Acceleration of murine hepatocyte proliferation by imazalil through the activation of nuclear receptor PXR

Abstract

-The nuclear receptor pregnane X receptor (PXR) plays a major role in the xenobiotic-induced expression of drug-metabolizing enzymes. PXR activation is also associated with several adverse events in the liver. Especially, the receptor enhances hepatocyte proliferation mediated by chemical liver tumor promoters, suggesting that exposure to PXR activators increases the risk of liver cancer. In this study, we have investigated the influences of food additives on PXR to understand their potential adverse effects when they are taken in combination with other chemical compounds. We first screened 25 food additives and related compounds for their PXR-activating ability using reporter assays in HepG2 cells expressing mouse PXR, and found that imazalil dose-dependently activated mouse PXR. Next, to investigate whether imazalil could activate mouse PXR in vivo, mice were treated with imazalil and we found that imazalil treatment increased hepatic mRNA levels of Cyp3a11, a PXR target gene. Finally, to investigate the influence of imazalil exposure on the hepatocyte proliferation induced by nuclear receptor constitutive active/androstane receptor (CAR), mice were treated with imazalil with or without mouse CAR activator TCPOBOP. Although imazalil alone did not induce hepatocyte proliferation, co-treatment with imazalil facilitated the TCPOBOP-dependent proliferation, indicated by the increases in cell proliferation marker levels, Ki-67-positive nuclei and Mcm2 mRNA levels. These results suggest that in mice imazalil activates PXR to enhance hepatocyte proliferation mediated by CAR-activating liver tumor promoters.

Role of Nuclear Receptors PXR and CAR in Xenobiotic-Induced Hepatocyte Proliferation and Chemical Carcinogenesis

“…Co-treatment with verteporfin, which is reported to inhibit the interaction between YAP and TEAD, 71) prevented the TCPOBOP-induced hepatomegaly and tended to reduce YAP target gene mRNA levels as well as those of Ccna2, Ccnb1, and Mcm2. Taken together with the report by Kowalik et al, 70) our results 33) suggest that the YAP/TEAD system plays a role in CAR-dependent hepatocyte proliferation at least in mice.…”

“…As expected, adenoviral expression of mPXR in combination with PCN treatment did not affect the cell number, but co-expression of mPXR and mCAR with respective ligand treatment increased the cell number more markedly than single expression of mCAR. 33) These results indicate that we are able to monitor and investigate CAR-induced hepatocyte proliferation using the in vitro system established.…”

“…We thus aimed to construct such an in vitro system. 33) Primary hepatocytes could be used for such a study since they retain various liver-related functions, but we found that expression levels of CAR in mouse hepatocytes were drasti- cally reduced within 24 h after plating. We thus expressed mouse CAR (mCAR) using an adenovirus vector and tested its influence on proliferation of mouse primary hepatocytes and AML12 cells.…”

“…We thus expressed mouse CAR (mCAR) using an adenovirus vector and tested its influence on proliferation of mouse primary hepatocytes and AML12 cells. 33) With mCAR expression, mouse primary hepatocytes proliferated in a multiplicity of infection (MOI)dependent manner. The number of hepatocytes expressing mCAR and treated with TCPOBOP became more than 2-fold 5 d after adenovirus infection compared with control cells.…”

“…In this manuscript, I will review the background and our recent results on the role of PXR in hepatocyte proliferation and the mechanism for CARmediated hepatocyte proliferation. [31][32][33][34]…”