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      Induction of lacZ Mutations in Muta™Mouse Primary Hepatocytes

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          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.

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          Most cited references54

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          Tobacco carcinogens, their biomarkers and tobacco-induced cancer.

          The devastating link between tobacco products and human cancers results from a powerful alliance of two factors - nicotine and carcinogens. Without either one of these, tobacco would be just another commodity, instead of being the single greatest cause of death due to preventable cancer. Nicotine is addictive and toxic, but it is not carcinogenic. This addiction, however, causes people to use tobacco products continually, and these products contain many carcinogens. What are the mechanisms by which this deadly combination leads to 30% of cancer-related deaths in developed countries, and how can carcinogen biomarkers help to reveal these mechanisms?
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            Preparation of isolated rat liver cells.

            P O Seglen (1976)
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              Comparison of primary human hepatocytes and hepatoma cell line Hepg2 with regard to their biotransformation properties.

              Cultures of primary hepatocytes and hepatoma cell line HepG2 are frequently used in in vitro models for human biotransformation studies. In this study, we characterized and compared the capacity of these model systems to indicate the presence of different classes of promutagens. Genotoxic sensitivity, enzyme activity, and gene expression were monitored in response to treatment with food promutagens benzo[a]pyrene, dimethylnitrosamine (DMN), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). DNA damage could be detected reliably with the comet assay in primary human hepatocytes, which were maintained in sandwich culture. All three promutagens caused DNA damage in primary cells, but in HepG2 no genotoxic effects of DMN and PhIP could be detected. We supposed that the lack of specific enzymes accounts for their inability to process these promutagens. Therefore, we quantified the expression of a broad range of genes coding for drug-metabolizing enzymes with real-time reverse transcription-polymerase chain reaction. The genes code for cytochromes p450 and, in addition, for a series of important phase II enzymes. The expression level of these genes in human hepatocytes was similar to those previously reported for human liver samples. On the other hand, expression levels in HepG2 differed significantly from that in human. Activity and expression, especially of phase I enzymes, were demonstrated to be extremely low in HepG2 cells. Up-regulation of specific genes by test substances was similar in both cell types. In conclusion, human hepatocytes are the preferred model for biotransformation in human liver, whereas HepG2 cells may be useful to study regulation of drug-metabolizing enzymes.
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                Author and article information

                Journal
                Environ Mol Mutagen
                em
                Environmental and Molecular Mutagenesis
                Wiley Subscription Services, Inc., A Wiley Company
                0893-6692
                1098-2280
                May 2010
                : 51
                : 4
                : 330-337
                Affiliations
                simpleEnvironmental Health Sciences and Research Bureau, Research and Radiation Directorate, Health Canada Ottawa, Ontario, Canada
                Author notes
                *Correspondence to: Paul A. White, Environmental Health Sciences and Research Bureau, Research and Radiation Directorate, Health Canada, Ottawa, Ontario, Canada

                Guosheng Chen is currently at Risk Assessment Bureau, Health Canada, 269 Laurier Avenue West (A/L 4904C), Ottawa, Ontario, Canada K1A 0K9.

                Grant sponsors: Health Canada, Natural Sciences and Engineering Research Council of Canada (NSERC), under the Visiting Fellowships Program.

                Article
                10.1002/em.20540
                2959491
                19953605
                191178c3-4601-4c0a-b48c-0da9c63bcea0
                Copyright © 2010 Wiley-Liss, Inc., A Wiley Company

                Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.

                History
                : 13 July 2009
                : 18 September 2009
                : 17 September 2009
                Categories
                Brief Communication

                Molecular biology
                muta™mouse,primary hepatocytes,mutation
                Molecular biology
                muta™mouse, primary hepatocytes, mutation

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