Inhibition of post-lanosterol biosynthesis by fentanyl: potential implications for Fetal Fentanyl Syndrome (FFS).

A recent study discovered a novel, complex developmental disability syndrome, most likely caused by maternal fentanyl use disorder. This Fetal Fentanyl Syndrome (FFS) is biochemically characterized by elevated 7-dehydrocholesterol (7-DHC) levels in neonates, raising the question if fentanyl inhibition of the dehydrocholesterol reductase 7 (DHCR7) enzyme is causal for the emergence of the pathophysiology and phenotypic features of FFS. To test this hypothesis, we undertook a series of experiments on Neuro2a cells, primary mouse neuronal and astrocytic cultures, and human dermal fibroblasts (HDFs) with DHCR7+/+ and DHCR7+/- genotype. Our results revealed that in vitro exposure to fentanyl disrupted sterol biosynthesis across all four in vitro models. The sterol biosynthesis disruption by fentanyl was complex, and encompassed the majority of post-lanosterol intermediates, including elevated 7-DHC and decreased desmosterol (DES) levels across all investigated models. The overall findings suggested that maternal fentanyl use in the context of an opioid use disorder leads to FFS in the developing fetus through a strong disruption of the whole post-lanosterol pathway that is more complex than a simple DHCR7 inhibition. In follow-up experiments we found that heterozygous DHCR7+/- HDFs were significantly more susceptible to the sterol biosynthesis inhibitory effects of fentanyl than wild-type DHCR7+/+ fibroblasts. These data suggest that DHCR7+/- heterozygosity of mother and/or developing child (and potentially other sterol biosynthesis genes), when combined with maternal fentanyl use disorder, might be a significant contributory factor to the emergence of FFS in the exposed offspring. In a broader context, we believe that evaluation of new and existing medications for their effects on sterol biosynthesis should be an essential consideration during drug safety determinations, especially in pregnancy.

Vaping Induced Cannabidiol (CBD) Oxidation Product CBD Quinone Forms Protein Adducts with KEAP1 and Activates KEAP1-Nrf2 Genes.

Cannabidiol (CBD) vaping products have become widely available in the U.S. since their legalization in 2018. However, little is known about their respiratory health effects. Here we show that aerosolization of commercial CBD vaping products generates a reactive CBD quinone (CBDQ) which forms adducts with protein cysteine residues. Using click chemistry and a novel in vitro vaping product exposure system (VaPES), we further demonstrate that CBDQ forms adducts with human bronchial epithelial cell proteins including Keap1 and activates KEAP1-Nrf2 stress response pathway genes. These results suggest that vaping CBD alters protein function and induces cellular stress pathways in the lung.

Glucocorticoids are induced while dihydrotestosterone levels are suppressed in 5-alpha reductase inhibitor treated human benign prostate hyperplasia patients.

BACKGROUND: The development of benign prostatic hyperplasia (BPH) and medication-refractory lower urinary tract symptoms (LUTS) remain poorly understood. This study attempted to characterize the pathways associated with failure of medical therapy for BPH/LUTS. METHODS: Transitional zone tissue levels of cholesterol and steroids were measured in patients who failed medical therapy for BPH/LUTS and controls. Prostatic gene expression was measured using qPCR and BPH cells were used in organoid culture to study prostatic branching. RESULTS: BPH patients on 5-α-reductase inhibitor (5ARI) showed low levels of tissue dihydrotestosterone (DHT), increased levels of steroid 5-α-reductase type II (SRD5A2), and diminished levels of androgen receptor (AR) target genes, prostate-specific antigen (PSA), and transmembrane serine protease 2 (TMPRSS2). 5ARI raised prostatic tissue levels of glucocorticoids (GC), whereas alpha-adrenergic receptor antagonists (α-blockers) did not. Nuclear localization of GR in prostatic epithelium and stroma appeared in all patient samples. Treatment of four BPH organoid cell lines with dexamethasone, a synthetic GC, resulted in budding and branching. CONCLUSIONS: After failure of medical therapy for BPH/LUTS, 5ARI therapy continued to inhibit androgenesis but a 5ARI-induced pathway increased tissue levels of GC not seen in patients on α-blockers. GC stimulation of organoids indicated that the GC receptors are a trigger for controlling growth of prostate glands. A 5ARI-induced pathway revealed GC activation can serve as a master regulator of prostatic branching and growth.

Oxysterols Modify NLRP2 in Epithelial Cells, Identifying a Mediator of Ozone-induced Inflammation.

Ozone (O3) is a prevalent air pollutant causing lung inflammation. Previous studies demonstrate that O3 oxidizes lipids, such as cholesterol, in the airway to produce oxysterols, such as secosterol A (SecoA), which are electrophiles that are capable of forming covalent linkages preferentially with lysine residues and that consequently modify protein function. The breadth of proteins modified by this oxysterol as well as the biological consequences in the lung are unknown. By using an alkynyl-tagged form of SecoA and shotgun proteomics, we identified 135 proteins as being modified in bronchial epithelial cells. Among them was NLRP2 (NLR family pyrin domain-containing protein 2), which forms an alkynyl-tagged SecoA-protein adduct at lysine residue 1019 (K1019) in the terminal leucine-rich repeat region, a known regulatory region for NLR proteins. NLRP2 expression in airway epithelial cells was characterized, and CRISPR-Cas9 knockout (KO) and shRNA knockdown of NLRP2 were used to determine its function in O3-induced inflammation. No evidence for NLPR2 inflammasome formation or an NLRP2-dependent increase in caspase-1 activity in response to O3 was observed. O3-induced proinflammatory gene expression for CXCL2 and CXCL8/IL8 was further enhanced in NLRP2-KO cells, suggesting a negative regulatory role. Reconstitution of NLRP2-KO cells with the NLRP2 K1019 mutated to arginine partially blocked SecoA adduction and enhanced O3-induced IL-8 release as compared with wild-type NLRP2. Together, our findings uncover NLRP2 as a highly abundant, key component of proinflammatory signaling pathways in airway epithelial cells and as a novel mediator of O3-induced inflammation.

Ozone-derived oxysterols impair lung macrophage phagocytosis via adduction of some phagocytosis receptors.

Inhalation of the ambient air pollutant ozone causes lung inflammation and can suppress host defense mechanisms, including impairing macrophage phagocytosis. Ozone reacts with cholesterol in the lung to form oxysterols, like secosterol A and secosterol B (SecoA and SecoB), which can form covalent adducts on cellular proteins. How oxysterol-protein adduction modifies the function of lung macrophages is unknown. Herein, we used a proteomic screen to identify lung macrophage proteins that form adducts with ozone-derived oxysterols. Functional ontology analysis of the adductome indicated that protein binding was a major function of adducted proteins. Further analysis of specific proteins forming adducts with SecoA identified the phagocytic receptors CD206 and CD64. Adduction of these receptors with ozone-derived oxysterols impaired ligand binding and corresponded with reduced macrophage phagocytosis. This work suggests a novel mechanism for the suppression of macrophage phagocytosis following ozone exposure through the generation of oxysterols and the formation of oxysterol-protein adducts on phagocytic receptors.

Small Molecule Antipsychotic Aripiprazole Potentiates Ozone-Induced Inflammation in Airway Epithelium.

Inhaled ground level ozone (O3) has well described adverse health effects, which may be augmented in susceptible populations. While conditions, such as pre-existing respiratory disease, have been identified as factors enhancing susceptibility to O3-induced health effects, the potential for chemical interactions in the lung to sensitize populations to pollutant-induced responses has not yet been studied. In the airways, inhaled O3 reacts with lipids, such as cholesterol, to generate reactive and electrophilic oxysterol species, capable of causing cellular dysfunction and inflammation. The enzyme regulating the final step of cholesterol biosynthesis, 7-dehydrocholesterol reductase (DHCR7), converts 7-dehydrocholesterol (7-DHC) to cholesterol. Inhibition of DHCR7 increases the levels of 7-DHC, which is much more susceptible to oxidation than cholesterol. Chemical analysis established the capacity for a variety of small molecule antipsychotic drugs, like Aripiprazole (APZ), to inhibit DHCR7 and elevate circulating 7-DHC. Our results show that APZ and the known DHCR7 inhibitor, AY9944, increase 7-DHC levels in airway epithelial cells and potentiate O3-induced IL-6 and IL-8 expression and cytokine release. Targeted immune-related gene array analysis demonstrates that APZ significantly modified O3-induced expression of 16 genes, causing dysregulation in expression of genes associated with leukocyte recruitment and inflammatory response. Additionally, we find that APZ increases O3-induced IL-6 and IL-8 expression in human nasal epithelial cells from male but not female donors. Overall, the evidence we provide describes a novel molecular mechanism by which chemicals, such as APZ, that perturb cholesterol biosynthesis affect O3-induced biological responses.

Identification and characterization of prescription drugs that change levels of 7-dehydrocholesterol and desmosterol.

Regulating blood cholesterol (Chol) levels by pharmacotherapy has successfully improved cardiovascular health. There is growing interest in the role of Chol precursors in the treatment of diseases. One sterol precursor, desmosterol (Des), is a potential pharmacological target for inflammatory and neurodegenerative disorders. However, elevating levels of the precursor 7-dehydrocholesterol (7-DHC) by inhibiting the enzyme 7-dehydrocholesterol reductase is linked to teratogenic outcomes. Thus, altering the sterol profile may either increase risk toward an adverse outcome or confer therapeutic benefit depending on the metabolite affected by the pharmacophore. In order to characterize any unknown activity of drugs on Chol biosynthesis, a chemical library of Food and Drug Administration-approved drugs was screened for the potential to modulate 7-DHC or Des levels in a neural cell line. Over 20% of the collection was shown to impact Chol biosynthesis, including 75 compounds that alter 7-DHC levels and 49 that modulate Des levels. Evidence is provided that three tyrosine kinase inhibitors, imatinib, ponatinib, and masitinib, elevate Des levels as well as other substrates of 24-dehydrocholesterol reductase, the enzyme responsible for converting Des to Chol. Additionally, the mechanism of action for ponatinib and masitinib was explored, demonstrating that protein levels are decreased as a result of treatment with these drugs.

Ozone-derived Oxysterols Affect Liver X Receptor (LXR) Signaling: A POTENTIAL ROLE FOR LIPID-PROTEIN ADDUCTS.

When inhaled, ozone (O3) interacts with cholesterols of airway epithelial cell membranes or the lung-lining fluid, generating chemically reactive oxysterols. The mechanism by which O3-derived oxysterols affect molecular function is unknown. Our data show that in vitro exposure of human bronchial epithelial cells to O3 results in the formation of oxysterols, epoxycholesterol-α and -ß and secosterol A and B (Seco A and Seco B), in cell lysates and apical washes. Similarly, bronchoalveolar lavage fluid obtained from human volunteers exposed to O3 contained elevated levels of these oxysterol species. As expected, O3-derived oxysterols have a pro-inflammatory effect and increase NF-κB activity. Interestingly, expression of the cholesterol efflux pump ATP-binding cassette transporter 1 (ABCA1), which is regulated by activation of the liver X receptor (LXR), was suppressed in epithelial cells exposed to O3 Additionally, exposure of LXR knock-out mice to O3 enhanced pro-inflammatory cytokine production in the lung, suggesting LXR inhibits O3-induced inflammation. Using alkynyl surrogates of O3-derived oxysterols, our data demonstrate adduction of LXR with Seco A. Similarly, supplementation of epithelial cells with alkynyl-tagged cholesterol followed by O3 exposure causes observable lipid-LXR adduct formation. Experiments using Seco A and the LXR agonist T0901317 (T09) showed reduced expression of ABCA1 as compared with stimulation with T0901317 alone, indicating that Seco A-LXR protein adduct formation inhibits LXR activation by traditional agonists. Overall, these data demonstrate that O3-derived oxysterols have pro-inflammatory functions and form lipid-protein adducts with LXR, thus leading to suppressed cholesterol regulatory gene expression and providing a biochemical mechanism mediating O3-derived formation of oxidized lipids in the airways and subsequent adverse health effects.

Inhibitors of 7-Dehydrocholesterol Reductase: Screening of a Collection of Pharmacologically Active Compounds in Neuro2a Cells.

A small library of pharmacologically active compounds (the NIH Clinical Collection) was assayed in Neuro2a cells to determine their effect on the last step in the biosynthesis of cholesterol, the transformation of 7-dehydrocholesterol (7-DHC) to cholesterol promoted by 7-dehydrocholesterol reductase, DHCR7. Of some 727 compounds in the NIH Clinical Collection, over 30 compounds significantly increased 7-DHC in Neuro2a cells when assayed at 1 µM. Active compounds that increased 7-DHC with a Z-score of +3 or greater generally gave rise to modest decreases in desmosterol and increases in lanosterol levels. Among the most active compounds identified in the library were the antipsychotic, antidepressant, and anxiolytic compounds that included perospirone, nefazodone, haloperidol, aripiprazole, trazodone, and buspirone. Fluoxetine and risperidone were also active at 1 µM, and another 10 compounds in this class of pharmaceuticals were identified in the screen at concentrations of 10 µM. Increased levels of 7-DHC are associated with Smith-Lemli-Opitz syndrome (SLOS), a human condition that results from a mutation in the gene that encodes DHCR7. The SLOS phenotype includes neurological deficits and congenital malformations, and it is linked to a higher incidence of autism spectrum disorder. The significance of the current study is that it identifies common pharmacological compounds that may induce a biochemical presentation similar to SLOS. Little is known about the side effects of elevated 7-DHC postdevelopmentally, and the elevated 7-DHC that results from exposure to these compounds may also be a confounder in the diagnosis of SLOS.

Assay of protein and peptide adducts of cholesterol ozonolysis products by hydrophobic and click enrichment methods.

Cholesterol undergoes ozonolysis to afford a variety of oxysterol products, including cholesterol-5,6-epoxide (CholEp) and the isomeric aldehydes secosterol A (seco A) and secosterol B (seco B). These oxysterols display numerous important biological activities, including protein adduction; however, much remains to be learned about the identity of the reactive species and the range of proteins modified by these oxysterols. Here, we synthesized alkynyl derivatives of cholesterol-derived oxysterols and employed a straightforward detection method to establish secosterols A and B as the most protein-reactive of the oxysterols tested. Model adduction studies with an amino acid, peptides, and proteins provide evidence for the potential role of secosterol dehydration products in protein adduction. Hydrophobic separation methods-Folch extraction and solid phase extraction (SPE)-were successfully applied to enrich oxysterol-adducted peptide species, and LC-MS/MS analysis of a model peptide-seco adduct revealed a unique fragmentation pattern (neutral loss of 390 Da) for that species. Coupling a hydrophobic enrichment method with proteomic analysis utilizing characteristic fragmentation patterns facilitates the identification of secosterol-modified peptides and proteins in an adducted protein. More broadly, these improved enrichment methods may give insight into the role of oxysterols and ozone exposure in the pathogenesis of a variety of diseases, including atherosclerosis, Alzheimer's disease, Parkinson's disease, and asthma.

GAPDH inhibition mediated by thiol oxidation in human airway epithelial cells exposed to an environmental peroxide.

Intracellular redox homeostasis in the airway epithelium is closely regulated through adaptive signaling and metabolic pathways. However, inhalational exposure to xenobiotic stressors such as secondary organic aerosols (SOA) can alter intracellular redox homeostasis. Isoprene hydroxy hydroperoxide (ISOPOOH), a ubiquitous volatile organic compound derived from the atmospheric photooxidation of biogenic isoprene, is a major contributor to SOA. We have previously demonstrated that exposure of human airway epithelial cells (HAEC) to ISOPOOH induces oxidative stress through multiple mechanisms including lipid peroxidation, glutathione oxidation, and alterations of glycolytic metabolism. Using dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition to tag intracellular protein thiol oxidation, we demonstrate that exposure of HAEC to micromolar levels of ISOPOOH induces reversible oxidation of cysteinyl thiols in multiple intracellular proteins, including GAPDH, that was accompanied by a dose-dependent loss of GAPDH enzymatic activity. These results demonstrate that ISOPOOH induces an oxidative modification of intracellular proteins that results in loss of GAPDH activity, which ultimately impacts the dynamic regulation of the intracellular redox homeostatic landscape in HAEC.

Probing lipid-protein adduction with alkynyl surrogates: application to Smith-Lemli-Opitz syndrome.

Lipid modifications aid in regulating (and misregulating) protein function and localization. However, efficient methods to screen for a lipid's ability to modify proteins are not readily available. We present a strategy to identify protein-reactive lipids and apply it to a neurodevelopmental disorder, Smith-Lemli-Opitz syndrome (SLOS). Alkynyl surrogates were synthesized for polyunsaturated fatty acids, phospholipids, cholesterol, 7-dehydrocholesterol (7-DHC), and a 7-DHC-derived oxysterol. To probe for protein-reactive lipids, we used click chemistry to biotinylate the alkynyl tag and detected the lipid-adducted proteins with streptavidin Western blotting. In Neuro2a cells, the trend in amount of protein adduction followed known rates of lipid peroxidation (7-DHC >> arachidonic acid > linoleic acid >> cholesterol), with alkynyl-7-DHC producing the most adduction among alkynyl lipids. 7-DHC reductase-deficient cells, which cannot properly metabolize 7-DHC, exhibited significantly more alkynyl-7-DHC-protein adduction than control cells. Model studies demonstrated that a 7-DHC peroxidation product covalently modifies proteins. We hypothesize that 7-DHC generates electrophiles that can modify the proteome, contributing to SLOS's complex pathology. These probes and methods would allow for analysis of lipid-modified proteomes in SLOS and other disorders exhibiting 7-DHC accumulation. More broadly, the alkynyl lipid library would facilitate exploration of lipid peroxidation's role in specific biological processes in numerous diseases.

Intramolecular H-Atom Transfers in Alkoxyl Radical Intermediates Underlie the Apparent Oxidation of Lipid Hydroperoxides by Fe(II).

The one-electron reduction of lipid hydroperoxides by low-valent iron species is believed to be a driver of cellular lipid peroxidation and associated ferroptotic cell death. We investigated reactions of cholesterol 7α-OOH, the primary cholesterol autoxidation product, with Fe2+ to find that 7-ketocholesterol (7-KC, an oxidation product) is the major product under these (reducing) conditions. Mechanistic studies reveal the intervention of a 1,2-H-atom shift upon formation of the 7-alkoxyl radical to yield a ketyl radical that can be oxidized by either Fe3+ or O2 to give 7-KC, the most abundant oxysterol in vivo. We also investigated the corresponding reduction of the isomeric cholesterol 5α-OOH and again found that an oxidation product (5-hydroxycholesten-3-one) predominates under reducing conditions. An intramolecular H-atom shift (this time 1,4-) in the initially formed 5-alkoxyl radical is suggested to yield a ketyl radical that is oxidized to give the observed product. It would appear that a 1,2-H shift also accounts for the predominance of ketones over alcohols when unsaturated fatty acid hydroperoxides are exposed to iron-based reductants, which had previously been reported with hematin and demonstrated here with Fe2+. The predominance of 7-KC over the corresponding alcohol is maintained when cholesterol 7α-OOH embedded in phospholipid liposomes is treated with Fe2+ or when ferroptosis is induced in mouse embryonic fibroblasts. Our observation that 7-KC accumulates in ferroptotic cells suggests that it may be a good biomarker for ferroptosis.

Plasma sterols and vitamin D are correlates and predictors of ozone-induced inflammation in the lung: A pilot study.

BACKGROUND: Ozone (O3) exposure causes respiratory effects including lung function decrements, increased lung permeability, and airway inflammation. Additionally, baseline metabolic state can predispose individuals to adverse health effects from O3. For this reason, we conducted an exploratory study to examine the effect of O3 exposure on derivatives of cholesterol biosynthesis: sterols, oxysterols, and secosteroid (25-hydroxyvitamin D) not only in the lung, but also in circulation. METHODS: We obtained plasma and induced sputum samples from non-asthmatic (n = 12) and asthmatic (n = 12) adult volunteers 6 hours following exposure to 0.4ppm O3 for 2 hours. We quantified the concentrations of 24 cholesterol precursors and derivatives by UPLC-MS and 30 cytokines by ELISA. We use computational analyses including machine learning to determine whether baseline plasma sterols are predictive of O3 responsiveness. RESULTS: We observed an overall decrease in the concentration of cholesterol precursors and derivatives (e.g. 27-hydroxycholesterol) and an increase in concentration of autooxidation products (e.g. secosterol-B) in sputum samples. In plasma, we saw a significant increase in the concentration of secosterol-B after O3 exposure. Machine learning algorithms showed that plasma cholesterol was a top predictor of O3 responder status based on decrease in FEV1 (>5%). Further, 25-hydroxyvitamin D was positively associated with lung function in non-asthmatic subjects and with sputum uteroglobin, whereas it was inversely associated with sputum myeloperoxidase and neutrophil counts. CONCLUSION: This study highlights alterations in sterol metabolites in the airway and circulation as potential contributors to systemic health outcomes and predictors of pulmonary and inflammatory responsiveness following O3 exposure.

Superoxide reaction with tyrosyl radicals generates para-hydroperoxy and para-hydroxy derivatives of tyrosine.

Tyrosine-derived hydroperoxides are formed in peptides and proteins exposed to enzymatic or cellular sources of superoxide and oxidizing species as a result of the nearly diffusion-limited reaction between tyrosyl radical and superoxide. However, the structure of these products, which informs their reactivity in biology, has not been unequivocally established. We report here the complete characterization of the products formed in the addition of superoxide, generated from xanthine oxidase, to several peptide-derived tyrosyl radicals, formed from horseradish peroxidase. RP-HPLC, LC-MS, and NMR experiments indicate that the primary stable products of superoxide addition to tyrosyl radical are para-hydroperoxide derivatives (para relative to the position of the OH in tyrosine) that can be reduced to the corresponding para-alcohol. In the case of glycyl-tyrosine, a stable 3-(1-hydroperoxy-4-oxocyclohexa-2,5-dien-1-yl)-L-alanine was formed. In tyrosyl-glycine and Leu-enkephalin, which have N-terminal tyrosines, bicyclic indolic para-hydroperoxide derivatives were formed ((2S,3aR,7aR)-3a-hydroperoxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indole-2-carboxylic acid) by the conjugate addition of the free amine to the cyclohexadienone. It was also found that significant amounts of the para-OH derivative were generated from the hydroxyl radical, formed on exposure of tyrosine-containing peptides to Fenton conditions. The para-OOH and para-OH derivatives are much more reactive than other tyrosine oxidation products and may play important roles in physiology and disease.

Dose-Response Effects of 7-Dehydrocholesterol Reductase Inhibitors on Sterol Profiles and Vesicular Stomatitis Virus Replication.

Cholesterol is ubiquitous in cells; it plays a critical role in membrane structure and transport as well as in intracellular trafficking processes. There are suggestions that cholesterol metabolism is linked to innate immunity with inhibitors of DHCR7, the last enzyme in the cholesterol pathway, suggested to have potential as viral therapeutics nearly a decade ago. In fact, there are a number of highly prescribed pharmaceuticals that are off-target inhibitors of DHCR7, causing increased cellular levels of 7-dehydrodesmosterol (7-DHD) and 7-dehydrocholesterol (7-DHC). We report here dose-response studies of six such inhibitors on late-stage cholesterol biosynthesis in Neuro2a cells as well as their effect on infection of vesicular stomatitis virus (VSV). Four of the test compounds are FDA-approved drugs (cariprazine, trazodone, metoprolol, and tamoxifen), one (ifenprodil) has been the object of a recent Phase 2b COVID trial, and one (AY9944) is an experimental compound that has seen extensive use as a DHCR7 inhibitor. The three FDA-approved drugs inhibit replication of a GFP-tagged VSV with efficacies that mirror their effect on DHCR7. Ifenprodil and AY9944 have complex inhibitory profiles, acting on both DHCR7 and DHCR14, while tamoxifen does not inhibit DHCR7 and is toxic to Neuro2a at concentrations where it inhibits the Δ7-Δ8 isomerase of the cholesterol pathway. VSV itself affects the sterol profile in Neuro2a cells, showing a dose-response increase of dehydrolathosterol and lathosterol, the substrates for DHCR7, with a corresponding decrease in desmosterol and cholesterol. 7-DHD and 7-DHC are orders of magnitude more vulnerable to free radical chain oxidation than other sterols as well as polyunsaturated fatty esters, and the effect of these sterols on viral infection is likely a reflection of this fact of Nature.

An azido-biotin reagent for use in the isolation of protein adducts of lipid-derived electrophiles by streptavidin catch and photorelease.

HNE (4-hydroxynonenal), a byproduct of lipid peroxidation, reacts with nucleophilic centers on proteins. A terminal alkynyl analog of HNE (alkynyl HNE, aHNE) serves as a surrogate for HNE itself, both compounds reacting with protein amine and thiol functional groups by similar chemistry. Proteins modified with aHNE undergo reaction with a click reagent that bears azido and biotin groups separated by a photocleavable linker. Peptides and proteins modified in this way are affinity purified on streptavidin beads. Photolysis of the beads with a low intensity UV light releases bound biotinylated proteins or peptides, i.e. proteins or peptides modified by aHNE. Two strategies, (a) protein catch and photorelease and (b) peptide catch and photorelease, are employed to enrich adducted proteins or peptide mixtures highly enriched in adducts. Proteomics analysis of the streptavidin-purified peptides by LC-MS/MS permits identification of the adduction site. Identification of 30 separate peptides from human serum albumin by peptide catch and photorelease reveals 18 different aHNE adduction sites on the protein. Protein catch and photorelease shows that both HSA and ApoA1 in human plasma undergo significant modification by aHNE.

Expressing the Human Cholesteryl Ester Transfer Protein Minigene Improves Diet-Induced Fatty Liver and Insulin Resistance in Female Mice.

Mounting evidence has shown that CETP has important physiological roles in adapting to chronic nutrient excess, specifically, to protect against diet-induced insulin resistance. However, the underlying mechanisms for the protective roles of CETP in metabolism are not yet clear. Mice naturally lack CETP expression. We used transgenic mice with a human CETP minigene (huCETP) controlled by its natural flanking region to further understand CETP-related physiology in response to obesity. Female huCETP mice and their wild-type littermates were fed a high-fat diet for 6 months. Blood lipid profile and liver lipid metabolism were studied. Insulin sensitivity was analyzed with euglycemic-hyperinsulinemic clamp studies combined with 3H-glucose tracer techniques. While high-fat diet feeding induced obesity for huCETP mice and their wild-type littermates lacking CETP expression, insulin sensitivity was higher for female huCETP mice than for their wild-type littermates. There was no difference in insulin sensitivity for male huCETP mice vs. littermates. The increased insulin sensitivity in females was largely caused by the better insulin-mediated suppression of hepatic glucose production. In huCETP females, CETP in the circulation decreased HDL-cholesterol content and increased liver cholesterol uptake and liver cholesterol and oxysterol contents, which was associated with the upregulation of LXR target genes in long-chain polyunsaturated fatty acid biosynthesis and PPARα target genes in fatty acid ß-oxidation in the liver. The upregulated fatty acid ß-oxidation may account for the improved fatty liver and liver insulin action in female huCETP mice. This study provides further evidence that CETP has beneficial physiological roles in the metabolic adaptation to nutrient excess by promoting liver fatty acid oxidation and hepatic insulin sensitivity, particularly for females.

Tyrosine-lipid peroxide adducts from radical termination: para coupling and intramolecular Diels-Alder cyclization.

Free radical co-oxidation of polyunsaturated lipids with tyrosine or phenolic analogues of tyrosine gave rise to lipid peroxide-tyrosine (phenol) adducts in both aqueous micellar and organic solutions. The novel adducts were isolated and characterized by 1D and 2D NMR spectroscopy as well as by mass spectrometry (MS). The spectral data suggest that the polyunsaturated lipid peroxyl radicals give stable peroxide coupling products exclusively at the para position of the tyrosyl (phenoxy) radicals. These adducts have characteristic (13)C chemical shifts at 185 ppm due to the cross-conjugated carbonyl of the phenol-derived cyclohexadienone. The primary peroxide adducts subsequently undergo intramolecular Diels-Alder (IMDA) cyclization, affording a number of diastereomeric tricyclic adducts that have characteristic carbonyl (13)C chemical shifts at ~198 ppm. All of the NMR HMBC and HSQC correlations support the structure assignments of the primary and Diels-Alder adducts, as does MS collision-induced dissociation data. Kinetic rate constants and activation parameters for the IMDA reaction were determined, and the primary adducts were reduced with cuprous ion to give a phenol-derived 4-hydroxycyclohexa-2,5-dienone. No products from adduction of peroxyls at the phenolic ortho position were found in either the primary or cuprous reduction product mixtures. These studies provide a framework for understanding the nature of lipid-protein adducts formed by peroxyl-tyrosyl radical-radical termination processes. Coupling of lipid peroxyl radicals with tyrosyl radicals leads to cyclohexenone and cyclohexadienone adducts, which are of interest in and of themselves since, as electrophiles, they are likely targets for protein nucleophiles. One consequence of lipid peroxyl reactions with tyrosyls may therefore be protein-protein cross-links via interprotein Michael adducts.

Screening ToxCast™ for Chemicals That Affect Cholesterol Biosynthesis: Studies in Cell Culture and Human Induced Pluripotent Stem Cell-Derived Neuroprogenitors.

BACKGROUND: Changes in cholesterol metabolism are common hallmarks of neurodevelopmental pathologies. A diverse array of genetic disorders of cholesterol metabolism support this claim as do multiple lines of research that demonstrate chemical inhibition of cholesterol biosynthesis compromises neurodevelopment. Recent work has revealed that a number of commonly used pharmaceuticals induce changes in cholesterol metabolism that are similar to changes induced by genetic disorders with devastating neurodevelopmental deficiencies. OBJECTIVES: We tested the hypothesis that common environmental toxicants may also impair cholesterol metabolism and thereby possibly contribute to neurodevelopmental toxicity. METHODS: Using high-throughput screening with a targeted lipidomic analysis and the mouse neuroblastoma cell line, Neuro-2a, the ToxCast™ chemical library was screened for compounds that impact sterol metabolism. Validation of chemical effects was conducted by assessing cholesterol biosynthesis in human induced pluripotent stem cell (hiPSC)-derived neuroprogenitors using an isotopically labeled cholesterol precursor and by monitoring product formation with UPLC-MS/MS. RESULTS: Twenty-nine compounds were identified as validated lead-hits, and four were prioritized for further study (endosulfan sulfate, tributyltin chloride, fenpropimorph, and spiroxamine). All four compounds were validated to cause hypocholesterolemia in Neuro-2a cells. The morpholine-like fungicides, fenpropimorph and spiroxamine, mirrored their Neuro-2a activity in four immortalized human cell lines and in a human neuroprogenitor model derived from hiPSCs, but endosulfan sulfate and tributyltin chloride did not. CONCLUSIONS: These data reveal the existence of environmental compounds that interrupt cholesterol biosynthesis and that methodologically hiPSC neuroprogenitor cells provide a particularly sensitive system to monitor the effect of small molecules on de novo cholesterol formation. https://doi.org/10.1289/EHP5053.