CYP1B1 mRNA Research Articles

Development of a strategy to identify and evaluate direct and indirect activators of constitutive androstane receptor in rats.

Constitutive androstane receptor (CAR) is a nuclear receptor that plays a key role in drug metabolism and disposition and in the development of liver tumors in rodents. CAR is activated by ligands and indirect activators, which do not bind to the receptor but activate it through cellular signaling. In this study, we sought to identify direct and indirect activators of rat CAR (rCAR). Assessment of the influence of mutations on the transcriptional activity of rCAR identified a mutant termed rCAR-3A-G354Q that displays low constitutive activity and high ligand responsiveness. Reporter assays using the mutant were performed with compounds that increased the mRNA levels of Cyp2b1, a CAR target gene, in rat primary hepatocytes. Several compounds activated rCAR-3A-G354Q and were implicated as rCAR ligands. Since indirect CAR activators are considered to display little species differences, we then determined CYP2B6 mRNA levels in human hepatocyte-like HepaRG cells after treatment with compounds that increased Cyp2b1 mRNA levels in rat hepatocytes but did not activate rCAR-3A-G354Q. The results demonstrated six compounds as possible rCAR indirect activators. Taken together, the combined measurement of Cyp2b1 mRNA levels in rat primary hepatocytes and rCAR-3A-G354Q activation in reporter assays can be useful for evaluating rCAR activation by chemicals.

Altered Serum Bile Acid Profile Associated with Chronic Allograft Dysfunction in Kidney Transplant Recipients.

BACKGROUND Chronic allograft dysfunction (CAD) is the leading cause of graft loss among kidney transplant recipients (KTRs). Bile acids (BAs) play an important role in regulating inflammatory process, which is the major contributor to the development of CAD. The aim of this study was to evaluate the association between BAs metabolic dysregulation and CAD in KTRs. MATERIAL AND METHODS Fifteen serum BA species were determined in 43 healthy controls (HCs) and 131 KTRs by UPLC-MS/MS. KTRs were grouped into stable renal function (STA) and CAD1 and CAD2 groups based on eGFR levels. Circulating CYP7A1, CYP7B1, CYP27A1, and SLCO2B1 mRNA levels were determined by RT-PCR. RESULTS Total BA concentrations were comparable among the 4 groups. However, KTRs showed significantly different BAs profiling compared to HCs. KTRs with severe CAD (CAD2) had significantly lower unconjugated BAs and secondary BAs (SBAs) compared to the other 3 groups. KTRs had significantly lower SBAs/primary BAs (PBAs) ratios than HCs, which were comparable among the 3 KTR groups. Conjugated/unconjugated BAs ratios increased significantly with the deterioration of allograft function, which was further confirmed by correlation analysis. Differential correlation network analysis revealed that perturbations in intraclass and interclass BA coregulation existed during CAD progression. Moreover, relative gene expressions of CYP7B1 and CYP27A1 were positively correlated with eGFR. CONCLUSIONS BA species profiling, but not total BA concentrations, was significantly altered in KTRs with CAD. The shifts from unconjugated BAs toward conjugated BAs, SBAs toward PBAs, and distinct pairwise BAs coregulation patterns were the main characteristics of KTRs with CAD.

Identification and Characterization of ACP-5862, the Major Circulating Active Metabolite of Acalabrutinib: Both Are Potent and Selective Covalent Bruton Tyrosine Kinase Inhibitors .

Acalabrutinib is a covalent Bruton tyrosine kinase (BTK) inhibitor approved for relapsed/refractory mantle cell lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma. A major metabolite of acalabrutinib (M27, ACP-5862) was observed in human plasma circulation. Subsequently, the metabolite was purified from an in vitro biosynthetic reaction and shown by nuclear magnetic resonance spectroscopy to be a pyrrolidine ring-opened ketone/amide. Synthesis confirmed its structure, and covalent inhibition of wild-type BTK was observed in a biochemical kinase assay. A twofold lower potency than acalabrutinib was observed but with similar high kinase selectivity. Like acalabrutinib, ACP-5862 was the most selective toward BTK relative to ibrutinib and zanubrutinib. Because of the potency, ACP-5862 covalent binding properties, and potential contribution to clinical efficacy of acalabrutinib, factors influencing acalabrutinib clearance and ACP-5862 formation and clearance were assessed. rCYP (recombinant cytochrome P450) reaction phenotyping indicated that CYP3A4 was responsible for ACP-5862 formation and metabolism. ACP-5862 formation Km (Michaelis constant) and Vmax were 2.78 μM and 4.13 pmol/pmol CYP3A/min, respectively. ACP-5862 intrinsic clearance was 23.6 μL/min per mg. Acalabrutinib weakly inhibited CYP2C8, CYP2C9, and CYP3A4, and ACP-5862 weakly inhibited CYP2C9 and CYP2C19; other cytochrome P450s, UGTs (uridine 5'-diphospho-glucuronosyltransferases), and aldehyde oxidase were not inhibited. Neither parent nor ACP-5862 strongly induced CYP1A2, CYP2B6, or CYP3A4 mRNA. Acalabrutinib and ACP-5862 were substrates of multidrug resistance protein 1 and breast cancer resistance protein but not OATP1B1 or OATP1B3. Our work indicates that ACP-5862 may contribute to clinical efficacy in acalabrutinib-treated patients and illustrates how proactive metabolite characterization allows timely assessment of drug-drug interactions and potential contributions of metabolites to pharmacological activity. SIGNIFICANCE STATEMENT: This work characterized the major metabolite of acalabrutinib, ACP-5862. Its contribution to the pharmacological activity of acalabrutinib was assessed based on covalent Bruton tyrosine kinase binding kinetics, kinase selectivity, and potency in cellular assays. The metabolic clearance and in vitro drug-drug interaction potential were also evaluated for both acalabrutinib and ACP-5862. The current data suggest that ACP-5862 may contribute to the clinical efficacy observed in acalabrutinib-treated patients and demonstrates the value of proactive metabolite identification and pharmacological characterization.

Identification and Expression Pattern of cyp26b1 Gene in Gonad of the Chinese Tongue Sole (Cynoglossus semilaevis).

Simple SummaryIn fish, it is obvious that the asynchronous development of the gonads and sexual dimorphism limit the development of aquaculture, so the research into sex-differentiation and gonadal growth is very important. Due to the sexual reversal phenomenon (genetic females becoming phenotypic males), the Chinese tongue sole (Cynoglossus semilaevis) is a great model for investigating sex-differentiation. Herein, we report one gene involved in sex-differentiation and gonadal growth of the Chinese Tongue Sole. The gene cyp26b1 (cytochrome P450 family 26 subfamily b member 1) is a metabolizing Retinoic Acid (RA) enzyme. Since it regulates RA to control sex determination and differentiation, cyp26b1 is considered a critical part of mammals’ ovary-antagonizing and testis-determining downstream passageway of Sry (sex-determining region Y) and Sox9 (sry- box transcription factor 9). In fish, the related research is reported only on the Japanese flounder (Paralichthys olivaceus) and zebrafish (Danio rerio). In the current investigation, the identification and expression pattern of the cyp26b1 gene in the Chinese tongue sole suggested that cyp26b1 might impact sex-differentiation and gonadal development.As an RA-metabolizing enzyme, cyp26b1 has a substantial impact on RA-signaling pathways. The cyp26b1 gene from the Chinese tongue sole was cloned and identified in this investigation. The cyp26b1 ORF was 1536 bp in length and encoded a 512 amino acid protein. A quantitative real-time PCR (qPCR) indicated that the cyp26b1 expression is no significant sexual dimorphism in the gonads at the 80 days post-hatching (dph) stages. After 4 months post-hatching (mph), the expression of cyp26b1 showed sexual dimorphism and lower level of expression in the ovaries than in the testes. An in situ hybridization demonstrated that cyp26b1 mRNA was primarily located in the testis. Interestingly, the cyp26b1 mRNA probe was also detected in the ovaries. These results suggested that cyp26b1 participates in the sex-differentiation and gonadal development of the Chinese tongue sole.

Adenosine N6-methylation upregulates the expression of human CYP2B6 by altering the chromatin status

N6-Methyladenosine (m6A) modification is the most prevalent RNA modification in mammals. We have recently demonstrated that inhibition of m6A modification by 3-deazaadenosine results in an increase in the expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, and CYP2C8 in human liver-derived cells. In the present study, we aimed to clarify the mechanism of m6A-mediated regulation of CYP2B6 expression. RNA immunoprecipitation using an anti-m6A antibody revealed that CYP2B6 mRNA in human liver and hepatocarcinoma-derived HepaRG cells was m6A-modified around the stop codon. In contrast to the treatment with 3-deazaadenosine, double knockdown of methyltransferase like (METTL) 3 and METTL14 (METTL3/14) resulted in a decrease in the levels of CYP2B6 mRNA in Huh-7 and HepaRG cells and a decrease in bupropion hydroxylase activity, a marker activity of CYP2B6, in HepaRG cells. The stability of CYP2B6 mRNA was not influenced by siMETTL3/14. Reporter assays using the plasmids containing the last exon or 5′-flanking region of CYP2B6 indicated that reporter activities were not influenced by knockdown of METTL3/14. The expression levels of the constitutive androstane receptor, pregnane X receptor, and retinoid X receptor, which are the nuclear receptors regulating the transcription of CYP2B6, were not influenced by siMETTL3/14. The chromatin immunoprecipitation and formaldehyde-assisted enrichment of regulatory elements assays revealed that H3K9me2, a repressive histone marker, was enriched in the vicinity of the upstream region of CYP2B6, and knockdown of METTL3/14 induced the condensation of the chromatin structure in this region. In conclusion, we demonstrated that METTL3/14 upregulated CYP2B6 expression by altering the chromatin status.

Isotretinoin and its Metabolites Alter mRNA of Multiple Enzyme and Transporter Genes In Vitro, but Downregulation of Organic Anion Transporting Polypeptide Does Not Translate to the Clinic.

Isotretinoin [13-cis-retinoic acid (13cisRA)] is widely used for the treatment of neuroblastoma and acne. It acts via regulating gene transcription through binding to retinoic acid receptors. Yet, the potential for isotretinoin to cause transcriptionally mediated drug-drug interactions (DDIs) has not been fully explored. We hypothesized that isotretinoin and its active metabolites all-trans-retinoic acid (atRA) and 4-oxo-13cisRA would alter the transcription of enzymes and transporters in the human liver via binding to nuclear receptors. The goal of this study was to define the DDI potential of isotretinoin and its metabolites resulting from transcriptional regulation of cytochrome P450 and transporter mRNAs. In human hepatocytes (n = 3), 13cisRA, atRA, and 4-oxo-13cisRA decreased OATP1B1, CYP1A2, CYP2C9, and CYP2D6 mRNA and increased CYP2B6 and CYP3A4 mRNA in a concentration-dependent manner. The EC50 values for OATP1B1 mRNA downregulation ranged from 2 to 110 nM, with maximum effect (Emax ) ranging from 0.17- to 0.54-fold. Based on the EC50 and Emax values and the known circulating concentrations of 13cisRA and its metabolites after isotretinoin dosing, a 55% decrease in OATP1B1 activity was predicted in vivo. In vivo DDI potential was evaluated clinically in participants dosed with isotretinoin for up to 32 weeks using coproporphyrin-I (CP-I) as an OATP1B1 biomarker. CP-I steady-state serum concentrations were unaltered following 2, 8, or 16 weeks of isotretinoin treatment. These data show that isotretinoin and its metabolites alter transcription of multiple enzymes and transporters in vitro, but translation of these changes to in vivo drug-drug interactions requires clinical evaluation for each enzyme. SIGNIFICANCE STATEMENT: Isotretinoin and its metabolites alter the mRNA expression of multiple cytochrome P450s (CYPs) and transporters in human hepatocytes, suggesting that isotretinoin may cause clinically significant drug-drug interactions (DDIs). Despite the observed changes in organic anion transporting polypeptide 1B1 (OATP1B1) mRNA in human hepatocytes, no clinical DDI was observed when measuring a biomarker, coproporphyrin-I. Further work is needed to determine whether these findings can be extrapolated to a lack of a DDI with CYP1A2, CYP2B6, and CYP2C9 substrates.

Further characterization of tryptophan metabolism and its dysregulation in fibroids

To determine the expression of enzymes in tryptophan (Trp) catabolism in fibroids and matched myometrium and determine the effects of race and mediator complex subunit 12 gene (MED12) mutation on their expression. Experimental laboratory study. Academic research laboratory. Women of reproductive age who underwent hysterectomy while on no hormonal medications before surgery. Fibroids and matched myometrium were obtained from patients who underwent hysterectomy from different race or ethnic groups. The expression of enzymes in the Trp catabolic pathway, tryptophan transporters, and the cytochrome P450 1B1 gene (CYP1B1) in the fibroids and matched myometrium of women from different race and ethnic groups and in tumors bearing the MED12 mutation and tumors without the mutation was determined using quantitative reverse-transcription polymerase chain reaction. The levels of serotonin, kynurenic acid (KYNA), and nicotinamide adenine dinucleotide (NAD) were determined using enzyme-linked immunosorbent assay. In fibroids, the expression of tryptophan hydroxylase 1 (TPH1), kynurenine amino transferase (KAT)2, large neutral amino acid transporter small subunit 2 (SLC7A8), and large neutral amino acid transporter small subunit 1 (SLC7A5) messenger RNA (mRNA) was high and that of kynureninase (KYNU) and tryptophanyl-tRNA ligase (WARS1) mRNA was low, with no changes in the expression of WARS2, kynurenine formamidase (AFMID), kynurenine 3-monooxygenase (KMO), KAT1, KAT3, and KAT4 compared with that in the matched myometrium (n = 81). The expression of CYP1B1 mRNA, a marker of the activation of the aryl hydrocarbon receptor, was higher in fibroids. Tumors bearing the MED12 mutation expressed higher levels of CYP1B1 and lower levels of WARS1, KAT1, KAT3, and KAT4 mRNAs compared with tumors without the MED12 mutation. Race or ethnicity affected the expression of KYNU, with tumors from African American and Hispanic patients expressing lower levels of KYNU mRNA compared with those from Caucasian patients. We also quantified the levels of serotonin, KYNA, and NAD, which are the end products of Trp catabolism. There were no significant differences in the levels of serotonin and KYNA, whereas the levels of NAD were lower in fibroids than in the paired myometrium. This reduction in the levels of NAD was independent of race or ethnicity. In addition to the expression of tryptophan 2,3-dioxygenase or indoleamine-pyrrole 2,3-dioxygenase, there was marked dysregulation in the expression of other enzymes in the Trp metabolic pathway and Trp transporters in fibroids. Both MED12 mutation status and race or ethnicity had selective effects on the expression of the components of this pathway. Additional functional studies are necessary to establish the physiologic significance of the tryptophan degradation pathway in the pathogenesis of fibroids and its potential as a target for novel therapies.

Expression of Cytochrome P450 Enzymes in Pediatric Non-Rhabdomyosarcoma Soft Tissue Sarcomas: Possible Role in Carcinogenesis and Treatment Response.

The 5-year relative survival rate estimate of treated patients with non-rhabdomyosarcoma soft tissue sarcomas (NRSTS) is ∼50% since they generally present with tumor progression, relapse, metastasis, and/or chemoresistance. The expression of cytochrome P450 (CYP) enzymes in malignancies can affect the pharmacology of drugs commonly used in chemotherapy or confer susceptibility to development of chemical carcinogenesis; in addition, their specific tumor expression can be used as a therapeutic target. Using qPCR and Western blot assays, the expression of CYP1B1, CYP2E1, CYP3A4, and CYP3A5 were analyzed in a cohort of tumor tissue paired with non-malignant adjacent tissue of patients with NRSTS. The mRNA and protein expression of CYP1B1, CYP2E1, and CYP3A4 were significantly increased in tumor tissue. We propose that the expression of these isoforms is related to carcinogenesis and chemoresistance frequently observed in these neoplasms.

MiR-27b inhibition contributes to cytotoxicity in patulin-exposed HEK293 cells

Patulin (PAT) is a mycotoxin produced by Penicillium and other fungi that contaminate fruit. PAT targets the kidney and is associated with nephrotoxicity. Micro-RNAs (miRNA) may offer new insights into PAT-induced nephrotoxicity. Cytochrome P450 family 1, subfamily B, polypeptide 1 (CYP1B1), involved in metabolism of dietary toxins is negatively regulated by miR-27b and linked with the nuclear factor kappa B (NF-κB) pathway and peroxisome proliferator activated receptor gamma (PPARɣ) in renal fibrosis. This study investigated the effects of PAT on miR-27b, CYP1B1, PPARɣ and cytotoxicity in human kidney (HEK293) cells.HEK293 cells were exposed to PAT (2.5 μM, 24h). Protein expression of CYP1B1, PPARɣ, NF-κB (p65), pNF-κB (p65) (phospho-Ser563) and cleaved PARP-1 was quantified using western blotting. QPCR evaluated mRNA levels of CYP1B1, IL-6, miR-27b, OGG1, mtDNA, TFAM and UCP2. Mitochondrial membrane potential and phosphatidylserine (PS) externalization was evaluated by flow cytometry while levels of ATP and caspase −9, −8, −3/7 activity was measured using luminometry.PAT significantly decreased miR-27b levels (p = 0.0014) and increased CYP1B1 mRNA (p = 0.0015) and protein (p = 0.0013) levels. PPARɣ protein expression was significantly increased (p = 0.0002) and associated with decreased NF-κB activation (p = 0.0273) and IL-6 mRNA levels (p = 0.0265). Finally, PAT significantly compromised mitochondrial repair mechanisms and increased apoptotic biomarkers.PAT altered miR-27b levels and PPARɣ, with associated changes to NF-κB activation, downstream IL-6 and CYP1B1 expression. These results show that PAT impairs detoxification mechanisms leading to mitochondrial damage and apoptosis. In conclusion, PAT altered the epigenetic environment and impaired detoxification processes, supporting a mechanism for nephrotoxic outcomes.

CYP2B6 allelic variants and non-genetic factors influence CYP2B6 enzyme function

Human CYP2B6 enzyme although constitutes relatively low proportion (1–4%) of hepatic cytochrome P450 content, it is the major catalyst of metabolism of several clinically important drugs (efavirenz, cyclophosphamide, bupropion, methadone). High interindividual variability in CYP2B6 function, contributing to impaired drug-response and/or adverse reactions, is partly elucidated by genetic polymorphisms, whereas non-genetic factors can significantly modify the CYP2B6 phenotype. The influence of genetic and phenoconverting non-genetic factors on CYP2B6-selective activity and CYP2B6 expression was investigated in liver tissues from Caucasian subjects (N = 119). Strong association was observed between hepatic S-mephenytoin N-demethylase activity and CYP2B6 mRNA expression (P < 0.0001). In less than one third of the tissue donors, the CYP2B6 phenotype characterized by S-mephenytoin N-demethylase activity and/or CYP2B6 expression was concordant with CYP2B6 genotype, whereas in more than 35% of the subjects, an altered CYP2B6 phenotype was attributed to phenoconverting non-genetic factors (to CYP2B6-specific inhibitors and inducers, non-specific amoxicillin + clavulanic acid treatment and chronic alcohol consumption, but not to the gender). Furthermore, CYP2B6 genotype–phenotype mismatch still existed in one third of tissue donors. In conclusion, identifying potential sources of CYP2B6 variability and considering both genetic variations and non-genetic factors is a pressing requirement for appropriate elucidation of CYP2B6 genotype–phenotype mismatch.

AHR agonistic effects of 6-PN contribute to potential beneficial effects of Hops extract

Hops (Humulus lupulus) is used as an alternative to hormone replacement therapy due to the phytoestrogen, 8-prenylnaringenin (8-PN). To examine the potential risks/benefits of hops extract and its compounds (8-PN and 6-prenylnaringenin, 6-PN), we aimed to evaluate the estrogen receptor α (ERα) and aryl hydrocarbon receptor (AHR) signaling pathways in human endometrial cancer cells. Hops extract, 8-PN and 6-PN showed estrogenic activity. Hops extract and 6-PN activated both ERα and AHR pathways. 6-PN increased the expression of the tumor suppressor gene (AHRR), and that of genes involved in the estrogen metabolism (CYP1A1, CYP1B1). Although 6-PN might activate the detoxification and genotoxic pathways of estrogen metabolism, hops extract as a whole only modulated the genotoxic pathway by an up-regulation of CYP1B1 mRNA expression. These data demonstrate the relevant role of 6-PN contained in the hops extract as potential modulator of estrogen metabolism due to its ERα and AHR agonist activity.

Decreased FABP5 and DSG1 protein expression following PAX6 knockdown of differentiated human limbal epithelial cells

PAX6 haploinsufficiency related aniridia is characterized by disorder of limbal epithelial cells (LECs) and aniridia related keratopathy. In the limbal epithelial cells of aniridia patients, deregulated retinoic acid (RA) signaling components were identified. We aimed to visualize differentiation marker and RA signaling component expression in LECs, combining a differentiation triggering growth condition with a small interfering RNA (siRNA) based aniridia cell model (PAX6 knock down).Primary LECs were isolated from corneoscleral rims of healthy donors and cultured in serum free low Ca2+ medium (KSFM) and in KSFM supplemented with 0.9 mmol/L Ca2+. In addition, LECs were treated with siRNA against PAX6. DSG1, PAX6, KRT12, KRT 3, ADH7, RDH10, ALDH1A1, ALDH3A1, STRA6, CYP1B1, RBP1, CRABP2, FABP5, PPARG, VEGFA and ELOVL7 expression was determined using qPCR and western blot.DSG1, FABP5, ADH7, ALDH1A1, RBP1, CRABP2 and PAX6 mRNA and FABP5 protein expression increased (p ≤ 0.03), PPARG, CYP1B1 mRNA expression decreased (p ≤ 0.0003) and DSG1 protein expression was only visible after Ca2+ supplementation. After PAX6 knock down and Ca2+ supplementation, ADH7 and ALDH1A1 mRNA and DSG1 and FABP5 protein expression decreased (p ≤ 0.04), compared to Ca2+ supplementation alone.Using our cell model, with Ca2+ supplementation and PAX6 knockdown with siRNA treatment against PAX6, we provide evidence that haploinsufficiency of the master regulatory gene PAX6 contributes to differentiation defect in the corneal epithelium through alterations of RA signalling. Upon PAX6 knockdown, DSG1 differentiation marker and FABP5 RA signaling component mRNA expression decreases. A similar effect becomes apparent at protein level though differentiation triggering Ca2+ supplementation in the siRNA-based aniridia cell model. Expression data from this cell model and from our siRNA aniridia cell model strongly indicate that FABP5 expression is PAX6 dependent. These new findings may lead to a better understanding of differentiation processes in LECs and are able to explain the insufficient cell function in AAK.

Prenatal exposure to the phthalate DEHP impacts reproduction-related gene expression in the pituitary

Phthalates are chemicals used in products including plastics, personal care products, and building materials, leading to widespread contact. Previous studies on prenatal exposure to Di-(2-ethylhexyl) phthalate (DEHP) in mice and humans demonstrated pubertal timing and reproductive performance could be affected in exposed offspring. However, the impacts at the pituitary, specifically regarding signaling pathways engaged and direct effects on the gonadotropins LH and FSH, are unknown. We hypothesized prenatal exposure to DEHP during a critical period of embryonic development (e15.5 to e18.5) will cause sex-specific disruptions in reproduction-related mRNA expression in offspring’s pituitary due to interference with androgen and aryl hydrocarbon receptor (AhR) signaling. We found that prenatal DEHP exposure in vivo caused a significant increase in Fshb specifically in males, while the anti-androgen flutamide caused significant increases in both Lhb and Fshb in males. AhR target gene Cyp1b1 was increased in both sexes in DEHP-exposed offspring. In embryonic pituitary cultures, the DEHP metabolite MEHP increased Cyp1a1 and Cyp1b1 mRNA in both sexes and Cyp1b1 induction was reduced by co-treatment with AhR antagonist. AhR reporter assay in GHFT1 cells confirmed MEHP can activate AhR signaling. Lhb, Fshb and Gnrhr mRNA were significantly decreased in both sexes by MEHP, but co-treatment with AhR antagonist did not restore mRNA levels in pituitary culture. In summary, our data suggest phthalates can directly affect the function of the pituitary by activating AhR signaling and altering gonadotropin expression. This indicates DEHP’s impacts on the pituitary could contribute to reproductive dysfunctions observed in exposed mice and humans.

Evaluation of Nigerian Medicinal Plants Extract on Human P-glycoprotein and Cytochrome P450 Enzyme Induction: Implications for Herb-drug Interaction.

Herbal medicine represents a significant component of disease prevention and therapy in most African countries. Herb-drug interactions (HDI) can arise from the co-administration of herbal and orthodox medicines. This study assessed the potential for HDI of V. amygdalina, O. gratissimum, M. oleifera, A. indica, and P. nitida extracts using in vitro assays. Little is known about these medicinal plants' potential for drug interaction despite their extensive use in Nigeria for several disease conditions. The medicinal plant crude extracts were evaluated for Cytochrome P450 (CYP) enzyme induction using cryopreserved human hepatocytes. Enzyme activity was determined by quantifying probe substrate metabolism and metabolite formation using liquid chromatography-mass spectrometry/mass spectrometry. The extracts were evaluated for the potential to inhibit P-glycoprotein (P-gp) activity using human embryonic kidney membrane vesicles over-expressing human P-gp. The herbal extracts in vivo drug interaction potential was predicted based on the USFDA drug interaction guidance. O. gratissimum and P. nitida methanol extracts induced CYP1A2 enzyme activity by greater than 3-fold. P. nitida methanol extracts showed over 2-fold induction of CYP1A2 mRNA expression. O. gratissimum methanol extract induced CYP2B6 mRNA expression over 2-fold. P. nitida and A. indica methanol extracts showed potent inhibition of P-gp activity (IC50: 3.8 and 5.4 μg/mL), respectively, while V. amygdalina and M. oleifera methanol extracts showed moderate P-gp inhibition (IC50: 12.1 and 37.2 μg/mL, respectively). Our studies suggested that the medicinal plants' extracts can modulate CYP enzymes and P-gp activity with the potential to cause herb-drug interaction in vivo.

Tris (2,3-Dibromopropyl) Isocyanurate (TDBP-TAZTO or TBC) Shows Different Toxicity Depending on the Degree of Differentiation of the Human Neuroblastoma (SH-SY5Y) Cell Line

Tris (2,3-dibromopropyl) isocyanurate (TDBP-TAZTO or TBC) is a heterocyclic hexabromated flame retardant. It is widely used during the production of many synthetic compounds. High concentrations of TDBP-TAZTO were found in river water, surface sediments, soil, earthworms, and carp tissues. Moreover, it has been shown that this compound can cross the blood–brain barrier and accumulate in the gut and brain of carp. The aryl hydrocarbon receptor (AhR) has been characterized as a multifunctional intracellular sensor and receptor. AhR is an activator of cytochrome P450 1A1 and 1A2, which metabolize various toxic compounds. The aim of the study was to explain how/whether TDBP-TAZTO increases the expression and/or activity of the CYP1A1 enzyme and the AhR and TUBB3 expression during SH-SY5Y cell differentiation. SH-SY5Y cells were differentiated for 7 and 14 days using retinoic acid. Cell viability, ethoxyresorufin-O-deethylase (EROD) activity, and mRNA expression of CYP1A1, AhR, and TUBB3 were assessed. Our experiment showed that, during the differentiation process, the ability of TDBP-TAZTO to induce EROD activity in SH-SY5Y cells subsequently decreased, which may have been an effect of cell differentiation into neurons. Moreover, the results suggest that TDBP-TAZTO can affect the differentiation process. Since no CYP2B6 mRNA expression was detected, the CAR receptor may not be involved in the TDBP-TAZTO mechanism of action. However, more research is needed in this field to elucidate this mechanism precisely.

The Influence of Long-Term Treatment with Asenapine on Liver Cytochrome P450 Expression and Activity in the Rat. The Involvement of Different Mechanisms.

Therapy of schizophrenia requires long-term treatment with a relevant antipsychotic drug to achieve a therapeutic effect. The aim of the present study was to investigate the influence of prolonged treatment with the atypical neuroleptic asenapine on the expression and activity of rat cytochrome P450 (CYP) in the liver. The experiment was carried out on male Wistar rats. Asenapine (0.3 mg/kg s.c.) was administered for two weeks. The levels of CYP mRNA protein and activity were determined in the liver and hormone concentrations were measured in the pituitary gland and blood serum. Asenapine significantly decreased the activity of CYP1A (caffeine 8-hydroxylation and 3-N-demethylation), CYP2B, CYP2C11 and CYP3A (testosterone hydroxylation at positions 16β; 2α and 16α; 2β and 6β, respectively). The neuroleptic did not affect the activity of CYP2A (testosterone 7α-hydroxylation), CYP2C6 (warfarin 7-hydroxylation) and CYP2E1 (chlorzoxazone 6-hydroxylation). The mRNA and protein levels of CYP1A2, CYP2B1, CYP2C11 and CYP3A1 were decreased, while those of CYP2B2 and CYP3A2 were not changed. Simultaneously, pituitary level of growth hormone-releasing hormone and serum concentrations of growth hormone and corticosterone were reduced, while that of triiodothyronine was enhanced. In conclusion, chronic treatment with asenapine down-regulates liver cytochrome P450 enzymes, which involves neuroendocrine mechanisms. Thus, chronic asenapine treatment may slow the metabolism of CYP1A, CYP2B, CYP2C11 and CYP3A substrates (steroids and drugs). Since asenapine is metabolized by CYP1A and CYP3A, the neuroleptic may inhibit its own metabolism, therefore, the plasma concentration of asenapine in patients after prolonged treatment may be higher than expected based on a single dose.

Oleuropein-Induced Acceleration of Cytochrome P450-Catalyzed Drug Metabolism: Central Role for Nuclear Receptor Peroxisome Proliferator-Activated Receptor α.

Oleuropein (OLE), the main constituent of Olea europaea, displays pleiotropic beneficial effects in health and disease, which are mainly attributed to its anti-inflammatory and cardioprotective properties. Several food supplements and herbal medicines contain OLE and are available without a prescription. This study investigated the effects of OLE on the main cytochrome P450s (P450s) catalyzing the metabolism of many prescribed drugs. Emphasis was given to the role of peroxisome proliferator-activated receptor α (PPARα), a nuclear transcription factor regulating numerous genes including P450s. 129/Sv wild-type and Ppara-null mice were treated with OLE for 6 weeks. OLE induced Cyp1a1, Cyp1a2, Cyp1b1, Cyp3a14, Cyp3a25, Cyp2c29, Cyp2c44, Cyp2d22, and Cyp2e1 mRNAs in liver of wild-type mice, whereas no similar effects were observed in Ppara-null mice, indicating that the OLE-induced effect on these P450s is mediated by PPARα. Activation of the pathways related to phosphoinositide 3-kinase/protein kinase B (AKT)/forkhead box protein O1, c-Jun N-terminal kinase, AKT/p70, and extracellular signal-regulated kinase participates in P450 induction by OLE. These data indicate that consumption of herbal medicines and food supplements containing OLE could accelerate the metabolism of drug substrates of the above-mentioned P450s, thus reducing their efficacy and the outcome of pharmacotherapy. Therefore, OLE-induced activation of PPARα could modify the effects of drugs due to their increased metabolism and clearance, which should be taken into account when consuming OLE-containing products with certain drugs, in particular those of narrow therapeutic window. SIGNIFICANCE STATEMENT: This study indicated that oleuropein, which belongs to the main constituents of the leaves and olive drupes of Olea europaea, induces the synthesis of the major cytochrome P450s (P450s) metabolizing the majority of prescribed drugs via activation of peroxisome proliferator-activated receptor α. This effect could modify the pharmacokinetic profile of co-administered drug substrates of the P450s, thus altering their therapeutic efficacy and toxicity.

Aryl hydrocarbon receptor modulates airway inflammation in mice with cockroach allergen-induced asthma by regulating Th17/Treg differentiation

To investigate whether aryl hydrocarbon receptor (AhR) modulates cockroach allergen (CRE)-induced asthma by regulating Th17/Treg differentiation. Mouse models of CRE-induced asthma established by sensitizing and challenging the mice with CRE were randomized into asthma model group, AhR agonist group treated with TCDD (10 μg/ kg), and AhR antagonist group treated with TCDD and CH223191 (10 mg/kg) (n=5), with 5 mice without CRE challenge as the control group. The expressions of AhR, Cyp1a1 and Cyp1b1 mRNA in the lung tissues of the mice were detected using RT-PCR, and pulmonary inflammation was evaluated with immumohistochemical staining. The expressions of inflammatory cytokines in the lungs were detected using ELISA, and the expression of Treg in the lung tissues and pulmonary lymph nodes was analyzed with flow cytometry. Both TCDD and CH223191 were capable of modulating pulmonary expressions of AhR and its downstream genes Cyp1a1 and Cyp1b1 in asthmatic mice (P < 0.002). TCDD treatment significantly decreased inflammatory cells and mucus production in the lungs of asthmatic mice, and BALFs from TCDD-treated mice with CRE challenge contained lowered levels of the proinflammatory factors including IL-4, IL-13 and IL-17A (P < 0.001) but increased anti-inflammatory factors including IL-10, IL-22 and TGF-β1 (P < 0.001). All these changes were significantly reversed by treatment with CH223191 to the levels comparable with those in the asthma model group (P>0.05). More importantly, TCDD treatment significantly increased the number of Tregs cells and FOXP3 expression and lowered RORγt mRNA expression in the lungs and pulmonary lymph nodes in asthmatic mice (P < 0.001); inhibition of AhR with CH223191, as compared with TCDD, significantly decreased the expression of CD4+CD25+Foxp3+Treg cells in the lungs and pulmonary lymph nodes and the expression of FOXP3 mRNA in lymphocytes and increased RORγt mRNA expression (P < 0.001) to the levels comparable with those in asthma model group (P>0.05). AhR activation modulates airway inflammation in mice with CRE-induced asthma by modulating the differentiation of Th17/Treg.

Negative Regulation of Human Hepatic Constitutive Androstane Receptor by Cholesterol Synthesis Inhibition: Role of Sterol Regulatory Element Binding Proteins.

The squalene synthase inhibitor squalestatin 1 (Squal1) is a potent and efficacious inducer of CYP2B expression in primary cultured rat hepatocytes and rat liver. To determine whether Squal1 is also an inducer of human CYP2B, the effects of Squal1 treatment were evaluated in primary cultured human hepatocytes, differentiated HepaRG cells, and humanized mouse livers. Squal1 treatment did not increase CYP2B6 mRNA levels in human hepatocytes or HepaRG cells and only slightly and inconsistently increased CYP2B6 mRNA content in humanized mouse liver. However, treatment with farnesol, which mediates Squal1's effect on rat CYP2B expression, increased CYP2B6 mRNA levels in HepaRG cells expressing the constitutive androstane receptor (CAR), but not in cells with knocked-down CAR. To determine the impact of cholesterol biosynthesis inhibition on CAR activation, the effects of pravastatin (Prava) were determined on CITCO-mediated gene expression in primary cultured human hepatocytes. Prava treatment abolished CITCO-inducible CYP2B6 expression, but had less effect on rifampicin-mediated CYP3A4 induction, and CITCO treatment did not affect Prava-inducible HMG-CoA reductase (HMGCR) expression. Treatment with inhibitors of different steps of cholesterol biosynthesis attenuated CITCO-mediated CYP2B6 induction in HepaRG cells, and Prava treatment increased HMGCR expression and inhibited CYP2B6 induction with comparable potency. Transfection of HepG2 cells with transcriptionally active sterol regulatory element binding proteins (SREBPs) reduced CAR-mediated transactivation, and inducible expression of transcriptionally active SREBP2 attenuated CITCO-inducible CYP2B6 expression in HepaRG cells. These findings suggest that Squal1 does not induce CYP2B6 in human hepatocytes because Squal1's inhibitory effect on cholesterol biosynthesis interferes with CAR activation. SIGNIFICANCE STATEMENT: The cholesterol biosynthesis inhibitor squalestatin 1 induces rat hepatic CYP2B expression indirectly by causing accumulation of an endogenous isoprenoid that activates the constitutive androstane receptor (CAR). This study demonstrates that squalestatin 1 does not similarly induce CYP2B6 expression in human hepatocytes. Rather, inhibition of cholesterol biosynthesis interferes with CAR activity, likely by activating sterol regulatory element binding proteins. These findings increase our understanding of the endogenous processes that modulate human drug-metabolizing gene expression.

Prenatal Exposure to the Endocrine Disrupting Chemical DEHP Impacts Reproduction-Related Gene Expression in the Pituitary

Abstract Phthalates are chemicals used in various common products including plastics and medical devices, leading to widespread contact. Phthalate exposure during embryonic development can cause changes in puberty timing, reduced fertility and genital abnormalities. Previous studies on prenatal exposure to Di-(2-ethylhexyl) phthalate (DEHP) in mice indicated that it disrupts pituitary-gonadal feedback and alters reproductive performance in the offspring, however, the mechanism behind this is unknown. We hypothesize that prenatal exposure to DEHP during a critical period of embryonic development (e15.5 to e18.5) will cause sex-specific disruptions in reproduction-related functions in the pituitary in offspring due to interference with androgen and aryl hydrocarbon receptor (AhR) signaling. In order to discover the direct effects of DEHP on the reproduction-related functions in the pituitary, we performed both in vivo dosing and in vitro pituitary culture experiments. First, we dosed pregnant CD-1 mice with corn oil, the antiandrogen flutamide or DEHP from gestational day 15.5 to 18.5, then collected the pituitaries of the offspring on postnatal day 0. We found that prenatal DEHP exposure caused a significant increase in Fshb specifically in males, and flutamide caused significant increases in both Lhb and Fshb in males. Besides, DEHP exposure significantly increased AhR pathway related gene Cyp1b1 in both males and females. In the in vitro experiment, we took whole pituitaries from e16.5 embryos and cultured them in media containing DEHP, MEHP and/or AhR antagonist for 72hrs. We found that the DEHP metabolite MEHP was actually the chemical that exerted the effects directly at the level of the pituitary. Similar to in vivo experiments, Cyp1a1 and Cyp1b1 mRNA level were increased in pituitaries treated with MEHP in both sexes and the induction could be reduced by co-treatment with AhR antagonist. The mRNA level of Lhb, Fshb and Gnrhr were significantly decreased in both sexes by MEHP and co-treatment with AhR antagonist did not restore mRNA levels. The induction of Cyp1a1/Cyp1b1 gene in both in vivo and in vitro experiments indicates the possible activation of AhR by DEHP/MEHP. The in vitro experiment with AhR antagonist further proved that the induction of Cyp1a1/Cyp1b1 was indeed due to AhR activation directly at the level of the pituitary. The difference between in vivo and in vitro experiments in terms of gonadotropin gene expression indicates multiple mechanisms should be involved in the regulation of gonadotropin gene expression in vivo including androgen-related pathways and possibly AhR-related pathways. In summary, our data suggest that phthalates can directly affect the function of the pituitary in terms of regulation of reproductive- related genes. This indicates that pituitary impacts of phthalates could contribute to reproductive dysfunction observed in exposed mice and humans.