Leibniz Research Centre for Working Environment and Human Factors at Dortmund TU, Dortmund, Germany.
Arch Toxicol. 2013 Feb;87(2):337-45. doi: 10.1007/s00204-012-0999-8. Epub 2012 Dec 29.
Cultivated hepatocytes represent a well-established in vitro system. However, the applicability of hepatocytes in toxicogenomics is still controversially discussed. Recently, an in vivo/in vitro discrepancy has been described, whereby the non-genotoxic rat liver carcinogen methapyrilene alters the expression of the metabolizing genes SULT1A1 and ABAT, as well as the DNA damage response gene GADD34 in vitro, but not in vivo. If the collagen sandwich cultures of hepatocytes really produce false-positive data, this would compromise its application in toxicogenomics. To revisit the putative in vivo/in vitro discrepancy, we first analyzed and modeled methapyrilene concentrations in the portal vein of rats. The relatively short half-life of 2.8 h implies a rapid decrease in orally administered methapyrilene in vivo below concentrations that can cause gene expression alterations. This corresponded to the time-dependent alteration levels of GADD34, ABAT and SULT1A1 RNA in the liver: RNA levels are altered 1, 6 and 12 h after methapyrilene administration, but return to control levels after 24 and 72 h. In contrast, methapyrilene concentrations in the culture medium supernatant of primary rat hepatocyte cultures decreased slowly. This explains why GADD34, ABAT and SULT1A1 were still deregulated after 24 h exposure in vitro, but not in vivo. It should also be considered that the earliest analyzed time point in the previous in vivo studies was 24 h after methapyrilene administration. In conclusion, previously observed in vitro/in vivo discrepancy can be explained by different pharmacokinetics present in vitro and in vivo. When the in vivo half-life is short, levels of some initially altered genes may have returned to control levels already 24 h after administration.
培养的肝细胞代表了一种成熟的体外系统。然而，肝细胞在毒理学基因组学中的应用仍然存在争议。最近，有人描述了一种体内/体外差异，即非遗传毒性大鼠肝致癌物甲吡咯烷在体外改变了代谢基因 SULT1A1 和 ABAT 的表达以及 DNA 损伤反应基因 GADD34 的表达，但在体内没有改变。如果肝细胞的胶原三明治培养真的产生了假阳性数据，这将影响其在毒理学基因组学中的应用。为了重新审视潜在的体内/体外差异，我们首先分析并模拟了大鼠门静脉中甲吡咯烷的浓度。相对较短的半衰期为 2.8 h，这意味着口服给予的甲吡咯烷在体内迅速下降到低于引起基因表达改变的浓度。这与 GADD34、ABAT 和 SULT1A1 RNA 在肝脏中的时间依赖性改变水平相对应：RNA 水平在甲吡咯烷给药后 1、6 和 12 h 发生改变，但在 24 和 72 h 后恢复到对照水平。相比之下，原代大鼠肝细胞培养物培养基上清液中的甲吡咯烷浓度下降缓慢。这解释了为什么 GADD34、ABAT 和 SULT1A1 在体外暴露 24 h 后仍被下调，但在体内没有被下调。还应考虑到，在之前的体内研究中最早分析的时间点是甲吡咯烷给药后 24 h。总之，以前观察到的体外/体内差异可以用体内和体外存在的不同药代动力学来解释。当体内半衰期较短时，一些最初改变的基因的水平可能在给药后 24 h 已经恢复到对照水平。
