Cytochrome P450 Pathway Research Articles

The Alexipharmic Mechanisms of Five Licorice Ingredients Involved in CYP450 and Nrf2 Pathways in Paraquat-Induced Mice Acute Lung Injury.

Oxidative stress is an important mechanism in acute lung injury (ALI) induced by paraquat (PQ), one of the most widely used herbicides in developing countries. In clinical prophylaxis and treatment, licorice is a widely used herbal medicine in China due to its strong alexipharmic characteristics. However, the corresponding biochemical mechanism of antioxidation and detoxification enzymes induced by licorice's ingredients is still not fully demonstrated. In this study, the detoxification effect of licorice was evaluated in vivo and in vitro. The detoxification and antioxidation effect of its active ingredients involved in the treatment was screened systematically according to Absorption, Distribution, Metabolism, and Excretion (ADME): predictions and evidence-based literature mining methods in silico approach. Data shows that licorice alleviate pulmonary edema and fibrosis, decrease Malondialdehyde (MDA) contents and increase Superoxide Dismutase (SOD) activity in PQ-induced ALI mice, protect the morphologic appearance of lung tissues, induce cytochrome 3A4 (CYA3A4) and Nuclear factor erythroid 2-related factor 2 (Nrf2) expression to active detoxification pathways, reduce the accumulation of PQ in vivo, protect or improve the liver and renal function of mice, and increase the survival rate. The 104 genes of PPI network contained all targets of licorice ingredients and PQ, which displayed the two redox regulatory enzymatic group modules cytochrome P450 (CYP450) and Nrf2 via a score-related graphic theoretic clustering algorithm in silico. According to ADME properties, glycyrol, isolicoflavonol, licochalcone A, 18beta-glycyrrhetinic acid, and licoisoflavone A were employed due to their oral bioavailability (OB) ≥ 30%, drug-likeness (DL) ≥ 0.1, and being highly associated with CYP450 and Nrf2 pathways, as potential activators to halt PQ-induced cells death in vitro. Both 3A4 inhibitor and silenced Nrf2 gene decreased the alexipharmic effects of those ingredients significantly. All these disclosed the detoxification and antioxidation effects of licorice on acute lung injury induced by PQ, and glycyrol, isolicoflavonol, licochalcone A, 18beta-glycyrrhetinic acid, and licoisoflavone A upregulated CYP450 and Nrf2 pathways underlying the alexipharmic mechanisms of licorice.

Oxidative stress and cell death induction by amitraz and its metabolite BTS-27271 mediated through cytochrome P450 and NRF2 pathway alteration in primary hippocampal cell

Amitraz is a neurotoxic formamidine pesticide that induces cell death in hippocampal neurons, although its mechanisms are unknown. Amitraz produces reactive oxygen species (ROS), which could lead to cell death. Amitraz was shown to induce different cytochrome P450 (CYP) isoenzymes involved with ROS and apoptotic cell death induction. Finally, amitraz was described to decrease the activity of antioxidant enzymes regulated through KEAP1/NRF2 pathway, thus likely leading to a reduction of ROS elimination and to cell death induction. We evaluated the effect of amitraz or BTS-27271 co-treatment with or without the antioxidant N-acetylcysteine and/or the unspecific CYP inhibitor 1-aminobenzotriazole on cell viability and its related mechanisms in wild type and silenced primary hippocampal neurons after 24 h treatment. We observed that amitraz produced oxidative stress and CYPs induction leading to apoptotic cell death. ROS generation was partially mediated by CYPs induction and downregulation of NRF2-pathway through KEAP1 overexpression. These data could help explain the mechanism by which amitraz induces cell death and oxidative stress and provide a therapeutic strategy to protect against this effect in case of poisoning.

Proteome analysis of type II alveolar epithelial cell in hyperoxia induced lung injury

To investigate the damage mechanism of type II alveolar epithelial cells (AEC II) after hyperoxia exposure by proteomics. The primary AEC II of preterm Sprague-Dawley (SD) rats were divided into normoxia and hyperoxia groups, and cultured in room air (21% O2) or hyperoxia (95% O2) condition, respectively. The cell morphology change was observed under an inverted contrast microscope; the protein expressions of Bcl-2 and caspase-3 were detected by Western Blot to ensure a successful model. Total protein in AEC II was collected, and mass spectrometry-based tandem mass tag (TMT)-labeled quantitative proteomics were used to detect the change of protein profile. Proteins with changes greater than 1.5-fold and P < 0.05 were considered differentially expressed, and bioinformatics analysis was performed. According to the proteomic results, AEC II were divided into three groups: normoxia group, hyperoxia group and hyperoxia+MW167 group (γ-secretase inhibitor MW167 was added to culture medium 30 minutes before they were placed into the chamber). The cell viability was detected by the cell proliferation and toxicity kit (CCK-8), and the expressions of Hes1, Bax mRNA were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR). (1) The cells in the normoxia group proliferated and prolonged significantly, and the cytoplasmic particulate matter was abundant. In the hyperoxia group, nucleus pyknosis and cytoplasmic particulate matter decreased significantly. Compared with the normoxia group, the expression of caspase-3 in the hyperoxia group was significantly increased, and the expression of Bcl-2 was significantly decreased (caspase-3/GAPDH: 1.352±0.086 vs. 0.769±0.080, Bcl-2/GAPDH: 0.614±0.060 vs. 1.361±0.078, both P < 0.01). (2) A total of 162 differentially expressed proteins were identified between normoxia and hyperoxia groups, the proteins up-regulated by hyperoxia were commonly associated with response processes to various stimuli, and located in the extracellular region; the proteins down-regulated by hyperoxia were commonly associated with synthesis of substances, and located in the cellular matrix. KEGG Pathway analyses suggested that metabolism by cytochrome P450, oxidative phosphorylation, and Notch signaling pathway were associated with the mechanism of hyperoxia injury on AEC II. (3) Compared with the normoxia group, the viability of cells in the hyperoxia group was significantly decreased, and the expressions of Hes1 and Bax mRNA were significantly increased [cell viability (A value): 0.060±0.003 vs. 1.058± 0.017, Hes1 mRNA (2-ΔΔCt): 2.235±0.606 vs. 1.144±0.107, Bax mRNA (2-ΔΔCt): 2.210±0.240 vs. 1.084±0.096, all P < 0.05]. Compared with the hyperoxia group, the viability of cells in the hyperoxia+MW167 group was significantly increased, and the expressions of Hes1 and Bax mRNA were significantly decreased [cell viability (A value): 0.271±0.025 vs. 0.060±0.003, Hes1 mRNA (2-ΔΔCt): 0.489±0.046 vs. 2.235±0.606, Bax mRNA (2-ΔΔCt): 1.289±0.041 vs. 2.210±0.240, all P < 0.05]. The mechanism of hyperoxia injury on AECII may be related to the metabolism by cytochrome P450, oxidative phosphorylation and activation of Notch signaling pathway.

Abnormal arachidonic acid metabolic network may reduce sperm motility via P38 MAPK.

Asthenozoospermia is a common cause of male infertility, the aetiology of which remains unclear in 50–60% of cases. The current study aimed to characterize metabolic alterations in asthenozoospermic seminal plasma and to explore the signalling pathways involved in sperm motility regulation. At first, high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry was used to detect the targeted metabolic network of arachidonic acid (AA). Metabolomic multivariate data analysis showed significant distinction of AA metabolites between asthenozoospermic and healthy seminal plasma. AA as well as its lipoxygenase (LOX) and cytochrome P450 metabolites were found to be abnormally increased, while cyclooxygenase (COX) metabolites were complicatedly disturbed in asthenozoospermic volunteers compared with those in healthy ones. In vitro experiments and western blot analysis of sperm cells revealed a decrease in sperm motility and upregulation of sperm phosphor-P38 induced by AA. P38 inhibitor could increase AA-reduced sperm motility. Also, all the inhibitors of the three metabolic pathways of AA could block AA-induced P38 mitogen-activated protein kinase (MAPK) activation and further improve sperm motility. We report here for the first time that an abnormal AA metabolic network could reduce sperm motility via P38 MAPK activation through the LOX, cytochrome P450 and COX metabolic pathways, which might be an underlying pathomechanism of asthenozoospermia.

Cytochrome P450 in Palliative Care and Hospice Kits.

As hospice and palliative care populations shift from the majority having a primary cancer diagnosis to most with a noncancer diagnosis, clinicians are challenged with caring for chronically ill patients with multiple comorbidities. In addition to traditional pain and symptom management, patients' comfort goals are frequently addressed by managing the underlying disease and comorbid conditions. As a result, many patients have extensive medication profiles. This raises the potential for drug-drug interactions at cytochrome P450 pathways that can interfere with anticipated drug response. Likewise, polypharmacy can be problematic when using palliative care order sets and hospice comfort kits to manage emergent symptoms or as the patient approaches death. This is further complicated when medications are administered before a pharmacist's review for drug interactions. This article provides an overview of cytochrome P450 and uses an unfolding case study approach to explore interactions that may occur within a patient's medication profile or in combination with medications commonly used by palliative care and hospice.

Pyrene degradation by marine-derived ascomycete: process optimization, toxicity, and metabolic analyses.

Marine-derived fungi are relevant genetic resources for bioremediation of saline environments/processes. Among the five fungi recovered from marine sponges able to degrade pyrene (Py) and benzo[a]pyrene (BaP), Tolypocladium sp. strainCBMAI 1346 and Xylaria sp. CBMAI 1464 presented the best removal rates of Py and BaP, respectively. Since the decrease in BaP was related to mycelial adsorption, a combined strategy was applied for the investigation of Py degradation by the fungus Tolypocladium sp. CBMAI 1346. The selected fungus was able to degrade about 95% of Py after 7days of incubation (optimized conditions), generating metabolites different from the ones found before optimization. Metabolites and transcriptomic data revealed that the degradation occurred mainly by the cytochrome P450 pathway. Putative monooxygenases and dioxygenases found in the transcriptome may play an important role. After 21days of degradation, no toxicity was found in the optimized culture conditions. The findings from the present study highlight the potential of marine-derived fungi to degrade environmental pollutants and convey innovative information related to the metabolism of pyrene.

Review of the many faces of synthetic cannabinoid toxicities.

IntroductionSynthetic cannabinoids (SCs) are psychoactive substances that are gaining popularity for their availability and lack of detection by standardized drug tests. Although some users may perceive SCs as safer alternatives to marijuana, some SCs are more potent and result in more severe toxicities.MethodsA search of the literature was conducted in the PubMed and SciFinder databases. Results in PubMed were limited to human studies, and only articles in English were included.ResultsReview of the literature illustrates the hazards associated with SC use. A range of severe toxicities affecting numerous systems has been identified, such as arrhythmias, myocardial infarction, sudden cardiac death, psychosis, suicidal ideation, seizures, acute tubular necrosis, and intracranial hemorrhage. Additionally, a recent outbreak of coagulopathies and at least 4 associated deaths due to SCs tainted with brodifacoum have been reported.DiscussionSynthetic cannabinoids may be perceived as a safer alternative to marijuana; however, SCs can be more potent at the cannabinoid receptors and in turn have greater toxicities. Limited information is available on the metabolism of SCs; however, cytochrome P450 pathways may be involved, which could result in drug interactions and unpredicted adverse effects. Toxicity with SC use is not just related to its effects, but also to additives that may taint these products and enhance their effects. Health care providers should be aware of the range of toxicities related to SC use, and tainted products such as these agents are not detected on routine drug screens.

Brain oxylipin concentrations following hypercapnia/ischemia: effects of brain dissection and dissection time

PUFAs are precursors to bioactive oxylipin metabolites that increase in the brain following CO2-induced hypercapnia/ischemia. It is not known whether the brain-dissection process and its duration also alter these metabolites. We applied CO2 with or without head-focused microwave fixation for 2 min to evaluate the effects of CO2-induced asphyxiation, dissection, and dissection time on brain oxylipin concentrations. Compared with head-focused microwave fixation (control), CO2 followed by microwave fixation prior to dissection increased oxylipins derived from lipoxygenase (LOX), 15-hydroxyprostaglandin dehydrogenase (PGDH), cytochrome P450 (CYP), and soluble epoxide hydrolase (sEH) enzymatic pathways. This effect was enhanced when the duration of postmortem ischemia was prolonged by 6.4 min prior to microwave fixation. Brains dissected from rats subjected to CO2 without microwave fixation showed greater increases in LOX, PGDH, CYP and sEH metabolites compared with all other groups, as well as increased cyclooxygenase metabolites. In nonmicrowave-irradiated brains, sEH metabolites and one CYP metabolite correlated positively and negatively with dissection time, respectively. This study presents new evidence that the dissection process and its duration increase brain oxylipin concentrations, and that this is preventable by microwave fixation. When microwave fixation is not available, lipidomic studies should account for dissection time to reduce these artifacts.

Alterations in fingerprints of polychlorinated biphenyls in benthic biota at the Portland Harbor Superfund Site (Oregon, USA) suggest metabolism

In order to understand the sources and fate of polychlorinated biphenyls (PCBs) in several species of benthic biota, including clams (Corbicula fluminea), oligochaetes (Lumbriculus variegatus), and mussels (Margaritifera falcata and Anodonta nuttalliana) at the Portland Harbor Superfund Site (PHSS), their congener fingerprints were examined. First, diagnostic ratios of congeners known to be metabolizable vs. recalcitrant in the cytochrome P450 (CYP) pathway were significantly lower in biota than in its co-located sediment, indicating metabolism may have occurred. Next, the congener patterns were analyzed using Positive Matrix Factorization (PMF). The dominant fingerprint (by mass) in benthic biota is related to Aroclor 1260 but displays differences in the fingerprint that are consistent with weathering via absorption, distribution, metabolism, and excretion (ADME). This fingerprint is similar to one isolated from PCBs in fish from Washington State, indicative of common metabolic pathways and consistent with CYP metabolism. When metabolism is taken into account, the spatial distribution of the PMF-isolated PCB fingerprints in biota matches well with those from co-located sediment samples, suggesting that the same mix of sources at one location partitions into biota and sediment. In accordance to their higher hydrophobicity, higher molecular weight (MW) PCB formulations were proportionately more abundant in biota than in sediment, although low MW PCBs (e.g., PCBs 4 and 11) do bioaccumulate in benthic organisms and should not be ignored in risk assessment efforts. Finally, fingerprinting suggests potential reasons why lab-based and field-based biota-sediment accumulation factors (BSAFs) differ substantially for bivalves.

Transcriptome analyses provide insights into the expression pattern and sequence similarity of several taxol biosynthesis-related genes in three Taxus species

BackgroundTaxol is an efficient anticancer drug; however, the accumulation of taxoids can vary hugely among Taxus species. The mechanism underlying differential accumulation of taxoids is largely unknown. Thus, comparative analysis of the transcriptomes in three Taxus species, including T. media, T. mairei and T. cuspidata, was performed.ResultsKEGG enrichment analysis revealed that the diterpenoid biosynthesis and cytochrome P450 pathways were significantly enriched in different comparisons. Differential expressions of these taxol biosynthesis related genes might be a potential explanation for the interspecific differential accumulation of taxol and its derivatives. Besides, the sequences of several MEP pathway-associated genes, such as DXS, DXR, MCT, CMK, MDS, HDS, HDR, IPPI, and GGPPS, were re-assembled based on independent transcriptomes from the three Taxus species. Phylogenetic analysis of these MEP pathway-associated enzymes also showed a high sequence similarity between T. media and T. cuspidata. Moreover, 48 JA-related transcription factor (TF) genes, including 10 MYBs, 5 ERFs, 4 RAPs, 3 VTCs, and 26 other TFs, were analyzed. Differential expression of these JA-related TF genes suggested distinct responses to exogenous JA applications in the three Taxus species.ConclusionsOur results provide insights into the expression pattern and sequence similarity of several taxol biosynthesis-related genes in three Taxus species. The data give us an opportunity to reveal the mechanism underlying the variations in the taxoid contents and to select the highest-yielding Taxus species.

RiboTag translatomic profiling of Drosophila oenocytes under aging and induced oxidative stress

BackgroundAging is accompanied with loss of tissue homeostasis and accumulation of cellular damages. As one of the important metabolic centers, liver shows age-related dysregulation of lipid metabolism, impaired detoxification pathway, increased inflammation and oxidative stress response. However, the mechanisms for these age-related changes still remain unclear. In the fruit fly, Drosophila melanogaster, liver-like functions are controlled by two distinct tissues, fat body and oenocytes. Compared to fat body, little is known about how oenocytes age and what are their roles in aging regulation. To characterize age- and stress-regulated gene expression in oenocytes, we performed cell-type-specific ribosome profiling (RiboTag) to examine the impacts of aging and oxidative stress on oenocyte translatome in Drosophila.ResultsWe show that aging and oxidant paraquat significantly increased the levels of reactive oxygen species (ROS) in adult oenocytes of Drosophila, and aged oenocytes exhibited reduced sensitivity to paraquat treatment. Through RiboTag sequencing, we identified 3324 and 949 differentially expressed genes in oenocytes under aging and paraquat treatment, respectively. Aging and paraquat exhibit both shared and distinct regulations on oenocyte translatome. Among all age-regulated genes, oxidative phosphorylation, ribosome, proteasome, fatty acid metabolism, and cytochrome P450 pathways were down-regulated, whereas DNA replication and immune response pathways were up-regulated. In addition, most of the peroxisomal genes were down-regulated in aged oenocytes, including genes involved in peroxisomal biogenesis factors and fatty acid beta-oxidation. Many age-related mRNA translational changes in oenocytes are similar to aged mammalian liver, such as up-regulation of innate immune response and Ras/MAPK signaling pathway and down-regulation of peroxisome and fatty acid metabolism. Furthermore, oenocytes highly expressed genes involving in liver-like processes (e.g., ketogenesis).ConclusionsOur oenocyte-specific translatome analysis identified many genes and pathways that are shared between Drosophila oenocytes and mammalian liver, highlighting the molecular and functional similarities between the two tissues. Many of these genes were altered in both oenocytes and liver during aging. Thus, our translatome analysis provide important genomic resource for future dissection of oenocyte function and its role in lipid metabolism, stress response and aging regulation.

Eicosanoids in carcinogenesis

Inflammation is the body's reaction to pathogenic (biological or chemical) stimuli and covers a burgeoning list of compounds and pathways that act in concert to maintain the health of the organism. Eicosanoids and related fatty acid derivatives can be formed from arachidonic acid and other polyenoic fatty acids via the cyclooxygenase and lipoxygenase pathways generating a variety of pro- and anti-inflammatory mediators, such as prostaglandins, leukotrienes, lipoxins, resolvins and others. The cytochrome P450 pathway leads to the formation of hydroxy fatty acids, such as 20-hydroxyeicosatetraenoic acid, and epoxy eicosanoids. Free radical reactions induced by reactive oxygen and/or nitrogen free radical species lead to oxygenated lipids such as isoprostanes or isolevuglandins which also exhibit pro-inflammatory activities. Eicosanoids and their metabolites play fundamental endocrine, autocrine and paracrine roles in both physiological and pathological signaling in various diseases. These molecules induce various unsaturated fatty acid dependent signaling pathways that influence crosstalk, alter cell–cell interactions, and result in a wide spectrum of cellular dysfunctions including those of the tissue microenvironment. Although the complete role of eicosanoids, including that of the recently elucidated anti-inflammatory specialized pro-resolving lipid mediators (SPMs), e.g. lipoxins, resolvins, protectins and maresins, is not completely understood, the result of unremitting chronic inflammation is fostering early stages of carcinogenesis. Chronic inflammation facilitates the transition from a normal cell to a cancerous one. The disruption of homeostasis across a wide, but identifiable, swath of diverse molecular pathways creates a micromilieu which constitutes an early and necessary step in the 6-step sequence of carcinogenesis for the vast majority of cancers, termed “sporadic cancers”.

\u201cOmics\u201d data integration and functional analyses link Enoyl-CoA hydratase, short chain 1 to drug refractory dilated cardiomyopathy

BackgroundLarge-scale “omics” datasets have not been leveraged and integrated with functional analyses to discover potential drivers of cardiomyopathy. This study addresses the knowledge gap.MethodsWe coupled RNA sequence (RNA-Seq) variant detection and transcriptome profiling with pathway analysis to model drug refractory dilated cardiomyopathy (drDCM) using the BaseSpace sequencing hub and Ingenuity Pathway Analysis. We used RNA-Seq case-control datasets (n = 6 cases, n = 4 controls), exome sequence familial DCM datasets (n = 3 Italians, n = 5 Italians, n = 5 Chinese), and controls from the HapMap project (n = 5 Caucasians, and n = 5 Asians) for disease modeling and putative mutation discovery. Variant replication datasets: n = 128 cases and n = 15 controls. Source of datasets: NCBI Sequence Read Archive. Statistics: Pairwise differential expression analyses to determine differentially expressed genes and t-tests to calculate p-values. We adjusted for false discovery rates and reported q-values. We used chi-square tests to assess independence among variables, the Fisher’s Exact Tests and overlap p-values for the pathways and p-scores to rank network.ResultsData revealed that ECHS1(enoyl-CoA hydratase, short chain 1(log2(foldchange) = 1.63329) hosts a mirtron, MIR3944 expressed in drDCM (FPKM = 5.2857) and not in controls (FPKM = 0). Has-miR3944-3p is a putative target of BAG1 (BCL2 associated athanogene 1(log2(foldchange) = 1.31978) and has-miR3944-5p of ITGAV (integrin subunit alpha V(log2(foldchange) = 1.46107) and RHOD (ras homolog family member D(log2(foldchange) = 1.28851). There is an association between ECHS1:11 V/A(rs10466126) and drDCM (p = 0.02496). The interaction (p = 2.82E-07) between ECHS1:75 T/I(rs1049951) and ECHS1:rs10466126 is associated with drDCM (p < 2.2e-16). ECHS1:rs10466126 and ECHS1:rs1049951 are in linkage disequilibrium (D’ = 1). The interaction (p = 7.84E-08) between ECHS1:rs1049951 and the novel ECHS1:c.41insT variant is associated with drDCM (p < 2.2e-16). The interaction (p = 0.001096) between DBT (Dihydrolipoamide branched chain transacylase E2):384G/S(rs12021720) and ECHS1:rs10466126 is associated with drDCM (p < 2.2e-16). At the mRNA level, there is an association between ECHS1 (log2(foldchange) = 1.63329; q = 0.013927) and DBT (log2(foldchange) = 0.955072; q = 0.0368792) with drDCM. ECHS1 is involved in valine (−log (p = 3.39E00)), isoleucine degradation (p = 0.00457), fatty acid β-oxidation (−log(p) = 2.83E00), and drug metabolism:cytochrome P450 (z-score = 2.07985196) pathways. The mitochondria (−log(p) = 8.73E00), oxidative phosphorylation (−log(p) = 5.35E00) and TCA-cycle II (−log(p) = 2.70E00) are dysfunctional.ConclusionsWe introduce an integrative data strategy that considers the interplay between the DNA, mRNA, and associated pathways, which represents a possible diagnostic, prognostic, biomarker, and personalized treatment discovery approach in genomically heterogeneous diseases.

Arachidonic Acid Induces Ferroptosis-Mediated Cell-Death in Multiple Myeloma

Mounting evidence suggests positive correlation of obesity and bone marrow (BM) adipogenesis with multiple myeloma (MM) initiation and progression. MM is more prevalent in patients with metabolic disorders including obesity and Gaucher's disease. Adipocytes constitute 70% of BM cellular volume in human adults making them one of the prominent players in MM progression. However, the underlying mechanisms involved remain unknown. Fat depots are increasingly recognized as the key endocrine organs for their ability to secrete various hormones (e.g. leptin, adiponectin, resistin), lipid substrates (polyunsaturated fatty acids), and cytokines (e.g. IL-6, IL-1, and TNFα). For example, leptin levels are increased in newly diagnosed MM patients that allow MM initiation by inhibiting NK T-cells (Favreaue 2017, Leukemia). Lipid substrates are also deregulated in the serum of cancer patients, including MM patients. These fatty acids are important initiators of many key downstream drug-targetable signaling pathways such as cyclooxygenase, cytochrome P450, and lipoxygenase pathways. In this study, we hypothesized that the changes in lipid profiles in MM patients contribute to disease progression.PPARγ is the key transcription factor that potentiates adipocyte differentiation and its key functions including lipid synthesis and transportation. We found that the BM adipose tissue (BMAT) fraction from MGUS, and SMM patients showed significantly increased PPARγ expression compared to the healthy donors. In vitro, MGUS and SMM BM stromal cells (BMSCs) showed increased adipogenic differentiation potential compared to the healthy donors. In-vitro, the proliferation of MM.1S human MM cells was significantly increased when co-cultured with BMSCs from MGUS/SMM patients compared to healthy donors. Taken together these data demonstrate a vicious cycle in which early stage MM cells and BM adipocytes support each other's growth.Multicomponent gene expression analysis of the BMAT from MGUS and MM patients showed altered genes involved mostly in fatty acid synthesis and metabolism. Lipidomics analysis revealed significantly decreased Arachidonic Acid (AA) levels in the BMSCs of SMM, the BM supernatant (BMS) of NDMM patients, and the blood serum of MGUS and SMM patients. This led us to hypothesize that increasing AA levels around MM cells may decrease MM progression.Physiological-range AA treatment of human MM cell lines, MM1S, H929, and U266, dose-dependently decreased proliferation and viability after 72-hours in all three cell lines. For in vivo studies, we generated humanized MM tumor growth in SCID mice by growing MM.1S cells in the intrascapular subcutaneous region for 3-weeks. Mice were then treated with either 100µg/g or 500µg/g of AA daily along with vehicle control via subcutaneous injection for 3-weeks near the tumor mass. Tumor volume continued to increase in vehicle control and 100 µg/g AA treatment groups but was significantly decreased in 500µg/g AA treatment group beginning 10-days after starting treatment. Gross examination of the tumor mass showed dramatically increased tumor necrosis. Flow cytometry analysis of CD138+ myeloma cells from non-necrotic part of the tumors showed significantly increased number of dead cells and the cells undergoing apoptosis in 500µg/g AA treatment group.AA is metabolized to primarily activate cyclooxygenase (COX), cytochrome P450 (CYP), and lipoxygenase (LOX) signaling pathways. Recent studies also show that AA induces ferroptosis, a programmed cell death in response to accumulation of lipid peroxides and mediated by iron (Kagan et al., 2017). To identify the primary apoptosis-inducing AA signaling pathway in MM cells, we used various inhibitors of each of these signaling pathways including ibuprofen, baicalein, BW B70C, 1-aminobenzotriazole, and ferrostatin. Among these compounds, ferrostatin completely rescued AA induced apoptosis in the human MM cells.Taken together, here we show that the adipogenesis is involved in myeloma pathogenesisis. Furthermore, AA signaling induces ferroptosis-mediated cell death in MM cells. Therefore, therapeutically targeting members of this signaling pathway is a potential novel treatment strategy for MM, especially in the MGUS and SMM stages. DisclosuresNo relevant conflicts of interest to declare.

Lipidomic profiling of subchronic As4S4 exposure identifies inflammatory mediators as sensitive biomarkers in rats.

Arsenic sulfide compounds provide nearly all of the world's supply of arsenic. However, the risk of arsenic trisulfide exposure is still not fully investigated. Here, we systemically assessed the toxicology of As4S4 in rats by combining arsenic metabolite detection, routine testing and lipidomic profiling. It was revealed that the oral administration of As4S4 for two months increased the total arsenic content in the liver reaching a saturation level. Further analysis by anion exchange chromatography coupled with inductively coupled plasma mass spectrometry (ICP-MS) technology showed no trace of inorganic arsenic, but there was significant presence of dimethylarsinic acid (DMA), in the livers of rats. This arsenic metabolite was less toxic to rats and did not induce overt liver pathology and functional injury. In contrast, lipidomic profiling provided a comprehensive map of lipids and uncovered a more complex inflammatory response, exhibiting more sensitive change to arsenic exposure. We observed that metabolites of cyclooxygenase, including PGF2α, dhk PGF2α, 15k PGF2α, 8-iso-PGF2a, PGE2, dhk PGE2, PGD2, 15d-PGD2, and PGJ2, were significantly elevated. But mediators from lipoxygenase, cytochrome P450, docosahexaenoic acid, and eicosapentaenoic acid pathways were not markedly affected. In summary, we identified DMA as the predominant arsenic species in the livers of rats, and found cyclooxygenase-derived lipids as the inflammatory mediators before the development of overt liver injury for subchronic As4S4 exposure. These mediators could translate into potential metabolic biomarkers in early arsenic risk assessment and as targets for therapeutic intervention.

Analysis of Potential Molecular Targets inMonochamus alternatus(Coleoptera: Cerambycidae) Inoculated withBeauveria bassiana(Deuteromycotina: Hyphomycetes)

Abstract Monochamus alternatus Hope, the Japanese pine sawyer (Coleoptera: Cerambycidae), is a longhorn beetle that is a known vector of the pinewood nematode Bursaphelenchus xylophilus (Steiner et Buhrer) Nickel. Beauveria bassiana (Balsamo) Vuillemin is an entomopathogenic fungus that is widely used as a microbial control agent because of its ease of mass production and safety to most vertebrates. To identify molecular targets that are potentially associated with B. bassiana toxicology, differentially expressed gene (DEG) libraries of M. alternatus contacted with B. bassiana have been prepared. The transcripts are sequenced using the Ion Proton platform; We identify 5,637, 9,181, and 1,787 sequences that involved cellular components, molecular functions, and biological processes, respectively. Fifty DEGs are enriched in the metabolism of xenobiotics by the cytochrome P450 pathway, and 33 DEGs are enriched in insect hormone biosynthesis by a Kyoto Encyclopedia of Genes and Genomes pathway enrichment ana...

Gene Regulatory Network Analysis Identifies Sex-Linked Differences in Colon Cancer Drug Metabolism.

Understanding sex differences in colon cancer is essential to advance disease prevention, diagnosis, and treatment. Males have a higher risk of developing colon cancer and a lower survival rate than women. However, the molecular features that drive these sex differences are poorly understood. In this study, we use both transcript-based and gene regulatory network methods to analyze RNA-seq data from The Cancer Genome Atlas for 445 patients with colon cancer. We compared gene expression between tumors in men and women and observed significant sex differences in sex chromosome genes only. We then inferred patient-specific gene regulatory networks and found significant regulatory differences between males and females, with drug and xenobiotics metabolism via cytochrome P450 pathways more strongly targeted in females. This finding was validated in a dataset of 1,193 patients from five independent studies. While targeting, the drug metabolism pathway did not change overall survival for males treated with adjuvant chemotherapy, females with greater targeting showed an increase in 10-year overall survival probability, 89% [95% confidence interval (CI), 78-100] survival compared with 61% (95% CI, 45-82) for women with lower targeting, respectively (P = 0.034). Our network analysis uncovers patterns of transcriptional regulation that differentiate male and female colon cancer and identifies differences in regulatory processes involving the drug metabolism pathway associated with survival in women who receive adjuvant chemotherapy. This approach can be used to investigate the molecular features that drive sex differences in other cancers and complex diseases.Significance: A network-based approach reveals that sex-specific patterns of gene targeting by transcriptional regulators are associated with survival outcome in colon cancer. This approach can be used to understand how sex influences progression and response to therapies in other cancers. Cancer Res; 78(19); 5538-47. ©2018 AACR.

Assessment of the Biotransformation of Low-Turnover Drugs in the HµREL Human Hepatocyte Coculture Model.

Metabolic profiles of four drugs possessing diverse metabolic pathways (timolol, meloxicam, linezolid, and XK469) were compared following incubations in both suspended cryopreserved human hepatocytes and the HμREL hepatocyte coculture model. In general, minimal metabolism was observed following 4-hour incubations in both suspended hepatocytes and the HμREL model, whereas incubations conducted up to 7 days in the HμREL coculture model resulted in more robust metabolic turnover. In the case of timolol, in vivo human data suggest that 22% of the dose is transformed via multistep oxidative opening of the morpholine moiety. Only the first-step oxidation was detected in suspended hepatocytes, whereas the relevant downstream metabolites were produced in the HµREL model. For meloxicam, both the hydroxymethyl and subsequent carboxylic acid metabolites were abundant following incubation in the HμREL model, while only a trace amount of the hydroxymethyl metabolite was observed in suspension. Similar to timolol, linezolid generated substantially higher levels of morpholine ring-opened carboxylic acid metabolites in the HμREL model. Finally, while the major aldehyde oxidase-mediated mono-oxidative metabolite of XK469 was minimally produced in hepatocyte suspension, the HμREL model robustly produced this metabolite, consistent with a pathway reported to account for 54% of the total urinary excretion in human. In addition, low-level taurine and glycine conjugates were identified in the HµREL model. In summary, continuous metabolite production was observed for up to 7 days of incubation in the HµREL model, covering cytochrome P450, aldehyde oxidase, and numerous conjugative pathways, while predominant metabolites correlated with relevant metabolites reported in human in vivo studies.

Characterization of the retina-induced relaxation in mice.

The retinal relaxing factor (RRF) is a continuously released factor from the retina that causes vasorelaxation, the identity and potential role in physiology of which remain largely unknown. Experiments were performed to find out whether the RRF-induced relaxation is influenced by serotonin, glutamate, L-cysteine, the cytochrome P450 pathway, the cyclooxygenase pathway, or oxidative stress. In addition, the sensitivity of retinal and non-retinal arteries towards the RRF was compared. In vitro tension measurements were performed on isolated mouse femoral or bovine retinal arteries to study the vasorelaxing effect of the RRF, induced by mouse or bovine retinas. The presence of serotonin, glutamate, or L-cysteine did not alter the RRF-induced relaxation. Increasing oxidative stress by hydroquinone and diethyldithiocarbamic acid sodium salt enhanced the RRF response. Inhibition of the cytochrome P450 or the cyclooxygenase pathway did not cause any alteration. Surprisingly, the RRF-induced relaxation was enhanced by the presence of flufenamic acid or carbenoxolone. Furthermore, bringing retinal tissue in close contact with retinal or non-retinal arteries induced comparable relaxations. Serotonin, glutamate, L-cysteine, the cytochrome P450, and the cyclooxygenase pathway do not influence the RRF-induced relaxation and the RRF-induced relaxation seems to be resistant to oxidative stress. The mechanism responsible for the enhanced RRF-induced relaxation in the presence of flufenamic acid or carbenoxolone remains elusive and the RRF does not show more effectivity on retinal arteries.

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.