Murine hepatic aldehyde dehydrogenase 1a1 is a major contributor to oxidation of aldehydes formed by lipid peroxidation

https://doi.org/10.1016/j.cbi.2011.01.013Get rights and content

Abstract

Reactive lipid aldehydes are implicated in the pathogenesis of various oxidative stress-mediated diseases, including non-alcoholic steatohepatitis, atherosclerosis, Alzheimer's and cataract. In the present study, we sought to define which hepatic Aldh isoform plays a major role in detoxification of lipid-derived aldehydes, such as acrolein and HNE by enzyme kinetic and gene expression studies. The catalytic efficiencies for metabolism of acrolein by Aldh1a1 was comparable to that of Aldh3a1 (Vmax/Km = 23). However, Aldh1a1 exhibits far higher affinity for acrolein (Km = 23.2 μM) compared to Aldh3a1 (Km = 464 μM). Aldh1a1 displays a 3-fold higher catalytic efficiency for HNE than Aldh3a1 (218 ml/min/mg vs 69 ml/min/mg). The endogenous Aldh1a1 gene was highly expressed in mouse liver and a liver-derived cell line (Hepa-1c1c7) compared to Aldh2, Aldh1b1 and Aldh3a1. Aldh1a1 mRNA levels was 34-fold and 73-fold higher than Aldh2 in mouse liver and Hepa-1c1c7 cells respectively. Aldh3a1 gene was absent in mouse liver, but moderately expressed in Hepa-1c1c7 cells compared to Aldh1a1. We demonstrated that knockdown of Aldh1a1 expression by siRNA caused Hepa-1c1c7 cells to be more sensitive to acrolein-induced cell death and resulted in increased accumulation of acrolein–protein adducts and caspase 3 activation. These results indicate that Aldh1a1 plays a major role in cellular defense against oxidative damage induced by reactive lipid aldehydes in mouse liver. We also noted that hepatic Aldh1a1 mRNA levels were significantly increased (≈3-fold) in acrolein-fed mice compared to control. In addition, hepatic cytosolic ALDH activity was induced by acrolein when 1 mM NAD+ was used as cofactor, suggesting an Aldh1a1-protective mechanism against acrolein toxicity in mice liver. Thus, mechanisms to induce Aldh1a1 gene expression may provide a useful rationale for therapeutic protection against oxidative stress-induced pathologies.

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 (O2radical dot) and hydroxyl radicals (OHradical dot) 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.

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