Nrf2 is essential for protection against acute pulmonary injury in mice

Abstract

Nrf2 is a member of the "cap 'n' collar" family of transcription factors. These transcription factors bind to the NF-E2 binding sites (GCTGAGTCA) that are essential for the regulation of erythroid-specific genes. Nrf2 is expressed in a wide range of tissues, many of which are sites of expression for phase 2 detoxification genes. Nrf2(-/-) mice are viable and have a normal phenotype under normal laboratory conditions. The NF-E2 binding site is a subset of the antioxidant response elements that have the sequence GCNNNGTCA. The antioxidant response elements are regulatory sequences found on promoters of several phase 2 detoxification genes that are inducible by xenobiotics and antioxidants. We report here that Nrf2(-/-) mice are extremely susceptible to the administration of the antioxidant butylated hydroxytoluene. With doses of butylated hydroxytoluene that are tolerated by wild-type mice, the Nrf2(-/-) mice succumb from acute respiratory distress syndrome. Gene expression studies show that the expression of several detoxification enzymes is altered in the Nrf2(-/-) mice. The Nrf2(-/-) mice may prove to be a good in vivo model for toxicological studies. As oxidative damage causes DNA breakage, these mice may also be useful for testing carcinogenic agents.

Figure 1

Survival rate of wild-type and Nrf2^−/− mice given BHT. Solid lines represent the wild-type mice; broken lines represent the Nrf2^−/− mice. (A and B) Mice were fed 0.5% BHT (n = 36 for wild-type and Nrf2^−/− mice) or 1% BHT (n = 48 for wild-type mice, and n = 46 for Nrf2^−/− mice). (C and D) i.p. injection of 600 mg of BHT per kg of body weight (n = 12 for wild-type mice, and n = 13 for Nrf2^−/− mice) or 700 mg of BHT per kg of body weight (n = 12 for wild-type mice, and n = 11 for Nrf2^−/− mice). P.O., per os.

Figure 2

Photomicrographs of lungs at day 5 of 1% dietary BHT. (A and B) No difference is found between the lungs of untreated wild-type (Nrf2^+/+) and Nrf2^−/− mice. (C) The lung of a wild-type mouse fed 1% BHT shows thickening of the alveolar septa and some congestion. (D) In the lung of a mouse fed BHT, the alveolar architecture is mostly destroyed; extensive capillary damage was evident, and erythrocytes are found in the alveolar space. (Bars = 50 μm.)

Figure 3

LD₅₀ and LD₁₀₀ values for Nrf2^−/− injected i.p. with BHT. Solid line, wild-type mice; broken line, Nrf2^−/− mice. The LD₅₀ and LD₁₀₀ of Nrf2^−/− are 530 mg/kg and 660 mg/kg, respectively.

Figure 4

Localization of Nrf2 mRNA in adult intestine. In situ hybridization was performed on sections of adult intestine by using antisense probe (Left) and sense probe (Right). Each section is shown under brightfield (Upper) and darkfield (Lower) illumination. l, lumen; v, villi; g, intestinal gland; m, muscle. (Bars = 100 μm.)

Figure 5

Northern blot analyses of differentially expressed genes in the lungs. The ratios of gene expressions between the Nrf2^−/− mice and wild-type mice shown are calculated after normalization to their respective β-actin gene expression. WT, wild type.