Warfarin

RNA samples of Senegalese sole juveniles were obtained from an experiment conducted for a previous publication [19 (link)]. In brief, specimens aged 57 dpf (with 80 ± 7 mg wet weight (WW) and 14.91 ± 1.79 mm of standard length) grown under standard rearing procedures were distributed into 4 experimental flat bottom plastic trays (40 specimens per tray), and further cultured under the environmental conditions previously described. Juveniles were initially fed with 125 mg kg⁻¹ DW VK1 inert diet (125VK1 diet) at 3% WW during 5 days, then: (i) fed with 125VK1 diet for 5 days (Control group); (ii) fasting during 2 days (Unfed group) and re-fed for 3 days with VK1 supplemented diet (1250 mg kg⁻¹ DW, 1250VK1 diet; Re-fed group); (iii) fed with 125VK1 diet and exposed to 25 mg L⁻¹ of warfarin (3-(α-acetonylbenzyl)-4-hydroxycoumarin sodium salt; Sigma-Aldrich, Madrid, Spain) during 2 days (warfarin group) and re-fed for 3 days with 1250VK1 diet while still exposed to warfarin (warfarin rescue group); or (iv) fed with 1250VK1 diet for 5 days (VK1 suppl group). Individual fish were sampled in triplicate from each experimental group at 2 and 5 days after each treatment initiated, euthanized with an overdose of MS-222, washed with sterile distilled water and stored in TRI-Reagent at −80 °C until RNA extraction.

Replicon experiments were conducted as previously described [72 (link)]. Briefly, Huh7 cells were detached by trypsin, washed twice in ice-cold DEPC-treated PBS and re-suspended at 1 x 10⁷ cells / mL in DEPC-treated PBS. Subsequently 400 μL of cells was mixed with 2 μg of RNA transcript, transferred to a 4 mm gap electroporation cuvette (SLS, UK) and pulsed at 260 V, 25 ms pulse length in a Bio-Rad Gene Pulser (Bio-Rad, USA). Electroporated cells were recovered into 4 mL media, seeded into replicate 6-well tissue culture vessels, and replication measured at 24 h intervals using Nano-Glo luciferase assay system (Promega, USA). For inhibitor treatment the electroporated cells were seeded into replicate 24-well plates, allowed to adhere for 24 h before the media was replaced with fresh media containing antipain, AEBSF, leupeptin, (Sigma-Aldrich, USA), warfarin, dabigatran etexilate (Merck Life Sciences, USA) or apixiban (Insight Biotechnology, UK), at the indicated concentrations. The cell viability experiments were conducted by seeding cells into 96-well plates, allowing to adhere for 24 h before addition of a serial dilution of protease inhibitors and measurement of cell viability 72 h later using the CellTiter AQueous One solution (Promega, USA), following manufacturer’s instructions.

Acetonitrile (≥ 99.9%), methanol (99.9%), fenofibrate, warfarin, porcine pancreatin (8 x USP specifications), bovine serum albumin, dimethyl sulfoxide (DMSO, ≥ 99.9%) D-α-Tocopherol polyethylene glycol succinate (TPGS), hexadecane (anhydrous, 95%), Tris-maleate, 4-bromophenol boronic acid, olive oil, Kolliphor EL (macrogolglycerol ricinoleate), Kolliphor RH40 (macrogolglycerol hydroxystearate), Tween 85, and Carbitol (diethylene glycol monoethyl ether) were purchased from Merck (Darmstadt, Germany). Felodipine was kindly donated by Lundbeck Pharma (Valby, Denmark). Captex 355 and Capmul MCM EP (Abitec, Janesville, WI, USA) were kindly donated by Barentz (Odense, Denmark). Miglyol 812 N was obtained from IOI Oleo (Wittenberge, Germany). FaSSIF/FeSSIF/FaSSGF powder were bought from Biorelevant.com (Croydon, UK). Lucifer Yellow CH dilithium salt was obtained from Biotium (Fremont, CA, USA). Lecithin 20% soy PC extract was obtained from Avanti Polar Lipids (Alabaster, AL, USA). GIT-0 lipid solution and Acceptor Sink Buffer were purchased from Pion (Billerica, MA, USA). N-dodecane (≥ 99%) was obtained from Alfa Aesar (Lancashire, UK). Ethanol (99.5%, denatured with 0.4% isopropyl alcohol) was obtained from Solveco (Rosersberg, Sweden). All water used was of grade I from a Milli-Q lab water purification system (Merck).

Ochratoxin
A (OTA), chrysin
(CHR), warfarin, iodipamide, hemin, bilirubin, human serum albumin
(HSA; product code: A1653), and rat serum albumin (RSA; product code:
A6272) were purchased from Merck (Darmstadt, Germany). Chyrsin-7-sulfate
(C7S) was synthesized as it has been previously reported.^{22 (link),27 (link)} All other reagents were analytical grade or HPLC grade.

Acetonitrile (≥99.9%), bovine serum
albumin (BSA), CARBITOL (diethylene glycol monoethyl ether), dimethyl
sulfoxide (DMSO, ≥ 99.9%), d-α-tocopherol polyethylene
glycol succinate (TPGS), hexadecane (anhydrous, 95%), Kolliphor RH40
(macrogolglycerol hydroxystearate), methanol (99.9%), olive oil, porcine
lipase Type II, Tris-maleate, Tween 85, and warfarin were purchased
from Merck (Darmstadt, Germany). Felodipine (FEL) was kindly donated
by AstraZeneca (Mölndal, Sweden). MIGLYOL 812 N was kindly
donated by IOI Oleo (Wittenberge, Germany). “FaSSIF/FeSSIF/FaSSGF”
(simulated intestinal fluid) powders were purchased from Biorelevant.com (Croydon,
UK). Lucifer yellow (LY) CH dilithium salt was obtained from Biotium
(Fremont, CA, USA). Lecithin 20% Soy PC extract and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine
rhodamine B sulfonyl) were obtained from Avanti Polar Lipids (Alabaster,
AL, USA). N-dodecane (≥99%) was obtained from
Alfa Aesar (Lancashire, UK). Ethanol (99.5%, denatured with 0.4% isopropyl
alcohol) was obtained from Solveco (Rosersberg, Sweden). All water
used was of grade I from a Milli-Q water purification system (Merck).

Fumonisin B1 (FB1), N-palmitoyl-fumonisin B1 (N-pal-FB1), 5-O-palmitoyl-fumonisin B1 (5-O-pal-FB1), and fumonisin B4 (FB4) were provided by Fumizol Ltd. (Szeged, Hungary). N-C17:0-FB1 and FB1-¹³C34 internal standards were from Fumizol Ltd. and Romer Labs (Tulln, Austria), respectively. Human serum albumin (HSA), palmitic acid, warfarin, naproxen, S-camptothecin, and tricaine methanesulfonate (MS-222) were obtained from Merck (Darmstadt, Germany). FB1, N-pal-FB1, 5-O-pal-FB1, palmitic acid, and FB4 (each 5 mM) were dissolved in dimethyl sulfoxide; these solutions were applied in spectroscopic, ultracentrifugation, and ultrafiltration studies.