The assessment of safety and the toxicity profile of chemical and biological entities remains a challenge. Despite ongoing efforts to better understand the mechanisms underlying safety and toxicity, ~30% of the attrition in drug discovery and development is still due to safety concerns [1]. Despite mandatory testing in preclinical in vivo models, the existing interspecies differences do not allow reliable prediction of human hepatic toxicity from animal studies. Widely used human hepatic cell lines and cultures show suboptimal outcomes due to the low activity of metabolizing enzymes, and primary human hepatocytes (PHHs) remain the best choice. PHHs have their own limitations, like limited supply volume and lot-to-lot variations. Thus, better in vitro and in vivo models are still required for the prediction of human hepatic toxicity and drug induced liver injury (DILI).
The recent paper “Detection of acute toxicity of aflatoxin B1 to human hepatocytes in vitro and in vivo using chimeric mice with humanized livers” shows results generated by using the PXB-mouse® and PXB-cells®. Both systems appear to be the advantageous, robust, and predictive models of human hepatic toxicity.
The technology of the chimeric mouse with a humanized liver (PXB-mouse®) gives researchers an opportunity to use a rodent model carrying human hepatocytes for the most relevant in vivo evaluation of human hepatotoxicity. Moreover, PXB-cells® overcome the limitations of PHHs: they are available on demand and allow superior lot-to-lot reproducibility due to the same donor source use.
Aflatoxins are a group of secondary metabolites produced by Aspergillus fungi. Among about 20 known aflatoxins, aflatoxin B1 (AFB1) is considered as one of the most potent inducers of acute and chronic liver injury and hepatocellular carcinoma. Examples of AFB1-contaminated food which caused human deaths in past decades are corn, peanuts, milo, sorghum, copra, and rice. Interspecies differences (i.e., mice are known to have low sensitivity to liver toxicity of aflatoxin B1) in the susceptibility to AFB-1 induced liver toxicity limit the use of experimental animals for human-related studies.
Authors examined the toxicity of aflatoxin B1 in human hepatocytes in chimeric mice with a humanized liver as compared to a negative control (corn oil) and carbon tetrachloride (CCl4) administration. Histological analysis revealed that AFB1 promoted hepatocyte vacuolation and infiltration of inflammatory cells in the areas of human hepatocytes in PXB-mice, but not in mouse hepatocytes. In contrast, CCl4 induced toxic effects in the remaining mouse hepatocytes in the PXB-mouse® model and in the SCID mouse liver.
To confirm the histology data, authors used a novel human alanine aminotransferase 1 (hALT1) specific sandwich enzyme-linked immunosorbent assay (ELISA). This assay was developed by PhoenixBio to evaluate the impact of the human specific biomarker in total ALT response. The developed hALT1 specific assay shows no cross reactivity with either hALT2 or mouse-derived ALT. Such a method allows for monitoring human hepatocyte injury throughout the in-life phase, which is fitting for use in drug-induced liver injury studies with humanized liver chimeric mice.
For the in vitro AFB1 toxicity evaluation, authors compared HepG2 cells, HepaRG cells, primary human hepatocytes (PHH) and PXB-cells®. Widely used cell lines, such as HepG2 cells, HepaRG cells, underperform and show lower sensitivity to aflatoxin B1 induced hepatotoxicity when compared to PHH and PXB-cells®. Even though PHH are considered a gold standard for in vitro toxicity, metabolizing enzyme activity in these cells usually quickly diminishes. In contrast, PXB-cells® showed high CYP3A activity three weeks after plating and even longer. The paper highlights that the quality of PXB-cells® allows the study of long-term exposure (6 or 14 days) at a low dose and shows time-dependent cytotoxicity of relatively low levels of AFB1. Authors showed that using a non-specific CYP inhibitor or transfection with CYP3A4 specific siRNA reduce aflatoxin B1 cytotoxicity. This supported the hypothesis that bioactivation of AFB1 catalyzed by CYPs is an essential step for cytotoxicity in human hepatocytes.
As a result of these studies, humanized liver chimeric mice and proprietary fresh human hepatocytes (PXB-cells®) were demonstrated to be reliable and relevant research tools for the assessment of human hepatic toxicity.
[1] Sahota T. et al. Mutagenesis 31(3): 359–374