Bacterial microcompartments (BMCs) are proteinaceous metabolic compartments found in a wide range of bacteria, whose function it is to encapsulate pathways for the breakdown of various carbon sources, whilst retaining toxic and volatile intermediates formed from substrate breakdown. Examples of these metabolic processes are the 1,2- propanediol-breakdown pathway in Salmonella enterica (Pdu microcompartment), as well as the ethanolamine breakdown pathway in Clostridium difficile (Eut microcompartment). Some of the major challenges to exploiting BMCs as a tool in biotechnology are understanding how enzymes are targeted to microcompartments, as well as being able to engineer the protein shell of BMCs to make synthetic microcompartments that allow specific enzyme pathways to be targeted to their interior. Finally, the metabolic burden imposed by the production of large protein complexes requires a detailed knowledge of how the expression of these systems are controlled. This project explores the structure and biochemistry of an essential BMC pathway enzyme, the acylating propionaldehyde dehydrogenase. With crystal structures of the enzyme with the cofactors in the cofactor binding site and biochemical data presented to confirm the enzyme's substrate. The project also focuses on the creation of synthetic biology tools to enable BMC engineering with a modular library of BMC shell protein parts; forward engineered ribosome binding sites (RBS) fused to BMC aldehyde dehydrogenase localisation sequences. The parts for this library were taken from the BMC loci found in Clostridium phytofermentans and Salmonella enterica. Using a synthetic biology toolkit will allow the rapid prototyping of BMC constructs for use in metabolic engineering. The shell protein parts were used to generate a number of transcriptional units, to assess the effect of overexpression of individual BMC shell components on the morphology of BMCs and the effect these had on their host chassis. Different strength forward engineered RBS and localisation constructs have been designed to assess the possibility of controlling the levels of heterologous proteins targeted to the interior of microcompartment shell to allow metabolic engineering of encapsulated pathways. Along with looking at overexpression of a single shell protein, to assess viability of BMCs as scaffold-like structures, recombinant BMCs can be explored for their utility in bioengineering and their potential role in generating biofuels.

Alkylierte polyzyklische aromatische Kohlenwasserstoffe (alk-PAK) kommen zusammen mit rein aromatischen polyzyklischen Kohlenwasserstoffen u.a. im Zigarettenrauch, Dieselabgasen sowie einigen Lebensmitteln (z.B. Freilandgemüse, planzliche Öle und Fette) vor. Benzylische Hydroxylierung und nachfolgende Sulfokonjugation ist ein wichtiger Bioaktivierungsweg für einige alk-PAK. Oxidation der benzylischen Alkohole durch Alkoholdehydrogenasen (ADH) und Aldehyddehydrogenasen (ALDH) zur Carbonsäure könnte einen wichtigen Detoxifizierungsweg in Konkurrenz zur Aktivierung durch Sulfotransferasen (SULT) darstellen, was für 1-Hydroxymethylpyren in der Ratte bereits gezeigt wurde (Ma, L., Kuhlow, A. & Glatt, H. (2002). Polycyclic Aromat Compnds 22, 933-946). Durch Hemmung der ADH und/oder ALDH ist eine verstärkte Aktivierung zu erwarten, wie in der besagten Studie ebenfalls nachgewiesen wurde. Insbesondere Ethanol kommt in diesem Zusammenhang eine Rolle als möglicher Risikofaktor für alk-PAK induzierte Kanzerogenese zu. Menschen konsumieren häufig große Mengen Ethanol und oft besteht eine Koexposition mit alk-PAK (z.B. durch Rauchen). Ähnliches gilt für 5-(Hydroxymethyl)-2-furfural (HMF), einem Pyrolyseprodukt reduzierender Zucker, dem gegenüber Menschen in recht hohen Mengen exponiert sind. Auch bei HMF steht der ADH- und ALDH-vermittelte oxidative Metabolismus in Konkurrenz zu einer Aktivierung durch Sulfokonjugation. Um die Bedeutung humaner ADH und ALDH im Metabolismus von alk-PAK und von HMF aufzuklären, wurden alle bekannten humanen ADH sowie die humanen ALDH2 und 3A1 (aus theoretischen Überlegungen heraus die vielversprechendsten Formen) für kinetische Analysen in Bakterien exprimiert. Als Enzymquelle dienten zytosolische Präparationen und durch Anionenaustauschchromatographie partiell gereinigte Enzyme. In der vorliegenden Arbeit wurde nachgewiesen, dass primäre benzylische Alkohole von Methyl- und Dimethylpyrenen gute Substrate humaner ADH sind. Sekundäre benzylische Alkohole und benzylische Alkohole von alk-PAK mit größerem Kohlenwasserstoffgrundgerüst erwiesen sich dagegen als schlechte Substrate. Vier Formen (ADH1C, 2, 3 und 4) wurden näher analysiert. Dazu wurden sie partiell gereinigt, primär um die störende endogene Bakterien-ADH zu eliminieren. Alle untersuchten ADH waren in der Lage Pyrenylmethanole zu oxidieren. Insbesondere ADH2 katalysierte die Oxidation der Pyrenylmethanole effizient, aber auch für ADH1C und 4 waren die Pyrenylmethanole gute Substrate. ADH3 oxidierte die Pyrenylmethanole mit geringer katalytischer Effizienz. Die Reduktion der entsprechenden Pyrenaldehyde durch ADH1C, 2 und 4 wurde mit noch höherer Effizienz katalysiert als die Oxidation der Pyrenylmethanole, was die Bedeutung von ALDH für die effiziente Detoxifizierung dieser Verbindungen unterstreicht. In einer an diese Arbeit angelehnten Diplomarbeit (Rost, K. (2007). Universität Potsdam, Mathematisch-Naturwissenschaftliche Fakultät) wurde auch tatsächlich gezeigt, dass humane ALDH2 aber auch ALDH3A1 in der Lage sind, die Pyrenaldehyde zu Pyrenylcarbonsäuren zu oxidieren. Die bestimmten kinetischen Parameter legen nahe, dass insbesondere ALDH2 von Bedeutung für die Detoxifizierung von Methyl- und Dimethylpyrenen ist. Schon allein auf Grund der an der Detoxifizierung beteiligten Enzyme ist Ethanolaufnahme bei Koexposition mit Pyrenderivaten als Risiokofaktor anzusehen. Es ist wahrscheinlich, dass Ethanol und, nach dessen Oxidation, Acetaldehyd als konkurrierende Substrate die ADH- und ALDH-katalysierte Oxidation von Pyrenylmethanolen bzw. Pyrenaldehyden inhibieren und somit zu einer verstärkten SULT-vermittelten Aktivierung der Pyrenylmethanole führen. In der Tat wurde eine effiziente Inhibition der ADH2-katalysierten Oxidation von 1-Hydroxymethylpyren und von 1-(Hydroxymethyl)-8-methylpyren durch physiologisch relevante Ethanolkonzentrationen nachgewiesen. Drei humane ADH (4, 2 und 3), die HMF effizient zum 2,5-Diformylfuran oxidieren können, wurden identifiziert. Durch ALDH-katalysierte Weiteroxidation dieser Substanz entsteht schließlich 2,5-Furandicarbonsäure, die nach HMF-Exposition auch tatsächlich im menschlichen Urin gefunden wurde (Jellum, E., Børresen, H. C. & Eldjarn, L. (1973). Clin Chim Acta 47, 191-201). Weiter wurde gezeigt, dass ALDH3A1, aber auch ALDH2 HMF effizient zur 5-(Hydroxymethyl)-2-furancarbonsäure (HMFA) oxidieren können, ein weiterer nachgewiesener HMF Metabolit in vivo. Dass die ADH-katalysierte Oxidation von HMFA und nachfolgende ALDH-katalysierte Oxidation zur Bildung von 2,5-Furandicarbonsäure einen nennenswerten Anteil beträgt, kann aufgrund der kinetischen Daten für HMFA als Substrat humaner ADH ausgeschlossen werden. Die beobachteten Enzymaktivitäten lassen den Schluss zu, dass Ethanolaufnahme zu einer Reduktion des oxidativen HMF Metabolismus führt und somit eine Aktivierung von HMF durch Sulfokonjugation begünstigt. / Alkylated polycyclic aromatic hydrocabons (alk-PAH), together with purely aromatic PAH, are present e.g. in tobacco smoke, diesel exhausts and also in some foods (e.g. outdoor vegetables, vegetable oils). Benzylic hydroxylation and subsequent sulfo conjugation is an important metabolic activation pathway for some of these compounds. Nevertheless, oxidation of the benzylic alcohols by alcohol dehydrogenases (ADH) and subsequently by aldehyde dehydrogenases (ALDH) can compete with the sulfo conjugation. Therefore, this pathway is probably important in the detoxification as could be shown for the representative compound 1-hydroxymethylpyrene in the rat (Ma, L., Kuhlow, A. & Glatt, H. (2002). Polycyclic Aromat Compnds 22, 933-946). Inhibition of ADH and/or ALDH should increase bioactivation as indeed was shown for 1-hydroxymethylpyrene in this study. Particularly ethanol, a competing ADH substrate, is of high interest in this context. Humans often consume large quantities of ethanol and often they are coexposed to alk-PAH (e.g. due to tobacco smoking). Similar relationships can be considered for 5-(hydroxymethyl)-2-furfural (HMF), a common pyrolysate of reducing sugars with high exposure to humans. Oxidative metabolism of HMF by ADH and ALDH also competes with its bioactivation by sulfotransferases (SULT). To clarify the importance of human ADH and ALDH in the metabolism of alk-PAH and HMF, all known human ADH as well as human ALDH2 and 3A1 (the most promising forms according to theoretical considerations) were expressed in bacteria for kinetic anlalyses. Cytosolic preparations or enzymes partially purified by anion exchange chromatography were used as enzyme source. In the present study it was shown that primary benzylic alcohols of methyl- and dimethylpyrenes were good substrates for human ADH. However, secondary benzylic alcohols and benzylic alcohols derived from alk-PAH with a bulkier hydrocarbon skeletal were poor substrates for human ADH. The most promising forms (ADH1C, 2, 3 and 4) were partially purified and further analysed. The purification step was necessary to eliminate the bacterial ADH. Particularly ADH2 was efficient for oxidation of pyrenylmethanols, although ADH1C and 4 were relatively efficient too. ADH3 was also capable of oxidising the tested pyrenylmethanols but with low catalytic efficiency. The reduction of the corresponding pyrene aldehydes was catalysed by ADH1C, 2 and 4 even with higher efficiency than the oxidation of the pyrenylmethanols emphasising the importance of ALDH for the detoxification of these compounds. In a diploma work related to the present study (Rost, K. (2007). University of Potsdam, Mathematisch-Naturwissenschaftliche Fakultät) it was shown that human ALDH2, but also ALDH3A1, can oxidise pyrene aldehydes to pyrenylcarboxylic acids. Particularly ALDH2 efficiently catalyse these reactions and, therefore, is probably of importance for the detoxification of methyl- and dimethylpyrenes. Due to the enzymes involved ethanol consumption could be a risk factor for methyl- and dimethylpyrene induced damage in the case of coexposure to methyl- and dimethylpyrenes. It is probable that ethanol and, after its oxidation, acetaldehyde will inhibit the ADH- and ALDH-catalysed oxidation of pyrenylmethanols and pyrenealdehydes. Indeed, it was shown that ADH2 catalysed oxidation of 1-hydroxymethylpyrene and of 1-(hydroxymethyl)-8-methylpyrene was efficiently inhibited by physiologically attainable concentrations of ethanol. Three human ADHs (4, 2 and 3) that efficiently oxidise HMF to 2,5-diformylfuran were identified. Further oxidation by ALDH leads to 2,5-furandicarboxylic acid, which was found in human urine after exposure to HMF (Jellum, E., Børresen, H. C. & Eldjarn, L. (1973). Clin Chim Acta 47, 191-201). Moreover, it was shown that human ALDH3A1 and also ALDH2 efficiently oxidise HMF to 5-(hydroxymethyl)-2-furancarboxylic acid (HMFA), which was also found in human urine. That 2,5-furandicarboxylic acid can be formed in significant amounts by ADH-catalysed oxidation of HMFA and subsequent oxidation by ALDH could be ruled out due to the kinetic data with HMFA as a substrate for human ADH. Due to the enzymes involved it is probable that ethanol consumption will inhibit the oxidative metabolism of HMF and, therefore, will increase the sulfo conjugation of HMF.

Organic nitrates, such as glyceryl trinitrate (GTN), have been used clinically for more than a century. However optimal nitrate therapy is hindered by the development of tolerance, which is associated with a desensitized response to GTN, oxidative stress, and the inactivation of aldehyde dehydrogenase 2 (ALDH2). This thesis evaluated the ALDH2 inactivation hypothesis of GTN tolerance and investigated the role of oxidative stress in GTN tolerance mediated by the lipid peroxidation product, 4-hydroxynonenal (HNE). Evidence for a direct role of ALDH2 in nitrate action was sought using a stably transfected cell line that overexpressed ALDH2, or siRNA to deplete endogenous ALDH2. Neither manipulation altered GTN-induced cGMP formation, indicating that ALDH2 does not mediate GTN bioactivation and tolerance. In a second study using an in vivo GTN tolerance model and a cell culture model of nitrate action, a marked increase in HNE adduct formation was detected in GTN-tolerant tissues, and treatment with HNE reduced the cGMP and vasodilator responses to GTN, thus mimicking GTN-tolerance. Together, the results suggest a primary role for HNE in the development of GTN tolerance, and provide the framework for a unified hypothesis that accommodates the previous findings of sulfhydryl depletion, ALDH2 inactivation and oxidative stress that are associated with nitrate tolerance. Studies have implicated oxidative stress and increased HNE formation in the pathogenesis of Alzheimer’s disease (AD). It was hypothesized that the gene deletion of ALDH2 would result in increased HNE-adduct formation leading to impaired cognitive function, and AD-like pathological changes. We observed a marked increase in HNE-adduct formation in Aldh2-/- mouse hippocampi as well as hyperphosphorylated tau, activated caspases, age-related changes in hippocampal amyloid βeta1-42 (Aβ1-42), post-synaptic density protein 95 (PSD95) and phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB) expression, endothelial dysfunction and other vascular pathologies. These data provide further evidence for the importance of HNE and oxidative stress in AD pathogenesis, and establish Aldh2-/- mice as a new, oxidative stress-based animal model of age-related cognitive impairment and AD. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-05-31 11:10:58.145

研究目的：以酒助眠是許多人認為可幫助睡眠的一種方式，但許多研究發現，飲酒後雖然在睡眠初期似有鎮靜鬆弛、縮短入睡時間的效果，但在入睡後的睡眠品質卻不一定良好，出現淺眠易醒的現象。但人體對酒精的反應存有明顯的個別差異，部分會對酒精產生敏感反應，出現頭暈、臉紅、心悸、噁心和嘔吐等不舒服的症狀，研究認為造成此種酒精敏感性（alcohol sensitivity）差異的原因主要來自一種乙醛脫氫酶（ALDH2）的基因型變異，導致個體在酒精代謝的過程中形成不同程度的乙醛堆積，進而影響到酒精攝取行為上的差異。然而不同個體對酒精敏感反應上的差異，是否也會影響到酒精造成睡眠狀況的變化，進而影響到個體對於酒精助眠效果的主觀感受的個別差異。因此，本研究的目的在於比較不同ALDH2基因型者，在飲酒和未飲酒的情境下，對睡眠的主觀知覺和實質睡眠參數測量的改變程度是否有差異，以了解ALDH2基因型對於酒精作用於睡眠歷程的影響。 研究方法：本研究共募集20位受試者，經基因型鑑定排除對酒精過於敏感的ALDH2*2/*2者和睡眠品質不佳者後，共14位納入正式分析。分別在睡前飲用中低劑量酒精（每公斤體重0.3克酒精）和非酒精飲料的情境下，採用多頻道睡眠記錄儀等儀器測量客觀的各項生理和睡眠指標，以及使用自填式量表評估睡前和早晨主觀的生理與心理感受。 研究結果：研究發現ALDH2*1/*1者飲酒後有入睡耗時、醒覺指數增加，以及Stage 2 睡眠潛伏期縮短的現象；ALDH2*1/*2者則有REM睡眠潛伏期延後的現象。在主觀感受上，飲酒後兩者皆在睡前誘發了明顯的生理負向感覺，早晨醒後則僅ALDH2*1/*2者有較高的生理激發程度。 結論：在給予中低劑量的酒精後，不同ALDH2基因型者其睡眠型態呈現不同的特性，也產生了生理心理反應上的差異，未來可再與睡眠相關的神經系統測量結合，釐清酒精和乙醛在影響人體睡眠中的角色，進一步了解酒精代謝對中樞神經的影響。 / Background: The effects of alcohol on sleep have been well documented. While alcohol consumption may decrease sleep onset latency, it may also lower sleep quality. Sleep problems, including prolonged sleep latency and decreased total sleep time, are more common among alcoholics than among nonalcoholics. But the effect of alcohol consumption has individual difference in the sensitivity of physiological reaction to alcohol, as reflected in facial flushing, palpitation, nausea, and other uncomfortable symptoms. The degree of physiological reaction was found to be associated with the efficiency in ethanol metabolism. Aldehyde dehydrogenase (ALDH) is the major enzymes involved in ethanol metabolism in humans. Homozygosity of the variant ALDH2*2 allele almost fully protects East Asian populations against the development of alcoholism. However, how the individual difference of alcohol sensitivity influence sleep pattern after alcohol consumption remains unknown. In this study, we aim to compare the changes of subjective report and objective measure of sleep following alcohol ingestion on variant ALDH2 genotypes. Methods: Fourteen nonalcohol-dependent subjects were recruited. After passing screening tests for ALDH2*2/*2 genotype and poor-sleep quality on PSG, subjects were required to complete two nights of PSG recording under the controlled condition (nonalcoholic beverage) and experimental condition (alcoholic beverage). Questionnaires were also used to evaluate subjective feelings before sleep and in the morning. Results: Decreased sleep onset latency and stage 2 latency as well as higher arousal index were observed in ALDH2*1/*1 subjects after challenge with a moderate dose of ethanol (0.3 g/kg of body weight), while only longer REM latency was showed in ALDH2*1/*2 individuals. As for subjective feelings, both genotypes showed more intense negative physiological responses before sleep, while only ALDH2*1/*2 individuals showed increased physical arousal the next morning. Conclusions: The variation of ALDH2*2 allele contributes to individual differences of alcohol sensitivity. The results indicate that sleep patterns and subjective perceptions following by alcohol administration were also affected by ALDH2 genotypes. This study suggests that sleep-related psychological factors may be crucial for the development of alcoholism.

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 144-161). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

The aldehyde dehydrogenase (ALDH) superfamily is gaining momentum in regard to stem cell and cancer research. However, their regulation and expression in the cancer microenvironment is poorly understood. The aim of this work was to understand the role of selected ALDH isoforms (1A1, 1A2, 1A3, 1B1, 2, 3A1 and 7A1) in colorectal cancer (CRC) and explore the impact of hypoxia on their expression. CRC cell lines (HT29, DLD-1, SW480 and HCT116) were grown under normoxic or hypoxic conditions (0.1% O2) and HT29 and DLD-1 in spinner flasks to generate multicellular spheroids (MCS). Hypoxia was demonstrated to have an impact on the ALDH expression, which appeared cell-specific. Notably, ALDH7A1 was induced upon exposure to hypoxia in both HT29 and DLD-1 cells, shown to be expressed in the hypoxic region of the MCS variants and in 5/5 CRC xenografts (HT29, DLD-1, HCT116, SW620, and COLO205). ALDH7A1 siRNA knockdown studies in DLD-1 cells resulted in significant reduction of viable cells and significant increase in ROS levels, suggesting ALDH7A1 to possess antioxidant properties. These findings were further supported using isogenic H1299/RFP and H1299/ALDH7A1 lung cancer cell lines. ALDH7A1, however, was found not to be involved in inhibiting the pharmacological effect or causing resistance to different cytotoxic and molecularly targeted anticancer drugs. To unravel the functional role of ALDH7A1, 9 compounds obtained from a virtual screening of 24,000 compounds from the Maybridge collection of compounds were used to probe ALDH7A1 functional activity. One compound, HAN00316, was found to inhibit the antioxidant properties of ALDH7A1 and thus could be a good starting point for further chemical tool development. Although this study underpins a potential important role of ALDH7A1 in hypoxic CRC, further work is required to fully validate its potential as a biomarker and/or pharmacological target.

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No. of bitstreams: 1 garcia_dp_me_sjrp.pdf: 1928934 bytes, checksum: 40dac212e7d7b6c4fb6e39b2972fc94f (MD5) Previous issue date: 2016-03-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Dentre os efeitos negativos da exposição a poluentes nos animais aquáticos podemos destacar o estresse oxidativo, uma produção exacerbada de espécies reativas de oxigênio, consequentemente, uma cascata de eventos bioquímicos denominados peroxidação lipídica (PL) ocorrem tendo como principal produto o malondialdeído (MDA). Assim, ainda que o aumento dos níveis de MDA esteja relacionado a intoxicação por poluentes, alguns trabalhos têm mostrado uma diminuição do MDA frente a essa exposição, mesmo com alterações em enzimas antioxidantes. Deste modo, este trabalho teve por objetivo avaliar se possíveis decréscimos nos níveis de MDA em peixes Astyanax altiparanae expostos a contaminantes ambientais pode ter relação com aumentos na atividade de defesas antioxidantes, aumentos na atividade da ALDH ou excreção do MDA na água. Assim, os lambaris foram expostos a misturas de metais (cádmio e cobre) nas concentrações 40 e 100 μg/L e a biodiesel B5 nas concentrações de 0,001, 0,01 e 0,1 mL/L para verificar a relação entre MDA e as enzimas antioxidantes, em amostras de brânquia e fígado. Um segundo experimento, injetando MDA nos espécimes foi realizado, nestes organismos, foram injetados intraperitonealmente doses de 10 mg/kg e 100 mg/kg de MDA com coleta do material biológico após 5 dias para avaliar o efeito do MDA no fígado e na brânquia do lambari. Um terceiro experimento foi realizado para avaliar a metabolização do MDA em brânquia e fígado e sangue com coleta de material biológico e água nos tempos zero (logo após receber a dose), 1 e 12 horas. Nestes experimentos a metabolização do MDA foi observada pela atividade da enzima aldeído desidrogenase (ALDH) e as enzimas antioxidantes catalase (CAT), glutationa peroxidase (GPx), glicose – 6- fosfato- desidrogenase (G6PDH) e a de biotransformação glutationa S-transferase (GST), peróxidos lipídicos (FOX), níveis de glutationa total (GSH-t) e ensaio do MDA. Por fim foi realizado um experimento in vitro para avaliar o efeito do MDA nas enzimas (CAT, GPx, G6PDH, GST e ALDH). Nossos resultados mostraram que o MDA tem vias de metabolização e que o aumento das enzimas antioxidantes não é responsável por sua diminuição. Sugerimos que o B5 foi capaz de induzir a atividade da ALDH e esta diminuiu a quantidade de MDA nos dois grupos de maior concentração de B5. O MDA não foi capaz de induzir a atividade da ALDH, entretanto observamos uma forte correlação entre o MDA e a ALDH. O experimento in vitro mostrou que em concentrações elevadas o MDA pode até mesmo inibir a atividade da ALDH, sugerindo que esta enzima atue na manutenção dos níveis basais deste aldeído. Por fim, o trabalho mostrou que o lambari foi capaz de eliminar o MDA em excesso por meio da excreção, fortalecendo nossa hipótese de que o organismo dos peixes utiliza vias de excreção e da ALDH para eliminar o excesso deste aldeído toxico produzido na PL por estressores ambientais / Among the adverse effects of exposure to pollutants in aquatic animals, we can highlight oxidative stress, which can be caused due to an exacerbated production of reactive oxygen species along with a cascade of biochemical events. During this cascade, one of oxidative consequences we can observe is called lipid peroxidation (LPO) with its main product malondialdehyde (MDA). Thus, although the increase in MDA levels is related to intoxication by pollutants, some studies have shown a decrease in MDA levels in aquatic organisms exposed to pollutants, even with significant improvment in antioxidant enzymes. Thus, this study aimed to assess the relationship among the reduced MDA levels in Astyanax altiparanae fish exposed to environmental contaminants, and increased antioxidant defenses and aldehyde dehydrogenase (ALDH) activity, or excretion of MDA in the water. In this way, the fishes were exposed to mixtures of metals (cadmium or copper) at concentrations of 40 g/L and 100 g/L and to B5 biodiesel in concentrations of 0.001 mL/L, 0.01 mL/L and 0.1 mL/L in order to verify the relation between MDA and antioxidant enzymes in the liver, and gill samples. We performed a second experiment by injecting MDA in the fish specimens. We intraperitoneally injected doses of 10 mg/kg and 100 mg/kg of MDA. After 5 days, we collected the liver and gill in order to evaluate the effect of MDA in the fishes. A third experiment was conducted to investigate MDA metabolization in blood, gill and liver samples, through collection of biological material and water at zero time (shortly after receiving the same intraperitoneal injections as in the second experiment), 1 and 12 hours after the injections. In these experiments, the MDA metabolization was monitored by the activity of ALDH and by the antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PDH) and glutathione-S-transferase biotransformation (GST), lipid peroxides by ferrous ion oxidation xylenol orange (FOX) assay, the total glutathione (GSH-t) and MDA assay. Finally, an experiment was conducted in vitro to evaluate the effect of enzymes on MDA (CAT, GPx, G6PDH, GST and ALDH). Our results showed that the MDA levels have metabolizing pathways and increased antioxidant enzymes are not responsible for their decrease. We suggest that the B5 was able to induce the ALDH activity, Moreover, ALDH was related to reduced amount of MDA in both groups of higher B5 concentration. The MDA was not able to induce the activity of ALDH, despite we found a strong correlation between the MDA levels and ALDH activity. In the in vitro experiment, we observed that high concentrations of MDA could even inhibit the activity of ALDH, suggesting that this enzyme acts in the maintenance of basal levels of this aldehyde. Finally, the study showed that the Astyanax altiparanae have been able to eliminate the MDA excess by excretion. This fact reinforces our hypothesis that the fish uses excretion routes and ALDH to eliminate excess of this toxic aldehyde produced during LPO cased by stressors environmental.

Indiana University-Purdue University Indianapolis (IUPUI) / The human aldehyde dehydrogenase superfamily consists of 19 distinct genetic loci that play key roles in both health and disease. Aldehyde dehydrogenases are primarily involved in the metabolism of reactive aldehyde substrates; the ALDH1A subfamily, in particular, metabolizes retinaldehyde and is involved in a pathway regulating tissue differentiation, cell proliferation, and apoptosis. Recently, ALDH1 isoenzymes have been implicated as significant elements in cancer progression. ALDH1 activity has been used as a marker of cancer stem cells, a subpopulation of cancer stem cells with high drug resistance, proliferative potential, and ability to differentiate into multiple cell types. In accordance with this, ALDH1 activity and expression has been shown to correlate with lower survival, increased chemoresistance, and increased chance of relapse in multiple solid cancer types, including breast, ovarian, lung, and colorectal. Despite the clear relevance of ALDH1 enzymes in cancer, the specific roles of individual isoenzymes are unclear. Isoenzyme-selective small molecule modulators of the ALDH1A subfamily would allow the probing of the function of individual isoenzymes in healthy and disease states. Two ALDH1A1 inhibitors, CM38 and C10, were previously identified in a high-throughput screen. In this study, CM38, an ALDH1A1-selective inhibitor, and CM10, an ALDH1A inhibitor, were characterized using kinetic assays, structural biology, and cell culture experiments. A structure-activity relationship was built for each series, and an X-ray crystallography structure was used to determine the binding mode. These approaches allowed the investigation of the ALDH1A active site and identification of structural features that can be used to design and improve selective modulators of this subfamily. CM38 and CM10 were also tested in a breast cancer cell line to determine their efficacy in a cellular environment. While the CM38 series showed warning signs of potential off-target toxicity, members of the CM10 compound series showed excellent initial characteristics as potential chemical tools. The results of this study may be useful in the design of new chemical tools to delineate the functions of individual ALDH1 isoenzymes in cancer biology, as well as in the development of drugs to selectively target cancer stem cells.

Petroleum Technology Development Fund (PTDF) Nigeria / The full text will be available at the end of the embargo period: 21st March 2026

No / Background/Aims: The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared liver slices has not been previously reported. The present investigation compares the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activities in the oxidation of vanillin, isovanillin and protocatechuic aldehyde with freshly prepared liver slices. Methods: Vanillin, isovanillin or protocatechuic aldehyde was incubated with liver slices in the presence/absence of specific inhibitors of each enzyme, followed by HPLC. Results: Vanillin was rapidly converted to vanillic acid. Vanillic acid formation was completely inhibited by isovanillin (aldehyde oxidase inhibitor), whereas disulfiram (aldehyde dehydrogenase inhibitor) inhibited acid formation by 16% and allopurinol (xanthine oxidase inhibitor) had no effect. Isovanillin was rapidly converted to isovanillic acid. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. Protocatechuic aldehyde was converted to protocatechuic acid at a lower rate than that of vanillin or isovanillin. Allopurinol only slightly inhibited protocatechuic aldehyde oxidation, isovanillin had little effect, whereas disulfiram inhibited protocatechuic acid formation by 50%. Conclusions: In freshly prepared liver slices, vanillin is rapidly oxidized by aldehyde oxidase with little contribution from xanthine oxidase or aldehyde dehydrogenase. Isovanillin is not a substrate for aldehyde oxidase and therefore it is metabolized to isovanillic acid predominantly by aldehyde dehydrogenase. All three enzymes contribute to the oxidation of protocatechuic aldehyde to its acid.