Showing papers on "Aldehyde dehydrogenase published in 1997"
TL;DR: Sequence comparisons of the class 3 ALDH with other ALDHs indicate a similar polypeptide fold, novel NAD-binding mode and catalytic site for this family, and a mechanism for enzymatic specificity and activity is postulated.
Abstract: The first structure of an aldehyde dehydrogenase (ALDH) is described at 2.6 A resolution. Each subunit of the dimeric enzyme contains an NAD-binding domain, a catalytic domain and a bridging domain. At the interface of these domains is a 15 A long funnel-shaped passage with a 6 × 12 A opening leading to a putative catalytic pocket. A new mode of NAD binding, which differs substantially from the classic β-α-β binding mode associated with the ‘Rossmann fold’, is observed which we term the β-α,β mode. Sequence comparisons of the class 3 ALDH with other ALDHs indicate a similar polypeptide fold, novel NAD-binding mode and catalytic site for this family. A mechanism for enzymatic specificity and activity is postulated.
293 citations
TL;DR: Results indicate that microsomal fatty aldehyde dehydrogenase is a distinct human aldealdehyde dehydrogenases isozyme that acts on a variety of medium- and long-chain aliphatic substrates.
131 citations
TL;DR: Asian-American men 21 to 25 years of age were recruited from advertisements in university newspapers for this randomized, double-blind, crossover study and measured blood levels of alcohol and acetaldehyde after ingestion of alcoholic or placebo beverages in Asian- American men who underwent genotyping at the ALDH2 locus.
Abstract: Background: About half of certain Asians have a deficiency of the low-Km aldehyde dehydrogenase (ALDH2) isoenzyme. This deficiency results from inheritance of mutant ALDH2*2 allele. Objective: To d...
108 citations
TL;DR: The stomach may contribute only a small portion of the alcohol metabolism observed in humans, and the liver may be the major site for first-pass metabolism, suggesting that different vulnerabilities to ethanol-induced mucosal injury may exist.
108 citations
TL;DR: Evidence for several newly identified antioxidant-inducible genes is presented, the proposed mechanisms of antioxidant signal transduction leading to enhanced expression of these enzymes are described, and the mechanisms and consequences of induction of phase 2 xenobiotic-metabolizing enzymes by antioxidants are described.
Abstract: Publisher Summary Antioxidants inhibit the propagation of free radical reactions Because of their widespread use as preservatives in processed foods, their biochemical effects have been investigated Early feeding studies indicated that butylated hydroxytoluene (BHT) increased liver weight, induced proliferation of smooth endoplasmic reticulum, and elevated several hepatic microsomal mono-oxygenase activities typical of phase 1 metabolism In addition to phase1, antioxidants also induce phase2 xenobiotic-metabolizing enzymes Phase2 enzymes detoxify activated electrophilic metabolites of xenobiotics via conjugation of endogenous substrates such as glutathione (GSH) Antioxidants act to directly terminate the propagation of free radical reactions, and increase the activity of enzymes those readily metabolize and aid in the elimination of potential cytotoxic chemicals Dietary administration of antioxidants induced phase 2 xenobiotic-metabolizing enzymes Induction of phase 2 enzymes by antioxidants was found in many organs and tissues, such as liver, lung, kidney, small intestine, colon, and spleen, thereby affording protection at many anatomical sites This chapter presents evidence for several newly identified antioxidant-inducible genes, describes the proposed mechanisms of antioxidant signal transduction leading to enhanced expression of these enzymes, and complements the information presented in related reviews concerning the mechanisms and consequences of induction of phase 2 xenobiotic-metabolizing enzymes by antioxidants The chapter discusses the genes induced by antioxidants—cytochrome P450s, Glutathione, S-transferases, NAD(P)H: quinone reductase, UDP-glucuronosyltransferases, microsomal epoxide hydrolase, aflatoxin BI-aldehyde reductase, dihydrodiol dehydrogenases, aldehyde dehydrogenases, enzymes of glutathione and reduced nicotinamide metabolism, and other proteins and enzymes The chapter delves into the mechanisms of gene induction by antioxidants—the antioxidant-response element (ARE), proteins binding and signal transduction through the ARE—and the consequences of antioxidant gene induction The regulation of antioxidant-inducible genes helps understand the signals leading to cellular transformation and carcinogenesis Antioxidants can be used to decipher the encrypted signal transduction pathways that prevent cell transformation and carcinogenesis
108 citations
TL;DR: Tobacco plants engineered to express a sugar beet betaine aldehyde dehydrogenase (BADH) cDNA acquired not only BADH activity, but also three other aldehydes activities (those measured with 3-dimethylsulfoniopropionaldehyde, 3-aminopropionde Hyde, and 4-aminobutyraldehyde), which shows that BADH is not, as believed up to now, a substrate-specific enzyme.
Abstract: Tobacco (Nicotianum tabacum L.) plants engineered to express a sugar beet (Beta vulgaris L.) betaine aldehyde dehydrogenase (BADH) cDNA acquired not only BADH activity, but also three other aldehyde dehydrogenase activities (those measured with 3-dimethylsulfoniopropionaldehyde, 3-aminopropionaldehyde, and 4-aminobutyraldehyde, all of which are natural products). This shows that BADH is not, as believed up to now, a substrate-specific enzyme and that its role may not be limited to glycine betaine synthesis.
103 citations
Journal Article•
TL;DR: This article discusses the possible physiological role of these enzymatic systems as responsible to synthesize or catabolize several endogenous metabolites that regulate growth, metabolism, differentiation and neuroendocrine function in mammals.
101 citations
TL;DR: The role of these two NAD+-ribose-binding residues was investigated, and a pre-steady state burst of NADH formation was observed for the E399Q/K and K192Q mutants with benzaldehyde, and p-nitrobenzaldehyde was oxidized faster than benzaldehyde so that when aromatic aldehydes were used as substrates, the rate-limiting step remained deacylation for all these mutants.
82 citations
TL;DR: There is still much to learn about factors affecting the non–P450 enzymes in the clinical situation, but oxidation via aldehyde oxidase and xanthine oxidase gives different metabolites to those resulting from P450 hydroxylation.
Abstract: In addition to cytochrome P450, oxidation of drugs and other xenobiotics can also be mediated by non–P450 enzymes, the most significant of which are flavin monooxygenase, monoamine oxidase, alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase and xanthine oxidase. This article highlights the importance of these non–P450 enzymes in drug metabolism. A brief introduction to each of the non–P450 oxidizing enzymes is given in this review and the oxidative reactions have been illustrated with clinical examples. Drug oxidation catalyzed by enzymes such as flavin monooxygenase and monoamine oxidase may often produce the same metabolites as those generated by P450 and thus drug interactions may be difficult to predict without a clear knowledge of the underlying enzymology. In contrast, oxidation via aldehyde oxidase and xanthine oxidase gives different metabolites to those resulting from P450 hydroxylation. Although oxidation catalyzed by non-P450 enzymes can lead to drug inactivation, oxidation may be essential for the generation of active metabolite(s). The activation of a number of prodrugs by non–P450 enzymes is thus described. It is concluded that there is still much to learn about factors affecting the non–P450 enzymes in the clinical situation.
80 citations
TL;DR: In this paper, Steinmetz et al. report the mutational analysis of other conserved residues possessing reactive side chains Arg84, Lys192, Lys 192, Thr384, Glu399, and Ser471, along with partially conserved Glu398 and Lys489.
74 citations
TL;DR: The use of specific inhibitors of ALDH and the pyruvate dehydrogenase (PDH) complex indicates that ALDH activity is important for pollen tube growth, and thus may have a function in biosynthesis or energy production.
Abstract: Acetaldehyde is one of the intermediate products of ethanolic fermentation, which can be reduced to ethanol by alcohol dehydrogenase (ADH). Alternatively, acetaldehyde can be oxidized to acetate by aldehyde dehydrogenase (ALDH) and subsequently converted to acetyl-CoA by acetyl-CoA synthetase (ACS). To study the expression of ALDHs in plants we isolated and characterized a cDNA coding for a putative mitochondrial ALDH (TobAldh2A) in Nicotiana tabacum. TobALDH2A shows 54-60% identity at the amino acid level with other ALDHs and shows 76% identity with maize Rf2, a gene involved in restoration of male fertility in cms-T maize. TobAldh2A transcripts and protein were present at high levels in the male and female reproductive tissues. Expression in vegetative tissues was much lower and no induction by anaerobic incubation was observed. This suggests that TobALDH expression is not part of the anaerobic response, but may have another function. The use of specific inhibitors of ALDH and the pyruvate dehydrogenase (PDH) complex indicates that ALDH activity is important for pollen tube growth, and thus may have a function in biosynthesis or energy production.
TL;DR: High intracolonic acetaldehyde may contribute to the pathogenesis of alcohol‐induced diarrhoea and the increased risk of colon polyps and colon cancer found to be associated with heavy alcohol consumption in man.
Abstract: Many bacteria possess marked alcohol dehydrogenase activity and in the presence of ethanol they produce reactive and toxic acetaldehyde. Acetaldehyde production mediated by microbial alcohol dehydrogenases has been demonstrated in the oropharynx and bronchopulmonary washings. Also the most important gastric pathogen, Helicobacter pylori, and many skin bacteria associating with pathological dermatological conditions, possess alcohol dehydrogenase activity and produce acetaldehyde from ethanol. The most richly colonized site of the human body, however, is the large intestine, and therefore bacterial acetaldehyde production is most important in this organ. Alcohol ingested orally is transported to the colon by blood circulation and, after the distribution phase, intracolonic ethanol levels are equal to those in the blood. In the large bowel ethanol is oxidized by a bacteriocolonic pathway. In this pathway intracolonic ethanol is at first oxidized by bacterial alcohol dehydrogenase to acetaldehyde. Then acetaldehyde is oxidized either by colonic mucosal or bacterial aldehyde dehydrogenase to acetate. Part of intracolonic acetaldehyde may also be absorbed via the portal vein and metabolized in the liver. Bacteriocolonic pathway offers a new explanation for the disappearance of a part of ethanol calories. Due to the low aldehyde dehydrogenase activity of colonic mucosa acetaldehyde accumulates in the colon. Accordingly, during ethanol oxidation highest acetaldehyde levels of the body are found in the colon and not in the liver. High intracolonic acetaldehyde may contribute to the pathogenesis of alcohol-induced diarrhoea. Acetaldehyde has been proven to be a carcinogen in experimental animals. It may therefore contribute to the increased risk of colon polyps and colon cancer found to be associated with heavy alcohol consumption in man. Intracolonic acetaldehyde may also be an important determinant of blood acetaldehyde level and a possible hepatotoxin. In addition to acetaldehyde, gut-derived endotoxin is another potential candidate in the pathogenesis of alcohol-related liver injury.
TL;DR: Observations are consistent with mechanisms where inhibition of aldehyde dehydrogenase by DSF in vitro involves oxidation of the active site, whereas MeDTC-SO forms a covalent adduct with the protein in vitro resulting in cessation of enzyme activity.
TL;DR: The results of the study indicate that MeDTC sulfoxide and sulfone are potent inhibitors of human ALDH and are reasonable candidates for the proximal inhibitors of ALDH following disulfiram administration.
Abstract: We expressed recombinant human cytosolic (ALDH1, high Km) and mitochondrial aldehyde dehydrogenase (ALDH2, low Km) in Escherichia coli and purified the enzymes to homogeneity to examine the nature of inhibition of human ALDH by disulfiram, its confirmed metabolite S-methyl N,N-diethylthiocarbamate (MeDTC) sulfoxide, and its proposed metabolite MeDTC sulfone. Disulfiram, MeDTC sulfoxide, and MeDTC sulfone, respectively, were potent inhibitors with IC50 values of 0.15 +/- 0.02 microM, 0.27 +/- 0.04 microM, and 0.12 +/- 0.02 microM for ALDH1, and 1.45 +/- 0.40 microM, 1.16 +/- 0.56, and 0.40 +/- 0.10 microM for ALDH2. Extensive dialysis did not restore the activity of the inactivated enzyme, indicating irreversible inhibition. Both the esterase and dehydrogenase activities of ALDH2 were inhibited to the same extent by MeDTC sulfone and sulfoxide, suggesting that both catalytic sites are closely linked. The time course of inhibition of ALDH appeared to be first-order for both MeDTC sulfone and MeDTC sulfoxide. Kitz and Wilson plots of the half-life of inactivation versus 1/[inhibitor] indicated that the reactions between ALDH and inhibitors were bimolecular. The pseudobimolecular rate constants (k3/KI) for the ALDH-inhibitor reactions were 1 x 10(5), 1 x 10(4), 3 x 10(3), and 1 x 10(3) s-1 M-1 ALDH1-sulfone, ALDH1-sulfoxide, ALDH2-sulfone, and ALDH2-sulfoxide, respectively. ALDH2 was not significantly protected from inactivation from either MeDTC sulfoxide or MeDTC sulfone by NAD alone, but high concentrations of NAD and acetaldehyde completely prevented inhibition. Since disulfiram is rapidly metabolized in vivo, it is believed that disulfiram is too short-lived to inhibit ALDH directly. The results of our study indicate that MeDTC sulfoxide and sulfone are potent inhibitors of human ALDH and are reasonable candidates for the proximal inhibitors of ALDH following disulfiram administration.
Journal Article•
TL;DR: The non-phosphorylating glyceraldehyde-3-ph phosphate dehydrogenase can be found in all three domains, archaea, bacteria and eukarya and is a member of the aldehyde dehydrogenases superfamily.
Abstract: The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase catalyses the irreversible reaction of glyceraldehyde-3-phosphate to 3-phosphoglycerate by the reduction of NADP to NADPH. This is in contrast to the extensively analysed phosphorylating glyceraldehyde-3-phosphate dehydrogenases which catalyse the reversible reaction of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. Sequence analysis revealed that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is not related to the phosphorylating glyceraldehyde-3-phosphate dehydrogenases but a member of the aldehyde dehydrogenase superfamily. The aldehyde dehydrogenases are of ancient origin and they have already existed in the progenote as indicated by phylogenetic analysis. Thus the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase can be found in all three domains, archaea, bacteria and eukarya. The catalytic mechanism of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase and the other aldehyde dehydrogenases resembles a thioester mechanism involving the universally conserved cysteine 298 (pea GAPN). The cofactor of the aldehyde dehydrogenases is bound in a new mode to a structure described as beta-alpha,beta-fold.
TL;DR: The cospecificity of P1 and RalDH(I) is established, show that retinoid status affects expression of its mRNA in a tissue-dependent manner, and illustrate that aldehyde dehydrogenase isozymes with extensive homology can participate in different metabolic paths.
TL;DR: Glutathione content was associated with progression of ovarian carcinomas but neither glutathione nor glutATHione S -transferases α and π or aldehyde dehydrogenase were independent factors of resistance to cyclophosphamide/carboplatin.
TL;DR: Dosimetric estimates suggest that the intracellular acid production rates that may occur in olfactory mucosa during acetaldehyde exposure may be sufficiently high to cause tissue damage, and may represent an important process in the production of tumors in this tissue.
Abstract: Acetaldehyde is a ubiquitous air pollutant. It is an important industrial chemical and is also produced during the combustion of wood or tobacco. In smoky indoor atmospheres concentrations of the aldehyde may reach 100 ppb. Acetaldehyde is metabolized to acetate (releasing hydrogen ion) by aldehyde dehydrogenase a process which, in most tissues, represents a detoxification pathway. In vitro, acetaldehyde forms DNA–DNA and DNA–protein crosslinks. It is a clastogen, and inducer of sister chromatid exchanges, and is, perhaps, a weak mutagen. Inhalation exposure to 1000 ppm may induce DNA–protein crosslink formation in nasal tissues in the rat in vivo. Inhalation toxicity studies have shown acetaldehyde vapor causes chronic tissue injury and tumor formation in nasal tissues at exposure concentrations of 750 ppm or higher, with nasal olfactory mucosa being more sensitive than respiratory mucosa. Dosimetric estimates suggest that marked tissue injury and carcinogenicity occurs only at inspired concentrations which are sufficiently high to overwhelm nasal aldehyde dehydrogenase detoxification capacity. The induction of squamous cell carcinomas in the respiratory mucosa by acetaldehyde displays many analogies to the induction of squamous cell carcinomas by formaldehyde. For both vapors, non-linear concentration response relationships are observed for DNA–protein crosslink formation, tissue injury, and carcinogenicity, suggesting these responses are associated. For both vapors it is possible to document an exposure concentration that produces nasal respiratory epithelial injury without increasing tumor incidence, suggesting that for respiratory mucosa-derived tumors, exposure to non-cytotoxic concentrations may pose limited carcinogenic risk. In addition to squamous cell carcinomas of the respiratory epithelium, acetaldehyde exposure also results in nasal olfactory injury and tumors (adenocarcinomas) in the rat. The studies performed to date have not demonstrated a no observable effect level for these responses, therefore, the precise role of cytotoxicity and regenerative cell proliferation in the carcinogenic process in olfactory tissues can not be evaluated. Acetaldehyde metabolism via aldehyde dehydrogenase results in the formation of two hydrogen ions. The olfactory mucosa is quite sensitive to acid and dosimetric estimates suggest that the intracellular acid production rates that may occur in olfactory mucosa during acetaldehyde exposure may be sufficiently high to cause tissue damage. Such acid-induced tissue damage may enhance the genotoxic and tumorigenic potential of acetaldehyde in olfactory mucosa, and may, therefore, represent an important process in the production of tumors in this tissue.
TL;DR: The concentration of NAD+ required for the reaction was high compared with the physiological level of NAD+, suggesting that the reaction does not occur in vivo NAD+ at physiological concentrations stimulated the aldehyde dehydrogenase reaction performed by FDH or its COOH-terminal domain using NADP+.
TL;DR: Identification of putative alcoholism vulnerability genes by direct analysis of candidate genes and genetic linkage may therefore help improve approaches to prevention and treatment.
Abstract: Recent human genetic studies suggest that a predisposition to alcohol abuse and/or to develop alcoholism may be inherited. Pedigree analysis, linkage, and association studies have helped to detect marker loci and candidate genes that may prove useful in identifying individuals at risk. In particular, molecular genetic research into the causes of alcoholism has drawn attention to the potentially important role of alcohol- and acetaldehyde-metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Functional polymorphisms have been observed at various genes encoding these enzyme proteins, all of which act to alter the rate of synthesis of the toxic metabolite acetaldehyde, or decrease its further oxidation. The occurrence of functional polymorphisms in alcohol-metabolizing enzymes makes them favored candidate genes suitable for further molecular genetic research. A positive selection of such genetic polymorphisms in some populations might act as a protective factor against alcohol abuse and alcohol-related disease outcomes. For example, individuals who show initial sensitivity to alcohol by virtue of their genetically controlled abnormality of ALDH2*2 allele are discouraged from excessive alcohol consumption. On the other hand, persons with the heterozygous ALDH2*2 genotype (ALDH2*1/2*2) are at higher risk for developing alcohol abuse-related end-organ damage than those with a homozygous ALDH2*1/2*1 genotype. Moreover, the frequency of C2 allele of cytochrome P45 02E1 was found to be higher in patients with nonfibrotic alcoholic liver disease than in patients with severe hepatic fibrosis or liver cirrhosis. Identification of putative alcoholism vulnerability genes by direct analysis of candidate genes and genetic linkage may therefore help improve approaches to prevention and treatment.
TL;DR: A bienzymic sensor for the determination of dithiocarbamate fungicides was developed based on aldehyde dehydrogenase inhibition and the sensitivity of the sensor was improved by lowering the amount of enzyme and by increasing the contact time between the pesticide and the enzyme.
TL;DR: Two highly fluorogenic aldehydes were examined as indicators of the aldehyde dehydrogenase (ALDH) activity in human tissue homogenates and accessible body fluids, and their activities can be measured selectively with the two fluorogenic substrates described.
TL;DR: Seizures caused by the glutamate analogs N-methyl-d-aspartate and methionine sulfoximine, or by hyperbaric oxygen, are prevented by DETC-MeSO, indicating that carbamoylation of glutamate receptors gives an antagonist effect.
TL;DR: Two flor yeast strains of Saccharomyces cerevisiae which form velum on the surface of sherry wine during biological aging have been used and found that this strain has a slower and prolonged growth in the flor film, which permits a continued accumulation of acetaldehyde in the wine.
Abstract: Two flor yeast strains of Saccharomyces cerevisiae (S. cerevisiae strains capensis and bayanus) which form velum on the surface of sherry wine during biological aging have been used. Aldehyde and alcohol (isoenzymes I and II) dehydrogenases were detected in vitro during the entire wine-aging process in the flor yeast strains. All enzymatic activities decreased during the first 155 days of wine aging, and after this period, an increase was observed. Ethanol consumption in the wine and the specific activity of alcohol dehydrogenase I were independent of the S. cerevisiae strain. The greater activity of alcohol dehydrogenase II is directly related to the higher acetaldehyde production by S. cerevisiae race bayanus in the wine. This strain has a slower and prolonged growth in the flor film, which permits a continued accumulation of acetaldehyde in the wine. The higher activity of aldehyde dehydrogenase in capensis strain during the flor formation may be related to the production and consumption of large amoun...
TL;DR: The results indicate that the evolution of DMSP biosynthesis in flowering plants could have been facile in that it required no new aldehyde dehydrogenase; BADH may simply have been recruited for a novel function.
TL;DR: It is concluded that the alcohol drinking preference between the B6 and D2 inbred mouse strains is genetically determined, in part, by the level of brain catalase activity which, in turn, regulates brain acetaldehyde concentrations.
Abstract: Genetic factors are known to influence the preference for drinking alcohol-in humans as well as certain inbred strains of laboratory animals. Here we examined the possible role of the aromatic hydrocarbon receptor (AHR) in alcohol-preferring C57BL/6J (B6, high-affinity AHR) and alcohol-avoiding DBA/2J (D2, low-affinity AHR) inbred mouse strains, and in the two congenic lines B6.D2-Ahrd (> 99% B6 genome with the D2 low-affinity AHR) and D2.B6-Ahrb-1 (> 99% D2 genome with the B6 high-affinity AHR). This laboratory had previously shown an association between resistance to intraperitoneal ethanol-induced toxicity and the high-affinity AHR. Offering the choice between drinking water and 10% ethanol, we found that alcohol preference is three- to four-fold greater in B6 than D2 mice, as well as three- to four-fold greater in B6.D2-Ahrd than D2.B6-Ahrb-1 mice-indicating that alcohol preference is AHR-independent. The prototype AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) did not affect the rates of chronic alcohol consumption in B6 or D2 mice, suggesting that dioxin-inducible metabolism does not play a major role in alcohol drinking preference. In B6 mice, we found that oral treatment with the aldehyde dehydrogenase (ALDH) inhibitor disulfiram decreased alcohol preference by 50%, whereas oral treatment of the catalase inhibitor 3-amino-1,2,4-triazole increased alcohol drinking preference by 15-20%. Although liver and brain ALDH activities were both significantly higher in D2 than B6, these activities were not related to alcohol consumption. Hepatic and brain catalase activities, on the other hand, were two- to three-fold higher in D2 and D2.B6-Ahrb-1 mice, compared with that in B6 and B6.D2-Ahrd. Furthermore, brain acetaldehyde levels were inversely related to the quantity of alcohol voluntarily consumed. We conclude that the alcohol drinking preference between the B6 and D2 inbred mouse strains is independent of the Ah receptor-but is genetically determined, in part, by the level of brain catalase activity which, in turn, regulates brain acetaldehyde concentrations.
TL;DR: The inhibition of Saccharomyces cerevisiae aldehyde dehydrogenase by gaseous nitric oxide in solution and by NO generated from diethylamine nonoate was time and concentration dependent and the presence of oxygen significantly reduced the extent of inhibition by NO, indicating that NO itself rather than an oxidation product of NO such as N2O3 is the inhibitory species under physiological conditions.
TL;DR: The results demonstrate that the co-culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.
Abstract: In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P-450 (CYP), including CYP1A1/2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase alpha (GST alpha), aldehyde dehydrogenase and epoxide hydrolase in co-cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin O-deethylation activity was well maintained and ethoxyresorufin O-deethylation activity gradually decreased during co-culture time. In addition, we showed that phenobarbital and 3-methylcholanthrene treatments resulted in a significant increase of these activities. Two general patterns of accumulation of liver-specific mRNAs were observed. CYP1A1/2, CYP2B1/2, CYP3A1/2, GST alpha, aldehyde dehydrogenase and epoxide hydrolase mRNAs were maintained at a stable level, whereas CYP2C6 and CYP2E1 mRNAs showed a continuous decline. In addition, we observed a strong increase of CYP1A1/2 (13.6-fold) and GST alpha (3.9-fold) mRNA expression in 3-methylcholanthrene-treated co-cultures and induction of CYP2B1/2 (19-fold), CYP2C6 (10-fold), CYP3A1/2 (11.2-fold), GST alpha (9-fold), aldehyde dehydrogenase (6-fold) and epoxide hydrolase (5-fold) mRNA expression in phenobarbital-treated co-cultures. Furthermore, we demonstrated that liver-specific gene expression was restricted to hepatocytes, with the notable exception of epoxide hydrolase and CYP2E1 which were expressed in both cell types during the co-culture, as shown by the selective recovery of both hepatocytes and rat liver epithelial cells. Finally, to investigate whether co-cultures could be used to study the molecular mechanisms regulating CYP transcription, we performed transfection of hepatocytes, before the establishment of the co-culture, with large CYP2B1 (3.9 kb) or CYP2B2 (4.5 kb) promoter chloramphenicol acetyltransferase constructs or with a construct containing a 163-bp DNA sequence element reported to confer phenobarbital responsiveness. A 2-3-fold increase over the basal level of chloramphenicol acetyltransferase activity was observed in phenobarbital-treated co-cultures transfected with the phenobarbital-responsive element construct, although phenobarbital had no effect on large CYP2B1 or CYP2B2 promoter fragments. Our results demonstrate that the co-culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.
TL;DR: The treatment of Caco-2 cells, a human colon adenocarcinoma cell line that closely resembles normal human small intestinal epithelial cells, with acetaldehyde resulted in significantly decreased activities of brush border enzymes sucrase, maltase, lactase, and gamma-glutamyltransferase; alkaline phosphatase activity was not affected.
Abstract: The treatment of Caco-2 cells, a human colon adenocarcinoma cell line that closely resembles normal human small intestinal epithelial cells, with acetaldehyde resulted in significantly decreased activities of brush border enzymes sucrase, maltase, lactase, and gamma-glutamyltransferase; alkaline phosphatase activity was not affected. In the case of sucrase and maltase, the activities were also decreased by a combination of acetaldehyde and ethanol, although ethanol alone markedly increased them. The possibility that intraintestinal acetaldehyde, formed by intestinal microbes, might play a role in some small intestinal enzyme deficiencies observed earlier in alcoholics should therefore be considered. The mechanism by which acetaldehyde alters these enzyme activities remains unclear. The observation that acetaldehyde also disturbed cell polarization, an initial step in the process of differentiation in Caco-2 cells, indicates that acetaldehyde might decrease these enzyme activities by interfering with cell differentiation. Because ethanol and acetaldehyde metabolizing enzymes have not been previously studied from Caco-2 cells, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities were also measured from these cells, and their ALDH isoenzyme pattern was characterized. Like many cancerous cell lines, Caco-2 cells were found to express no ADH. They, however, possessed ALDH activity that was comparable with normal colonic mucosal activity and also expressed the same ALDH classes (ALDHs 1 to 3) than normal human colonic mucosa.
Journal Article•
TL;DR: An important in vivo role for erythrocyte ALDH-1 in systemic aldophosphamide detoxification is suggested since a wide variety of aldehydes are known to be substrates for AL DH-1.
Abstract: Detoxification of cyclophosphamide is effected, in part, by hepatic class 1 aldehyde dehydrogenase (ALDH-1)-catalyzed oxidation of aldophosphamide, a pivotal aldehyde intermediate, to the nontoxic metabolite, carboxyphosphamide. This enzyme is found in erythrocytes as well. Detoxification of aldophosphamide may also be effected by enzymes, viz. certain aldo-keto reductases, that catalyze the reduction of aldophosphamide to alcophosphamide. Such enzymes are also found in erythrocytes. Not known at the onset of this investigation was whether the contribution of erythrocyte ALDH-1 and/or aldo-keto reductases to the overall systemic detoxification of circulating aldophosphamide is significant. Thus, NAD-linked oxidation and NADPH-linked reduction of aldophosphamide catalyzed by relevant erythrocyte enzymes were quantified. ALDH-1-catalyzed oxidation of aldophosphamide (160 microM) to carboxyphosphamide occurred at a mean (+/- SD) rate of 5.0 +/- 1.4 atmol/min/rbc (red blood cell). Aldo-keto reductase-catalyzed reduction of aldophosphamide (160 microM) to alcophosphamide occurred at a much slower rate, viz. 0.3 +/- 0.2 atmol/min/rbc. Thus, at a pharmacologically relevant concentration of aldophosphamide, viz. 1 microM, estimated aggregate erythrocyte ALDH-1-catalyzed aldophosphamide oxidation, viz. 2.0 micromol/min, was only about 3% of estimated aggregate hepatic enzyme-catalyzed aldophosphamide oxidation, viz. 72 micromol/min; however, this rate is greater than the estimated flow-limited rate of aldophosphamide delivery to the liver by the blood, viz. 1.5 micromol/min. These observations/considerations suggest an important in vivo role for erythrocyte ALDH-1 in systemic aldophosphamide detoxification. Erythrocyte ALDH-1-effected oxidation of other aldehydes to their corresponding acids, e.g. retinaldehyde to retinoic acid, may also be of pharmacological and/or physiological significance since a wide variety of aldehydes are known to be substrates for ALDH-1.