Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Prostaglandin E 2 inhibits NLRP3 inflammasome activation through EP4 receptor and intracellular cAMP in human macrophages

doi: 10.4049/jimmunol.1401343

Figure Lengend Snippet: (A and B) primary MDM were treated with/without LPS (100 ng/ml) for 4 h in complete medium, followed by 30 min treatment with EP2 agonist (butaprost, free acid, 0.5 μM), EP4 agonist (CAY10598, 0.1 μM), PGE2 (0.1 μM) or vehicle (EtOH), then stimulated with alum (400 μg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for WB (A) or IL-1β ELISA (B). IL-1β release data represent the mean ± SEM from 3 independent experiments from 3 healthy donors, each performed in duplicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 111.5 to 139.9 pg/ml.* P < .05 as indicated, as assessed by Kruskal-Wallis ANOVA on ranks, followed by Dunn's post hoc test (B). (C and D) primary MDM were treated with/without LPS (100 ng/ml) for 4 h in complete culture medium, then 30 min with EP2 inhibitor (PF-04418948, 0.5 μM), EP4 inhibitor (GW627368X, 2 μM), both inhibitors or vehicle (DMSO), followed by 30 min treatment with PGE2 (0.1 μM) or vehicle (EtOH), and stimulation with alum (400 μg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for WB (C) or IL-1β ELISA (D). IL-1β release data represent the mean ± SEM from 5 independent experiments from 5 healthy donors, each performed in duplicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 36.6 to 372.6 pg/ml (D). (E, F and G), MDM were transfected with EP2 siRNA (1 μM), EP4 siRNA (1 μM) or both. After 48 h cells were treated with/without LPS (100 ng/ml) for 4 h in complete culture medium, followed by 30 min treatment with PGE2 (0.1 μM) or vehicle (EtOH), and then stimulated with alum (400 μg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for RT-PCR (E), WB (F) or IL-1β ELISA (G). Gene expression was normalized to GAPDH transcripts and represented as a relative quantification (RQ) compared with vehicle-treated control siRNA transfected cells. Data represent the mean ± SEM from 3 independent experiments from 3 healthy donors, each performed in duplicate. * P < .05 as indicated, as assessed Kruskal-Wallis ANOVA on ranks, followed by Dunn's post hoc test (E). IL-1β release data represent the mean ± SEM from 3 independent experiments from 3 healthy donors, performed in duplicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 34.6 to 491.5 pg/ml (G). * P < .05 as indicated, as assessed by Kruskal-Wallis ANOVA on ranks, followed by Dunn's post hoc test (D and G). The WB are representative of 3 independent experiments from 3 healthy donors, each showing similar results (A, C and F).

Article Snippet: ON-TARGET plus SMART pool small interfering RNA (siRNA) (Dharmacon, Thermo Scientific, Lafayette, CO) against human NLRP3 (L-017367-00-0005), or sc-45469, Santa Cruz), EP2 (PTGER2) (L-005712-00-0005), EP4 (PTGER4) (L-005714-00-0005) and cPLA 2 α (PLA2G4A) (L-009886-00-005) together with ON-TARGETplus Control Non-targeting pool (D-00181970-05) were used to perform knockdown experiments in human primary MDM or THP-1 cells.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Quantitative Proteomics, Control

Journal: Toxicology letters

Article Title: The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity ✩

doi: 10.1016/j.toxlet.2016.04.016

Figure Lengend Snippet: Cytotoxicity of amiodarone (AM) and its major metabolite desethylamiodarone (DEA) in HepG2 cells. HepG2 cells were treated with 6.25–100 μM of amiodarone or desethylamiodarone for 6 or 24 h. The cell viability was measured by a CellTiter-Glo assay at the end of treatments. Data were presented as mean±SD of three independent experiments. *P < 0.05 compared with the cell viability of amiodarone at the same concentration.

Article Snippet: Desethylamiodarone hydrochloride, amiodarone-d4 hydrochloride, and desethylamiodarone-d4 hydrochloride were obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Techniques: Glo Assay, Concentration Assay

Journal: Toxicology letters

Article Title: The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity ✩

doi: 10.1016/j.toxlet.2016.04.016

Figure Lengend Snippet: HPLC-QDa mass spectrometry chromatograms for amiodarone (AM) and its metabolite desethylamiodarone (DEA) in an extract from a human microsomal incubation of 12.5 μM amiodarone for 60 min. The analytes were separated using a Waters e2695 HPLC System and monitored with an ACQUITY QDa detector in a positive-electrospray ionization mode. The chromatogram shows selected ion recording (SIR) channels for (a) amiodarone (m/z 646), its internal standard (b) amiodarone-d4 (m/z 650), (c) desethylamiodarone (m/z 618), and its internal standard (d) desethylamiodarone-d4 (m/z 622).

Article Snippet: Desethylamiodarone hydrochloride, amiodarone-d4 hydrochloride, and desethylamiodarone-d4 hydrochloride were obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Techniques: Mass Spectrometry, Incubation

Journal: Toxicology letters

Article Title: The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity ✩

doi: 10.1016/j.toxlet.2016.04.016

Figure Lengend Snippet: Desethylamiodarone formation in HepG2Cells overexpressing human CYPs. Different human CYPs overexpressed or empty vector-transduced HepG2 cells were treated with 12.5 μM of amiodarone for 6 h. At the end of treatment, cell pellets were harvested and extracted with ice-cold acetonitrile containing 100 ng/ml of deuterated internal standard desethylamiodarone-d4. Desethylamiodarone was quantified with LC–MS as described in Section 2. Data were presented as mean±SD of three independent experiments. *P < 0.05 compared with that of HepG2/empty vector.

Article Snippet: Desethylamiodarone hydrochloride, amiodarone-d4 hydrochloride, and desethylamiodarone-d4 hydrochloride were obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Techniques: Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy

Journal: Toxicology letters

Article Title: The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity ✩

doi: 10.1016/j.toxlet.2016.04.016

Figure Lengend Snippet: (A) Effects of CYP1A1 or CYP3A4 inhibition on the formation of desethylamiodarone in human microsomal incubation of amiodarone. Human microsomes were incubated with 12.5 μM of amiodarone or coincubated with α-naphthoflavone (50 μM) or ketoconazole (50 μM) in a total volume of 200 μl for 60 min. The incubations were quenched by adding 1 ml of ice-cold acetonitrile containing deuterated internal standards. The amount of amiodarone and desethylamiodarone in extractions was measured with LC–MS as described in Materials and Methods. Data are presented as mean±SD of three independent experiments. *P < 0.05 amiodarone amount compared to that of cells without CYP inhibitor addition; #P < 0.05 desethylamiodarone formation compared to that of cells without CYP inhibitor addition. (B) Effects of CYP inhibition on the formation of desethylamiodarone in HepG2 cells overexpressing CYP1A1 or CYP3A4. HepG2 cells overexpressing CYP1A1 were treated with 12.5 μM of amiodarone (AM) or co-incubated with α-naphthoflavone (10 μM) for 6 h; and HepG2 cells overexpressing CYP3A4 were treated with 12.5 μM of amiodarone or co-incubated with ketoconazole (10 μM) for 6 h. Cell pellets were harvested and extracted with ice-cold acetonitrile containing deuterated internal standards. The extractions were measured by LC–MS for the amounts of amiodarone and desethylamiodarone amounts. The bar graphs show as mean±SD of three independent experiments. *P < 0.05 versus without inhibition.

Article Snippet: Desethylamiodarone hydrochloride, amiodarone-d4 hydrochloride, and desethylamiodarone-d4 hydrochloride were obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Techniques: Inhibition, Incubation, Liquid Chromatography with Mass Spectroscopy