Dietary phytochemicals in the protection against oxysterol-induced damage

Accepted Manuscript Title: Dietary phytochemicals in the protection against oxysterol-induced damage Authors: Antonio Cilla, Amparo Alegr´ıa, Alessandro Attanzio, Guadalupe Garcia-Llatas, Luisa Tesoriere, Maria A. Livrea PII: DOI: Reference:

S0009-3084(16)30189-X http://dx.doi.org/doi:10.1016/j.chemphyslip.2017.03.001 CPL 4530

To appear in:

Chemistry and Physics of Lipids

Received date: Accepted date:

22-11-2016 2-3-2017

Please cite this article as: Cilla, Antonio, Alegr´ıa, Amparo, Attanzio, Alessandro, Garcia-Llatas, Guadalupe, Tesoriere, Luisa, Livrea, Maria A., Dietary phytochemicals in the protection against oxysterol-induced damage.Chemistry and Physics of Lipids http://dx.doi.org/10.1016/j.chemphyslip.2017.03.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Dietary phytochemicals in the protection against oxysterol-induced

damage

Antonio Cillaa*, Amparo Alegríaa, Alessandro Attanzio b, Guadalupe Garcia-Llatasa, Luisa Tesoriereb, Maria A. Livreab*

a

Nutrition and Food Science Area. Faculty of Pharmacy, University of Valencia, Avda.

Vicente Andrés Estellés s/n, 46100 - Burjassot (Valencia), Spain.

b

Dipartimento Scienze e Technologie Biologiche Chimiche e Farmaceutiche

(STEBICEF), Università di Palermo, Palermo, Italy.

* To whom correspondence should be addressed (Telephone: +34-963544972; Fax:

+34-963544954; E-mail: [email protected]) (E-mail: [email protected])

1

Highlights 

Sterol oxidation products (SOPs) implied in the origin of different pathophysiological conditions.



Description of distinct studies in various cell models evidencing protection against SOPs toxicity by dietary phytochemicals.



Prevention of SOPs injury through modulation of intracellular signaling cascades beyond direct antioxidant activity.



Perspectives and recommendations on further research needed in the field of oxysterol injury prevention.

Abstract The intake of fruits and vegetables is associated with reduced incidence of many chronic diseases. These foods contain phytochemicals that often possess antioxidant and free radical scavenging capacity and show anti-inflammatory action, which are also the basis of other bioactivities and health benefits, such as anticancer, anti-aging, and protective action for cardiovascular diseases, diabetes mellitus, obesity and neurodegenerative disorders. Many factors can be included in the etiopathogenesis of all of these multifactorial diseases that involve oxidative stress, inflammation and/or cell death processes, oxysterols, i.e. cholesterol oxidation products (COPs) as well as phytosterol oxidation products (POPs), among others. These oxidized lipids result from either spontaneous and/or enzymatic oxidation of cholesterol/phytosterols on the steroid nucleus or on the side chain and their critical roles in the pathophysiology of the abovementioned diseases has become increasingly evident. In this context, many studies investigated the potential of dietary phytochemicals (polyphenols, carotenoids and vitamins C and E, among others) to protect against oxysterol toxicity in various cell models mimicking pathophysiological conditions. This review, summarizing the mechanisms involved in the chemopreventive effect of phytochemicals against the injury by oxysterols may constitute a step forward to consider the importance of preventive strategies on a nutritional point of view to decrease the burden of many agerelated chronic diseases.

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Keywords: Antioxidants, phytochemicals, oxysterols, cholesterol oxidation products, phytosterol oxidation products, human chronic diseases.

Abbreviations COPs: cholesterol oxidation products; POPs: phytosterol oxidation products; SOPs: sterol oxidation products; ROS: reactive oxygen species; MDA: malonyldialdehyde; GSH:

reduced

glutathione;

GPx:

glutathione

peroxidase;

HP:

fatty

acids

hydroperoxides; ICAM-1: Intercellular adhesion molecule-1; VCAM-1: vascular cell adhesion molecule-1; 7kc: 7-ketocholesterol; 7βOHc: 7β-hydroxycholesterol; 6βOHc: 6β-hydroxycholesterol; 7αOHc: 7β-hydroxycholesterol; Triolc: cholestan-3β,5α,6βtriol; α-epox-c: 5α,6α-epoxycholesterol; β-epox-c: 5β,6β-epoxycholesterol; VEGF: vascular endothelial growth factor; Nox1: colonic NADPH oxidase isoform; 24OHc: 24-hydroxycholesterol; 25OHc: 25-hydroxycholesterol; 27OHc: 25-hydroxycholesterol. 7kc:7-ketocholesterol. 24OHc: 24-hydroxycholesterol. 25OHc: 25-hydroxycholesterol. AA: arachidonic acid; PKC: protein kinase C; 7βOHsit: 7β-hydroxysitosterol; 7ksit:7ketositosterol; PDK-1: 3’ phosphoinositide-regulated kinase-1; PI3-K: phosphoinositide 3-kinase; 7βOHstig: 7β-hydroxystigmasterol; Epoxy-stig: 5,6-epoxystigmasta-22,23diol; Diepoxy-stig: 5,6,22,23-diepoxystigmastane; GSK3β: glycogen synthase kinase 3 isoform β; Mcl-1: myeloid cell leukemia-1; DHA: docosahexaenoic acid; PS: phosphatidylserine; PGE2: prostaglandin E2; ERK: extracellular regulated kinase; JNK: Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; NF-κB: nuclear factor-kappaB; PPAR: peroxisome proliferator-activated receptor; TNF-α: tumor necrosis factor α; BAD: Bcl-2 antagonist of cell death; NOX-4: NADPH oxidase-4; PARP: poly(ADP-ribose) polymerase; ER: endoplasmic reticulum; IRE1α: inositolrequiring enzyme 1α; PERK: protein kinase RNA-like ER kinase; T-AOC: total antioxidant capacity; SOD: superoxide dismutase; TR: thioredoxin reductase; PGI 2: prostacyclin; TXA2: tromboxane A2.

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1. Introduction Human beings have always exploited the plant kingdom as a food source to survive, and as a means to cure diseases as they realized of health benefits and toxicity of medicinal plants. The two concepts, nutrition and cure, have recently merged in “nutraceutical”, when a number among the huge amount of components from edible plants commonly referred to as phytochemicals, have appeared to possess both dietary importance and potential therapeutic activity. Secondary plant metabolites, synthesized by every part of the plant to cope with and adapt to a variety of environmental stressors, when ingested by animals the phytochemicals can trigger responses through still largely unknown mechanistic pathways. Their capacity to maintain the cell redox homeostasis (Forman et al., 2014), appears as a fundamental aspect of their bioactivities. The phenol/polyphenol nature of the large majority of phytochemicals at first generated the idea that reducing chemistry and metal-chelating properties had a role in their benefits. Subsequent studies definitely showing the limited bioavailability and transformations of the ingested compounds in the body (Visioli et al., 2011), almost completely ruled out direct effects of plant phenolics as antioxidants, with the possible exception of some phytochemicals acting at the level of the gastro-intestinal tract (Halliwell et al., 2005). Rather, these molecules may control oxidative processes and help maintaining the cell redox homeostasis by affecting transcription factors related to the expression of antioxidant proteins, detoxification enzymes and xenobiotic transporters (Fraga & Oteiza, 2011; Niture et al., 2014). This is now considered to be true for other nonphenol phytochemicals and even for dietary antioxidant vitamins including vitamin E that, in addition to its lipoperoxyl radical-scavenging action in membranes and protection of LDL (Niki, 2014), may trigger pathways of signal transduction (Niture et al., 2014). Then dietary “antioxidants”, either phenolics, vitamins and other phytochemicals

may

share

the

property

of

providing

protection

against

oxidative/electrophile stress and associated diseases. Hypercholesterolemia, a quite common condition in the western world has a number of consequences from moderate to severe, the latest including pathogenesis of cardiovascular and other inflammation-based diseases. The cholesterol excess has long been considered as a major problem, however evidence has been accumulated proving the deleterious effects of some cholesterol oxidation products, toxic oxysterols (Hwang, 1991), the level of which can easily grow under such a condition (Addis et al., 1989; 4

Sevanian et al., 1994; Dzeletovic et al., 1995; Chang et al., 1997). Research groups long studying these molecules and their bioactivities have often been comprehensively reviewed and updated literature data (Otaegui-Arrazola et al. 2010; Poli et al., 2013; Zarrouk et al. 2014; Kulig et al. 2016), analyzing various aspects, from the involvement of oxysterols in the pathogenesis of chronic diseases to the impact of usual therapeutic treatments of hypercholesterolemia on their blood level, and of either natural, or chemical antioxidants on their formation from cholesterol (Csullany et al., 2001; Belinki et al., 1998). Nevertheless, although oxysterols have conclusively been demonstrated to cause extensive oxidation generating reactive oxygen species (ROS) through upregulation of NADPH oxidase family enzymes (Poli et al., 2004), which can provide a rationale for most of their toxic effects, to the best of our knowledge a comprehensive review of studies treating with the protection from dietary redox-active phytochemicals and antioxidant vitamins on the oxysterol-induced damage in various disease models does not exist. In the attempt to promote further studies that consider potentially protective nutritional approaches against several degenerative and chronic diseases, this review gathered the major data reporting the protection by natural antioxidants, either vitamins or various phytochemicals, against oxysterol injury in a number of cell lines under various experimental contexts modelling pathophysiological conditions.

2. Oxysterols 2.1 Origin and formation Cholesterol and phytosterols from animal and plant origin, respectively, are abundantly present in western type diets, with ~300 mg/day as an average daily intake (Vanmierlo et al. 2013). Both sterols share a very similar structure composed of three regions: a hydrocarbon tail (or lateral chain), a ring structure region composed by four hydrocarbon rings, and a hydroxyl group in C-3. The difference between these sterols relies on the lateral chain located in C-17, since compared to cholesterol, phytosterols possess different substitutions in C-24 (Hovenkamp et al. 2008; Vejux et al. 2008). The oxidation of the double bond present in C5-C6, or those located in other positions in the steroid ring or the lateral chain of sterols, results in the formation of sterol oxidation products (SOPs) or oxysterols, either cholesterol oxidation products (COPs), or phytosterols oxidation products (POPs) (Otaegui-Arrazola et al. 2010; Brzeska et al. 2016). 5

COPs and POPs may have a dietetic origin, mainly formed by auto-oxidation due to heat, light exposure, storage and freeze-drying in fat-rich foodstuffs (i.e. red meat, egg, milk, cheese, ham), or in vegetable oils and phytosterol-enriched products, respectively (Otaegui-Arrazola et al. 2010; García-Llatas & Rodríguez-Estrada, 2011; Poli et al., 2013; Rodriguez-Estrada et al., 2014; Barriuso et al. 2016). The most abundant oxysterols detected in diet are 7/β-hydroxysterols, 7-ketosterols, /β epoxysterols and triols (Otaegui-Arrazola et al. 2010), that have been found in foods at concentrations ranging from 10 µM to 150 µM (Kanner 2007; Incani et al. 2016). In the case of POPs, they can be also a source of SOPs from diet, but it has been reported an estimated dietary intake lower than that of COPs (Hovenkamp et al. 2008). Dietary oxysterols can contribute to the onset and development of inflammation related intestinal diseases or evoke injury in other organs after absorption in the gut (Kanner 2007). Endogenous formation of COPs, which may be relevant to human pathology, may occur through non-enzymatic and enzymatic pathways, the first by radical mechanism, which mainly affects the sterol ring, and the second which mostly forms sterol ring and lateral chain hydroxylated oxysterols (Zarrouk et al. 2014; Kulig et al. 2016). Non-enzymatic formation of COPs is a multi-step chain process initiated by ROS, peroxyl and alkoxyl radicals, or the leukocyte/H 2O2/HOCl system during inflammatory processes (Poli et al. 2013), abstracting one allylic hydrogen atom from the C-7 position of the sterol ring; the carbon radical generated can react with oxygen to form a cholesterol peroxyl radical which further abstracts hydrogen, generating relatively stable 7 /β-hydroperoxides; hydroperoxides may continue oxidizing to form 7α- or 7-β-hydroxycolesterol (7αOHc or 7βOHc)

and 7-ketocholesterol (7kc) as the

main non-enzymatically generated oxysterols present in most tissues. Formation of other oxysterols such as 5α, 6α- or 5β,6β-epoxycholesterol (α-epoxy-c or β-epoxy-c) as well as cholestan-3β,5α,6β-triol (triolc) has also been reported (Otaegui-Arrazola et al. 2010; Zarrouk et al. 2014; Kulig et al. 2016). Similarly, POPs can be formed by autoxidation giving rise mainly to the formation of 7α/β-hydroxy and 7-keto derivatives, but also epoxy- and triol-derivatives (Hovenkamp et al. 2008; García-Llatas & Rodríguez-Estrada, 2011). The enzymatic formation of COPs implies the activity of microsomal cytochrome P450 monooxygenases, dehydrogenases, epoxydases, hydroxylases and 6

oxidases, some of them tissue specific. 24-S-hydroxycholesterol (24OHc), 25hydroxycholesterol

(25OHc),

27-hydroxycholesterol

(27OHc)

and

4β-

hydroxycholesterol are the main oxysterols formed via cholesterol 24-hydroxylase (CYP46A1), cholesterol 25-hydroxylase (not CYP450 enzyme), cholesterol 27hydroxylase (CYP27A1) and cholesterol 4β-hydroxylase (CYP3A4), respectively. In addition, 7αOHc can be enzymatically formed by CYP7A1 (Vejux et al. 2008). Concerning POPs, CYP450 hydroxylases and other enzymes could be involved in their formation. Alkyl groups at position C24 of the lateral chain enable stereospecific 24Shydroxylation and might hamper the formation of 25- or 27-hydroxyphytosterols (Hovenkamp et al. 2008). Blood concentration of COPs under physiological conditions has been indicated to be several orders of magnitude lower than that of cholesterol (approximately 0.01-0.1 µM versus approximately 5000 µM) (Zarrouk et al. 2014). In hypercholesterolemia, however, higher amounts of oxysterols have been reported. The major plasma oxysterols in these subjects, i.e. 7kc, triolc, α-epox-c, 7αOHc, 7βOHc, and β-epox-c, occur at concentrations of 7 µM, 2 µM, 4 µM, 1 µM, 2 µM, 4 µM, respectively, with a total amount around 20 µM (Chang et al., 1997; Leonarduzzi et al., 2007; Gargiulo et al., 2012) In the case of POPs, usual concentrations found in normal human plasma range between 0.3 and 4.5 ng/mL, i.e. 10-100 times lower values than typical ones observed for COPs (Vanmierlo et al. 2013).

2.2 Cytotoxicity Long time known as products of cholesterol metabolism (Smith et al., 1989; Hwang, 1991), oxysterols attracted the interest of researchers during the latest fifteen years, after it was suggested their involvement in the pathogenetic mechanisms supporting

inflammation-driven

pathologies

like

cancer,

atherosclerosis

and

neurodegenerative disorders (Sottero, et al. 2009; Zarrouk, et al. 2014; Poli, et al., 2013; Poli & Biasi, 2016). Data from these studies rapidly pointed to the importance of controlling the body levels of these compounds, either endogenously generated from parent cholesterol or introduced with food, the amount of which can be remarkable (Otaegui-Arrazola et al., 2010; Rodriguez-Estrada et al., 2014). Toxicity and effects of oxysterols in the initiation and progression of major chronic diseases are stronger than

7

unoxidised cholesterol by at least one or two orders of magnitude (Otaegui-Arrazola et al. 2010). The most cytotoxic have appeared 7-hydroxy, 7-keto and triol derivatives (Ryan et al. 2005). A number of potential molecular mechanisms of COPs toxicity have emerged (Vejux & Lizard, 2009). Cytotoxic effects of oxysterols associated with human diseases include the induction of cell death (apoptosis and/or oncosis), oxidative and inflammatory activities, phospholipidosis, variations of cytoplasmatic levels of calcium, mitochondrial and microsomal membrane perturbations, and polyamine metabolism, with the intracellular overproduction of ROS as a common point (Roussi et al. 2006, Roussi et al. 2007, Lordan et al. 2009, Sottero et al. 2009). In this context COPs are going to be considered as “typical Janus molecules” (Poli & Biasi, 2016), that may behave as positive stressors, or induce toxic effects, possibly depending on their amount and the extent of oxidative redox imbalance elicited. A network of redox-regulated pathways and activation of receptors and transcription factors appear crucial in the outcome of their actions (Vurusaner et al. 2016). As pointed out in the preceding section (2.1), POPs may be considered as potentially toxic dietary components. A role of POPs on atherogenicity, inflammation and cytotoxicity cannot be ruled out (Alemany et al., 2014), however concentrations higher than COPs were needed to exert comparable deleterious effects in vitro (O’Callaghan et al. 2014) and in an in vivo model (Meyer et al., 1998).

3. Phytochemicals and prevention of oxysterols-induced cytotoxicity Dietary phytochemicals (polyphenols, carotenoids and vitamins C and E, among others), that display anti-inflammatory and antioxidant effects may have important roles in preventing oxidative stress-induced injury which characterizes onset and/or progression of a number of chronic diseases. Thus, antioxidant phytochemicals could play an important role in the prevention and treatment of chronic diseases (Zhang et al. 2015). Since generation of ROS, through up-regulation of NADPH oxidases of the Nox family enzymes (Sottero et al. 2009), has been shown as a pathogenetic mechanism of oxysterols toxicity, the eventual preventive and/or therapeutic intervention with bioactive redox phytochemicals, from polyphenols to carotenoids, antioxidant vitamins and other redox-active plant-derived compounds, has been postulated as a natural means to counteract COPs action in various pathologies. The following sections summarize 8

literature data concerning a number of pathophysiological cell models, including intestinal bowel diseases (IBD), atherosclerosis, neurodegenerative diseases and eye disease (i.e. age-related macular degeneration). A few phytochemicals, either pure compounds or food extracts, have been tested in these studies, almost all of them occurring in foods from a typical Mediterranean dietary pattern, such as red wine, olive oil, and characteristic fruits and vegetables. With regards to antioxidant vitamins, the lipid-soluble vitamin E and the water-soluble vitamin C have been studied in the context of oxysterol-induced damage. Finally, a couple of studies treated with phytochemicals highly represented in green tea and extracts of herbs from China or other East countries. It should be mentioned that in some of these studies toxic oxysterols have been assayed at concentrations higher, or much higher, than those relevant to conditions of hypercholesterolemia, and the counteracting activity of either antioxidant vitamins or other phytochemicals investigated at concentrations of pharmacologic more than physiologic or dietary magnitude orders. Then, while a few results will fall in the field of eventual prevention and/or possible adjuvant activity of phytochemicals in oxysterolrelated pathophysiologic conditions, other data, while disclosing molecular pathways of oxysterol toxicity, may suggest potential intervention sites. 3.1 Polyphenols There are many thousands of phenolic or polyphenolic compounds found in plant derived foods and beverages. Polyphenols possess at least one aromatic ring with one or more hydroxyl groups, conferring these compounds remarkable reducing capacities. As a function of the number of phenol rings and on the basis of structural elements that bind these rings to one another, various subgroups are considered. The main classes of polyphenols include phenolic acids, flavonoids, stilbenes and lignans (Crozier et al. 2009). Polyphenols have been reported to express beneficial properties in the prevention of cancer, cardiovascular disease and neurodegeneration, through their interaction with cellular signaling pathways rather than by direct antioxidant effects (Vauzour et al. 2010). Typical levels measured in plasma can vary a lot and has been reported to be in the nM to low µM range, reaching higher µM levels in the gastrointestinal environment (Espín et al. 2007). In recent years, the link of oxysterols in the origin and development of major chronic diseases has been established (Poli et al. 2013), however, the protection exerted by polyphenols in this specific context has received less attention. Table 1 shows the studies quoting chemopreventive 9

mechanisms exerted by polyphenols against oxysterol-induced damage in different pathophysiological cell models.

3.1.1. Inflammatory bowel disease (IBD) IBD is characterized by auto-immune and inflammation related complications of the large intestine (ulcerative colitis) and additional parts of the digestive tract (Crohn’s disease). Recently, SOPs of dietary origin have been involved in the pathogenesis of IBD due to excessive production of pro-inflammatory cytokines and ROS resulting in persistent inflammation (Biasi et al. 2013). Polyphenols may serve as a preventive or adjuvant therapy due to their anti-inflammatory, anti-oxidant, immuno-modulatory and apoptotic properties. Indeed, they can reduce local oxidative stress, and also act on various cellular targets, altering gene-expression related to the progression of IBD, including NF-κB, Nrf-2, Jak/STAT and MAPKs, suppressing the formation of downstream cytokines (e.g. IL-8, IL-1ß, TNF-α), and increasing endogenous antioxidant enzyme systems (HO-1, SOD, GPX) (Kaulmann & Bohn, 2016). In tune with this, pretreatment of differentiated Caco-2 cells (oxysterolchallenged enterocyte-like cells used as an IBD model) with individual polyphenols at physiological relevant concentrations have shown cytoprotection against damage caused by dietary COPs mixtures. Mascia et al. (2010) reported the protective action of epigallocatechin-3-gallate at 1 µM against the pro-inflammatory effect caused by a oxysterol mixture (30 µM) by means of the decrease of pro-inflammatory and chemotactic genes (IL-8, IL-1α, IL-6, IL-23, MCP-1, TGF-β-1, TLR2 and TLR9) and caspase-3 activation, all mediated by the overexpression of the ROS generating NADPH oxidase isoform NOX1. Similarly, Atzeri et al. (2016) showed that the most biologically active phenolic alcohols present in olive oil (hydroxytyrosol and tyrosol) and their sulfate metabolites that can be originated during the process of crossing enterocytes, displayed a protective effect against cell death, lipid peroxidation, ROS production and decrease of GSH levels and GPx activity evoked by an oxidized cholesterol mixture (75-100 µg/mL). Likewise, the use of polyphenol mixtures (more realistic than individual compounds from a nutritional point of view) from extracts of Sardinian wines and extra virgin olive oil also counteracted the pro-oxidant effects derived from dietary oxysterol mixtures. Biasi et al. (2013) and Guina et al. (2015) have shown that polyphenolic extracts from Cannonau (red wine) and Vermentino (white wine) at 25 µg/mL prevented activation of NOX1/p38/MAPK/NF-κB inflammation 10

signaling axis caused by a COPs mixture (60 µM), decreasing the production of IL-6 and IL-8 cytokines (more efficiently in red wine). In addition, Incani et al. (2016) reported that the phenolic fraction of extra virgin olive oil at 5-25 µg/mL protected Caco-2 cells from the oxidant action of a COPs mixture (150 µM) diminishing ROS formation, GSH decrease and limiting membrane lipid oxidation.

3.1.2. Atherosclerosis Oxysterols have been shown to contribute to the development of endothelial cell dysfunction and consequently atherosclerosis due to pro-inflammatory actions and foam cell formation (Poli et al. 2013). In addition, it has been indicated that inactivation of NADPH-oxidase can be a suitable target for the prevention of oxysterol-induced atherosclerosis (Zarrouk et al. 2014). Various polyphenols have been reported to decrease inflammation in cell cultures and animal studies, reduce pro-inflammatory cytokines and inhibit inflammatory mediators such as NF-κB, COX-2 and iNOS. Furthermore, several studies in animals and humans have additionally shown that polyphenols may decrease critical biomarkers of endothelial inflammation such as VCAM and ICAM, further supporting their anti-inflammatory effects (Goya et al. 2016). However, studies that have addressed the protective effect of polyphenols against oxysterols-induced atherosclerosis in relevant cell models are limited. Naito et al. (2004) reported that pretreatment with phenolic red wine extracts at 1-100 ng/mL for 10 h decreased the 10 µM 7βOHc or 25OHc –induced expression and mRNA levels of adhesion molecules (VCAM-1 and ICAM-1), in human aortic endothelial cells, together with a descent of human monocytic U937 cells adhesion to endothelial cells, indicating a protection of blood vessels from atherosclerotic processes. On the other hand, a polyphenol rich extract of Danshen Chinese herb (Salvia miltiorrhiza) containing polyphenols showed a preventive effect against triolc-induced endothelial cell apoptosis both in vitro (human endothelial cells, HUVEC) and in vivo (Sprague-Dawley rats aorta endothelial cells) (Nakazawa et al. 2005). Similarly, pretreatment with epicatechin at 510 µM for 1h prevented intracellular ROS increase derived from NADPH-oxidase induction and caspase-3 upregulation evoked by 7kc at 20 µM in murine macrophages J774A.1 cells (Leonarduzzi et al. 2006). In connection with this, it was reported that pretreatment with kaempferol at 1-30 µM for 18h prevented the degradation of the antiapoptotic BcL-xL, caspase-3 activation and DNA fragmentation caused by high doses

11

of 7βOHc (100 µM) in primary cultures of male Wistar Kyoto rat vascular smooth muscle cells (Ruiz et al. 2006).

3.1.3. Eye disease Several pharmacological actions of polyphenols have been described to be potentially useful in the prevention or treatment of ocular diseases responsible for vision loss such as diabetic retinopathy, macular degeneration and cataract (Majumdar & Srirangam, 2010). In the case of age-related macular degeneration (AMD), reported to be the leading cause of blindness in the elderly population and might have some similarities with atherosclerosis (Malvitte et al. 2006). Indeed, it has been indicated that by analogy to atherosclerosis, oxysterols such as 7βOHc, 7kc or 25OHc at 75 µM may have cytotoxic (caspase-independent cell death associated with phospholipidosis), oxidative (increased ROS production), inflammatory (IL-8 secretion) and/or angiogenic (enhanced VEGF secretion) activities in human retinal ARPE-19 cells. However, when co-incubated with resveratrol at 1 µM, for 24 and 40h, cell death induced by 7βOHc and 7kc was prevented, as well as the pro-angiogenic VEGF secretion induced by 7β-OHc and 25OHc, indicating that this phenolic compound may be considered as a valuable treatment in AMD (Dugas et al. 2010).

3.1.4. Neurodegenerative disease Oxysterols have been long considered as the driving force for developing of neurodegenerative diseases such as Alzheimer’s disease and possibly Parkinson’s disease, since they can permeate the blood-brain barrier and exert multifactorial damaging actions including neuroinflammation, oxidative stress, altered cholesterol metabolism, glial cell activation and dysregulation of intercellular communication among brain cells (Gamba et al. 2015). In this context, non-enzymatic COPs such as 24OHc and 27OHc have shown involvement in both initiation and progression of brain degenerative disease events, with 27OHc displaying lower neurotoxicity than the 24OH counterpart (Poli et al. 2013). In addition, it has been reviewed that dietary polyphenols may exert neuroprotective activity through their ability to suppress neuroinflammation and the potential to promote memory, learning and cognitive functions (Spagnuolo et al. 2016). However, only one study has been found where protective effects of polyphenols have been investigated in relation with neurodegenerative activity of oxysterols (Testa et al. 2014). The study reports that neuroinflammatory activity exerted by 24OHc, 12

27OHc and 7βOHc alone (5 µM) or as a mixture (15 µM), in human neuroblastoma SHSY5Y

cells,

through

Toll-like

receptor-4/cyclooxygenase-2/membrane

bound

prostaglandin E synthase signaling cascade, was prevented by pretreatment with quercetin (5 µM for 1h) loaded in β-cyclodextrin nanoparticles (Testa et al. 2014).

3.2 Vitamins E and C Occurrence in food, absorption and accumulation in human body and functional cycling with vitamin C make α-tocopherol (plasma level 11-37 µM) the most important lipophilic radical quencher in membranes (Wang & Quinn, 1999). By scavenging lipoperoxyl radicals, vitamin E breaks the sequela of events leading to destruction of membrane function and inactivation of membrane enzymes and receptors, which in turn preserves biological responses and activity of pathways controlling cell functions (Azzi & Stocker, 2000). On the other hand, the water-soluble vitamin C, either cytoplasmatic or present in the extracellular fluids (plasma level 43-75 µM), concurs to maintain the cell redox environment by reducing radicals (Niki, 2014) and interplaying with vitamin E and thiol antioxidants, glutathione and lipoic acid (Costantinescu et al., 1993; Hathcock et al. 2005). Due their pivotal importance in maintaining the entire body redox balance, studies investigating the potential of vitamins E and C in preventing and/or treating free radical-mediated degenerative diseases and conditions including cancer, atherosclerosis and neurodegenerative disorders, are numberless, sometimes with contradictory results (Lonn et al., 2012; Wada, 2012, Lopes da Silva et al., 2014). On the basis that pathogenetic mechanisms of oxysterols are associated with production of ROS, a number of investigations have been carried out to assess the effects of these antioxidants against the toxicity of oxysterols in experimental systems mimicking the conditions of various degenerative diseases. Table 2 is a synopsis of literature data, most of which report toxicity of SOPs in atherosclerosis and in neurodegeneration cell models, whereas a few others treat with induction of cell death.

3.2.1 Atherosclerosis In a number of studies aimed at assessing the impact of SOPs on the onset of the atherogenetic process, U937 promonocytic cells were challenged with individual toxic oxysterols at high concentrations (20 µM to 100 µM) for various time lengths (6 to 30 h), after pretreatment of cells with vitamin E (10 µM to 100 µM) (Lizard et al., 2000; 13

Lyons et al., 2001; Miguet-Alfonsi et al., 2002; Vejux et al., 2009), or vitamin C (50 µM) (Lizard et al., 2000). In spite of the different experimental settings, vitamin E (αtocopherol) exhibited protection of various apoptosis features, including loss of mitochondrial transmembrane potential, release of cytochrome C, and overproduction of superoxide. Vitamin E also prevented GSH depletion, lipid peroxidation and increase in apoptotic nuclei (Lyons et al, 2001; Miguet-Alfonsi et al., 2002). On the other hand, vitamin C, though was inhibitor of superoxide production, did not prevent 7kc-induced apoptotic cell death of U937 cells (Lizard et al., 2000). Two studies (Ryan et al., 2005; O’Callaghan et al., 2010) challenged U937 cells with oxidized phytosterols (7βOHsit or 7ksit, 120 µM; 7βOHstig, epoxy-stig, diepoxystig, 60 µM), pre-treating or co-incubating with 10 µM α-tocopherol or 2 µM βcarotene. In the study by Ryan et al (2005), cells were also submitted to 7βOHc. Whereas β-carotene was ineffective in all experiments, α-tocopherol protected against cytotoxicity and apoptosis induced by the 7βOHc, and by epoxy-stig, but was ineffective against all other oxidized phytosterols, suggesting different cytotoxic pathways for POPs. Endothelial dysfunction associated to oxidized lipids and low density lipoprotein is a key event in atherogenesis (Stocker & Keany, 2004). Pretreatment with αtocopherol prevented COPs–induced cytotoxicity in HUVEC (Uemura et al., 2002), and tocotrienols inhibited adhesion of monocytic cells to human aortic endothelial cells (HAECs) (Naito et al., 2005) by inhibiting VCAM-1 expression. An oxysterol mixture qualitatively and quantitatively consistent with the levels of oxysterols in hypercholesterolemia subjects was used to cause apoptosis of red blood cells, or eryptosis (Tesoriere et al., 2014), a process contributing to thromboembolic complications (Chung et al., 2007; Andrews & Low, 1999; Closse et al., 1999), finally enhancing the pathogenetic potential of oxysterols in atherogenesis. The COPs-induced eryptosis goes through production of ROS, followed by release of prostaglandin (PGE2) and opening of PGE2-dependent Ca channels, membrane phosphatidylserine externalisation, and cell shrinkage. Vitamin E, at a physiological blood level (20 µM), co-incubated with either the oxysterol mixture, or the individual 7kc, or triolc, entirely prevented eryptotic cell death, loss of GSH and membrane lipid oxidation, stressing the importance of early ROS production and control in the COPs-induced eryptotic process. Finally, a pionieristic study in rats (Rosenblat & Aviram, 2002) investigated the effect of accumulation of oxysterols in macrophages on the cell-mediated oxidation of 14

LDL. For the first time, the authors showed that the enrichment of peritoneal macrophages with the major oxysterols found in apo-E-deficient mouse, a condition associated with hypercholesterolemia, caused translocation of p47phox from the cytosol to plasma membrane, forming an active NOX complex which produces superoxide anion and facilitates cell-mediated oxidation of LDL. The increased oxysterol content and activity of NOX in macrophages were substantially reduced by supplementing rats with suitable amounts of vitamin E, being this effect associated with decreased release of arachidonate, PKC activity, p47phox translocation, superoxide anion release, and macrophage-related LDL oxidation.

3.2.2 Neurodegenerative diseases Oxysterols that, unlike cholesterol, can permeate the blood brain barrier (Lutjohann et al. 1996), are now considered important for the brain pathophysiology (Solomon et al. 2007; Hu et al. 2008). In this context, studies aimed at reducing neurotoxicity of these compounds have explored antioxidant molecules and vitamins E and C. Old reports examined toxicity of oxysterols against neuronal cells in a differentiated PC12 cell line, as a model of sympathetic neurons, by challenging cells with 25OHc (0-50 µM) (Chang et al., 1998a). This treatment induced dose- and timedependent cell death, which was prevented by co-incubation with high amounts (2-8 mM) vitamin E. Similar observations were then reported by the same group (Chang et al., 1998b) utilizing primary cell cultures of rat sympathetic neuronal cells, and by Kolsch et al., (2001), who assayed 24OHc in differentiated human neuroblastoma SHSY5Y cells, by co-incubating with vitamin E 10-100 µM. Vitamin C, at 1 mM was not effective. Other literature data on this topic fall in the latest five years. In spite of much more complex experimental set-ups and mechanistic pathways revealed, the fundamental protection of vitamin E is confirmed in all studies. 7kc (50 µM) caused apoptosis in 158N murine oligodendrocytes, characterized by condensed and/or fragmented nuclei, caspase activation, internucleosomal DNA fragmentation, loss of transmembrane mitochondrial potential, dephosphorylation of Akt and GSK3 and Mcl1 degradation. Tocopherol (400 µM) counteracted all these effects in the cell line considered (Ragot et al., 2011). 7kc, 7βOHc and 27OHc were assayed on the same 158N murine oligodendrocytes (Ragot et al., 2013) and the apoptotic mode of cell death compared. 27OHc did not cause apoptosis, but accumulated in lipid rafts as well as 7kc but not 7βOHc. α-tocopherol counteracted the 7kc and 7βOHc induced cell death and 15

accumulation of oxysterols in rafts. Other studies (Nury et al., 2014, 2015) revealed oxoapoptophagy of 158N murine oligodendrocytes treated with 7kc, or 7βOHc or 24OHc, as a particular mode of apoptotic death triggered by oxysterols, involving oxidative stress, apoptosis and autophagy. The cytotoxic effects are reduced by vitamin E (400 µM), and docosahexaenoic acid (50 µM) potentiates the effects of vitamin E.

3.2.3 Others A few other studies have been published on the effects of tocopherol/vitamin C in oxysterol-induced toxicity in various cells. Ohtani et al. (1996) studied the toxicity by 7kc (100 µM) in rat hepatocytes showing formation of large amounts of reactive species (•NO and superoxide anion) and decreased cell viability (50% at 24h). α-tocopherol, at 50 and 100 µM, dose-dependently prevented cell death (60% and 80%, respectively) by suppressing the incorporation of 7kc in the hepatocytes and by scavenging O2•-. In the same system vitamin C (50 and 100 µM) had little effect on cell viability when alone, but totally prevented cell death (100%) when added together with equivalent amounts of vitamin E. Rat hepatocytes were co-incubated with 0-2 mg/mL 7kc or triolc, which resulted in increase of the level of superoxide dismutase and catalase, with variable effects on GSH peroxidase (Cantwell & Devery, 1998). Lipid peroxidation was not observed, indicating the effectiveness of the liver cell antioxidant system (possibly vitamin E in membranes). A natural tocopherol blend (Covi-ox), at 0.8 mg/mL, however reduced the effects of both oxysterols on the antioxidant enzymes, and provided an indirect evidence for oxysterol-dependent superoxide production, since TBA-reactive substances appeared to be reduced with respect to cells that were not treated with the blend. Then, though redox-dependent mechanistic pathways leading to defensive antioxidant cell response were less known at the time of the study, the paper revealed that acting as stressors at the membrane level, 7kc or triolc activated production of ROS in turn triggering pathways leading to expression of cytoprotective antioxidant enzymes. Pretreatment with 10 or 100 µM α- or γ-tocopherol was used to investigate eventual protective activity against toxicity induced by four oxysterols (25OHc, 7βOHc, β-epoxyc, α-epoxyc) in colonic adenocarcinoma Caco-2 cells and HepG2 cells (O’Sullivan et al., 2003). While all oxysterols were toxic, with 25OHc exhibiting the highest cytotoxicity in both cell lines, α- and γ-tocopherol were not protective.

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A study in human colon cancer Caco-2 cell line (Roussi et al., 2007) reports that loss of mitochondrial membrane potential and cell death induced by 7βOHc (30 µM) and 7βOHsit (60 µM) were prevented by co-treatment with vitamin C (50 µM). As pointed out in the preceding section (2.2), activity and effects of oxysterols may largely vary according the type of cells, cell environment, oxysterol concentration and exposure time (Vurusaner et al., 2016). In common with the activities of oxysterols in healthy cells, and in spite of the molecular pathways involved in the apoptotic cell death, variation of cell redox equilibrium toward oxidation is a common aspect in cytotoxicity even in cancer cells. The protective antiapoptotic effect observed for vitamin C would support this evidence, although it is difficult to reconcile with the absence of protective effects observed for vitamin C in normal cells. In this scenery, however, while eventual pro-apoptotic effects in healthy cells are considered negative, a similar behavior in transformed cells might in principle be promising for cancer therapy (Poli & Biasi, 2016) and activity of antioxidant vitamins in this context should be monitored. The chemistry of oxysterols allows these compounds to interact with and rapidly move in membrane bilayers, affecting membrane–associated functional proteins (Massey & Pownall, 2006; Olkkonen & Hynynen, 2009), which may relate cytotoxic effects of oxysterols to their capacity of generating ROS through perturbing the activity of signaling enzymes including NADPH oxidases (Han et al., 2008; Shao et al., 2003). At the same level cells are protected by the lipid-soluble and amphipathic vitamin E, whose location in the membrane bilayer allows scavenging and reduction of oxidants, including lipoperoxyl and superoxide radicals, by releasing protons from the hydroxyl group on its chromanol ring. At the same time, as observed in liver membranes that store high amounts of vitamin E, its presence may suppress oxysterol incorporation (Ohtani et al. 1996) and block subsequent adverse effects. Ascorbic acid may protect membrane either intercepting water-soluble radicals before they attack the membrane, or indirectly, through regeneration of the vitamin E radical. As for the activity observed in oxysterol-stimulated cell models, it has been shown scarcely effective in protecting cells when alone, but, as expected, its ability to act in concert with vitamin E causes synergistic protective effects.

3.3 Carotenoids Carotenoids are the most abundant lipophilic phytochemicals, especially present in high amounts in yellow-orange fruits and vegetables and in dark green leafy 17

vegetables. The structure of carotenoids is based on a C40 isoprenoid backbone that may be acyclic or have one or both ends modified into rings. The hydrocarbon carotenoids are classified as carotenes, and those containing at least one oxygen atom are the xanthophylls (Domonkos et al. 2013). These compounds can reach plasma concentrations as high as 2 µM, being the most abundant lycopene, β-carotene and lutein, with higher plasma half-life than polyphenols (days to weeks compared with 230 h). In addition, it has been recently reviewed evidence from in vitro and in vivo studies in animals and humans about their beneficial health effects through influencing transcription factors involved in inflammation and oxidative stress such as NF-κB or Nrf2, beyond mere direct antioxidant actions (Kaulmann & Bohn, 2014). Despite the health benefits indicated in relation to chronic disease prevention, very few studies have addressed the protective effect of carotenoids against oxysterol damage in human relevant pathophysiological cell models, mainly with focus on atherosclerosis (table 3).

3.3.1. Atherosclerosis Accumulation of oxidized LDL and oxysterols in the sub-endothelium and relevant production of ROS, may trigger pro-oxidative, pro-inflammatory and cytotoxic reactions, thus contributing to the progression of vascular dysfunction and development of atheromas (Colles et al. 2001). Antioxidants have been reported to prevent the formation of oxidized LDL during atherogenesis (Zhang et al. 2014). In this sense, and taking into account oxysterols as one of the main actors in atherosclerosis progression, lycopene, β-carotene and astaxanthin have revealed cytoprotection in vitro. Accordingly, the co-incubation of β-carotene (1 µM) (Palozza et al. 2007) or lycopene (2 µM) (Palozza et al. 2010 and 2011) with 7kc (15-25 µM) counteracted the oxidative stress, apoptosis and proinflammatory cytokine cascade evoked by this oxysterol in human THP-1 macrophages, through the prevention of cell growth inhibition, cell cycle arrest and apoptosis induction, decrease of ROS formation by NADPH-oxidase NOX-4, blocking of redox-sensitive MAP kinases and pro-inflammatory NF-κB transcription factor, and increase of PPARγ levels (known for its anti-inflammatory effects). In contrast, it was generally reported that lycopene or astaxanthin (0.1-1 µM) or apigenin (0.5-50 µM) were ineffective against apoptosis caused by 7βOHc (30 µM) in human monocytic U937 cells, as a result of down-regulation of Akt activity (a protein kinase involved in survival signaling) (Lordan et al. 2008).

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3.4 Others In the context of SOPs and diseases, the activity of other plant components, i.e. selenium, the betalain pigment indicaxanthin and the phytosterol stigmasterol, in contrasting oxysterol toxicity have been investigated in models of atherosclerosis and intestinal cytotoxicity (table 4).

3.4.1 Atherosclerosis Selenium is a trace element essential for humans in that it is part of selenoproteins involved in maintaining of the cell redox balance. An inverse correlation has been found between Se and risk of cardiovascular disease (Huttunen, 1997). The relationship between COPs-derived ROS and their role in initiation and/or development of atherosclerosis led a group of researchers to investigate the protective activity of Se against oxysterol toxicity in vivo and in cell systems. Rats, fed a Se-deficient or Seadequate diet, received triolc intravenously (Huang et al., 2002). In the status of deficiency, triolc injured arterial endothelium much more than in the state of Se adequate, indicating that the higher level of blood Se and Se-enzymes may be the main protective factor. In other studies, carried out by the same group in models of endothelial cells and smooth muscle cells (Tang & Hang, 2004; Tang et al., 2005), the antioxidant effect of selenoproteins was shown as a mechanism of Se protection. Betalains, red betacyanins and yellow betaxanthins, are nitrogen-containing pigments with the structure of betalamic acid, occurring in very few edible species including beetroot and cactus pear (Strack et al., 2003; Stintzing & Carle, 2005). Indicaxanthin is a reducing and amphipathic molecule, can interact with and partition in membranes, penetrate cells and counteract oxidative damage in various cell environments in vitro (Tesoriere et al., 2006; Tesoriere et al., 2007) and modulate specific redox-driven signalling pathways involved in the inflammatory response by affecting activity of NADPH oxidase (Tesoriere et al., 2013a). Remarkably, in comparison with most of dietary phytochemicals (Manach et al., 2005), it is highly bioavailable in humans, with plasma levels of 7 μM after ingestion of a dietaryconsistent amount of cactus pear fruit pulp (4 fruits, 28 mg of indicaxanthin) (Tesoriere et al., 2004). This betaxanthin has been investigated in two models of oxysterol-induced damage relevant to atherogenesis, the 7kc-induced apoptosis of human monocytemacrofage THP1 cell line (Tesoriere et al., 2013b) and the red blood cell apoptosis (eryptosis), induced by either an oxysterol mixture consistent with the plasma oxysterol 19

composition of hypercholesterolemic subjects, or the individual 7kc or triolc (Tesoriere et al., 2015). The eryptotic activity of oxysterols may cause additional injury to the pathogenetic pattern of atherosclerosis in pathophysiologic condition of hypercholesterolaemia, because of the adhesion of eryptotic erythrocytes to endothelial cells and eventual thrombo-occlusive complications. In both models indicaxanthin prevented cell death by inhibiting a number of proapoptotic events and ROS overproduction. Inhibition of NOX-4 basal activity and over-expression in THP-1 cells, and of ROScontrolled PGE2 formation in erythrocytes, has appeared fundamental for the protective activity. It is remarkable that these observations were done with concentrations of indicaxanthin in the range of its bioavailability (Tesoriere et al., 2004), suggesting potential health benefits from dietary consumption of this phytochemical in preventing atherogenesis-related pathologies.

3.4.2 Intestinal bowel diseases Bioactivities of POPs, including cytotoxic effects, are less known than COPs, with contradictory results (Hovenkamp et al., 2008; Ryan et al., 2009; García-Llatas and Rodríguez-Estrada, 2011). In general, the effects of POPs have appeared less severe (Ryan et al., 2005; Roussi et al., 2005; Roussi et al., 2007). Interestingly, one study evaluating cytotoxicity of 7k-stigmasterol, a POP formed from one among the most represented dietary plant sterols, in comparison with 7kc, in differentiated Caco-2 cells, with varying concentrations (0-120 µM) and incubation times (4-24 hours), reports that negative effects caused by 7kc on mitochondria and cell cycle were reduced by 7kstigmasterol as well as unoxidized stigmasterol (Alemany et al., 2012). Then, in addition to their known cholesterol-lowering effects, plant sterols and their oxidized derivatives deserve further studies and consideration for eventual protective action against the most toxic oxysterols at the level of the intestinal epithelium, the first physiological barrier after the oral intake of these compounds, the dysfunction of which may start dangerous chronic inflammatory responses.

4. Conclusion and perspectives The capacity to maintain the cell redox homeostasis through various means involving receptors, enzymes, and transcription factors among others, appears as a fundamental aspect of bioactivities of phytochemicals. Although they do not accumulate

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and usually are present in body at very low micromolar concentrations, the role of these molecules in the entire cell redox balance seems to be not less important than that of antioxidant vitamins. From a nutritional and public health standpoint, dietary phytochemicals and antioxidant vitamins have shown, in a number of in vitro mechanistic studies with pathophysiological cell models, their potential beneficial effects in the prevention of diverse oxysterol-driven chronic degenerative diseases mainly through the modulation of intracellular signaling cascades responsible for oxidative stress, inflammation and apoptosis, beyond their direct and stoichiometric antioxidant activity. Although this is true, caution is needed before these promising results can be extrapolated to the in vivo situation in humans. Thus, there is a need for more studies to unravel still unknown molecular effects on transcription factors and some other considerations such as: (i) use of combination of phytochemicals as found in foods or even their metabolites at dietary/physiological relevant proportions and concentrations to evaluate additive, synergistic and/or antagonistic effects; in this context, glucosinolates from Brassica and their products isothiocyanates, known for anti-cancer and neuroprotective effects, deserve attention; (ii) perform assays with patho-physiologic amounts of individual oxysterols and/or followed by analogous experiments with oxysterol mixtures; (iii) test biological actions of esterified oxysterols with fatty acids as occurs in vivo versus solutions of free unesterified oxysterols; and (iv) consider the search for new and more sophisticated cell models (i.e. co-cultures, 3D cell culture, stem cells…). Future research will aid toward an improved understanding of cell bio-signaling pathways and molecular mechanisms through which these phytochemicals could positively affect cell function and/or defend cells from either endogenous or exogenous injury.

Contributions Antonio Cilla, Maria A. Livrea and Luisa Tesoriere have participated in the conception and design of the review; all atuhors have contributed in the drafting and critically revision of the intellectual content and approved the final version.

Fundings This study has been financially supported by the Spanish Ministry of Economy and Competitiveness through Project AGL2015-68006-C2-1-R (MINECO-FEDER). 21

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Table 1. Mechanisms involved in the chemopreventive effect of polyphenolic compounds against oxysterol-induced damage in different pathophysiological cell models. Sample (Target compound/s)

Cell type

Cytoprotective pretreatment

Oxysterol treatment

Cellular mechanism

References

Inflammatory bowel disease Oxysterol mixture 30 µM

Epigallocatechin-3gallate

Sardinian wine extracts and phenolics (gallic and caffeic acids, catechin, epicatechin and quercetin) Sardinian wine extracts and phenolics (gallic and caffeic acids, catechin, epicatechin and quercetin)

Differentiated Caco-2 cells (human enterocyte-like cells)

1 µM for 1h

Differentiated Caco-2 cells

25µg/mL with Cannonau (red wine) and Vermentino (white wine) extracts or phenolics 10µM (quercetin 1µM) for 1h

(12.9 µM 7kc; 9.7 µM α-epox-c; 1.7 µM β-epox-c; 1.3 µM 7αOHc; 4.4 µM 7βOHc) or individual oxysterols 30 min (NOX1 activity), 2h (mRNA expression) and 24h (cytokine synthesis and caspase-3) Oxysterol mixture 60 µM

Protective action against both IL-8 mRNA and protein synthesis and caspase-3 activation by inhibiting NOX1 activity. Also inhibition of pro-inflammatory and chemotactic genes (IL-1α, IL-6, IL-23, MCP1, TGF-β-1, TLR2 and TLR9)

(25.8 µM 7kc; 19.4 µM α-epox-c; 3.4 µM β-epox-c; 2.6 µM 7αOHc; 8.8 µM 7βOHc)

Pretreatment with phenolic wine extracts counteracted NOX1 activation and IL-6 and IL-8 expression and synthesis more efficiently in red wine

30 min (NOX1 activity and IL-6 and IL-8 synthesis) 4h (IL-6 and IL-8 mRNA expression)

Gallic and caffeic acids exerted more significant reduction of IL-6 and IL-8 synthesis than the other phenolics

Mascia et al. (2010)

Biasi et al. (2013)

Oxysterol mixture 60 µM

Differentiated Caco-2 cells

25µg/mL with Cannonau (red wine) and Vermentino (white wine) extracts or phenolics 10µM (quercetin 1µM) for 1h

(25.8 µM 7kc; 19.4 µM α-epox-c; 3.4 µM β-epox-c; 2.6 µM 7αOHc; 8.8 µM 7βOHc) 2h (p38 MAPK and JNK activation), 3h (NF-κB activation) and 24h (IL-8 synthesis)

Mainly red wine extract prevented p38 and NF-κB activation Only epicatechin and caffeic acid prevented NOX1, p38 and NF-κB activation and consequently IL-8 production

Guina et al. (2015)

Oxidized cholesterol mixture

Hydroxytyrosol, tyrosol and their sulfate metabolites

Differentiated Caco-2 cells

2.5-25 µM in water solution for 30 min

(58.9% cholesterol; 13.5% 7kc; 9.5% 7βOHc; 5.6% 6βOHc; 5.9% 7α-OHc; 4.5% cholesta-4,6-dien-3-ol; 2.1% 3keto-4-cholestene)

Protection against cell death (neutral red) from 5µM; inhibition of MDA increase from 2.5 µM; ROS production lower from 10 µM; preservation of control GSH levels from 2.5 µM; prevention of GPx rise from 10 µM

Atzeri et al. (2016)

100 µg/mL 24h (neutral red and MDA), 75 µg/mL 30 min (ROS and GSH), and 18h (GPx activity)

35

Table 1. (continued-I). Sample Cell type

(Target compound/s)

Cytoprotective pretreatment

Oxysterol treatment

Cellular mechanism

References

Oxidized cholesterol mixture (150 µM)

Protection against oxidant action preventing ROS formation from 5 µg/mL and GSH level decrease from 10 µg/mL. Protection of cell membrane oxidation with decreased levels MDA and HP from 5 µg/mL

Incani et al.

Inhibition of surface protein expression and mRNA levels of adhesion molecules (VCAM1 and ICAM-1) and adhesion to monocytes

Naito et al. (2004)

Prevention of triol-induced endothelial cell apoptosis both in vitro and in vivo

Nakazawa et al. (2005)

Prevention of intracellular ROS increase and caspase-3 upregulation

Leonarduzzi et al. (2006)

Kaempferol, unlike rutin, prevented BcL-xL degradation, caspase-3 activation and DNA fragmentation

Ruiz et al. (2006)

Inflammatory bowel disease

Phenolic fraction from Sardinian extra virgin olive oil

Differentiated Caco-2 cells

5-25 µg/mL for 30 min

(42.96% 7kc; 4.26% 7α-OHc; 14.71% 7βOHc; 32.3% α-epox-c ; 5.76% βepox-c)

(2016)

60 min (ROS and GSH), and 24h (MDA and HP levels)

Atherosclerosis

Red wine phenolic extracts

Human aortic endothelial cells (HAEC) and human monocytic U937 cells

1-100 ng/mL for 10h

7βOHc or 25OHc 10 µM for 3, 4 or 6h

In vitro: Extract of Danshen (Salvia Miltiorrhiza) Chinese herb containing polyphenols

Epicatechin

Kaempferol rutin

and

Human umbilical vein endothelial cells (HUVECs)

In vitro: 20 or 200 µg/mL 48h

In vivo:

In vivo:

Sprague-Dawley rats aorta endothelial cells

700 mg/kg 6h

Murine macrophages J774A.1 cells

5-10 µM for 1h

Primary cultures of male Wistar Kyoto rat vascular smooth muscle cells

1-30 µM for 18h

In vitro: 5 µg/mL triolc 6h In vivo: 30 mg/kg triolc 6h just after Danshen administration

7kc 20 µM (3h ROS and 18h caspase-3)

7βOHc 100 µM 6-48h

36

Table 1. (continued-II). Sample (Target compound/s)

Cell type

Cytoprotective pretreatment

Oxysterol treatment

Cellular mechanism

Co-incubation

7βOHc, 7kc or 25OHc

Prevention of cell death induced by 7βOHc and 7-kc not reaching control values

1 µM for 24 and 40h

75 µM for 24 and 40h

References

Eye disease (age-related macular degeneration)

Resveratrol

Human retinal ARPE-19 cells

Decrease on VEGF secretion (pro-angiogenic) induced by 25OHc and 7 βOHc

Dugas et al. (2010)

Neurodegenerative disease 24OHc, 27OHc and 7βOHc at 5 µM Better decrease of the proinflammatory alone or 15 µM as mixture for 3h (TLR- mediator expression via Toll-like receptor5 µM for 1h 4 expression), 6h (CD36, β-integrin, IL- 4/cyclooxygenase-2/membrane bound Testa et al. (2014) 8, MCP-1, MMP-9 and mPGES-1 prostaglandin E synthase (TLR4/COXexpression) and 48h (COX-2 synthesis) 2/mPGES-1) with quercetin nanoparticles MDA: malonyldialdehyde. ROS: reactive oxygen species. GSH: reduced glutathione. GPx: glutathione peroxidase. HP: fatty acids hydroperoxides. ICAM-1: Intercellular adhesion molecule-1 VCAM-1: vascular cell adhesion molecule-1; 7kc: 7-ketocholesterol. 7βOHc: 7β-hydroxycholesterol. 6βOHc: 6β-hydroxycholesterol. 7αOHc: 7β-hydroxycholesterol. Triolc: cholestan3β,5α,6β-triol. α-epox-c: 5α,6α-epoxycholesterol. β-epox-c: 5β,6β-epoxycholesterol. VEGF: vascular endothelial growth factor. Nox1: colonic NADPH oxidase isoform. 24OHc: 24hydroxycholesterol. 25OHc: 25-hydroxycholesterol. 27OHc: 25-hydroxycholesterol. Quercetin: free or loaded into nanoparticles

Human neuroblastoma SH-SY5Y cells

37

Table 2. Mechanisms involved in the chemopreventive effect of vitamins E and C against oxysterol-induced damage in different pathophysiological cell models. Sample (Target compound/s)

Cell type

Cytoprotective pretreatment

Oxysterol treatment

Cellular mechanism

References

7kc 40µg/mL for 24h

Prevention of apoptosis by decrease of mitochondrial transmembrane potential loss (only vit. E), cytocrome c release from mitochondria to cytosol (only vit. E) and reduction of O2• levels (both vitamins)

Lizard et al. (2000)

Atherosclerosis

Vitamins E and C

Human promonocytic leukemia cells (U937)

100 µM (vit. E) or 50 µM (vit C) for 30 min

α-tocopherol, αtocopherol acetate and γ-tocopherol

Human promonocytic leukemia cells (U937)

Co-incubation with 10µM vit. E isoforms for 48h

7βOHc 30 µM for 48h

Vitamin E

Human promonocytic leukemia cells (U937)

100 µM for 1h

7βOHc 50 µM for 18h or 7kc 100 µM for 24h

Vitamin E

Macrophages isolated from atherosclerotic apolipoprotein E deficient mice

α-tocopherol

Only α-tocopherol able to prevent GSH depletion, decrease of membrane integrity, increase in apoptotic nuclei and reduction of DNA fragmentation Protection of apoptosis features such as loss of mitochondrial transmembrane potential, overproduction of O2•, lipid peroxidation, fragmented/condensed nuclei and myelin figures (autophagic vacuoles)

Lyons et al. (2001)

Miguet-Alfonsi et al. (2002)

40 mg/kg per day for 2 months

Major oxysterols found in peritoneal macrophages (7kc, β-epox-c and 7βOHc)

Vitamin E reduction of macrophage oxysterols was associated with decreased macrophage AA release, PKC activity, p47phox translocation to plasma membrane, superoxide anion release and macrophagemediated LDL oxidation

Human umbilical vein endothelial cells (HUVEC)

100 µM for 24h

7kc or 7βOHc 50 µM for 24h

Prevention of cytotoxicity, ROS production, DNA fragmentation and caspase-3 activation

Uemura et al. (2002)

α-tocopherol, γtocopherol and βcarotene

Human promonocytic leukemia cells (U937)

10 µM (tocopherols) or 2 µM (carotene) for 1h

7βOHc 30 µM or 7βOHsit or 7ksit 120 µM for 24h

Only α- and γ-tocopherol protected against cytotoxicity and apoptosis induced by COPs but not POPs. β-carotene exerted no protection against any oxysterol

Ryan et al. (2005)

α-tocopherol and α-, β-, γ-, and δtocotrienol

Human aortic endothelial cells (HAEC) and human monocytic U937 cells

25OHc 20 µM for 18h

Tocotrienols had a profound inhibitory effect on monocytic cell adherence to HAECs relative to α-tocopherol via inhibition of VCAM-1 expression

Naito et al. (2005)

In vivo:

10 µM for 18h

Rosenblat & Aviram (2002)

38

Table 2. (continued-I). Sample

Cytoprotective pretreatment

Oxysterol treatment

Cellular mechanism

References

α-tocopherol

Human promonocytic leukemia cells (U937)

100 µM for 30 min

7kc 100 µM for 6, 14, 18, 24 and/or 30h

Counteraction of phospholipidosis and certain associated apoptotic events (caspase activation, lysosomal degradation) to restore PI3-K activity and to prevent PDK-1 and Akt phosphorylation

Vejux et al. (2009)

α-tocopherol, γtocopherol and βcarotene

Human promonocytic leukemia cells (U937)

Co-incubation with 10 µM (tocopherols) or 2 µM (carotene) for 24h

7βOHstig, epoxy-stig and diepoxy-stig 60 µM for 24h

Only α-tocopherol decreased the percent of apoptotic nuclei induced by epoxy-stig, but all three antioxidants reduced caspase-3 activity induced by this oxysterol. No protective effects with other oxysterols

O’Callaghan et al. (2010)

Inhibition of eryptosis induced by the mixture of oxysterols or individual 7kc or triol by preventing Ca2+ entry to cell, PGE2 production, externalization of membrane PS, cell shrinkage, ROS production, GSH depletion and membrane lipid oxidation

Tesoriere et al. (2014)

Chang et al. (1998a)

(Target compound/s) Atherosclerosis

Cell type

Oxysterol mixture 20 µM α-tocopherol

Erythrocytes from healthy humans

(7 µM 7kc; 2 µM triolc; 4 µM α-epoxc; 4 µM β-epox-c; 1 µM 7αOHc; 2 µM 7βOHc) or individual oxysterols for 48h

20 µM for 1h

Neurodegenerative disease PC12

Co-incubation with 1-8 mM for 72h

25OHc 10µg/mL for 72h

Vitamin E dose-dependent neurotoxicity prevention by bringing the viability from 50% to 100% of control. No protective effect of vitamin C

25OHc 2µg/mL for 48h

Neurotoxicity prevention by bringing the viability from 50% to 95% of control

Chang et al. (1998b)

24OHc 25 µM for 30h

Partially prevention of neurotoxicity with 50100 µM reaching 75% viability (only with vit. E)

Kölsch et al. (2001)

Vitamins E and C

Neuronal cells

Vitamin E

Sympathetic neurons from Sprague-Dawley rats

Co-incubation 2mM for 48h

Vitamins E and C

Differentiated human neuroblastoma SH-SY5Y cells

Co-incubation with 10100 µM (vit. E) or 101000 µM (vit. C) for 30h

with

39

Table 2. (continued-II). Sample Cell type

(Target compound/s)

Cytoprotective pretreatment

Oxysterol treatment

Cellular mechanism

References

7kc 25 µM for 24 and 48h

Inhibition of 7kc accumulation in lipid rafts that counteracts Akt phosphorylation, restoration of GSK3β and Mcl-1 phosphorylation and inhibition associated cascade of apoptotic events (mitochondrial membrane potential loss, release of cytochrome c into cytosol and caspase-3 activation)

Ragot et al. (2011)

7kc, 7βOHc or 27OHc 25 µM for 24 and 48h

α-tocopherol at 400 µM (but no ellagic acid or resveratrol) was able to counteract the Akt/GSK3/Mcl-1/caspase-3 dependent apoptotic cell death signaling pathway induced by 7kc and 7βOHc independent of the rise in Ca2+ level and their accumulation in lipid raft microdomains

Ragot et al. (2013)

7kc 12.5-100 µM for 6, 14, 18 and/or 24h

α-tocopherol prevented 7kc-induced oxiapoptophagy by decreasing ROS overproduction, acidic vesicles formation (autophagy), mitochondrial depolarized cells, apoptotic cells and conversion of microtubuleassociated protein ligh chain 3 (LC3-I) to LCE-II (typical of autophagy)

Nury et al. (2014)

7kc, 7βOHc or 24OHc 50 µM for 24h

DHA enhanced the protective effects of αtocopherol against oxiapoptophagy induced by oxysterols through reduction of cytotoxic effects (inhibition of cell growth, loss of mitochondrial membrane potential, induction of apoptosis, ROS overproduction, rise in caspase-3 activity, PARP degradation, downregulation of Bcl-2 and conversion of LC3-I to LC3-II)

Nury et al. (2015)

Neurodegenerative disease

158N murine oligodendrocytes

α-tocopherol

α-tocopherol, ellagic acid resveratrol

or

158N murine oligodendrocytes

α-tocopherol

α-tocopherol DHA

158N murine oligodendrocytes

and

158N murine oligodendrocytes

400 µM for 2h

α-tocopherol 50-400 µM or with ellagic acid or resveratrol 10-50 µM for 2h

400 µM for 2h

α-tocopherol 400 µM, DHA 50 µM or mixture of both compounds for 2h

40

Table 2. (continued-III). Sample Cell type

Cytoprotective pretreatment

Oxysterol treatment

Cellular mechanism

References

Vitamin E

Hepatocytes from Sprague-Dawley rats

100 µM for 1h

7kc 100 µM for 24h

Prevention of cell death by suppressing the incorporation of 7kc into cell membranes and by scavenging O2•

Ohtani et al. (1996)

Covi-ox (blend of tocopherols)

Hepatocytes from Sprague-Dawley rats

Co-incubation with 0.8 mg/mL for 3h

7kc or triolc 0-2 mg/mL for 3h

Prevention of lipid peroxidation and restoration of antioxidant enzyme levels (SOD, CAT and GPx) to control values

Cantwell & Devery (1998)

α-tocopherol and γtocopherol

Human colonic adenocarcinoma Caco-2 cells and human hepatoma HepG2 cells

10 or 100 µM for 24h

25OHc, 7βOHc, β-epox-c or α-epox-c (0-20 µg/mL) for 24h. Then replaced with complete media and additional incubation 72h

Pretreatment with vit. E isoforms did not protect against oxysterols-induced cytotoxicity using neutral red uptake assay

O’Sullivan et al. (2003)

Vitamin C

Human colon adenocarcinoma Caco-2 cells

Co-treatment with 50 µM for 24 and 48h (hypodiploid cells) or 12 and 24h (mitochondrial membrane potential)

Co-treatment with 7βOHc 30 µM or 7βOHsit 60 µM at the times indicated

Abolishment of the pro-apoptotic effects of both oxysterols decreasing hypodiploid bodies formation and mitochondrial membrane depolarization

Roussi et al. (2007)

(Target compound/s) Other

7kc:7-ketocholesterol. 24OHc: 24-hydroxycholesterol. 25OHc: 25-hydroxycholesterol. 27OHc: 27-hydrpxycholesterol. 7βOHc: 7β-hydroxycholesterol. Triolc: cholestan-3β,5α,6β-triol. β-epoxc: 5β,6β-epoxycholesterol. AA: arachidonic acid. PKC: protein kinase C. α-epox-c: 5α,6α-epoxycholesterol. 7βOHsit: 7β-hydroxysitosterol. 7ksit:7-ketositosterol. PDK-1: 3’ phosphoinositideregulated kinase-1. PI3-K: phosphoinositide 3-kinase. 7βOHstig: 7β-hydroxystigmasterol. Epoxy-stig: 5,6-epoxystigmasta-22,23-diol. Diepoxy-stig: 5,6,22,23-diepoxystigmastane. GSK3β: glycogen synthase kinase 3 isoform β. Mcl-1: myeloid cell leukemia-1. DHA: docosahexaenoic acid. PS: phosphatidylserine. PGE2: prostaglandin E2.

41

Table 3. Mechanisms involved in the chemopreventive effect of carotenoids against oxysterol-induced damage in different pathophysiological cell models. Sample Cell type

(Target compound/s)

Cytoprotective pretreatment

Oxysterol treatment

Cellular mechanism

References

7kc 15 µM for 24h

Prevention of cell growth inhibition by restoring cell cycle arrest in G0/G1 phase (by decreasing p53 a p21 proteins), protection on apoptosis induction (decrease of caspase-3 activity, avoiding Akt, Bcl-2 and Bcl-xL down-regulation and Bax up-regulation), prevention of ROS formation decreasing the expression of NOX-4, and blocking the expression of MAP kinases p38, JNK and ERK1/2

Palozza et al. (2007)

7βOHc 30 µM for 24h

Astaxanthin protected against apoptosis only at 0.1 µM, while lycopene did not protect as well as apigenin at low concentrations, but exacerbated cell death at higher concentrations. These effects can be attributed by their down-regulation of Akt activity

Lordan et al. (2008)

7kc 25 µM for 24h

Prevention of cell growth inhibition by restoring cell cycle arrest in G0/G1 phase (by decreasing p53 a p21 proteins), protection on apoptosis induction (decrease of caspase-3 activity, avoiding Akt, Bcl-2 and Bcl-xL down-regulation and Bax up-regulation), prevention of ROS formation decreasing the expression of NOX-4, avoinding induction of hsp70 and hsp90 proteins and 8-OHdG formation, and blocking the expression of MAP kinases p38, JNK and ERK1/2

Palozza et al. (2010)

7kc 16 µM or 25OHc 4 µM for 3, 6 or 24h

Prevention of proinflammatory cytokine cascade (IL-1β, IL-6, IL-8 and TNF-α) by inhibiting NADPH-oxidase NOX-4 which decreased ROS production and MAP kinases p38, JNK and ERK1/2 expression. Also induction of PPARγ which further inhibited NF-κB activation

Palozza et al. (2011)

Atherosclerosis

THP-1 human macrophages

β-carotene

Lycopene, astaxanthin apigenin

Lycopene

Lycopene

and

Human monocytic U937 cells

THP-1 human macrophages

THP-1 human macrophages

Co-incubation µM for 24h

with 1

Lycopene or astaxanthin (0.1-1 µM) or apigenin (0.5-50 µM) for 1h

Co-incubation µM for 24h

with 2

Co-incubation with 2 µM for 3, 6 or 24h

42

7kc:7-ketocholesterol. 7βOHc: 7β-hydroxycholesterol. 25OHc: 25-hydroxycholesterol. ERK: extracellular regulated kinase. JNK: Jun N-terminal kinase. MAPK: mitogen-activated protein kinase. NF-κB: nuclear factor-kappaB. PPAR: peroxisome proliferator-activated receptor. TNF-α: tumor necrosis factor α.

43

Table 4. Mechanisms involved in the chemopreventive effect of other antioxidants/phytochemicals against oxysterol-induced damage in different pathophysiological cell models. Sample (Target compound/s)

Cell type

Cytoprotective treatment

Blood and vascular wall of Wistar rats Rat vascular smooth muscle cells (VSMCs)

Oxysterol treatment

Cellular mechanism

References

Se-deficient (0.038 mg/kg diet) and Seadequate (0.326 mg/kg diet) diets for 13 weeks

Triolc 5mg/kg for 24h

Se-deficient diet showed lower PGI2 (vasodilator) and higher TXA2 (vasoconstrictor) and more injured endothelium than Se-adequate diet

Huang et al. (2002)

Pretreatment with 50 nM for 12 or 24h

Triolc 1-50 µM for 24h

Protection through increase of cell viability, descent of apoptosis and rise in GPx activity

Tang et al. (2004)

Triolc 10 µM for 24h

Inhibition of apoptosis due to the decrease of GPx activity, T-AOC, SOD activity, fluidity of cell membrane, changes in mitochondrial membrane potential, mRNA expression of cmyc, bcl-2, GPx, TR, and the increase of MDA, ROS and intracellular Ca2+

Tang et al. (2005)

Co-treatment with 7kc 16 µM for 124h depending on the assay

Prevention of cell death associated events (cell cycle arrest, externalization of phosphatidylserine, mitochondrial membrane depolarization and PARP cleavage), and inhibition of NOX-4 induction, ROS overproduction, decrease of total thiols, NFκB activation, cytosolic Ca2+ increase and activation of pro-apoptotic BAD protein

Tesoriere et al. (2013)

Inhibition of oxysterol-induced eryptosis by decreasing: externalization of phosphatidylserine, loss of cell volume (forward scatter), cytosolic Ca2+ increase, formation of PGE2, ROS production, GSH depletion and endothelial adherence of eryptotic cells

Tesoriere et al. (2015)

Atherosclerosis

Selenium

Selenium (sodium selenite)

Selenium (sodium selenite)

Rat vascular smooth muscle cells (VSMCs)

Indicaxanthin

Human monocyte/macrophage THP-1 cells

Indicaxanthin

Healthy human erythrocytes and human umbilical vein entothelial (HUVEC) cells

Pretreatment with 50 nM for 12 or 24h

Co-treatment with 2.5 µM for 1-24h depending on the assay

Oxysterol mixture 20 µM for 48h Pretreatment with 1-5 µM for 1h

(7 µM 7kc; 2 µM triolc; 4 µM α-epoxc; 4 µM β-epox-c; 1 µM 7αOHc; 2 µM 7βOHc)

44

Table 4. (continued-I). Sample (Target compound/s)

Cell type

Cytoprotective treatment

Oxysterol treatment

Cellular mechanism

References

Inflammatory bowel disease Prevention of cell viability decrease, mitochondrial membrane depolarization and Stigmasterol Co-treatment with 7kc 120 µM for 24h Alemany et al. (2012) decrease of total RNA content in G1 phase of cell cycle exerted by 7kc alone 7kc:7-ketocholesterol. 7βOHc: 7β-hydroxycholesterol. 7βOHsit: 7β-hydroxysitosterol. Triolc: cholestan-3β,5α,6β-triol. BAD: Bcl-2 antagonist of cell death. NOX-4: NADPH oxidase-4. PARP: Differentiated human colon adenocarcinoma Caco-2 cells

Co-treatment with 120 µM for 24h

poly(ADP-ribose) polymerase. PGE2: prostaglandin E2. ER: endoplasmic reticulum. IRE1α: inositol-requiring enzyme 1α. PERK: protein kinase RNA-like ER kinase. T-AOC: total antioxidant capacity. GPx: glutathione peroxidase. SOD: superoxide dismutase. MDA: malodialdehyde. TR: thioredoxin reductase. PGI2: prostacyclin. TXA2: tromboxane A2.

45