenic aglycone of cycasin, a natural component of the cycad plant. In the present study, the stable acetate ester of MAM, MAM acetate, was tested in combination with porcine liver esterase and Salmonella typhimurium His G46 to study the comparative mutagenicity of this compound in the presence of rat hepatic alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and rat liver microsomes. In the presence of rat liver microsomes and an NADPH-generating system, mutagenicity of MAM acetate was not significantly altered. However, addition of rat liver 105,000g supernatant fraction and/or NAD+ significantly increased the number of his+ revertants above control. A concentration-dependent increase in mutagenicity of MAM acetate was observed for NAD+ from 50 to 200 microM, while NADP+ caused a decrease in mutagenicity of MAM acetate in this same concentration range. Pyrazole (100-500 microM) had no significant effect on mutagenicity of MAM acetate in the presence of rat liver 105,000g supernatant, while disulfiram at 500 microM resulted in a significant decrease in mutagenicity of MAM acetate. The results of this study implicate ALDH as essential in activation of MAM acetate to a mutagenic species in this system, while the role of ADH and microsomes appears to be minimal.