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T Koivisto

Publications and source records attributed to T Koivisto.

28 records · Page 2Linked to original sources
PubMed1997

Immunohistochemical determination of hepatic cytochrome P-4502E1 in formalin-fixed, paraffin-embedded sections.

Cytochrome P-4502E1 (2E1) is inducible by chronic ethanol consumption that results in enhanced activation of anesthetics and commonly used drugs (such as acetaminophen) to hepatotoxins. Therefore, assessment of hepatic 2E1 is needed in prescribing these drugs for the management of alcoholic patients. Currently, measurement of 2E1 requires either immunohistochemistry on frozen sections or Western blot (WB) analysis of homogenized tissue in excess of that needed for pathology. To obtain a more widely applicable method, we developed a procedure to detect 2E1 by immunohistochemistry in formalin-fixed, paraffin-embedded liver biopsies obtained routinely for diagnosis. Data were collected from rats fed ethanol-containing or control liquid diets for 3 weeks. Immunostaining was performed using anti-human rabbit 2E1 antibody as the primary antibody, and the immunoreaction was detected by the avidin-biotin immunoperoxidase method after treating sections with target unmasking fluid, an antigen retrieval buffer that enhanced the staining of 2E1. In control rats, 2E1 staining was weak and perivenular. After ethanol feeding, it showed a lobular gradient, strongest perivenular and weakest periportal, similar to that seen in frozen sections. The staining intensity was scored as: 0 (no staining) to 3 (strong staining). The zonal staining was scored as follows: 1 = perivenular zonal staining, 2 = midzonal, and 3 = panlobular. With the product of the two scores, a significant difference was found between alcohol-fed and control rats (5.1 +/- 0.3 vs. 0.8 +/- 0.2, p < 0.001). 2E1 assessments by WB were also significantly different for these rat pairs (68.5 +/- 2.1 vs. 7.9 +/- 0.8 arbitrary units/mg protein, p < 0.001), with a parallel increase of immunostaining scores and WB measurement of 2E1 content. This immunohistochemical method was then validated in 14 paraffin-embedded percutaneous human liver biopsy samples. In livers of nonalcoholics, 2E1 staining was seen in the perivenular zone only, whereas in samples of alcoholics, the staining was perivenular to midzonal and sometimes periportal. A significant correlation between the zonal staining scores (rs = 0.67, p < 0.005) or intensity x zonal staining scores (rs = 0.79, p < 0.001) and WB analysis was found. The immunohistochemical assessments of 2E1 expression in formalin-fixed, paraffin-embadded sections from livers of alcoholics was found to correlate with WB analysis, and lobular distribution was consistent with that seen in frozen sections. The proposed method should therefore be useful for the assessment of 2E1 content in paraffin-embedded liver samples, thereby aiding in the management of heavy drinkers.

Adult↗
PubMed1997

Effects of acetaldehyde on brush border enzyme activities in human colon adenocarcinoma cell line Caco-2.

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.

Acetaldehyde↗
PubMed1996

The determination of cytochrome P450 2E1-dependent p-nitrophenol hydroxylation by high-performance liquid chromatography with electrochemical detection.

A highly sensitive, simple assay for the determination of 4-nitrocatechol formed during the P450-dependent hydroxylation of p-nitrophenol has been developed. The assay utilizes high-performance liquid chromatography with electrochemical detection. The procedure consists of enzymatic reaction, quenching with trifluoroacetic acid, precipitation of the protein by centrifugation, and direct injection of supernatant aliquots for analysis on a reverse-phase C18 column. The electrochemical detection makes it possible to detect 0.5 pmol of 4-nitrocatechol injected, 30 times less than previously reported for an ultraviolet detector. The detector response was linear with time and with protein content, down to as little as 1.0 microgram of microsomal protein. The usefulness of the method was demonstrated with hepatic microsomal as well as S-9 fractions from both ethanol-treated and control rats, and the dependency of the reaction on cytochrome P450 2E1 activity was proven by immunoinhibition experiments.

Animals↗
PubMed1996

Aldehyde dehydrogenases of the rat colon: comparison with other tissues of the alimentary tract and the liver.

Intracolonic bacteria have previously been shown to produce substantial amounts of acetaldehyde during ethanol oxidation, and it has been suggested that this acetaldehyde might be associated with alcohol-related colonic disorders, as well as other alcohol-induced organ injuries. The capacity of colonic mucosa to remove this bacterial acetaldehyde by aldehyde dehydrogenase (ALDH) is, however, poorly known. We therefore measured ALDH activities and determined ALDH isoenzyme profiles from different subcellular fractions of rat colonic mucosa. For comparison, hepatic, gastric, and small intestinal samples were studied similarly. Alcohol dehydrogenase (ADH) activities were also measured from all of these tissues. Rat colonic mucosa was found to possess detectable amounts of ALDH activity with both micromolar and millimolar acetaldehyde concentrations and in all subcellular fractions. The ALDH activities of colonic mucosa were, however, generally low when compared with the liver and stomach, and they also tended to be lower than in small intestine. Mitochondrial low K(m) ALDH2 and cytosolic ALDH with low K(m) for acetaldehyde were expressed in the colonic mucosa, whereas some cytosolic high K(m) isoenzymes found in the small intestine and stomach were not detectable in colonic samples. Cytosolic ADH activity corresponded well to ALDH activity in different tissues: in colonic mucosa, it was approximately 6 times lower than in the liver and about one-half of gastric ADH activity. ALDH activity of the colonic mucosa should, thus, be sufficient for the removal of acetaldehyde produced by colonic mucosal ADH during ethanol oxidation. It may, however, be insufficient for the removal of the acetaldehyde produced by intracolonic bacteria. This may lead to the accumulation of acetaldehyde in the colon and colonic mucosa after ingestion of ethanol that might, at least after chronic heavy alcohol consumption, contribute to the development of alcohol-related colonic morbidity, diarrhea, and cancer.

Acetaldehyde↗
PubMed1996

Induction of cytochrome P-4502E1 by ethanol in rat Kupffer cells.

Ethanol has been shown to affect several Kupffer cell functions, but the mechanisms underlying these changes are unknown. One possible mediator is cytochrome P-4502E1 (CYP2E1), an ethanol-inducible enzyme that has been associated with toxic effects in the liver, as well as in many extrahepatic organs. To assess whether CYP2E1 can be induced by ethanol in Kupffer cells, male rats pair-fed ethanol-containing or control Lieber-DeCarli diets for 3 weeks were studied. Immunoblotting experiments showed that ethanol-treatment caused a 7-fold increase in CYP2E1 content both in Kupffer cells and hepatocytes. When expressed per milligram of S9 protein, the content of CYP2E1 in Kupffer cells was, however, 10 times lower than in hepatocytes. Immunohistochemical studies revealed that CYP2E1 is located in the endoplasmic reticulum of Kupffer cells in vivo and that it is also present in isolated Kupffer cells. In both Kupffer cells and hepatocytes, ethanol feeding increased the hydroxylation of p-nitrophenol, a relatively specific substrate for CYP2E1, demonstrating that the induced CYP2E1 was catalytically active. This reaction was significantly inhibited by anti-CYP2E1 IgG in both types of cells. Although CYP2E1 may not be the predominant pathway for ethanol metabolism in hepatocytes, it is possibly the major one in Kupffer cells. Thus, the induction of CYP2E1 by ethanol in these cells could cause significant changes in intracellular acetaldehyde concentrations which, together with increased lipid peroxidation, may contribute to the development of alcoholic liver injury.

Acetaldehyde↗
PubMed1994

Hepatic aldehyde and alcohol dehydrogenases in alcohol-preferring and alcohol-avoiding rat lines.

The alcohol-avoiding ANA (Alko, Non-Alcohol) and alcohol-preferring AA (Alko, Alcohol) rat lines are known to differ in their acetaldehyde metabolism and were originally found to differ in hepatic alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities in the 1970s. At the beginning of the 1980s, these rat lines were revitalized and some previously found line differences were lost. Thus, the purpose of this study was to determine whether these enzymatic line differences still exist and, if so, to study them further at the isoenzyme level. ADH and ALDH activities were measured from liver homogenates and different subcellular fractions of the rats. The ANA rats were found to have lower hepatic ALDH and higher ADH activities than AA rats, in accordance with the previous study. The line difference in ALDH activity was observed in all fractions, but was more apparent with millimolar than micromolar substrate concentrations and generally more pronounced in females than in males. The line difference in the microsomal ALDH activity was found to be quantitative, and it seemed to concern both microsomal ALDH isoenzymes. A qualitative line difference concerning mitochondrial high Km ALDH isoenzyme was found, and three different cytosolic ALDH isoenzyme patterns were observed, the frequencies of which were found to be different in the two lines. In conclusion, the results of the present study support the assumption that line differences in hepatic ADH and ALDH activities may be relevant to the acetaldehyde accumulation and the particularly low ethanol consumption of the ANA rats.

Alcohol Dehydrogenase↗
PubMed1993

Characteristics of Helicobacter pylori alcohol dehydrogenase.

BACKGROUND: Helicobacter pylori shows alcohol dehydrogenase activity, which in the presence of ethanol leads to in vitro production of acetaldehyde, a toxic and highly reactive substance. The present study was undertaken to further define H. pylori-related ethanol and acetaldehyde metabolism by characterizing H. pylori alcohol dehydrogenase and by determining whether the organism possesses aldehyde dehydrogenase. METHODS: Cytosolic alcohol and aldehyde dehydrogenase activities were determined spectrophotometrically. Acetaldehyde produced by cytosol during incubation with ethanol was measured by head space gas chromatography. Isoenzyme pattern was studied using isoelectric focusing. RESULTS: Significant alcohol dehydrogenase activity was observed at a neutral pH known to occur in gastric mucus. The Km for ethanol oxidation was approximately 100 mmol/L for the two strains tested. Acetaldehyde was formed already from a low ethanol concentration known to prevail in the stomach endogenously. Isoelectric focusing of the enzyme showed activity bands with pI at 7.1-7.3, a pattern different from that of gastric mucosal alcohol dehydrogenase. 4-methylpyrazole inhibited enzyme activity in a competitive manner and suppressed the growth of the organism during culture. Neither Helicobacter strain studied showed aldehyde dehydrogenase activity and can thus not remove acetaldehyde by that pathway. CONCLUSIONS: Acetaldehyde production by H. pylori from exogenous or endogenous ethanol may be a pathogenetic mechanism behind mucosal injury associated with the organism.

Acetaldehyde↗
PubMed1993

Mitochondrial aldehyde dehydrogenase (ALDH2) polymorphism in AA and ANA rats: lack of genotype and phenotype line differences.

Polymorphism of the gene coding for mitochondrial ALDH2 in humans is known to be associated with differences in alcohol drinking behavior. Recently, two different alleles of the ALDH2 gene, ALDH2R and ALDH2Q, have been found in rats also and a possible relationship between the frequencies of the two alleles and drinking behavior has been proposed. In this study, we examined whether this polymorphism of ALDH2 was the underlying cause for the previously reported acetaldehyde accumulation in the alcohol-avoiding ANA rat line and, thus, could be one of the factors explaining the differences in alcohol drinking behavior between the ANA and the alcohol-preferring AA rat lines. The experimental animals were genotyped and their mitochondrial ALDH activities and blood acetaldehyde concentrations after ethanol injection were measured. The two lines did not differ in their frequencies of ALDH2R and ALDH2Q alleles. Thus, the polymorphism in the ALDH2 gene does not explain the acetaldehyde accumulation in ANA rats and it does not seem to be associated with differences in the alcohol drinking behavior in these rat lines.

Acetaldehyde↗
PubMed1993

Detection of a CA repeat polymorphism in the rat class I alcohol dehydrogenase gene.

Differences in the amount of alcohol dehydrogenase (ADH) expressed in liver could play a role in altering ethanol metabolic rates and thus influence alcohol consumption. There is an alternating purine-pyrimidine repeat length polymorphism in the first intron of mouse Adh-1. Mouse strains that lack 101 basepairs of this 288 basepairs alternating sequence express only half as much ADH mRNA and have lower ADH-A2 activity, suggesting that this alternating sequence might modify transcription of the gene. The rat class I ADH gene also has a CA repeat in the first intron. A polymerase chain reaction (PCR) method was used to amplify the CA repeat region in different rat lines to determine whether this CA repeat is polymorphic in the rat and if so, if different length repeats correlate with the drinking behavior of rats selectively bred for high [preferring (P); high alcohol drinking (HAD); Alko, Alcohol (AA) lines] and low [non-preferring (NP); low alcohol drinking (LAD); Alko, Non-Alcohol (ANA) rat lines] alcohol drinking. A CA repeat polymorphism was detected in rat class I ADH of these different rat lines, but there was no difference in the length of the repeat between the high and low drinking rats in each line. Liver ADH activity was also not significantly different between two rat lines that have different CA repeat lengths. Thus, there is CA repeat length polymorphism in the first intron of rat class I ADH, which may be useful in genomic mapping, but it is not associated with differences in ADH activity or drinking behavior in these rat lines.

Alcohol Dehydrogenase↗