The antioxidant response element (ARE) and Nrf2 are known to regulate the 
expression and coordinated induction of genes encoding detoxifying enzymes 
including NAD(P)H:quinone oxidoreductase1 (NQO1) in response to antioxidants. In 
this report, we demonstrate that overexpression of the transcription factor 
Bach1 in Hep-G2 cells negatively regulated NQO1 gene expression and induction in 
response to antioxidant t-BHQ. Bandshift and supershift assays revealed that 
Bach1 binds to the ARE as a heterodimer with small Maf proteins but not as a 
homodimer or heterodimer with Nrf2. The transfection and ChIP assays revealed 
that Bach1 and Nrf2 competed with each other to regulate ARE-mediated gene 
expression. Heme, a negative regulator of Bach1 relieved the Bach1 repression of 
NQO1 gene expression in transfected cells. The transcription of Bach1 and Nrf2 
did not change in response to t-BHQ. Immunofluorescence assays and Western blot 
analysis revealed that both Bach1 and Nrf2 localized in the cytoplasm and 
nucleus of the untreated cells. The treatment of cells with t-BHQ resulted in 
the nuclear accumulation of both Bach1 and Nrf2. Interestingly, the 
t-BHQ-induced nuclear accumulation of Bach1 was significantly delayed over that 
of Nrf2. These results led to the conclusion that a balance of Nrf2 versus Bach1 
inside the nucleus influences up- or down-regulation of ARE-mediated gene 
expression. The results further suggest that antioxidant-induced delayed 
accumulation of Bach1 contributes to the down-regulation of ARE-regulated genes, 
presumably to reduce the antioxidant enzymes to normal levels.