An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure
Abstract
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 (5 min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC{sub 50} 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 HLMmore »
- Authors:
-
- School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4 (Switzerland)
- Clinical Pharmacology and Toxicology Service, Geneva University Hospitals, Rue Gabrielle Perret-Gentil, 1211 Genève 14 (Switzerland)
- Publication Date:
- OSTI Identifier:
- 22689193
- Resource Type:
- Journal Article
- Journal Name:
- Toxicology and Applied Pharmacology
- Additional Journal Information:
- Journal Volume: 302; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0041-008X
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 60 APPLIED LIFE SCIENCES; ABUNDANCE; ACTIVITY LEVELS; ACUTE EXPOSURE; CYTOCHROMES; HAZARDS; IN VITRO; INCUBATION; INHIBITION; INSECTICIDES; LIVER; MICROSOMES; PROBES; RISK ASSESSMENT; SUBSTRATES; XENOBIOTICS
Citation Formats
Spaggiari, Dany, Daali, Youssef, Rudaz, Serge, and Swiss Centre for Applied Human Toxicology, University of Geneva, Boulevard d'Yvoy 20, 1211 Geneva 4. An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure. United States: N. p., 2016.
Web. doi:10.1016/J.TAAP.2016.04.013.
Spaggiari, Dany, Daali, Youssef, Rudaz, Serge, & Swiss Centre for Applied Human Toxicology, University of Geneva, Boulevard d'Yvoy 20, 1211 Geneva 4. An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure. United States. https://doi.org/10.1016/J.TAAP.2016.04.013
Spaggiari, Dany, Daali, Youssef, Rudaz, Serge, and Swiss Centre for Applied Human Toxicology, University of Geneva, Boulevard d'Yvoy 20, 1211 Geneva 4. 2016.
"An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure". United States. https://doi.org/10.1016/J.TAAP.2016.04.013.
@article{osti_22689193,
title = {An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure},
author = {Spaggiari, Dany and Daali, Youssef and Rudaz, Serge and Swiss Centre for Applied Human Toxicology, University of Geneva, Boulevard d'Yvoy 20, 1211 Geneva 4},
abstractNote = {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 (5 min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC{sub 50} 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 IC{sub 50} 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. - Highlights: • Ten P450 isoforms activities assessed simultaneously with only one incubation. • P450 activity levels measured using the metabolic ratio approach. • IC{sub 50} values generated 10-fold faster and cheaper compared to individual assays. • P450 2B6 was the most affected by pesticides with IC{sub 50} in the nanomolar range. • Cumulative inhibition of P450 2B6 by mixtures of four low-dosed insecticides.},
doi = {10.1016/J.TAAP.2016.04.013},
url = {https://www.osti.gov/biblio/22689193},
journal = {Toxicology and Applied Pharmacology},
issn = {0041-008X},
number = ,
volume = 302,
place = {United States},
year = {Fri Jul 01 00:00:00 EDT 2016},
month = {Fri Jul 01 00:00:00 EDT 2016}
}