Efficacy of theobromine in preventing intestinal CaCo-2 cell damage induced by oxysterols.

The alteration of the intestinal barrier function is currently believed to be involved in the pathogenesis of gut diseases mainly associated with the activation of inflammation processes. Diet plays an important role in the control of human gut integrity. Theobromine is a natural methylxanthine present in dark chocolate particularly abundant in cocoa bean shell. This is a polyphenol rich by-product generated in cocoa industrial processing, which is gaining value as a functional ingredient. This study aims to highlight for the first time the capability of theobromine in protecting the intestinal cell monolayer from a mixture of dietary oxysterols showing an inflammatory action in terms of IL-8 and MCP-1 overproduction. Differentiated CaCo-2 cells were treated with 60 µM oxysterol mixture and pre-incubated with 10 µM theobromine. Intestinal barrier damage was investigated in terms of tight junction claudin 1, occludin and JAM-A protein levels, matrix metalloproteinase (MMP) -2 and -9 activation and anti/pro-apoptotic protein changes. The observed cell monolayer permeability protection by theobromine may be due to its ability to inhibit the production of cytokines and MMPs that can be responsible for tight junction loss and apoptosis in intestinal cells. Our findings provide additional mechanistic hints on the healthy effect of theobromine cocoa component as an attractive natural molecule in the prevention of inflammatory gut diseases.

Oxysterols of helminth parasites and pathogenesis of foodborne hepatic trematodiasis caused by Opisthorchis and Fasciola species.

The foodborne trematodiases refer to a cluster of zoonotic neglected tropical diseases caused by trematodes, with transmission involving ingestion of contaminated plants, fishes, and crustaceans. Over 40 million people are infected with foodborne trematodes and 750 million are at risk of infection. From a public health point of view, important species include Clonorchis sinensis, Opisthorchis viverrini, Opisthorchis felineus, Fasciola hepatica, and Fasciola gigantica. Infection with C. sinensis and O. viverrini is classified as a group 1 biological carcinogen and a major risk factor for cholangiocarcinoma. The carcinogenic potential of the infection with O. felineus is less clear but recent biochemical and histopathological findings revealed that opisthorchiasis felinea also fits this pattern. By contrast, evidence of carcinogenic potential of infection with F. hepatica or F. gigantica, close phylogenetics relatives of Opisthorchis, is less certain. Oxysterols have been essentially described in animal model of opisthorchiasis and associated cholangiocarcinoma. Several oxysterol-like metabolites have been detected not only on developmental stages of O. viverrini and O. felineus but also on biofluids from experimentally infected hamsters as products of the activities of the liver flukes. These sterol derivatives are metabolized to active quinones that can modify host DNA. We have postulated that helminth parasite-associated sterols might induce tumor-like phenotypes in biliary epithelia, the cells of origin of liver fluke infection-associated cholangiocarcinoma, through the formation of DNA adducts, dysregulation of apoptosis, and other homeostatic pathways. Here we review, interpret, and discuss findings of oxysterol-like metabolites detected in liver flukes and their role in carcinogenesis, aiming to enhance understanding the pathogenesis of foodborne trematodiasis caused by Opisthorchis and Fasciola species. In future, further investigations will be necessary in order to comprehend relationship between liver flukes' oxysterols and their role in infection-associated diseases in humans.

Activation of LXRs Reduces Oxysterol Lipotoxicity in RPE Cells by Promoting Mitochondrial Function.

Effective treatments for age-related macular degeneration (AMD), the most prevalent neurodegenerative form of blindness in older adults, are lacking. Genome-wide association studies have identified lipid metabolism and inflammation as AMD-associated pathogenic changes. Liver X receptors (LXRs) play a critical role in intracellular homeostases, such as lipid metabolism, glucose homeostasis, inflammation, and mitochondrial function. However, its specific role in AMD and its underlying molecular mechanisms remain unknown. In this study, we investigated the effects of lipotoxicity in human retinal pigmental epithelial (ARPE-19) cells and evaluated how LXRs reduce 7-ketocholesterol (7KCh) lipotoxicity in RPE cells using models, both in vivo and in vitro. A decrease in oxidative lipid accumulation was observed in mouse retinas following the activation of the LXRs; this result was also confirmed in cell experiments. At the same time, LXRs activation reduced RPE cell apoptosis induced by oxysterols. We found that oxysterols decreased the mitochondrial membrane potential in ARPE-19 cells, while LXR agonists counteracted these effects. In cultured ARPE-19 cells, activating LXRs reduced p62, mTOR, and LC3I/II levels, and the knockdown of LXRs elevated the expression of these proteins, indicating that activating LXRs could boost mitophagy. The findings of this study suggest LXR-active pharmaceuticals as a potential therapeutic target for dry AMD.

Oxysterols in stored powders as potential health hazards.

Cholesterol oxidation products (COPs) have greater biological activity than cholesterol itself. Oxysterols reduce the nutritional value of foods and exhibit a wide range of biological activity, including pro-oxidant, carcinogenic, and cytotoxic properties. The most commonly detected oxysterols in foods are 7α-HC, 7ß-HC, a product of their dehydrogenation 7-KC and α-CE, ß-CE. The main dietary sources of oxysterols are eggs and egg-derived products, thermally processed milk and milk-based products, fried meat. This study aimed to measure the amount of cholesterol oxidation products in milk powder, egg powder and milk-egg powder during 24 months of storage. The changes in the selected oxysterols (determined by gas chromatography) were recorded. In milk powder, after the production process, the amount of cholesterol was 0.2 g 100 g-1 fat and in egg powder it was 3.4 g 100 g-1. After 6 months of storage, the dominant oxysterol in milk and egg powder was 7α-HC and in milk-egg powder it was 7-KC. After the storage period, oxysterols in powdered milk reached 1.81% of total cholesterol.  The most stable cholesterol was in the milk-egg mixture and its oxidation was the slowest. This study showed the presence of COPs in milk powder, egg powder and milk-egg powder and the effect of storage on cholesterol oxidation.

Dietary phytochemicals in the protection against oxysterol-induced damage.

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 age-related chronic diseases.

Genotoxicity of lipid oxidation compounds.

Lipid peroxidation, the oxidative degradation of membrane lipids by reactive oxygen species generates a large variety of breakdown products such as alkanes, aldehydes, ketones, alcohols, furans and others. Due to their reactivity aldehydes (alkanals, 2-alkenals, 2,4-alkadienals, 4-hydroxyalkenals) received a lot of attention, in particular because they can diffuse from the site of formation and interact with proteins and nucleic acids thus acting as second toxic messengers. The major aldehydic peroxidation product of membrane lipids is 4-hydroxynonenal (HNE). Since HNE and other 4-hydroxyalkenals are strong alkylating agents they have therefore been considered to be the biologically most important peroxidation products. Although initially research focused on the toxicological potential of these compounds it is now well known that they play also a crucial role in cell signaling under physiological and pathophysiological conditions. Thus, it is obvious that the biological effects will be determined by the intracellular concentrations which can trigger adaptation, DNA damage and cell death. This review will not cover all these aspects but will concentrate on the genotoxic properties of selected lipid oxidation products important in the context of pathophysiological developments together with a chapter on epigenetic modifications.

P2X7-pannexin-1 and amyloid ß-induced oxysterol input in human retinal cell: Role in age-related macular degeneration?

Age-related macular degeneration (AMD) is the most common cause of severe vision loss worldwide. Amyloid ß involvement in degenerative diseases such as AMD is well known and its toxicity has been related to P2X7 receptor-pannexin-1. Recently, oxysterols (oxidized derivatives of cholesterol) have been implicated in AMD pathogenesis. The aim of our study was to highlight amyloid ß/oxysterols relationship and to describe P2X7 receptor-pannexin-1 role in oxysterols toxicity. Using retinal epithelial cells, we first quantified sterols levels after amyloid ß incubation and second we investigated the cytotoxic effects induced by oxysterols. For the first time, our results showed that amyloid ß induced oxysterols formation in human retinal pigmented epithelial cells. We showed that oxysterol toxicity is mediated by P2X7 receptor activation. This activation was dependent on pannexin-1 with 25-hydroxycholesterol whereas P2X7 receptor signaling pathway was pannexin-1-independent for 7-ketocholesterol. Taken together our data suggest a pivotal role of P2X7 receptor-pannexin-1 in oxysterols toxicity in retinal cells which could be an important target to develop new treatments for AMD.