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Aim: Cholesterol homeostasis is associated with Alzheimer's disease (AD). Despite the multitude of cholesterol metabolites, little is known about which metabolites are directly involved in AD pathogenesis and can serve as its potential biomarkers. Methods: To identify "hit" metabolites, steroid profiling was conducted in mice with different age, diet, and genotype and also in humans with normal cognition, mild cognitive impairment, and AD using gas chromatography–mass spectrometry. Then, using one of the "hit" molecules (7β‐hydroxycholesterol; OHC), molecular and histopathological experiment and behavioral testing were conducted in normal mice following its intracranial stereotaxic injection to see whether this molecule drives AD pathogenesis and causes cognitive impairment. Results: The serum levels of several metabolites, including 7β‐OHC, were increased by aging in the 3xTg‐AD unlike normal mice. Consistently, the levels of 7β‐OHC were increased in the hairs of patients with AD and were correlated with clinical severity. We found that 7β‐OHC directly affects AD‐related pathophysiology; intrahippocampal injection of 7β‐OHC induced astrocyte and microglial cell activation, increased the levels of pro‐inflammatory cytokines (TNF‐alpha, IL‐1β, IL‐6), and enhanced amyloidogenic pathway. Mice treated with 7β‐OHC also exhibited deficits in memory and frontal/executive functions assessed by object recognition and 5‐choice serial reaction time task, respectively. Conclusions: Our results suggest that 7β‐OHC could serve as a convenient, peripheral biomarker of AD. As directly involved in AD pathogenesis, 7β‐OHC assay may help actualize personalized medicine in a way to identify an at‐risk subgroup as a candidate population for statin‐based AD treatment. [ABSTRACT FROM AUTHOR]

Background: Mitochondrial dysregulation in skeletal myocytes is considered a major factor in aged sarcopenia. In this study, we aimed to study the effects of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) on Sestrin2-mediated mechanistic target of rapamycin complex 1 (mTORC1) in aged skeletal muscles. Methods: C2C12 myoblasts were stimulated by 50 μM 7β-hydroxycholesterol (7β-OHC) to observe the changes of DNA damage, mitochondrial membrane potential (Δψm), mitochondrial ROS and PGC-1α protein. The PGC-1α silence in the C2C12 cells was established by siRNA transfection. The levels of DNA damage, Δψm, mitochondrial ROS, Sestrin2 and p-S6K1/S6K1 proteins were observed after the PGC-1α silence in the C2C12 cells. Recombinant Sestrin2 treatment was used to observe the changes of DNA damage, Δψm, mitochondrial ROS and p-S6K1/S6K1 protein in the 7β-OHC-treated or PGC-1α siRNA-transfected C2C12 cells. Wild-type (WT) mice and muscle-specific PGC-1α conditional knockout (MKO) mice, including young and old, were used to analyse the effects of PGC-1α on muscle function and the levels of Sestrin2 and p-S6K1 in the white gastrocnemius muscles. Recombinant Sestrin2 was administrated to analyse its effects on muscle function in the old WT mice and old MKO mice. Results: 7β-OHC treatment induced DNA damage, mitochondrial dysfunction and decrease of PGC-1α protein in the C2C12 cells. PGC-1α silence also induced DNA damage and mitochondrial dysfunction in the C2C12 cells. Additionally, PGC-1α silence or 7β-OHC treatment decreased the levels of Sestrin2 and p-S6K1/S6K1 protein in the C2C12 cells. Recombinant Sestrin2 treatment significantly improved the DNA damage and mitochondrial dysfunction in the 7β-OHC-treated or PGC-1α siRNA-transfected C2C12 cells. At the same age, muscle-specific PGC-1α deficiency aggravated aged sarcopenia and decreased the levels of Sestrin2 and p-S6K1 in the white gastrocnemius muscles when compared to the WT mice. Recombinant Sestrin2 treatment improved muscle function and increased p-S6K1 levels in the old two genotypes. Conclusion: This research demonstrates that PGC-1α participates in regulating mitochondrial function in aged sarcopenia through effects on the Sestrin2-mediated mTORC1 pathway.

Mitochondrial dysregulation in skeletal myocytes is considered a major factor in aged sarcopenia. In this study, we aimed to study the effects of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) on Sestrin2-mediated mechanistic target of rapamycin complex 1 (mTORC1) in aged skeletal muscles. C2C12 myoblasts were stimulated by 50 μM 7β-hydroxycholesterol (7β-OHC) to observe the changes of DNA damage, mitochondrial membrane potential (Δψm), mitochondrial ROS and PGC-1α protein. The PGC-1α silence in the C2C12 cells was established by siRNA transfection. The levels of DNA damage, Δψm, mitochondrial ROS, Sestrin2 and p-S6K1/S6K1 proteins were observed after the PGC-1α silence in the C2C12 cells. Recombinant Sestrin2 treatment was used to observe the changes of DNA damage, Δψm, mitochondrial ROS and p-S6K1/S6K1 protein in the 7β-OHC-treated or PGC-1α siRNA-transfected C2C12 cells. Wild-type (WT) mice and muscle-specific PGC-1α conditional knockout (MKO) mice, including young and old, were used to analyse the effects of PGC-1α on muscle function and the levels of Sestrin2 and p-S6K1 in the white gastrocnemius muscles. Recombinant Sestrin2 was administrated to analyse its effects on muscle function in the old WT mice and old MKO mice. 7β-OHC treatment induced DNA damage, mitochondrial dysfunction and decrease of PGC-1α protein in the C2C12 cells. PGC-1α silence also induced DNA damage and mitochondrial dysfunction in the C2C12 cells. Additionally, PGC-1α silence or 7β-OHC treatment decreased the levels of Sestrin2 and p-S6K1/S6K1 protein in the C2C12 cells. Recombinant Sestrin2 treatment significantly improved the DNA damage and mitochondrial dysfunction in the 7β-OHC-treated or PGC-1α siRNA-transfected C2C12 cells. At the same age, muscle-specific PGC-1α deficiency aggravated aged sarcopenia and decreased the levels of Sestrin2 and p-S6K1 in the white gastrocnemius muscles when compared to the WT mice. Recombinant Sestrin2 treatment improved muscle function and increased p-S6K1 levels in the old two genotypes. This research demonstrates that PGC-1α participates in regulating mitochondrial function in aged sarcopenia through effects on the Sestrin2-mediated mTORC1 pathway. • 7β-OHC treatment induced mitochondrial dysfunction and PGC-1α reduction in C2C12. • PGC-1α affects the Sestrin2-mediated mTORC1 pathway. • Recombinant Sestrin2 treatment could improve aged sarcopenia. [ABSTRACT FROM AUTHOR]

Oxysterols (OXY) are oxidized derivatives of cholesterol associated with oxidation and can increase the risk of cardiovascular diseases. The aim of the current study is to examine the relationships between OXY profile, lipids, lipoprotein(a) [Lp(a)] and paraoxonase1 (PON1) with coronary heart disease (CHD) in patients with diabetes mellitus type1 (T1DM) and type2 (T2DM). 120 diabetic patients (T1DM=40, T2DM=80) and 60 healthy subjects were recruited in the study. OXY profile (7-KChol, 7ß-OHChol and Chol-triol) was measured using liquid chromatography-mass spectrometry. The clinical profile of the study participants was also collected. 7-KChol, 7ß-OHChol and Chol-triol and Lp(a), FBG and glycation parameters were higher in diabetic patients compared to controls (p<0.01), whereas PON1 was lower in patients compared to controls (p<0.01). Within the T2DM group, 7-KChol and 7ß-OHChol levels were associated with CHD, obesity, and smoking (p<0.05). In addition, KChol, 7ß-OHChol and Chol-triol levels were associated with smoking in T1DM (p<0.05). In both diabetic types, 7-KChol, 7ß-OHChol and Chol-triol were significantly correlated with TC, LDL, ApoB and Lp(a), glycation parameters and inversely with PON1 (p<0.05). OXY profile in diabetic patients can be used as a reliable biomarker of CHD, particularly in T2DM. [ABSTRACT FROM AUTHOR]

More and more attention is nowadays given to the possible translational application of a great number of biochemical and biological findings with the involved molecules. This is also the case of cholesterol oxidation products, redox molecules over the last years deeply investigated for their implication in human pathophysiology. Oxysterols of non-enzymatic origin, the excessive increase of which in biological fluids and tissues is of toxicological relevance for their marked pro-oxidant and pro-inflammatory properties, are increasingly applied in clinical biochemistry as molecular markers in the diagnosis and monitoring of several human and veterinary diseases. Conversely, oxysterols of enzymatic origin, the production of which is commonly under physiological regulation, could be considered and tested as promising pharmaceutical agents because of their antiviral, pro-osteogenic and antiadipogenic properties of some of them. Very recently, the quantification of oxysterols of non-enzymatic origin has been adopted in a systematic way to evaluate, monitor and improve the quality of cholesterol-based food ingredients, that are prone to auto-oxidation, as well as their industrial processing and the packaging and the shelf life of the finished food products. The growing translational value of oxysterols is here reviewed in its present and upcoming applications in various industrial fields.

• We collected follicular fluid samples of PCOS patients and controls without PCOS. • Metabolomic analysis identified 21 differential metabolites. • The Vitamin B6 metabolism, phenylalanine metabolism and carnitine synthesis were related to PCOS. • 7β-Hydroxycholesterol was a potential biomarker of PCOS. The polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disorder syndrome of women in reproductive age. Metabolomic studies of the follicular fluid can reveal the potential metabolic pathways related to PCOS. The objection of this study was to explore the changes of metabolites in the follicular fluid of PCOS. We collected follicular fluid samples of 35 patients with PCOS and 33 controls without PCOS for metabolomic analysis with UPLC Q-Exactive. The identified metabolites were annotated with KEGG and HMDB to determine the disturbances of metabolic pathways in PCOS. Based on the regression model, we conducted the ROC analysis to find the biomarker of PCOS in the follicular fluid. Metabolomic analysis identified 21 differential metabolites in PCOS, which revealed that the Vitamin B6 metabolism, phenylalanine metabolism and carnitine synthesis were the key changed pathways. We found that 7β-Hydroxycholesterol was potential biomarker of PCOS based on the ROC analysis. We identified metabolic alterations and biomarker in the follicular fluid of PCOS, providing novel ways for the diagnosis and treatment of PCOS. [ABSTRACT FROM AUTHOR]

Aging is characterized by a progressive increase in oxidative stress, which favors lipid peroxidation and the formation of cholesterol oxide derivatives, including 7β-hydroxycholesterol (7β-OHC). This oxysterol, which is known to trigger oxidative stress, inflammation, and cell death, could contribute to the aging process and age-related diseases, such as sarcopenia. Identifying molecules or mixtures of molecules preventing the toxicity of 7β-OHC is therefore an important issue. This study consists of determining the chemical composition of Tunisian Pistacia lentiscus L. seed oil (PLSO) used in the Tunisian diet and evaluating its ability to counteract the cytotoxic effects induced by 7β-OHC in murine C2C12 myoblasts. The effects of 7β-OHC (50 µM; 24 h), associated or not with PLSO, were studied on cell viability, oxidative stress, and on mitochondrial and peroxisomal damages induction. α-Tocopherol (400 µM) was used as the positive control for cytoprotection. Our data show that PLSO is rich in bioactive compounds; it contains polyunsaturated fatty acids, and several nutrients with antioxidant properties: phytosterols, α-tocopherol, carotenoids, flavonoids, and phenolic compounds. When associated with PLSO (100 µg/mL), the 7β-OHC-induced cytotoxic effects were strongly attenuated. The cytoprotection was in the range of those observed with α-tocopherol. This cytoprotective effect was characterized by prevention of cell death and organelle dysfunction (restoration of cell adhesion, cell viability, and plasma membrane integrity; prevention of mitochondrial and peroxisomal damage) and attenuation of oxidative stress (reduction in reactive oxygen species overproduction in whole cells and at the mitochondrial level; decrease in lipid and protein oxidation products formation; and normalization of antioxidant enzyme activities: glutathione peroxidase (GPx) and superoxide dismutase (SOD)). These results provide evidence that PLSO has similar antioxidant properties than α-tocopherol used at high concentration and contains a mixture of molecules capable to attenuate 7β-OHC-induced cytotoxic effects in C2C12 myoblasts. These data reinforce the interest in edible oils associated with the Mediterranean diet, such as PLSO, in the prevention of age-related diseases, such as sarcopenia.

Oxysterols are assumed to be the driving force behind numerous neurodegenerative diseases. In this work, we aimed to study the ability of 7β-hydroxycholesterol (7β-OHC) to trigger oxidative stress and cell death in human neuroblastoma cells (SH-SY5Y) then the capacity of Nigella sativa and Milk thistle seed oils (NSO and MTSO, respectively) to oppose 7β-OHC-induced side effects. The impact of 7β-OHC, associated or not with NSO or MTSO, was studied on different criteria: cell viability; redox status, and apoptosis. Oxidative stress was assessed through the intracellular reactive oxygen species (ROS) production, levels of enzymatic and non-enzymatic antioxidants, lipid, and protein oxidation products. Our results indicate that 7β-OHC (40 µg/mL) exhibit pr-oxidative and pro-apoptotic activities shown by a decrease of the antioxidant enzymatic activities and an increase of ROS production, lipid, and protein oxidation end products as well as nitrotyrosine formation and caspase 3 activation. However, under the pre-treatment with NSO, and especially with MTSO (100 µg/mL), a marked attenuation of oxidative damages was observed. Our study suggests harmful effects of 7β-OHC consisting of pro-oxidative, anti-proliferative, and pro-apoptotic activities that may contribute to neurodegeneration. NSO and especially MTSO showed potential cytoprotection against the cytotoxicity of 7β-OHC.

Mitochondrial dysfunction and oxidative stress are involved in neurodegenerative diseases associated with an enhancement of lipid peroxidation products such as 7β-hydroxycholesterol (7β-OHC). It is, therefore, important to study the ability of 7β-OHC to trigger mitochondrial defects, oxidative stress, metabolic dysfunctions and cell death, which are hallmarks of neurodegeneration, and to identify cytoprotective molecules. The effects of biotin were evaluated on 158N murine oligodendrocytes, which are myelin synthesizing cells, exposed to 7β-OHC (50 µM) with or without biotin (10 and 100 nM) or α-tocopherol (positive control of cytoprotection). The effects of biotin on 7β-OHC activities were determined using different criteria: cell adhesion; plasma membrane integrity; redox status. The impact on mitochondria was characterized by the measurement of transmembrane mitochondrial potential (ΔΨm), reactive oxygen species (ROS) overproduction, mitochondrial mass, quantification of cardiolipins and organic acids. Sterols and fatty acids were also quantified. Cell death (apoptosis, autophagy) was characterized by the enumeration of apoptotic cells, caspase-3 activation, identification of autophagic vesicles, and activation of LC3-I into LC3-II. Biotin attenuates 7β-OHC-induced cytotoxicity: loss of cell adhesion was reduced; antioxidant activities were normalized. ROS overproduction, protein and lipid oxidation products were decreased. Biotin partially restores mitochondrial functions: attenuation of the loss of ΔΨm; reduced levels of mitochondrial O2•− overproduction; normalization of cardiolipins and organic acid levels. Biotin also normalizes cholesterol and fatty acid synthesis, and prevents apoptosis and autophagy (oxiapoptophagy). Our data support that biotin, which prevents oligodendrocytes damages, could be useful in the treatment of neurodegeneration and demyelination. [ABSTRACT FROM AUTHOR]

Age-related diseases, including neurodegenerative diseases, are associated with oxidative stress and lipid peroxidation, and increase the levels of cholesterol auto-oxidation products such as 7β-hydroxycholesterol (7β-OHC). Thus, it is imperative to identify agents that can prevent 7β-OHC-induced side-effects. We evaluated the potential protective effects of Carpobrotus edulis ethanol-water extract (EWe) on murine oligodendrocytes (158N) cultured in the absence or presence of 7β-OHC (20 μg/mL, 24 h). The cells were incubated with EWe (20-200 µg/mL) 2 h before 7β-OHC treatment. Mitochondrial activity and cell growth were evaluated with the MTT assay. Photometric methods were used to analyze antioxidant enzyme [catalase (CAT) and glutathione peroxidase (GPx)] activities and the generation of lipid and protein oxidation products [malondialdehyde (MDA), conjugated diene (CD), and carbonylated proteins (CPs)]. Treatment with 7β-OHC induced cell death and oxidative stress (reflected by alteration in CAT and SOD activities). Overproduction of lipid peroxidation products (MDA and CDs) and CPs was also reported. The cytotoxic effects associated with 7β-OHC were attenuated by 160 μg/mL of EWe of C. edulis. Cell death induced by 7β-OHC treatment was ameliorated, GPx and CAT activities were restored to normal, and MDA, CD, and CP levels were reduced following C. edulis extract treatment. These data demonstrate the protective activities of C. edulis EWe against 7β-OHC-induced disequilibrium in the redox status of 158N cells, indicative of the potential role of this plant extract in the prevention of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Aging is a complex biological process which can be associated with skeletal muscle degradation leading to sarcopenia. The aim of this study consisted i) to determine the oxidative and inflammatory status of sarcopenic patients and ii) to clarify the impact of oxidative stress on myoblasts and myotubes. To this end, various biomarkers of inflammation (C-reactive protein (CRP), TNF-α, IL-6, IL-8, leukotriene B4 (LTB4)) and oxidative stress (malondialdehyde, conjugated dienes, carbonylated proteins and antioxidant enzymes: catalase, superoxide dismutase, glutathione peroxidase) as well as oxidized derivatives of cholesterol formed by cholesterol autoxidation (7-ketocholesterol, 7β-hydroxycholesterol), were analyzed. Apelin, a myokine which contributes to muscle strength, was also quantified. To this end, a case-control study was conducted to evaluate the RedOx and inflammatory status in 45 elderly subjects (23 non-sarcopenic; 22 sarcopenic) from 65 years old and higher. SARCopenia-Formular (SARC-F) and Timed Up and Go (TUG) tests were used to distinguish between sarcopenic and non-sarcopenic subjects. By using red blood cells, plasma and/or serum, we observed in sarcopenic patients an increased activity of major antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) associated with lipid peroxidation and protein carbonylation (increased level of malondialdehyde, conjugated dienes and carbonylated proteins). Higher levels of 7-ketocholesterol and 7β-hydroxycholesterol were also observed in the plasma of sarcopenic patients. Significant differences were only observed with 7β-hydroxycholesterol. In sarcopenic patients comparatively to non-sarcopenic subjects, significant increase of CRP, LTB4 and apelin were observed whereas similar levels of TNF-α, IL-6 and IL-8 were found. The increased plasma level of 7-ketocholesterol and 7β-hydroxycholesterol in sarcopenic patients led us to study the cytotoxic effect of these oxysterols on undifferentiated (myoblasts) and differentiated (myotubes) murine C2C12 cells. With the fluorescein diacetate and sulforhodamine 101 assays, an induction of cell death was observed both on undifferentiated and differentiated cells: the cytotoxic effects were less pronounced with 7-ketocholesterol. In addition, IL-6 secretion was never detected whatever the culture conditions, TNF-α secretion was significantly increased on undifferentiated and differentiated C2C12 cells treated with 7-ketocholesterol- and 7β-hydroxycholesterol, and IL-8 secretion was increased on differentiated cells. 7-ketocholesterol- and 7β-hydroxycholesterol-induced cell death was strongly attenuated by α-tocopherol and Pistacia lentiscus L. seed oil both on myoblasts and/or myotubes. TNF-α and/or IL-8 secretions were reduced by α-tocopherol and Pistacia lentiscus L. seed oil. Our data support the hypothesis that the enhancement of oxidative stress observed in sarcopenic patients could contribute, especially via 7β-hydroxycholesterol, to skeletal muscle atrophy and inflammation via cytotoxic effects on myoblasts and myotubes. These data bring new elements to understand the pathophysiology of sarcopenia and open new perspectives for the treatment of this frequent age-related disease. [Display omitted] • Increase of inflammatory and oxidative stress biomarkers in sarcopenic patients. • Increase of 7-ketocholesterol and 7β-hydroxycholesterol in the serum of sarcopenic patients. • 7-ketocholesterol and 7β-hydroxycholesterol induced cell death on myoblasts and myotubes (C2C12 cells). • 7-ketocholesterol and 7β-hydroxycholesterol induced inflammation on myoblasts and myotubes (C2C12 cells). • Attenuation of 7-ketocholesterol and 7β-hydroxycholesterol induced cell death and inflammation by α-tocopherol and Pistacia lentiscus L. seed oil. [ABSTRACT FROM AUTHOR]

Abstract Some oxysterols resulting either from enzymatic oxidation or autoxidation of cholesterol are associated with age-related diseases including neurodegenerative diseases. Among these oxysterols, 7β-hydroxycholesterol (7β-OHC) is often found at increased levels in patients. It is therefore important to identify molecules or mixtures of molecules to prevent 7β-OHC-induced side effects. Consequently, murine oligodendrocytes (158N) were cultured in the absence or presence of 7β-OHC (20 μg/mL, 24 h) with or without a natural oil extracted from sea urchin (Paracentrotus lividus) eggs known for its biological activity. Firstly, the chemical composition of this oil was determined using 31P NMR and GC-MS. Secondly, this oil was used to reduce 7β-OHC-induced side effects. To this end, the oil (160 μg/mL) was added to the culture medium of 158N cells 2 h before 7β-OHC. The effects of 7β-OHC with or without the oil on cell viability were studied with the MTT test. Photometric methods were used to analyze antioxidant enzyme activities, superoxide dismutase (SOD) and glutathione peroxidase (GPx), as well as the generation of lipid peroxidation products (malondialdehyde (MDA), conjugated dienes (CDs)) and protein oxidation product (carbonylated proteins (CPs)). Gas chromatography was used to determine the fatty acid profile. With 7β-OHC, an induction of cell death associated with oxidative stress (alteration of GPx and SOD activities) was observed; an overproduction of lipid peroxidation products (MDA and CDs) and CPs was also revealed. Sea urchin egg oil attenuated 7β-OHC-induced cytotoxicity: 7β-OHC-induced cell death was reduced, GPx and SOD activities were normalized, and lower levels of MDA, CDs and CPs were produced. In addition, whereas a disturbed fatty acid profile was observed with 7β-OHC, similar fatty acid profiles were found in control cells and in cells cultured with 7β-OHC associated with sea urchin egg oil. These data demonstrate the protective activities of sea urchin egg oil against 7β-OHC-induced side effects on 158N cells, supporting the concept that this oil may have benefits in the prevention of neurodegenerative diseases. Highlights • 7β-hydroxycholesterol (7β-OHC) induces 158N cell death associated with cellular fatty acid profile alteration. • 7β-OHC induces an enhancement of Gluthation peroxidase (GPx) and superoxide dismutase (SOD) activities. • 7β-OHC induces an overproduction of lipid peroxidation products (MDA and CD) and carbonylated proteins (CP). • Sea urchin egg oil attenuates 7β-OHC induced side effects: it reduces cell death, and normalize the fatty acid profile. • Sea urchin egg oil modulates the antioxidant enzymes activities and attenuates the production of MDA and CD. [ABSTRACT FROM AUTHOR]

Altered cholesterol metabolism could be associated with cognitive impairment. The quantitative profiling of 19 hair sterols was developed using gas chromatography–mass spectrometry coupled to multivariate data analysis. The limit of quantification of all sterols ranged from 5 to 20 ng/g, while the calibration linearity was higher than 0.98. The precision (% CV) and accuracy (% bias) ranged from 3.2% to 9.8% and from 83.2% to 119.4%, respectively. Among the sterols examined, 8 were quantitatively detected from two strands of 3-cm-long scalp hair samples of female participants, including mild cognitive impairment (MCI, n = 15), Alzheimer’s disease (AD, n = 31), and healthy controls (HC, n = 36). The cognitive impairment (MCI or AD) was correlated with a higher metabolic rate than that of HCs based on 7β-hydroxycholesterol ( P < 0.005). Significant negative correlations ( r = −0.822) were detected between Mini-Mental State Examination (MMSE) scores and hair sample metabolic ratios of 7β-hydroxycholesterol to cholesterol, which is an accepted, sensitive, and specific tool for discriminating HCs from individuals with MCI or AD. In conclusion, improved diagnostic values can be obtained using hair sterol signatures coupled with MMSE scores. This method may prove useful for predictive diagnosis in population screening of cognitive impairment. [ABSTRACT FROM AUTHOR]

In demyelinating or non-demyelinating neurodegenerative diseases, increased levels of 7-ketocholesterol (7KC), 7β-hydroxycholesterol (7β-OHC) and 24(S)-hydroxycholesterol (24S-OHC) can be observed in brain lesions. In 158N murine oligodendrocytes, 7KC triggers a complex mode of cell death defined as oxiapoptophagy, involving simultaneous oxidative stress, apoptosis and autophagy. In these cells, 7KC as well as 7β-OHC and 24S-OHC induce a decrease of cell proliferation evaluated by phase contrast microscopy, an alteration of mitochondrial activity quantified with the MTT test, an overproduction of reactive oxygen species revealed by staining with dihydroethidium and dihydrorhodamine 123, caspase-3 activation, PARP degradation, reduced expression of Bcl-2, and condensation and/or fragmentation of the nuclei which are typical criteria of oxidative stress and apoptosis. Moreover, 7KC, 7β-OHC and 24S-OHC promote conversion of microtubule-associated protein light chain 3 (LC3-I) to LC3-II which is a characteristic of autophagy. Consequently, 7β-OHC and 24S-OHC, similarly to 7KC, can be considered as potent inducers of oxiapoptophagy. Furthermore, the different cytotoxic effects associated with 7KC, 7β-OHC and 24S-OHC-induced oxiapoptophagy are attenuated by vitamin E (VitE, α-tocopherol) and DHA which enhances VitE protective effects. In 158N murine oligodendrocytes, our data support the concept that oxiapoptophagy, which can be inhibited by VitE and DHA, could be a particular mode of cell death elicited by cytotoxic oxysterols. [ABSTRACT FROM AUTHOR]

[Display omitted] Peroxisomes play an important role in regulating cell metabolism and RedOx homeostasis. Peroxisomal dysfunctions favor oxidative stress and cell death. The ability of 7β-hydroxycholesterol (7β-OHC; 50 μM, 24 h), known to be increased in patients with age-related diseases such as sarcopenia, to trigger oxidative stress, mitochondrial and peroxisomal dysfunction was studied in murine C2C12 myoblasts. The capacity of milk thistle seed oil (MTSO, 100 μg/mL) as well as α-tocopherol (400 µM; reference cytoprotective agent) to counteract the toxic effects of 7β-OHC, mainly at the peroxisomal level were evaluated. The impacts of 7β-OHC, in the presence or absence of MTSO or α-tocopherol, were studied with complementary methods: measurement of cell density and viability, quantification of reactive oxygen species (ROS) production and transmembrane mitochondrial potential (ΔΨm), evaluation of peroxisomal mass as well as topographic, morphologic and functional peroxisomal changes. Our results indicate that 7β-OHC induces a loss of cell viability and a decrease of cell adhesion associated with ROS overproduction, alterations of mitochondrial ultrastructure, a drop of ΔΨm, and several peroxisomal modifications. In the presence of 7β-OHC, comparatively to untreated cells, important quantitative and qualitative peroxisomal modifications were also identified: a) a reduced number of peroxisomes with abnormal sizes and shapes, mainly localized in cytoplasmic vacuoles, were observed; b) the peroxisomal mass was decreased as indicated by lower protein and mRNA levels of the peroxisomal ABCD3 transporter; c) lower mRNA level of Pex5 involved in peroxisomal biogenesis as well as higher mRNA levels of Pex13 and Pex14, involved in peroxisomal biogenesis and/or pexophagy, was found; d) lower levels of ACOX1 and MFP2 enzymes, implicated in peroxisomal β-oxidation, were detected; e) higher levels of very-long-chain fatty acids, which are substrates of peroxisomal β-oxidation, were found. These different cytotoxic effects were strongly attenuated by MTSO, in the same range of order as with α-tocopherol. These findings underline the interest of MTSO and α-tocopherol in the prevention of peroxisomal damages (pexotherapy). [ABSTRACT FROM AUTHOR]

11β-Hydroxysteroid dehydrogenase 1 (11βHSD1; EC 1.1.1.146) generates active glucocorticoids from inert 11-keto metabolites. However, it can also metabolize alternative substrates, including 7β-hydroxy- and 7-keto-cholesterol (7βOHC, 7KC). This has been demonstrated in vitro but its consequences in vivo are uncertain. We used genetically modified mice to investigate the contribution of 11βHSD1 to the balance of circulating levels of 7KC and 7βOHC in vivo, and dissected in vitro the kinetics of the interactions between oxysterols and glucocorticoids for metabolism by the mouse enzyme. Circulating levels of 7KC and 7βOHC in mice were 91.3±22.3 and 22.6±5.7nM respectively, increasing to 1240±220 and 406±39nM in ApoE −/− mice receiving atherogenic western diet. Disruption of 11βHSD1 in mice increased (p <0.05) the 7KC/7βOHC ratio in plasma (by 20%) and also in isolated microsomes (2 fold). The 7KC/7βOHC ratio was similarly increased when NADPH generation was restricted by disruption of hexose-6-phosphate dehydrogenase. Reduction and oxidation of 7-oxysterols by murine 11βHSD1 proceeded more slowly and substrate affinity was lower than for glucocorticoids. in vitro 7βOHC was a competitive inhibitor of oxidation of corticosterone (K i =0.9μM), whereas 7KC only weakly inhibited reduction of 11-dehydrocorticosterone. However, supplementation of 7-oxysterols in cultured cells, secondary to cholesterol loading, preferentially slowed reduction of glucocorticoids, rather than oxidation. Thus, in mouse, 11βHSD1 influenced the abundance and balance of circulating and tissue levels of 7βOHC and 7KC, promoting reduction of 7KC. In health, 7-oxysterols are unlikely to regulate glucocorticoid metabolism. However, in hyperlipidaemia, 7-oxysterols may inhibit glucocorticoid metabolism and modulate signaling through corticosteroid receptors. [ABSTRACT FROM AUTHOR]

Abstract: There is some evidence that oxidized derivatives of cholesterol, 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7βOHC), are increased in the plasma of patients with neurodegenerative diseases associated with demyelinization of the central nervous system (CNS). It was therefore of interest to investigate the effects of these oxysterols on oligodendrocytes, the myelin-forming cells in the CNS. To this end, 158N murine oligodendrocytes were treated with 7KC or 7βOHC inducing an apoptotic mode of cell death characterized by condensation/fragmentation of the nuclei, dephosphorylation of Akt and GSK3, mitochondrial depolarization involving Mcl-1, and caspase-3 activation. In contrast, under treatment with 27-hydroxycholesterol (27OHC), no cell death was observed. When the cells were stained with Fura-2, no significant Ca2+ rise was found with the different oxysterols, whereas strong signals were detected with ionomycin used as positive control. At concentrations which induced apoptosis, 7KC but not 7βOHC accumulated in lipid rafts. Although not cytotoxic, 27OHC was mainly detected in lipid rafts. It is noteworthy that α-tocopherol (but not ellagic acid and resveratrol) was able to counteract 7KC- and 7βOHC-induced apoptosis and to decrease the accumulation of 7KC and 27OHC in lipid rafts. Thus, in 158N cells, the ability of oxysterols to trigger a mode of cell death by apoptosis involving GSK-3 and caspase-3 activation is independent of the increase in the Ca2+ level and of their accumulation in lipid raft microdomains. [Copyright &y& Elsevier]

The atherogenic potential of oxidized low-density lipoproteins (oxLDL) has been correlated to their 7β-hydroxycholesterol (7βOHC) content; oxLDLs have a dual effect on endothelial cell viability, inducing apoptosis or proliferation depending on the concentration. Considering that 7βOHC is apoptotic for endothelial cells at concentrations ≥20 μg/ml, a study on the effect of lower concentrations of 7βOHC on human umbilical vein endothelial cells (HUVECs) was undertaken. 7βOHC (1–10 μg/ml) increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction of growth-factor-deprived HUVECs. This effect was due to the increased cell proliferation, determined by [3H]thymidine incorporation, and the reduction of apoptosis, revealed by the decreased caspase-3 activation and annexin V staining. 7βOHC also protected against staurosporine apoptosis. 7βOHC induced an increase in intracellular ROS antagonized by N-acetylcysteine; however, HUVECs treatment with the antioxidant did not inhibit the effects of 7βOHC. 7βOHC produced an increase in extracellular signal-regulated kinase (ERK) phosphorylation that was blocked by inhibitors of store-operated calcium entry 2-aminoethoxydiphenyl borate and gadolinium. MEK inhibition with PD98059 or U0126 as well as store-operated calcium entry inhibition antagonized the effect of 7βOHC. The results suggest that 7βOHC promotes HUVECs survival and proliferation by a mechanism independent of ROS production and involving calcium-dependent activation of ERK. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Peripheral natural killer (NK) cells are reduced in patients with coronary artery disease and highly susceptible to apoptosis induced by oxidized lipids including 7β-hydroxycholesterol (7βOH) in vitro. The present study aimed to further explore the mechanisms behind 7βOH-mediated cytotoxicity to human NK cells. Human NK cells were purified and treated with 7βOH in different concentrations and times. Cell death, lysosomal and mitochondrial permeabilization and reactive oxygen species (ROS) production were then analysed. The 7βOH induced time and dose dependent apoptosis and necrosis in human NK cells, which was preceded by loss of lysosomal integrity and enhanced ROS production. At later time points, the mitochondrial membrane permeability in 7βOH-treated cells was significantly increased. The findings indicate that 7βOH induces human NK cell death through early lysosomal permeabilization and consequent oxidative stress. The data further suggest that 7βOH may induce immune disturbances in clinical settings such as atherosclerosis. [ABSTRACT FROM AUTHOR]

Purpose: To characterize the possible cytotoxic effects of oxysterols (7β-hydroxycholesterol (7β-OH), 25-hydroxycholesterol (25-OH)) in human retinal pigment epithelial cells (ARPE-19) and to detail the relationships between some of these effects. Methods: ARPE-19 cells were treated with 7β-OH and 25-OH. Cell viability was measured with the MTT assay. Membrane permeability, mitochondrial potential, and lysosomal integrity were measured by flow cytometry with propidium iodide, DiOC6(3), and acridine orange, respectively. Cell death was characterized by staining with Hoechst 33342, transmission electron microscopy, and analysis of the DNA fragmentation pattern. Caspase activity was examined with fluorochrome-labeled inhibitors of caspases (FLICA) and Western blotting. Immunofluorescence staining was used to visualize the cellular distribution of cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF). The effect of the cathepsin inhibitor (z-FA-fmk) on oxysterol-induced cell death was evaluated. Results: Cell viability of ARPE-19 cells was decreased with 7β-OH, whereas 25-OH had no cytotoxic effects. Loss of mitochondrial potential and lysosomal destabilization was associated with 7β-OH-induced cell death, few morphologically apoptotic cells were identified, and no internucleosomal DNA fragmentation was found. Slight caspase activation was detected with FLICA, and no caspase-3 activation was revealed. A pronounced relocalization of Cyt-c and AIF was observed. Noteworthy, z-FA-fmk was able to prevent cell death. Conclusion: 7β-OH induced a caspase-3-independent mode of cell death associated with lysosomal destabilization, which could play a key role in the signaling pathways leading to cell death, as shown by the ability of z-FA-fmk to counteract the cytotoxic effects of 7β-OH. [ABSTRACT FROM AUTHOR]

Oxysterols arise from the enzymic or non-enzymic oxidation of cholesterol and have been shown to be cytotoxic to certain cell lines. In particular, apoptosis induced by the oxysterol 7β-hydroxycholesterol (7β-OH) has been associated with the generation of oxidative stress, cytochrome c release and caspase activation. Due to the fundamental importance of apoptosis in pathological processes, the identification of substances capable of modulating this form of cell death is now actively researched. The objective of the present study was to investigate if apigenin, lycopene and astaxanthin could inhibit 7β-OH-induced apoptosis in U937 cells. Pretreatment with 0·1 μm-astaxanthin protected against apoptosis, while lycopene did not oppose the adverse effects of 7β-OH. At low concentrations, apigenin did not protect against oxysterol-induced apoptosis; however, at higher concentrations it intensified cell death. Additionally, we investigated the effect of 7β-OH, apigenin and astaxanthin on the activation of the serine threonine kinase Akt (phosphorylated Akt:Akt ratio) to determine whether the effect on cell viability and growth was linked to the Akt signalling pathway. Akt activation was decreased in the oxysterol-treated cells compared with control cells; however, this did not attain significance. Interestingly, activation of Akt was significantly reduced compared with control cells following incubation with apigenin and astaxanthin both in the absence and in the presence of 7β-OH. Our data suggest that apigenin, lycopene and astaxanthin failed to protect against 7β-OH-induced apoptosis, and the decrease in cell viability and the increase in apoptotic nuclei induced by the antioxidants appear to be associated with down regulation of Akt activity. [ABSTRACT FROM PUBLISHER]

The induction of apoptosis in cells of the arterial wall is a critical event in the development of atheroma. 7β-Hydroxycholesterol (7β-OH) and cholesterol-5β,6β-epoxide (β-epoxide) are components of oxLDL and have previously been shown to be potent inducers of apoptosis. However, the exact mechanisms through which these oxysterols induce apoptosis remains to be fully elucidated. The specific interaction of the Fas death receptor with Fas ligand (FasL) initiates a caspase cascade culminating in apoptosis. The purpose of the present study was to determine the involvement of Fas signalling in 7β-OH-and β-epoxide-induced apoptosis. To this end we employed the Fas/FasL antagonist, Kp7-6, and examined the effect of Fas inhibition on oxysterol-induced cell death in U937 cells. Fas levels were increased following 24 h exposure to 30 μ M 7β-OH while treatment with 30 μ M β-epoxide had no effect. Kp7-6 reduced the Fas content of 7β-OH-treated cells to control levels and partially protected against 7β-OH-induced apoptosis. This coincided with a decrease in cytochrome c release along with a reduction in caspase-3 and caspase-8 activity. Our data implicate Fas signalling in the apoptotic pathway induced by 7β-OH and also highlight differences between apoptosis induced by 7β-OH and β-epoxide. [ABSTRACT FROM AUTHOR]

Abstract: Oxysterols result from cholesterol by enzymatic or oxidative processes. Some exert cytotoxic effects leading to necrosis or apoptosis. Detoxification of these compounds mainly occurs in the liver and requires transport from peripheral tissues towards it. Some ATP-binding cassette transporters are involved in export of cytotoxic compounds. In the current study, we investigated whether ABC transporter family member G1 (ABCG1) may be involved in oxysterol transport, since its gene expression is highly responsive to oxysterol loading. TetOff HeLa cells stably expressing ABCG1 showed decreased mass uptake of 7β-hydroxycholesterol (7β-HC) whereas that of other physiologically relevant oxysterols was unaffected. Application of 7β-HC to ABCG1 expressing cells induced hyperpolarization of mitochondrial membrane potential and production of reactive oxygen species, indicating energy consumption by the ATP-binding cassette transporter when it is activated by its correct substrate. Our study points to detoxification as one of potential cellular functions of ABCG1. We assume that ABCG1 protects against 7β-HC-induced cell death, an important role in prevention of neurodegenerative and cardiovascular disease. [Copyright &y& Elsevier]

We reported previously that 7β-hydroxysitosterol and 7β-hydroxycholesterol induced apoptosis in Caco-2 cells. Apoptosis caused by 7β-hydroxysitosterol but not by 7β-hydroxycholesterol was related to a caspase-dependent process. In the present report, we compared the effects of both compounds on mitochondria integrity and on various modulators of apoptosis. When Caco-2 cells were exposed to both hydroxysterols, no changes in Bcl-2 and Bax expressions were detected indicating a Bcl-2/Bax-independent cell death pathway, whereas loss of mitochondrial membrane potential and cytochrome c release were observed. Endonuclease G expression and enhanced production of reactive oxygen species were detected in 7β-hydroxycholesterol treated cells, but not with 7β-hydroxysitosterol. Loss of mitochondrial membrane potential and cell death produced by both hydroxysterols were prevented by vitamin C. Lysosomal membrane integrity was altered with both hydroxysterols, but 7β-hydroxysitosterol was significantly more active on than 7β-hydroxycholesterol. Both hydroxysterols induced apoptosis by mitochondrial membrane permeabilization. However, 7β-hydroxycholesterol exhibited a specific enhancement of oxidative stress and of endonuclease G expression despite its closely related chemical structure with 7β-hydroxysitosterol. The two hydroxysterols exhibit different lipophilic properties which may explain their different biological effects. [ABSTRACT FROM AUTHOR]

Abstract: Antioxidants such as flavonoids afford protection against oxysterols-induced toxicity. We have investigated the effect of kaempferol and rutin, active components of red wine, in the apoptosis induced by 7β-hydroxycholesterol in rat vascular smooth muscle cells. 7β-Hydroxycholesterol induced apoptosis in vascular smooth muscle which include BcL-xL degradation, caspase-3 activation and DNA fragmentation. The apoptosis induced by 7β-hydroxycholesterol was prevented by pretreatment with kaempferol (10–30 μM), but not with rutin. Interestingly preincubation with the estrogen receptor α antagonist ICI 182,780 (1 μM) prior to kaempferol partially reverted the antiapoptotic effect of this flavonoid on caspase-3 activation and DNA fragmentation induced by 7β-hydroxycholesterol. In conclusion, the flavonoid kaempferol, unlike rutin, diminished the apoptosis induced by a component of oxidized low-density lipproteins (oxLDL). This effect was partially mediated by the estrogen receptor α. [Copyright &y& Elsevier]

Oxidized low-density lipoprotein (oxLDL) is believed to play a central role in the development of atherosclerosis. The induction of apoptosis in cells of the arterial wall is a critical event in the development of atheroma. 7ß -Hydroxycholesterol (7ß -OH) and cholesterol-5ß,6ß -epoxide (ß -epoxide) are components of oxLDL and have previously been shown to be potent inducers of apoptosis. The exact mechanism through which these oxysterols induce apoptosis remains to be fully elucidated. A perturbation of intracellular calcium homeostasis has been found to trigger apoptosis in many experimental systems. The aim of the present study was to determine the involvement of calcium signaling in 7ß -OH and ß -epoxide–induced apoptosis. To this end, the authors employed the calcium channel blockers verapamil and nifedipine and inhibitors of calpain activation, ALLM and ALLN. Verapamil protected against the decrease in viability induced by 7ß -OH whereas nifedipine had a protective effect in both 7ß -OH and ß -epoxide–treated cells, though these compounds did not restore viability to control levels. Verapamil, nifedipine, and ALLM prevented apoptosis induced by ß -epoxide. None of the compounds employed in the current study protected against 7ß -OH–induced apoptosis. Our results implicate calcium signaling in the apoptotic pathway induced by ß -epoxide and also highlight differences between apoptosis induced by 7ß-OH and ß -epoxide. [ABSTRACT FROM AUTHOR]

Plant sterols are found in fruits and vegetables. Their cholesterol-lowering effect is well documented. Our study aimed at comparing antiproliferative effects of 7ß-hydroxysitosterol (7ß-OHsito) versus 7ß-hydroxycholesterol (7ß-OHchol) on the human colon cancer cell line Caco-2. When cells were exposed for 32?h to 60?µM 7ß-OHsito or to 30?µM 7ß-OHchol, both compounds caused 50%growth inhibition. Cells treated with 7ß-OHsito showed enhanced caspase-9 and -3 activities followed by DNA fragmentation. In contrast, 7ß-OHchol did not activate caspase-3 and activation of caspase-9 and DNA fragmentation were delayed. The treatment of cells with the caspase inhibitor Z-VAD.fmk retarded the 7ß-OHsito-induced apoptotic process but not that triggered by 7ß-OHchol. Our data suggest that the two compounds in spite of their structural similarities target different cellular pathways, which lead to cell death.Cell Death and Differentiation (2005) 12, 128-135. doi:10.1038/sj.cdd.4401530 Published online 19 November 2004 [ABSTRACT FROM AUTHOR]

7β-Hydroxysteroid dehydrogenase (7β-HSD), a specific enzyme active in the metabolization of 7β-hydroxycholesterol, was purified about 300-fold from male rabbit liver microsomes using ion exchange, hydroxylapatite, 2′5′ADP Sepharose 4B, and high-performance liquid chromatography on the basis of its catalytic activity.The specific activity of the purified enzyme was 276 nmol/min/mg protein. The molecular weight of the purified enzyme was 34,000. The preferred coenzyme was β-NADP+. The optimum pH for oxidation was around 7.7 in potassium phosphate buffer, and 11.0 in glycine–NaOH buffer. The purified enzyme catalyzed the synthesis of not only 7β-hydroxycholesterol but also corticosterone and hydrocortisone. Enzyme activities toward these three substrates accompanied all purification steps of 7β-HSD. The amino acid sequence of the N-terminal of the purified enzyme showed that 7β-HSD had sequence similarity to rabbit type I 11β-hydroxysteroid dehydrogenase (11β-HSD), indicating that 7β-HSD may belong to the rabbit type I 11β-HSD family and may play the same role in the metabolism of 11-hydroxysteroids and 7-hydroxysterols. [Copyright &y& Elsevier]

Atherosclerosis involves inflammatory processes, as well as cytotoxic and oxidative reactions. In atherosclerotic plaques, these phenomena are revealed by the presence of dead cells, oxidized lipids, and oxidative DNA damage, but the molecules triggering these events are still unknown. As 7β-hydroxycholesterol and 7-ketocholesterol, which are present at elevated concentrations in atherosclerotic lesions, are strongly cytotoxic and pro-oxidative, their effects were determined on cell death, superoxide anion and nitric oxide production, lipid peroxidation, and oxidative DNA damage. 7-Ketocholesterol- and 7β-hydroxycholesterol-induced cell death leads to a loss of mitochondrial potential, to increased permeability to propidium iodide, and to morphological nuclear changes (swelling, fragmentation, and/or condensation of nuclei). These effects are preceded by the formation of cytoplasmic monodansylcadaverine-positive structures and are associated with a rapid enhancement of cells overproducing superoxide anions, a decrease in cells producing nitric oxide, lipid peroxidation (formation of malondialdehyde and 4-hydroxynonenal adducts, low ratio of [unsaturated fatty acids]/[saturated fatty acids]) as well as oxidative DNA damage (8-oxoguanine formation). Noteworthy, none of the cytotoxic features previously observed with 7β-hydroxycholesterol and 7-ketocholesterol were noted with cholesterol, 7β-hydroxycholesteryl-3-oleate and 7-ketocholesteryl-3-oleate, with the exception of a slight increase in superoxide anion production with 7β-hydroxycholesteryl-3-oleate. This finding supports the theory that 7β-hydroxycholesterol and 7-ketocholesterol could induce cytotoxic and oxidative processes observed in atherosclerotic lesions and that esterification of these compounds may contribute to reducing atherosclerosis progression. [Copyright &y& Elsevier]

This study was conducted to determine the interaction between the type of dietary fat (coconut oil or salmon oil) and the vitamin E concentration of the diet [10, 20, 40 or 240 mg alpha-tocopherol equivalents (alpha-toc)/kg] in relation to the concentration of 7β-hydroxycholesterol (7β-OH) in liver, plasma, LDL and erythrocytes of rats. In the rats whose diet contained salmon oil, the concentration of 7β-OH was dependent on the dietary vitamin E concentration. Rats whose diet contained 10 mg alpha-toc/kg had significantly higher concentrations of 7β-OH in all samples studied than those whose diet contained 20, 40 or 240 mg alpha-toc/kg. Increasing the dietary vitamin E concentration from 40 to 240 mg alpha-toc/kg did not reduce the concentration of 7β-OH in any samples. In the rats whose diet contained coconut oil, the concentration of 7β-OH was independent of the dietary vitamin E concentration in all samples. The study shows that insufficient vitamin E in the diet increases the formation of 7β-OH in rats fed salmon oil, whereas a dietary vitamin E supply in excess of the requirement does not lower 7β-OH concentrations compared with an adequate vitamin E supply. [ABSTRACT FROM AUTHOR]

The metabolism of epimeric 7-hydroxycholesterol was studied in vitro. 7α-Hydroxycholesterol or 7β-hydroxycholesterol were incubated with rabbit, guinea pig, rat, hamster, and chicken microsomal suspensions and then extracted and analyzed using high-performance liquid chromatography (HPLC). 7α-Hydroxy-4-cholesten-3-one was the main product from 7α-hydroxycholesterol in the rabbit, guinea pig, and rat. A considerable amount of 7-ketocholesterol was also produced in the hamster and chicken. In all vertebrates, 7β-hydroxycholesterol was converted only to 7-ketocholesterol in all vertebrates. 7β-Hydroxy-4-cholesten-3-one was not detected. Reduction of 7-ketocholesterol was also studied in the rat and hamster. Whereas 7-ketocholesterol was converted to 7β-hydroxycholesterol in the rat, it was converted to both 7α- and 7β-hydroxycholesterol in the hamster. These results suggest that 7α-hydroxycholesterol is converted not only to 7α-hydroxy-4-cholesten-3-one but also to 7-ketocholesterol in the hamster and chicken. 7β-Hydroxycholesterol was converted to 7-ketocholesterol in all vertebrates tested. The interconversion between 7α- and 7β-hydroxycholesterol via 7-ketocholesterol was observed in the hamster in this in vitro study. [Copyright &y& Elsevier]

The aim of this study is to evaluate oxidative stress in man after paraquat ingestion by analyzing 7α- and 7β-hydroperoxycholest-5-en-3β-ol (7α- and 7β-OOH) as well as oxysterols, cholesterol oxidation products, as indices of lipid peroxidation. Lung, kidney, and liver were collected at autopsy from seven patients with paraquat poisoning and seven controls matched for age and sex. We identified for the first time 7-ketocholesterol (7-keto) and 7-hydroxycholesterol (7α-OH and 7β-OH) in human kidney by LC-MS. Next, we quantified 7α-OOH and 7β-OOH by HPLC with postcolumn chemiluminescence as well as oxysterols by HPLC-UV. Both 7α-OOH and 7β-OOH detected in lung and kidney from the controls were as low as the paraquat group. In contrast, we found both 7-keto and 7β-OH in lung and 7-keto in kidney from the paraquat group were significantly higher than from the controls. This is the first report on accumulated oxysterols in lung and kidney from human paraquat poisoning. It seems to reflect greater oxidative stress in the pathology of paraquat intoxication. [ABSTRACT FROM AUTHOR]

Background: Misfolded proteins are formed in the endoplasmic reticulum (ER) due to diverse stimuli including oxidant production, calcium disturbance, and inflammatory factors. Accumulation of these non-native proteins in the ER evokes cellular stress involving the activation of unfolded protein response (UPR) and the execution of ER-associated degradation (ERAD). Naturally-occurring plant compounds are known to interfere with UPR due to their antioxidant and anti-inflammatory activities, leading to inhibition of ER stress. However, there are few studies dealing with the protective effects of natural compounds on the functionality of ERAD.Purpose: The current study examined whether asaronic acid enhanced ubiquitin-proteasomal degradation in J774A.1 murine macrophages exposed to 7β-hydroxycholesterol, a risk factor for atherosclerosis. Asaronic acid (2,4,5-trimethoxybenzoic acid), identified as one of purple perilla constituents, has anti-diabetic and anti-inflammatory effects. Little is known regarding the effects of asaronic acid on the ERAD process and the ubiquitin-proteasomal degradation.Methods and Results: Murine macrophages were incubated with 28 μM 7β-hydroxycholesterol in absence and presence of 1-20 μΜ asaronic acid for up to 24 h. Nontoxic asaronic acid in macrophage diminished the activation of the ER stress sensors of ATF6, IRE1 and PERK stimulated by 7β-hydroxycholesterol. This methoxybenzoic acid down-regulated the oxysterol-induced expression of EDEM1, OS9, Sel1L-Hrd1 and p97/VCP1, all required for the recognition, recruitment and dislocation of misfolded proteins. On the other hand, asaronic acid enhanced the ubiquitin-proteasomal degradation of non-native proteins dislocated to the cytosol by 7β-hydroxycholesterol, which entailed the induction of the chaperones of Hsp70 and CHIP and the increased colocalization of ubiquitin and proteasomes. Taken together, asaronic acid attenuated the induction of the UPR-associated sensors and the dislocation-linked transmembrane components in the ER. Conversely, this compound enhanced the proteasomal degradation of dislocated non-native proteins in concert with the chaperones of Hsp70 and CHIP through ubiquitination.Conclusion: These observations demonstrate that asaronic acid may be a potent atheroprotective agent as a natural chaperone targeting ER stress-associated macrophage injury. [ABSTRACT FROM AUTHOR]

More and more attention is nowadays given to the possible translational application of a great number of biochemical and biological findings with the involved molecules. This is also the case of cholesterol oxidation products, redox molecules over the last years deeply investigated for their implication in human pathophysiology. Oxysterols of non-enzymatic origin, the excessive increase of which in biological fluids and tissues is of toxicological relevance for their marked pro-oxidant and pro-inflammatory properties, are increasingly applied in clinical biochemistry as molecular markers in the diagnosis and monitoring of several human and veterinary diseases. Conversely, oxysterols of enzymatic origin, the production of which is commonly under physiological regulation, could be considered and tested as promising pharmaceutical agents because of their antiviral, pro-osteogenic and antiadipogenic properties of some of them. Very recently, the quantification of oxysterols of non-enzymatic origin has been adopted in a systematic way to evaluate, monitor and improve the quality of cholesterol-based food ingredients, that are prone to auto-oxidation, as well as their industrial processing and the packaging and the shelf life of the finished food products. The growing translational value of oxysterols is here reviewed in its present and upcoming applications in various industrial fields., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

[Display omitted] • 7-ketocholesterol and 7β-hydroxycholesterol are increased in several age-related diseases. • 7-ketocholesterol and 7β-hydroxycholesterol induce oxidative stress, apoptosis and autophagy (oxiapoptophagy). • Inhibition of 7-ketocholesterol- and 7β-hydroxycholesterol-induced cell death is an important health challenge. • Natural and synthetic molecules, including nutrients, mixture of molecules, plant extracts and oils attenuate 7-ketocholesterol- and 7β-hydroxycholesterol-induced cell death. 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. [ABSTRACT FROM AUTHOR]

Oxysterols are assumed to be the driving force behind numerous neurodegenerative diseases. In this work, we aimed to study the ability of 7β-hydroxycholesterol (7β-OHC) to trigger oxidative stress and cell death in human neuroblastoma cells (SH-SY5Y) then the capacity of Nigella sativa and Milk thistle seed oils (NSO and MTSO, respectively) to oppose 7β-OHC-induced side effects. The impact of 7β-OHC, associated or not with NSO or MTSO, was studied on different criteria: cell viability; redox status, and apoptosis. Oxidative stress was assessed through the intracellular reactive oxygen species (ROS) production, levels of enzymatic and non-enzymatic antioxidants, lipid, and protein oxidation products. Our results indicate that 7β-OHC (40 µg/mL) exhibit pr-oxidative and pro-apoptotic activities shown by a decrease of the antioxidant enzymatic activities and an increase of ROS production, lipid, and protein oxidation end products as well as nitrotyrosine formation and caspase 3 activation. However, under the pre-treatment with NSO, and especially with MTSO (100 µg/mL), a marked attenuation of oxidative damages was observed. Our study suggests harmful effects of 7β-OHC consisting of pro-oxidative, anti-proliferative, and pro-apoptotic activities that may contribute to neurodegeneration. NSO and especially MTSO showed potential cytoprotection against the cytotoxicity of 7β-OHC. [ABSTRACT FROM AUTHOR]

The cytotoxicity of 7β-hydroxycholesterol (7β-OHC) was investigated on rat astrocyte primary cultures and spontaneously transformed cell lines derived from them. Confluent astrocyte primary cultures (normal cells) were unaffected by 20 µM 7(3-OHC over a period of 72 h whereas 30 µM markedly affected the viability of the transformed cells within the first 72 h. Both cell types incorporated 18% of the total amount of 7β-OHC added to the cultures at concentrations of 20 µM or 30 µM. Cellular fractionation after incubation with 20 µM or 30 µM 7β-OHC indicated that the plasma membrane incorporated 2 or 6 fold more 7β-OHC than the intracellular one's respectively. Plasma membrane cholesterol (CH) and phospholipid (PL) analysis showed that 20 µM 7β-OHC did not affect CH/PL in normal cells; in contrast, plasma membranes of transformed cells displayed a significant CH/PL decrease, which was more pronounced with 30 µM 7β-OHC treatment. Fluorescence anisotropy measurements indicated that 20 µM 7β-OHC slightly fluidified the plasma membrane of normal cells whereas it has not effect on that of the transformed cells one; however, an increase in plasma membrane fluidity was observed when the transformed cells were treated with 30 µM 7β-OHC. Lactoperoxidase catalyzed radioiodination of cell surface proteins and subsequent autoradioelectrophoretic analysis demonstrated that the labelled protein pattern was unchanged when both cell types were incubated with 30 µM 7β-OHC. [ABSTRACT FROM AUTHOR]

• Oxysterol association with serum neurofilaments in MS were studied. • Baseline 7-ketocholesterol and 7β-hydroxycholesterol levels were positively associated serum neurofilaments at 5-year follow-up. • 7-ketocholesterol was associated with serum neurofilaments at 5-year follow-up. • Associations remained significant after adjusting for lipids. Serum neurofilament light chain (sNfL) is an established marker of neuroaxonal injury in multiple sclerosis (MS). To investigate if oxysterols produced from non-enzymatic and enzymatic cholesterol oxidation are differentially associated with sNfL measurements in MS. This longitudinal study included 62 relapsing-remitting (RR-MS) and 36 progressive MS (PMS) patients with baseline and 5-year follow-up measures of serum levels of 6 oxysterols, sNfL and lipids. The oxysterols, 24-hydroxycholesterol (24HC), 25HC, 27HC, 7αHC, 7βHC and 7-ketocholesterol (7KC), were measured using liquid chromatography-mass spectrometry. sNfL was measured using single molecular array assay. Serum high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were obtained from a lipid profile. The enzymatically produced oxysterols 24HC, 25HC, 27HC and 7αHC were not associated with sNfL. However, baseline levels of reactive oxygen species (ROS) produced oxysterols, 7KC (p = 0.032) and 7βHC (p = 0.0025), were positively associated with sNfL levels at follow-up. Follow-up 7KC (p = 0.038) levels were also associated with follow-up sNfL levels. The associations of 7KC or 7βHC with sNfL remained significant after adjusting for LDL-C or HDL-C. 7KC and 7βHC, produced by ROS-mediated cholesterol oxidation are associated with neuroaxonal injury as assessed by sNfL in MS. [ABSTRACT FROM AUTHOR]