Abstract
We have developed an in vitro mutation assay using primary hepatocytes from the transgenic Muta™Mouse. Primary hepatocytes were isolated using a two-step perfusion method with purification by Percoll, cultured, and treated with benzo[a]pyrene (BaP), 2-amino-1-methyl-6-phenyl- imidazo[4,5-b]pyridine (PhIP), 3-nitrobenzoanthrone (3-NBA), and cigarette smoke condensate (CSC). The mean lacZ mutant frequency (MF) for the solvent control was approximately twofold greater than the spontaneous MF observed in liver tissue. A concentration-dependent increase in MF (up to 3.7-fold above control) was observed following exposure to BaP. Fourfold and twofold increases in mutant frequency were observed for 3-NBA and PhIP exposures, respectively, without the addition of any exogenous metabolic activation. A slight but statistically significant increase in lacZ MF was observed for CSC, but only at the lowest concentration. This is the first report demonstrating that mutations can be detected in cultured primary hepatocytes from Muta™Mouse. The preliminary results presented suggest that the Muta™Mouse primary hepatocyte mutagenicity assay can be used as a cost-effective tool for screening of environmental mutagens and therapeutic products. Environ. Mol. Mutagen. 51:330–337, 2010. © 2009 Wiley-Liss, Inc.
Highlights
Transgenic rodent (TGR) mutation models such as MutaTMMouse and Big Blue1 rat/mouse provide efficient methods for quantitative assessments of in vivo gene mutation
We demonstrate that cultured primary hepatocytes derived from the MutaTMMouse can be employed to assess the mutagenic activity of selected test mutagens that require metabolic activation by cytochrome P450 isozymes
This study introduces a novel in vitro assay system for mutagenicity assessment that takes simultaneous advantage of the P-gal–positive selection system to score mutations at the lacZ transgene, and the metabolic capacity of primary hepatocytes
Summary
Transgenic rodent (TGR) mutation models such as MutaTMMouse and Big Blue rat/mouse provide efficient methods for quantitative assessments of in vivo gene mutation. Such transgenic mutation assays involve scoring of mutations at transgenic lacZ or lacI sequences carried on a lambda phage shuttle vector that has been stably integrated into the rodent genome. A major advantage of the transgenic mutation system lies in its ability to provide reliable and reproducible assessments of in vivo mutagenicity in any organ or tissue [Heddle et al, 2000; Nohmi et al, 2000; Thybaud et al, 2003] In their detailed review paper, Lambert et al concluded that TGR mutation models showed excellent concordance (77%) with rodent carcinogenicity that meets or exceeds what has been observed for other genotoxicity.
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