Cytochromes P450 Catalyze the Reduction of α,β-Unsaturated Aldehydes

Department of Biochemistry and Molecular Biology, The University of Louisville School of Medicine , Louisville, KY 40292, USA.
Chemical Research in Toxicology (Impact Factor: 3.53). 08/2011; 24(8):1223-30. DOI: 10.1021/tx200080b
Source: PubMed


The metabolism of α,β-unsaturated aldehydes, e.g., 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently, we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O(2), and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ≅ P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 and rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice a diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and α-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of the reduction of α,β-unsaturated aldehydes in the liver.

  • Source
    • "Detoxification of 4-HNE is an important process that has been shown to protect against tissue injury and disease progression (Galligan et al., 2012; Hartley et al., 1999; Terneus et al., 2008). Distinct enzymes have been identified that detoxify 4-HNE, including alcohol dehydrogenase, aldehyde dehydrogenase, aldo-keto reductase, alkenal/one oxidoreductase , cytochrome P450's and various glutathione S-transferases (Amunom et al., 2011; Burczynski et al., 2001; Dick et al., 2001; Forman, 2010; Hartley et al., 1995; Srivastava et al., 2000). Although metabolism of 4-HNE has been studied extensively in the liver, much less is known about its metabolism in the lung and brain, and this represents the focus of the present studies. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The lipid peroxidation end-product 4-hydroxynonenal (4-HNE) is generated in tissues during oxidative stress. As a reactive aldehyde, it forms Michael adducts with nucleophiles, a process that disrupts cellular functioning. Liver, lung and brain are highly sensitive to xenobiotic-induced oxidative stress and readily generate 4-HNE. In the present studies, we compared 4-HNE metabolism in these tissues, a process that protects against tissue injury. 4-HNE was degraded slowly in total homogenates and S9 fractions of mouse liver, lung and brain. In liver, but not lung or brain, NAD(P)+and NAD(P)H markedly stimulated 4-HNE metabolism. Similar results were observed in rat S9 fractions from these tissues. In liver, lung and brain S9 fractions, 4-HNE formed protein adducts. When NADH was used to stimulate 4-HNE metabolism, the formation of protein adducts was suppressed in liver, but not lung or brain. In both mouse and rat tissues, 4-HNE was also metabolized by glutathione S-transferases. The greatest activity was noted in livers of mice and in lungs of rats; relatively low glutathione S-transferase activity was detected in brain. In mouse hepatocytes, 4-HNE was rapidly taken up and metabolized. Simultaneously, 4-HNE-protein adducts were formed, suggesting that 4-HNE metabolism in intact cells does not prevent protein modifications. These data demonstrate that, in contrast to liver, lung and brain have a limited capacity to metabolize 4-HNE. The persistence of 4-HNE in these tissues may increase the likelihood of tissue injury during oxidative stress.
    Full-text · Article · May 2014 · Toxicology and Applied Pharmacology
  • Source
    • "At low levels, α,β-unsaturated aldehydes are oxidized by mammalian P450 enzymes to their carboxylic acid derivatives[27]. Additionally , P450 has been shown to catalyze the reduction of α,β-unsaturated aldehydes to their alcohol form in vivo[28]. Thus, redundant mechanisms for the detoxification and clearance of reactive lipid electrophiles exist to efficiently scavenge such reactive species and prevent cytotoxicity. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Oxygen is essential for the growth and function of mammalian cells. However, imbalances in oxygen or abnormalities in the ability of a cell to respond to oxygen levels can result in oxidative stress. Oxidative stress plays an important role in a number of diseases including atherosclerosis, rheumatoid arthritis, cancer, neurodegenerative diseases and asthma. When membrane lipids are exposed to high levels of oxygen or derived oxidants, they undergo lipid peroxidation to generate oxidized phospholipids (oxPL). Continual exposure to oxidants and decomposition of oxPL results in the formation of reactive electrophiles, such as 4-hydroxy-2-nonenal (HNE). Reactive lipid electrophiles have been shown to covalently modify DNA and proteins. Furthermore, exposure of cells to lipid electrophiles results in the activation of cytoprotective signaling pathways in order to promote cell survival and recovery from oxidant stress. However, if not properly managed by cellular detoxification mechanisms, the continual exposure of cells to electrophiles results in cytotoxicity. The following perspective will discuss the biological importance of lipid electrophile protein adducts including current strategies employed to identify and isolate protein adducts of lipid electrophiles as well as approaches to define cellular signaling mechanisms altered upon exposure to electrophiles. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.
    Full-text · Article · Apr 2012 · Biochimica et Biophysica Acta
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k(cat) of ∼25 min(-1) was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. The reduction was caused by a direct hydride transfer from preferentially R-NADP(2)H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP(2)H but not D(2)O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.
    No preview · Article · Feb 2012 · Biochemical and Biophysical Research Communications
Show more