Search Results (14)

Enzymes of the cytochrome P450 monooxygenase family 4 (CYP4) in mammals are generally involved either in endobiotic metabolism (e.g., acting on fatty acids or eicosanoids), or the modification of xenobiotics including therapeutic drugs. CYP4B1 is special, as it is an enigmatic enzyme acting at the interface between xenobiotic and endobiotic metabolism. However, a systematic analysis of CYP4B1’s substrate scope has not yet been reported. Herein, a three-step approach to identify novel substrates for three CYP4B1 orthologs (namely from rabbits, green monkeys, and mouse lemurs) is described. First, screening of a library containing 502 natural products revealed potential novel substrate groups for CYP4B1. Second, based on these results, a systematic library was defined consisting of 63 compounds representing 10 compound groups. Third, in vitro conversion of these compounds by CYP4B1 and identification of conversion products were conducted, supported by in silico docking, allowing the prediction of binding probabilities and potential oxidation sites. We report five new substrate groups (acyclic, monocyclic and bicyclic terpenoids, stilbenoids, and vanilloids), twenty-eight new substrates (inter alia capsaicin, gingerol, genistein, stilbene, myristicin, thioanisole), and two new reaction types for CYP4B1 (S-oxidation, O-demethylation). Consequently, CYP4B1 is a far more promiscuous enzyme than previously thought. Full article

Background: Cytochrome P450 2B6 (CYP2B6) is a sexually dimorphic, anti-obesity CYP enzyme responsible for the metabolism of xeno- and endobiotics, including the metabolism of polyunsaturated fatty acids (PUFAs) into 9-hydroxyoctadecadienoic acid (9-HODE) and 9-hydroxyoctadecatrienoic acid (9-HOTrE). However, humanized CYP2B6 transgenic (hCYP2B6-Tg) mice are sensitive to diet-induced hepatic steatosis despite their resistance to obesity. The purpose of this study was to determine if 9-HODE, 9-HOTrE, or other factors contribute to the sexually dimorphic steatosis observed in hCYP2B6-Tg mice. Results: Cyp2b9/10/13-null (Cyp2b-null) mice were injected with either 9-HODE or 9-HOTrE for 2 days and were then subjected to a fasting period of 20 h to induce steatosis. Serum lipids were moderately increased, especially in females, after 9-HODE (triglycerides (TGs), very low-density lipoproteins (VLDLs)) and 9-HOTrE (high-density lipoproteins (HDLs), low-density lipoproteins (LDLs), cholesterol) treatment. No change in hepatic lipids and few changes in hepatic gene expression were observed in mice treated with either oxylipin, suggesting that these oxylipins had minimal to moderate effects. Therefore, to further investigate CYP2B6’s role in steatosis, hCYP2B6-Tg and Cyp2b-null mice were subjected to a 20 h fast and compared. Both male and female hCYP2B6-Tg mice exhibited increased steatosis compared to Cyp2b-null mice. Serum cholesterol, triglycerides, HDLs, and VLDLs were increased in hCYP2B6-Tg males. Serum triglycerides and VLDLs were decreased in hCYP2B6-Tg females, suggesting the greater hepatic retention of lipids in females. Hepatic oxylipin profiles revealed eight perturbed oxylipins in female hCYP2B6-Tg mice and only one in males when compared to Cyp2b-null mice. RNA-seq also demonstrated greater effects in females in terms of the number of genes and gene ontology (GO) terms perturbed. There were only a few overlapping GO terms between sexes, and lipid metabolic processes were enriched in hCYP2B6-Tg male mice but were repressed in hCYP2B6-Tg females compared to Cyp2b-nulls. Conclusions: hCYP2B6-Tg mice are sensitive to fasting-mediated steatosis in males and females, although the responses are different. In addition, the oxylipins 9-HODE and 9-HOTrE are unlikely to be the primary cause of CYP2B6’s pro-steatotic effects. Full article

The mammalian cytochrome P450 monooxygenase CYP4B1 can bioactivate a wide range of xenobiotics, such as its defining/hallmark substrate 4-ipomeanol leading to tissue-specific toxicities. Similar to other members of the CYP4 family, CYP4B1 has the ability to hydroxylate fatty acids and fatty alcohols. Structural insights into the enigmatic role of CYP4B1 with functions in both, xenobiotic and endobiotic metabolism, as well as its unusual heme-binding characteristics are now possible by the recently solved crystal structures of native rabbit CYP4B1 and the p.E310A variant. Importantly, CYP4B1 does not play a major role in hepatic P450-catalyzed phase I drug metabolism due to its predominant extra-hepatic expression, mainly in the lung. In addition, no catalytic activity of human CYP4B1 has been observed owing to a unique substitution of an evolutionary strongly conserved proline 427 to serine. Nevertheless, association of CYP4B1 expression patterns with various cancers and potential roles in cancer development have been reported for the human enzyme. This review will summarize the current status of CYP4B1 research with a spotlight on its roles in the metabolism of endogenous and exogenous compounds, structural properties, and cancer association, as well as its potential application in suicide gene approaches for targeted cancer therapy. Full article

The human UDP-glycosyltransferase (UGTs) superfamily has a critical role in the metabolism of anticancer drugs and numerous pro/anti-cancer molecules (e.g., steroids, lipids, fatty acids, bile acids and carcinogens). Recent studies have shown wide and abundant expression of UGT genes in human cancers. However, the extent to which UGT genes acquire somatic mutations within tumors remains to be systematically investigated. In the present study, our comprehensive analysis of the somatic mutation profiles of 10,069 tumors from 33 different TCGA cancer types identified 3427 somatic mutations in UGT genes. Overall, nearly 18% (1802/10,069) of the assessed tumors had mutations in UGT genes with huge variations in mutation frequency across different cancer types, ranging from over 25% in five cancers (COAD, LUAD, LUSC, SKCM and UCSC) to less than 5% in eight cancers (LAML, MESO, PCPG, PAAD, PRAD, TGCT, THYM and UVM). All 22 UGT genes showed somatic mutations in tumors, with UGT2B4, UGT3A1 and UGT3A2 showing the largest number of mutations (289, 307 and 255 mutations, respectively). Nearly 65% (2260/3427) of the mutations were missense, frame-shift and nonsense mutations that have been predicted to code for variant UGT proteins. Furthermore, about 10% (362/3427) of the mutations occurred in non-coding regions (5′ UTR, 3′ UTR and splice sites) that may be able to alter the efficiency of translation initiation, miRNA regulation or the splicing of UGT transcripts. In conclusion, our data show widespread somatic mutations of UGT genes in human cancers that may affect the capacity of cancer cells to metabolize anticancer drugs and endobiotics that control pro/anti-cancer signaling pathways. This highlights their potential utility as biomarkers for predicting therapeutic efficacy and clinical outcomes. Full article

Opioids are widely used in cancer and non-cancer pain management. However, many transporters at the blood–brain barrier (BBB), such as P-glycoprotein (P-gp, ABCB1/MDR1), may impair their delivery to the brain, thus leading to opioid tolerance. Nonetheless, opioids may regulate P-gp expression, thus altering the transport of other compounds, namely chemotherapeutic agents, resulting in pharmacoresistance. Other kinds of painkillers (e.g., acetaminophen, dexamethasone) and adjuvant drugs used for neuropathic pain may act as P-gp substrates and modulate its expression, thus making pain management challenging. Inflammatory conditions are also believed to upregulate P-gp. The role of P-gp in drug–drug interactions is currently under investigation, since many P-gp substrates may also act as substrates for the cytochrome P450 enzymes, which metabolize a wide range of xenobiotics and endobiotics. Genetic variability of the ABCB1/MDR1 gene may be accountable for inter-individual variation in opioid-induced analgesia. P-gp also plays a role in the management of opioid-induced adverse effects, such as constipation. Peripherally acting mu-opioid receptors antagonists (PAMORAs), such as naloxegol and naldemedine, are substrates of P-gp, which prevent their penetration in the central nervous system. In our review, we explore the interactions between P-gp and opioidergic drugs, with their implications in clinical practice. Full article

The pregnane X receptor (PXR) regulates the metabolism of many xenobiotic and endobiotic substances. In consequence, PXR decreases the efficacy of many small-molecule drugs and induces drug-drug interactions. The prediction of PXR activators with theoretical approaches such as machine learning (ML) proves challenging due to the ligand promiscuity of PXR, which is related to its large and flexible binding pocket. In this work we demonstrate, by the example of random forest models and support vector machines, that classifiers generated following classical training procedures often fail to predict PXR activity for compounds that are dissimilar from those in the training set. We present a novel regularization technique that penalizes the gap between a model’s training and validation performance. On a challenging test set, this technique led to improvements in Matthew correlation coefficients (MCCs) by up to 0.21. Using these regularized ML models, we selected 31 compounds that are structurally distinct from known PXR ligands for experimental validation. Twelve of them were confirmed as active in the cellular PXR ligand-binding domain assembly assay and more hits were identified during follow-up studies. Comprehensive analysis of key features of PXR biology conducted for three representative hits confirmed their ability to activate the PXR. Full article

Caenorhabditis elegans is an important model used for many aspects of biological research. Its genome contains 76 genes coding for cytochromes P450 (P450s), and few data about the biochemical properties of those P450s have been published so far. However, an increasing number of articles have appeared on their involvement in the metabolism of xenobiotics and endobiotics such as fatty acid derivatives and steroids. Moreover, the implication of some P450s in various biological functions of C. elegans, such as survival, dauer formation, life span, fat content, or lipid metabolism, without mention of the precise reaction catalyzed by those P450s, has been reported in several articles. This review presents the state of our knowledge about C. elegans P450s. Full article

Hepatic cytochrome P450 CYP2E1 is an enzyme engaged in the metabolic biotransformation of various xenobiotics and endobiotics, resulting in both detoxification and/or metabolic activation of its substrates to more therapeutic or toxic products. Elevated hepatic CYP2E1 content is implicated in various metabolic diseases including alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH), diabetes and obesity. While hepatic CYP2E1 elevation is considered essential to the pathogenesis of these liver diseases, our findings in two mouse models of E3 ubiquitin ligase genetic ablation fed a regular lab chow diet, argue that it is not sufficient for triggering NAFLD/NASH. Thus, albeit comparable hepatic CYP2E1 elevation and functional stabilization in these two models upon E3 ubiquitin ligase genetic ablation and consequent disruption of its ubiquitin-dependent proteasomal degradation, NAFLD/NASH was only observed in the mouse livers that exhibited concurrent SREBP1c-transcriptional upregulation of hepatic lipogenesis. These findings reinforce the critical complicity of an associated prolipogenic scenario induced by either an inherently upregulated hepatic lipogenesis or a high fat/high carbohydrate diet in CYP2E1-mediated NAFLD/NASH. Full article

The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice. Full article

ADME genes are a group of genes that are involved in drug absorption, distribution, metabolism, and excretion (ADME). The expression profiles of ADME genes within tumours is proposed to impact on cancer patient survival; however, this has not been systematically examined. In this study, our comprehensive analyses of pan-cancer datasets from the Cancer Genome Atlas (TCGA) revealed differential intratumoral expression profiles for ADME genes in 21 different cancer types. Most genes also showed high interindividual variability within cancer-specific patient cohorts. Using Kaplan-Meier plots and logrank tests, we showed that intratumoral expression levels of twenty of the thirty-two core ADME genes were associated with overall survival (OS) in these cancers. Of these genes, five showed significant association with unfavourable OS in three cancers, including SKCM (ABCC2, GSTP1), KIRC (CYP2D6, CYP2E1), PAAD (UGT2B7); sixteen showed significant associations with favourable OS in twelve cancers, including BLCA (UGT2B15), BRCA (CYP2D6), COAD (NAT1), HNSC (ABCB1), KIRC (ABCG2, CYP3A4, SLC22A2, SLC22A6), KIRP (SLC22A2), LIHC (CYP2C19, CYP2C8, CYP2C9, CYP3A5, SLC22A1), LUAD (SLC15A2), LUSC (UGT1A1), PAAD (ABCB1), SARC (ABCB1), and SKCM (ABCB1, DYPD). Overall, these data provide compelling evidence supporting ADME genes as prognostic biomarkers and potential therapeutic targets. We propose that intratumoral expression of ADME genes may impact cancer patient survival by multiple mechanisms that can include metabolizing/transporting anticancer drugs, activating anticancer drugs, and metabolizing/transporting a variety of endogenous molecules involved in metabolically fuelling cancer cells and/or controlling pro-growth signalling pathways. Full article

Pregnane X receptor (PXR, NR1I2) is a member of the ligand-activated nuclear receptor superfamily. This receptor is promiscuous in its activation profile and is responsive to a broad array of both endobiotic and xenobiotic ligands. PXR is involved in pivotal cellular detoxification processes to include the regulation of genes that encode key drug-metabolizing cytochrome-P450 enzymes, oxidative stress response, as well as enzymes that drive steroid and bile acid metabolism. While PXR clearly has important regulatory roles in the liver and gastrointestinal tract, this nuclear receptor also has biological functions in breast tissue. In this review, we highlight current knowledge of PXR’s role in mammary tumor carcinogenesis. The elevated level of PXR expression in cancerous breast tissue suggests a likely interface between aberrant cell division and xeno-protection in cancer cells. Moreover, PXR itself exerts positive effect on the cell cycle, thereby predisposing tumor cells to unchecked proliferation. Activation of PXR also plays a key role in regulating apoptosis, as well as in acquired resistance to chemotherapeutic agents. The repressive role of PXR in regulating inflammatory mediators along with the existence of genetic polymorphisms within the sequence of the PXR gene may predispose individuals to developing breast cancer. Further investigations into the role that PXR plays in driving tumorigenesis are needed. Full article

Biological organisms are constantly exposed to an immense repertoire of molecules that cover environmental or food-derived molecules and drugs, triggering a continuous flow of stimuli-dependent adaptations. The diversity of these chemicals as well as their concentrations contribute to the multiplicity of induced effects, including activation, stimulation, or inhibition of physiological processes and toxicity. Metabolism, as the foremost phenotype and manifestation of life, has proven to be immensely sensitive and highly adaptive to chemical stimuli. Therefore, studying the effect of endo- or xenobiotics over cellular metabolism delivers valuable knowledge to apprehend potential cellular activity of individual molecules and evaluate their acute or chronic benefits and toxicity. The development of modern metabolomics technologies such as mass spectrometry or nuclear magnetic resonance spectroscopy now offers unprecedented solutions for the rapid and efficient determination of metabolic profiles of cells and more complex biological systems. Combined with the availability of well-established cell culture techniques, these analytical methods appear perfectly suited to determine the biological activity and estimate the positive and negative effects of chemicals in a variety of cell types and models, even at hardly detectable concentrations. Metabolic phenotypes can be estimated from studying intracellular metabolites at homeostasis in vivo, while in vitro cell cultures provide additional access to metabolites exchanged with growth media. This article discusses analytical solutions available for metabolic phenotyping of cell culture metabolism as well as the general metabolomics workflow suitable for testing the biological activity of molecular compounds. We emphasize how metabolic profiling of cell supernatants and intracellular extracts can deliver valuable and complementary insights for evaluating the effects of xenobiotics on cellular metabolism. We note that the concepts and methods discussed primarily for xenobiotics exposure are widely applicable to drug testing in general, including endobiotics that cover active metabolites, nutrients, peptides and proteins, cytokines, hormones, vitamins, etc. Full article

Stroke represents one of the main causes of disability and death worldwide. The pathological subtypes of stroke are ischemic stroke, the most frequent, and hemorrhagic stroke. Nrf2 is a transcription factor that regulates redox homeostasis. In stress conditions, Nrf2 translocates inside the nucleus and induces the transcription of enzymes involved in counteracting oxidative stress, endobiotic and xenobiotic metabolism, regulators of inflammation, and others. Different natural compounds, including food and plant-derived components, were shown to be able to activate Nrf2, mediating an antioxidant response. Some of these compounds were tested in stroke experimental models showing several beneficial actions. In this review, we focused on the studies that evidenced the positive effects of natural bioactive compounds in stroke experimental models through the activation of Nrf2 pathway. Interestingly, different natural compounds can activate Nrf2 through multiple pathways, inducing a strong antioxidant response associated with the beneficial effects against stroke. According to several studies, the combination of different bioactive compounds can lead to a better neuroprotection. In conclusion, natural bioactive compounds may represent new therapeutic strategies against stroke. Full article

The constitutive androstane receptor (CAR) is a critical nuclear receptor in the gene regulation of xenobiotic and endobiotic metabolism. The LanthaScreenTM TR-FRET CAR coactivator assay provides a simple and reliable method to analyze the affinity of a ligand to the human CAR ligand-binding domain (LBD) with no need to use cellular models. This in silico assay thus enables the study of direct CAR ligands and the ability to distinguish them from the indirect CAR activators that affect the receptor via the cell signaling-dependent phosphorylation of CAR in cells. For the current paper we characterized the pharmacodynamic interactions of three known CAR inverse agonists/antagonists—PK11195, clotrimazole and androstenol—with the prototype agonist CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3] thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) using the TR-FRET LanthaScreenTM assay. We have confirmed that all three compounds are inverse agonists of human CAR, with IC50 0.51, 0.005, and 0.35 μM, respectively. All the compounds also antagonize the CITCO-mediated activation of CAR, but only clotrimazole was capable to completely reverse the effect of CITCO in the tested concentrations. Thus this method allows identifying not only agonists, but also antagonists and inverse agonists for human CAR as well as to investigate the nature of the pharmacodynamic interactions of CAR ligands. Full article