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In Vitro Methods: Alternatives to Animal Testing
Published in Heather A.E. Benson, Michael S. Roberts, Vânia Rodrigues Leite-Silva, Kenneth A. Walters, Cosmetic Formulation, 2019
Dayane Pifer Luco, Vânia Rodrigues Leite-Silva, Heather A.E. Benson, Patricia Santos Lopes
At least three test concentrations that meet the acceptability criteria (appropriate cytotoxicity, number of cells, etc.) should be evaluated. A cytokinesis blocker (cytochalasin B [cytoB]) may also be included. S9 fraction (an exogenous metabolic activation system containing cytosol and microsomes) is added to activate metabolism in cells, although the exogenous metabolic activation system does not entirely mimic in vivo conditions (OECD 487, 2014). It is mandatory to include clastogenic and aneugenic positive controls, with and without metabolic activation, as well as negative controls, to demonstrate both the ability of the laboratory to identify clastogens and aneugens under the conditions of the test protocol used, and the effectiveness of the exogenous metabolic activation system. Micronuclei in interphase cells can be assessed objectively (Figure 26.3), therefore the slides can be scored relatively quickly and analysis can be automated. This makes it practical to score thousands of cells per treatment, increasing the power of the test.
General toxicology
Published in Timbrell John, Study Toxicology Through Questions, 2017
The compound requires metabolism by the S9 fraction which is the supernatant fraction of liver homogenate that remains after the homogenate is centrifuged at . This contains the microsomal enzymes (including cytochrome P450) which may be required for the metabolic activation. The compound is therefore not carcinogenic itself but requires an activation step.
The Study of Drug Metabolism Using Radiotracers
Published in Graham Lappin, Simon Temple, Radiotracers in Drug Development, 2006
A crude preparation of liver homogenate, centrifuged at 9000 g to remove particulate matter, is known as the S9 fraction. The S9 fraction contains cytosolic enzymes as well as fragments of membrane with membrane-bound enzymes such as CYP P450s (these enzymes are explained in Appendix 1). The preparation can be stored frozen (cryopreserved) over reasonable periods.
Three-dimensional liver models: state of the art and their application for hepatotoxicity evaluation
Published in Critical Reviews in Toxicology, 2020
Xihui Zhang, Tianyan Jiang, Dandan Chen, Qi Wang, Leshuai W. Zhang
While regular 2D cell culture was commonly used for traditional genotoxicity test, cell spheroids have not been attempted for genotoxicant evaluation previously. HepG2 spheroids were generated by hanging drop method and applied for genotoxicity assessment of benzo(a)pyrene (BaP). Greater micronucleus induction by BaP was found in HepG2 spheroids than 2D format after four days of treatment, indicating the sensitivity of 3D culture format for prediction on chemical induced hepatic mutation (Shah et al. 2018). S9 fraction is the liver homogenate responsible for drug metabolic activation and is required for genotoxicity testing of pharmaceuticals intended for human use (ICH guideline genotoxicity 2011). The advantage of 3D liver spheroid culture is that S9 fraction is not required due to the inherent availability of PhaseI/II enzymatic expression and activities in liver spheroids. Likely in the future, liver spheroids can partially replace S9 fraction or microsomes as metabolic activation reagents for genotoxicity studies.
Effect of biotransformation by liver S9 enzymes on the mutagenicity and cytotoxicity of melanin extracted from Aspergillus nidulans
Published in Pharmaceutical Biology, 2016
Rita de Cássia Ribeiro Gonçalves, Rodrigo Rezende Kitagawa, Eliana Aparecida Varanda, Maria Stella Gonçalves Raddi, Carla Andrea Leite, Sandra Regina Pombeiro Sponchiado
In toxicity and mutagenicity assays, biotransformation is one of the most important factors that can affect the toxic profile of a drug; it can lead to the detoxification and excretion of the drug, as well as its bioactivation. The hepatic microsomal S9 fraction is part of the cytochrome P-450 family and is associated with the endoplasmic reticulum membrane. The S9 fraction is rich in soluble enzymes that possess catalytic abilities against specific substrates. Accordingly, this fraction contains a wide variety of phase I and phase II enzymes (Xiaodong and Lee 2007). In vitro studies of metabolism-mediated cytotoxicity and mutagenicity have used cell viability assays that included the test mixture and the hepatic microsomal S9 fraction because the majority of cell lines are currently used in vitro (Brandon et al. 2003; Kouvelis et al. 2011; Kitagawa et al. 2015).
2-Naphthalenemethanol participates in metabolic activation of 2-methylnaphthalene
Published in Xenobiotica, 2022
Kunna Li, Ying Zou, Yang Wang, Mengyue Zhou, Jing Li, Rong Tan, Shiyu Zhang, Weiwei Li, Jiang Zheng
We did not observe the GSH conjugate in GSH-fortified microsomal incubations, indicating that other enzymes participated in the metabolic activation of 2-MN in addition to P450 enzymes. Hepatic S9 fraction is the de-mitochondrial supernatant of liver homogenate and contains a large number of drug metabolising enzymes such as P450 enzymes and sulfotransferases for metabolising activating mutagens (Yoshihara et al. 2001). To probe the participation of sulphation pathway in the metabolic activation of 2-MN, we incubated 2-MN with hepatic S9 in the presence of NADPH, PAPS and GSH, and we detected the GSH conjugate in the incubation mixture. This further supports that sulphation of 2-NM was involved in the metabolic activation of 2-MN.