Isothiocyanates Activate the Constitutive Androstane Receptor in an Oxidative Stress Dependent Manner in hUGT1 Mice

Abstract

Our laboratory has previously developed a humanized UDP‐glucuronosyltransferase 1 (hUGT1) mouse model, creating an in vivo model to examine UGT1A metabolism and gene regulation. Mirroring human development, neonatal hUGT1 mice experience a developmental delay in expression of UGT1A1, the sole enzyme capable of bilirubin glucuronidation, resulting in an increase in unconjugated bilirubin. The accumulation of total serum bilirubin (TSB) is readily quantifiable and creates an observable phenotype linked to the expression of UGT1A1. The UGT1A1 gene is regulated by a host of nuclear receptors allowing drugs to be examined in vivo for their effects on nuclear receptor activity. By examination of the relationship between oxidative stress and UGT1A1 expression, we discovered a novel association between the constitutive androstane receptor (CAR) and oxidative stress. In order to induce oxidative stress, hUGT1 mice were treated with phenylethyl isothiocyanate (PEITC), a natural pesticide found in cruciferous vegetables with known anticancer and antioxidant response properties. It is well established that PEITC reacts with glutathione, depleting a major cellular antioxidant, which then activates antioxidant response pathways in response to the change in the cells redox potential. Neonatal hUGT1 mice experienced a drastic decrease in TSB levels following oral PEITC treatment. Concurrently, a significant increase in hepatic and intestinal UGT1A1 expression was observed. In characterizing a mechanism responsible for UGT1A1 induction, we determined CAR to be the major contributor by examining nuclear receptor target genes. CAR's activation was observed by the significant induction CYP2B10, with the Cyp2b10 gene being a consensus CAR target. This suggested a novel association between CAR activation and oxidative stress. In order to confirm CAR's role in the induction of UGT1A1 and CYP2B10, we treated hUGT1/Car(−/−) and hUGT1/Car(+/−) mice orally with PEITC and demonstrated CAR dependence for intestinal and hepatic CYP2B10 induction. We further identified PEITC induction of UGT1A1 was significantly dependent on CAR in liver. PEITC's ability to directly activate CAR was examined using an in vitro transactivation assay, and determined that PEITC is incapable of directly activating CAR by binding to its ligand binding domain. To probe the role of oxidative stress on UGT1A1 induction/CAR activation, hUGT1 mice were pretreated with N‐acetyl cysteine (NAC), an antioxidant and precursor to glutathione. NAC effectively prevented PEITC induced oxidative stress and a concurrent drop in hepatic UGT1A1 and CYP2B10 induction was observed. This suggests a direct correlation between oxidative stress and CAR activation.Support or Funding InformationU.S. Public Health Service Grants ES010337, GM100481, GM086713 (R.H.T.) and R21‐ES024818 (S.C)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.