Identification of Nrf2-regulated genes induced by the chemopreventive agent sulforaphane by oligonucleotide microarray

Electrophiles formed during metabolic activation of chemical carcinogens and reactive oxygen species generated from endogenous and exogenous sources play a significant role in carcinogenesis. Cancer chemoprevention by induction of phase 2 proteins to counteract the insults of these reactive intermediates has gained considerable attention. Nuclear factor E2 p45-related factor 2 (Nrf2), a bZIP transcription factor, plays a central role in the regulation (basal and or inducible expression) of phase 2 genes by binding to the "antioxidant response element" in their promoters. Identification of novel Nrf2-regulated genes is likely to provide insight into cellular defense systems against the toxicities of electrophiles and oxidants and may define effective targets for achieving cancer chemoprevention. Sulforaphane is a promising chemopreventive agent that exerts its effect by strong induction of phase 2 enzymes via activation of Nrf2. In the present study, a transcriptional profile of small intestine of wild-type (nrf2 +/+) and knock out (nrf2 -/-) mice treated with vehicle or sulforaphane (9 μmol/day for 1 week, p.o.) was generated using the Murine Genome U74Av2 oligonucleotide array (representing ∼6000 well-characterized genes and nearly 6000 expressed sequence tags). Comparative analysis of gene expression changes between different treatment groups of wild-type and nrf2-deficient mice facilitated identification of numerous genes regulated by Nrf2 including previously reported Nrf2-regulated genes such as NAD(P)H: quinone reductase (NQO1), glutathione S-transferase (GST), γ-glutamylcysteine synthetase (GCS), UDP-glucuronosyltransferases (UGT), epoxide hydrolase, as well as a number of new genes. Also identified were genes encoding for cellular NADPH regenerating enzymes (glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme), various xenobiotic metabolizing enzymes, antioxidants (glutathione peroxidase, glutathione reductase, ferritin, and haptaglobin), and biosynthetic enzymes of the glutathione and glucuronidation conjugation pathways. The data were validated by Northern blot analysis and enzyme assays of selected genes. This investigation expands the horizon of Nrf2-regulated genes, highlights the cross-talk between various metabolic pathways, and divulges the pivotal role played by Nrf2 in regulating cellular defenses against carcinogens and other toxins.