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Graphical abstract Abstract 7-Ketocholesterol (or 7-oxocholesterol) is an oxysterol essentially formed by cholesterol autoxidation. It is often found at enhanced levels in the body fluids and/or target tissues of patients with age-related diseases (cardiovascular, neuronal, and ocular diseases) as well as in subjects concerned with civilization diseases (type 2 diabetes, bowel diseases, and metabolic syndrome). The involvement of increased 7-ketocholesterol levels in the pathophysiology of these diseases is widely suspected. Indeed, 7-ketocholesterol at elevated concentrations is a powerful inducer of oxidative stress, inflammation, and cellular degeneration which are common features of all these diseases. It is important to better know the origin of 7-ketocholesterol (diet, incidence of environmental factors, and endogenous formation (autoxidation and enzymatic synthesis)) and its inactivation mechanisms which include esterification, sulfation, oxidation, and reduction. This knowledge will make it possible to act at different levels to regulate 7-ketocholesterol level and counteract its toxicity in order to limit the incidence of diseases associated with this oxysterol. These different points as well as food and biomedical applications are addressed in this review.

Malignant brain tumors are among the most devastating types of cancer. Glioblastoma is the most common and serious form of brain cancer. Most glioblastomas are surgically unresectable and are typically diagnosed at an advanced stage. The high level of resistance to chemotherapy, radiotherapy and immunotherapy makes glioblastoma one of the most difficult cancers to treat. In brain tumors, the challenges of targeted therapy also include the blood-brain barrier, which often contributes to treatment failure. Therefore, developments of new treatment strategies are required. Metabolic treatments could be an alternative to conventional therapies. Metabolic approaches aim at suppressing glioblastoma tumorigenicity leading to glioblastoma cell death. Since cholesterol metabolism is deregulated in these tumors, this is a promising potential target for therapy. As glioblastoma cells draw on cholesterol from the central nervous system to survive, their growth is theoretically unlimited. Targeting the metabolism of cholesterol by different strategies using, among others, targets of LXRs (Liver X Receptors) or toxic cholesterol analogues could potentially oppose the growth of glial tumors. This chapter discusses the potential of targeting cholesterol metabolism using cholesterol derivatives as a pharmacological alternative to current therapeutic strategy.

Direct translation of findings achieved in experimental cell or animal models to humans is quite a difficult task. We focused here only on the epidemiological and ex vivo human studies so far available about the role of 27-hydroxycholesterol (27OHC) and related metabolism in cancer development. Some studies point to an adverse effect of 27OHC in breast cancer, based on the oxysterol's recognized ability to bind to and modulate estrogen receptors. The detrimental role of this side chain oxysterol would be evident in cancer progression, mainly in post-menopausal women and in an advanced stage of the disease. Other human researches, however, would rather correlate 27OHC intra-tumoral levels to a better prognosis. The analyses on human prostate cancer specimens performed to date are all against a detrimental contribution of 27OHC, rather suggesting interesting anti-prostate cancer effects exerted by this oxysterol. Finally, an increased 27OHC synthesis on the contrary seems to favour progression of late stage cancers in colon, brain and thyroid tissues, as found for breast cancer, possibly due to pro-inflammatory and pro-survival signalling triggered by disproportionate amounts of this oxysterol.

Age-related diseases for which there are no effective treatments include cardiovascular diseases; neurodegenerative diseases such as Alzheimer's disease; eye disorders such as cataract and age-related macular degeneration; and, more recently, Severe Acute Respiratory Syndrome (SARS-CoV-2). These diseases are associated with plasma and/or tissue increases in cholesterol derivatives mainly formed by auto-oxidation: 7-ketocholesterol, also known as 7-oxo-cholesterol, and 7β-hydroxycholesterol. The formation of these oxysterols can be considered as a consequence of mitochondrial and peroxisomal dysfunction, leading to increased in oxidative stress, which is accentuated with age. 7-ketocholesterol and 7β-hydroxycholesterol cause a specific form of cytotoxic activity defined as oxiapoptophagy, including oxidative stress and induction of death by apoptosis associated with autophagic criteria. Oxiaptophagy is associated with organelle dysfunction and in particular with mitochondrial and peroxisomal alterations involved in the induction of cell death and in the rupture of redox balance. As the criteria characterizing 7-ketocholesterol- and 7β-hydroxycholesterol-induced cytotoxicity are often simultaneously observed in major age-related diseases (cardiovascular diseases, age-related macular degeneration, Alzheimer's disease) the involvement of these oxysterols in the pathophysiology of the latter seems increasingly likely. It is therefore important to better understand the signalling pathways associated with the toxicity of 7-ketocholesterol and 7β-hydroxycholesterol in order to identify pharmacological targets, nutrients and synthetic molecules attenuating or inhibiting the cytotoxic activities of these oxysterols. Numerous natural cytoprotective compounds have been identified: vitamins, fatty acids, polyphenols, terpenes, vegetal pigments, antioxidants, mixtures of compounds (oils, plant extracts) and bacterial enzymes. However, few synthetic molecules are able to prevent 7-ketocholesterol- and/or 7β-hydroxycholesterol-induced cytotoxicity: dimethyl fumarate, monomethyl fumarate, the tyrosine kinase inhibitor AG126, memantine, simvastatine, Trolox, dimethylsufoxide, mangafodipir and mitochondrial permeability transition pore (MPTP) inhibitors. The effectiveness of these compounds, several of which are already in use in humans, makes it possible to consider using them for the treatment of certain age-related diseases associated with increased plasma and/or tissue levels of 7-ketocholesterol and/or 7β-hydroxycholesterol.

Cholesterol oxidation products, also named oxysterols, can be formed either by cholesterol auto-oxidation, enzymatically or both. Among these oxysterols, 7-ketocholesterol (7KC) is mainly formed during radical attacks that take place on the carbon 7 of cholesterol. As increased levels of 7KC have been found in the tissues, plasma and/or cerebrospinal fluid of patients with major diseases, especially age-related diseases (cardiovascular diseases, eye diseases, neurodegenerative diseases), some cancers, and chronic inflammatory diseases, it is suspected that 7KC, could contribute to their development. Since 7KC, provided by the diet or endogenously formed, is not or little efficiently metabolized, except in hepatic cells, its cellular accumulation can trigger numerous side effects including oxidative stress, inflammation and cell death. To counteract 7KC-induced side effects, it is necessary to characterize the metabolic pathways activated by this oxysterol to identify potential targets for cytoprotection and geroprotection. Currently, several natural compounds (tocopherols, fatty acids, polyphenols, etc) or mixtures of compounds (oils) used in traditional medicine are able to inhibit the deleterious effects of 7KC. The different molecules identified could be valued in different ways (functional foods, recombinant molecules, theranostic) to prevent or treat diseases associated with 7KC.

Graphical abstract, 7-Ketocholesterol, which is one of the earliest cholesterol oxidization products identified, is essentially formed by the auto-oxidation of cholesterol. In the body, 7-ketocholesterol is both provided by food and produced endogenously. This pro-oxidant and pro-inflammatory molecule, which can activate apoptosis and autophagy at high concentrations, is an abundant component of oxidized Low Density Lipoproteins. 7-Ketocholesterol appears to significantly contribute to the development of age-related diseases (cardiovascular diseases, age-related macular degeneration, and Alzheimer's disease), chronic inflammatory bowel diseases and to certain cancers. Recent studies have also shown that 7-ketocholesterol has anti-viral activities, including on SARS-CoV-2, which are, however, lower than those of oxysterols resulting from the oxidation of cholesterol on the side chain. Furthermore, 7-ketocholesterol is increased in the serum of moderately and severely affected COVID-19 patients. In the case of COVID-19, it can be assumed that the antiviral activity of 7-ketocholesterol could be counterbalanced by its toxic effects, including pro-oxidant, pro-inflammatory and pro-coagulant activities that might promote the induction of cell death in alveolar cells. It is therefore suggested that this oxysterol might be involved in the pathophysiology of COVID-19 by contributing to the acute respiratory distress syndrome and promoting a deleterious, even fatal outcome. Thus, 7-ketocholesterol could possibly constitute a lipid biomarker of COVID-19 outcome and counteracting its toxic effects with adjuvant therapies might have beneficial effects in COVID-19 patients.

International audience; Steroidal maleic anhydrides were prepared in one step: lithocholic, chenodeoxicholic,deoxicholic, ursocholic, and hyodeoxicholic acid derivatives. Their capability to induce celldeath was studied on C6 rat glioma cells, and 7β-hydroxycholesterol was used as positivecytotoxic control. The highest cytotoxicity was observed with lithocholic andchenodeoxicholic acid derivatives (23-(4-methylfuran-2,5-dione)-3α-hydroxy-24-nor-5β-cholane (compound 1a), and 23-(4-methylfuran-2,5-dione)-3α,7α-dihydroxy-24-nor-5β-cholane (compound 1b), respectively), which induce a non-apoptotic mode of cell deathassociated with mitochondrial membrane potential loss and reactive oxygen speciesoverproduction. No cells with condensed and/or fragmented nuclei, no PARP degradation andno cleaved-caspase-3, which are apoptotic criteria, were observed. Similar effects were foundwith 7β-hydroxycholesterol. The cell clonogenic survival assay showed that compound 1bwas more cytotoxic than compound 1a and 7β-hydroxycholesterol. Compound 1b and 7β-hydroxycholesterol also induce cell cycle modifications. In addition, compounds 1a and 1b,and 7β-hydroxycholesterol favour the formation of large acidic vacuoles revealed by stainingwith acridine orange and monodansylcadaverine evocating autophagic vacuoles; they alsoinduce an increased ratio of [LC3-II / LC3-I], and modify the expression of mTOR, Beclin-1,Atg12, and Atg5-Atg12 which is are autophagic criteria. The ratio [LC3-II / LC3-I] is alsostrongly modified by bafilomycin acting on the autophagic flux. Rapamycin, an autophagicinducer, and 3-methyladenine, an autophagic inhibitor, reduce and increase 7β-hydroxycholesterol-induced cell death, respectively, supporting that 7β-hydroxycholesterolinduces survival autophagy. Alpha-tocopherol also strongly attenuates 7β-hydroxycholesterol-induced cell death. However, rapamycin, 3-methyladenine, and α-tocopherol have no effecton compounds 1a and 1b-induced cell death. It is concluded that these compounds trigger anon apoptotic mode of cell death, involving the mitochondria and associated with severalcharacteristics of autophagy.

This work was presented as an oral presentation at the 7th ENOR (European Network for Oxysterol Research) Symposium ‘Oxysterols and Sterol Derivatives in Health and Disease’, September 21–22, 2017, Université catholique de Louvain, Brussels, Belgium (https://www.oxysterols.net/).; International audience; The involvement of organelles in cell death is well established especially for endoplasmic reticulum, lysosomes and mitochondria. However, the role of the peroxisome is not well known, though peroxisomal dysfunction favors a rupture of redox equilibrium. To study the role of peroxisomes in cell death, 158 N murine oligodendrocytes were treated with 7-ketocholesterol (7 KC: 25-50 mu M, 24 h). The highest concentration is known to induce oxiapoptophagy (OXIdative stress + APOPTOsis + autoPHAGY), whereas the lowest concentration does not induce cell death. In those conditions (with 7 KC: 50 mu M) morphological, topographical and functional peroxisome alterations associated with modifications of the cytoplasmic distribution of mitochondria, with mitochondrial dysfunction (loss of transmembrane mitochondrial potential, decreased level of cardiolipins) and oxidative stress were observed: presence of peroxisomes with abnormal sizes and shapes similar to those observed in Zellweger fibroblasts, lower cellular level of ABCD3, used as a marker of peroxisomal mass, measured by flow cytometry, lower mRNA and protein levels (measured by RT-qPCR and western blotting) of ABCD1 and ABCD3 (two ATP-dependent peroxisomal transporters), and of ACOX1 and MFP2 enzymes, and lower mRNA level of DHAPAT, involved in peroxisomal beta-oxidation and plasmalogen synthesis, respectively, and increased levels of very long chain fatty acids (VLCFA: C24:0, C24:1, C26:0 and C26:1, quantified by gas chromatography coupled with mass spectrometry) metabolized by peroxisomal beta-oxidation. In the presence of 7 KC (25 mu M), slight mitochondrial dysfunction and oxidative stress were found, and no induction of apoptosis was detected; however, modifications of the cytoplasmic distribution of mitochondria and clusters of mitochondria were detected. The peroxisomal alterations observed with 7 KC (25 mu M) were similar to those with 7 KC (50 mu M). In addition, data obtained by transmission electron microcopy and immunofluorescence microscopy by dual staining with antibodies raised against p62, involved in autophagy, and ABCD3, support that 7 KC (25-50 mu M) induces pexophagy. 7 KC (25-50 mu M)-induced side effects were attenuated by alpha-tocopherol but not by alpha-tocotrienol, whereas the anti-oxidant properties of these molecules determined with the FRAP assay were in the same range. These data provide evidences that 7 KC, at concentrations inducing or not cell death, triggers morphological, topographical and functional peroxisomal alterations associated with minor or major mitochondrial changes.

Oxysterols are oxidized derivatives of cholesterol that are formed enzymatically or via reactive oxygen species or both. Cholesterol or oxysterols ingested as food are absorbed and packed into lipoproteins that are taken up by hepatic cells. Within hepatic cells, excess cholesterol is metabolised to form bile acids. The endoplasmic reticulum acts as the main organelle in the bile acid synthesis pathway. Metabolised sterols originating from this pathway are distributed within other organelles and in the cell membrane. The alterations to membrane oxysterol:sterol ratio affects the integrity of the cell membrane. The presence of oxysterols changes membrane fluidity and receptor orientation. It is well documented that hydroxylase enzymes located in mitochondria facilitate oxysterol production via an acidic pathway. More recently, the presence of oxysterols was also reported in lysosomes. Peroxisomal deficiencies favour intracellular oxysterols accumulation. Despite the low abundance of oxysterols compared to cholesterol, the biological actions of oxysterols are numerous and important. Oxysterol levels are implicated in the pathogenesis of multiple diseases ranging from chronic inflammatory diseases (atherosclerosis, Alzheimer's disease and bowel disease), cancer and numerous neurodegenerative diseases. In this article, we review the distribution of oxysterols in sub-cellular organelles and in biological fluids.