Northern blotting has shown that mouse small intestine contains relatively large amounts of the nuclear factor-E2 p45-related factor (Nrf) 2
transcription factor but relatively little Nrf1. Regulation of intestinal
antioxidant and detoxication
enzymes by Nrf2 has been assessed using a mouse line bearing a targeted disruption of the gene encoding this factor. Both Nrf2-/- and Nrf2+/+ mice were fed a control diet or one supplemented with either synthetic
cancer chemopreventive agents [
butylated hydroxyanisole (
BHA),
ethoxyquin (EQ), or
oltipraz] or
phytochemicals [
indole-3-
carbinol,
cafestol and
kahweol palmitate,
sulforaphane,
coumarin (CMRN), or
alpha-angelicalactone]. The constitutive level of
NAD(P)H:
quinone oxidoreductase (NQO) and
glutathione S-transferase (GST)
enzyme activities in cytosols from small intestine was typically found to be between 30% and 70% lower in samples prepared from Nrf2 mutant mice fed a control diet than in equivalent samples from Nrf2+/+ mice. Most of the chemopreventive agents included in this study induced NQO and GST
enzyme activities in the small intestine of Nrf2+/+ mice. Increases of between 2.7- and 6.2-fold were observed in wild-type animals fed diets supplemented with
BHA or EQ; increases of about 2-fold were observed with a mixture of
cafestol and
kahweol palmitate, CMRN, or
alpha-angelicalactone; and increases of 1.5-fold were measured with
sulforaphane. Immunoblotting confirmed that in the small intestine, the constitutive level of NQO1 is lower in the Nrf2-/- mouse, and it also showed that induction of the
oxidoreductase was substantially diminished in the mutant mouse. Immunoblotting class-alpha and class-mu GST showed that constitutive expression of most
transferase subunits is also reduced in the small intestine of Nrf2 mutant mice. Significantly, induction of class-alpha and class-mu GST by EQ,
BHA, or CMRN is apparent in the gene knockout animal. No consistent change in the constitutive levels of the catalytic heavy subunit of gamma-glutamylcysteinyl
synthetase (GCS(h)) was observed in the small intestine of Nrf2-/- mice. However, although the expression of GCS(h) was found to be increased dramatically in the small intestine of Nrf2+/+ mice by dietary
BHA or EQ, this induction was essentially abolished in the knockout mice. It is apparent that Nrf2 influences both constitutive and inducible expression of intestinal
antioxidant and detoxication
proteins in a gene-specific fashion. Immunohistochemistry revealed that induction of NQO1, class-alpha GST, and GCS(h) occurs primarily in epithelial cells of the small intestine. This suggests that the variation in inducibility of NQO1, Gsta1/2, and GCS(h) in the mutant mouse is not attributable to the expression of the
enzymes in distinct cell types but rather to differences in the dependency of these genes on Nrf2 for induction.