Hydroxytyrosol protects against oxidative damage by simultaneous activation of mitochondrial biogenesis and phase II detoxifying enzyme systems in retinal pigment epithelial cells

Abstract

Studies in this laboratory have previously shown that hydroxytyrosol, the major antioxidant polyphenol in olives, protects ARPE-19 human retinal pigment epithelial cells from oxidative damage induced by acrolein, an environmental toxin and endogenous end product of lipid oxidation, that occurs at increased levels in age-related macular degeneration lesions. A proposed mechanism for this is that protection by hydroxytyrosol against oxidative stress is conferred by the simultaneous activation of two critically important pathways, viz., induction of phase II detoxifying enzymes and stimulation of mitochondrial biogenesis. Cultured ARPE-19 cells were pretreated with hydroxytyrosol and challenged with acrolein. The protective effects of hydroxytyrosol on key factors of mitochondrial biogenesis and phase II detoxifying enzyme systems were examined. Hydroxytyrosol treatment simultaneously protected against acrolein-induced inhibition of nuclear factor-E2-related factor 2 (Nrf2) and peroxisome proliferator-activated receptor coactivator 1 alpha (PPARGC1α) in ARPE-19 cells. The activation of Nrf2 led to activation of phase II detoxifying enzymes, including γ-glutamyl-cysteinyl-ligase, NADPH (nicotinamide adenine dinucleotide phosphate)-quinone-oxidoreductase 1, heme-oxygenase-1, superoxide dismutase, peroxiredoxin and thioredoxin as well as other antioxidant enzymes, while the activation of PPARGC1α led to increased protein expression of mitochondrial transcription factor A, uncoupling protein 2 and mitochondrial complexes. These results suggest that hydroxytyrosol is a potent inducer of phase II detoxifying enzymes and an enhancer of mitochondrial biogenesis. Dietary supplementation of hydroxytyrosol may contribute to eye health by preventing the degeneration of retinal pigment epithelial cells induced by oxidative stress.

Introduction

Age-related macular degeneration (AMD) is the leading cause of vision loss in the Western world among people over 65 y of age [1], and worldwide, it is the third most common cause of blindness [2]. AMD is characterized by an age-related degeneration of retinal pigment epithelium (RPE) and the photoreceptors in the macular area of the retina. The underlying cause of the disease is unknown, but oxidative stress is involved [3], suggesting that consumption of diets rich in antioxidants may be of benefit.

The Mediterranean diet has been associated with a lower incidence of not only certain cancers, but also cardiovascular disease, which is the most common and serious complication of diabetes [4], [5], [6], all conditions associated with oxidative stress. Olives and olive oil are considered an important part of the Mediterranean diet. Evidence has accumulated recently that in addition to olive lipids, which are rich in monounsaturated fatty acids, antioxidant polyphenols such as hydroxytyrosol also contribute to the health effects of olives [7], [8], [9], [10].

Work in our laboratory has led to the proposal that oxidative damage to mitochondria in RPE cells may contribute to the retinal degeneration observed in AMD and the compounds that protect mitochondrial function may prevent or alleviate this damage. Our work also showed that acrolein, a lipid oxidation end product and mitochondrial toxin [11], causes oxidative mitochondrial damage in RPE cells; moreover, hydroxytyrosol protects RPE cells against this acrolein-induced oxidative stress [12]. To investigate the underlying mechanisms, the same acrolein model is now used to study the effects of hydroxytyrosol on the induction of phase II detoxifying enzymes and stimulation of mitochondrial biogenesis, two of the most important pathways for cells to fight against oxidative stress.

When cells are subjected to a variety of oxidative environmental stresses, they typically respond by inducing a coordinated expression of genes encoding the set of phase II detoxifying enzymes (Fig. 13) , principally mediated by activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) [15], [16]. Nrf2 controls the orchestrated expression of phase II enzymes and genes involved in oxidative defense, although normally Nrf2 protein is kept inactive in the cytoplasm by complexing with its cytosolic inhibitor keap-1. Upon activation and release from keap-1, Nrf2 protein translocates to the nucleus, where it binds to promoters containing antioxidant response elements, resulting in the transactivation of the respective genes for phase II detoxifying enzymes. Key phase II detoxifying enzymes include glutathione (GSH) S-transferase (GST), heme oxygenase-1 (HO-1), NAD(P)H quinone oxido-reductase-1 and γ-glutamyl cysteine ligase (GCL), enhanced expression of which leads to an increase in levels of endogenous antioxidants such as the major thiol antioxidant GSH and reduced quinones [13], [15], [17].

Phase II enzymes perform a variety of vital cellular functions important for protecting against oxidative damage. GCL controls the production of GSH, the major endogenous antioxidant thiol. GSH reductase (GR) catalyses the NADP-dependent reduction of GSSG (oxidized GSH) to GSH to maintain a high cytoplasmic GSH:GSSG ratio. GSH peroxidase (GPx), an enzyme widely present in many tissues, is thought to be an important cellular H₂O₂ detoxifier in neurons [18] and mice lacking GPx develop cataracts at a young age [19]. NAD(P)H:quinone oxidoreductase (NQO1) reduces quinones via a two-electron reduction and converts the dopamine quinones into less toxic hydroquinones that may be further detoxified via conjugation to sulfate or glucuronic acid [20]. Therefore, NQO1 is likely to play a crucial role in the protection of cells against oxidative damage. HO-1 produces the antioxidant bilirubin; it is typically associated with an increased production of ferritin, which results in reduced amounts of free iron [21], the main catalyst of the Fenton reaction. HO-1 expression is ubiquitous and its activity is increased by many types of agents, particularly those involved in oxidative stress such as heme, metalloporphyrins and transition metals.

The cytoplasmic antioxidant system [including NQO1, GST, GCL and Cu/Zn superoxide dismutase (SOD)] is mainly controlled by Nrf2. In contrast, the mitochondrial antioxidant system [thioredoxin-2, peroxiredoxin (Prdx)3, Prdx5 and Mn SOD] is modulated through the transcription factor FOXO3a.

Inducing phase II enzymes and stimulating mitochondrial biogenesis may also enhance other antioxidant defense systems, such as the antioxidant enzyme catalase. The functions of catalase include catalyzing the decomposition of hydrogen peroxide to water and oxygen to remove free radicals and protect cells from oxidative damage.

The role of mitochondrial dysfunction in the aging process [22] and in the development of chronic degenerative diseases, such as Type 2 diabetes [23] and neurodegenerative diseases [24], is being increasingly acknowledged. One underlying mechanism of mitochondrial dysfunction is the loss of mitochondria. For example, mitochondrial loss in adipose tissue is correlated with the development of Type 2 diabetes [25].

Thus, an effective strategy for preventing and treating mitochondrial dysfunction-related disease should be the effective stimulation of mitochondrial biogenesis. This may be achieved by activation of the key factor promoting mitochondrial biogenesis, peroxisome proliferator-activated receptor coactivator 1 alpha (PPARGC1α) [26]. PPARGC1α possesses dual activities — stimulation of mitochondrial electron transport while enforcing suppression of reactive oxygen species (ROS) — and may serve as an adaptive set-point regulator, capable of providing an accurate balance between metabolic requirements and cytotoxic protection [27]. Therefore, its dual activities of inducing mitochondrial biogenesis and suppressing ROS make PPARGC1α an almost ideal target protein for the control or limiting of damage associated with mitochondrial dysfunction.

Signalling molecules upstream of PPARGC1, such as adenosine monophosphate kinase (AMPK) [28], nitric oxide [29], and calcium [30], can also promote mitochondrial biogenesis. Of these, AMPK also regulates other metabolic pathways, including the cellular uptake of glucose, the β-oxidation of fatty acids and the biogenesis of glucose transporters [31]. The enzyme nitric oxide synthase (NOS) produces NO, and one isoform, endothelial cell NOS (eNOS), is an upstream regulating factor for mitochondrial biogenesis [32].

Finally, uncoupling protein 2 (UCP2), a mitochondrial factor controlled by PPARGC1α, is involved in maintaining acceptable ROS levels and is neuroprotective during ischemia/reperfusion [33]; it therefore may play a role in preventing and correcting mitochondrial dysfunction.

Polyphenols have been shown to protect RPE from oxidative-stress-induced death [34] and to induce phase II detoxifying enzymes [35]. Hydroxytyrosol is the major antioxidant polyphenol in olives and has been shown to have beneficial effects on human health. Results from our previous experiments have shown that hydroxytyrosol exhibits protective effects against acrolein-induced toxicity in the human retinal pigment epithelial cell line ARPE-19 [12]. Pretreatment with hydroxytyrosol dose-and time-dependently protected the ARPE-19 cells from acrolein-induced oxidative damage and mitochondrial dysfunction. A short-term pretreatment (48 h) with over 75 μmol/L hydroxytyrosol was required for protection while a long-term pretreatment (7 days) showed protective effects with as little as 5 μmol/L or more, suggesting that lower long-term doses of hydroxytyrosol treatment can achieve similar protective effects as the higher short-term doses. These results suggest that hydroxytyrosol may be a mitochondrial protecting nutrient even at a relatively low concentration when given for an extended period of time. Our hypothesis is that the mechanism behind hydroxytyrosol's protective effects against acrolein-induced RPE damage may be related to its capability to activate simultaneously both mitochondrial biogenesis and phase II detoxifying enzyme systems. In the present study, these pathways are investigated using acrolein-challenged ARPE-19 cells.