Hyperoxia-mediated transcriptional activation of cytochrome P4501A1 (CYP1A1) and decreased susceptibility to oxygen-mediated lung injury in newborn mice

Abstract

Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. In this study, we tested the hypothesis that newborn transgenic mice carrying the human CYP1A1-Luc promoter will display transcriptional activation of the human CYP1A1 promoter in vivo upon exposure to hyperoxia, and that these mice will be less susceptible to hyperoxic lung injury and alveolar simplification than similarly exposed wild type (WT) mice. Newborn WT (CD-1) or transgenic mice carrying a 13.2 kb human CYP1A1 promoter and the luciferase (Luc) reporter gene (CYP1A1-luc) were maintained in room air or exposed to hyperoxia (85% O₂) for 7-14 days. Hyperoxia exposure of CYP1A1-Luc mice for 7 and 14 days resulted in 4- and 30-fold increases, respectively, in hepatic Luc (CYP1A1) expression, compared to room air controls. In lung, hyperoxia caused a 2-fold induction of reporter Luc at 7 days, but the induction declined after 14 days. The newborn CYP1A1-Luc mice were less susceptible to lung injury and alveolar simplification than similarly exposed wild type (WT) CD-1 mice. Also, the CYP1A1-Luc mice showed increased levels of hepatic and pulmonary CYP1A1 expression and hepatic CYP1A2 activity after hyperoxia exposure. Hyperoxia also increased NADP(H) quinone reductase (NQO1) pulmonary gene expression in both CD-1 and CYP1A1-Luc mice at both time points, but this was more pronounced in the latter at 14 days. Our results support the hypothesis that hyperoxia activates the human CYP1A1 promoter in newborn mice, and that increased endogenous expression of CYP1A1 and NADP(H) quinone reductase (NQO1) contributes to the decreased susceptibilities to hyperoxic lung injury in the transgenic animals. This is the first report providing evidence of hyperoxia-mediated transcriptional activation of the human CYP1A1 promoter in newborn mice, and this in conjunction with decreased lung injury, suggests that these phenomena have important implications for BPD.

Keywords: Bronchopulmonary dysplasia (BPD); Cytochrome P450; Human CYP1A1-luciferase; Hyperoxia; Lung injury; Newborn.

Fig. 1

Effect of hyperoxia on luciferase expression in liver (A) and lung (B) tissues of CYP1A1-Luc mice, and endogenous hepatic (C) and pulmonary (D) Cyp1a1 gene expression in WT as well as CYP1A1-Luc mice at 7 and 14 day time points. Exposure to hyperoxia and determination of luciferase and mRNA expression are described under Materials and Methods. Values represent means ± SE of 3–5 mice from each group. *,**, and ***, Statistically significant differences between air and hyperoxia exposed newborn mice at P < 0.05, 0.01, and 0.001, respectively, by two-way ANOVA.

Fig. 2

Histological analysis of Hematoxylin and Eosin (H&E) stained lungs from WT (CD-1) and CYP1A1-Luc newborn mice exposed to room air and hyperoxia as described in methods. Zinc-formalin fixed, and paraffin-embedded sections were stained using H& E. Panels A and B are newborn WT (CD-1), and C and D are representative sections from CYP1A1-Luc exposed to air or hyperoxia for 14 days at 20X magnification. WT (CD1) group showed increased lung injury with perivascular edema and increased alveolar simplification after hyperoxia exposure (B). However, CYP1A1-Luc newborn mice even after hyperoxia exposure showed lesser lung injury and alveolar simplification (D). LW/BW ratio, which is an index of pulmonary edema, and RAC and MLI, which are quantitative indexes of alveolar simplification are depicted in panels E, F, and G, respectively. Values are means ± S.E.M. from 5 to 8 individual mice in air or hyperoxia groups exposed for 7 or 14 days. Significant differences in air and hyperoxia exposed newborn mice of different genotypes are indicated by *, **** at P < 0.05 or 0.0001, respectively.

Fig. 3

Effect of hyperoxia on hepatic EROD (A), MROD (B) and pulmonary EROD (C) activities, and apoprotein contents of endogenous pulmonary CYP1A1 (D,E,F) of WT (CD-1) and CYP1A1-Luc mice at 7 and 14 day time points. Values represent means ± SE of 3–5 mice from each group. *, **, and ****, Statistically significant differences between air and hyperoxia exposed newborn mice at P < 0.05, 0.01, and 0.0001, respectively, by two-way ANOVA.

Fig. 4

Effect of hyperoxia on hepatic (A) and pulmonary (B) NQO1 mRNA and pulmonary NQO1 protein levels (C,D,E) in WT (CD-1) and CYP1A1-Luc mice. NQO1 mRNA levels were determined by real time-PCR as described in Materials and Methods. Western blot analysis was performed using NQO1 specific antibodies with β-actin as a loading control. Values represent means ± SE of 3–5 mice from each group. *, **, ***, and ****, Statistically significant differences between air and hyperoxia exposed newborn mice at P < 0.05, 0.01, 0.001, and 0.0001, respectively, by two-way ANOVA.