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NRF2-Independent Regulation of Drug Metabolism by CAR and Prooxidants


Exposure to toxicants that create oxidative stress, such as isothiocyanates (ITCs) and heavy metals, initiate inductions of antioxidant response genes and detoxification genes such as glutathione S-transferases (GSTs) and UDP-glucuronosyltransferases (UGTs) by the NRF2-KEAP1 pathway. UGTs act on a wide range of hydrophobic compounds, including the heme-degradation product, bilirubin, which can only be conjugated by UGT1A1. Newborn humans commonly exhibit an accumulation of bilirubin in the serum known as hyperbilirubinemia due to a developmental delay in the transcription of UGT1A1. This effect is replicated in neonatal mice that lack a functional mouse Ugt1 locus but contain the human UGT1 locus. These hUGT1 mice experience neonatal hyperbilirubinemia that can be ameliorated by the induction of UGT1A1 by active nuclear/xenobiotic receptors. The crossing of hUGT1 mice with nuclear/xenobiotic receptor knockout mice creates a mouse model that links a readily observed phenotype, hyperbilirubinemia, with nuclear/xenobiotic receptor function following exposure to a desired compound. Phenylethyl isothiocyanate (PEITC) treated neonates experienced a reduction of total serum bilirubin levels, an induction of hepatic and intestinal UGT1A1 and the CAR target gene, CYP2B10. Utilizing hUGT1/Car-/-, it was determined that PEITC induced hepatic UGT1A1 in a CAR-dependent manner. It was also shown that some CAR-independent induction of intestinal UGT1A1 occurred. Inductions of CYP2B10 and UGT1A1 could be blocked by pretreatment with the antioxidant, N-acetyl cysteine (NAC), which indicates oxidative stress as a component to CAR activation. To examine the role of NRF2 on intestinal induction of UGT1A1 by PEITC, hUGT1/Nrf2-/- mice were generated. Intestinal UGT1A1 induction by PEITC was determined to be partially dependent upon NRF2, while Cyp2b10 and hepatic UGT1A1 induction was retained in hUGT1/Nrf2-/- mice. This indicated that CAR activity following PEITC exposure does not require NRF2. Similar results were obtained with the heavy metals arsenic and cadmium in the small intestine. Western blot analysis of intestinal fractions of hUGT1/Nrf2-/- mice treated with CdCl2 demonstrated increases in nuclear CAR accumulation, nuclear P38 content, and the rise and fall of ERK1/2 phosphorylation. Mitogen activated protein kinase activity may serve as the regulatory mechanism for CAR by oxidative stress.

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