Augmented oxygen-mediated transcriptional activation of cytochrome P450 (CYP)1A expression and increased susceptibilities to hyperoxic lung injury in transgenic mice carrying the human CYP1A1 or mouse 1A2 promoter in vivo

Abstract

Supplemental oxygen administration is frequently administered to pre-term and term infants having pulmonary insufficiency. However, hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Cytochrome P450 (CYP)A enzymes have been implicated in hyperoxic lung injury. In this study, we tested the hypothesis that hyperoxia induces CYP1A1 and 1A2 enzymes by transcriptional activation of the corresponding promoters in vivo, and transgenic mice expressing the human CYP1A1 or the mouse 1A2 promoter would be more susceptible to hyperoxic lung injury than wild type (WT) mice. Adult WT (CD-1) (12week-old) mice, transgenic mice carrying a 10kb human CYP1A1 promoter and the luciferase (luc) reporter gene (CYP1A1-luc), or mice expressing the mouse CYP1A2 promoter (CYP1A2-luc) were maintained in room air or exposed to hyperoxia for 24-72h. Hyperoxia exposure of CYP1A1-luc mice for 24 and 48h resulted in 2.5- and 1.25-fold increases, respectively, in signal intensities, compared to room air controls. By 72h, the induction had declined to control levels. CYP1A2-luc mice also showed enhanced luc expression after 24-48h, albeit to a lesser extent than those expressing the CYP1A1 promoter. Also, these mice showed decreased levels of endogenous CYP1A1 and 1A2 expression after prolonged hyperoxia, and were also more susceptible to lung injury than similarly exposed WT mice, with CYP1A2-luc mice showing the greatest injury. Our results support the hypothesis that hyperoxia induces CYP1A enzymes by transcriptional activation of its corresponding promoters, and that decreased endogenous expression of these enzymes contribute to the increased susceptibilities to hyperoxic lung injury in the transgenic animals. In summary, this is the first report providing direct evidence of hyperoxia-mediated induction of CYP1A1 and CYP1A2 expression in vivo by mechanisms entailing transcriptional activation of the corresponding promoters, a phenomenon that has implications for hyperoxic lung injury, as well as other pathologies caused by oxidative stress.

Figure 1

Bioluminescent imaging of CYP1A1-luc (A, B) or CYP1A2-luc (C,D) mice following hyperoxia exposures. CYP1A1-luc (A) or 1A2-luc mice (C) were maintained in room air or exposed to hyperoxia (> 95% O₂) for 24–72 h, and luciferase expression was analyzed by bioluminescent imaging in real time at the indicated time points Quantitation of bioluminescent imaging data of CYP1A1-luc (B) or 1A2-luc (D) mice was conducted using IVIS imaging software. Values represent mean ± S.E. (n = 5). *, Statistically significant differences between room air and hyperoxic mice at P < 0.05, as determined by two-way ANOVA.

Figure 2

Effect of hyperoxia on endogenous hepatic EROD (CYP1A1) (A), MROD (B), activities and apoprotein contents (C), and pulmonary EROD (D) and apoprotein expression (E). Adult male WT (CD-1), CYP1A1-luc or CYP1A2-luc mice were maintained in room air or exposed to hyperoxia at the indicated time points, and hepatic EROD (A), MROD (B) activities were determined in the liver microsomes. Data represent mean ± SE of fold induction versus room air controls from at least 4 individual animals. C, Representative Western blot showing effect of hyperoxia on endogenous hepatic CYP1A1 and 1A2 (C) expression in mice. PC, positive control showing induction of CYP1A1/1A2 by 3-methylcholanthrene. D and E represent pulmonary EROD and apoprotein expression, respectively, in microsomes of WT, CYP1A1-luc, and CYP1A2-luc mice.

Figure 3

Effect of hyperoxia on endogenous hepatic CYP1A1 (A), CYP1A2 (B), and pulmonary mRNA (C) in mice. Adult male WT (CD-1), CYP1A1-luc or CYP1A2-luc mice were maintained in room air or exposed to hyperoxia for 24–72 h, and hepatic CYP1A1 (A), CYP1A2 (B), and pulmonary CYP1A1 (C) mRNA levels were determined by real time RT-PCR at the indicated time points. Data represents mean ± S.E. of fold induction versus controls from at least four individual animals. *, Statistically significant differences between room air and hyperoxic mice at P < 0.05, as determined by two-way ANOVA.

Figure 4

Effect of hyperoxia on lung injury in WT, CYP1A1-Luc, and CYP1A2-mice. WT, CYP1A1-luc, and CYP1A2-luc mice were exposed to hyperoxia as described under Materials and Methods, and lung tissues were processed for histological analyses. The figure shows representative light micrographs of lung sections of mice maintained in room air or exposed to hyperoxia for 72 h. Air-breathing mice (upper panels) showed normal lung architecture. Hyperoxia for 72 h showed increased lung injury in WT mice. Single arrow shows infiltration of inflammatory cells, and double arrow shows areas of intra-alveolar edema. CYP1A1-luc mice showed even more injury after 72 h of hyperoxia, with lungs showing perivascular and intra-alveolar edema and marked widening of the adventitia of the pulmonary artery. CYP1A2-luc mice showed the greatest susceptibility to lung injury, as evidenced by intra-alveolar edema and proteinaceous materials filling the alveoli.