Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

No description available.

Abstract In the search for more reliable blood markers for excessive alcohol consumption, considerable effort has been devoted to measurements of carbohydrate-deficient transferrin (CDT), which increases in body fluids as a result of prolonged alcohol intake. In the present work, three CDT methods, CDTect (Pharmacia & Upjohn), %CDT radioimmunoassay (%CDT RIA) by Axis (Oslo, Norway), and Axis %CDT turbidimetric immunoassay (%CDT TIA) were examined for their diagnostic performance in cases of alcohol abuse with or without liver disease. The diagnostic performance of CDT as a marker of alcohol abuse correlates positively with alcohol consumption. As compared with g-glutamyltransferase (GGT) and mean corpuscular volume of erythrocytes (MCV), which are conventionally used as laboratory markers of excessive ethanol consumption, CDT (CDTect) has the highest sensitivity (64%) at the specificity level of 100% in heavy drinkers consuming >100 g ethanol/day, but its sensitivity decreases to 34% in cases with an alcohol intake of <100 g/day, which hampers the use of CDT as a community screening method. Patients with alcoholic liver disease (ALD) have significantly higher CDT values than alcoholics with non-liver pathology. However, CDT is primarily increased in cases with an early stage of ALD, so that there is a weak negative correlation between CDT and disease severity, which may prove to be of diagnostic value. Especially in men, CDTect seems to achieve greater sensitivity than %CDT RIA or %CDT TIA for detecting recent alcohol abuse among heavy drinkers, but it does have a significant correlation with serum transferrin, especially in individuals reporting social drinking or no alcohol intake. This should be considered when interpreting the assay results in patients with increased serum transferrin. %CDT methods achieve greater specificity than CDTect when analyzing samples from patients with high serum transferrin concentrations. Acetaldehyde-protein adducts are formed in the body after excessive ethanol intake, and their formation triggers antibody production, which may contribute to some forms of tissue damage seen in alcohol abusers. To obtain more information on the association between serum antibodies against acetaldehyde adducts, ALD and alcohol consumption, assays for antibodies against albumin and haemoglobin adducts were performed. Antibodies of the immunoglobulin (Ig) isotypes A, G, and M against acetaldehyde-adducts are formed in patients with prolonged heavy alcohol consumption. IgA titres in ALD patients are significantly higher than those found in patients with non-alcoholic liver disease, non-drinking controls, or heavy drinkers with no signs of liver disease. Anti-adduct IgG titres, in turn, are increased both in ALD and in heavy drinkers with no signs of liver disease as compared with non-alcoholic liver disease patients or non-drinking controls. It appears that anti-adduct IgA, IgG and IgM titres in ALD patients correlate with the severity of the liver disease. Although this association is a limitation for the usefulness of these antibodies as markers of alcohol abuse, it may serve as a basis for the differential diagnosis of alcohol-induced liver disease.

Alcohol abuse and dependence are complex disorders that are influenced by many genetic and environmental factors. Acute behavioral responses to ethanol have predictive value for determining an individual’s long-term susceptibility to alcohol abuse and dependence. These behavioral responses are strongly influenced by genetics. Here, we have explored the role of genetic influences on acute behavioral responses to ethanol using the nematode worm, Caenorhabditis elegans. First, we explored the role of ethanol metabolism in acute behavior responses to ethanol. Natural variation in human ethanol metabolism machinery is one of the most reported and reproducible associations found to alter drinking behavior. Ethanol metabolism is conserved across phyla and alteration in this pathway alters acute behavioral responses to ethanol in humans, mice, rats, and flies. We have extended these findings to the worm and have shown that loss of either alcohol dehydrogenase or aldehyde dehydrogenase results in an increase in sensitivity to the acute effects of ethanol. Second, we explored the influence of differences in basal and ethanol-induced gene expression in ethanol responsive behaviors. We identified a set of candidate genes using the basal gene expression differences in npr-1(ky13) mutant animals to enrich for genes involved in AFT. This analysis revealed ethanol changes to the expression of genes involved in a variety of biological processes including lipid metabolism. We focused on a gene involved in the metabolism of fatty acids, acs-2. acs-2 encodes an acyl-CoA synthetase that activates fatty acids for mitochondrial beta-oxidation. Animals carrying mutant acs-2 have significantly reduced AFT and we explored the role of genes in the mitochondria beta-oxidation pathway for alterations in ethanol responsive behaviors. We have shown that knockdown of ech-6, an enoyl-CoA hydratase, enhances the development of AFT. This work has uncovered a role for fatty acid utilization pathways in acute ethanol responses and we suggest that natural variation in these pathways in humans may impact the acute alcohol responses to alcohol that in turn influence susceptibility to alcohol abuse and dependence.

O consumo de álcool vem sendo associado a um aumento do risco de câncer e a uma situação de estresse oxidativo. Os metabólitos responsáveis por tais processos permanecem em discussão. Neste trabalho, caracterizamos novos metabólitos radicalares do etanol e examinamos suas interações com ácidos nucléicos. Primeiramente, demonstramos que os radicais 1-hidroxietila e 2-hidroxietila produzidos durante a oxidação do etanol por sistemas Fenton alquilam DNA e RNA in vitro produzindo os adutos 8-(1-HE)Gua e 8-(2-HE)Gua, respectivamente. Esses adutos foram sintetizados e caracterizados quimicamente. Também, demonstramos que acetaldeído, o principal metabólito do etanol, é oxidado por sistemas Fenton, peroxinitrito, xantina oxidase, partículas submitocondriais e ratos a radicais acetila e metila. Esses radicais foram caracterizados e seus mecanismos de formação elucidados, pelo menos in vitro. A possibilidade do radical 1-hidroxietila alquilar ácidos nucléicos in vivo foi também examinada. Inesperadamente, o aduto 8-(1-HE)Gua foi detectado em RNA e DNA do fígado de ratos controle e seus níveis não foram significativamente alterados após administração aguda de etanol. Esses resultados sugerem que os radicais 1-hidroxietila, acetila e metila são importantes metabólitos do etanol in vivo mas atacam preferencialmente outras biomoléculas que não ácidos nucléicos. / Alcohol consumption has been associated with increased cancer risk and an oxidative stress condition. Ethanol metabolites responsible for these processes remain debatable. Here, we characterized novel radical metabolites of ethanol and examined their interactions with nucleic acids. First, we demonstrated that the 1-hydroxyethyl and 2-hydroxyethyl radical produced from ethanol oxidation by Fenton systems alkylated DNA and RNA in vitro to produce 8-(1HE)Gua and 8-(2-HE)Gua, respectively. Both adducts were synthesized and structurally characterized. Next, we demonstrated that acetaldehyde, the main ethanol metabolite, is oxidized by Fenton systems, peroxynitrite, xanthine oxidase, submitochondrial particles and whole rats to acetyl and methyl radicals. These radicals were characterized and their production mechanisms in vitro elucidated. The possibility of the 1-hydroxyethyl radical alkylating nucleic acids in vivo was also examined. Unexpectedly, the adduct 8-(1-HE)Gua was detected in RNA and DNA from liver of control rats and their levels were not increased by acute ethanol treatment. Overall, the results suggest that the radicals 1-hydroxyethyl, acetyl and methyl are important ethanol metabolites in vivo but they preferentially attack biomolecules other than nucleic acids.

O consumo de álcool vem sendo associado a um aumento do risco de câncer e a uma situação de estresse oxidativo. Os metabólitos responsáveis por tais processos permanecem em discussão. Neste trabalho, caracterizamos novos metabólitos radicalares do etanol e examinamos suas interações com ácidos nucléicos. Primeiramente, demonstramos que os radicais 1-hidroxietila e 2-hidroxietila produzidos durante a oxidação do etanol por sistemas Fenton alquilam DNA e RNA in vitro produzindo os adutos 8-(1-HE)Gua e 8-(2-HE)Gua, respectivamente. Esses adutos foram sintetizados e caracterizados quimicamente. Também, demonstramos que acetaldeído, o principal metabólito do etanol, é oxidado por sistemas Fenton, peroxinitrito, xantina oxidase, partículas submitocondriais e ratos a radicais acetila e metila. Esses radicais foram caracterizados e seus mecanismos de formação elucidados, pelo menos in vitro. A possibilidade do radical 1-hidroxietila alquilar ácidos nucléicos in vivo foi também examinada. Inesperadamente, o aduto 8-(1-HE)Gua foi detectado em RNA e DNA do fígado de ratos controle e seus níveis não foram significativamente alterados após administração aguda de etanol. Esses resultados sugerem que os radicais 1-hidroxietila, acetila e metila são importantes metabólitos do etanol in vivo mas atacam preferencialmente outras biomoléculas que não ácidos nucléicos. / Alcohol consumption has been associated with increased cancer risk and an oxidative stress condition. Ethanol metabolites responsible for these processes remain debatable. Here, we characterized novel radical metabolites of ethanol and examined their interactions with nucleic acids. First, we demonstrated that the 1-hydroxyethyl and 2-hydroxyethyl radical produced from ethanol oxidation by Fenton systems alkylated DNA and RNA in vitro to produce 8-(1HE)Gua and 8-(2-HE)Gua, respectively. Both adducts were synthesized and structurally characterized. Next, we demonstrated that acetaldehyde, the main ethanol metabolite, is oxidized by Fenton systems, peroxynitrite, xanthine oxidase, submitochondrial particles and whole rats to acetyl and methyl radicals. These radicals were characterized and their production mechanisms in vitro elucidated. The possibility of the 1-hydroxyethyl radical alkylating nucleic acids in vivo was also examined. Unexpectedly, the adduct 8-(1-HE)Gua was detected in RNA and DNA from liver of control rats and their levels were not increased by acute ethanol treatment. Overall, the results suggest that the radicals 1-hydroxyethyl, acetyl and methyl are important ethanol metabolites in vivo but they preferentially attack biomolecules other than nucleic acids.

Indiana University-Purdue University Indianapolis (IUPUI) / Seven human alcohol dehydrogenase genes (which encode the primary enzymes involved in alcohol metabolism) are grouped into classes based on function and sequence identity. While the Class I ADH isoenzymes contribute significantly to ethanol metabolism in the liver, Class IV ADH isoenzymes are involved in the first-pass metabolism of ethanol. It has been suggested that the ability to efficiently oxidize ethanol occurred late in primate evolution. Kinetic data obtained from the Class I ADH isoenzymes of marmoset and brown lemur, in addition to data from resurrected ancestral human Class IV ADH isoenzymes, supports this proposal--suggesting that two major events which occurred during primate evolution resulted in major adaptations toward ethanol metabolism. First, while human Class IV ADH first appeared 520 million years ago, a major adaptation to ethanol occurred very recently (approximately 15 million years ago); which was caused by a single amino acid change (A294V). This change increases the catalytic efficiency of the human Class IV enzymes toward ethanol by over 79-fold. Secondly, the Class I ADH form developed 80 million years ago--when angiosperms first began to produce fleshy fruits whose sugars are fermented to ethanol by yeasts. This was followed by the duplication and divergence of distinct Class I ADH isoforms--which occurred during mammalian radiation. This duplication event was followed by a second duplication/divergence event which occurred around or just before the emergence of prosimians (some 40 million years ago). We examined the multiple Class I isoforms from species with distinct dietary preferences (lemur and marmoset) in an effort to correlate diets rich in fermentable fruits with increased catalytic capacity toward ethanol oxidation. Our kinetic data support this hypothesis in that the species with a high content of fermentable fruit in its diet possess greater catalytic capacity toward ethanol.

The proven ability to ferment Saccharomyces cerevisiae on a large scale presents an attractive target for producing chemicals and fuels from sustainable sources. Efficient and predominant carbon flux through to ethanol is a significant engineering issue in the development of this yeast as a multi-product cell chassis used in biorefineries. In order to evaluate diversion of carbon flux away from ethanol, combinatorial deletions were investigated in genes encoding the six isozymes of alcohol dehydrogenase (ADH), which catalyse the terminal step in ethanol production. The scarless, dominant and counter- selectable amdSYM gene deletion method was optimised for generation of a combinatorial ADH knockout library in an industrially relevant strain of S. cerevisiae. Current understanding of the individual ADH genes fails to fully evaluate genotype-by-genotype and genotype-by-environment interactions: rather, further research of such a complex biological process requires a multivariate mathematical modelling approach. Application of such an approach using the Design of Experiments (DoE) methodology is appraised here as essential for detailed empirical evaluation of complex systems. DoE provided empirical evidence that in S. cerevisiae: i) the ADH2 gene is not associated with producing ethanol under anaerobic culture conditions in combination with 25 g l-1 glucose substrate concentrations; ii) ADH4 is associated with increased ethanol production when the cell is confronted with a zinc-limited [1 μM] environment; and iii) ADH5 is linked with the production of ethanol, predominantly at pH 4.5. A successful metabolic engineering strategy is detailed which increases the product portfolio of S. cerevisiae, currently used for large-scale production of bioethanol. Heterologous expression of the cytochrome P450 fatty acid peroxygenase from Jeotgalicoccus sp., OleTJE, fused to the RhFRED reductase from Rhodococcus sp. NCIMB 978 converted free fatty acid precursors to C13, C15 and C17 alkenes (3.81 ng μl-1 total alkene concentration).