An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure.
Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In this work, an extensive cocktail mixture was developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS method were critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLM concentration, (iii) fast incubation (5min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC50 values for model inhibitors with those generated with the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC50 values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at low concentrations showed a cumulative inhibitory in vitro effect on P450 2B6.
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address: dany.spaggiari@unige.ch.
Dany
D
Spaggiari
Clinical Pharmacology and Toxicology Service, Geneva University Hospitals, Rue Gabrielle Perret-Gentil, 1211 Genève 14, Switzerland. Electronic address: youssef.daali@hcuge.ch.
Youssef
Y
Daali
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland; Swiss Centre for Applied Human Toxicology, University of Geneva, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address: serge.rudaz@unige.ch.
Serge
S
Rudaz
D004390:Chlorpyrifos / Q000633:toxicity; D065607:Cytochrome P-450 Enzyme Inhibitors / Q000633:toxicity*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004347:Drug Interactions; D005278:Fenitrothion / Q000633:toxicity; D006801:Humans; D007306:Insecticides / Q000633:toxicity*; D008743:Methyl Parathion / Q000633:toxicity; D008862:Microsomes, Liver / Q000378:metabolism; D063086:Organothiophosphates / Q000633:toxicity; D018570:Risk Assessment; D015262:Xenobiotics / Q000633:toxicity*
D065607:Cytochrome P-450 Enzyme Inhibitors; D007306:Insecticides; D063086:Organothiophosphates; D015262:Xenobiotics; D008743:Methyl Parathion; C024273:profenofos; D003577:Cytochrome P-450 Enzyme System; D004390:Chlorpyrifos; D005278:Fenitrothion
Cytochrome P450; Human liver microsomes; IC(50); Pesticides; Risk assessment; UHPLC-MS/MS