Ronald Lindahl

Comparative subcellular distribution of aldehyde dehydrogenase in rat, mouse and rabbit liver

The subcellular distribution of hepatic aldehyde dehydrogenase (ALDH) activity was determined in Buffalo, Fischer 344, Long-Evans, Sprague-Dawley, Wistar and Purdue/Wistar rats. These subcellular distributions were compared to the distribution of mouse and rabbit liver ALDH. For the six rat strains, at millimolar propionaldehyde concentrations, NAD-dependent ALDH activity was associated primarily with mitochondria (51%) and microsomes (30%). At millimolar acetaldehyde concentrations, NAD-dependent ALDH was primarily mitochondrial (up to 80%). Less than 1% of total NAD-dependent aldehyde dehydrogenase was found in the cytosol. The highly inbred Purdue/Wistar line possessed significantly less acetaldehyde-NAD ALDH activity as well as less total NADP-dependent ALDH activity than the other strains. In CD-1 mouse liver, millimolar Km, NAD-dependent ALDH activity was found in mitochondria (60%), microsomes (23%) and cytosol (5%). In rabbit liver, millimolar Km, NAD-dependent ALDH was also distributed among mitochondria (36%), microsomes (19%) and cytosol (28%). At micromolar substrate concentrations, mitochondria possessed the majority of rat, mouse and rabbit liver ALDH activity. In all three species, NADP-dependent ALDH activity was found predominantly in the microsomal fraction (up to 65%). The cytosol possessed little NADP-dependent ALDH in any species. We conclude that there are significant species differences in the subcellular distribution of aldehyde dehydrogenase between rat, mouse and rabbit liver. In all three species, mitochondria and microsomes possessed the majority of hepatic aldehyde dehydrogenase activity. However, the cytosol of mouse and rabbit liver also made a significant contribution to total ALDH activity. For the six rat strains examined, liver cytosol possessed little or no ALDH activity.

Characterization of rat cornea aldehyde dehydrogenase

Aldehyde dehydrogenase has been purified from rat cornea in a single step. The enzyme is a class 3 aldehyde dehydrogenase. Cornea aldehyde dehydrogenase is a 100-kDa dimer composed of 51-kDa subunits, prefers NADP+ as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes as well as medium chain length (4-9 carbons) aliphatic aldehydes. The substrate and coenzyme specificity, immunochemical properties, effect of disulfiram, pH profile, and isoelectric point of cornea aldehyde dehydrogenase are identical to those of tumor-associated aldehyde dehydrogenase, the prototype class 3 enzyme. The substrate and coenzyme preferences are consistent with a role for cornea aldehyde dehydrogenase in the oxidation of a variety of aldehydes generated by lipid metabolism, including lipid peroxidation.

Substrate preference of a cytosolic aldehyde dehydrogenase inducible in rat liver by treatment with 3-methylcholanthrene

The substrate preference of an aldehyde dehydrogenase induced in rat liver cytosol by 3-methylcholanthrene was examined. This enzyme, T-ALDH, is identical to the aldehyde dehydrogenase inducible in rat liver by 2,3,7,8-tetrachloro-dibenzo-p-dioxin and the tumor-associated aldehyde dehydrogenase found in rat hepatocellular neoplasms. With either NAD or NADP as coenzyme, the preferred substrates were the aliphatic aldehydes n-hexanal, n-nonanal, and isobutyraldehyde and the aromatic aldehydes 2,5-dihydroxybenzaldehyde, benzaldehyde, and 3-hydroxybenzaldehyde. The results indicate that T-ALDH may play a role in oxidizing a variety of aldehydes produced in physiological lipid metabolism. On the contrary, this isozyme does not seem to participate in the oxidation of small aliphatic aldehydes generated during lipid peroxidation. Similarly, no significant activity could be detected when the enzyme was tested with aldehydes produced in carbohydrate, amino acid, polyamine, steroid, and vitamin metabolism.

Subcellular distribution and properties of rabbit liver aldehyde dehydrogenases

Abstract In rabbit liver, both NAD + - and NADP + -dependent aldehyde dehydrogenases were identified. The activities were distributed among at least three major groups of isozymes identifiable by gel electrophoresis. These isozymes also differed in their substrate and coenzyme preferences, subcellular distributions, and/or responses to effectors. The NAD + -dependent aldehyde dehydrogenase activity was distributed among the mitochondrial, microsomal, and cytosolic fractions. The NADP + -dependent aldehyde dehydrogenase activity was largely microsomal, with little true cytosolic NADP + -dependent activity demonstrable. Aliphatic aldehydes were oxidized equally well by aldehyde dehydrogenases in all three fractions. Aromatic aldehydes, however, were preferentially oxidized by microsomal aldehyde dehydrogenases. Disulfiram significantly inhibited mitochondrial (45 per cent) and cytosolic (93 per cent) NAD + -dependent aldehyde dehydrogenase, but it did not cause significant inhibition of microsomal NAD + -dependent activity. Disulfiram inhibited the NADP + -dependent aldehyde dehydrogenase activity (>71 per cent) in all subcellular fractions. Diethylstilbestrol activated both NAD + - and NADP + -dependent aldehyde dehydrogenases in mitochondria and cytosol. Microsomal aldehyde dehydrogenases were not affected by diethylstilbestrol.

Characterization of aldehyde dehydrogenase from HTC rat hepatoma cells

We have proposed developing rat hepatoma cell lines as an in vitro model for studying the regulation of changes in aldehyde dehydrogenase activity occurring during hepatocarcinogenesis. Aldehyde dehydrogenase purified in a single step from HTC rat hepatoma cells is identical to the aldehyde dehydrogenase isolated from rat hepatocellular carcinomas. HTC aldehyde dehydrogenase is a 100 kDa dimer composed of 54-kDa subunits, prefers NADP+ as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes but not phenylacetaldehyde. The substrate and coenzyme specificity, effects of disulfiram, pH profile and isoelectric point of HTC aldehyde dehydrogenase are also identical to these same properties of the tumor aldehyde dehydrogenase. In immunodiffusion, both isozymes are recognized with complete identity by anti-HTC aldehyde dehydrogenase antibodies. Having established that HTC aldehyde dehydrogenase is very similar, if not identical, to the aldehyde dehydrogenase found in hepatocellular carcinomas, simplifies the development of molecular probes for examination of the regulation of tumor aldehyde dehydrogenase activity in vivo and in vitro.

Aldehyde dehydrogenase heterogeneity in rat hepatic cells.

In normal rat liver, aldehyde dehydrogenase (Aldehyde:NAD+ oxidoreductase, EC 1.2.1.3; ALDH) is found primarily in mitochondrial and microsomal fractions. During hepatocarcinogenesis, an additional tumor-associated aldehyde dehydrogenase (T-ALDH) is detectable in the cytosol of preneoplastic and neoplastic cells. We report here differences in the ALDH distribution pattern in different rat hepatoma cell lines compared to normal rat hepatocytes. Of the four basal ALDH enzymes, one mitochondrial ALDH and one microsomal ALDH account for 96% of total ALDH molecules detectable with our probes in normal hepatocytes. The other two mitochondrial and microsomal ALDH enzymes are only detectable in the appropriate subcellular fraction from large populations of cells. The tumor-associated ALDH is not detectable in normal hepatocytes. In addition to varying amounts of T-ALDH in the six different rat hepatoma cell lines examined, differences in the amounts of mitochondrial and microsomal ALDHs also occur in both high and low T-ALDH activity hepatoma cell lines. Each of five ALDH enzymes examined has a characteristic half-life varying from 45 min to 95 h.

Characteristics and aldehyde dehydrogenase activity of four rat hepatoma cell lines produced by diethylnitrosamine-phenobarbital treatment

SummaryRecent studies in our laboratory have shown that five established rat hepatoma cell lines provide a wide spectrum of tumor-associated aldehyde dehydrogenase (ALDH) activity representative of the range of activities of this enzyme seen in primary rat hepatocellular carcinomas. Four newly established rat hepatoma cell lines, RLT-2M, RLT-3C, RLT-9F, and RLT-5G, were derived from a primary hepatocellular carcinoma. The primary tumor was induced by a single injection of diethylnitrosamine (15 μM/g body weight) to a 1-d-old female S-D rat followed at weaning by chronic phenobarbital treatment. RLT-2M was established from outgrowths of minced tumor pieces. RLT-3C, RLT-9F, and RLT-5G were cloned from RLT-2M by the serial endpoint dilution. All four lines have been maintained in culture for over 100 passages. The ALDH phenotype in both the primary tumor and the four new cell lines was determined by total activity assay, gel electrophoresis, and histochemistry. By total activity assay, the primary tumor did not possess significant tumor-ALDH activity. In contrast, the four new cell lines expressed tumor-ALDH activity. However, they differed in their basal ALDH activities and in ALDH inducibility by 3-methycholanthrene, benzo(a)pyrene, and phenobarbital. Additionally, significant decreases in tumor-ALDH activity occurred when cells from each line were passaged in vivo. The four lines have been characterized by light and electron microscopic morphology, tumorigenicity, chromosome number, doubling time, and colony formation efficiency, in soft agar.

Aldehyde dehydrogenase in 2-acetylaminofluorene-induced rat hepatomas. Characterization of antigens recognized by anti-hepatoma aldehyde dehydrogenase sera

Abstract The material in normal rat liver which cross-reacts with anti-hepatoma aldehyde dehydrogenase sera has been partially purified. The normal liver cross-reacting material is a series of proteins electrophoretically identical to the series of hepatoma-specific aldehyde dehydrogenases (aldehyde:NAD(P) + oxidoreductase, EC 1.2.1.5) induced by 2-acetylaminofluorene. The molecular weights (100 000) and the isoelectric points (6.8–7.2) of the cross-reacting material are identical to the molecular weights and pI values of the hepatoma-specific isozymes. Absorption of anti-hepatoma aldehyde dehydrogenase sera with the cross-reacting material removes an antibody population which recognizes a normal liver aldehyde dehydrogenase. This isozyme accounts for 35% of the total normal liver aldehyde dehydrogenase. A second antibody population recognizes an antigen which accounts for 10% of the total normal liver aldehyde dehydrogenase. Approx. 55% of the normal liver aldehyde dehydrogenase activity is not recognized by any antibody population. In hepatomas, 15% of the total aldehyde dehydrogenase activity is immunochemically identical to the normal liver cross-reacting material. The remaining 85% of the tumor aldehyde dehydrogenase is characterizable as hepatoma specific on the basis of these immunochemical studies.

Peroxisome-associated aldehyde dehydrogenase in normal rat liver

We have combined subcellular fractionation and cytochemical staining techniques to study the distribution of aldehyde dehydrogenase in rat liver. In addition to confirming the mitochondrial and microsomal localization of aldehyde dehydrogenase, this combined approach has allowed us to demonstrate that peroxisome-like organelles possess significant aldehyde dehydrogenase. When peroxisomal fractions are cytochemically stained for aldehyde dehydrogenase, activity is observed along membranes of structures resembling peroxisomal ghosts. These bodies lack a matrix but many appear to enclose peroxisomal cores. Moderate to dense reaction product is also located in single membrane-limited structures present in fractions containing morphologically recognizable peroxisomes. On occasion, the osmiophilic precipitate is also present in the matrix of intact peroxisomes. The aldehyde dehydrogenase activity in these peroxisome-like organelles prefers aliphatic aldehydes, including acetaldehyde in both millimolar and micromolar concentrations, and NAD. Aromatic aldehydes and NADP are also metabolized, but to a lesser extent. These results indicate that peroxisome-like organelles contain an aldehyde dehydrogenase activity possessing properties compatible with a role in ethanol metabolism.