Cytochrome P4501A Induction Research Articles

Effects of benzo[a]pyrene, aromatic amines, and a combination of both on CYP1A1 activities in RT-4 human bladder papilloma cells

ABSTRACTThe interaction of arylamines and polycyclic aromatic hydrocarbons (PAH) is of particular interest in the etiology of bladder cancer. The aim of this study was to (1) examine the metabolic capacity of RT-4 human bladder papilloma cells and (2) investigate the influence of aromatic amines on the induction of cytochrome P-450 1A1 (CYP1A1) activity and their effects on benzo[a]pyrene (BaP)-induced CYP1A1 activities. Cells were incubated for 24 h with different concentrations of BaP, 1- or 2-naphthylamine (NA), 2-, 3-, or 4-aminobiphenyl (ABP), and binary mixtures consisting of 1 µM BaP and different concentrations of each arylamine. Changes in CYP1A1 activities were measured at concentrations with no or only low cytotoxicity and accompanied by specific protein detection. Several phase I and II enzymes relevant to metabolism of PAH and arylamines were present in RT-4 cells. Concentration-dependent elevation in CYP1A1 activities accompanied by increasing protein levels was found after treating cells with BaP and 1- or 2-NA. The majority of synergistic effects in binary mixtures were less than additive. In contrast, concentration-dependent inhibition was observed for 2-, 3-, and 4-ABP and in both the presence and absence of BaP. Our results suggest that RT-4 cells represent a reliable model cell line to study arylamine- and PAH-induced effects in vitro and that BaP-induced CYP1A1 activities are modulated by aromatic amines. In general, the direction of the effect depends upon the aromatic amine, rather than being unidirectional for aromatic amines.

Tissue‐specific bioconcentration and biotransformation of cypermethrin and chlorpyrifos in a native fish (Jenynsia multidentata) exposed to these insecticides singly and in mixtures

The aim of the present study was to evaluate the accumulation of cypermethrin and chlorpyrifos when the fish Jenynsia multidentata was exposed to these pesticides singly and in technical and commercial mixtures. Adult female fish were exposed over 96 h to 0.04 μg/L of cypermethrin; 0.4 μg/L of chlorpyrifos; 0.04 μg/L of cypermethrin + 0.4 μg/L of chlorpyrifos in a technical mixture; and 0.04 μg/L of cypermethrin + 0.4 μg/L of chlorpyrifos in a mixture of commercial products. Fish exposed to cypermethrin accumulated this compound only in muscle, probably because of the low biotransformation capacity of this organ and the induction of cytochrome P4501A1 (CYP1A1) expression in the liver. The accumulation of chlorpyrifos occurred in fish exposed to the insecticide (intestine > liver > gills) even when these fish had higher gluthatione-S-transferase (GST) activity in gills and P-glycoprotein (P-gp) expression in the liver, compared with the control. Fish exposed to the technical mixture showed cypermethrin accumulation (liver > intestine > gills) with higher levels than those measured in fish after only cypermethrin exposure. Higher expression levels of CYP1A1 in the liver were also observed compared with the Control. Fish exposed to the commercial mixture accumulated both insecticides (cypermethrin: intestine > gills and chlorpyrifos: liver > intestine > gills > muscle). In the organs where accumulation occurred, biotransformation enzymes were inhibited. Consequently, the commercial formulation exposure provoked the highest accumulation of cypermethrin and chlorpyrifos in J. multidentata, possibly associated with the biotransformation system inhibition. Environ Toxicol Chem 2017;36:1764-1774. © 2016 SETAC.

Cytochrome P450 1A mRNA in the guppy Phalloceros caudimaculatus and response to beta-naphthoflavone and environmental samples

The cytochrome P450 1A (CYP1A) mRNA is induced by environmental contaminants such as PAHs, PCBs and dioxins. The present study cloned the CYP1A transcript from the guppy Phalloceros caudimaculatus, which represents a potential fish for toxicological studies in South America. The newly identified CYP1A encodes a protein with 521 amino acids that shared 96–70% identity with other fishes. The characterization of organ- and time-dependent induction of CYP1A using RT-qPCR was evaluated after waterborne exposure to beta-naphthoflavone (BNF; 1μM). The minimum exposure time that elicited significant CYP1A induction was 1h for liver, gill, gut, brain, anal fin and fingerlings; 2h for dorsal fin; and 4h for kidney and tail fin. CYP1A tended to reach peak induction in the first few hours (4h–8h) of experiment in most organs, although levels remained induced until the end of the experiment (96h). Validation of CYP1A use in environmental sample was performed by exposing P. caudimaculatus to elutriate made from sediment of three streams located in adjacent areas of the Patos Lagoon Estuary (RS, Brazil). CYP1A in liver, gills and anal fin was induced by elutriate made from urban (S1) and industrial (S2) sites; and not induced by a reference site located 22 Km from potential contaminant sources, suggesting that environmental contamination plays a role in this induction. The results suggest that fins could be used for CYP1A biomarker analysis and employed in non-lethal biopsy methods for environmental monitoring. The responsiveness of the newly identified CYP1A to BNF and elutriate indicates that the guppy P. caudimaculatus could be used for environmental toxicology investigations in South American environments.

Prolactin alters blood pressure by modulating the activity of endothelial nitric oxide synthase

Increased levels of a cleaved form of prolactin (molecular weight 16 kDa) have been associated with preeclampsia. To study the effects of prolactin on blood pressure (BP), we generated male mice with a single-copy transgene (Tg; inserted into the hypoxanthine-guanine phosphoribosyltransferase locus) that enables inducible hepatic production of prolactin and its cleavage product. The Tg is driven by the indole-3-carbinol (I3C)-inducible rat cytochrome P450 1A1 promoter. When the Tg mice were fed normal chow (NC), plasma prolactin concentrations were comparable to those in female WT mice in the last third of pregnancy, and BP was lower than in WT mice (∼95 mm Hg vs. ∼105 mm Hg). When the Tg mice were fed chow containing IC3, plasma prolactin concentrations increased threefold, BP increased to ∼130 mm Hg, and cardiac function became markedly impaired. IC3 chow did not affect the WT mice. Urinary excretion of nitrite/nitrate and the amount of Ser1177-phosphorylated endothelial nitric oxide (NO) synthase (eNOS) were significantly greater in the Tg mice fed NC than in WT mice, as they are during pregnancy. However, when I3C was fed, these indicators of NO production became significantly less in the Tg mice than in WT mice. The effects of increased plasma prolactin were abolished by a genetic absence of eNOS. Thus, a threefold increase in plasma prolactin is sufficient to increase BP significantly and to markedly impair cardiac function, with effects mediated by NO produced by eNOS. We suggest that pregnant women with abnormally high prolactin levels may need special attention.

Clinical Pharmacokinetics and Pharmacodynamics of Cabozantinib.

Cabozantinib inhibits receptor tyrosine kinases involved in tumor angiogenesis and metastasis. The capsule formulation (Cometriq®) is approved for the treatment of progressive metastatic medullary thyroid cancer at a 140-mg free base equivalent dose. The tablet formulation (Cabometyx™, 60-mg free base equivalent dose) is approved for the treatment of renal cell carcinoma following anti-angiogenic therapy. Cabozantinib displays a long terminal plasma half-life (~120 h) and accumulates ~fivefold by day 15 following daily dosing based on area under the plasma concentration-time curve (AUC). Four identified inactive metabolites constitute >65 % of total cabozantinib-related AUC following a single 140-mg free base equivalent dose. Cabozantinib AUC was increased by 63-81 % or 7-30 % in subjects with mild/moderate hepatic or renal impairment, respectively; by 34-38 % with concomitant cytochrome P450 3A4 inhibitor ketoconazole; and by 57 % following a high-fat meal. Cabozantinib AUC was decreased by 76-77 % with concomitant cytochrome P450 3A4 inducer rifampin, and was unaffected following administration of proton pump inhibitor esomeprazole. Cabozantinib is a potent in vitro inhibitor of P-glycoprotein, and multidrug and toxin extrusion transporter 1 and 2-K, and is a substrate for multidrug resistance protein 2. No clinically significant covariates affecting cabozantinib pharmacokinetics were identified in a population pharmacokinetic analysis. Patients with medullary thyroid cancer with low model-predicted apparent clearance were more likely to dose hold/reduce cabozantinib early, and had a lower average dose through day 85. However, longitudinal tumor modeling suggests that cabozantinib dose reductions from 140 to 60 mg/day did not markedly reduce tumor growth inhibition in medullary thyroid cancer patients.

Hemopericardium with Tamponade Following Rivaroxaban Administration and Its Attenuation by Cyp3A4 Inhibitors

Novel oral anticoagulants including the factor Xa inhibitor rivaroxaban are important alternatives to warfarin for the prevention of thromboembolic stroke in patients with nonvalvular atrial fibrillation. The pharmacology and metabolism of these agents differ from those of the vitamin K antagonists used over the decades preceding their introduction. We present a case of spontaneous hemopericardium and cardiac tamponade following administration of rivaroxaban. A review of the patient's medications revealed a total of seven agents known to be metabolized through cytochrome P450 3A4 (CYP3A4), the major pathway for rivaroxaban metabolism. While most physicians are familiar with recommendations to monitor renal function in patients prescribed rivaroxaban, we suspect that many fail to evaluate possible interactions with other agents having CYP3A4 inhibitory or inducer activity.

Proton-pump inhibitor omeprazole attenuates hyperoxia induced lung injury.

BackgroundThe administration of supplemental oxygen to treat ventilatory insufficiency may lead to the formation of reactive oxygen species and subsequent tissue damage. Cytochrome P4501A1 (CYP1A1) can modulate hyperoxic lung injury by a currently unknown mechanism. Our objective was to evaluate the effect of administration of omeprazole on the induction of CYP1A1 and its influence on hyperoxic lung injury in an established preterm rabbit model.MethodsOmeprazole was administered either (1) directly to the fetus, (2) to the mother or (3) after birth to the pups in different doses (2–10 or 20 mg/kg). Controls were injected with the same amount of saline. Pups were housed in normoxia (21 %) or hyperoxia (>95 %) for 5 days. Outcome parameters were induction of CYP1A1 measured by real-time polymerase chain reaction (RT-PCR) immediately after delivery, at day 3 and day 5 as well as lung function, morphometry and immunohistochemistry assessed at day 5 of life. Transcriptome analysis was used to define the targeted pathways.ResultsDaily neonatal injections demonstrated a dose-dependent increase in CYP1A1. Lung function tests showed a significant improvement in tissue damping, tissue elasticity, total lung capacity, static compliance and elastance. Morphometry revealed a more developed lung architecture with thinned septae in animals treated with the highest dose (20 mg/kg) of omeprazole. Surfactant protein B, vascular endothelial growth factor and its receptor were significantly increased on immunohistochemical stainings after omeprazole treatment.ConclusionsNeonatal administration of omeprazole induces CYP1A1 in a dose-dependent matter and combined pre- and postnatal administration attenuates hyperoxic lung injury in preterm rabbits, even with the lowest dose of omeprazole without clear CYP1A1 induction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1009-3) contains supplementary material, which is available to authorized users.

Dietary Omega-3 Polyunsaturated Fatty Acids Prevent Vascular Dysfunction and Attenuate Cytochrome P4501A1 Expression by 2,3,7,8-Tetrachlorodibenzo-P-Dioxin.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) found in fish protect against cardiovascular morbidity and mortality; however, many individuals avoid fish consumption due to concerns about pollutants. We tested the hypothesis that n-3 PUFAs would prevent vascular dysfunction induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). C57Bl/6 male mice were fed a chow or n-3 PUFA diet for 10 weeks and were exposed to vehicle or 300 ng/kg/d TCDD during the final 2 weeks on each diet. Aortic vasoconstriction mediated by arachidonic acid (AA) ± SKF525 (P450 inhibitor) or SQ29548 (thromboxane/prostanoid [TP] receptor antagonist) was assessed. RBC fatty acids and expression of n-3 and n-6 PUFA metabolites were analyzed. Cytochrome P4501A1 (CYP1A1), CYP1B1, and aryl hydrocarbon receptor (AHR) expression was measured. TCDD significantly increased AA-mediated vasoconstriction on a chow diet by increasing the contribution of P450s and TP receptor to the constriction response. In contrast, the n-3 PUFA diet prevented the TCDD-induced increase in AA vasoconstriction and normalized the contribution of P450s and TP receptor. Although TCDD increased the levels of AA vasoconstrictors on the chow diet, this increase was prevent by the n-3 PUFA diet. Additionally, the n-3 PUFA diet significantly increased the levels of n-3 PUFA-derived vasodilators and TCDD increased these levels further. Interestingly, the n-3 PUFA diet significantly attenuated CYP1A1 induction by TCDD without a significant effect on AHR expression. These data suggest that n-3 PUFAs can prevent TCDD-induced vascular dysfunction by decreasing vasoconstrictors, increasing vasodilators, and attenuating CYP1A1 induction, which has been shown previously to contribute to TCDD-induced vascular dysfunction.

Effects of inducers of cytochrome P450s on enrofloxacin N-deethylation in crucian carp Carassius auratus gibelio.

In this study with crucian carp (Carassius auratus gibelio), the effect on enrofloxacin (EF) and its metabolite ciprofloxacin (CF) and on the activity of cytochrome P450 1A (CYP1A) and cytochrome P450 3A (CYP3A) was estimated following the oral administration of rifampicin (RIF) (12mg/kg) and β-naphthoflavone (BNF) (12mg/kg), respectively. First, reversed-phase high-performance liquid chromatography (RP-HPLC) was used to detect the pharmacokinetics of EF with continual blood sampling. In RIF-treated, BNF-treated and control groups, the value of the CmaxCF/CmaxEF ratio was 4.41, 0.81 and 0.95, and the corresponding value of the AUC0-t-CF/AUC0-t-EF ratio was 3.69, 1.84 and 1.76, respectively. In the RIF-treated, BNF-treated and control groups, the MRT values of EF were 26.57, 27.45 and 30.88h, and the corresponding values for CF were 5.79, 35.18 and 38.11h, respectively. Based on these results for crucian carp, the accumulation and elimination of EF and CF in the RIF-treated group were more rapid than in BNF-treated and control groups. Second, liver microsomes were pretreated with the inducer of CYP1A for BNF and that of CYP3A for RIF, and then the enzymatic activities of CYP1A and CYP3A were measured, respectively. The activities of ethoxyresorufin-O-deethylation (EROD) and erythromycin-N-demethylation (ERND) increased significantly (P<0.05) for CYP1A and CYP3A, respectively. However, in further experiments on the formation of CF, the level of EF N-deethylation was significantly induced by RIF and inhibited by ketoconazole (KTZ) for CYP3A but had no influence for CYP1A, BNF and berberine chloride (BER). We concluded that CYP3A might be responsible for the N-deethylation of EF and because of this activity, could also serve as a toxicity biomarker in crucian carp.

INFLUENCE OF POMEGRANATE JUICE ON THE CYP3A4-MEDIATED METABOLISM AND P-GLYCOPROTEIN MEDIATED TRANSPORT OF SAQUINAVIR IN VIVO AND EX VIVO MODELS

Cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) play an important role in the first pass metabolism thereby limits the oral bioavailability of many clinically important and frequently prescribed drugs. The absolute oral bioavailability of saquinavir is very low (i. e. 4%) due to its extensive first pass metabolism by the major metabolizing isozyme CYP3A4 and it is also a substrate of P-gp. Pomegranate juice (PGJ) was known to be a modulator of CYP3A4 and P-gp. Therefore, the aim of this study was to evaluate the influence of PGJ on the pharmacokinetics (PK) of saquinavir in wistar rats and on the P-gp mediated intestinal transport of saquinavir in everted gut sacs ex vivo. Rats were treated orally with saquinavir (100 mg/kg) alone and in combination with PGJ (0.5, 1.0 and 2.0 mL/200g, body weight) for 15 consecutive days. Blood samples were collected on 1 st day in single dose pharmacokinetic study (SDS) and on 15 th day in multiple dose pharmacokinetic study (MDS). The peak plasma concentration (C max )and area under the plasma concentration-time curve (AUC 0-24 ) of saquinavir was increased with PGJ in SDS (p&lt;0.001) may be due to inhibition of CYP3A4 and P-gp. But interestingly, the C max and AUC 0-24 of saquinavir was decreased significantly with PGJ in MDS. This is may be due to induction of CYP3A4. The transport of saquinavir was increased in presence of PGJ and known P-gp inhibitors (Verapamil, Ketoconazole and Quinindine) across the rat everted gut sacs ex vivo. The present study results suggested that PGJ has both effects (inhibition, in SDS and induction, in MDS) on CYP3A4-mediated saquinavir metabolism in vivo and inhibitory effect on the P-gp mediated intestinal transport of saquinavir ex vivo. Further studies are needed to confirm this interaction at cellular level using cell lines and in humans.

Ultrafine Particles of Diesel Exhaust Induces Cytochrome P450 1A1 Mediated Oxidative Stress and DNA Damage in Cultured Blood and Lung Cells

&lt;div class="page" title="Page 1"&gt;&lt;div class="layoutArea"&gt;&lt;div class="column"&gt;&lt;p&gt;&lt;span&gt;Attempts were made to investigate the role of cytochrome P450 1A1 (CYP1A1) on similarities in the generation of reactive oxygen species (ROS) and DNA damage in IM9, a human B lymphoblastic cell line with A549, the human lung adenocarcinoma epithelial cell line on exposure of diesel exhaust particles (DEP). &lt;/span&gt;&lt;span&gt;A suspension of ultrafine particles (&amp;lt; 0.2 μM) of DEP (1mg/ml) in DMEM-F12 medium, at a concentration range of 1-100 μg/ml, was added to the cells for 6-48h. Expression studies revealed that DEP induced similar &lt;/span&gt;&lt;span&gt;increase in the expression of CYP1A1, generation of ROS and DNA damage in both the cells. Pre-incubation with 3-methylcholanthrene (MC), a CYP1A1 inducer resulted in higher magnitude of induction of CYP1A1, ROS &lt;/span&gt;&lt;span&gt;and DNA damage. This synergistic effect was lowered when α-naphthoflavone (α-NF), an inhibitor of CYP1A1 &lt;/span&gt;&lt;span&gt;catalysed reactions, was added to these cells. Though the magnitude of alterations was lower in IM9 cells when compared to A549 cells, similarities in the alterations in blood and lungs cells has further suggested that blood lymphocytes can be used as a surrogate to monitor toxicity of vehicular emissions.&lt;/span&gt;&lt;/p&gt;&lt;/div&gt;&lt;/div&gt;&lt;/div&gt;

Enhancing bio-availability of β-naphthoflavone by supramolecular complexation with 6,6′-thiobis(methylene)-β-cyclodextrin dimer

The aryl hydrocarbon receptor (AhR) is a ligand activated transcriptional regulator, which governs key biological processes including detoxification of carcinogens. β-Naphthoflavone (β-NF) is a non-toxic flavonoid, and a potent AhR agonist. Thus, β-NF can induce the representative detoxifying enzyme cytochrome P4501A1, thereby enhancing the detoxification potential. However, its low water solubility hampers the use. We found that supramolecular complexation of β-NF with the synthetic 6,6′-thiobis(methylene)-β-cyclodextrin (β-CD-S) dimer significantly enhanced β-NF’s role as an AhR agonist. The water solubility of β-NF was increased to 469 fold by effective supramolecular complexation with the β-CD-S dimer, and caused significant induction of cytochrome P4501A1. Stable formation of the supramolecular complex of β-NF with β-CD-S-dimer was verified by various analyses. In summary, supramolecular complexation of β-NF with β-CD-S dimer greatly enhanced bio-availability of β-NF as an AhR agonist. Our findings provide an easy, non-destructive, and alternative approach to enhance the bio-availability of therapeutics.

Clinical efficacy of perampanel for partial-onset and primary generalized tonic-clonic seizures.

Background and purposePerampanel, a selective noncompetitive antagonist at the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, is highly effective in a wide range of experimental models. Although initially licensed as adjunctive therapy for partial seizures with or without secondary generalization in patients aged 12 years or older, the US Food and Drug Administration has recently approved its use in the treatment of primary generalized tonic–clonic seizures (PGTCS). This paper reviews the pharmacokinetics, efficacy, and tolerability of perampanel as an antiepileptic drug.ResultsAfter oral ingestion, perampanel is rapidly absorbed (Tmax, 0.5–2.5 hours), has a bioavailability of ~100%, and is highly protein bound (~95%) in plasma. It undergoes extensive (>90%) hepatic metabolism, primarily via cytochrome P450 3A4 (CYP3A4), with a half-life of 48 hours. Carbamazepine and other antiepileptic drugs can enhance its metabolism via induction of CYP3A4. Efficacy of perampanel in focal seizures has been extensively evaluated in Phase II and randomized, placebo-controlled Phase III trials. The efficacy in PGTCS has been reported in one class I study. In the treatment of focal seizures, perampanel showed significant dose-dependent median seizure reductions: 4 mg/d, 23%; 8 mg/d, 26%–31%; 12 mg/d, 18%–35%; and placebo, 10%–21%. The 50% responder rates were 15%–26%, 29%, 33%–38%, and 34%–36% for placebo, 4 mg/d, 8 mg/d, and 12 mg/d perampanel, respectively. Freedom from seizures was recorded in 0%–1.7% of the placebo group, 1.9% of the 2 mg group, 2.6%–4.4% of the 8 mg group, and 2.6%–6.5% of the 12 mg group. For PGTCS, the median seizure reduction was 76.5% for perampanel and 38.4% for placebo. The 50% responder rate was 64.2% for perampanel and 39.5% for placebo. Seizure freedom during maintenance phase was 30.9% for perampanel and 12.3% for placebo. Adverse effects included dose-dependent increases in the frequency of dizziness, somnolence, fatigue, irritability, falls, and probably nausea.ConclusionPerampanel is effective in treating both partial-onset seizures and PGTCS.

Modeling of drug-mediated CYP3A4 induction by using human iPS cell-derived enterocyte-like cells

Many drugs have potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in small intestinal enterocytes. Therefore, a model that can accurately evaluate drug-mediated CYP3A4 induction is urgently needed. In this study, we overlaid Matrigel on the human induced pluripotent stem cells-derived enterocyte-like cells (hiPS-ELCs) to generate the mature hiPS-ELCs that could be applied to drug-mediated CYP3A4 induction test. By overlaying Matrigel in the maturation process of enterocyte-like cells, the gene expression levels of intestinal markers (VILLIN, sucrase-isomaltase, intestine-specific homeobox, caudal type homeobox 2, and intestinal fatty acid-binding protein) were enhanced suggesting that the enterocyte-like cells were maturated by Matrigel overlay. The percentage of VILLIN-positive cells in the hiPS-ELCs found to be approximately 55.6%. To examine the CYP3A4 induction potential, the hiPS-ELCs were treated with various drugs. Treatment with dexamethasone, phenobarbital, rifampicin, or 1α,25-dihydroxyvitamin D3 resulted in 5.8-fold, 13.4-fold, 9.8-fold, or 95.0-fold induction of CYP3A4 expression relative to that in the untreated controls, respectively. These results suggest that our hiPS-ELCs would be a useful model for CYP3A4 induction test.

Molecular Regulation of Cytochrome P450 1A1 Induction By Hyperoxia in Human Pulmonary Cells

Molecular Regulation of Cytochrome P450 1A1 Induction By Hyperoxia in Human Pulmonary Cells

Effect of Rifampin on the Pharmacokinetics of Apixaban, an Oral Direct Inhibitor of Factor Xa.

Apixaban is a substrate of cytochrome P450 3A4 (CYP3A4) and P-glycoprotein. The effects of rifampin, a strong inducer of CYP3A4 and P-glycoprotein, on the pharmacokinetics of oral and intravenous apixaban were evaluated in an open-label, randomized, sequential crossover study. Twenty healthy participants received single doses of apixaban 5 mg intravenously on day 1 and 10 mg orally on day 3, followed by rifampin 600 mg once daily on days 5-15. Finally, participants received single doses of apixaban 5 mg intravenously and 10 mg orally separately on days 12 and 14 in one of two randomized sequences. Apixaban, given intravenously and orally, was safe and well tolerated when administered in the presence and absence of rifampin. Apixaban absolute oral bioavailability was 49 % when administered alone and 39 % following induction by rifampin. Rifampin reduced apixaban area under the plasma concentration-time curve from time zero to infinity (AUC∞) by 39 % after intravenous administration and by 54 % after oral administration. Rifampin induction increased mean clearance by 1.6-fold for intravenous apixaban and mean apparent clearance by 2.1-fold for oral apixaban, indicating rifampin affected both pre-systemic and systemic apixaban elimination pathways. Co-administration of apixaban with rifampin reduced apixaban exposure via both decreased bioavailability and increased systemic clearance.

Use of the rainbow trout cell lines, RTgill-W1 and RTL-W1 to evaluate the toxic potential of benzotriazoles

Epithelial cell lines, RTgill-W1 and RTL-W1 from respectively gill and liver of rainbow trout, Onchorhynchus mykiss (Walbaum), were used to evaluate the toxic potential of six benzotriazoles (BTRs) and tolytriazole (TT), which is a commercial mixture of 4-methyl-1H-benzotriazole (4MBTR) and 5-methyl-1H-benzotriazole (5MBTR). The other BTRs were 1H-benzotriazole (1H-BTR), 5-chlorobenzotriazole (5CBTR), 1-hydroxybenzotriazole (1OHBTR) and 5,6-dimethyl-1H-benzotriazole monohydrate (DM). Except for DM, all BTRs were cytotoxic at concentrations above 15mg/L and transitorily elevated reactive oxygen species (ROS) levels. Neither N-acetyl cysteine (NAC) nor IM-54 inhibited cytotoxicity, suggesting that ROS were not the major cause of the cell death. Cell death was not blocked by Necrostatin nor accompanied by DNA laddering, suggesting that the cell death mechanism was neither necroptosis nor apoptosis. As judged by the comet assay, DNA strand breaks were detected with three BTRs: 4MBTR, 5MBTR and 5CBTR. In RTL-W1, the BTRs weakly induced cytochrome P4501A, suggesting that they have the potential to alter xenobiotic metabolism and activate the aryl hydrocarbon receptor. In summary, the toxic potential of BTRs appears to be limited to only high concentrations, which are higher than have been measured in the environment to date.

Generation of enterocyte-like cells from human induced pluripotent stem cells for drug absorption and metabolism studies in human small intestine.

Enterocytes play an important role in drug absorption and metabolism. However, a widely used enterocyte model, Caco-2 cell, has difficulty in evaluating both drug absorption and metabolism because the expression levels of some drug absorption and metabolism-related genes in these cells differ largely from those of human enterocytes. Therefore, we decided to generate the enterocyte-like cells from human induced pluripotent stem (iPS) cells (hiPS-ELCs), which are applicable to drug absorption and metabolism studies. The efficiency of enterocyte differentiation from human iPS cells was significantly improved by using EGF, SB431542, and Wnt3A, and extending the differentiation period. The gene expression levels of cytochrome P450 3A4 (CYP3A4) and peptide transporter 1 in the hiPS-ELCs were higher than those in Caco-2 cells. In addition, CYP3A4 expression in the hiPS-ELCs was induced by treatment with 1, 25-dihydroxyvitamin D3 or rifampicin, which are known to induce CYP3A4 expression, indicating that the hiPS-ELCs have CYP3A4 induction potency. Moreover, the transendothelial electrical resistance (TEER) value of the hiPS-ELC monolayer was approximately 240 Ω*cm2, suggesting that the hiPS-ELC monolayer could form a barrier. In conclusion, we succeeded in establishing an enterocyte model from human iPS cells which have potential to be applied for drug absorption and metabolism studies.

A Highly Sensitive Liquid Chromatography–Electrospray Ionization–Time of Flight/Mass Spectrometric Assay for the Quantitation of 4-Beta-Hydroxycholesterol and Its Application to in vivo Cytochrome P450 3a Induction by AGM-130

A liquid chromatography–electrospray ionization-time-of-flight/mass spectrometry (LC–ESI-TOF/MS) method was developed for the determination of 4-beta-hydroxycholesterol (4β-HC) in rat plasma, to evaluate cytochrome P450 3A (CYP3A) induction liability of a novel cyclin dependent kinases (CDKs) inhibitor, AGM-130. The method consisted of sample preparation without derivatization reagents for high sensitivity such as picolinyl ester, which could be labor-intensive and time consuming. The 4% bovine serum albumin in phosphate-buffered saline as a surrogate matrix was used in the calibration curve and quality control samples for the determination of 4β-HC. Stable isotope-labeled (SIL) 4β-HC (d7-4β-HC) was used as an internal standard. A quadratic regression (weighted 1/concentration), with an equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 3.01–2220 ng/mL for 4β-HC. For quality control samples at 15.0, 165, and 1820 ng/mL from the qualification experiment, the within-run accuracy ranged from 95% to 105% with precision values ≤10% for 4β-HC. This novel LC–ESI-TOF/MS method was successfully applied to evaluate the potential of CYP3A induction by 5′-hydroxy-5-nitro-indirubin oxime (AGM-130) in rat.

Measurement of human CYP1A2 induction by inhalation exposure to benzo(a)pyrene based on in vivo isotope breath method

Cytochrome P450 1A2 (CYP1A2) is an enzyme involved in the metabolic activation of certain carcinogens, and inducible by toxic substrates. To date, few studies have investigated in vivo CYP1A2 induction in humans and its relationship to polycylic aromatic hydrocarbons (PAHs) like benzo(a)pyrene (BaP). Non-smoking healthy male coke-oven workers (n = 30) were recruited as ‘exposure’ group, and non-smoking healthy office workers in the same city (n = 10) were selected as ‘control’ group, to test whether high inhalation exposure to PAHs can induce CYP1A2 activity in human livers. Significantly higher inhalation exposure of PAHs were found among the exposure group compared to the control. Inhalation BaP exposure concentration in the exposure group was more than 30 times higher than the control group (p < 0.001). However, the exposure group did not exhale significant higher levels of 13CO2/12CO2 in breath samples (p = 0.81), and no significant relationship was found between the inhaled BaP concentration and the 13CO2/12CO2 ratio (p = 0.91). A significant association was found between the 13CO2/12CO2 exhalation and dietary BaP intake level. Hepatic CYP1A2 activity/induction level was not effected by inhaled BaP but was altered by ingestion of BaP.