Broccardo Carolyn J, Billings Ruth E, Chubb Laura S, Andersen Melvin E, Hanneman William H
Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523-1680, USA.
Toxicol Sci. 2004 Apr;78(2):287-94. doi: 10.1093/toxsci/kfh077. Epub 2004 Feb 19.
The shape of the dose-response curve may vary depending on whether one examines response at a population or a single cell level. Populations of cells may exhibit a graded response whereas single cell responses may have threshold or switch-like behavior. Studies in vivo and in vitro using primary hepatocyte cultures have shown that induction of CYP1A1 in the liver exhibits switch-like behavior in response to PCB 126 (3,3',4,4',5-pentachlorobiphenyl). The goal of the present study was to determine if two liver cell lines (H4IIE rat hepatoma and Hepa 1c1c7 mouse hepatoma) also show switch-like behavior and develop experimental models for studying mechanisms of these switch-like responses. Both cell lines were analyzed via concentration-response and time-course studies using quantitative real-time PCR, revealing a sigmoidal concentration-response curve for CYP1A1 mRNA induction at the population level. To study CYP1A1 protein induction on a single cell level, flow cytometry was employed. In both cell lines the distribution of fluorescence increased with increasing concentrations of PCB 126. The switch behavior was more pronounced in the H4IIE cells than in the Hepa 1c1c7 cells, exhibiting a well-defined shift of induction from the "off" to the "on" state. The concentration-response curve at the single cell level appeared more switch-like with two populations of cells-basal levels and maximally induced. Immunocytochemistry studies of individual cells also support these conclusions. Our data support the hypothesis that PCB 126 induces CYP1A1 in a switch-like fashion in H4IIE rat hepatoma cells. These cells can now be used to study the mechanism of the biological switch.
剂量反应曲线的形状可能因观察的是群体水平还是单细胞水平的反应而有所不同。细胞群体可能表现出分级反应，而单细胞反应可能具有阈值或类似开关的行为。使用原代肝细胞培养物进行的体内和体外研究表明，肝脏中CYP1A1的诱导对多氯联苯126（3,3',4,4',5-五氯联苯）的反应呈现类似开关的行为。本研究的目的是确定两种肝细胞系（H4IIE大鼠肝癌细胞和Hepa 1c1c7小鼠肝癌细胞）是否也表现出类似开关的行为，并建立研究这些类似开关反应机制的实验模型。通过使用定量实时PCR的浓度反应和时间进程研究对两种细胞系进行分析，揭示了群体水平上CYP1A1 mRNA诱导的S形浓度反应曲线。为了在单细胞水平上研究CYP1A1蛋白的诱导，采用了流式细胞术。在两种细胞系中，荧光分布均随多氯联苯126浓度的增加而增加。H4IIE细胞中的开关行为比Hepa 1c1c7细胞中更明显，表现出从“关闭”状态到“开启”状态的明确诱导转变。单细胞水平的浓度反应曲线在具有两个细胞群体（基础水平和最大诱导水平）时显得更类似开关。对单个细胞的免疫细胞化学研究也支持这些结论。我们的数据支持这样的假设，即多氯联苯126以类似开关的方式在H4IIE大鼠肝癌细胞中诱导CYP1A1。这些细胞现在可用于研究生物开关的机制。
