Changes In Aldehyde Dehydrogenase Activity Research Articles

Proliferating pancreatic beta-cells upregulate ALDH.

High levels of aldehyde dehydrogenase (ALDH) activity have been regarded as a specific feature of progenitor cells and stem cells. Hence, as an indicator of ALDH activity, aldefluor fluorescence has been widely used for the identification and isolation of stem and progenitor cells. ALDH activity was recently detected in embryonic mouse pancreas, and specifically and exclusively in adult centroacinar and terminal duct cells, suggesting that these duct cells may harbor cells of endocrine and exocrine differentiation potential in the adult pancreas. Here, we report the presence of aldefluor+ beta-cells in a beta-cell proliferation model, partial pancreatectomy. The aldefluor+ beta-cells are essentially all positive for Ki-67 and expressed high levels of cell-cycle activators such as CyclinD1, CyclinD2, and CDK4, suggesting that they are mitotic cells. Our data thus reveal a potential change in ALDH activity of proliferating beta-cells, which provides a novel method for the isolation and analysis of proliferating beta-cells. Moreover, our data also suggest that aldefluor lineage-tracing is not a proper method for analyzing progenitor or stem activity in the adult pancreas.

ALDH expression characterizes G1-phase proliferating beta cells during pregnancy.

High levels of aldehyde dehydrogenase (ALDH) activity have been detected in various progenitor and stem cells. Thus, Aldefluor fluorescence, which represents precisely the ALDH activity, has been widely used for the identification, evaluation, and isolation of stem and progenitor cells. Recently, ALDH activity was detected in embryonic and adult mouse pancreas, specifically in adult centroacinar and terminal duct cells supposed to harbor endocrine and exocrine progenitor cells in the adult pancreas. Nevertheless, ALDH activity and aldeflour fluorescence have not been examined in beta cells. Here, we report a dynamic increase in the number of aldeflour+ beta cells during pregnancy. Interestingly, nearly all these aldeflour+ beta cells are positive for Ki-67, suggesting that they are in an active cell cycle (G1, S and M phases). To determine precisely at which phase beta cells activate ALDH activity and thus become aldeflour+, we co-stained insulin with additional proliferation markers, phosphohistone3 (PHH3, a marker for M-phase proliferating cells) and Bromodeoxyuridine (BrdU, a marker for S-phase proliferating cells). Our data show little aldeflour+ beta cells that were positive for either PHH3, or BrdU, suggesting that beta cells activate ALDH and become Aldefluor+ when they enter G1-phase of active cell cycle, but may downregulate ALDH when they leave G1-phase and enter S phase. Our data thus reveal a potential change in ALDH activity of proliferating beta cells during pregnancy, which provides a novel method for isolation and analysis of proliferating beta cells. Moreover, our data also suggest that caution needs to be taken on interpretation of Aldefluor lineage-tracing data in pancreas.

Heterogeneity of aldehyde dehydrogenase expression in lung cancer cell lines is revealed by Aldefluor flow cytometry‐based assay

We have been interested in studying the roles of two aldehyde dehydrogenases in the biology of lung cancer. In this study, we seek to apply Aldefluor flow cytometry-based assay for the measurement of aldehyde dehydrogenase (ALDH) activity in lung cancer cell lines, which may become a new tool that will facilitate our continued research in this field. Several established lung cancer cell lines were used, including A549 cell line expressing siRNA against aldehyde dehydrogenase class-1A1 (ALDH1A1). Western blot analysis, spectrophotometry assay, and Aldefluor staining were used to measure protein or enzyme activity in these cell lines. For the purpose of measurement of ALDH activity by Aldefluor in cells with known high ALDH levels, cells were mixed 1:10 with immortalized lung epithelial cell line (Beas-2B), which is known to lack ALDH activity. To delineate dead cells, double staining using Aldefluor and propidium iodide (PI) was done. Double staining was also used to detect changes in ALDH activity in two different cell lines after treatment with 4-hydroperoxycyclophosphamide (4-HC). Our results show a very good correlation between Aldefluor, Western blot, and spectrophotometry assays. Mixing experiments with Beas-2B cells allowed accurate assessment of ALDH activity in A549 cells at baseline and after siRNA expression, thus establishing an approach that facilitates the measurement of very high ALDH using the Aldefluor assay. Aldefluor staining was able to detect heterogeneity in ALDH expression among as well as within the same cell lines and better assess viability after 4-HC treatment when combined with PI. Aldefluor assay can be adapted successfully to measure ALDH activity in lung cancer cells and may have the advantage of providing real time changes in ALDH activity in viable cells treated with siRNA or chemotherapy.

Comparative analysis of hepatic ethanol metabolism in Fawn-Hooded and Wistar-Kyoto rats

Results of a number of studies have supported the suggestion that a correlation exists between voluntary ethanol consumption and enhanced ethanol metabolism in some (but not all) rodent strains. However, as yet, the capacity for alcohol-preferring Fawn-Hooded (FH) rats to metabolize ethanol has not been investigated. Hence, the aim of the current study was to compare the activities of the major hepatic enzymes involved in ethanol metabolism—cytosolic alcohol dehydrogenase (ADH) and mitochondrial aldehyde dehydrogenase (ALDH)—in the FH rat and its alcohol-nonpreferring counterpart, the Wistar-Kyoto (WKY) rat. In addition, the effect of chronic (5 weeks in vivo) ethanol pretreatment on the activity of these enzymes was investigated. Alcohol-naive FH rats were found to have significantly higher ADH activity (+61%) and no significant change in ALDH activity when compared with findings for WKY rats. In addition, chronic ethanol self-administration produced a small increase in ADH activity (+14%) in WKY rats only. Taken as a whole, these findings are the first to demonstrate an increased in vitro hepatic ethanol metabolism in alcohol-preferring FH rats and further demonstrate an association between hepatic ethanol metabolism and voluntary ethanol self-administration in rodents.

Acetaldehyde metabolism by isolated hepatic mitochondria from rats consuming different amounts of ethanol

We have previously reported that in rats, ethanol intake as low as 4 g/kg per day induced mitochondrial alterations, detected by a decrease in mitochondrial O 2 uptake supported by substrates entering at the three sites of coupled phosphorylation. Since it has been reported that acetaldehyde oxidation occurs mainly inside the mitochondria, linked to the respiration chain, the effect of daily previous administration of low doses of ethanol during a month, on the acetaldehyde oxidation rate by intact rat liver mitochondria was studied. Determination of acetaldehyde oxidation rate and O 2 uptake accompanying acetaldehyde removal by liver mitochondria indicates that the mean value of these parameters studied in rats consuming low amount of ethanol (0.5—3.0 g/kg per day) was significantly higher than that of controls drinking only water ( P<0.001, in state 3, and P<0.05, in state 4). This enhancing effect of ethanol cannot be ascribed to an uncoupling of oxidative phosphorylation and also not to a change in aldehyde dehydrogenase activity which was measured in disrupted mitochondria. On the other hand, high ethanol consumption (4–7 g/kg per day), which alters mitochondrial function, did not decrease mitochondrial acetaldehyde oxidation, as compared to rats drinking water only. This result differs from the decrease induced by higher levels of previous ethanol intake (12–14 g/kg per day) as reported by several authors, showing that possibly this effect is dose dependent.

Effect of acute ethanol administration on human blood aldehyde dehydrogenase activity.

The aldehyde dehydrogenase (EC 1.2.1.3) activity in human blood was measured after acute oral administration of two different doses of ethanol to healthy subjects. No changes in aldehyde dehydrogenase activity were observed in either of the two dose groups, as compared to control subjects receiving no ethanol. Likewise, incubation of human whole blood samples with 20 mM ethanol for 2 hr caused no change in aldehyde dehydrogenase activity. The results show that human blood aldehyde dehydrogenase activity is not affected by acute ethanol administration.

Changes in aldehyde dehydrogenase activity during diethylnitrosamine-initiated rat hepatocarcinogenesis.

Diethylnitrosamine following partial hepatectomy followed by phenobarbital promotion was used to study changes in aldehyde dehydrogenase (ALDH) activity during rat hepatocarcinogenesis. Over a period of 350 days, animals were killed at intervals and the ALDH phenotype of normal liver and any lesions was characterized by histochemical analysis, total activity assays and gel electrophoresis using propionaldehyde and NAD+ to detect normal liver ALDH activities, and benzaldehyde and NADP+ for tumor-associated ALDH. In contrast to previously tested protocols, no significant changes in ALDH activity were demonstrable by histochemistry or total activity assays in preneoplastic livers. However, nine of 16 (56%) of the hepatocellular carcinomas examined expressed the tumor-associated ALDH phenotype. The present results are integrated with previous observations as a hypothesis explaining the roles of initiation and promotion in expression of the tumor-associated aldehyde dehydrogenase phenotype.

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 duringhepatocarcinogenesis. 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 110 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 immunodiffusions, 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.

Expression of tumor-associated aldehyde dehydrogenase during rat hepatocarcinogenesis using the resistant hepatocyte model.

The resistant hepatocyte model was used to study expression of tumor-associated aldehyde dehydrogenase (ALDH) activity during the course of rat hepatocarcinogenesis. The hepatic ALDH phenotype was determined at intervals over 280 days by histochemical analysis, total ALDH activity assays and gel electrophoresis, using propionaldehyde and NAD (P/NAD) to characterize normal liver ALDH activity or benzaldehyde and NADP (B/NADP) to determine tumor-associated ALDH activity. By total activity assays and gel electrophoresis, no significant changes in ALDH activity occurred until day 70. However, histochemical analysis clearly demonstrated changes in ALDH activity early in neoplastic development. Intense focal hepatocyte staining with P/NAD and/or B/NADP was first detectable at day 28. The number of P/NAD-positive foci increased until day 35 then declined until day 70. The number of B/NADP-positive foci also increased until day 35, but then remained relatively constant for the remainder of the experiment. GGT activity of serial sections indicated that early ALDH-positive lesions represent a small subpopulation (9%) of all GGT-positive foci. However, by day 168 a significant portion (80%) of persistent GGT-positive neoplastic nodules were also B/NADP-positive histochemically. In addition, virtually all hepatocellular carcinomas (96%) generated by this protocol possessed significantly elevated levels of tumor-associated ALDH by histochemical analysis, total ALDH activity and gel electrophoresis. These results indicate that early appearing ALDH-positive lesions may define one early subpopulation of all initiated cells that have a high probability of progressing to the ultimate neoplasm.

Age-related changes in aldehyde dehydrogenase activity of rat brain, liver, and heart.

Aldehyde dehydrogenase (ALDH) activity was measured in brains, livers, and hearts of 23-26-month-old and 3-month-old rats. A significant increase of ALDH activity was found in whole brain of old rats with both acetaldehyde (39%) and propionylaldehyde (15%) used as substrates. In different brain areas of old rats, with acetaldehyde used as substrate, a significant increase of ALDH activity was found in striatum (30-50%) and cerebral cortex (37%). However, no significant difference in ALDH activity was found in livers and hearts of young and old rats. Preliminary experiments showed a significant increase of aldehyde reductase activity (52%) with p-nitrobenzaldehyde used as substrate in whole brain of old rats compared with young rats. The present work indicates that an increase of ALDH activity in brain of old rats may be an adaptive phenomenon.

Effects of long-term ethanol treatment on aldehyde and alcohol dehydrogenase activities in rat liver

Administration of intoxicating doses of ethanol by gavage for 3 weeks caused weight loss and reduced hepatic aldehyde dehydrogenase activity in the soluble, mitochondrial and microsomal fractions. Rats receiving equivalent amounts of ethanol as a constituent of a liquid diet for 5 weeks gained weight and showed no changes in aldehyde dehydrogenase activity. Alcohol dehydrogenase activity was decreased in the rats treated by gavage and unchanged in those given ethanol in the diet, but in spite of this the rate of ethanol elimination was accelerated in both groups. In the livers of two strains of rats genetically selected for their difference in voluntary alcohol consumption, the mitochondrial and microsomal aldehyde dehydrogenase activities had previously been shown to be significantly higher in the alcohol-consuming (AA) than in the alcohol-avoiding (ANA) rats. Similar differences were now found after long-term intragastric ethanol administration, although in both strains the absolute levels of aldehyde dehydrogenase were reduced. Profound reduction of mitochondrial low- K m aldehyde dehydrogenase activity and high blood acetaldehyde were observed, especially in the ANA rats. This suggests a possible connection between the low activity of this enzyme and the increased acetaldehyde level.