Murine hepatic aldehyde dehydrogenase 1a1 is a major contributor to oxidation of aldehydes formed by lipid peroxidation
Research highlights
► Murine Aldh1a1 is an excellent catalyst for oxidation of (,β-unsaturated aldehydes. ► Ablation of Aldh1a1 expression in Hepa1c1c7 cells leads to increased acrolein–protein adducts and acrolein-induced cell death. ► Acrolein exposure of mice leads to induction of Aldh1a1 mRNA and enzyme activity.
Introduction
The liver as the major site for metabolism and biotransformation of drugs and foreign compounds is constantly exposed to reactive oxygen species (ROS), resulting in oxidative stress. During oxidative stress, these ROS which include hydrogen peroxide (H2O2), superoxide radicals (O2−) and hydroxyl radicals (OH) can covalently modify proteins, lipids and DNA. The peroxidation of polyunsaturated fatty acids in membrane lipid by ROS produces unstable lipid hydroperoxides which decompose into aldehydes such as malondialdehyde (MDA), hexanal, trans-2-hexenal, propen-2 al (acrolein) and 4-hydroxy-2-nonenal (HNE) [1], [2], [3]. Among these aldehydes, HNE and acrolein are highly electrophilic α,β-unsaturated aldehyde and can undergo Schiff base and Michael addition reactions with nucleophilic groups on proteins and DNA [4]. There is increasing evidence that the pathophysiological effects of ROS in cells is mediated by these cytotoxic aldehydes because they are very stable and can diffuse to distant sites of formation [5], [6].
In addition, humans are exposed to these aldehydes as environmental pollutants and by endogenous processes that generate reactive aldehydes in the liver. For example, chronic alcohol consumption, high fat diet or exposure to foreign compounds such as carbon tetrachloride (CCl4), allyl alcohol and the widely used anticancer drug cyclophosphamide markedly elevate the intracellular concentrations of cytotoxic aldehydes [7], [8], [9], [10]. Reactive α,β-unsaturated aldehyde such as acrolein and crotonaldehyde are also present in cigarette smoke, vehicle exhaust emission, overheated foods and oil, drinking water and in effluents from industrial plants [10], [11]. It is estimated that the maximum daily human consumption of unsaturated aldehydes is 5 mg/kg, while the total aldehyde consumption has been suggested to be ≈7 mg/kg [12], [13].
The levels of reactive lipid aldehydes are elevated in various oxidative stress-mediated diseases, including steatohepatitis [14], [15], atherosclerosis [16], Alzheimer's [17], cataract [18], diabetes [19] and cancer [20]. In fact, accumulation of reactive aldehydes is associated with the pathogenesis of these diseases. The toxicity of α,β-unsaturated aldehyde lies in their ability to form Michael adducts with thiol and amino groups of proteins resulting in alteration of several cellular processes. For example, enzymes such as glyceraldehyde-3-phosphate, glucose-6-phosphate dehydrogenases and cytochrome c oxidase containing lysine and cysteine residues in their active sites are readily inactivated by reactive aldehydes [21], [22], [23], [24], [25]. In addition, α,β-unsaturated aldehyde can induce oxidative stress in cells by depleting cellular reduced glutathione, thereby altering signal transduction pathways in cells. At low concentrations, acrolein is known to trigger apoptotic cell death by mechanisms that involve activation of mitochondrial death pathways and caspases [10], [26], [27]. Caspases, particularly caspase 3, which can cleave substrates such as poly(ADP-ribose) polymerase (PARP), actin, laminin are widely used as marker for apoptosis in different cell types. However, at high concentrations acrolein causes necrotic cell death. This phenomenon has also been observed with HNE [28], [29], [30].
Despite their toxicity, many cytotoxic lipid-derived aldehydes can be successfully metabolized to less toxic compounds by the action of oxidative, reductive and conjugative enzymes. These enzymes include glutathione S-transferases (GST), aldehyde dehydrogenases (ALDH), aldo-keto reductases (AKR), alcohol dehydrogenases (ADH) and cytochrome P450 (CYP) [31], [32], [33], [34]. The relative importance of these enzymes in reactive aldehydes metabolism is cell type- and species-dependent. It is now known that the conjugation of acrolein with glutathione is not a true detoxification process, since acrolein–glutathione conjugates can undergo renal processing to form reactive species [35]. In addition, the conjugation process is compromised when GSH concentrations are depleted, such as may occur during oxidative stress. ALDH oxidizes a range of toxic aldehydes to their corresponding non toxic carboxylic acids using either NAD+ or NADP+ as cofactors. They play a critical role in the cellular protection against these toxic species. There are 19 ALDH genes in the human genome [33]. To date three isozymes, the cytosolic ALDH1A1 and ALDH3A1, and the mitochondrial ALDH2 are the main lipid aldehyde-oxidizing enzymes expressed in the mouse liver.
The role of these enzymes in the cellular defense against oxidative damage induced by cytotoxic aldehydes is controversial. Previous studies by Townsend et al. revealed that ALDH1A1 overexpression provides only moderate protection against trans-2-nonenal and not against other lipid aldehydes [36]. However, ALDH3A1 could protect cells against HNE-induced apoptosis. Aldh3a1 is poorly expressed in normal liver and highly expressed in cancerous cells [37]. It is also abundantly expressed in the cornea and protects the cornea against cytotoxic lipid peroxidation-derived aldehydes [38]. However, recent experiments with Aldh1a1−/− mice indicate that Aldh1a1 protects the eye from cataract formation induced by oxidative stress by detoxifying cytotoxic lipid aldehydes [39], [40]. Moreover, the human lens ALDH1A1 efficiently oxidizes lipid-derived aldehydes, including HNE (Km 4.8 μM), trans-2-heptenal (Km 177 μM) and MDA (Km 3.5 μM) [41]. In addition, over-expression of ALDH1 in neuroblastoma cells reduces production of protein–HNE adducts and activation of caspase-3 [30]. Aldh1a1 is known to decrease the effectiveness of the anticancer drugs cyclophosphamide by detoxifying its major active metabolites acrolein [42]. These results indicate that Aldh1a1 has the potential to protect against aldehyde produced as a result of lipid peroxidation. However, it is unknown whether Aldh1a1 can protect against acrolein-induced toxicity in mouse liver. We hypothesized that Aldh1a1 is the major enzymes involved in acrolein and other lipid-derived aldehyde detoxification in mouse liver.
Although considerable characterization of the rat Aldh's in cytosol and mitochondria has been published, the relative contribution of different Aldh isozymes in detoxification of reactive aldehydes in mice liver is unknown. Moreover to date, the kinetic properties of murine Aldh orthologs for reactive lipid aldehydes oxidation especially acrolein have not been biochemically characterized. Mouse is a more common laboratory model for research in medicine because of the availability of transgenic and gene-knockout mice. In the present study, we examine the role of hepatic Aldh isozymes in detoxification of lipid-derived aldehydes such as acrolein and HNE by enzyme kinetic and gene expression studies. We showed by substrate preferences, gene expression patterns and in vitro knockdown experiments in Hepa-1c1c7 that murine Aldh1a1 is the major cytosolic Aldh in mice liver involved in cellular defense against reactive aldehydes-induced toxicity.
Section snippets
Chemicals and reagents
Propionaldehyde, benzaldehyde, trans-2-hexenal, acetaldehyde, dithiothreitol, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), isopropyl β-d-1-thiogalactopyranoside (IPTG), N-ethylmaleimide (NEM), propen-2-al (acrolein) and malonaldehyde bis-(dimethyl acetal) were purchased from Sigma–Aldrich Company, Inc. (St Louis, MO). 4-Hydroxy-2-nonenal (HNE) was obtained from Cayman Chemical Co (Ann Arbor, MI). Oxidized β-NAD+ and β-NADP+ were purchased from Codexis (Redwood City, CA).
Biochemical characterization of recombinant Aldh1a1 and Aldh3a1
We examined the kinetic parameters (Km, Vmax and Vmax/Km) of recombinant mouse Aldh1a1 and Aldh3a1 (Table 1) for oxidative metabolism of a wide range of aldehyde substrates using either NAD+ or NADP+ as pyridine nucleotide cofactor. The cofactor preference of recombinant mouse ALDH was also assessed using varying concentrations of either NAD+ or NADP+. Kinetic parameters indicate that mouse Aldh1a1 exhibited high affinity for short chain aldehydes, such as propionaldehyde (Km = 141 μM) and
Comparison of substrate specificity for murine Aldh's
The relative contribution of Aldh isozymes in protection against aldehyde toxicity is controversial. While earlier studies indicated that ALDH3A1 but not ALDH1A1 protects against cytotoxic lipid derived-aldehydes, most recent studies in eye lens and neuroblastoma cell lines showed that ALDH1A1 can protect against these reactive aldehydes [36], [40]. The contribution of the different isozymes of Aldh to oxidation of lipid derived-aldehydes has not been examined in the liver with the liver being
Conclusion
Our studies confirm that Aldh1a1 quantitatively is a major Aldh in mouse liver at mRNA and protein level. Its kinetic parameters also indicate that catalytically it is a major factor in cytosolic oxidation of α,β-unsaturated aldehydes in liver. Ablation of Aldh1a1 increases sensitivity of Hepa1c1c7 cells to acrolein toxicity, acrolein–protein adducts and caspase 3 cleavage leading to apoptosis. These results indicate that induction of Aldh1a1, on the other hand, significantly decreased acrolein
Conflict of interest
None.
Acknowledgements
Supported in part from NIH Grants ES11860 (DJC/RAP), HL89380 (DJC), and 5P30ES014443. This publication is in partial completion of the Ph.D. degree for N.L. Makia and he was also supported by USPHS NIH grant R13-AA019612 to present this work at the 15th International Meeting on Enzymology and Molecular Biology of Carbonyl Metabolism in Lexington, KY, USA.
References (53)
- et al.
Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes
Free Radic. Biol. Med.
(1991) - et al.
Acrolein is a product of lipid peroxidation reaction. Formation of free acrolein and its conjugate with lysine residues in oxidized low density lipoproteins
J. Biol. Chem.
(1998) Reactive carbonyls and oxidative stress: potential for therapeutic intervention
Pharmacol. Ther.
(2007)Role of reactive aldehyde in cardiovascular diseases
Free Radic. Biol. Med.
(2000)- et al.
The course of CCl4 induced hepatotoxicity is altered in mGSTA4-4 null (−/−) mice
Toxicology
(2006) - et al.
Ethanol-induced modulation of hepatocellular extracellular signal-regulated kinase-1/2 activity via 4-hydroxynonenal
J. Biol. Chem.
(2007) - et al.
Aldehydes: occurrence, carcinogenic potential, mechanism of action and risk assessment
Mutat. Res.
(1991) - et al.
Acrolein consumption exacerbates myocardial ischemic injury and blocks nitric oxide-induced PKCepsilon signaling and cardioprotection
J. Mol. Cell. Cardiol.
(2008) - et al.
Acrolein consumption induces systemic dyslipidemia and lipoprotein modification
Toxicol. Appl. Pharmacol.
(2010) - et al.
In situ detection of lipid peroxidation and oxidative DNA damage in non-alcoholic fatty liver diseases
J. Hepatol.
(2002)
Effect of oxidative stress by iron on 4-hydroxynonenal formation and proliferative activity in hepatomas of different degrees of differentiation
Free Radic. Biol. Med.
Role of 4 hydroxynonenal in modification of cytochrome c oxidase in the ischemic/reperfused rat heart
J. Mol. Cell. Cardiol.
Thiolation of protein-bound carcinogenic aldehyde. An electrophilic acrolein–lysine adduct that covalently binds to thiols
J. Biol. Chem.
Inactivation of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal. Selective modification of an active-site lysine
J. Biol. Chem.
In vitro inactivation of glucose-6-phosphate dehydrogenase from human red blood cells by acrolein: a possible biomarker of exposure
Toxicol. Lett.
Covalent attachment of 4-hydroxynonenal to glyceraldehyde-3-phosphate dehydrogenase. A possible involvement of intra- and intermolecular cross-linking reaction
J. Biol. Chem.
Transfection of mGSTA4 in HL-60 cells protects against 4-hydroxynonenal-induced apoptosis by inhibiting JNK-mediated signaling
Arch. Biochem. Biophys.
Cytochromes P450 catalyze oxidation of alpha,beta-unsaturated aldehydes
Arch. Biochem. Biophys.
Involvement of aldose reductase in the metabolism of atherogenic aldehydes
Chem. Biol. Interact.
Metabolism of the lipid peroxidation product, 4-hydroxy-trans-2-nonenal, in isolated perfused rat heart
J. Biol. Chem.
Selective protection by stably transfected human ALDH3A1 (but not human ALDH1A1) against toxicity of aldehydes in V79 cells
Chem. Biol. Interact.
Multiple and additive functions of ALDH3A1 and ALDH1A1: cataract phenotype and ocular oxidative damage in Aldh3a1(−/−)/Aldh1a1(−/−) knock-out mice
J. Biol. Chem.
Lipid aldehyde oxidation as a physiological role for class 3 aldehyde dehydrogenases
Biochem. Pharmacol.
Cellular lipid peroxidation end-products induce apoptosis in human lens epithelial cells
Free Radic. Biol. Med.
Gene expression profile and cytotoxicity of human bronchial epithelial cells exposed to crotonaldehyde
Toxicol. Lett.
Acrolein causes transcriptional induction of phase II genes by activation of Nrf2 in human lung type II epithelial (A549) cells
Toxicol. Lett.
Cited by (42)
Genomic expansion of Aldh1a1 protects beavers against high metabolic aldehydes from lipid oxidation
2021, Cell ReportsCitation Excerpt :To exclude influence from Aldh2, we tested aldehyde dehydrogenase activity using cytosolic protein extracts from liver cells. Crude cytosolic protein extracts were prepared from wild beaver or mouse (C57/Bl6) livers using phosphate buffer similar to a previously described method (Makia et al., 2011). Specifically, tissue was resuspended in K-Phos Buffer (50 mM potassium phosphate pH7.4, 250 mM sucrose, 1 mM EDTA) at 1g/3.0ml.
Formation and repair of unavoidable, endogenous interstrand cross-links in cellular DNA
2021, DNA RepairCitation Excerpt :To date, ICLs derived from α,β-unsaturated aldehydes have not been detected in cellular DNA. Acrolein, crotonaldehyde, and HNE are detoxified in cells by ALDH enzymes including ALDH2, ALDH1A1 and ALDH7A1 [253–255,269,270]. Thus, the cellular levels of these α,β-unsaturated aldehydes are expected to increase in ALDH2-deficient individuals and in Aldh2–/– cell lines [235].
Impaired Hepatic Vitamin A Metabolism in NAFLD Mice Leading to Vitamin A Accumulation in Hepatocytes
2021, Cellular and Molecular Gastroenterology and HepatologyCarotenoids and carotenoid conversion products in adipose tissue biology and obesity: Pre-clinical and human studies
2020, Biochimica et Biophysica Acta - Molecular and Cell Biology of LipidsCitation Excerpt :In fact, evidence has been provided that ALDH1A1 functions as a promoter of adiposity in young mice (and of adipogenesis in preadipose cells) independently of effects on retinaldehyde or atRA concentrations in WAT (measured using gold-standard liquid chromatography with tandem mass spectrometry assays) [105]. To be noted, ALDH1A1 recognizes multiple aldehyde substrates besides retinaldehyde, such as aldehyde lipid peroxidation products (malondialdehyde, 4-hydroxy-2-nonenal) and others [124,125]. Altogether, a proadipogenic effect of ALDH1A1 is well established, yet its molecular bases remain to be clarified.
6-(Methylsulfinyl)hexyl isothiocyanate protects acetaldehyde-caused cytotoxicity through the induction of aldehyde dehydrogenase in hepatocytes
2020, Archives of Biochemistry and BiophysicsVitamin A signaling and homeostasis in obesity, diabetes, and metabolic disorders
2019, Pharmacology and Therapeutics