Contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydro-genase on the oxidation of aromatic aldehydes
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AbstractAliphatic aldehydes have a high affinity toward aldehyde dehydrogenase activity but are relatively poor substrates of aldehyde oxidase and xanthine oxidase. In addition, the oxidation of xenobiotic-derived aromatic aldehydes by the latter enzymes has not been studied to any great extent. The present investigation compares the relative contribution of aldehyde dehydrogenase, aldehyde oxidase, and xanthine oxidase activities in the oxidation of substituted benzaldehydes in separate preparations. The incubation of vanillin, isovanillin, and protocatechuic aldehyde with either guinea pig liver aldehyde oxidase, bovine milk xanthine oxidase, or guinea pig liver aldehyde dehydrogenase demonstrated that the three aldehyde oxidizing enzymes had a complementary substrate specificity. Incubations were also performed with specific inhibitors of each enzyme (isovanillin for aldehyde oxidase, allopurinol for xanthine oxidase, and disulfiram for aldehyde dehydrogenase) to determine the relative contribution of each enzyme in the oxidation of these aldehydes. Under these conditions, vanillin was rapidly oxidized by aldehyde oxidase, isovanillin was predominantly metabolized by aldehyde dehydrogenase activity, and protocatechuic aldehyde was slowly oxidized, possibly by all three enzymes. Thus, aldehyde oxidase activity may be a significant factor in the oxidation of aromatic aldehydes generated from amines and alkyl benzenes during drug metabolism. In addition, this enzyme may also have a role in the catabolism of biogenic amines such as dopamine and noradrenaline where 3-methoxyphenylacetic acids are major metabolites.
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CitationBeedham, C., Panoutsopoulos, G.I. and Kouretas, D. (2004). Contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydro-genase on the oxidation of aromatic aldehydes. Chemical Research in Toxicology. Vol. 17, No. 10, pp. 1368-1376.
Link to publisher’s versionhttp://dx.doi.org/10.1021/tx030059u
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Enzymatic oxidation of vanillin, isovanillin and protocatechuic aldehyde with freshly prepared Guinea pig liver slices.Panoutsopoulos, Georgios I.; Beedham, Christine (2005)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.
Towards the development of fluorescent probes targeting aldehyde dehydrogenase (ALDH) in cancer. Expression and epigenetic modulation of ALDH1A1, ALDH2 and ALDH3A1 in selected in vitro models.Pors, Klaus; Cosentino, Laura (University of BradfordInstitute of Cancer Therapeutics, 2012)The cancer stem cell (CSC) concept is still very controversial; therefore identification and isolation of this specific population remain challenging. A variety of putative markers have been described and measurement of high aldehyde dehydrogenase (ALDH) activity has been defined as a characteristic of stem cells (SCs). In this study, a library of novel small molecules (1,4-di-substituted acetalanthraquinones, AAQs), containing an acetal group as protected aldehyde functionality, was designed with the aim of probing affinity for ALDH metabolism and demonstrating their potential as molecular fluorescent probes to identify CSCs. The AAQs were shown to be subjective to acidic hydrolysis using 2M HCl at 37ºC; however compounds containing secondary or tertiary amine functionalities in their sidechain were only partly hydrolysed at 70 ºC. Metabolism studies were conducted using cytosolic fractions from rat liver enriched in ALDHs, yeast ALDH and human recombinant ALDH1A1. Some evidence was demonstrated which linked ALDH metabolism with aldehyde functionalities of hydrolysed AAQs (HAAQs). The AAQs were shown to emit far-red fluorescence (600-750 nm). A close relationship between structure modifications and alteration of cellular localisation, with gained specificity for selected sub-cellular compartments were achieved when assessed in A549 and U-2 OS cell lines. Thermal DNA denaturation and chemosensitivity assays were used to obtain information about DNA binding properties and cytotoxicity of AAQs and HAAQ congeners. All compounds were shown to be weak*to*moderately binding to DNA, and symmetrical 1,4-di-substituted compounds were shown to be non*toxic (IC50 = 100 :/! with non-symmetrical analogues generating IC50 values in the 1-100 :/ range. No fundamental variation in the biological activity was observed when comparing AAQs with HAAQs in the A549 (+ALDH) and MCF7 (-ALDH) cell lines. A pilot investigation revealed that aberrant gene methylation was cell-type dependent for three ALDH isoforms (1A1, 2, 3A1). Decitabine treatment led to enhanced protein expression for ALDH1A1 (A549), ALDH2 (MCF7) and ALDH3A1 (A549). In contrast, the protein level was reduced for ALDH1A1 in HT29 cells after decitabine treatment. ALDH1A1, ALDH2 and ALDH3A1 were highly expressed in prostate cell lines, with expression linked to promoter methylation. In contrast, low levels of DNA methylation were found in primary prostate cancer cells and benign prostatic hyperplasia. Interestingly, ALDH1A1, considered a SC marker, was found to be expressed at low levels in CD133+/ α2β1hi stem cell fraction and upregulated in CD133-= α2β1lo differentiated prostate cancer cells. In summary, the results in this thesis demonstrate the complexity and tumour type specificity of ALDH expression. This creates challenges for the development of selective probes for CSC isolation, such as the AAQs discussed in this thesis. Although inconclusive results were obtained in regard to AAQs and their potential in targeting ALDHs, selected AAQs were shown to reveal interesting biological features highlighting them as potential non-invasive cytometric probes for tracking molecular interactions in live cells.
Metabolism of isovanillin by aldehyde oxidase, xanthine oxidase, aldehyde dehydrogenase and liver slices.Panoutsopoulos, Georgios I.; Beedham, Christine (2005)Aromatic aldehydes are good substrates of aldehyde dehydrogenase activity but are relatively poor substrates of aldehyde oxidase and xanthine oxidase. However, the oxidation of xenobiotic-derived aromatic aldehydes by thelatter enzymes has not been studied to any great extent. The present investigation compares the relative contribution of aldehyde dehydrogenase, aldehyde oxidase and xanthine oxidase activities in the oxidation of isovanillin in separate preparations and also in freshly prepared and cryopreserved liver slices. The oxidation of isovanillin was also examined in the presence of specific inhibitors of each oxidizing enzyme. Minimal transformation of isovanillin to isovanillic acid was observed in partially purified aldehyde oxidase, which is thought to be due to residual xanthine oxidase activity. Isovanillin was rapidly metabolized to isovanillic acid by high amounts of purified xanthine oxidase, but only low amounts are present in guinea pig liver fraction. Thus the contribution of xanthine oxidase to isovanillin oxidation in guinea pig is very low. In contrast, isovanillin was rapidly catalyzed to isovanillic acid by guinea pig liver aldehyde dehydrogenase activity. The inhibitor studies revealed that isovanillin was predominantly metabolized by aldehyde dehydrogenase activity. The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared or cryopreserved liver slices has not been previously reported. In freshly prepared liver slices, isovanillin was rapidly converted to isovanillic acid, whereas the conversion was very slow in cryopreserved liver slices due to low aldehyde dehydrogenase activity. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. It is therefore concluded that isovanillin is predominantly metabolized by aldehyde dehydrogenase activity, with minimal contribution from either aldehyde oxidase or xanthine oxidase.