Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice

Abstract

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50-80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.

Figure 1

Effect of nrf2 genotype on gastric and hepatic activities of GST and NQO1. GST (1-chloro-2,4-dinitrobenzene) and NQO1 (menadione) activities were measured in gastric and hepatic cytosols prepared from wild-type and nrf2-disrupted mice treated with vehicle or oltipraz (500 mg/kg, p.o.) 48 h before being killed. a, P < 0.05 compared with wild-type vehicle-treated control; b, P < 0.05 compared with wild-type mice treated with oltipraz. Values are mean ± SE for three to four animals in each group.

Figure 2

Nuclear Nrf2 levels after treatment of mice with oltipraz. Hepatic and gastric extracts were prepared from livers of mice treated for 6 h with either vehicle (Veh) or 500 mg/kg oltipraz (Olt). Std, purified recombinant Nrf2.

Figure 3

Effect of oltipraz on benzo[a]pyrene-induced neoplasia of the forestomach in female wild-type and nrf2-deficient mice. Female mice (7–9 weeks of age) were treated weekly with 500 mg/kg oltipraz 48 h before dosing with benzo[a]pyrene (120 mg/kg in corn oil, p.o.) for 4 consecutive weeks and killed 30 weeks after the initial treatment. Gastric tumors are reported as number of gastric tumors in the entire group/number of mice at risk at termination of experiment (number in parentheses). Open bar, vehicle-treated; shaded bar, oltipraz-treated.