Croubels, Siska

Devreese, Mathias

Schelstraete, Wim

Ghent University Academic Bibliography

IMPACT OF FUSARIUM MYCOTOXINS ON IN VITRO ACTIVITY OF MAJOR HEPATIC CYTOCHROME P450 BIOTRANSFORMATION ENZYMES IN PIGSIntroductionCytochrome P450 enzymes (CYP450) are catalytic oxido-reductases capable of metabolising a wide variety of endogenous and xenobioticcompounds. A principal function of these CYP450 is to improve elimination of such substances by biotransformation to more polar and watersoluble metabolites. However, some xenobiotics can inhibit or induce CYP450 activity, and co-ingestion of these compounds with substratedrugs can lead to an altered disposition of these substrate drugs. This has been associated with a number of clinically relevant drug-drug ordrug-food interactions. Nonetheless, regarding drug-food contaminant interactions, literature reports are scarce. Mycotoxins are highlyprevalent food and feed contaminants produced by several fungal species1. Pigs are very sensitive to the toxic effects of mycotoxins, inparticular deoxynivalenol (DON) and zearalenone (ZEA). Moreover, the inhibitory impact of T-2 toxin (T-2) on the hepatic CYP3A activity in pigswas previously demonstrated2. In addition, the similarities between porcine and human CYP450 enzymes suggest that the pig can serve as asuitable animal model for drug metabolism and safety studies in humans3,4. Therefore, the aim of the study was to investigate the impact ofDON, ZEA, T-2 and fumonsin B1 (FB1) on six important drug metabolising CYP450 enzymes in a porcine in vitro model.METHODOLOGYMycotoxinsZEA, DON, FB1, T-2+Substratesmidazolam, tolbutamide, coumarin, dextromethorphan, chlorzoxazone, phenacetinLC-MS analysis of metabolites(Schelstraete et al., submitted)Experimental methods Inhibition profile analysisMycotoxin + SubstrateSelection of mycotoxins-substrate pairsActivity (pmol/min/mg protein)Substrate concentrationControl[I]-1[I]-2[I]-3- Using nonlinear regression analysis with Sigmaplot® version 13- Eight different inhibition profiles fitted for each selected mycotoxin-substrate pair- Determination of the model parameters: Km (Michaelis-Mentenconstant), Vmax (maximal biotransformation rate), Ki (inhibitoryconstant) and modification constant α and β where applicable- Selection of model based on Akaike information criterion (AIC),passing statistical tests for homogenity of residuals and significanceof the parameter estimates.- Based on Ki values, in vivo direct inhibition potential of themycotoxins was estimated, taking into account maximal guidancecontamination levels of mycotoxins and daily feed intake of pigs.RESULTS0200400600800100012001400160018000 20 40 60 80 100 120Rate (pmol/min/mg proteinS (µM)T-2-MDZT-2-MDZ-1T-2-MDZ-2T-2-MDZ-3050010001500200025000 100 200 300 400 500 600Rate (pmol/min/mg proteinS (µM)ZEA-DEXZEA-DEX-1ZEA-DEX-2ZEA-DEX-301020304050600 100 200 300 400 500 600Rate (pmol/min/mg proteinS (µM)T-2-TBT-2-TB-1T-2-TB-2T-2-TB-301020304050600 100 200 300 400 500 600Rate (pmol/min/mg proteinS (µM)ZEA-TBZEA-TB-1ZEA-TB-2ZEA-TB-3050010001500200025000 20 40 60 80 100 120Rate (pmol/min/mg proteinS (µM)ZEA-MDZZEA-MDZ-1ZEA-MDZ-2ZEA-MDZ-3Fig 1. Non-competitive inhibition of T-2 and midazolam afterpre-incubation of microsomes with T-2. Concentrations were0 µM (T-2-MDZ), 1 µM (T-2-MDZ-1), 10 µM (T-2-MDZ-2) and100 µM (T-2-MDZ-3). Km= 11.3 ± 1.32 µM; Vmax= 1,899 ±88.0 pmol/min/mg protein; Ki= 27 ± 3.97 µM.Fig 2. Mixed partial inhibition of ZEA and midazolam,Concentrations were 0 µM (ZEA-MDZ), 0.5 µM (ZEA-MDZ-1), 5 µM (ZEA-MDZ-2) and 20 µM (ZEA-MDZ-3).Km= 12.3 ± 1.1 µM; Vmax= 2,155 ± 73.2 pmol/min/mgprotein; Ki= 1.1 ± 0.22 µM; α= 3.8 ± 1.43; β= 0.3 ±6.37*10-2.Fig 3. Full competitive inhibition of T-2 and tolbutamide(TB). Concentrations were 0 µM (T-2-TB), 1 µM (T-2-TB-1), 10 µM (T-2-TB-2) and 50 µM (T-2-TB-3). Km= 805 ±150.0 µM; Vmax= 132 ± 17.9 pmol/min/mg protein; Ki=14.8 ± 1.45 µM.Fig 4. Partial competitive inhibition of ZEA and tolbutamide(TB). Concentrations were 0 µM (ZEA-TB), 0.1 µM (ZEA-TB-1), 1 µM (ZEA-TB-2) and 10 µM (ZEA-TB-3). Km=1,074 ± 137.9 µM; Vmax= 160.8± 15.98 pmol/min/mgprotein; Ki= 0.5 ± 4.27*10-2 µM; α= 9.5 ± 1.05.0501001502002503003504000 50 100 150 200 250Reactiesnelheid (pmol/mg protein/ min)S (µM)FB1-CMFB1-CM-1FB1-CM-2FB1-CM-3Fig 5. Non-competitive inhibition of ZEA anddextromethorphan (DEX). Concentrations were 0 µM(ZEA-DEX), 1 µM (ZEA-DEX-1), 10 µM (ZEA-DEX-2) and100 µM (ZEA-DEX-3). Km= 2.0 ± 0.17 µM; Vmax= 1,971±39.7 pmol/min/mg protein; Ki= 55.4 ± 5.77 µM.Fig 6. Full mixed inhibition of FB1 and coumarin (CM).Concentrations were 0 µM (FB1-CM), 0.1 µM (FB1-CM-1), 1 µM (FB1-CM-2) and 5 µM (FB1-CM-3). Km= 0.9 ±5.10*10-2 µM; Vmax= 359 ± 4.1 pmol/min/mg protein; Ki=2.3 ± 0.34 µM; α= 14 ± 4.3.CONCLUSIONS- ZEA can directly inhibit CYP3A, CYP2C and CYP2D enzymes with high potency towards CYP3A and CYP2C as reflected in their Ki values (1.1 and 0.5 µM respectively).- FB1 is a potent inhibitor of coumarin hydroxylase indicating inhibition potential towards CYP2A.- T-2 is a time dependent inhibitor of CYP3A and a competitive inhibitor for CYP2C.- DON could not inhibit any of the reactions significantly.- Based on the volume of distribution, daily feed intake, maximalguidance levels of mycotoxins and assuming complete oralbioavailability, expected in vivo mycotoxin plasma concentrations are20-400 times lower then Ki values. Therefore, direct inhibition in vivoseems unlikely. However, hepatic cellular concentrations can behigher then estimated plasma concentrations. In addition, the micro-environment can influence interactions between toxin and enzymesignificantly. Moreover, mycotoxins can possibly alter CYP450 in vivoby exerting a regulatory effect, depending on the mycotoxin.- Future trials are needed to investigate the in vivo impactACKNOWLEDGEMENTSSchelstraete W., Devreese M., Croubels S.Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, BelgiumMicrosomalCYP3A CYP2C CYP2A CYP2DCYP2E CYP1AContactWim.Schelstraete@ugent.beSiska.Croubels@ugent.bewwwwww.ugent.be/di/ftbwww.mytox.bewww.mytoxsouth.orgREFERENCES1. Lee, H. J. et al. J. Agric. Food Chem. A-R (2016)2, Goossens, J. et al. Food Chem. Toxicol. 57, 54–6 (2013)3. Skaanild, M. T. Curr. Pharm. Des. 12, 1421–7 (2006)4. Puccinelli, E. et al. Curr. Drug Metab. 12, 507–525 (2011)This research is supported by the Special Research Fund (BOF.DOC.2015.0075) from Ghent University.The assistance of Sanne Vandelanotte during the execution of theexperiments is kindly appreciated.

Impact of Fusarium mycotoxins on in vitro activity of major hepatic cytochrome P450 biotransformation enzymes in pigs

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Impact of Fusarium mycotoxins on in vitro activity of major hepatic cytochrome P450 biotransformation enzymes in pigs

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