Mice that lack the Nrf2 basic-region leucine-zipper transcription factor are more sensitive than wild-type (WT) animals to the cytotoxic and genotoxic effects of foreign chemicals and oxidants. To determine the basis for the decrease in tolerance of the Nrf2 homozygous null mice to xenobiotics, enzyme assay, Western blotting and gene-specific real-time PCR (TaqMan®) have been used to examine the extent to which hepatic expression of GSH-dependent enzymes is influenced by the transcription factor. The amounts of protein and mRNA for class Alpha, Mu and Pi glutathione S-transferases were compared between WT and Nrf2 knockout (KO) mice of both sexes under both constitutive and inducible conditions. Among the class Alpha and class Mu transferases, constitutive expression of Gsta1, Gsta2, Gstm1, Gstm2, Gstm3, Gstm4 and Gstm6 subunits was reduced in the livers of Nrf2 mutant mice to between 3% and 60% of that observed in WT mice. Induction of these subunits by butylated hydroxyanisole (BHA) was more marked in WT female mice than in WT male mice. TaqMan® analyses showed the increase in transferase mRNA caused by BHA was attenuated in Nrf2−/− mice, with the effect being most apparent in the case of Gsta1, Gstm1 and Gstm3. Amongst class Pi transferase subunits, the constitutive hepatic level of mRNA for Gstp1 and Gstp2 was not substantially affected in the KO mice, but their induction by BHA was dependent on Nrf2; this was more obvious in female mutant mice than in male mice. Nrf2 KO mice exhibited reduced constitutive expression of the glutamate cysteine ligase catalytic subunit, and, to a lesser extent, the expression of glutamate cysteine ligase modifier subunit. Little variation was observed in the levels of glutathione synthase in the different mouse lines. Thus the increased sensitivity of Nrf2−/− mice to xenobiotics can be partly attributed to a loss in constitutive expression of multiple GSH-dependent enzymes, which causes a reduction in intrinsic detoxification capacity in the KO animal. These data also indicate that attenuated induction of GSH-dependent enzymes in Nrf2−/− mice probably accounts for their failure to adapt to chronic exposure to chemical and oxidative stress.
- antioxidant responsive element
- cancer chemoprevention
- glutamate cysteine ligase (also called γ-glutamylcysteine synthetase)
↵1 These individuals contributed equally to this work, and should therefore be regarded as joint first authors.
The nucleotide sequence data reported for mouse Gstm4 will appear in DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AF501320.
Abbreviations used: AGLN, α-angelicalactone; ARE, antioxidant-responsive element; BaP, benzo[a]pyrene; BHA, butylated hydroxyanisole; t-BHQ, t-butylhydroquinone; bZIP, basic-region leucine-zipper; CDNB, 1-chloro-2,4-dinitrobenzene; C+K, cafestol and kahweol palmitate; CMRN, coumarin; 3-OH CMRN, 3-hydroxy-CMRN; 7-OH CMRN, 7-hydroxy-CMRN; ECL, enhanced chemiluminescence; EQ, ethoxyquin; EST, expressed sequence tag; GCL(C), glutamate cysteine ligase (catalytic subunit); GCLM, GCL modifier subunit; GST, glutathione S-transferase; Gsta1/2, GST class Alpha 1/2; Gstm1/2/3/4/5/6, GST class Mu 1/2/3/4/5/6; Gstp1/2, Gst class Pi 1/2; I3C, indole-3-carbinol; KO, knockout; LMTN, limettin; Nrf2, Nuclear factor-erythroid 2 p45-related factor 2; OPZ, oltipraz; RACE, rapid amplification of cDNA ends; SUL, sulforaphane; WT, wild-type.
- The Biochemical Society, London ©2002