Xenobiotic Receptor Research Articles

Tox21-Based Comparative Analyses for the Identification of Potential Toxic Effects of Environmental Pollutants.

Chemical pollution has become a prominent environmental problem. In recent years, quantitative high-throughput screening (qHTS) assays have been developed for the fast assessment of chemicals' toxic effects. Toxicology in the 21st Century (Tox21) is a well-known and continuously developing qHTS project. Recent reports utilizing Tox21 data have mainly focused on setting up mathematical models for in vivo toxicity predictions, with less attention to intuitive qHTS data visualization. In this study, we attempted to reveal and summarize the toxic effects of environmental pollutants by analyzing and visualizing Tox21 qHTS data. Via PubMed text mining, toxicity/structure clustering, and manual classification, we detected a total of 158 chemicals of environmental concern (COECs) from the Tox21 library that we classified into 13 COEC groups based on structure and activity similarities. By visualizing these COEC groups' bioactivities, we demonstrated that COECs frequently displayed androgen and progesterone antagonistic effects, xenobiotic receptor agonistic roles, and mitochondrial toxicity. We also revealed many other potential targets of the 13 COEC groups, which were not well illustrated yet, and that current Tox21 assays may not correctly classify known teratogens. In conclusion, we provide a feasible method to intuitively understand qHTS data.

Novel fluorescent and secreted transcriptional reporters for quantifying activity of the xenobiotic sensor aryl hydrocarbon receptor (AHR)

Aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that plays a critical role in diverse biological processes, including xenobiotic metabolism, carcinogenesis, and physiological functions such as regulation of the immune system and cell differentiation. To improve studies of AHR activity, we constructed two new reporter genes: a fluorescent GFP-tagged histone 2B (XRE-H2B-eGFP) and a secreted nanoluciferase (XRE-pNL1.3[secNluc]). Here, we demonstrate how these reporters can be used to monitor AHR activity in different types of cells, including human primary trophoblasts and cell lines, following incubation with a strong AHR ligand, benzo[a]pyrene (B[a]P), or an AHR inhibitor (CH223191). Compared to vehicle control cells, a significant increase in AHR activity was observed in cells treated with 0.5 and/or 2 µM B[a]P and a significant decrease was detected in response to treatment with 3 µM CH223191. These new plasmids have great potential for use in a variety of applications, such as screening for endogenous or exogenous ligands of AHR.

Deletion of Constitutive Androstane Receptor Led to Intestinal Alterations and Increased Imidacloprid in Murine Liver.

Imidacloprid (IMI) is the most frequently detected neonicotinoid pesticide in the environment. Despite typically low toxicity in vertebrates, IMI exposure is associated with liver and gastrointestinal toxicity. The mechanism underlying IMI toxicity in mammals is unclear. Pesticide exposure frequently activates xenobiotic nuclear receptors, such as the constitutive androstane receptor (CAR), to induce detoxification phase I and phase II genes. This study examined the role of CAR in mediating IMI off-target toxicity. Female Car-/- and wild-type (WT) mice were orally administered imidacloprid (50 mg/kg, twice daily) for 21 days, following which serum, liver, and intestinal tissues were collected. Liver tissue analysis indicated mild inflammation and induction of detoxification gene Cyp2b10 in IMI-exposed WT mice. The absence of CAR increased hepatic IMI accumulation. Microbiome analysis of ileal samples revealed IMI altered microbial diversity in a genotype-specific manner, with increased α-diversity in Car-/- mice while decreased α-diversity in WT mice. We observed Car-/- mice exhibit intestinal alterations with decreased CYP-P450 expression, blunted villi height, and increased small intestine length and weight independent of IMI exposure. Our results suggest that IMI is not overtly toxic. However, the absence of xenobiotic nuclear receptor CAR allows increased accumulation of IMI in the liver and disrupts the villi structure and Cyp gene expression in the intestine.

Regulation of Nuclear Receptors PXR and CAR by Small Molecules and Signal Crosstalk: Roles in Drug Metabolism and Beyond.

Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are ligand-activated transcription factors that regulate the expression of drug metabolizing enzymes and drug transporters. Since their discoveries, they have been studied as important factors for regulating processes related to drug efficacy, drug toxicity, and drug-drug interactions. However, their vast ligand-binding profiles extend into additional spaces, such as endogenously produced chemicals, microbiome metabolites, dietary compounds, and environmental pollutants. Therefore, PXR and CAR can respond to an enormous abundance of stimuli, resulting in significant shifts in metabolic programs and physiologic homeostasis. Naturally, PXR and CAR have been implicated in various diseases related to homeostatic perturbations, such as inflammatory bowel disorders, diabetes, and certain cancers. Recent findings have injected the field with new signaling mechanisms and tools to dissect the complex PXR and CAR biology and have strengthened the potential for future PXR and CAR modulators in the clinic. Here, we describe the historical and ongoing importance of PXR and CAR in drug metabolism pathways and how this history has evolved into new mechanisms that regulate and are regulated by these xenobiotic receptors, with a specific focus on small molecule ligands. To effectively convey the impact of newly emerging research, we have arranged five diverse and representative key recent advances, four specific challenges, and four perspectives on future directions. SIGNIFICANCE STATEMENT: PXR and CAR are key transcription factors that regulate homeostatic detoxification of the liver and intestines. Diverse chemicals bind to these nuclear receptors, triggering their transcriptional tuning of the cellular metabolic response. This minireview revisits the importance of PXR and CAR in pharmaceutical drug responses and highlights recent results with implications beyond drug metabolism.

Gefitinib Inhibits Rifampicin-Induced CYP3A4 Gene Expression in Human Hepatocytes.

During multidrug combination chemotherapy, activation of the nuclear receptor and the transcription factor human pregnane xenobiotic receptor (hPXR) has been shown to play a role in the development of chemoresistance. Mechanistically, this could occur due to the cancer drug activation of hPXR and the subsequent upregulation of hPXR target genes such as the drug metabolism enzyme, cytochrome P450 3A4 (CYP3A4). In the context of hPXR-mediated drug resistance, hPXR antagonists would be useful adjuncts to PXR-activating chemotherapy. However, there are currently no clinically approved hPXR antagonists in the market. Gefitinib (GEF), a tyrosine kinase inhibitor used for the treatment of advanced non-small-cell lung cancer and effectively used in combinational chemotherapy treatments, is a promising candidate owing to its hPXR ligand-like features. We, therefore, investigated whether GEF would act as an hPXR antagonist when combined with a known hPXR agonist, rifampicin (RIF). At therapeutically relevant concentrations, GEF successfully inhibited the RIF-induced upregulation of endogenous CYP3A4 gene expression in human primary hepatocytes and human hepatocells. Additionally, GEF inhibited the RIF induction of hPXR-mediated CYP3A4 promoter activity in HepG2 human liver carcinoma cells. The computational modeling of molecular docking predicted that GEF could bind to multiple sites on hPXR including the ligand-binding pocket, allowing for potential as a direct antagonist as well as an allosteric inhibitor. Indeed, GEF bound to the ligand-binding domain of the hPXR in cell-free assays, suggesting that GEF directly interacts with the hPXR. Taken together, our results suggest that GEF, at its clinically relevant therapeutic concentration, can antagonize the hPXR agonist-induced CYP3A4 gene expression in human hepatocytes. Thus, GEF could be a potential candidate for use in combinational chemotherapies to combat hPXR agonist-induced chemoresistance. Further studies are warranted to determine whether GEF has sufficient hPXR inhibitor abilities to overcome the hPXR agonist-induced chemoresistance.

Potential Pharmacokinetic Effect of Chicken Xenobiotic Receptor Activator on Sulfadiazine: Involvement of P-glycoprotein Induction.

Studies on pharmacokinetic drug–drug interactions have highlighted the importance of P-glycoprotein (P-gp) because of its involvement in substrate drug transport. This study aimed to investigate the role of chicken xenobiotic receptor (CXR) in the regulation of P-gp and its influences on pharmacokinetics of P-gp substrate sulfadiazine. ALAS1 and CYP2C45, the prototypical target genes of CXR, were used as a positive indicator for CXR activation in this study. Results show that ABCB1 gene expression was upregulated, and transporter activity was increased when exposed to the CXR activator metyrapone. Using ectopic expression techniques and RNA interference to manipulate the cellular CXR status, we confirmed that ABCB1 gene regulation depends on CXR. In vivo experiments showed that metyrapone induced ABCB1 in the liver, kidney, duodenum, jejunum and ileum of chickens. In addition, metyrapone significantly changed the pharmacokinetic behavior of orally administered sulfadiazine, with a Cmax (8.01 vs. 9.61 μg/mL, p < 0.05) and AUC0-t (31.46 vs. 45.59 h·mg/L, p < 0.01), as well as a higher T1/2λ (2.42 vs.1.67 h, p < 0.05), Cl/F (0.62 vs. 0.43 L/h/kg, p < 0.01) and Vz/F (2.16 vs.1.03 L/kg, p < 0.01). Together, our data suggest that CXR is involved in the regulation of P-gp, and, consequently, the CXR activator can affect, at least in part, the pharmacokinetic behavior of orally administered sulfadiazine.

Computational Tools to Expedite the Identification of Potential PXR Modulators in Complex Natural Product Mixtures: A Case Study with Five Closely Related Licorice Species.

The genus Glycyrrhiza, comprising approximately 36 spp., possesses complex structural diversity and is documented to possess a wide spectrum of biological activities. Understanding and finding the mechanisms of efficacy or safety for a plant-based therapy is very challenging, yet it is crucial and necessary to understand the polypharmacology of traditional medicines. Licorice extract was shown to modulate the xenobiotic receptors, which might manifest as a potential route for natural product-induced drug interactions. However, different mechanisms could be involved in this phenomenon. Since the induced herb–drug interaction of licorice supplements via Pregnane X receptor (PXR) is understudied, we ventured out to analyze the potential modulators of PXR in complex mixtures such as whole extracts by applying computational mining tools. A total of 518 structures from five species of Glycyrrhiza: 183 (G. glabra), 180 (G. uralensis), 100 (G. inflata), 33 (G. echinata), and 22 (G. lepidota) were collected and post-processed to yield 387 unique compounds. Visual inspection of top candidates with favorable ligand–PXR interactions and the highest docking scores were identified. The in vitro testing revealed that glabridin (GG-14) is the most potent PXR activator among the tested compounds, followed by licoisoflavone A, licoisoflavanone, and glycycoumarin. A 200 ns molecular dynamics study with glabridin confirmed the stability of the glabridin-PXR complex, highlighting the importance of computational methods for rapid dereplication of potential xenobiotic modulators in a complex mixture instead of undertaking time-consuming classical biological testing of all compounds in a given botanical.

Adverse outcome pathway from activation of the AhR to breast cancer-related death.

Adverse outcome pathways (AOPs) are formalized and structured linear concepts that connect one molecular initiating event (MIE) to an adverse outcome (AO) via different key events (KE) through key event relationships (KER). They are mainly used in eco-toxicology toxicology, and regulatory health issues. AOPs must respond to specific guidelines from the Organization for Economic Co-operation and Development (OECD) to weight the evidence between each KE. Breast cancer is the deadliest cancer in women with a poor prognosis in case of metastatic breast cancer. The role of the environments in the formation of metastasis has been suggested. We hypothesized that activation of the AhR (MIE), a xenobiotic receptor, could lead to breast cancer related death (AO), through different KEs, constituting a new AOP. An artificial intelligence tool (AOP-helpfinder), which screens the available literature, was used to collect all existing scientific abstracts to build a novel AOP, using a list of key words. Four hundred and seven abstracts were found containing at least a word from our MIE list and either one word from our AO or KE list. A manual curation retained 113 pertinent articles, which were also screened using PubTator. From these analyses, an AOP was created linking the activation of the AhR to breast cancer related death through decreased apoptosis, inflammation, endothelial cell migration, angiogenesis, and invasion. These KEs promote an increased tumor growth, angiogenesis and migration which leads to breast cancer metastasis and breast cancer related death. The evidence of the proposed AOP was weighted using the tailored Bradford Hill criteria and the OECD guidelines. The confidence in our AOP was considered strong. An in vitro validation must be carried out, but our review proposes a strong relationship between AhR activation and breast cancer-related death with an innovative use of an artificial intelligence literature search.

Gestational Diabetes Sensitizes Mice to Future Metabolic Syndrome That Can Be Relieved by Activating CAR.

Diabetes and related metabolic syndrome are common metabolic disorders. Gestational diabetes mellitus (GDM) is rather prevalent in the clinic. Although most GDM resolves after therapeutic intervention and/or after delivery, the long-term health effect of GDM remains to be better understood. The constitutive androstane receptor (CAR), initially characterized as a xenobiotic receptor, was more recently proposed to be a therapeutic target for obesity and type 2 diabetes mellitus (T2DM). In this study, high-fat diet (HFD) feeding was used to induce GDM. Upon delivery, GDM mice were returned to chow diet until the metabolic parameters were normalized. Parous non-GDM control females or metabolically normalized GDM females were then subjected to HFD feeding to induce nongestational obesity and T2DM. Our results showed that GDM sensitized mice to metabolic abnormalities induced by a second hit of HFD. Treatment with the CAR agonist 1,4-bis [2-(3,5 dichloropyridyloxy)] benzene efficiently attenuated GDM-sensitized and HFD-induced obesity and T2DM, including decreased body weight, improved insulin sensitivity, inhibition of hyperglycemia and hepatic steatosis, increased oxygen consumption, and decreased adipocyte hypertrophy. In conclusion, our results have established GDM as a key risk factor for the future development of metabolic disease. We also propose that CAR is a therapeutic target for the management of metabolic disease sensitized by GDM.

Clinical Relevance of the Constitutive Androstane Receptor.

Constitutive androstane receptor (CAR) (NR1I3), a xenobiotic receptor, has long been considered a master mediator of drug disposition and detoxification. Accumulating evidence indicates that CAR also participates in various physiologic and pathophysiological pathways regulating the homeostasis of glucose, lipid, and bile acids, and contributing to cell proliferation, tissue regeneration and repair, as well as cancer development. The expression and activity of CAR can be regulated by various factors, including small molecular modulators, CAR interaction with other transcription factors, and naturally occurring genetic variants. Given that the influence of CAR has extended beyond the realm of drug metabolism and disposition and has expanded into a potential modulator of human diseases, growing efforts have centered on understanding its clinical relevance and impact on human pathophysiology. This review highlights the current information available regarding the contribution of CAR to various metabolic disorders and cancers and ponders the possible challenges that might arise from pursuing CAR as a potential therapeutic target for these diseases. SIGNIFICANCE STATEMENT: The growing importance of the constitutive androstane receptor (CAR) in glucose and lipid metabolism as well as its potential implication in cell proliferation emphasizes a need to keenly understand the biological function and clinical impact of CAR. This minireview captures the clinical relevance of CAR by highlighting its role in metabolic disorders and cancer development.

The Role of Vitamin K in CKD-MBD.

Purpose of ReviewWe describe the mechanism of action of vitamin K, and its implication in cardiovascular disease, bone fractures, and inflammation to underline its protective role, especially in chronic kidney disease (CKD).Recent FindingsVitamin K acts as a coenzyme of y-glutamyl carboxylase, transforming undercarboxylated in carboxylated vitamin K-dependent proteins. Furthermore, through the binding of the nuclear steroid and xenobiotic receptor, it activates the expression of genes that encode proteins involved in the maintenance of bone quality and bone remodeling. There are three main types of K vitamers: phylloquinone, menaquinones, and menadione.SummaryCKD patients, for several conditions typical of the disease, are characterized by lower levels of vitamin K than the general populations, with a resulting higher prevalence of bone fractures, vascular calcifications, and mortality. Therefore, the definition of vitamin K dosage is an important issue, potentially leading to reduced bone fractures and improved vascular calcifications in the general population and CKD patients.

Chinese herbal component, Praeruptorin E, enhances anti-asthma efficacy and prevents toxicity of aminophylline by targeting the NF-κB/PXR/CYP3A4 pathway

BackgroundAminophylline is widely used for the treatment of asthma, but the therapeutic dose is very close to the toxic dose, which makes this drug prone to accumulation poisoning. In the present study, we explored whether the Chinese herbal component, Praeruptorin E (PE), enhances anti-asthma efficacy and prevents the toxicity of aminophylline.MethodsFirst, an ovalbumin (OVA)-induced mouse model of asthma, immunohistochemistry, pathological staining, and bronchoalveolar lavage fluid (BALF) were used to detect the lung condition of asthmatic mice. The content of Th2 cytokines in serum was measured by enzyme-linked immunosorbent assay (ELISA), and the expression of related proteins was detected by Western blotting and immunofluorescence. Concentrations of theophylline and its metabolites in rat serum were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). siRNA transfection and chromatin immunoprecipitation (ChIP) were used to investigate the mechanism of PE.ResultsPE was found to synergize with aminophylline to reduce the infiltration of inflammatory cells, collagen deposition, and mucus hyperplasia in the lungs of asthmatic mice. It inhibited the expression of Th2 cytokines, interleukin (IL)-4, IL-5, and IL-13; promoted lung tissue repair; and reduced the toxic effect of aminophylline on the heart. Moreover, LC-MS/MS analysis showed that PE reduced the plasma concentration of the parent theophylline and its metabolite 1,3-dimethyluric acid (1,3-DMU). PE facilitated aminophylline’s suppression of nuclear factor-κB (NF-κB), and increased the expression of the xenobiotic nuclear receptor pregnane X receptor (PXR) and its primary target gene, CYP3A11 [this is the mouse homolog of cytochrome P450 3A (CYP3A)] in the asthmatic mouse liver and in the L-02 human fetal hepatocyte cell culture model. In addition, the ChIP assay revealed that PE attenuated the binding of NF-κB to the promoter region of the PXR gene and prevented the suppression of PXR gene expression by NF-κB.ConclusionsPE has a dual function in enhancing the immune regulation and anti-inflammatory effects of theophylline, as well as preventing theophylline toxicity by targeting the NF-κB/PXR/CYP3A4 axis. PE is a promising herbal medicine that will benefit asthmatics taking theophylline.

Reconstruction of evolutionary changes in fat and toxin consumption reveals associations with gene losses in mammals: A case study for the lipase inhibitor PNLIPRP1 and the xenobiotic receptor NR1I3.

The inactivation of ancestral protein-coding genes (gene loss) can be associated with phenotypic modifications. Within placental mammals, repeated losses of PNLIPRP1 (gene inhibiting fat digestion) occurred preferentially in strictly herbivorous species, whereas repeated NR1I3 losses (gene involved in detoxification) occurred preferentially in strictly carnivorous species. It was hypothesized that lower fat contents of herbivorous diets and lower toxin contents of carnivorous diets cause relaxed selection pressure on these genes, resulting in the accumulation of mutations and ultimately to convergent gene losses. However, because herbivorous and carnivorous diets differ vastly in their composition, a fine-grained analysis is required for hypothesis testing. We generated a trait matrix recording diet and semi-quantitative estimates of fat and toxin consumption for 52 placental species. By including data from 31 fossil taxa, we reconstructed the ancestral diets in major lineages (grundplan reconstruction). We found support that PNLIPRP1 loss is primarily associated with low levels of fat intake and not simply with herbivory/carnivory. In particular, PNLIPRP1 loss also occurred in carnivorous lineages feeding on a fat-poor diet, suggesting that the loss of this gene may be beneficial for occupying ecological niches characterized by fat-poor food resources. Similarly, we demonstrated that carnivorous species are indeed less exposed to diet-related toxins, suggesting that the loss of NR1I3 and related genes (NR1I2 and UGT1A6) resulted from relaxed selection pressure. This study illustrates the need of detailed phenotype studies to obtain a deeper understanding of factors underlying gene losses and to progress in understanding genomic causes of phenotypic variation in mammals.

Synthetic neuroactive steroids as new sedatives and anaesthetics: Back to the future.

Since the 1990s, there has been waning interest in researching general anaesthetics (anaesthetics). Although currently used anaesthetics are mostly safe and effective, they are not without fault. In paediatric populations and neonatal animal models, they are associated with learning impairments and neurotoxicity. In an effort to research safer anaesthetics, we have gone back to re-examine neuroactive steroids as anaesthetics. Neuroactive steroids are steroids that have direct, local effects in the central nervous system. Since the discovery of their anaesthetic effects, neuroactive steroids have been consistently used in human or veterinary clinics as preferred anaesthetic agents. Although briefly abandoned for clinical use due to unwanted vehicle side effects, there has since been renewed interest in their therapeutic value. Neuroactive steroids are safe sedative/hypnotic and anaesthetic agents across various animal species. Importantly, unlike traditional anaesthetics, they do not cause extensive neurotoxicity in the developing rodent brain. Similar to traditional anaesthetics, neuroactive steroids are modulators of synaptic and extrasynaptic γ-aminobutyric acid type A (GABAA ) receptors and their interactions at the GABAA receptor are stereo- and enantioselective. Recent work has also shown that these agents act on other ion channels, such as high- and low-voltage-activated calcium channels. Through these mechanisms of action, neuroactive steroids modulate neuronal excitability, which results in characteristic burst suppression of the electroencephalogram, and a surgical plane of anaesthesia. However, in addition to their interactions with voltage and ligand gated ions channels, neuroactive steroids interact with membrane bound metabotropic receptors and xenobiotic receptors to facilitate signaling of prosurvival, antiapoptotic pathways. These pathways play a role in their neuroprotective effects in neuronal injury and may also prevent extensive apoptosis in the developing brain during anaesthesia. The current review explores the history of neuroactive steroids as anaesthetics in humans and animal models, their diverse mechanisms of action, and their neuroprotective properties.

Xenobiotic receptors and the regulation of intestinal homeostasis: harnessing the chemical output of the intestinal microbiota.

The commensal bacteria that reside in the gastrointestinal tract exist in a symbiotic relationship with the host, driving the development of the immune system and maintaining metabolic and tissue homeostasis in the local environment. The intestinal microbiota has the capacity to generate a wide array of chemical metabolites to which the cells of the intestinal mucosa are exposed. Host cells express xenobiotic receptors, such as the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR), that can sense and respond to chemicals that are generated by nonhost pathways. In this review, we outline the physiological and immunological processes within the intestinal environment that are regulated by microbial metabolites through the activation of the AhR and the PXR, with a focus on ligands generated by the stepwise catabolism of tryptophan.

Cadmium induced Fak -mediated anoikis activation in kidney via nuclear receptors (AHR/CAR/PXR)-mediated xenobiotic detoxification pathway

Cadmium (Cd) is a toxic heavy metal of considerable toxicity, possessing a serious environmental problem that threatening food safety and human health. However, the underlying mechanisms of Cd-induced nephrotoxicity and detoxification response remain largely unclear. Cd was administered at doses of 35, 70, and 140 mg/kg diet with feed for 90 days and produced potential damage to chickens' kidneys. The results showed that Cd exposure induced renal anatomical and histopathological injuries. Cd exposure up-regulated cytochrome P450 enzymes (CYP450s), activated nuclear xenobiotic receptors (NXRs) response, including aryl hydro-carbon receptor (AHR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR) by low and moderate doses of Cd, and induced an increase in CYP isoforms expression. Cd exposure down-regulated phase II detoxification enzymes (glutathione-S-transferase (GST), glutathione peroxidase (GSH-PX) activities, and glutathione (GSH) content), and GST isoforms transcription . Furthermore, ATP-binding cassette (ABC) transporters, multidrug resistance protein (MRP1), and P-glycoprotein (P-GP) levels were elevated by low dose, but high dose inhibited the P-GP expression. Activation of detoxification enzymes lost their ability of resistance as increasing dose of Cd, afterwards brought into severe renal injury. Additionally, Cd suppressed focal adhesion kinase (Fak) and integrins protein expression as well as activated extrinsic pathway and intrinsic pathways, thereby producing anoikis. In conclusion, these results indicated that Cd induced Fak-mediated anoikis activation in the kidney via nuclear receptors (AHR/CAR/PXR)-mediated xenobiotic detoxification pathway.

Role of miR-653 and miR-29c in downregulation of CYP1A2 expression in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major contributor to the worldwide cancer burden. Recent studies on HCC have demonstrated dramatic alterations in expression of several cytochrome P450 (CYP) family members that play a crucial role in biotransformation of many drugs and other xenobiotics; however, the mechanisms responsible for their deregulation remain unclear. We investigated a potential involvement of miRNAs in downregulation of expression of CYPs observed in HCC tumors. We compared miRNA expression profiles (TaqMan Array Human MicroRNA v3.0 TLDA qPCR) between HCC human patient tumors with strong (CYP-) and weak/no (CYP+) downregulation of drug-metabolizing CYPs. The role of significantly deregulated miRNAs in modulation of expression of the CYPs and associated xenobiotic receptors was then investigated in human liver HepaRG cells transfected with relevant miRNA mimics or inhibitors. We identified five differentially expressed miRNAs in CYP- versus CYP+ tumors, namely miR-29c, miR-125b1, miR-505, miR-653 and miR-675. The two most-upregulated miRNAs found in CYP- tumor samples, miR-29c and miR-653, were found to act as efficient suppressors of CYP1A2 or AHR expression. Our results revealed a novel role of miR-653 and miR-29c in regulation of expresion of CYPs involved in crucial biotransformation processes in liver, which are often deregulated during liver cancer progression.

A Novel Candidate Gene for Predisposition of Hodgkin Lymphoma; Pregnan X Receptor (PXR)

ObjectiveHodgkin lymphoma (HL) is a lymphoproliferative malignancy of B-cell origin and one of the common tumors in adolescent and young adults. Less than one percent of HL patients have a family history and siblings of the affected individual may have an increased risk of developing the disease. HL risk is associated with immunodeficiency syndromes. Clustering of cases of HL within families may also suggest a genetic predisposition. In cases with familial clustering, recognition of genetic predisposition genes in the pathogenesis of HL may offer therapeutic benefits, and provide genetic counseling and surveillance for the family members for early diagnosis. We had previously reported CD70 signaling defect in an extended family with multiple consanguineous marriages and individuals affected with Hodgkin lymphoma and/or immune deficiency (J Clin Immunol.2020, 40(6):883-892). The aim of this study is to investigate for a cancer predisposition gene/condition in two siblings with HL, whose parents had a consanguineous marriage.Material and MethodsA 10 year-old girl with progressive HL was referred for further evaluation and treatment. She had been diagnosed with stage IIIBs HL, nodular sclerosing subtype and had received chemotherapy (ABVD) where she was reported to have partial response, followed by progressive disease. The tumor was CD30 positive. At admission, the patient had a massive enlarged mediastinum due to conglomerate lymph nodes, nodules in the lung, lymph adenomegalies in the cervical, supraclavicular, axillary regions. She was treated with ifosfamide+mesna, etoposide, carboplatinum (ICE) and brentuximab and achieved a complete metabolic response in PET-CT evaluation after six courses. She is transferred to receive stem cell transplantation. Her 24 year-old sister was also diagnosed with early stage HL during pregnancy, had a healthy delivery, was treated with chemotherapy and was in remission.In this study, we performed exome sequencing of the index case (IV-7). The elder sister who was diagnosed with HL and the parents, whose informed consents were taken. There were five children in the family; two of the girls were (IV-7 and IV-11) diagnosed with HL, a boy (IV-8) with inflammatory skin findings, a sister (IV-9) and a cousin (IV-13) with suspected lymphoma, and a healthy girl (IV-10) (Figure 1). After quality control and alignment steps, variants were annotated using ANNOVAR, dbSNP, and ExAC. Variant evaluation was performed by in-house filtering steps based on MAF<0.1%, CADD score >10, SIFT, PolyPhen, MutationTaster, FATHMM scores. We also used Pediatric Cancer Variant Pathogenicity Information Exchange (PeCanPIE) tool for identification and classification of the variants.ResultsA germline homozygous missense gene variation (c.928 G>A, p.D310N, rs144833620) was identified on the ligand binding domain of the Pregnane X Receptor Gene (PXR or NR1I2) gene. Sanger sequencing confirmed the familial segregation; IV-7 and IV-11 HL cases were homozygous and parents (III-13 and III-14) were heterozygous. The index case did not have elevated EBV-DNA PCR levels. Index case had increased CD3, CD2 and CD8 and reduced CD4 levels.(Conclusion)The PXR encodes a member of the nuclear receptor (NR) superfamily and functions in the metabolism and elimination of xenobiotics and several commonly used drugs. Heterodimerization of PXR and RXR (functional partner) lead to conformational changes upon ligand binding, functions as the transcriptional regulator of PXR target genes including metabolizing enzymes, drug transporters, inflammation and cell cycle associated genes. Previously reported Pxr-/- animal model showed that mice lacking the steroid and xenobiotic receptor develop B cell lymphoma. However, there is no case of HL published linked to variation in PXR gene. Clustering of cases of HL within families, especially in families with consanguineous marriages should suggest a genetic predisposition. The PXR seems to be a novel candidate gene for predisposition of HL.Funding: This study was funded by Scientific Research Projects Coordination Unit of Istanbul University with a project number TDK-2020-37127. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genomic Characterization of in Vitro Acquired-Resistance to Proteasome Inhibitors

Multiple myeloma (MM) is the second most common hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novels agents that have significantly improved clinical outcomes, MM patients invariably relapse. A better understanding of the drug resistance mechanisms and development of biomarkers remain of major interest to improve the treatment of patients.In order to further investigate the mechanisms involved in resistance to proteasome inhibitors (PI), we have derived and characterized PI-resistant human myeloma cells lines (HMCLs) from different molecular subgroups including XG2BR t(12;14), XG7BR t(4;14), XG19BR t(14;16), and XG1BR t(11;14). These cell lines were cultured continuously with escalating concentrations of Bortezomib (Btz) during 12 months and showed a significant resistance to Bortezomib compared to their parental cell lines (mean IC50: Btz-resistant HMCLs =5.5nM vs parental HMCLs=2.5nM, p<0.05). Of interest, we demonstrated that Btz-resistant HMCLs are also significantly more resistant to Carfilzomib (Cfz) and Ixazomib (Ixa) PIs (mean IC50: Btz-resistant HMCLs =6nM for Cfz and =70nM for Ixa vs parental HMCLs=3nM, for Cfz, p<0.05 ; and =21nM for Ixa, p<0.05. No significant cross-resistance was observed with other therapeutic agents including melphalan, dexamethasone and IMIDs indicating that the observed drug resistance mechanisms are specifically related to PIs.In order to understand the PIs resistance mechanisms acquired by MM cells, we used a combination of genomic approaches including whole genome sequencing, and comparative transcriptomic analysis.Among the 40 mutations identified in Btz-resistant HMCLs compared to the parental ones, a mutation residing in the Btz-binding pocket in the proteasome beta5-subunit (PSMB5) gene was identified. This mutation has already been found in other models of PIs-resistant MM cell lines and in relapsed MM patients, and is associated to PIs- resistance by reducing the PI binding capacity and impairing the chemotrypsin-like catalytic activity of the 20S proteasome.When we compared the gene expression profiling of Btz-resistant HMCLs with the parental ones, we identified a gene expression signature significantly enriched in nucleotide excision repair (NER) pathway with an increased expression of ERCC1, ERCC5, LIG1, POLD1 in the Btz-resistant HMCLs (FC >1.5). Since, protein ubiquitination is essential in regulation and coordination of various pathways of DNA damage recognition, signaling and repair, proteasome inhibitors affect DNA repair, overexpression of DNA repair pathways may participate in drug resistance mechanisms.Using gene expression profiling data, we also identified a significant downregulation of 8 solute carrier protein (SLC) intake transporters (SLC6A6, SLC16A1, SLC16A14, SLC16A10, SLC25A13, SLC5A6, SLCO3A1, SLCO4A1) together with a significant upregulation of xenobiotic receptors (RXRA, RXRB) in Btz-resistant HMCLs compared to parental HMCLs (FC> or < 1.5; p value <0.05). In addition, several genes involved in antioxidant response (NQO1), and in glutathione regulation (MGST1, MGST2, GSTO1) were also overexpressed in Btz-resistant HMCLs (FC>1.5 p value <0.05).Investigating the deregulated genes involved on energy metabolism that is often associated with resistance, we identified an upregulation of glycolytic enzymes directly involved in glycolytic metabolism (ALDOC, ENO3, HK1, PDK1, PDK3, PFKB3, PFKB4, PFKL, SLC2A1 (FC>1.5 p value <0.05) in the Btz-resistant HMCLs.Altogether our data underline a significant deregulation of genes involved in cell metabolism and drug clearance system that allow the PI resistant-MM cells to maintain metabolic homeostasis and survival in stringent redox conditions. This is in accordance with described mechanisms linking drug resistance and glycolytic metabolism in cancers cells. Metabolomic analyzes are currently ongoing for functional validation.Altogether, drug-resistant cell lines represent an attractive preclinical model to test molecules targeting these pathways in order to identify new therapeutic strategies to overcome PI resistance in MM. DisclosuresDe Boussac: Diag2Tec: Current Employment. Kassambara: Diag2Tec: Consultancy. Machura: Diag2Tec: Current Employment. Chemlal: Diag2Tec: Current Employment. Vincent: Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Herbaux: Abbvie: Honoraria, Research Funding; Roche: Honoraria; Janssen: Honoraria; Takeda: Honoraria, Research Funding. Bruyer: Diag2Tec: Current Employment. Moreaux: Diag2Tec: Consultancy.

Profiling Tryptophan Catabolites of Human Gut Microbiota and Acute-Phase Protein Levels in Neonatal Dried Blood Specimens.

National screening programs use dried blood specimens to detect metabolic disorders or aberrant protein functions that are not clinically evident in the neonatal period. Similarly, gut microbiota metabolites and immunological acute-phase proteins may reveal latent immune aberrations. Microbial metabolites interact with xenobiotic receptors (i.e., aryl hydrocarbon and pregnane-X) to maintain gastrointestinal tissue health, supported by acute-phase proteins, functioning as sensors of microbial immunomodulation and homeostasis. The delivery (vaginal or cesarean section) shapes the microbial colonization, which substantially modulates both the immune system’s response and mucosal homeostasis. This study profiled microbial metabolites of the kynurenine and tryptophan pathway and acute-phase proteins in 134 neonatal dried blood specimens. We newly established neonatal blood levels of microbial xenobiotic receptors ligands (i.e., indole-3-aldehyde, indole-3-butyric acid, and indole-3-acetamide) on the second day of life. Furthermore, we observed diverse microbial metabolic profiles in neonates born vaginally and via cesarean section, potentially due to microbial immunomodulatory influence. In summary, these findings suggest the supportive role of human gut microbiota in developing and maintaining immune system homeostasis.