Environmental Health Sciences and Research Bureau, Research and Radiation Directorate, Health Canada, Ottawa, Ontario, Canada.
Environ Mol Mutagen. 2010 May;51(4):330-7. doi: 10.1002/em.20540.
We have developed an in vitro mutation assay using primary hepatocytes from the transgenic MutaMouse. 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 MutaMouse. The preliminary results presented suggest that the MutaMouse primary hepatocyte mutagenicity assay can be used as a cost-effective tool for screening of environmental mutagens and therapeutic products.
我们使用转基因 MutaMouse 的原代肝细胞开发了一种体外突变检测方法。原代肝细胞采用两步灌流法分离，用 Percoll 进行纯化，培养后用苯并[a]芘（BaP）、2-氨基-1-甲基-6-苯基咪唑[4,5-b]吡啶（PhIP）、3-硝基苯并蒽酮（3-NBA）和香烟烟雾冷凝物（CSC）处理。溶剂对照的平均 lacZ 突变频率（MF）比肝脏组织中观察到的自发 MF 高约两倍。暴露于 BaP 后，MF 呈浓度依赖性增加（比对照高 3.7 倍）。3-NBA 和 PhIP 暴露分别导致突变频率增加 4 倍和 2 倍，而无需添加任何外源性代谢激活。CSC 导致 lacZ MF 略有但统计学上显著增加，但仅在最低浓度下。这是首次报道表明可以在 MutaMouse 的培养原代肝细胞中检测到突变。初步结果表明，MutaMouse 原代肝细胞致突变性检测可以作为筛选环境诱变剂和治疗产品的一种具有成本效益的工具。
