Dose-Response Modeling of Early Molecular and Cellular Key Events in the CAR-Mediated Hepatocarcinogenesis Pathway

Abstract

Low-dose extrapolation and dose-related transitions are paramount in the ongoing debate regarding the quantification of cancer risks for nongenotoxic carcinogens. Phenobarbital (PB) is a prototypical nongenotoxic carcinogen that activates the constitutive androstane receptor (CAR) resulting in rodent liver tumors. In this study, male and female CD-1 mice administered dietary PB at 0, 0.15, 1.5, 15, 75, or 150 mg/kg-day for 2 or 7 days to characterize multiple apical and molecular endpoints below, at (∼75 mg/kg-day), and above the carcinogenic dose level of PB and examine these responses using benchmark dose modeling. Linear toxicokinetics were observed for all doses. Increased liver weight, hepatocellular hypertrophy, and mitotic figures were seen at 75 and 150 mg/kg-day. CAR activation, based on Cyp2b qPCR and pentoxyresorufin dealkylase activity, occurred at doses ≥ 1.5 mg/kg-day. The no-observable transcriptional effect level for global gene expression was 15 mg/kg-day. At 2 days, several xenobiotic metabolism and cell protective pathways were activated at lower doses and to a greater degree in females. However, hepatocellular proliferation, quantified by bromodeoxyuridine immunohistochemistry, was the most sensitive indicator of PB exposure with female mice more sensitive than males, contrary to sex-specific differences in sensitivity to hepatocarcinogenesis. Taken together, the identification of low-dose cellular and molecular transitions in the subtumorigenic dose range aids the understanding of early key events in CAR-mediated hepatocarcinogenesis.

Dose-response analysis of epigenetic, metabolic, and apical endpoints after short-term exposure to experimental hepatotoxicants

“…2A), which correlated with hepatomegaly and hepatocellular hypertrophy at both dose levels and increased hepatocellular proliferation at 250 mg/kg/day. In the PB treated animals, Cyp2b1 mRNA was induced in a dose-dependent manner, consistent with CAR activation (Elcombe et al, 2014; Geter et al, 2014); in the low-, mid-, and high-dose groups, Cyp2b1 mRNA was induced 11-, 48-, and 180-fold, respectively (Fig. 2A).…”

“…It has been reported that following 7-day exposure to PB, the range of BMD for apical endpoints (e.g. liver weight, hepatic biomarkers, and hepatocyte cell proliferation) in male CD-1 mice was 2.4–30 mg/kg/day (Geter et al, 2014). For CF, the lowest tumorigenic dose is 250 mg/kg/day in rats (Reddy et al, 1979; Sargent et al, 2002).…”

Pilot studies evaluating the nongenotoxic rodent carcinogens phenobarbital and clofibrate in the rat Pig‐a assay

“…These observations are consistent with previous publications, which suggested similar increases in relative liver weights, hepatomegaly, and hepatocellular hypertrophy following PB and CF exposures (Corton et al, 2014;Elcombe et al, 2014;Miousse et al, 2017). Following oral administration, both PB and CF are rapidly absorbed and transported via the blood, consistent with their pharmaceutical applications (Cayen et al, 1977;Geter et al, 2014). Furthermore, a recent publication has evaluated the in vivo mutagenic response of genotoxic chemicals in a 28-day dosing paradigm at fractions of the carcinogenic dose (Dertinger et al, 2014).…”

Mode of action analysis for pesticide‐induced rodent liver tumours involving activation of the constitutive androstane receptor: relevance to human cancer risk

“…29 In a recent study, male and female CD-1 mice were treated with PB at dose levels of 0.15-150 mg kg −1 day −1 for periods of 2 and 7 days. 50 Treatment with PB produced clear dose-dependent effects on a number of key and associative events for the MOA for PB-induced liver tumour formation. Some information on the concordance of dose-response relationships for key and associative events in the MOA for PB-induced tumour formation was observed in a study on the low spontaneous liver tumour incidence C57BL/10 J mouse strain.…”

“…For example, PB has been reported to produce liver tumours in CD‐1 mice at a dose level of 75 mg kg −1 day −1 , but not at a dose level of 10 mg kg −1 day −1 . In a recent study, male and female CD‐1 mice were treated with PB at dose levels of 0.15–150 mg kg −1 day −1 for periods of 2 and 7 days . Treatment with PB produced clear dose‐dependent effects on a number of key and associative events for the MOA for PB‐induced liver tumour formation.…”