OXYGEN-MEDIATED LUNG injury in mice Lacking the gene for NRF2: Rescue with the cytochrome P4501A-INDUCER, beta-naphthoflavone (BNF), and differential sex-specific effects

Background: Acute respiratory distress syndrome (ARDS) leads to progressive lung injury, which significantly impacts patient morbidity and mortality but may differ clinically between the sexes. Cytochrome P450 (CYP) 1A enzymes are protective against hyperoxic lung injury and may contribute to sex-dependent pathology. NRF2 is a critical transcriptional regulator of antioxidants and loss of NRF2 leads to severe hyperoxic lung injury and mortality in mice. NRF2 deficiencies and polymorphisms have been observed in patients with pulmonary diseases such as chronic obstructive pulmonary disease and severe asthma. No prior studies have evaluated whether there are sex-specific differences in oxygen-mediated lung injury in Nrf2^-/- mice and there are few rescue studies.

Objective: To test the hypothesis that hyperoxia induces greater lung injury and inflammation in Nrf2^-/- mice compared to wild type (WT) that differs between sexes, and that this phenotype will be rescued by the administration of the cytochrome P450 (CYP) 1A inducer beta-naphthoflavone (BNF).

Design: /Methods: Male and female 8-10-week-old WT or Nrf2^-/- C57BL/6 mice were pre-treated with BNF (40 mg/kg) or corn oil control and exposed to hyperoxia (95% O₂) for 68 hours. Survival, pulmonary edema, neutrophil recruitment, and lung injury scores were evaluated. Gene expression of phase II detoxification enzymes, pulmonary cytokines, and Cyp1a1/2 was quantified. CYP1A1/2 protein expression and catalytic activities were also measured.

Results: Hyperoxia exposure greatly reduced survival in Nrf2^-/- mice, particularly in females. BNF treatment improved survival by 182.8% in Nrf2^-/- females and by 41.4% in Nrf2^-/- males as well as in WT females by 85.7%. Females had greater pulmonary edema as measured by lung weight to body weight ratios but was attenuated in all groups except Nrf2^-/- females by BNF. Neutrophils doubled in Nrf2^-/- lungs compared to WT in hyperoxia but were decreased in BNF-treated females of both genotypes. Pulmonary cytokine gene expression including Il-6 and Tnf-α increased in hyperoxia especially in Nrf2^-/- mice and was unaffected by BNF. Pulmonary and hepatic Nqo1 gene expression w-as decreased in Nrf2^-/- mice and was largely unaffected by BNF; however pulmonary Ho-1 did not vary significantly between the genotypes and was decreased in WT animals treated with BNF. Activities and protein expression of pulmonary and hepatic CYP1A1/2 were induced via BNF across all groups. Although hepatic Cyp1a2 gene expression was higher in Nrf2^-/- males, the catalytic activity was higher in Nrf2^-/- females.

Conclusions: Hyperoxia augmented lung injury in Nrf2^-/- mice, and pre-treatment with BNF was protective against mortality and injury, eliminating the sex-dependent survival difference in both genotypes. Our results support the hypothesis that NRF2 protects mice against lung injury, and the fact that BNF rescues the lung injury phenotype in Nrf2^-/- mice suggests that augmented CYP1A expression by BNF may contribute to the beneficial effects. Further studies could lead to the development of BNF and other flavonoids for the prevention/treatment of hyperoxic lung injury, particularly in vulnerable patients with relative NRF2 deficiency, regardless of sex.

Keywords: ARDS; CYP1A; NRF2; hyperoxia; oxygen-mediated lung injury; sex differences.