Original Contribution
The oxidative stress mediator 4-hydroxynonenal is an intracellular agonist of the nuclear receptor peroxisome proliferator-activated receptor-β/δ (PPARβ/δ)

https://doi.org/10.1016/j.freeradbiomed.2007.01.003Get rights and content

Abstract

Liver insufficiency and damage are major causes of death and disease worldwide and may result from exposure to environmental toxicants, specific combinations or dosages of pharmaceuticals, and microbial metabolites. The generation of reactive intermediates, in particular 4-hydroxynonenal (4-HNE), is a common event in liver damage caused by a variety of hepatotoxic drugs and solvents. The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that are involved in the transcriptional regulation of lipid metabolism as well as other biological functions. Importantly, we have observed that the PPARβ/δ−/− mouse is more susceptible to chemically induced hepatotoxicity than its wild-type counterpart, and our objective in this study was to elucidate the mechanism(s) by which PPARβ/δ confers protection to hepatocytes. We hypothesized that PPARβ/δ plays a protective role by responding to toxic lipids and altering gene expression accordingly. In support, oxidized-VLDL and constituents including 13-S-hydroxyoctadecadienoic acid (13-S-HODE) and 4-HNE are PPARβ/δ ligands. A structure–activity relationship was established where 4-HNE and 4-hydroperoxynonenal (4-HpNE) enhanced the activity of the PPARβ/δ subtype while 4-hyroxyhexenal (4-HHE), 4-oxo-2-Nonenal (4-ONE), and trans-4,5-epoxy-2(E)-decenal did not activate this receptor. Increasing PPARβ/δ activity with a synthetic agonist decreased sensitivity of hepatocytes to 4-HNE and other toxic agents, whereas inhibition of this receptor had the opposite result. Gene expression microarray analysis identified several important PPARβ/δ-regulated detoxification enzymes involved in 4-HNE metabolism that are regulated at the transcript level. This research established 4-HNE as an endogenous modulator of PPARβ/δ activity and raises the possibility that agonists of this nuclear receptor may be utilized to prevent or treat liver disease associated with oxidative damage.

Introduction

Greater than 2.2 million hospitalized Americans suffer adverse drug reactions each year, with liver toxicity presenting as the most common adverse effect, and approximately 100,000 individuals die unintentionally from administration of medications [1], [2]. Many additional cases of liver failure occur due to acute, chronic, and degenerative disease processes, including those related to acetaminophen overdose, alcohol consumption, and solvent exposures. Reactive oxygen intermediates (ROI) elicit oxidative decomposition of polyunsaturated fatty acids (i.e., lipid peroxidation), leading to the formation of a complex mixture of aldehydic end products, including malondialdehyde (MDA), 4-HNE, and other alkenals [3]. These aldehydic molecules have been considered the ultimate mediators of toxic effects elicited by oxidative stress but may also affect cellular function at nontoxic levels via signal transduction, gene expression, and cell proliferation. Although the overt toxicity caused by aldehydic end products is due primarily to covalent binding to cellular macromolecules, the effects on signal transduction are not well characterized. Since millions of individuals suffer adverse drug reactions each year it is important to understand how the cell responds to intracellular insults such as production of ROI and 4-HNE.

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that exist as three subtypes (α, β/δ, and γ), which exhibit tissue-specific expression, preferential ligand recognition, and distinct biological functions [4], [5], [6], [7], [8], [9]. Although important as targets of pharmaceutical intervention, there is increasing evidence that the biological niche occupied by the PPARs is that of a receptor for fatty acid and their metabolites. Of the three PPAR genes (α, β/δ, and γ), the PPARβ/δ isoform is the least well studied in terms of its biological functions and endogenous ligands. PPARβ/δ plays an important role in differentiation of epithelial tissues, fatty acid catabolism in skeletal muscle, improvement of insulin sensitivity, attenuated weight gain, and elevated HDL levels [10]. Emerging evidence suggests that the presence of this receptor is important in ameliorating the effects of hepatotoxicants. For example, histological examination of liver and analysis of markers of overt damage to this organ (serum GPT) after treatment with the xenobiotics azoxymethane (AOM), arsenic, or carbon tetrachloride demonstrated that the extent of liver toxicity in PPARβ/δ-null mice was more severe than in wild-type mice.2 While it is remotely possible that the metabolic fate of these hepatotoxicants could be influenced by PPARβ/δ, it is more likely that regulation of oxidative stress underlies the protective role of this receptor in liver. These chemicals share a common mechanism of overt toxicity via production of ROI and oxidized lipid intermediates. For example, CCl4 affects eicosanoid pathways [11], [12] and increases circulating prostaglandin E2 (PGE2) levels [13] and 4-HNE and 4-HNE-protein adducts [3], [14], [15]. The purpose of this study was to determine the extent to which oxidized lipids and their metabolites interact with PPARβ/δ and influence gene regulation. We hypothesized that PPARβ/δ acts as an oxidative stress sensor in hepatocytes and, upon interaction with products of lipid peroxidation, regulates detoxification genes accordingly to ameliorate the toxic insult.

One possible explanation for the increased susceptibility of PPARβ/δ−/− mice to hepatotoxicity is that oxidative damage increases the production of an endogenous ligand for PPARβ/δ. This putative agonist would in turn stimulate lipid metabolism and degradation of lipid peroxidation intermediates. PPARs are well recognized as transcriptional regulators of lipid metabolism, transport, storage, and other activities [16]. In the absence of PPARβ/δ the signaling cascade would be disrupted and accumulation of toxic lipids such as 4-HNE would result. If our hypothesis were correct, endogenous ligands of PPARβ/δ should include oxidized lipids, in particular those derived from fatty acids. In support, we discovered that oxidized-VLDL and constituents including 13-S-HODE and 4-HNE are PPARβ/δ agonists. In addition, modulating PPARβ/δ activity, either by activation with synthetic PPARβ/δ-selective agonist tetradecylthioacetic acid (TTA) or inhibition with PPAR pan-antagonist GW9662 [17], affects the sensitivity of hepatocytes to 4-HNE and other toxic agents. This research raises the possibility that PPARβ/δ agonists may be utilized to prevent or treat liver disease associated with the generation of ROIs.

Section snippets

Reagents

VLDL (human plasma) was purchased from Calbiochem (La Jolla, CA), LPL was purchased from Sigma (lyophilized powder) and reconstituted in PBS (10 mg/mL), and 13-S-HODE, 13-S-HpODE, 4-HHE, 4-ONE, trans-4,5-epoxy-2E-decenal, 4-HpNE, and 4-HNE were purchased from Cayman Chemical (Ann Arbor, MI) and used without further purification. The semienzymatic synthesis and purification of some of the linoleic and arachidonic acid oxidation products such as 9-HODE, 12-HpODE, 5-HETE, 9-HETE, 12-HETE, 15-HETE,

Results

We hypothesized that endogenous ligands for PPARβ/δ would include oxidized lipids, such as those derived from VLDL. The VLDL particle is a mixture of free and esterified cholesterols, triglycerides (formed from various fatty acids), and apolipoproteins. As seen in Fig. 1A, the oxidation of VLDL with CuSO4 (oxVLDL) and subsequent incubation with LpL greatly increased PPARβ/δ activity in a dose-dependent manner (Fig. 1B). To determine which potential products released by oxVLDL are playing a role

Discussion

The PPARs are ligand-activated transcription factors that sense a variety of lipophilic molecules and control gene expression to maintain cellular homeostasis. Fatty acids and their metabolites are known endogenous agonists of all PPARs, with PPARβ/δ exhibiting similar structural and geometric preference as PPARα, whereas PPARγ tends to prefer long-chain polyunsaturated fatty acids [26]. PPARβ/δ agonists include linoleic acid, oleic acid, arachidonic acid, and EPA, which has been cocrystallized

Acknowledgments

This research was supported by the Penn State University competitive grants program sponsored by the Huck Institute of Life Sciences and the Institutes of the Environment (to J.V.H., J.M.P., and K.S.P.). Portions of this research were presented at the Society of Toxicology annual meeting in San Diego, CA.

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    These authors contributed equally to this work.

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