M. Mcmahon et al., The cap 'n' collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression ofintestinal detoxification and glutathione biosynthetic enzymes, CANCER RES, 61(8), 2001, pp. 3299-3307
Northern blotting has shown that mouse small intestine contains relatively
large amounts of the nuclear factor-E2 p45-related factor (Nrf) 2 transcrip
tion factor but relatively little Nrf1, Regulation of intestinal antioxidan
t and detoxication enzymes by Nrf2 has been assessed using a mouse line bea
ring 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), e
thoxyquin (EQ), or oltipraz] or phytochemicals [indole-3-carbinol, cafestol
and kahweol palmitate, sulfora-phane, coumarin (CMRN), or alpha -angelical
actone]. The constitutive level of NAD(P)H:quinone oxidoreductase (NQO) and
glutathione S-transferase (GST) enzyme activities in cytosols from small i
ntestine was typically found to be between 30% and 70% lower in samples pre
pared from Nrf2 mutant mice fed a control diet than in equivalent samples f
rom Nrf2(+/+) mice. Most of the chemopreventive agents included in this stu
dy 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 a
nimals fed diets supplemented with BHA or EQ; increases of about 2-fold wer
e observed with a mixture of cafestol and kahweol palmitate, CMRN, or alpha
-angelicalactone; and increases of 1.5-fold were measured dth sulfora-phan
e. Immunoblotting confirmed that in the small intestine, the constitutive l
evel of NQO1 is lower in the Nrf2(-/-) mouse, and it also showed that induc
tion of the oxidoreductase was substantially diminished in the mutant mouse
. Immunoblotting class-alpha and class-mu GST showed that constitutive expr
ession of most transferase subunits is also reduced in the small intestine
of Nrf2 mutant mice. Significantly, induction of class-alpha and class-mu G
ST by EQ, BHA, or CMRN is apparent in the gene knockout animal. No consiste
nt change in the constitutive levels of the catalytic heavy submit 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 diet
ary BHA or EQ, this induction was essentially abolished in the knockout mic
e. It is apparent that Nm influences both constitutive and inducible expres
sion 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 intestin
e. This suggests that the variation in inducibility of NQO1, Gsta1/2, and G
CS(h) in the mutant mouse is not attributable to the expression of the enzy
mes in distinct cell types but rather to differences in the dependency of t
hese genes on Nrf2 for induction.