Newborn Mice Lacking the Gene for Cyp1a1 Are More Susceptible to Oxygen-Mediated Lung Injury, and Are Rescued by Postnatal β-Naphthoflavone Administration: Implications for Bronchopulmonary Dysplasia in Premature Infants

Abstract

Prolonged hyperoxia contributes to bronchopulmonary dysplasia (BPD) in preterm infants. β-Naphthoflavone (BNF) is a potent inducer of cytochrome P450 (CYP)1A enzymes, which have been implicated in hyperoxic injuries in adult mice. In this investigation, we tested the hypothesis that newborn mice lacking the Cyp1a1 gene would be more susceptible to hyperoxic lung injury than wild-type (WT) mice and that postnatal BNF treatment would rescue this phenotype by mechanisms involving CYP1A and/or NAD(P)H quinone oxidoreductase (NQO1) enzymes. Newborn WT or Cyp1a1-null mice were treated with BNF (10 mg/kg) or the vehicle corn oil (CO) i.p., from postnatal day (PND) 2 to 14 once every other day, while being maintained in room air or hyperoxia (85% O2) for 14 days. Both genotypes showed lung injury, inflammation, and alveolar simplification in hyperoxia, with Cyp1a1-null mice displaying increased susceptibility compared to WT mice. BNF treatment resulted in significant attenuation of lung injury and inflammation, with improved alveolarization in both WT and Cyp1a1-null mice. BNF exposed normoxic or hyperoxic WT mice showed increased expression of hepatic CYP1A1/1A2, pulmonary CYP1A1, and NQO1 expression at both mRNA and protein levels, compared with vehicle controls. However, BNF caused greater induction of hepatic CYP1A2 and pulmonary NQO1 enzymes in the Cyp1a1-null mice, suggesting that BNF protects against hyperoxic lung injury in WT and Cyp1a1-null mice through the induction of CYP1A and NQO1 enzymes. Further studies on the protective role of flavonoids against hyperoxic lung injury in newborns could lead to novel strategies for the prevention and/or treatment of BPD.

Keywords: bronchopulmonary dysplasia.; cytochrome P4501A1; lung; newborn; oxidative injury; β-naphthoflavone.

FIG. 1

A, Hematoxylin and eosin (H&E) staining of lungs from newborn mice treated with hyperoxia and postnatal BNF. Newborn WT (C57BL/6J) and Cyp1a1-null mice were treated the vehicle corn oil (CO) or BNF (10 mg/kg), and exposed to hyperoxia and room air as described in Materials and Methods section. On PND 15, the mice were sacrificed and lungs were inflated at constant pressure with buffered zinc formalin, and paraffin-embedded sections were stained using H& E. Upper panels are representative sections from WT mice and lower panels of lungs from newborn Cyp1a1-null mice postnatally exposed to CO or BNF followed by exposure to room air or hyperoxia (×200 magnification). In this, the CO + hyperoxia group showed increased lung injury with perivascular edema and increased alveolar simplification in both genotypes with Cyp1a1-null mice displayed more damage. But, BNF treatment (BNF + hyperoxia) resulted in significant attenuation and improved alveolarization. B and C, Effect of hyperoxia and postnatal BNF on lung/body Wt ratios in newborn mice. On PND 15, animals were sacrificed and LW/BW ratios which are indexes of lung injury were determined as described. D–G, Effect of hyperoxia and postnatal BNF on radial alveolar count (RAC) and mean linear intercept (MLI) or Lm in newborn mice. Alveolar simplification was quantified by RAC (D, E) and MLI (F, G). Values are means ± SEM from 3 to 5 individual WT and Cyp1a1-null newborn lung mice in air or hyperoxia groups with and without BNF treatment. *, Statistically significant differences between BNF and vehicle-treated mice at P ≤.05 by two-way analyses of variance.

FIG. 2

Differential expression of von Willebrand factor (VWF) in newborn mice lungs treated with hyperoxia and postnatal BNF. Representative immune-histochemical expression of VWF in newborn lungs from WT (A, B, C, and D) and Cyp1a1-null mice (E, F, G, and H) showed that BNF treatment restored the endothelial cells with increased angiogenesis in both genotypes. Ten to fifteen representative high-power field images were collected at ×200 magnification for each group.

FIG. 3

Hyperoxia-induced inflammation was decreased after BNF treatment. The immunohistochemistry of neutrophils was performed on newborn lungs of WT and Cyp1a1-null mice as described in Materials and Methods section with neutrophil specific antibody. Representative images of anti-neutrophil antibody immunostained lung sections (×400 magnification) obtained from WT (A, B, C, and D) or Cyp1a1-null mice (E, F, G, and H) are shown. Further, neutrophil infiltration was quantified after collecting 20 representative high-power field images at ×200 magnification for WT (I) and Cyp1a1-null (J) group.*, Statistically significant differences between BNF and vehicle-treated mice at P ≤.05 by two-way analyses of variance.

FIG. 4

Effect of hyperoxia and postnatal BNF on liver CYP1A, CYP1A2, CYP1B1, and NQO1 mRNA levels in newborn mice. The postnatal treatment of newborn WT and Cyp1a1-null mice was conducted as described in Materials and Methods section and legend to Figure 1. Livers were excised and total RNA was isolated, and CYP1A1 (A) CYP1A2 (B and C), CYP1B1 (D and E), and NQO1 mRNA (F and G) levels were determined by real-time PCR after cDNA synthesis, as described under Materials and Methods section. Values represent means ± SE of 3–5 mice from each group. *, Statistically significant differences between BNF and vehicle-treated mice at P ≤.05 by two-way analyses of variance.

FIG. 5

Real-time (Q-PCR) analysis showing the effects of postnatal BNF and hyperoxia exposure on lung CYP1A1, CYP1B1, and NQO1 mRNA levels in newborn mice. The mRNA expression in newborn lungs exposed to BNF and hyperoxia postnatally was described in detail in Materials and Methods section. The Q-PCR data for lung mRNA levels of CYP1A1 (A), CYP1B1 (B and C), and NQO1 mRNA (D and E) levels were determined by real-time-PCR after cDNA synthesis in different genotypes. Values represent means ± SE at least three mice from each group. *, Statistically significant differences between BNF and vehicle-treated mice at P ≤0.05 by two-way analyses of variance.

FIG. 6

Effect of postnatal BNF and hyperoxia exposure on hepatic EROD (A and B), MROD (C and D), and pulmonary EROD (E and F) activities of WT and Cyp1a1-null mice. After the postnatal treatment of newborn mice with hyperoxia and BNF as described in Materials and Methods section, at PND15 mice were sacrificed and collected tissues. From the excised liver and lung tissues, tissue homogenates were prepared, and EROD and MROD activities were measured as described under Materials and Methods section. Values represent means ± SE of 3–5 mice from each group. *, Statistically significant differences between BNF and vehicle-treated mice at P ≤.05 by two-way analyses of variance.

FIG. 7

Effect of postnatal BNF and hyperoxia exposure on hepatic and pulmonary CYP1A1/A2 apo-protein expression. The liver and lung homogenates were prepared from WT and Cyp1a1-null newborn mice exposed to BNF or hyperoxia. Western blot analysis was performed as described in Materials and Methods section. Representative western blots showing the expression of CYP1A1/1A2 in lungs and livers were shown with β-actin as a loading control.

FIG. 8

ChIP assays showing the binding of BNF–AHR–ARNT complex to the CYP1A1 promoter region. Lung tissues from WT newborn mice were collected after PND15. ChIP assays were performed using AhR-specific antibody and analyzed by end point PCR using forward and reverse primers designed in the promoter region of CYP1A1. Data indicate that hyperoxia also induces the CYP1A1 by binding of AhR to the promoter region of CYP1A1.

FIG. 9

Effect of hyperoxia on 8-iso-PGF levels. Hyperoxia induced 8 iso-PGF₂ levels in WT and Cyp1a1-null new born mice lungs. The lung homogenates prepared from WT and Cyp1a1-null mice were analyzed for ROS-mediated lipid peroxidative product by 8-iso-PGF₂ by LCMS. Values represent means ± SE of 3–5 mice from each group. *, Statistically significant differences between room air and hyperoxia groups in both genotypes at P ≤.05 by two-way analyses of variance.