Human Hepatocytes Support the Hypertrophic but not the Hyperplastic Response to the Murine Nongenotoxic Hepatocarcinogen Sodium Phenobarbital in an In Vivo Study Using a Chimeric Mouse with Humanized Liver

Abstract

High doses of sodium phenobarbital (NaPB), a constitutive androstane receptor (CAR) activator, have been shown to produce hepatocellular tumors in rodents by a mitogenic mode of action (MOA) involving CAR activation. The effect of 1-week dietary treatment with NaPB on liver weight and histopathology, hepatic CYP2B enzyme activity and CYP2B/3A mRNA expression, replicative DNA synthesis and selected genes related to cell proliferation, and functional transcriptomic and metabolomic analyses was studied in male CD-1 mice, Wistar Hannover (WH) rats, and chimeric mice with human hepatocytes. The treatment of chimeric mice with 1000-1500-ppm NaPB resulted in plasma levels around 3-5-fold higher than those observed in human subjects given therapeutic doses of NaPB. NaPB produced dose-dependent increases in hepatic CYP2B activity and CYP2B/3A mRNA levels in all animal models. Integrated functional metabolomic and transcriptomic analyses demonstrated that the responses to NaPB in the human liver were clearly different from those in rodents. Although NaPB produced a dose-dependent increase in hepatocyte replicative DNA synthesis in CD-1 mice and WH rats, no increase in replicative DNA synthesis was observed in human hepatocyte-originated areas of chimeric mice. In addition, treatment with NaPB had no effect on Ki-67, PCNA, GADD45β, and MDM2 mRNA expression in chimeric mice, whereas significant increases were observed in CD-1 mice and/or WH rats. However, increases in hepatocyte replicative DNA synthesis were observed in chimeric mice both in vivo and in vitro after treatment epidermal growth factor. Thus, although NaPB could activate CAR in both rodent and human hepatocytes, NaPB did not increase replicative DNA synthesis in human hepatocytes of chimeric mice, whereas it was mitogenic to rat and mouse hepatocytes. As human hepatocytes are refractory to the mitogenic effects of NaPB, the MOA for NaPB-induced rodent liver tumor formation is thus not relevant for humans.

Evaluation of the human relevance of the constitutive androstane receptor-mediated mode of action for rat hepatocellular tumor formation by the synthetic pyrethroid momfluorothrin

“…We have provided some experimental data showing a key species difference; those were that while PB stimulates replicative DNA synthesis in rodent hepatocytes, such proliferative effects are not observed in cultured human hepatocytes (Yamada et al, 2015;Hirose et al, 2009) nor in human hepatocytes of chimeric mice (Yamada et al, 2014). The lack of this key event in humans strongly supports the previous conclusion that the rodent carcinogenicity by CAR activators such as PB is not relevant to humans based on experimental and epidemiology data .…”

“…In keeping with the properties of the prototypic CAR activator PB, two closely structurally related pyrethroid insecticides metofluthrin (Yamada et al, 2015;Hirose et al, 2009) and momfluorothrin (present study) were demonstrated not to stimulate replicative DNA synthesis in cultured human hepatocytes. In addition, no stimulation of replicative DNA synthesis by NaPB in human hepatocytes has already been demonstrated in vivo in the study utilizing chimeric mice with human hepatocytes (Yamada et al, 2014). The present study demonstrated that momfluorothrin and metofluthrin did not increase in vivo human hepatocyte replicative DNA synthesis in chimeric mice employing hepatocytes from three different donors.…”

“…The treatment of chimeric mice with human growth hormone (hGH) increases the repopulation speed and the replacement index of transplanted human hepatocytes, as determined by increased replicative DNA synthesis and the up-regulation of hGH-related signalling molecules (Masumoto et al, 2007). Furthermore, treatment with epidermal growth factor (Yamada et al, 2014) and partial hepatectomy (personal communication, Dr. Chise Tateno) also enhances hepatocellular proliferation in this chimeric mouse model. These findings demonstrated that transplanted human hepatocytes in the chimeric mice are responsive to hepatocyte mitogens.…”

“…The cell proliferation rate was evaluated as replicative DNA synthesis determined as the BrdU labeling index as previously described (Yamada et al, 2014). Four hundred milligrams of BrdU was dissolved in 1.0 mL DMSO and diluted with distilled water to 10.0 mL total for injection.…”

“…Hepatocyte replicative DNA synthesis was determined in the livers from all surviving animals by an immunohistochemical method using BrdU monoclonal antibody (Yamada et al, 2014). An image analysis system was used to evaluate the replicative DNA synthesis of human hepatocytes in chimeric mice.…”