Cytochrome P450 Products Research Articles

Polyunsaturated fatty acids stimulate immunity and eicosanoid production in Drosophila melanogaster

AbstractEicosanoids are a class of molecules derived from C20 polyunsaturated fatty acids (PUFAs) that play a vital role in mammalian and insect biological systems, including development, reproduction, and immunity. Recent research has shown that insects have significant but lower levels of C20 PUFAs in circulation in comparison to C18 PUFAs. It has been previously hypothesized in insects that eicosanoids are synthesized from C18 precursors, such as linoleic acid (LA), to produce downstream eicosanoids. In this study, we show that introduction of arachidonic acid (AA) stimulates production of cyclooxygenase, lipoxygenase, and cytochrome P450–derived eicosanoids. Downstream immune readouts showed that LA stimulates phagocytosis by hemocytes, while both LA and AA stimulate increased antimicrobial peptide production when D. melanogaster is exposed to a heat-killed bacterial pathogen. In totality, this work identifies PUFAs that are involved in insect immunity and adds evidence to the notion that Drosophila utilizes immunostimulatory lipid signaling to mitigate bacterial infections. Our understanding of immune signaling in the fly and its analogies to mammalian systems will increase the power and value of Drosophila as a model organism in immune studies.

Cytochrome p450 and innovative nutraceutical products

Dietary supplements are products that are ingested in addition to the regular diet to provide additional health-promoting nutrients. Dietary supplements are defined and regulated differently in the European Union (EU) and the United States (US). A fundamental aspect, besides the one related to the composition of the various products on the market, is linked to their quality, both from a nutritional and a pharmacological point of view. Concerning the knowledge of the metabolic aspects, the analysis of the interference, as an inductive or an inhibitory effect, of the p450 enzyme on individual preparations of supplements, is crucial. In this study, we present the results of the interference analysis of a new nutraceutical product based on 38% Bergamot Polyphenolic Fraction BPF® (Citrus bergamia Risso et Poit.), Pomegranate (Punica granatum) and Citrus fruits (Citrus aurantium var. dulcis, Citrus maxima Burm. Merr, Citrus paradisi Macfad) extract with cytochrome p450, showing that the product has limited activity on the cytochromes involved in most of human drug metabolism. This nutraceutical product is to be considered safe and potentially useful in the context of multiple treatments, not interfering with the traditional chronic therapies of patients. These findings open the door to modern "pharma-grade" nutraceuticals, expanding the safety and quality profiles of these new products.

Abstract 14554: Novel Upstream Targets for Atrial Fibrillation

Introduction: Atrial fibrillation (AF) represents one of the most common arrhythmias seen clinically and is associated with a significant increase in morbidity and mortality, yet, current treatment paradigms have proven largely inadequate. One of the main contributors to the pathophysiology of AF is inflammation. Hence, reduction of inflammation associated atrial remodeling represents a novel therapeutic strategy for the treatment of AF. One of the most biologically important groups of oxylipins (oxygenated lipids) is the eicosanoids that are potent modulators of immune responses and are derived from arachidonic acid, linoleic acid among others, which are metabolized through three pathways including the cytochrome P450 (CYP) pathway. The CYP products are major anti-inflammatory metabolites with several cardioprotective effects. Hypothesis: We hypothesize that lipid metabolites may represent a new therapeutic target for AF Methods: In the cross-sectional study, human atrial appendage specimens and blood from informed and consented patients were obtained in accordance with the approved UC Davis IRB protocol. Metabolomic profiling for over 100 metabolites was assessed from the plasma of patients using LC-MS/MS. We used Functional Connectome, an extension of the singular value decomposition for matrices, to determine how the metabolites were functionally related to each other. The underlying mechanisms of AF were determined using an integrated approach with molecular biology, flow cytometry, and electrophysiology. Results: Metabolomic profiling shows a significant difference in several metabolites from the arachidonic and linoleic acid pathways in the cohort with AF compared to patients in NSR. There was a significant increase in oxidative stress, endoplasmic reticulum stress, inflammatory cytokines, nuclear translocation of NF-κB, and the activation of MAPK and TGF-b pathways from patients with AF compared to NSR cohort. Conclusions: The study provides important insights into the functional relatedness of the metabolites from the CYP pathway in patients with AF and reveals the prospect of arachidonic and linoleic acid metabolites being novel therapeutic targets in the treatment of AF.

Altered oxylipin levels in human vitreous indicate imbalance in pro-/anti-inflammatory homeostasis in proliferative diabetic retinopathy

Proliferative diabetic retinopathy (PDR) is an advanced stage of diabetic retinopathy (DR), characterized by retinal neovascularization. It is a progressive fundus disease and a severe complication of diabetes that causes vision impairment. Hyperglycemia-induced persistent low-grade inflammation is a crucial factor underlying the pathogenesis of DR-associated damage and contributing to the progression of PDR. Highly enriched polyunsaturated fatty acids (PUFAs) in the retina are precursors to oxidized metabolites, namely, oxylipins, which exert pro-inflammatory or anti-inflammatory (resolving) effects under different pathological conditions and have been implicated in diabetes. To evaluate differences in oxylipin levels in the vitreous obtained from PDR and non-diabetic subjects, we performed a targeted assessment of oxylipins. A total of 41 patients with PDR and 22 non-diabetic control subjects were enrolled in this study. Vitreous humor obtained during routinely scheduled vitrectomy underwent a targeted but unbiased screening for oxylipins using mass spectrometry-based lipidomics. We found 21 oxylipins showing statistically significant differences in their levels between PDR and non-diabetic subjects (p < 0.05). Lipoxygenase (LOX)- and cytochrome P450 (CYP)- derived oxylipins were the most affected, while cyclooxygenase (COX) oxylipins were affected to a lesser extent. When categorized by their precursor PUFAs, ±19,20-EpDPE, a CYP product of docosahexaenoic acid (DHA) and 12S-HETE, a LOX product of arachidonic acid (ARA), were increased by the largest magnitude. Moreover, of these 21 oxylipins, 7 were considered as potential biomarkers for discriminating PDR patients from the non-diabetic controls. Our results indicate that altered oxylipin levels in the vitreous implicate an underlying imbalanced inflammation-resolution homeostasis in PDR.

Abstract P476: Determinants Of Atrial Fibrillation Mechanisms Using Metabolomic Profiling

Introduction: Atrial fibrillation (AF) represents one of the most common arrhythmias seen clinically, yet, current treatment paradigms have proven largely inadequate. One of the main contributors to the pathophysiology of AF is inflammation. Hence, reduction of inflammation associated atrial remodeling represents a novel therapeutic strategy for the treatment of AF. Oxylipins are derived from arachidonic acid, which is metabolized through three pathways including the cytochrome P450 (CYP450) pathway. The CYP450 products, epoxyeicosatrienoic acids (EETs), are major anti-inflammatory metabolites with several cardioprotective effects. The central hypothesis to be tested is that patients with AF will demonstrate an increase in oxylipin profiles towards inflammatory features compared to patients with normal sinus rhythm (NSR). We further hypothesize that lipid mediators may represent a new therapeutic target for AF. Methods: In the cross sectional study, human atrial appendage specimens and blood from informed and consented patients undergoing coronary artery bypass graft surgery were obtained in accordance with the approved UC Davis IRB protocol. Metabolomic profiling and inflammatory cytokines were assessed from the plasma of patients using LC-MS/MS. The underlying mechanisms of AF were determined using an integrated approach with molecular biology, flow cytometry, and electrophysiology. Results: Metabolomic profiling shows a significant decrease in the EETs/DHETs ratios and EpOMEs/DiHOMEs ratios in the cohort with AF compared to patients in NSR. There was a significant increase in inflammatory cytokine, chemokine levels and oxidative stress in patients with AF compared to patients in NSR. There was a significant increase in nuclear translocation of NF-κB (nNF-κB) from patients with AF compared to NSR using western blot analysis. Assessment of total IκB and phosphorylated-IκB (pIκB) levels showed a decrease in IκB level and an increase in the pIκB levels associated with the activation of NF-κB in AF compared to the NSR cohort. Conclusions: The study provides important insights into metabolomic profiles in patients with AF and reveals the prospect of oxylipins being a novel therapeutic target in the treatment of AF.

Linalool reverses benzene-induced cytotoxicity, oxidative stress and lysosomal/mitochondrial damages in human lymphocytes

Benzene exposure results in bone marrow suppression, leading to a decrease in the number of circulating white blood cells, an increased risk of chronic lymphocytic leukemia, acute myeloid leukemia and aplastic anemia. Since the mechanism of induction of benzene toxicity is due to active metabolites through cytochrome p450 enzymes and production of reactive oxygen species (ROS), we hypothesized that natural compound such linalool with anti-inflammatory/antioxidant properties could be effective in reducing its toxicity. Lymphocytes isolated from healthy individuals were simultaneously cotreated with different concentrations of LIN (10, 25 and 50 µM) and benzene (50 µM) for 4 h at 37 °C. After incubation, the toxicity parameters such cytotoxicity, ROS formation, lysosomal membrane integrity, mitochondria membrane potential (ΔΨm) collapse, oxidized/reduced glutathione (GSH/GSSG) and malondialdehyde (MDA) were analyzed using biochemical and flow cytometry evaluations. Our data showed that benzene (50 µM) induced a significant increase in cytotoxicity, ROS formation, mitochondrial membrane potential (MMP) collapse, lipid peroxidation and oxidative stress while LIN with antioxidant potential reversed the toxic effects of benzene on isolated human lymphocytes. Our results suggest that LIN reduces and reverses benzene-induced cytotoxicity, oxidative stress and lysosomal/mitochondrial damages in human lymphocyte. This study demonstrated that cotreatment of LIN with benzene can reduce several parameters indicative of oxidative stress. As such, LIN could represent a potential therapeutic agent in reducing certain aspects of benzene-induced toxicity.

TRPV4 channels mediate the mechanoresponse in retinal microglia.

The physiological and neurological correlates of plummeting brain osmolality during edema, traumatic CNS injury, and severe ischemia are compounded by neuroinflammation. Using multiple approaches, we investigated how retinal microglia respond to challenges mediated by increases in strain, osmotic gradients, and agonists of the stretch-activated cation channel TRPV4. Dissociated and intact microglia were TRPV4-immunoreactive and responded to the selective agonist GSK1016790A and substrate stretch with altered motility and elevations in intracellular calcium ([Ca2+ ]i ). Agonist- and hypotonicity-induced swelling was associated with a nonselective outwardly rectifying cation current, increased [Ca2+ ]i , and retraction of higher-order processes. The antagonist HC067047 reduced the extent of hypotonicity-induced microglial swelling and inhibited the suppressive effects of GSK1016790A and hypotonicity on microglial branching. Microglial TRPV4 signaling required intermediary activation of phospholipase A2 (PLA2), cytochrome P450, and epoxyeicosatrienoic acid production (EETs). The expression pattern of vanilloid thermoTrp genes in retinal microglia was markedly different from retinal neurons, astrocytes, and cortical microglia. These results suggest that TRPV4 represents a primary retinal microglial sensor of osmochallenges under physiological and pathological conditions. Its activation, associated with PLA2, modulates calcium signaling and cell architecture. TRPV4 inhibition might be a useful strategy to suppress microglial overactivation in the swollen and edematous CNS.

Culture density contributes to hepatic functions of fresh human hepatocytes isolated from chimeric mice with humanized livers: Novel, long-term, functional two-dimensional in vitro tool for developing new drugs.

Chimeric mice with humanized livers are considered a useful animal model for predicting human (h-) drug metabolism and toxicity. In this study, the characteristics of fresh h-hepatocytes (cFHHs, PXB-cells®) isolated from chimeric mice (PXB-mice®) were evaluated in vitro to confirm their utility for drug development. cFHHs cultured at high density (2.13 × 105 cells/cm2) displayed stable production of h-albumin and cytochrome P450 (CYP) 3A activities for at least 21 days. The mRNA expression levels of 10 of 13 CYP, UDP-glucuronosyltransferase (UGT), and transporters were maintained at >10% of the levels of freshly isolated cFHHs after 21 days. From 1 week, many bile canaliculi were observed between cFHHs, and the accumulation of the multidrug resistance-associated protein and bile salt export pump substrates in these bile canaliculi was clearly inhibited by cyclosporin A. Microarray analysis of cFHHs cultured at high density and at low density (0.53 × 105 cells/cm2) revealed that high density culture maintained high expressions of some transcription factors (HNF4α, PXR, and FXR) perhaps involved in the high CYP, UGT and transporter gene expressions of cFHHs. These results strongly suggest that cFHHs could be a novel in vitro tool for drug development studies.

The Presence of Active Brown Adipose Tissue Determines Cold-Induced Energy Expenditure and Oxylipin Profiles in Humans.

Accumulating evidence links brown adipose tissue (BAT) to increased cold-induced energy expenditure (CIEE) and regulation of lipid metabolism in humans. BAT has also been proposed as a novel source for biologically active lipid mediators including polyunsaturated fatty acids (PUFAs) and oxylipins. However, little is known about cold-mediated differences in energy expenditure and various lipid species between individuals with detectable BAT positive (BATpos) and those without BAT negative (BATneg). Here we investigated a unique cohort of matched BATpos and BATneg individuals identified by 18F-fluorodeoxyglucose positron emission tomography combined with computed tomography ([18F]-FDG PET/CT). BAT function, CIEE, and circulating oxylipins, were analyzed before and after short-term cold exposure using [18F]-FDG PET/CT, indirect calorimetry, and high-resolution mass spectrometry, respectively. We found that active BAT is the major determinant of CIEE since only BATpos individuals experienced significantly increased energy expenditure in response to cold. A single bout of moderate cold exposure resulted in the dissipation of an additional 20 kcal excess energy in BATpos but not in BATneg individuals. The presence of BAT was associated with a unique systemic PUFA and oxylipin profile characterized by increased levels of anti-inflammatory omega-3 fatty acids as well as cytochrome P450 products but decreased concentrations of some proinflammatory hydroxyeicosatetraenoic acids when compared with BATneg individuals. Notably, cold exposure raised circulating levels of various lipids, including the recently identified BAT-derived circulating factors (BATokines) DiHOME and 12-HEPE, only in BATpos individuals. In summary, our data emphasize that BAT in humans is a major contributor toward cold-mediated energy dissipation and a critical organ in the regulation of the systemic lipid pool.

Prediction and associations of preterm birth and its subtypes with eicosanoid enzymatic pathways and inflammatory markers

Endogenous signaling molecules derived from lipids, peptides, and DNA, are important regulators of physiological processes during pregnancy. The effect of their collective impact on preterm birth (delivery < 37 weeks gestation) is understudied. We aimed to characterize the associations and predictive capacity of an extensive panel of eicosanoids, immune biomarkers, oxidative stress markers, and growth factors towards preterm birth and its subtypes. We conducted a cross-sectional study of pregnant women (recruited < 15 weeks gestation) in the LIFECODES birth cohort, which included 58 cases of preterm birth and 115 controls that delivered term. Among the cases there were 31 cases who had a spontaneous preterm birth (cases who had spontaneous preterm labor and/or preterm premature rupture of membranes) and 25 that had preterm birth associated with aberrant placentation (cases who had preeclampsia and/or intrauterine growth restriction) and 2 cases that could not be sufficiently categorized as either. We analyzed single biomarker associations with each preterm birth outcome using multiple logistic regression. Adaptive elastic-net was implemented to perform a penalized multiple logistic regression on all biomarkers simultaneously to identify the most predictive biomarkers. We then organized biomarkers into biological groups and by enzymatic pathways and applied adaptive elastic-net and random forest to evaluate the accuracy of each group for predicting preterm birth cases. The majority of associations we observed were for spontaneous preterm birth, and adaptive elastic-net identified 5-oxoeicosatetraenoic acid, resolvin D1, 5,6-epoxy-eicsatrienoic acid, and 15-deoxy-12,14-prostaglandin J2 as most predictive. Overall, lipid biomarkers performed the best at separating cases from controls compared to other biomarker categories (adaptive elastic-net AUC = 0.78 [0.62, 0.94], random forest AUC = 0.84 [0.72, 0.96]). Among the enzymatic pathways that differentiate eicosanoid metabolites, we observed the highest prediction of overall preterm birth by lipoxygenase metabolites using random forest (AUC = 0.83 [0.69, 0.96]), followed by cytochrome p450 metabolites using adaptive elastic-net (AUC = 0.74 [0.52, 0.96]). In this study we translate biological hypothesis into the language of modern machine learning. Many lipid biomarkers were highly associated with overall and spontaneous preterm birth. Among eicosanoids, lipoxygenase and cytochrome p450 products performed best in identifying overall and spontaneous preterm birth. The combination of lipid biomarkers may have good utility in clinical settings to predict preterm birth.

Abstract P2029: Cytochrome P450 (CYP) 1B1-Estradiol Metabolite 2-Methoxyestradiol Protects Against Angiotensin II-induced Hypertension By Inhibiting CYP4A1 Expression &lt;And&gt; 20-Hydroxyeicosatetraenoic Acid (20-HETE) Production In The Female Mice

Previously we reported that angiotensin (Ang) II-induced hypertension is mediated by group IV cytosolic phospholipase A 2 α (cPLA 2 α) activation resulting in arachidonic acid (AA) release, and generation predominantly of eicosanoids with prohypertensive effects. Androgen dihydrotestosterone increases the expression of AA metabolizing enzyme cytochrome P450 (CYP) 4A1 and production of 20-HETE in the male rats and mice and an increase in blood pressure (BP). However, female mice that are resistant to Ang II-induced hypertension produce lower renal levels of 20-HETE, compared with males. Cyp4a12a, which has been identified as the predominant 20-HETE producing synthase in the mouse kidney, is expressed in very low levels in the female compared to male mice. Previously, we showed that the protection against Ang II-induced hypertension and associated pathogenesis in the female mice are mediated by CYP1B1-estradiol (E2) generated metabolite 2-methoxyestradiol (2-ME). These observations raised the possibility that CYP1B1-E2 metabolite 2-ME might protect against Ang II-induced hypertension by inhibiting expression of CYP4A1 and 20-HETE production. To test this hypothesis, we examined the effect of 20-HETE receptor antagonist 20-SOLA on Ang II (700 ng/kg/min, s.c. for two weeks)-induced hypertension in 8 weeks old wild type intact, ovariectomized (OVX), and intact Cyp1b1 -/- female mice. The systolic blood pressure (SBP, mmHg) was measured by tail-cuff. At the end of 2 weeks, Ang II caused greater increase in SBP in OVX- Cyp1b1 +/+ (161±6) and Cyp1b1 -/- intact female mice (165±2) compared to the intact Cyp1b1 +/+ mice (128±7) (p&lt;0.05, n=4-5). 20-SOLA (10 mg/kg/day, s.c.), the 20-HETE receptor antagonist minimized Ang II-induced increase in SBP in OVX- Cyp1b1 +/+ (116±4, Δ45) and Cyp1b1 -/- female mice (135±9, Δ30) (P&lt;0.05, n= 4-5). The expression of CYP4A1 was increased in the kidney of OVX- Cyp1b1 +/+ and intact Cyp1b1 -/- compared to intact Cyp1b1 +/+ mice, which was inhibited by E2 in the former but not the later mice; however, 2-ME inhibited CYP4A1 expression in the intact Cyp1b1 -/- mice. These data suggest that CYP1B1-E generated metabolite 2-ME protects against Ang II-induced hypertension by inhibiting the expression of CYP4A1 and production of 20-HETE.

Abstract 495: Determinants Of Atrial Fibrillation Mechanisms Using Metabolomic Profiling

Introduction: Atrial fibrillation (AF) represents one of the most common arrhythmias seen clinically, yet, current treatment paradigms have proven largely inadequate. One of the main contributors to the pathophysiology of AF is inflammation. Hence, reduction of inflammation associated atrial remodeling represents a novel therapeutic strategy for the treatment of AF. Oxylipins are derived from arachidonic acid, which is metabolized through three pathways including the cytochrome P450 (CYP450) pathway. The CYP450 products, epoxyeicosatrienoic acids (EETs), are major anti-inflammatory metabolites with several cardioprotective effects. The central hypothesis to be tested is that patients with AF will demonstrate an increase in oxylipin profiles towards inflammatory features compared to patients with normal sinus rhythm (NSR). We further hypothesize that lipid mediators may represent a new therapeutic target for AF. Methods: In the cross sectional study, human atrial appendage specimens and blood from informed and consented patients undergoing coronary artery bypass graft surgery were obtained in accordance with the approved UC Davis IRB protocol. Metabolomic profiling and inflammatory cytokines were assessed from the plasma of patients using LC-MS/MS. The underlying mechanisms of AF were determined using an integrated approach with molecular biology, flow cytometry, and electrophysiology. Results: Metabolomic profiling shows a significant decrease in the EETs/DHETs ratios (Fig 1A) and EpOMEs/DiHOMEs ratios (Fig 1B) in the cohort with AF compared to patients in NSR. There was a significant increase in inflammatory cytokine, chemokine levels and oxidative stress in patients with AF compared to patients in NSR. There was a significant increase in nuclear translocation of NF-κB (nNF-κB) from patients with AF compared to NSR using western blot analysis. Assessment of total IκB and phosphorylated-IκB (pIκB) levels showed a decrease in IκB level and an increase in the pIκB levels associated with the activation of NF-κB in AF compared to the NSR cohort. Conclusions: The study provides important insights into metabolomic profiles in patients with AF and reveals the prospect of EETs/DHETs being a novel therapeutic target in the treatment of AF.

Hepatoprotective efficacy of Shanxi aged vinegar extract against oxidative damage in vitro and in vivo

• SAVE exhibited hepatoprotective effects in vitro and in vivo . • SAVE protected against liver oxidative damage. • SAVE activated Nrf2-dependent antioxidant defences. Shanxi aged vinegar, a traditional fermented food, has beneficial effects on human health. Shanxi aged vinegar extract (SAVE) is rich in antioxidant components, which can prevent free radical-induced diseases. In the present study, we investigated the protective effects and molecular mechanisms of SAVE on oxidative stress-induced liver damage in vitro and in vivo . The results showed that SAVE attenuated H 2 O 2 -induced cytotoxicity and exhibited protective activity in hepatocyte by inhibiting reactive oxygen species (ROS) generation and cell apoptosis. In addition, SAVE alleviated CCl 4 -induced liver damage in mice through inhibiting cytochrome P450 2E1 (CYP2E1) expression, ROS generation and production of malondialdehyde (MDA), 4-hydroxyalkenals (4-HNE) and 8-hydroxy-2′-deoxyguanosine (8-OHdG). Moreover, SAVE protected against oxidative stress-induced liver damage by elevating antioxidant enzymes activities and glutathione level and upregulating Nrf2-antioxidant response element (ARE) pathway. The findings suggest that SAVE exhibits hepatoprotective effect and could be a potential alternative for hepatic oxidative damage.

Novel Electrotopological Atomic Descriptors for the Prediction of Xenobiotic Cytochrome P450 Reactions.

Cytochrome P450 (CYP) is an enzyme family that plays a crucial role in metabolism, mainly metabolizing xenobiotics to produce non-toxic structures, however, some metabolized products can cause hepatotoxicity. Hence, predicting the structures of CYP products is an important task in designing non-hepatotoxic drugs. Here, we have developed novel atomic descriptors to predict the sites of metabolism (SoM) in CYP substrates. We proposed descriptors that describe topological and electrostatic characteristics of CYP substrates using Gasteiger charge. The proposed descriptors were applied to CYP3A4 data analysis as a case study. As a result of the descriptor selection, we obtained a gradient boosting decision tree-based SoM classification model that used 139 existing descriptors and the proposed 45 descriptors, and the model performed well in terms of the Matthews correlation coefficient. We also developed a structure converter to predict CYP products. This converter correctly generated 51 structural formulas of experimentally observed CYP3A4 products according to a manual evaluation.

Coexpression of 5-Aminolevulinic Acid Synthase Gene Facilitates Heterologous Production of Thermostable Cytochrome P450, CYP119, in Holo Form in Escherichia coli.

Cytochrome P450 enzymes are heme-containing monooxygenases that exhibit potential as biocatalysts for practical applications. The Escherichia coli expression system is frequently used for biocatalyst production; however, heterologous production of hemeproteins in their holo form is difficult due to insufficient heme synthesis by the host. In this study, 5-aminolevulinic acid synthase (ALAS) from Rhodobacter capsulatus is used to accelerate intracellular heme biosynthesis in E. coli; this demonstrates that coexpression of the ALAS gene (ALAS) improves the heterologous production of cytochrome P450, CYP119, from Sulfolobus acidocaldarius. Coexpression of ALAS increased the amount of heterologous CYP119 isolated and the ratio of its holo form. The ratio of holo-CYP119 resulting from the coexpression of ALAS in E. coli was 99 %, whereas that from cells expressing CYP119 exclusively was 66 %. Coexpression of ALAS is a promising alternative for the efficient heterologous production of hemeproteins by using E. coli.

20-Hydroxyeicosatetraenoic acid antagonist attenuates the development of malignant hypertension and reverses it once established: a study in Cyp1a1-Ren-2 transgenic rats.

We hypothesized that vascular actions of 20-hydroxyeicosatetraenoic acid (20-HETE), the product of cytochrome P450 (CYP450)-dependent ω-hydroxylase, potentiate prohypertensive actions of angiotensin II (ANG II) in Cyp1a1-Ren-2 transgenic rats, a model of ANG II-dependent malignant hypertension. Therefore, we evaluated the antihypertensive effectiveness of 20-HETE receptor antagonist (AAA) in this model. Malignant hypertension was induced in Cyp1a1-Ren-2 transgenic rats by activation of the renin gene using indole-3-carbinol (I3C), a natural xenobiotic. Treatment with AAA was started either simultaneously with induction of hypertension or 10 days later, during established hypertension. Systolic blood pressure (SBP) was monitored by radiotelemetry, indices of renal and cardiac injury, and kidney ANG II levels were determined. In I3C-induced hypertensive rats, early AAA treatment reduced SBP elevation (to 161 ± 3 compared with 199 ± 3 mmHg in untreated I3C-induced rats), reduced albuminuria, glomerulosclerosis index, and cardiac hypertrophy (P<0.05 in all cases). Untreated I3C-induced rats showed augmented kidney ANG II (405 ± 14 compared with 52 ± 3 fmol/g in non-induced rats, P<0.05) which was markedly lowered by AAA treatment (72 ± 6 fmol/g). Remarkably, in TGR with established hypertension, AAA also decreased SBP (from 187 ± 4 to 158 ± 4 mmHg, P<0.05) and exhibited organoprotective effects in addition to marked suppression of kidney ANG II levels. In conclusion, 20-HETE antagonist attenuated the development and largely reversed the established ANG II-dependent malignant hypertension, likely via suppression of intrarenal ANG II levels. This suggests that intrarenal ANG II activation by 20-HETE is important in the pathophysiology of this hypertension form.

Elevated lipoxygenase and cytochrome P450 products predict progression of chronic kidney disease.

The clinical relevance of arachidonic acid (AA) metabolites in chronic kidney disease (CKD) progression is poorly understood. We aimed to compare the concentrations of 85 enzymatic pathway products of AA metabolism in patients with CKD who progressed to end-stage kidney disease (ESKD) versus patients who did not in a subcohort of Chronic Renal Insufficiency Cohort (CRIC) and to estimate the risk of CKD progression and major cardiovascular events by levels of AA metabolites and their link to enzymatic metabolic pathways. A total 123 patients in the CRIC study who progressed to ESKD were frequency matched with 177 nonprogressors and serum eicosanoids were quantified by mass spectrometry. We applied serum collected at patients' Year 1 visit and outcome of progression to ESKD was ascertained over the next 10 years. We used logistic regression models for risk estimation. Baseline 15-hydroxyeicosatetraenoate (HETE) and 20-HETE levels were significantly elevated in progressors (false discovery rate Q ≤ 0.026). The median 20-HETE level was 7.6 pmol/mL [interquartile range (IQR) 4.2-14.5] in progressors and 5.4 pmol/mL (IQR 2.8-9.4) in nonprogressors (P < 0.001). In an adjusted model, only 20-HETE independently predicted CKD progression. Each 1 standard deviation increase in 20-HETE was independently associated with 1.45-fold higher odds of progression (95% confidence interval 1.07-1.95; P = 0.017). Principal components of lipoxygenase (LOX) and cytochrome P450 (CYP450) pathways were independently associated with CKD progression. We found higher odds of CKD progression associated with higher 20-HETE, LOX and CYP450 metabolic pathways. These alterations precede CKD progression and may serve as targets for interventions aimed at halting progression.

Osh6p, a homologue of the oxysterol-binding protein, is involved in production of functional cytochrome P450 belonging to CYP52 family in n-alkane-assimilating yeast Yarrowia lipolytica

In this study, we investigated the role of OSH6, which encodes a homolog of the oxysterol-binding protein, in the assimilation of n-alkanes in the yeast Yarrowia lipolytica. The deletion mutant of OSH6 showed growth defects on n-alkanes of 10–16 carbons. In the deletion mutant, production of the functional cytochrome P450 was not observed. However, transcription of ALK1, encoding a major P450 belonging to the CYP52 family that plays a critical role in n-alkane hydroxylation, and further translation of its transcript were noted in the deletion mutant as well as in the wild-type strain. The phospholipid composition was altered and, the ratio of phosphatidylserine (PS) was reduced by the deletion of OSH6. Residues involved in the transport of PS and phosphatidylinositol-4-phosphate in Osh6 of Saccharomyces cerevisiae are conserved in Y. lipolytica Osh6p and substitutions of these residues resulted in a defect in the n-alkane assimilation by Y. lipolytica. From these results, we propose a hypothesis that Osh6p provides an ideal endoplasmic reticulum membrane environment for Alk proteins to have a functional conformation via lipid transport activity in Y. lipolytica.

Site‐Specific Dueteration Decreases Cytrochrome P450 Production of 12‐Hydroxy‐Nevirapine through the Kinetic Isotope Effect

The non‐nucleoside reverse transcriptase inhibitor nevirapine (NVP), classified as an essential medication by the World Health Organization, has been shown to cause life‐threatening hepatotoxicity and skin rash in patients. The 12‐hydroxyNVP (12‐OHNVP) metabolite of NVP has been implicated in these toxicities and here we aimed to gain a detailed understanding of utility of introducing deuteriums at the 12‐position of NVP as an approach to decreasing the formation of this metabolite. To do so, an ultra‐high performance liquid chromatograph tandem high‐resolution mass spectrometry assay was developed and employed to detect 12‐OHNVP formation following incubation of NVP with human liver microsomes and individual cDNA‐expressed enzymes. Through this work, we found that formation of 12‐OHNVP was most abundant following incubation with cytochrome P450 (P450; CYP) 2C19 although each of the P450s tested, with the exception of CYP3A5, catalyzed the formation of 12‐OHNVP to some extent. In line with this, formation of 12‐OHNVP by human liver microsomes was not abrogated in the presence of any established small molecule inhibitors of P450 activity including ketoconazole (CYP3A inhibitor). In order to test whether the deuterium kinetic isotope effect could be employed to control 12‐OHNVP production by all P450s we investigated the impact of 12th position deuteration of NVP on the formation of this metabolite. Human liver microsomes were incubated with 10 μM NVP or 10 μM 12‐d3NVP for 30 minute and the production of the 12‐OHNVP was measured. The abundance of 12‐OHNVP was 6.8‐fold lower when human liver microsomes were incubated with 12‐d3NVP, as compared to undeuterated NVP incubations. Of note, no difference in the formation of other P450‐dependent metabolites of NVP was observed. In order to test the impact of deuterium kinetic isotope effects on the metabolism of NVP in a whole cell system, we measured metabolite levels in the medium of primary mouse hepatocytes treated with 10 μM NVP and 12‐d3NVP for 24 hours. A 4.7‐fold decrease was observed in 12‐OHNVP production in the medium of cells treated with deuterated NVP as compared to undeuterated NVP. As with the human liver microsome experiments, no significant difference was observed in the production of other P450‐dependent metabolites between treatments. These results demonstrate that dueteration at the 12th position can markedly decrease cytochrome P450 production of 12‐OHNVP through the kinetic isotope effect. The impact of this controlled metabolism on NVP toxicity will be a subject of further research. This work was funded by NSF GRFP DGE‐1232825 awarded to CJSH and NIH R01 GM103853.Support or Funding InformationThis work was funded by NSF GRFP DGE‐1232825 awarded to CJSH and NIH R01 GM103853.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fenofibrate Attenuates Hypertension in Goldblatt Hypertensive Rats: Role of 20-Hydroxyeicosatetraenoic Acid in the Nonclipped Kidney

BackgroundThere is vast evidence that the renin-angiotensin system is not the sole determinant of blood pressure (BP) elevation in human renovascular hypertension or the relevant experimental models. This study tested the hypothesis that kidney deficiency of 20-hydroxyeicosatetraenoic acid (20-HETE), a product of cytochrome P450 (CYP)-dependent ω-hydroxylase pathway of arachidonic acid metabolism, is important in the pathophysiology of the maintenance phase of 2-kidney, 1-clip (2K1C) Goldblatt hypertension. Materials and MethodsIn 2K1C Goldblatt rats with established hypertension, angiotensin II, angiotensin 1-7, 20-HETE concentrations and gene expression of CYP4A1 enzyme (responsible for 20-HETE formation) of the nonclipped kidney were determined. We examined if 14 days׳ administration of fenofibrate, a lipid-lowering drug, would increase CYP4A1 gene expression and renal 20-HETE formation, and if increased 20-HETE concentrations in the nonclipped kidney would decrease BP (telemetric measurements). ResultsCYP4A1 gene expression, 20-HETE and angiotensin 1-7 concentrations were lower and angiotensin II levels were higher in the nonclipped kidney of 2K1C rats than in sham-operated rats. Fenofibrate increased CYP4A1 gene expression and 20-HETE concentration in the nonclipped kidney and significantly decreased BP in 2K1C rats but did not restore it to normotensive range. The treatment did not change BP in sham-operated rats. ConclusionsOur results suggest that alterations in the RAS and CYP-dependent ω-hydroxylase metabolites of arachidonic acid in the nonclipped kidneys are both important in the pathophysiology of the maintenance phase of 2K1C Goldblatt hypertension. Therefore, fenofibrate treatment effectively attenuated hypertension, probably via stimulation of 20-HETE formation in the nonclipped kidney.