Characterization of C-26 aminotransferase, indispensable for steroidal glycoalkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

7-Ketocholesterol disturbs RPE cells phagocytosis of the outer segment of photoreceptor and induces inflammation through ERK signaling pathway.

7-Ketocholesterol (7KCh), an oxidized form of cholesterol, is present at a high level in drusen and has been believed to play a role in the pathogenesis of age-related macular degeneration (AMD). Therefore, we developed a rat model to study the direct impact of 7KCh on retina. We delivered 7KCh to the rat retina by intravitreal injection using hydroxypropyl-ß-cyclodextrin as a vehicle. We observed that 7KCh mainly deposited in the retinal pigment epithelial (RPE) cells and induced marked photoreceptor apoptosis. Transmission electron microscope examination demonstrated cytoplasmic vacuoles in RPE cells and the microvilli detached from the outer segment after 7KCh treatment. In vitro experiments also revealed that RPE cells could take up 7KCh in culture. Moreover, 7KCh up-regulated IL-1ßmRNA, TNF-αmRNA, IL-6 mRNA, and IL-1ß secretion of RPE. U0126, a MEK1/2 inhibitor, down regulated the expression of these inflammation factors. Our findings may help elucidate the potential role of 7KCh in the pathogenesis of AMD.

Influence of the lung mechanical ventilation with injurious parameters on 7-ketocholesterol synthesis in Sus Scrofa.

The aim of work was to investigate changes of 7-ketocholesterol synthesis in alveolar macrophages in the dynamic of lung mechanical ventilation with injurious parameters. The goal of in vitro part of work was to observe influence of 7-ketocholesterol on iNOS and MIP1 Beta production in bronchoalveolar lavage fluid (BALF) cells. We used 17 healthy domestic pigs randomly assigned into two treatment groups: group I with mechanical ventilation with physiological parameters; group || underwent injurious ventilation with high volume tidal (VT) and low positive end expiratory pressure (PEEP). ells were analyzed for CYP27A1 protein and gene expression levels, 7-ketocholesterol production. In alveolar macrophages of group ||, we obtained increase of production of CYP27A1 protein and 7-ketocholesterol, as well as the expression of the CYP27A1 gene at the 2nd hour of ventilation. In the in vitro experiments we show dose-dependent increase of MIP1 Beta and decrease of CYP27A1, iNOS protein production after 7-ketocholesterol treatment.

Photodamage generates 7-keto- and 7-hydroxycholesterol in the rat retina via a free radical-mediated mechanism.

Albino Sprague-Dawley rats are known to undergo photoreceptor degeneration after exposure to constant light, but the molecular mechanism(s) by which the photoreceptors degenerate is not fully understood. We hypothesized that cytotoxic oxysterols are generated in situ in the retina under such conditions and may be involved in the degenerative mechanism. Thus, photodamaged and control rat retinas were analyzed for oxysterols by liquid chromatography mass spectroscopy. Elevated levels of two known cytotoxic oxysterols, 7-ketocholesterol (7KCh) and 7alphabeta-hydroxycholesterol (7HCh), were found in the photodamaged retinas, at levels six-fold and 50-fold greater, respectively, than those found in non photodamaged controls. Notably, two key intermediates, 5,6alpha,beta-epoxycholesterol (5,6-epoxyCh) and 7alphabeta-hydroperoxy-cholesterol, were also identified, indicating that the formation of 7KCh and 7HCh is mediated by a free radical mechanism. By immunohistochemistry, 7KCh was localized to the ganglion cell layer, photoreceptor inner segments and retinal pigment epithelium (RPE), which coincides with the localization of ferritin in the retina. Exposure of a mixture of ferritin and low-density lipoprotein to intense white light in vitro produced similar oxysterol species as seen in vivo. We propose that the increased levels of 7KCh and 7HCh, especially in photoreceptor inner segments and RPE, may arise due to ferritin-catalyzed reactions and may be important contributors to the photoreceptor degeneration observed in photodamaged rats.

Low density lipoprotein undergoes oxidation within lysosomes in cells.

The oxidized low density lipoprotein (LDL) hypothesis of atherosclerosis proposes that LDL undergoes oxidation in the interstitial fluid of the arterial wall. We have shown that aggregated (vortexed) nonoxidized LDL was taken up by J774 mouse macrophages and human monocyte-derived macrophages and oxidized intracellularly, as assessed by the microscopic detection of ceroid, an advanced lipid oxidation product. Confocal microscopy showed that the ceroid was located in the lysosomes. To confirm these findings, J774 macrophages were incubated with acetylated LDL, which is internalized rapidly to lysosomes, and then incubated (chase incubation) in the absence of any LDL. The intracellular levels of oxysterols, measured by HPLC, increased during the chase incubation period, showing that LDL must have been oxidized inside the cells. Furthermore, we found that this oxidative modification was inhibited by lipid-soluble antioxidants, an iron chelator taken up by fluid-phase pinocytosis and the lysosomotropic drug chloroquine, which increases the pH of lysosomes. The results indicate that LDL oxidation can occur intracellularly, most probably within lysosomes.

Estabilidade oxidativa do colesterol em ovo integral em pó / Oxidative stability of cholesterol in whole egg powder

Ovos são importantes como alimento e com matéria-prima industrial. Ao mesmo tempo, têm elevada concentração de colesterol, principalmente quando desidratado. O colesterol, por sua vez, está sujeito à oxidação durante o processamento e/ou estocagem, formando, em conseqüência, derivados oxidados com atividades tóxicas, entre as quais a aterogenicidade. Foi avaliada, neste trabalho, a estabilidade do colesterol em ovo integral em pó comercial, através da ocorrência do 7-cetocolesterol livre, quantificado por cromatografia líqüida de alta eficiência, depois da extração dos lípides totais e separação em coluna de Florisil. O 7-cetocolesterol livre ocorreu em todos os três lotes das cinco marcas analisadas de ovo integral em pó, em teor médio de 84,01±5,34 µg/g de lípides...

Consecutive administration of paraquat to rats induces enhanced cholesterol peroxidation and lung injury.

It is our hypothesis that as a consequence of increased oxidative stress, rats develop lung injury with increased cholesterol-derived hydroperoxides and oxysterols in lung after consecutive exposure of the rats to paraquat. To test this we administered 10 mg/kg of paraquat i.p. once or seven times (once a day) to Wistar rats. Rats were killed, and lung tissue was collected 24 h after the last paraquat injection. We found that in response to consecutive paraquat doses, there were significant increases in 7alpha- and 7beta-hydroperoxycholest-5-en-3beta-ol (7alpha-OOH and 7beta-OOH; P=0.01) as well as 7alpha- and 7beta-hydroxycholesterol (7alpha-OH and 7beta-OH; P=0.01), and 7-ketocholesterol (7-keto; P=0.03). In addition, pulmonary hemorrhage, thickening of alveolar septum, and inflammatory cell infiltration of macrophages were observed. This is the first report showing enhanced cholesterol peroxidation and lung injury of rats due to consecutive doses of paraquat.

Protection of superoxide-induced cholesterol oxidation by antioxidants in protic conditions.

It was found in previous experiments that superoxide in the presence of added hydrogen peroxide in protic conditions produces oxysterols. The oxysterols formed under these conditions were 7beta-ketocholesterol, 7alpha-hydroxycholesterol and 7beta-hydroxycholesterol. In the present experiments, the inhibitory effects of three antioxidants, alpha-tocopherol (alpha-T), butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), on the oxidation of cholesterol in the presence of superoxide anion, water and hydrogen peroxide were investigated. It was found that BHA had the highest antioxidant activity on cholesterol oxidation, followed by alpha-T and BHT. The presence of antioxidants markedly retarded the formation of 7-ketocholesterol. The formation of 7beta-hydroxycholesterol or 7alpha-hydroxycholesterol was also reduced, but to a lesser degree.

Major differences in oxysterol formation in human low density lipoproteins (LDLs) oxidized by *OH/O2*- free radicals or by copper.

The aim of our study was to determine the oxysterol formation in low density lipoproteins (LDLs) oxidized by defined oxygen free radicals (*OH/O2*-). This was compared to the oxysterol produced upon the classical copper oxidation procedure. The results showed a markedly lower formation of oxysterols induced by *OH/O2*- free radicals than by copper and thus suggested a poor ability of these radicals to initiate cholesterol oxidation in LDLs. Moreover, the molecular species of cholesteryl ester hydroperoxides produced by LDL copper oxidation seemed more labile than those formed upon *OH/O2*(-)-induced oxidation, probably due to their degradation by reaction with copper ions.

7-Ketocholesterol.

7-Ketocholesterol is a major oxidation product of cholesterol found in human atherosclerotic plaque and is more atherogenic than cholesterol in some animal studies. 7-Ketocholesterol can inhibit cholesterol 7 alpha-hydroxylase, the rate-limiting step in bile acid biosynthesis, as well as strongly inhibiting HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. It has even been suggested that 7-ketocholesterol is formed enzymically as an endogenous regulator of cholesterol biosynthesis. However, when tested as a pharmacological cholesterol-lowering agent, inhibition of HMG-CoA reductase was rapidly overcome and the 7-ketocholesterol metabolised. In vitro, 7-ketocholesterol has wide-ranging and potent effects, most of which have the potential to contribute to atherosclerosis. For example, 7-ketocholesterol can be cytotoxic and can induce apoptosis in vascular cells. These effects, either individually or more likely, in combination, all implicate 7-ketocholesterol in the initiation and development of atherosclerosis, but further work is needed to establish whether or not its role is a direct causal one.

Oxysterols: chemical synthesis, biosynthesis and biological activities.

As a class of compounds, oxysterols have demonstrated a wide variety of biological properties. Due to the general interest in these compounds, new methods of chemical synthesis have been developed to provide them for biological investigation. The specific inhibition by oxysterols of cholesterol biosynthesis in mammalian cells has been shown to result primarily from a decrease in cellular levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. Recent evidence suggests these cellular responses may be mediated by an oxysterol binding protein found in the cytosol of many lines of cultured cells. In certain instances, oxysterols have been shown to be produced in biological systems. These results support the supposition that oxysterols may regulate sterol biosynthesis at the cellular level. Included herein are the inhibitory effects of 9 alpha, 11 alpha-epoxycholest-7-en-3 beta-ol cholest-8-en-3 beta-ol-7-one and cholest-8-en-3 beta-ol-11-one on HMG-CoA reductase activity and their relative affinities for a cytosolic binding protein.