Screening Reveals Sterol Derivatives with Pro-Differentiation, Pro-Survival, or Potent Cytotoxic Effects on Oligodendrocyte Progenitor Cells.

Inducing the formation of new oligodendrocytes from oligodendrocyte progenitor cells (OPCs) represents a potential approach to repairing the loss of myelin observed in multiple sclerosis and other diseases. Recently, we demonstrated that accumulation of specific cholesterol precursors, 8,9-unsaturated sterols, is a dominant mechanism by which dozens of small molecules enhance oligodendrocyte formation. Here, we evaluated a library of 56 sterols and steroids to evaluate whether other classes of bioactive sterol derivatives may also influence mouse oligodendrocyte precursor cell (OPC) differentiation or survival. From this library, we identified U-73343 as a potent enhancer of oligodendrocyte formation that induces 8,9-unsaturated sterol accumulation by inhibition of the cholesterol biosynthesis enzyme sterol 14-reductase. In contrast, we found that mouse OPCs are remarkably vulnerable to treatment with the glycosterol OSW-1, an oxysterol-binding protein (OSBP) modulator that induces Golgi stress and OPC death in the low picomolar range. A subsequent small-molecule suppressor screen identified mTOR signaling as a key effector pathway mediating OSW-1's cytotoxic effects in mouse OPCs. Finally, evaluation of a panel of ER and Golgi stress-inducing small molecules revealed that mouse OPCs are highly sensitive to these perturbations, more so than closely related neural progenitor cells. Together, these studies highlight the wide-ranging influence of sterols and steroids on OPC cell fate, with 8,9-unsaturated sterols positively enhancing differentiation to oligodendrocytes and OSW-1 able to induce lethal Golgi stress with remarkable potency.

Candida albicans gains azole resistance by altering sphingolipid composition.

Fungal infections by drug-resistant Candida albicans pose a global public health threat. However, the pathogen's diploid genome greatly hinders genome-wide investigations of resistance mechanisms. Here, we develop an efficient piggyBac transposon-mediated mutagenesis system using stable haploid C. albicans to conduct genome-wide genetic screens. We find that null mutants in either gene FEN1 or FEN12 (encoding enzymes for the synthesis of very-long-chain fatty acids as precursors of sphingolipids) exhibit resistance to fluconazole, a first-line antifungal drug. Mass-spectrometry analyses demonstrate changes in cellular sphingolipid composition in both mutants, including substantially increased levels of several mannosylinositolphosphoceramides with shorter fatty-acid chains. Treatment with fluconazole induces similar changes in wild-type cells, suggesting a natural response mechanism. Furthermore, the resistance relies on a robust upregulation of sphingolipid biosynthesis genes. Our results shed light into the mechanisms underlying azole resistance, and the new transposon-mediated mutagenesis system should facilitate future genome-wide studies of C. albicans.

New steroidal alkaloid and furostanol glycosides isolated from Solanum lyratum with cytotoxicity.

Two previously undescribed steroidal compounds, 16, 23-epoxy-22, 26-epimino-cholest-22(N), 23, 25(26)-trien-3ß-ol-3-O-ß-D-glucopyranosyl-(1→2)-ß-D-glucopyranosyl-(1→4)-ß-D-galactopyranoside (1) and 26-O-ß-D-glucopyranosyl-(25R)-5α-furost-20(22)-en-3ß, 26-diol (2), together with 7 known ones including 26-O-ß-D-glucopyranosyl-(25R)-5, 20(22)-dien-furost-3ß, 26-diol (3), (25R)-5-en-spirost-3ß-ol-O-ß-D-glucopyranosyl-(1→4)-[α-L-rhmanopyranosyl-(1→2)]-ß-D-galactopyranoside (4), funkioside D (5), aspidistrin (6), tigogenin-3-O-ß-D-lucotrioside (7), desglucolanatigonin II (8), and degalactotigonin (9), were isolated from Solanum lyratum Thunb. Their cytotoxic activities were tested in two cancer cell lines by MTT method. One of the steroidal glycosides (6) showed significant cytotoxic activity against gastric cancer SGC7901 and liver cancer BEL-7402 cells.

Autoinhibitory sterol sulfates mediate programmed cell death in a bloom-forming marine diatom.

Cell mortality is a key mechanism that shapes phytoplankton blooms and species dynamics in aquatic environments. Here we show that sterol sulfates (StS) are regulatory molecules of a cell death program in Skeletonema marinoi, a marine diatom-blooming species in temperate coastal waters. The molecules trigger an oxidative burst and production of nitric oxide in a dose-dependent manner. The intracellular level of StS increases with cell ageing and ultimately leads to a mechanism of apoptosis-like death. Disrupting StS biosynthesis by inhibition of the sulfonation step significantly delays the onset of this fatal process and maintains steady growth in algal cells for several days. The autoinhibitory activity of StS demonstrates the functional significance of small metabolites in diatoms. The StS pathway provides another view on cell regulation during bloom dynamics in marine habitats and opens new opportunities for the biochemical control of mass-cultivation of microalgae.

Preclinical Safety Assessment of Furostanol Glycoside-Based Standardized Fenugreek Seed Extract in Laboratory Rats.

The present work is aimed at studying acute oral toxicity (AOT), subchronic oral toxicity, mutagenicity, and genotoxicity of furostanol glycosides-based standardized fenugreek seed extract (Fenu-FG) using the Organization for Economic Co-operation and Development (OECD) guidelines. The AOT and subchronic (90-day repeated dose) toxicity studies were performed on Wistar rats as per OECD 423 and OECD 408 guidelines, respectively. The mutagenicity (reverse mutation assay, Ames test) and genotoxicity (mammalian chromosome aberration test) were assessed in vitro using OECD 471 and OECD 473 guidelines, respectively. At an acute oral limit dose of 2,000 mg/kg, Fenu-FG did not show any mortality or treatment-related adverse signs. Ninety days of subchronic oral administration of Fenu-FG (250, 500, or 1,000 mg/kg) in rats did not induce any treatment-related significant changes with respect to body weight, hematology, blood biochemistry, urinalysis, gross pathology, or histopathology. The no-observed-adverse-effect-level of Fenu-FG was 1,000 mg/kg/day. Furthermore, Fenu-FG did not demonstrate mutagenic potential up to a concentration of 5,000 µg/plate (Ames test) and did not induce structural chromosome aberrations up to 2,000 µg/ml (in human lymphocyte cells in vitro). In conclusion, Fenu-FG was found safe during preclinical safety assessments.

Ultrahigh-Performance Liquid Chromatography-High-Resolution Quadrupole Time-of-Flight Mass Spectrometry Based Metabolomics Reveals Key Differences between Brachiaria decumbens and B. brizantha, Two Similar Pastures with Different Toxicities.

Several species of Brachiaria (Poaceae) currently cover extensive grazing areas in Brazil, providing valuable source of feed for a large cattle population. However, numerous cases of toxicity outbreaks in livestock have raised concerns on safety of using these plants, especially B. decumbens. In this study, chemometric analysis of ultrahigh-performance liquid chromatography-high-resolution quadrupole time-of-flight mass spectrometry (UHPLC-HR-QTOF-MS) data has for the first time uncovered qualitative and quantitative differences between metabolomes of toxic B. decumbens and nontoxic B. brizantha. The steroidal saponin protoneodioscin was established as the main biomarker for B. decumbens when compared to B. brizantha, and therefore the key explanation for their phytochemical differentiation. Quantification of protodioscin in both plants showed no significant differences; consequently, the idea that this compound is solely responsible for toxicity outbreaks must be discarded. Instead, we propose that the added occurrence of its stereoisomer, protoneodioscin, in B. decumbens, can be considered as the probable cause of these events. Interestingly, the greatest concentrations of saponins for both species were reached during winter (B. decumbens = 53.6 ± 5.1 mg·g(-1) dry weight (D.W.); B. brizantha = 25.0 ± 1.9 mg·g(-1) D.W.) and spring (B. decumbens = 49.4 ± 5.0 mg·g(-1) D.W.; B. brizantha = 27.9 ± 1.4 mg·g(-1) D.W.), although in the case of B. decumbens these values do not vary significantly among seasons.

A retrospective evaluation of species-specific sensitivity for neurological signs in toxicological studies: Is the dog more sensitive than the non-human primate?

Selection of the appropriate non-rodent species in preclinical programs is crucial for good translatability and human safety. There is no data available in the literature which provides exact comparison of dog and non-human primate (NHP) sensitivity regarding neurological signs in toxicological studies. We performed a retrospective analysis of 174 toxicity studies with 15 neuroscience substances. Neurological signs in dogs and NHPs were evaluated in correlation to exposure data. Overall incidence of substance induced convulsions was similar in both species and no gender differences were observed. The reported liability of beagles to spontaneous convulsions was not confirmed in our studies. The symptom tremor showed the best inter-species translatability. The current toxicological study design does not include exposure assessment at the time-point of neurological signs, therefore, we propose to include additional toxicokinetic samples. Our analysis revealed factors including housing, handling, and behavior, which prevents direct species comparison. In addition only one non-rodent species is routinely tested in development programs, therefore data for both species is rare. We however, had sufficient data which enabled comparison for one compound. In the spirit of 3Rs further examples should be evaluated.

Cytotoxic 5α,8α-epidioxy sterols from the marine sponge Monanchora sp.

Three new sterols, 5α,8α-epidioxy-24-norcholesta-6,9(11),22-trien-3ß-ol (1), 5α,8α-epidioxy-cholesta-6,9(11),24-trien-3ß-ol (2), and 5α,8α-epidioxy-cholesta-6,23-dien-3ß,25-diol (3), with four known sterols (4-7) were isolated from a marine sponge Monanchora sp. Their chemical structures were elucidated by extensive spectroscopic analysis. Compounds 1 and 3-7 showed moderate cytotoxicity against several human carcinoma cell lines including renal (A-498), pancreatic (PANC-1 and MIA PaCa-2), and colorectal (HCT 116) cancer cell lines.

IPS-1 plays an essential role in dsRNA-induced stress granule formation by interacting with PKR and promoting its activation.

The formation of cytoplasmic stress granules and the innate immune response are two distinct cellular stress responses. Our study investigated the involvement of four innate immune proteins - retinoic-acid-inducible gene I (RIG-I, also known as DDX58), melanoma differentiation-associated gene 5 (MDA5, also known as IFIH1), IFN-ß promoter stimulator (IPS-1, also known as MAVS) and protein kinase regulated by dsRNA (PKR, also known as EIF2AK2) in the formation of stress granules. Knockdown of IPS-1 or PKR significantly decreased the formation of stress granules induced by double-stranded (ds)RNA. IPS-1 depletion markedly attenuated the phosphorylation of PKR and eIF2α that was triggered by dsRNA, and IPS-1 facilitated the in vitro autophosphorylation of PKR. In IPS-1-depleted cells, the dsRNA-mediated dimerization of PKR through its dsRNA-binding domains was significantly abrogated, suggesting that IPS-1 might be involved in PKR dimerization. By co-immunoprecipitation and pulldown assays, our data demonstrate that IPS-1 directly binds to PKR through the IPS-1 caspase activation and recruitment domain (CARD), suggesting that the effect of IPS-1 on the formation of stress granules might be exerted through interacting with PKR and mediating its activation. PKR was recruited into stress granules upon activation, whereas the majority of IPS-1 protein formed clusters on mitochondrial membranes. Our work provides the first evidence that the innate signaling molecule IPS-1 plays an essential role in stress granule formation.

Steroid toxicity and detoxification in ascomycetous fungi.

In the last couple of decades fungal infections have become a significant clinical problem. A major interest into fungal steroid action has been provoked since research has proven that steroid hormones are toxic to fungi and affect the host/fungus relationship. Steroid hormones were found to differ in their antifungal activity in ascomycetous fungi Hortaea werneckii, Saccharomyces cerevisiae and Aspergillus oryzae. Dehydroepiandrosterone was shown to be the strongest inhibitor of growth in all three varieties of fungi followed by androstenedione and testosterone. For their protection, fungi use several mechanisms to lower the toxic effects of steroids. The efficiency of biotransformation in detoxification depended on the microorganism and steroid substrate used. Biotransformation was a relatively slow process as it also depended on the growth phase of the fungus. In addition to biotransformation, steroid extrusion out of the cells contributed to the lowering of the active intracellular steroid concentration. Plasma membrane Pdr5 transporter was found to be the most effective, followed by Snq2 transporter and vacuolar transporters Ybt1 and Ycf1. Proteins Aus1 and Dan1 were not found to be involved in steroid import. The research of possible targets of steroid hormone action in fungi suggests that steroid hormones inhibit ergosterol biosynthesis in S. cerevisiae and H. werneckii. Results of this inhibition caused changes in the sterol content of the cellular membrane. The presence of steroid hormones most probably causes the degradation of the Tat2 permease and impairment of tryptophan import.

Sterols with antileishmanial activity isolated from the roots of Pentalinon andrieuxii.

A new cholesterol derivative, pentalinonsterol (cholest-4,20,24-trien-3-one, 1), and a new polyoxygenated pregnane sterol glycoside, pentalinonside (2), together with 18 known compounds, including 14 sterols (3-16), three coumarins (17-19), and a triterpene (20), were isolated from a n-hexane partition of a methanol extract of the roots of the Mexican medicinal plant Pentalinon andrieuxii. Structure elucidation of compounds 1 and 2 was accomplished by spectroscopic data interpretation. All isolates were evaluated in vitro for their antileishmanial activity. Among these compounds, 6,7-dihydroneridienone (15) was found to be the most potent principle against promastigotes of Leishmania mexicana (L. mexicana). The cholesterol analogue, pentalinonsterol (1), together with two known sterols, 24-methylcholest-4,24(28)-dien-3-one (3) and neridienone (16), also exhibited significant leishmanicidal activity in this same bioassay. Compounds 1, 3, 15, 16, cholest-4-en-3-one (4), and cholest-5,20,24-trien-3ß-ol (7), showed strong antileishmanial activity against amastigotes of L. mexicana, and 4 was found to be the most potent agent with an IC(50) value of 0.03µM. All the isolates were also evaluated for their cytotoxicity in non-infected bone marrow-derived macrophages, but none of these compounds was found active towards this cell line. The intracellular parasites treated with compounds 1, 3, 4, 15, and 16 were further studied by electron microscopy; morphological abnormalities and destruction of the amastigotes were observed, as a result of treatment with these compounds.

Predicting the concentration range of unmonitored chemicals in wastewater-dominated streams and in run-off from biosolids-amended soils.

Organic compounds such as sterols and hormones have been detected in surface waters at ecologically relevant concentrations with sources including effluent discharged from publicly owned treatment works (POTWs) as well as leachate and runoff from land amended with municipal sludge (biosolids). Greater than 20% of regulated effluents discharged into U.S. surface waters experience in-stream dilution of <10-fold and potential impacts are particularly likely in receiving waters dominated by POTW effluents. The increasing use of biosolids on agricultural land exerts additional stress, thereby necessitating environmental monitoring for potential ecological and human health effects. Alternatively or in addition to monitoring efforts, screening for potentially hazardous chemicals can be performed using empirical models that are scalable and can deliver results rapidly. The present study makes use of data from U.S. EPA's Targeted National Sewage Sludge Survey (TNSSS) to predict the aqueous-phase concentrations and removal efficiencies of 10 sterols (campesterol, ß-sitosterol, stigmasterol, ß-stigmastanol, cholesterol, desmosterol, cholestanol, coprostanol, epicoprostanol, and ergosterol) as well as the putative toxicity posed by four specific hormones based on their reported biosolids concentrations using published empirical models. Model predictions indicate that removal efficiencies for sterols are uniformly high (~99%) and closely match removal rates calculated from chemical monitoring at POTWs (paired t-test; p=0.01). Results from toxicity modeling indicate that the hormones estrone, estradiol and estriol had the highest leaching potentials amongst the compounds considered here and that 17 ß-ethinylestradiol was found to pose a potentially significant threat to fathead minnows (Pimephales promelas) via run-off or leaching from biosolids-amended fields. This study exemplifies the use of in silico analysis to (i) identify potentially problematic organic compounds in biosolids, (ii) predict influent and effluent levels for hydrophobic organic compounds (HOCs) of emerging concern, and (iii) provide initial estimates of runoff concentrations, in this case for four prominent hormones known to act as endocrine disruptors.

Sterols from Sargassum oligocystum, a brown algae from the Persian Gulf, and their bioactivity.

Sargassum oligocystum (Heterokontophyta) is one of the most abundant algae distributed in the Persian Gulf. In this study, the cytotoxic effects of this algae on brine shrimp larvae were evaluated and the main sterols of the algae identified. Separation and purification of the compounds was carried out using silica gel column chromatography and HPLC to obtain eight pure compounds, 1-8. Structural elucidation of the constituents was based on the data obtained from (1)H-NMR, (13)C-NMR, HSQC, HMBC, DEPT and EI-MS. The compounds separated from S. oligocystum were identified as 22-dehydrocholesterol (1), cholesterol (2), fucosterol (3), 29-hydroperoxystigmasta-5,24(28)-dien-3ß-ol (4), 24-hydroperoxy-24-vinylcholesterol (5), a mixture of 24(S)-hydroxy-24-vinylcholesterol (6) and 24(R)-hydroxy-24-vinylcholesterol (7), and ostreasterol (8) based on their spectral data and from comparison with those previously reported in the literature.

Brominated aliphatic hydrocarbons and sterols from the sponge Xestospongia testudinaria with their bioactivities.

Four brominated aliphatic hydrocarbons (1-4), including a novel brominated ene-tetrahydrofuran named as mutafuran H (1), and five sterols (5-9) were isolated from the South China Sea sponge Xestospongia testudinaria. The structure of 1 was determined on the basis of NMR ((1)H, (13)C NMR, HSQC, HMBC, (1)H-(1)H COSY, and NOESY), MS, and optical rotation analysis. Known compounds were identified by comparison of their NMR data with those reported in the literature. Compounds 1-4, and 6-9 were evaluated for their toxicity against Artemia salina larvae, and anti-acetylcholinesterase activity.

Cytotoxic and PPARs transcriptional activities of sterols from the Vietnamese soft coral Lobophytum laevigatum.

A new unusual sterol, named lobophytosterol (1), and five known metabolites (2-6) were isolated from the methanol extract of the soft coral Lobophytum laevigatum. Their chemical structures were elucidated by extensive spectroscopic analysis and comparison with those reported in the literature. The absolute stereochemistry of 1 was determined using a modified Mosher's method. Compounds 1-3 showed cytotoxic activity against HCT-116 cells with IC(50) values of 3.2, 6.9 and 18.1 µM, respectively. Compound 1 additionally displayed cytotoxic effects on A549 and HL-60 cells with IC(50) values of 4.5 and 5.6 µM, respectively. Treatment of these cells with compound 1 resulted in an induction of apoptosis evident by chromatin condensation in treated cells. Besides, compounds 2, 4, and 6 significantly upregulated PPARs transcriptional activity dose-dependently in Hep-G2 cells. Taken together, these data suggest that compound 1 might inhibit the growth of the cancer cells by the induction of apoptosis, and compounds 2, 4, and 6 might act as specific agonists for PPARα, PPARδ, and PPARγ and may therefore regulate cellular glucose, lipid, and cholesterol metabolism.

Oxysterols: A world to explore.

Oxysterols (oxidized derivatives of cholesterol and phytosterols) can be generated in the human organism through different oxidation processes, some requiring enzymes. Furthermore, oxysterols are also present in food due to lipid oxidation reactions caused by heating treatments, contact with oxygen, exposure to sunlight, etc., and they could be absorbed from the diet, at different rates depending on their side chain length. In the organism, oxysterols can follow different routes: secreted into the intestinal lumen, esterified and distributed by lipoproteins to different tissues or degraded, mainly in the liver. Cholesterol oxidation products (COPs) have shown cytotoxicity, apoptotic and pro-inflammatory effects and they have also been linked with chronic diseases including atherosclerotic and neurodegenerative processess. In the case of phytosterol oxidation products (POPs), more research is needed on toxic effects. Nevertheless, current knowledge suggests they may also cause cytotoxic and pro-apoptotic effects, although at higher concentrations than COPs. Recently, new beneficial biological activities of oxysterols are being investigated. Whereas COPs are associated with cholesterol homeostasis mediated by different mechanisms, the implication of POPs is not clear yet. Available literature on sources of oxysterols in the organism, metabolism, toxicity and potential beneficial effects of these compounds are reviewed in this paper.

Detection of estrogenic activity from kraft mill effluents by the yeast estrogen screen.

Estrogenic activity of kraft pulp mill effluents (P. radiata, E. globulus and mixed -50% E. globulus and 50% P. radiata) was evaluated by the yeast estrogen screen assay. The estrogenic activity values were relatively low, ranking between 1.475 and 0.383 ng/L of EE2 eq. (Estrogenic equivalent of 17 alpha-ethynylestradiol), where the highest value corresponds to the E. globulus effluent and the lowest value to the P. radiata effluent. Analysis by solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) of chemical compounds present in all three effluents detected at least five major groups of organic compounds, corresponding to fatty acids, hydrocarbons, phenols, sterols and triterpenes. Comparison of analytical and biological data suggests that sterols could be the cause of the estrogenic activity in the evaluated effluent.

Synthesis and cytotoxicities of icogenin analogues with disaccharide residues.

For further structure-activity relationships (SAR) research of furostan saponin, two icogenin analogues: (25R)-22-O-methyl-furost-5-en-3beta,26-diol-3-O-alpha-l-rhamnopyranosyl-(1-->2)-beta-d-glucopyranoside 1 and (25R)-22-O-methyl-furost-5-en-3beta,26-diol-3-O-alpha-l-rhamnopyranosyl-(1-->2)-alpha-d-glucopyranoside 2, with valuable disaccharide moieties, were synthesized from diosgenin through eight steps. Both of the analogues behaved the similar cytotoxic activities with icogenin, towards nine types of human tumor cells herein.

Medical bioremediation: a concept moving toward reality.

Abstract A major driver of aging is catabolic insufficiency, the inability of our bodies to break down certain substances that accumulate slowly throughout the life span. Even though substance buildup is harmless while we are young, by old age the accumulations can reach a toxic threshold and cause disease. This includes some of the most prevalent diseases in old age-atherosclerosis and macular degeneration. Atherosclerosis is associated with the buildup of cholesterol and its oxidized derivatives (particularly 7-ketocholesterol) in the artery wall. Age-related macular degeneration is associated with carotenoid lipofuscin, primarily the pyridinium bisretinoid A2E. Medical bioremediation is the concept of reversing the substance accumulations by using enzymes from foreign species to break down the substances into forms that relieve the disease-related effect. We report on an enzyme discovery project to survey the availability of microorganisms and enzymes with these abilities. We found that such microorganisms and enzymes exist. We identified numerous bacteria having the ability to transform cholesterol and 7-ketocholesterol. Most of these species initiate the breakdown by same reaction mechanism as cholesterol oxidase, and we have used this enzyme directly to reduce the toxicity of 7-ketocholesterol, the major toxic oxysterol, to cultured human cells. We also discovered that soil fungi, plants, and some bacteria possess peroxidase and carotenoid cleavage oxygenase enzymes that effectively destroy with varied degrees of efficiency and selectivity the carotenoid lipofuscin found in macular degeneration.

Halichondria sulfonic acid, a new HIV-1 inhibitory guanidino-sulfonic acid, and halistanol sulfate isolated from the marine sponge Halichondria rugosa Ridley & Dendy.

A new sulfur-containing guanidino derivative, halichondria sulfonic acid (1) showing anti-HIV-1 activity, and halistanol trisulfate (2) with anti-tumor activity have been isolated from the marine sponge Halichondria rugosa Ridley & Dendy collected in the Chinese Southern Sea. The structure of 1 was elucidated by analysis of spectroscopic and crystal data.