Elevated levels of alcohol dehydrogenase aggravate ethanol-evoked cardiac remodeling and contractile anomalies through FKBP5-yap-mediated regulation of ferroptosis and ER stress.

Alcohol intake provokes severe organ injuries including alcoholic cardiomyopathy with hallmarks of cardiac remodeling and contractile defects. This study examined the toxicity of facilitated ethanol metabolism in alcoholism-evoked changes in myocardial morphology and contractile function, insulin signaling and various cell death domains using cardiac-selective overexpression of alcohol dehydrogenase (ADH). WT and ADH mice were offered an alcohol liquid diet for 12 weeks prior to assessment of cardiac geometry, function, ER stress, apoptosis and ferroptosis. Alcohol intake provoked pronounced glucose intolerance, cardiac remodeling and contractile anomalies with apoptosis, ER stress, and ferroptosis, the effects were accentuated by ADH with the exception of global glucose intolerance. Hearts from alcohol ingesting mice displayed dampened insulin-stimulated phosphorylation of insulin receptor (tyr1146) and IRS-1 (tyrosine) along with elevated IRS-1 serine phosphorylation, the effect was augmented by ADH. Alcohol challenge dampened phosphorylation of Akt and GSK-3ß, and increased phosphorylation of c-Jun and JNK, the effects were accentuated by ADH. Alcohol challenge promoted ER stress, FK506 binding protein 5 (FKBP5), YAP, apoptosis and ferroptosis, the effects were exaggerated by ADH. Using a short-term ethanol challenge model (3 g/kg, i.p., twice in three days), we found that inhibition of FKBP5-YAP signaling or facilitated ethanol detoxification by Alda-1 alleviated ethanol cardiotoxicity. In vitro study revealed that the ethanol metabolite acetaldehyde evoked cardiac contractile anomalies, lipid peroxidation, and apoptosis, the effects of which were mitigated by Alda-1, inhibition of ER stress, FKBP5 and YAP. These data suggest that facilitated ethanol metabolism via ADH exacerbates alcohol-evoked myocardial remodeling, functional defects, and insulin insensitivity possibly through a FKBP5-YAP-associated regulation of ER stress and ferroptosis.

Alcohol dehydrogenase-1B represses the proliferation, invasion and migration of breast cancer cells by inactivating the mitogen-activated protein kinase signalling pathway.

Breast cancer (BRCA) is a serious life-threatening cancer, especially triple-negative breast cancer (TNBC). Alcohol dehydrogenase-1B (ADH1B) has recently been revealed to be associated with poor prognosis of BRCA patients. This study identified the exact function of ADH1B on the progression of BRCA and TNBC. ADH1B effect on the prognosis of BRCA and TNBC patients was researched based on online databases and clinical samples. The function of ADH1B on the proliferation, invasion and migration, and growth of BRCA and TNBC cells was investigated by cell counting kit-8, Transwell, and in vivo assays. Western blot was utilized to determine the effect of ADH1B on the mitogen-activated protein kinase (MAPK) signalling pathway activity. As a result, ADH1B was down-regulated in BRCA and TNBC patients and cells, predicting unfavorable prognosis (P<0.05). ADH1B overexpression suppressed the proliferation, invasion and migration, and inactivated the MAPK signalling pathway in BRCA and TNBC cells (P<0.01). ADH1B synergized with Selumetinib (inhibitor of the MAPK signalling pathway) to attenuate the proliferation, invasion and migration of BRCA and TNBC cells (P<0.001). Conversely, Vacquinol-1 (activator of the MAPK signalling pathway) abolished the suppression of ADH1B on the proliferation, invasion and migration of BRCA and TNBC cells (P<0.05). ADH1B suppressed in vivo growth of TNBC cells (P<0.001). Thus, ADH1B may inhibit the proliferation, invasion and migration of BRCA and TNBC cells by inactivating the MAPK signalling pathway. It may be a promising target for the clinical treatment of BRCA and TNBC.

Reduced acute functional tolerance and enhanced preference for ethanol in Caenorhabditis elegans exposed to lead during development: Potential role of alcohol dehydrogenase.

Despite its relative simplicity, the invertebrate Caenorhabditis elegans (C. elegans) has become a powerful tool to evaluate toxicity. Lead (Pb) persistence in the environment and its distinctive characteristic as a neurodevelopmental toxicant determine the potential effects of this metal against challenging events later in life. Additionally, among other psychoactive substances, low to moderate ethanol (EtOH) doses have been pointed out to induce behaviors such as acute functional tolerance (AFT) and drug-induced chemotaxis. In the present study, we aimed to study the impact of early-life Pb exposure on EtOH-induced motivational and stimulant effects in C. elegans by assessing the preference for EtOH and the participation of alcohol dehydrogenase (ADH, sorbitol dehydrogenase -SODH in worms) in the AFT response. Thus, N2 (wild type) and RB2114 (sod-1 -/-) strains developmentally exposed to 24 µM Pb were evaluated in their AFT to 200 mM EtOH alone and in combination with acetaldehyde (ACD). We ascribed the enhanced EtOH-induced AFT observed in the N2 Pb-exposed animals to a reduced ADH functionality as evaluated by both, ADH activity determination and the allyl alcohol test, which altogether suggest excess EtOH accumulation rather than low ACD formation in these animals. Moreover, the Pb-induced preference for EtOH indicates enhanced motivational effects of this drug as a consequence of early-life exposure to Pb, results that resemble our previous reports in rodents and provide a close association between EtOH stimulant and motivational effects in these animals.

Phosphodiesterase 4 mRNA Level Suppression is Important for Extract of Black Carrot to Protect Against Hepatic Injury Induced by Ethanol.

Excessive alcohol use often results in alcoholic liver disease (ALD). An early change in the liver due to excessive drinking is hepatic steatosis, which may ultimately progress to hepatitis, liver fibrosis, cirrhosis, and liver cancer. Among these debilitating processes, hepatic steatosis is reversible with the appropriate treatment. Therefore, it is important to find treatments and foods that reverse hepatic steatosis. Black carrot has antioxidant and anti-inflammatory effects. In this study, we examined the effectiveness of black carrot extract (BCE) on hepatic steatosis in in vivo and in vitro ethanol-induced liver injury models. For the in vivo experiments, serum aminotransferase activities enhanced by ethanol- and carbon tetrachloride were significantly suppressed by the BCE diet. Furthermore, morphological changes in the liver hepatic steatosis and fibrosis were observed in the in vivo ethanol-induced liver injury model, however, BCE feeding resulted in the recovery to an almost normal liver morphology. In the in vitro experiments, ethanol treatment induced reactive oxygen species (ROS) levels in hepatocytes at 9 h. Conversely, ROS production was suppressed to control levels and hepatic steatosis was suppressed when hepatocyte culture with ethanol were treated with BCE. Furthermore, we investigated enzyme activities, enzyme protein levels, and messenger RNA levels of alcohol dehydrogenase (ADH), cytochrome p450 2E1 (CYP2E1), and aldehyde dehydrogenase (ALDH) using enzyme assays, western blot, and quantitative reverse transcription-polymerase chain reaction analyses. We found that the activities of ADH, CYP2E1, and ALDH were regulated through the cAMP-PKA pathway at different levels, namely, translational, posttranslational, and transcriptional levels, respectively. The most interesting finding of this study is that BCE increases cAMP levels by suppressing the Pde4b mRNA and PDE4b protein levels in ethanol-treated hepatocytes, suggesting that BCE may prevent ALD.

Functionalized Allopurinols Targeting Amyloid-Binding Alcohol Dehydrogenase Rescue Aß-Induced Mitochondrial Dysfunction.

Alzheimer's disease (AD) is the most common dementia affecting one in nine people over 65. Only a handful of small-molecule drugs and the anti-ß amyloid (Aß) antibody aducanumab are approved to treat AD. However, they only serve to reduce symptoms of advanced disease. Novel treatments administered early in disease progression before the accumulation of Aß and tau reaches the threshold where neuroinflammation is triggered and irreversible neuronal damage occurs are more likely to provide effective therapy. There is a growing body of evidence implying that mitochondrial dysfunction occurs at an early stage of AD pathology. The mitochondrial enzyme amyloid-binding alcohol dehydrogenase (ABAD) binds to Aß potentiating toxicity. Moreover, ABAD has been shown to be overexpressed in the same areas of the brain most affected by AD. Inhibiting the Aß-ABAD protein-protein interaction without adversely affecting normal enzyme turnover is hypothesized to be a potential treatment strategy for AD. Herein, we conduct structure-activity relationship studies across a series of functionalized allopurinol derivatives to determine their ability to inhibit Aß-mediated reduction of estradiol production from ABAD. The lead compound resulting from these studies possesses potent activity with no toxicity up to 100 µM, and demonstrates an ability to rescue defective mitochondrial metabolism in human SH-SY5Y cells and rescue both defective mitochondrial metabolism and morphology ex vivo in primary 5XFAD AD mouse model neurons.

ADH1C Facilitates Cisplatin Resistance of Lung Adenocarcinoma Cells.

Lung adenocarcinoma (LUAD) is a common form of lung cancer. Although cisplatin chemotherapy is an effective treatment option, some patients with LUAD can develop drug resistance. Modulated ADH1C expression has been reported in various cancer types. However, the mechanism by which ADH1C potentially influences progression and cisplatin resistance of LUAD remains poorly understood. In this study, we aimed to explore the role of ADH1C with respect to cisplatin resistance and to uncover the clinical significance of methionine adenosyltransferase (MAT1A). Compared with cisplatin-sensitive A549 cells, ADH1C was highly enriched in cisplatin-resistant A549/cis-dichlorodiammineplatinum II (DDP) cells. Inhibition of ADH1C expression in the latter suppressed cell proliferation and decreased their resistance to cisplatin. Furthermore, the proliferative capacity under cisplatin stimulation was reduced. Downregulation of ADH1C expression inhibited the expression of proliferating cell nuclear antigen and excision repair cross-complementing 1 (ERCC1). Knockdown of ADH1C resulted in arrested cell cycle (in G2/M phase). The proliferative capacity and cisplatin sensitivity induced by ADH1C upregulation in A549 cells were reversed upon knockdown of ADH1C. Bioinformatic analyses revealed ADH1C to be mainly enriched in cell cycle, RNA transport, biosynthesis of amino acids, and platinum drug resistance pathways. Meanwhile, the gene MAT1A with considerable positive association with ADH1C was identified. Furthermore, expression of MAT1A was upregulated in LUAD tissues relative to the paired adjacent normal specimens. Human Protein Atlas, The university of alabama at birmingham cancer data analysis portal (UALCAN), and Kaplan-Meier Plotter analysis indicated that upregulated MAT1A expression is correlated with poor prognosis of LUAD. Our results indicate that the ADH1C/MAT1A axis possibly increases cisplatin resistance in LUAD cells. The experiment was repeated three times and approved by the Medical Ethical Committee of the First Affiliated Hospital of Wenzhou Medical University (approval No.YS2018001).

[Molecular evidences of non-ADH pathway in alcohol metabolism and Class III alcohol dehydrogenase (ADH3)].

Class I alcohol dehydrogenase (ADH1), a key enzyme of alcohol metabolism, contributes around 70% to the systemic alcohol metabolism and also to the acceleration of the metabolism due to chronic alcohol consumption by increasing its liver content, if the liver damage or disease is not apparent. However, the contribution of ADH1 to alcohol metabolism decreases in case of acute alcohol poisoning or chronic alcohol consumption inducing liver damage or disease. On the contrary, non-ADH pathway, which is independent of ADH1, increases the contribution to alcohol metabolism in these cases, by complementing the reduced role of ADH1. The molecular substantiality of non-ADH pathway has been still unknown in spite of the long and hot controversy between two candidates of microsomal ethanol oxidizing system (MEOS) and catalase. This research history suggests the existence of other candidates. Among ADH isozymes, Class III (ADH3) has the highest Km for ethanol and the highest resistance to pyrazole reagents of specific ADH inhibitors. This ADH3 was demonstrated to increase the contribution to alcohol metabolism in vivo dose-dependently, therefore, is a potent candidate of non-ADH pathway. Moreover, ADH3 is considered to increase the contribution to alcohol metabolism in case of alcoholic liver diseases, because the enzyme content increases in damaged tissues with increased hydrophobicity or the activity of the liver correlates with the accumulated alcohol consumptions of patients with alcoholic liver diseases. Such adaptation of ADH3 to alcohol metabolism in these pathological conditions makes patients possible to keep drinking a lot in spite of decrease of ADH1 activity and develops alcoholism seriously.

Aggregates of denatured proteins stimulate nitric oxide and superoxide production in macrophages.

OBJECTIVE: Denatured proteins are deposited in damaged tissues, around implanted biomaterials, during natural aging as well as in a heterogeneous group of amyloid diseases, such as Alzheimer's disease. There is evidence that tissue damage observed in amyloidosis is mediated mainly by factors released from activated macrophages, such as superoxide and nitric oxide (NO), as opposed to direct interaction between amyloid fibrils and nonimmune cells. METHODS: Mouse resident peritoneal macrophages were stimulated in serum-free medium with different preparations of nonamyloidogenic proteins: alcohol dehydrogenase (AD), bovine serum albumin (BSA) or fibrinogen (FG). Intra- and extracellular superoxide production was measured by, respectively, nitro blue tetrazolium (NBT) reduction and lucigenin-enhanced chemiluminescence. Levels of nitrite (reflecting NO release) were measured in culture supernatants. RESULTS: Aggregates of denatured, nonamyloidogenic proteins, but not their native or denatured but not aggregated counterparts, stimulated superoxide and/or NO production in macrophages. The NO production was mediated by beta(1) and beta(2) integrins, with a possible contribution of receptor for advanced glycation end products (RAGE). It was catalyzed by inducible NO synthase (iNOS), enhanced synergistically by interferon-gamma (IFN-gamma), and inhibited by covalently modified proteins-components of advanced glycation end products. Although intracellular superoxide production was stimulated significantly by denatured BSA and AD, but not by FG, both denatured BSA and FG strongly enhanced zymosan-stimulated extracellular release of reactive oxygen species. CONCLUSION: Our results point at similarities in macrophage responses to denatured nonamyloidogenic proteins and to amyloid fibrils. Thus, the tissue injury observed in amyloidosis may result from overstimulation of mechanisms that, under physiological conditions, enable macrophages to recognize and remove denatured proteins.

Involvement of p42/44 MAPK in the effects of ethanol on secretion of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-1 in primary cultured rat hepatocytes.

This article investigates the effects of ethanol on Insulin-like growth factor (IGF)-I secretion, p42/44 mitogen-activated protein kinase (MAPK) activity, and IGF binding protein (IGFBP-1 secretion) in primary cultured rat hepatocytes. The p42/44 MAPK activity increased with the ethanol concentration compared to control after ethanol treatment. The secretion of IGF-I significantly increased compared to control, but IGFBP-1 secretion was inhibited. Treatment with 4-methylpyrazole blocked the IGF-I and IGFBP-1 secretion and p42/44 MAPK activity. Increased IGF-I secretion and inhibited IGFBP-1 secretion due to ethanol-induced p42/44 MAPK activity (at 30 min) was blocked by treatment with PD98059. Taken together, these results suggest that ethanol is involved in the modulation of the secretion of IGF-I and IGFBP-1 by p42/44 MAPK in primary cultured rat hepatocytes. In addition, inhibition of p42/44 MAPK activity by ethanol occurs via the activity of ADH.

Alcohol dehydrogenase 3 and risk of squamous cell carcinomas of the head and neck.

In order to examine the association between alcohol dehydrogenase 3 (ADH3) genotypes and risk of head and neck squamous cell carcinomas (HNSCC), we conducted a hospital based case-control study including 348 cases and 330 controls. DNA isolated from exfoliated cells from the oral cavity were genotyped for ADH3 polymorphisms using PCR followed by SspI digestion. Odds ratios (OR) and hazards ratios (HR) were done by unconditional logistic regression and Cox regression. Relative to ADH3(2-2) carriers, ADH3(1-1) [OR, 0.7; 95% confidence interval (CI), 0.4-1.1] and ADH3(1-2) (OR, 0.8; 95% CI, 0.5-1.2) had a nonsignificant reduced risk of HNSCC. ADH(1-2) smokers of >30 pack-years were at decreased risk of oral cavity squamous cell carcinomas compared with ADH3(2-2) (OR, 0.3, 0.1-0.9), whereas ADH3(1-1) smokers were not. After adjustment, those with ADH3(1-2) had significantly worse overall survival compared with ADH3(1-1) (HR, 0.3, 0.2-0.6) or ADH3(2-2) (HR, 0.4, 0.2-0.9) and increased recurrence (ADH3(1-1), 0.2, 0.1-0.6; ADH3(2-2), 0.6, 0.2-1.3). Our data did not show that ADH3 genotypes had a significantly independent effect on the risk of HNSCC, nor did they modify the risks increased by alcohol or tobacco consumption and high-risk human papillomavirus infection. However, participants with ADH3(1-2) genotype were associated with poorer survival compared with those who had the other two ADH3 genotypes and a higher rate of recurrence than participants with ADH3(1-1) genotype.

Markov entropy backbone electrostatic descriptors for predicting proteins biological activity.

The spherical truncation of electrostatic interactions between aminoacids makes it possible to break down long-range spatial electrostatic interactions, resulting in short-range interactions. As a result, a Markov Chain model may be used to calculate the probabilities with which the effect of a given interaction reaches aminoacids at different distances within the backbone. The entropies of a Markov Chain model of this type may then be used to codify information about the spatial distribution of charges in the protein used in this study exploring the structure-activity relationship. In this paper, a linear discriminant analysis is reported, which correctly classified 92.3% of 26 under investigation in training and leave-one-out cross validation, purely for illustrative purposes. Classification was carried out for three possible activities: lysozymes, dihydrofolate reductases, and alcohol dehydrogenases. The discriminant analysis equations were contracted into two canonical roots. These simple canonical roots have high regression coefficients (R(c1)=0.903 and R(c2)=0.70). Root1 explains the biological activity of alcohol dehydrogenases while Root2 discriminates between lysozymes and dihydrofolate reductases. It was possible to profile the effect of core, middle, and surface aminoacids on biological activity. In contrast, a model considering classic physicochemical parameters such as: polarizability, refractivity, and partition coefficient classify correctly only the 80.8% of the proteins.

Energization of Comamonas testosteroni ATCC 17454 for indicating toxic effects of chlorophenoxy herbicides.

The toxicity of chlorophenoxy herbicides to a bacterium, strongly related to the well-known species Delftia (formerly Comamonas) acidovorans that are able to detoxify these xenobiotics, was investigated. The oxidation of n-hexanol via alcohol dehydrogenases, coupled with the generation of ATP by electron transport phosphorylation (ETP), was used as an indicator for energy-toxic effects on the growth of Comamonas testosteroni ATCC 17454. Uncoupling--reductions in ATP synthesis accompanied by increased respiration--was found to be induced by 1 mM of the classic uncoupler 2,4-dinitrophenol (2,4-DNP) at pH 7.0 and 8.0. At pH 5.4 and 6.0, the ATP synthesis and respiration were strongly inhibited by both 2,4-DNP and the chlorophenoxy herbicides tested. In contrast, 5 mM of 2,4-dichlorophenoxyacetic acid (2,4-D) and of 2-(2,4-dichlorophenoxy)-propanoic acid (2,4-DCPP) were required for detectable uncoupling effects--reduction of the P/O ratios by about 30%--at pH 7.0. These chemicals may have less uncoupling power because the concentration of their protonated (undissociated) forms (pKa values 2.7 and 3.0) is an order of magnitude lower than that of 2,4-DNP (pKa = 4.0) at this pH value. Strong uncoupling accompanied by increased respiration, like that induced by 1 mM 2,4-DNP, was also caused by 5 mM 4-(2,4-dichlorophenoxy)-butyric acid (2,4-DCPB), which correlates with its high pKa value of 4.6. The order of toxicity of the chlorophenoxy herbicides (2,4-D < 2,4-DCPP < 2,4-DCPB) to the ETP, which correlates well with the lipophilicity of their undissociated forms (log P 2.7 < 3.4 < 3.5, respectively), was confirmed by measuring their capacity to inhibit the growth of Comamonas testosteroni ATCC 17454. The results show that energization via alcohol dehydrogenases can be used as an indicator for investigating energy-toxic effects of organics on the ETP and growth of chlorophenoxy herbicide-detoxifying bacteria.

Methylenetetrahydrofolate reductase, alcohol dehydrogenase, diet, and risk of colorectal adenomas.

An increased occurrence of colorectal cancer and its adenoma precursor is observed among individuals with low intakes or circulating levels of folate, especially if alcohol intake is high, although results have not been statistically significant in all studies. We examined folate and alcohol intake and genetic polymorphisms in methylenetetrahydrofolate reductase [MTHFR 667-->T (ala-->val) and MTHFR 1298A-->C (gln-->ala)] (associated with reduced MTHFR activity) and in alcohol dehydrogenase 3 [ADH3 (2-2) associated with decreased alcohol catabolism] in relation to risk of colorectal adenoma in the Health Professionals Follow-Up Study. Among 379 cases and 726 controls, MTHFR genotypes were not appreciably related to risk of adenoma, but a suggestive interaction (P = 0.09) was observed between MTHFR 677C-->T and alcohol intake; men with TT homozygotes who consumed 30+ g/day of alcohol had an odds ratio (OR) of 3.52 [95% confidence interval (CI), 1.41-8.78] relative to drinkers of < or =5 g/day with the CC/CT genotypes. ADH3 genotype alone was not appreciably related to risk, but its influence was modified by alcohol intake. Compared with fast alcohol catabolizers [ADH3(1-1)] with low intakes of alcohol (< or =5 g/day), high consumers of alcohol (30+ g/day) had a marked increase in risk if they had the genotype associated with slow catabolism [ADH3(2-2); OR, 2.94; 95% CI, 1.24-6.92] or intermediate catabolism [ADH3(1-2)] of alcohol (OR, 1.83; 95% CI, 1.03-3.26) but not if they were fast catabolizers [ADH3(1-1); OR = 1.27; 95% CI = 0.63-2.53). In addition, an increased risk of colorectal adenoma (OR, 17.1; 95% CI, 2.1-137) was observed for those with the ADH3(2-2) genotype and high alcohol-low folate intake compared with those with low alcohol-high folate intake and the ADH3(1-1) genotype (P for interaction = 0.006). Our results indicate that high intake of alcohol is associated with an increased risk of colorectal adenoma, particularly among MTHFR 677TT and ADH3(2-2) homozygotes. The findings that alcohol interacts with a folate-related gene (MTHFR) and that the interaction between alcohol and ADH3 is stronger among those with low folate intake support the hypothesis that the carcinogenic influence of alcohol in the large bowel is mediated through folate status.

Effects of the ADH3, CYP2E1, and GSTP1 genetic polymorphisms on their expressions in Caucasian lung tissue.

Individual differences in lung cancer susceptibility should be considered for effective lung cancer prevention. We investigated the CYP2E1, ADH3, and GSTP1 genetic polymorphisms that biotransform xenobiotic carcinogens, and variations of their enzyme activity in Caucasian lung tissues (N=28), and found a variant distribution in pulmonary ADH and CYP2E1 activity. The ADH3*1/*1 subjects (N=8) showed significantly higher ADH activity than ADH3*2/*2 (N=3) subjects (P<0.01). On the other hand, we found a 5-fold variation in the pulmonary CYP2E1 activity using a sensitive HLPC/EC based technique. A subject with the CYP2E1-c/t allele showed 2-fold higher CYP2E1 activity than subjects with the c/c allele (N=14). GSTP1 expression comprised 83% of the total pulmonary GSTs. However, neither the GSTP1 polymorphism, nor other lifestyle factors, such as age, gender, smoking status, were found to be associated with pulmonary GST expression. In conclusion, subjects with the ADH3*1 allele showed higher ADH activity and acetaldehyde-DNA adducts in lung than other subjects; thus, the ADH3*1 allele could be considered a risk factor for lung cancer.

Alcohol dehydrogenase-I from horse liver stimulates immunoglobulin production by human hybridoma and human peripheral blood lymphocytes.

Immunoglobulin production stimulating activity of alcohol dehydrogenase [EC 1.1.1.1] was assessed. Alcohol dehydrogenase-I (ADH-I) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells producing human lung cancer specific monoclonal IgM. IgM production of HB4C5 cells was enhanced more than 6 fold by the addition of ADH-I at 400 microg/ml under serum-free condition. However, yeast derived ADHs, such as ADH-II and -III were ineffective to accelerate immunoglobulin production of the hybridoma line. These results imply that the immunoglobulin production stimulating effect of ADH-I is irrelevant to its enzymatic function, and defined as a novel feature of ADH-I. This enzyme also stimulated IgM and IgG production by human peripheral blood lymphocytes 2.9 fold and 1.4 fold, respectively. This fact suggests that ADH-I stimulates immunoglobulin production not only by specific hybridoma cell line, but also by non-specific immunoglobulin producers.

Chemoenzymatic synthesis of chiral biologically active heterocycles.

The chemoenzymatic approach to the preparation of some chiral biologically active heterocycles is discussed. Synthetic strategies took advantage of enantioselective bioconversion processes carried out on suitable reaction intermediates. Reductions of carbonyl compounds catalyzed by different alcohol dehydogenases (TBADH from Thermoanaerobium brockii, 20 beta-HSDH from Streptomyces hydrogenans, beta-HSDH from Pseudomonas testosteroni) allowed the preparation with high enantiomeric purity of the eutomer of broxaterol (a selective beta 2-adrenergic agonist) and six out of the eight muscarine stereoisomers. On the other hand, hydrolyses, catalyzed by lipase PS (from Pseudomonas cepacia), of racemic butyrates were the key step in the synthesis of both the enantiomers of two muscarinic antagonists. Finally, the preparation of acetyl cycloserine antipodes was attained by means of a highly enantioselective hydrolysis catalyzed by lipase from Chromobacterium viscosum.

Oxidation of 3-butene-1,2-diol by alcohol dehydrogenase.

3-Butene-1,2-diol (BDD) is a metabolite of the carcinogenic petrochemical 1,3-butadiene. BDD is produced by cytochrome P450-mediated oxidation of 1,3-butadiene to butadiene monoxide, followed by enzymatic hydrolysis by epoxide hydrolase. The metabolic disposition of BDD is unknown. The current work characterizes BDD oxidation by purified horse liver alcohol dehydrogenase (ADH) and by cytosolic ADH from mouse, rat, and human liver. BDD is oxidized by purified horse liver ADH in a stereoselective manner, with (S)-BDD oxidized at approximately 7 times the rate of (R)-BDD. Attempts to detect and identify metabolites of BDD using purified horse liver ADH demonstrated formation of a single stable metabolite, 1-hydroxy-2-butanone (HBO). A second possible metabolite, 1-hydroxy-3-butene-2-one (HBONE), was tentatively identified by GC/MS, but HBONE formation could not be clearly attributed to BDD oxidation, possibly due to its rapid decomposition in the incubation mixture. Formation of HBO by ADH was dependent upon reaction time, protein concentration, substrate concentration, and the presence of NAD. Inclusion of GSH or 4-methylpyrazole in the incubation mixture resulted in inhibition of HBO formation. Based on these results and other lines of evidence, a mechanism is proposed for HBO formation involving generation of several potentially reactive intermediates which could contribute to toxicity of 1,3-butadiene in exposed individuals. Comparison of kinetics of BDD oxidation in rat, mouse, and human liver cytosol did not reveal significant differences in catalytic efficiency (Vmax/K(m)) between species. These results may contribute to a better understanding of 1,3-butadiene metabolism and toxicity.

4-Chlorothreonine is substrate, mechanistic probe, and mechanism-based inactivator of serine hydroxymethyltransferase.

Serine hydroxymethyltransferase catalyzes the cleavage of a variety of beta-hydroxy-L-amino acids to form glycine and aldehyde products. 4-chloro-L-threonine has been synthesized and shown to be both a substrate and a mechanism-based inactivator of serine hydroxymethyltransferase. kcat values for the formation of glycine in the absence of tetrahydrofolate were determined for 4-chloro-L-threonine and other beta-hydroxyamino acid substrates; an inverse relationship between the rate of cleavage of the amino acid and the electrophilicity of the product aldehyde was demonstrated. 4-Chloro-L-threonine inactivates serine hydroxymethyltransferase in a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations. Our evidence suggests that 4-chlorothreonine undergoes aldol cleavage, and generation of chloroacetaldehyde at the active site of the enzyme results in inactivation. Serine or glycine protect the enzyme against inactivation by chlorothreonine, while tetrahydrofolate does not. The enzyme is also protected from inactivation by 2-mercaptoethanol or by alcohol dehydrogenase and NADH. These studies suggest that halothreonine derivatives that generate electrophilic aldehyde products will be effective inhibitors of serine hydroxymethyltransferase and might be potentially useful chemotherapeutic agents.

Stimulatory effect of ethanol on weak organic acid uptake in rat renal tubules.

Ethanol at relatively low concentrations (10-40 mM) significantly stimulated the uphill uptake of a weak organic acid, fluorescein, in the superficial proximal tubules of rat renal cortex slices, but it did not affect the rate of glucose production from lactate or pyruvate in rat renal cortex fragment suspension. In a low Na+ medium, ethanol failed to stimulate fluorescein uptake, although under the conditions employed in the present study, the baseline weak organic acid uptake was not dependent on external Na+. The stimulation of fluorescein uptake by ethanol (20 mM) was abolished by an inhibitor of alcohol dehydrogenase (EC 1.1.1.1), pyrazole (1 mM), or an inhibitor of aldehyde dehydrogenase (EC 1.2.1.3), cyanamide (0.3 mM), suggesting that oxidation of ethanol mediated its effect on the uptake. Among gluconeogenesis inhibitors tested, only D-malate (2 mM) abolished the stimulatory effect of ethanol, while the rest either did not affect (quinolinate) or even slightly augmented (alpha-cyano-4-hydroxycinnamate and phenylpyruvate) it. The effect of ethanol was markedly increased by an inhibitor of the tricarboxylic acid cycle, fluoroacetate. It was concluded that the stimulation by ethanol of weak organic acid uptake in rat renal tubules was mediated by the production of acetate.

Metabolism and cytotoxicity of trans,trans-muconaldehyde and its derivatives: potential markers of benzene ring cleavage reactions.

trans,trans-Muconaldehyde (MA) has been proposed to be a myelotoxic metabolite of benzene, although it has not been isolated from benzene administration in vivo. Since the reactivity and further metabolism of MA may preclude its isolation, we have examined the metabolism of MA by: (a) mixtures of yeast alcohol and aldehyde dehydrogenases, (b) mouse liver cytosol, and (c) isolated rat hepatocytes. In all three systems, MA was metabolized rapidly and the major stable end-product of metabolism was the hydroxy/acid (OH/COOH) derivative of MA. The major route of metabolism involved initial reduction to the hydroxy/aldehyde (OH/CHO) derivative. trans,trans-Muconic acid (COOH/COOH), which is used as a marker of benzene ring cleavage reactions in vivo, was also formed from MA albeit to a much lesser extent compared to the OH/COOH. The thiol reactivity, metabolism, and cytotoxicity of MA and its different redox forms (i.e., OH/OH, OH/CHO, COOH/CHO, COOH/COOH, OH/COOH) were also investigated. MA was found to react most rapidly with reduced glutathione (GSH) in a cell-free system and was also the most cytotoxic to rat hepatocytes. Apart from MA, only the OH/CHO demonstrated GSH-reactivity and cytotoxicity. The OH/CHO was a major initial metabolite in all three systems and, thus, could represent a less reactive but more diffusible derivative of MA. These studies define the metabolism and cytotoxicity of MA and its redox derivatives and suggest that the OH/COOH metabolite of MA may have relevance as a marker of ring cleavage reactions of benzene in vivo.