Comparison of the Hepatic Effects of Phenobarbital in Chimeric Mice Containing Either Rat or Human Hepatocytes With Humanized Constitutive Androstane Receptor and Pregnane X Receptor Mice

Abstract

Using a chimeric mouse humanized liver model, we provided evidence that human hepatocytes are refractory to the mitogenic effects of rodent constitutive androstane receptor (CAR) activators. To evaluate the functional reliability of this model, the present study examined mitogenic responses to phenobarbital (PB) in chimeric mice transplanted with rat hepatocytes, since rats are responsive to CAR activators. Treatment with 1000 ppm PB for 7 days significantly increased replicative DNA synthesis (RDS) in rat hepatocytes of the chimeric mice, demonstrating that the transplanted hepatocyte model is functionally reliable for cell proliferation analysis. Treatment of humanized CAR and pregnane X receptor (PXR) mice (hCAR/hPXR mice) with 1000 ppm PB for 7 days significantly increased hepatocyte RDS together with increases in several mitogenic genes. Global gene expression analysis was performed with liver samples from this and from previous studies focusing on PB-induced Wnt/β-catenin signaling, and showed that altered genes in hCAR/hPXR mice clustered most closely with liver tumor samples from a diethylnitrosamine/PB initiation/promotion study than with wild type mice. However, different gene clusters were observed for chimeric mice with human hepatocytes for Wnt/β-catenin signaling when compared to those of hCAR/hPXR mice, wild type mice and liver tumor samples. The results of this study demonstrate clear differences in the effects of PB on hepatocyte RDS and global gene expression between human hepatocytes of chimeric mice and hCAR/hPXR mice, suggesting that the chimeric mouse model is relevant to humans for studies on the hepatic effects of rodent CAR activators, whereas the hCAR/hPXR mouse is not.

Application of humanized mice to toxicology studies: Evaluation of the human relevance of the mode of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR)

“…More recently, we provided data showing that treatment with 1000 ppm PB significantly increased RDS, together with a small increase in MKI67 mRNA levels, in chimeric rat hepatocyte mice 68 , which is consistent with a large number of previous in vivo and in vitro studies in rats showing increased RDS.…”

“…Hence, the chimeric mice with transplanted rat hepatocytes retained the original characteristics of normal rat hepatocytes by increasing RDS in response to stimulation by the CAR-activator PB 68 . These results strongly support the conclusion that the lack of proliferation of human hepatocytes in the chimeric mouse model is not due to any functional defect in the mouse liver environment, but because human hepatocytes are truly refractory to the mitogenic effects of PB and other rodent CAR-activators, as observed in cultured human hepatocyte studies.…”

“…Overall, unlike mouse hepatocytes, exposure of human hepatocytes to nongenotoxic CAR-activators appears to have little effect on the genes associated with the Wnt/β-catenin signaling pathway 68,93 . These findings support our consideration that although the hCAR/hPXR genes have been inserted genetically, the downstream genes are still mouse and may be the basis for the increased hepatocyte RDS, Wnt/β-catenin signaling, and tumor production observed in hCAR/hPXR mouse studies.…”

“…This apparent lack of effect is most likely attributable to the treatment time and/or dose levels of PB administered. Overall, these studies demonstrate that the treatment of either hCAR or hCAR/hPXR mice with PB can result in increased hepatocyte RDS, although the effects of PB treatment are less marked in hCAR/hPXR mice than in wild-type mice68,69 Braeuning et al (2014). performed an initiation/promotion study in which wild-type and hCAR/hPXR mice were administered with a single dose of DEN followed by treatment with 500 ppm PB in the diet for 40 weeks.…”

Critical evaluation of the human relevance of the mode of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR)

“…For example, while PB has been shown to induce hepatocyte RDS in wild-type (i.e. normal) mice and rats, no increases in hepatocyte RDS were observed following the treatment of CAR KO mice (Wei et al 2000;Huang et al 2005), CAR KO/PXR KO mice (Ross et al 2010;Haines et al 2019), CAR KO rats (Okuda et al 2017b;Haines et al 2018a;Yamada et al 2020), and CAR KO/PXR KO rats (Haines et al 2019) with PB. The lack of effect of PB on hepatocyte RDS in receptor KO mice and rats is not due to PB exposure as in studies where terminal plasma PB levels were analyzed, these were significantly higher in mice and rats lacking either hepatic CAR or CAR and PXR (Luisier et al 2014;Haines et al 2018a;Haines et al 2019;Yamada et al 2020).…”