Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2019 Jan 15.
Published in final edited form as: Toxicol Appl Pharmacol. 2017 Nov 26;339:133–142. doi: 10.1016/j.taap.2017.11.017

β-Naphthoflavone treatment attenuates neonatal hyperoxic lung injury in wild type Cyp1a2-knockout mice

Krithika Lingappan, Paramahamsa Maturu, Yanhong Wei Liang, Weiwu Jiang, Lihua Wang a, Bhagavatula Moorthy, Xanthi I Couroucli
PMCID: PMC5758404  NIHMSID: NIHMS928925  PMID: 29180065

Abstract

Exposure to supraphysiological concentrations of oxygen (hyperoxia) leads to bronchopulmonary dysplasia (BPD), one of the most common pulmonary morbidities in preterm neonates, which is more prevalent in males than females. Beta-naphthoflavone (BNF) is protective against hyperoxic lung injury in adult and neonatal wild type (WT) mice and in and mice lacking Cyp1a1gene. In this investigation, we tested the hypothesis that BNF treatment will attenuate neonatal hyperoxic lung injury in WT and Cyp1a2−/− mice, and elucidated the effect of sex-specific differences. Newborn WT or Cyp1a2−/− mice were treated with BNF (10 mg/kg) or the vehicle corn oil (CO) i.p., from postnatal day (PND) 2 to 8 once every other day, while being maintained in room air or hyperoxia (85% O2) for 14 days. Hyperoxia exposure lead to alveolar simplification and arrest in angiogenesis in WT as well as Cyp1a2−/− mice No significant differences were seen between WT and Cyp1a2−/− mice. Cyp1a2−/− female mice had better preservation of pulmonary angiogenesis at PND15 compared to similarly exposed males. BNF treatment attenuated lung injury and inflammation in both genotypes, and this was accompanied by a significant induction of hepatic and pulmonary CYP1A1 in WT but not in Cyp1a2−/− mice. BNF treatment increased NADPH quinone oxidoreductase (NQO1) mRNA levels in Cyp1a2−/− mouse livers compared to WT mice. These results suggest that BNF is protective in neonatal mice exposed to hyperoxia independent of CYP1A2 and this may entail the protective effect of phase II enzymes like NQO1.

Keywords: Hyperoxia, Neonate, Bronchoplumonary Dysplasia, CYP1A2, beta-naphthoflavone

INTRODUCTION

Exposure to supraphysiological concentrations of oxygen (hyperoxia) leads to arrest in alveolarization and angiogenesis in the developing lung. These are the hallmarks of bronchopulmonary dysplasia (BPD), one of the most common pulmonary morbidities in preterm neonates(Bhandari, 2010; Jobe, 2011). The alveoli are fewer in number and larger in size, decreasing the gas-exchange area in the lung. Also, the dysmorphic pulmonary vasculature leads to the development of pulmonary hypertension and additional long-term morbidities (Baker and Abman, 2015; Stenmark and Abman, 2005). Unfortunately, the incidence of BPD remains high despite advances in neonatal intensive care and has long –term consequences on lung function. There is an urgent need for therapeutic strategies that ameliorate the adverse effects of hyperoxia on the developing lung to decrease the incidence of BPD.

Hyperoxia exposure leads to the generation of reactive oxygen species (ROS), which in turn cause cellular damage in the lung by oxidizing nucleic acids, proteins, and membrane lipids. Previous studies have established the role of the cytochrome P450 (CYP) 1A subfamily in modulating hyperoxic lung injury (Bhakta et al., 2008; Couroucli et al., 2002; Okamoto et al., 1993; Shertzer et al., 2004; Sindhu et al., 2000). In mouse and humans, the CYP1A subfamily comprises of two genes; namely, CYP1A1 (Cyp1a1 in mice) and CYP1A2 (Cyp1a2 in mice). The CYP1A subfamily of enzymes metabolizes numerous endogenous and exogenous chemicals and their induction is regulated by the aryl hydrocarbon receptor (AHR). The AHR gene battery consists of many phase I and phase II metabolizing enzymes including CYP1A1, CYP1A2, CYP1B1, NQO1 and GSTA1. CYP1A1 is expressed mainly in the extra-hepatic tissues (rodent and human lung, intestine, placenta and kidney), while CYP1A2 is expressed mainly in the rodent and human liver. In a model of acute hyperoxic lung injury in adult mice, both Cyp1a1−/− and Cyp1a2−/− mice display a phenotype of exaggerated lung injury, inflammation and oxidative stress, thus highlighting the protective role of these enzymes in adult mice (Lingappan et al., 2014; 2017; Wang et al., 2015). In adult mice, hepatic CYP1A2 plays a critical role in the attenuation against hyperoxic lung injury by decreasing lipid peroxidation and oxidative stress in vivo (Wang et al., 2015). In neonatal mice, we have shown that prenatal induction of the CYP1A subfamily with BNF attenuated lung injury and that mice lacking the Cyp1a1 gene have greater arrest in alveolarization and angiogenesis when exposed to postnatal hyperoxia (Couroucli et al., 2011; Maturu et al., 2017). Exposure to postnatal hyperoxia for varying duration leads to a lung phenotype comprising of alveolar simplification similar to that observed in human neonates with BPD (Berger and Bhandari, 2014).

The expression of CYP1A2 is minimal in the fetal and neonatal liver, and the expression gradually increases beyond the second week of life (Hart et al., 2009). However, the enzyme is inducible in newborn mice (Couroucli et al., 2011). Prenatal administration of beta-naphthoflavone for three days on gestational days (E17–19) protected neonatal mice against lung injury, when subjected to hyperoxia postnatally. This was accompanied by induction in Cyp1a2 gene expression both in the fetal and neonatal liver (Couroucli et al., 2011). The role of CYP1A2 in modulating hyperoxic lung injury in the newborn period has not been investigated. Sex-specific differences have been observed in the incidence of BPD in premature neonates with male infants showing a greater predilection for the development of this disease in multiple clinical studies (Shim et al., 2017). In adult and neonatal mice exposed to hyperoxia, similar pattern was observed with increased lung injury observed in male mice(Lingappan et al., 2013; 2016). Differential expression of CYP1A1 and CYP1A2 was also observed (Lingappan et al., 2015). We hypothesized that in the absence of the Cyp1a2 gene BNF will not have protective effect in the setting of neonatal hyperoxia exposure. We also tested the effect of sex as a biological variable on the outcome measures related to lung development and angiogenesis in this experimental model.

MATERIALS AND METHODS

Animals

This study was conducted in accordance with the federal guidelines for the humane care and use of laboratory animals, and was approved by the Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine. Breeding pairs of WT mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Breeding pairs of Cyp1a2−/− mice of mixed background (C57BL/6J/Sv129) were obtained from Dr. Frank J. Gonzalez (National Cancer Institute, Bethesda, MD). The mice were backcrossed with C57BL/6J mice for 12 generations to generate theoretically a >99.2% C57BL/6J background. The dams were fed standard mice food (Purina Rodent Lab Chow 5001 from Purina Mills, Inc., Richmond, IN) and water ad libitum. Animals were maintained in 12-h day/night cycles.

Hyperoxia exposure and tissue collection

Newborn pups and dams of either genotype were placed in plexi-glass chambers within 12 hours of birth and were exposed to either 21% O2 (room air) or 85% O2 from PND 1–14. This period extends through the saccular and alveolar stages of lung development in the mouse. This model has been validated to produce a phenotype similar to the human BPD (Hilgendorff et al., 2013). Oxygen was delivered through a humidified circuit at a flow rate of 5 L/min through a blender to was monitored continuously by achieve an inspired oxygen concentration of 85%. FiO2 means of in-line analyzers at the out port of the chambers. The dams were rotated between room air and hyperoxia-exposed litters every 24 h to prevent oxygen toxicity in the dams. Mouse pups were treated with BNF (10 mg/kg) dissolved in corn oil (CO) or CO, intraperitoneally (i.p.) once every other day with, from postnatal days (PND) 2 to 8. The mice were sacrificed on PND 15. The animals were anesthetized with sodium pentobarbital (200 mg/kg i.p.) and euthanized by exsanguination while under deep pentobarbital anesthesia. Six animals from each group (three males and three females) were used for lung histopathology. Lung and liver tissues were harvested for mRNA and protein isolation. Lung weight and body weight were also measured on PND15.

Analysis of Lung Morphometry

Lungs were inflated through an intratracheal catheter with buffered zinc formalin (10%) and fixed in with the same solution at a constant pressure of 25 cm H20 for at least 10 min. Samples were left in solution for 24 h in 10% zinc formalin, and then transferred to 70% ethanol for long-term storage, following which they were embedded in paraffin for subsequent histological analyses. Lungs were sectioned at 4 um thickness on a rotary microtome and were stained with hematoxylin and eosin (H&E). Alveolar development was evaluated at PND15 (n=6/group) by radial alveolar counts (RAC) (Cooney and Thurlbeck, 1982) and mean linear intercept (MLI) (McGowan et al., 2000) as described before (Nicola et al., 2009). Fifteen randomly chosen areas were photographed (200x magnification). Fields containing large airways and vessels were not included. Analysis of each section was carried out in a blinded fashion

Immunohistochemistry and quantitation for assessment of angiogenesis and inflammatory cell infiltration in the lungs

Pulmonary vascular density was measured in lung sections stained for Von Willebrand factor (VWF) with anti-Von Willebrand Factor polyclonal antibody (Abcam-Cat# ab6994) at 1:500 dilution. Ten random non-overlapping fields were assessed at 200x magnification for each animal (n=6/group; 3 males and 3 females). Neutrophils were immunostained with rat anti-mouse ly-6b.2 antibody (AbD Serotec; catalog number: MCA771G) at 1:200 dilution and macrophages using the anti-F4/80 antibody (1:500 dilution, Bio Rad laboratories; catalog number: MCA497GA). Quantitation of neutrophils and macrophages was performed by averaging the counts from at least 20 random high power fields.

Liver and Lung tissue western blotting

The snap frozen lung samples were weighed and added to 1 ml of lysis buffer (PBS+ cocktail of protease inhibitor). The tissues were ground and the samples were incubated on a rocker at 4°C for 15 minutes followed by centrifugation at 10,000 G for 15 minutes. The supernatants were removed and protein concentrations were determined using the BCA (bicinchoninic acid) method. Liver and lung whole protein homogenates (20 μg) prepared from individual animals (n=3/group) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 10% acrylamide gels. The separated proteins on the gels were transferred to polyvinylidene difluoride membranes, followed by Western blotting using monoclonal antibodies to detect CYP1A1 (1:1500 dilution), which cross-reacts with CYP1A2 (Couroucli et al., 2002; Jiang et al., 2004; Moorthy et al., 2000) and NQO1 mouse monoclonal antibody (Santa Cruz-Cat#393736; 1:1000 dilution). β-actin was used as the loading control. This was followed by electrochemical detection of bands. Band intensities were quantified using Image Lab ™ 6.0 Software (Bio Rad laboratories) (Taylor et al., 2013).

CYP1A1 (EROD) and CYP1A2 (MROD) assays

Lung and liver samples at the time of dissection were frozen immediately with liquid nitrogen and maintained at a temperature of − 80°C. Homogenized lung and liver tissues were mixed with Tris-sucrose buffer (0.25M sucrose, 80 mM Tris, 5 mM MgCl2, 0.25 mM KCl, 1 mM EDTA) pH 7.4 and centrifuged at 12 000 rpm for 10 min. The supernatant was collected and after estimation of protein concentration was subjected to EROD and MROD assays (n=3/group). Ethoxyresorufin O-deethylase (EROD) (CYP1A1) activities in lung and liver and methoxyresorufin O-demethylase (MROD) (CYP1A2) activities in liver tissues were assayed as described previously (Moorthy et al., 1997).

Quantitative Real time PCR assays

RNA was isolated from snap frozen liver and lung samples using Direct-Zol RNA mini prep kit (Zymo research, Cat#R2052) with a DNase step, according to the instructions of the manufacturer. For quantitative real-time PCR, 1 μg of total RNA from livers and lungs with different treatment conditions was reverse-transcribed in 20 μL of using iScript Reverse Transcription super mix (Biorad-Cat#170-8841) according to manufacturer’s instructions. The cDNA (2μl) and the real-time PCR primers for CYP1A1 (Forward 5′-GGTTAACCATGACCGGGAACT-3′ Reverse 5′-TGCCCAAACCAAAGAGAGTGA-3′) and NQO1 (Forward 5′-GGAAGCTGCAGACCTGGTGA-3′ Reverse 5′ CCTTTCAGAATGGCTGGCA-3′) were used in final 20 μL qPCR reaction with a SYBR-green master mix (Qiagen-Cat#2014143). Real-time qPCR was performed in an ABI-Prism7700 sequence detection system (n=3/group). PCR conditions were with denaturation at 95°C for 15 sec and annealing and extension at 60°C for 1 minute, with 40 PCR cycles. Data was analyzed by ΔΔ Ct method; the expression of the target genes such as CYP1A1 and NQO1 were normalized to β-Actin as an endogenous control.

Statistical analysis

Based on preliminary results of hyperoxia-induced alveolar simplification in WT neonatal mice, it was estimated that 5 animals per group would provide 80% power at a significance level of 0.05 for the effect size of 5 μm in mean linear intercept (mean difference) between the two genotypes (WT and Cyp1a2−/−). Data are expressed as means ± SEM. 3-way analyses of variance (ANOVA) (effect of genotype and BNF-treatment or sex and hyperoxia exposure), followed by modified t-tests, were used to assess significant differences arising from exposure to hyperoxia and room air, WT and Cyp1a2−/− mice, male and female mice and BNF and corn-oil treated mice. P-values of < 0.05 were considered significant.

RESULTS

Effect of hyperoxia on Body weight (BW) and Lung weight (LW)

Cyp1a2−/− mice weighed less than WT mice on PND15 in room air. Chronic hyperoxia exposure led to a significant weight loss in WT mice and BNF treatment prevented this. In Cyp1a2−/− mice, BNF treatment led to an increase in body weight both in room air and under hyperoxic conditions compared to unexposed controls (Figure 1A). Lung weights did not show a statistically significant increase in any group after hyperoxia exposure; however BNF treatment decreased lung weight under hyperoxic conditions in W T mice compared to unexposed controls (Figure 1B).

Figure 1. Effect of hyperoxia on Body weight (BW) and Lung weight (LW).

Figure 1

Open in a new tab

Figure 1A shows the effect of hyperoxia exposure (PND 1–14; 85% FiO2) on body weights of WT and Cyp1a2 −/− mice treated with corn-oil or BNF. 1B: Effect of hyperoxia exposure (PND 1–14; 85% FiO2) on body weights of WT and Cyp1a2 −/− mice treated with corn-oil or BNF. Values are means ± SEM from at least 5–10 individual animals. Significant differences between room air and hyperoxia exposed mice are indicated by *, p<0.05. Differences between WT and Cyp1a2−/− mice are indicated by δ, p <0.05 and those between corn-oil and BNF treated are indicated by ##, p <0.01 and ###, p<0.001.

Neonatal WT and Cyp1a2−/− mice exposed to hyperoxia had increased alveolar simplification, which was ameliorated by BNF

WT and Cyp1a2−/− newborn mice were exposed to room air or 85% O2 from PND1–14 and lung morphometry was assessed to quantitate the effect on lung development. Representative lung fields from room air and hyperoxia exposed WT (Figure 2A–D) and Cyp1a2−/− mice (Figure 2E–H) are shown at 20x magnification. Exposure to hyperoxia caused dramatic alveolar simplification as seen in the lung sections with fewer and larger alveoli in both WT and Cyp1a2−/− mice. Alveolar size was quantified using mean linear intercept (MLI) and radial alveolar count (RAC) using established methods. These results are shown in Figure 3A–F. Hyperoxia significantly increased MLI and decreased RAC in hyperoxia-exposed mice of either genotype. Analysis by 3-way ANOVA showed that for MLI, effect of hyperoxia treatment was significant; while the effects of sex and genotype were not significant. In mice with BNF administration, radial alveolar count was decreased to a greater extent in Cyp1a2−/− males compared to WT males. There were no sex-specific differences noted. When sexes were combined, BNF treatment attenuated the adverse impact of hyperoxia on alveolarization and preserved MLI in both WT and Cyp1a2−/− mice and preserved RAC in WT mice (Figure 3C and F).

Figure 2. Effect of hyperoxia on lung histopathology.

Figure 2

Open in a new tab

Representative hematoxylin and eosin stained images from the lungs of WT and Cyp1a2−/− mice (n=6 mice per group) WT and Cyp1a2−/− mice were exposed to hyperoxia (PND 1–14; 85% FiO2) and treated with corn-oil or BNF. Hyperoxia exposed WT and Cyp1a2−/− mice show more alveolar simplification compared to mice in room-air conditions. Treatment with BNG ameliorates the adverse effects of hyperoxia on lung alveolarization.

Figure 3. Effect of hyperoxia on lung morphometry.

Figure 3

Open in a new tab

To quantitate the effects of hyperoxia exposure on alveolarization, mean linear intercept (Figures 3A–C) and radial alveolar count (Figures 3D–F) was measured in WT and Cyp1a2−/− mice. Figure 3A: Mean linear intercept in male and female mice (WT and Cyp1a2−/−) in the corn-oil group. Figure 3B: Mean linear intercept in male and female mice (WT and Cyp1a2−/−) that received treatment with BNF. Figure 3C: Effect of BNF treatment on MLI under hyperoxia and room air conditions compared to corn-oil treated controls. Figure 3D: Radial alveolar count (RAC) in male and female mice (WT and Cyp1a2−/−) in the corn-oil group. Figure 3E: RAC in male and female mice (WT and Cyp1a2−/−) that received treatment with BNF. Figure 3F: Effect of BNF treatment on RAC under hyperoxia and room air conditions compared to corn-oil treated controls. Values are means ± SEM from at least 3 individual animals of each sex and genotype. Significant differences between room air and hyperoxia exposed mice are indicated by *, p<0.05 and ***, p<0.001. Differences between WT and Cyp1a2−/− mice are indicated by δ, p <0.05 and those between corn-oil and BNF treated are indicated by ###, p<0.001.

Postnatal hyperoxia exposure leads to arrest in angiogenesis in both WT and Cyp1a2−/− mice and is recued by BNF treatment

To assess differences in angiogenesis between WT and Cyp1a2−/− neonatal mice with or without BNF treatment, we quantified vessel number using ant-vWF antibodies. Representative immunostained lung sections are shown in figures 4A–H. Hyperoxia significantly decreased vessel development in the lung in both WT and Cyp1a2−/− mice of either sex (Figure 4I); however, Cyp1a2−/− males had a greater arrest in angiogenesis compared to Cyp1a2−/− females, upon exposure to hyperoxia. Treatment with BNF ameliorated this process to a greater extent in WT mice compared to Cyp1a2−/− mice (Figure 4K). Among WT mice, males displayed no significant arrest in angiogenesis with BNF treatment, while this was still observed in Cyp1a2−/− males (Figure 4J).

Figure 4. Effect of hyperoxia on lung vessel development.

Figure 4

Open in a new tab

To assess differences in angiogenesis among WT and Cyp1a2−/− neonatal mice with or without BNF treatment, we quantified vessel number using ant-vWF antibodies with immunohistochemistry on PND15 after the completion of hyperoxia exposure. Representative lung sections from all the experimental groups are shown in figure panels A–H (4A–D in WT and 4E–H in Cyp1a2−/− mice). Arrows point to brown-staining vessels. Figures 4I–K: Quantitative analyses showing number of vessels per high-power field in lungs of WT and Cyp1a2−/− neonatal mice. Figure 4I: Vessel number/high power field (hpf) in male and female mice (WT and Cyp1a2−/−) in the corn-oil group. Figure 4J: Vessel number/hpf in male and female mice (WT and Cyp1a2−/−) that received treatment with BNF. Figure 4K: Effect of BNF treatment on vessel number/hpf under hyperoxia and room air conditions compared to corn-oil treated controls. Values are means ± SEM from at least 3 individual animals of each sex and genotype. Significant differences between room air and hyperoxia exposed mice are indicated by *, p<0.05, **, p<0.01 and ***, p<0.001. Differences between WT and Cyp1a2−/− mice are indicated by δ, p <0.05 and those between corn-oil and BNF treated are indicated by #, p<0.05 and ###, p<0.001.

Effect of hyperoxia on pulmonary macrophage and neutrophil infiltration

Exposure to hyperoxia caused a significant increase in macrophage infiltration in the lungs of WT mice of either sex (Figure 5A), this was significantly decreased by BNF treatment compared to corn-oil treated controls (Figure 5C). Cyp1a2−/− mice did not show increased macrophage infiltration in the lungs upon exposure to hyperoxia. Among BNF treated mice, no difference was seen due to hyperoxia treatment or between WT mice and Cyp1a2−/− mice (Figure 5B). Representative lung sections from room-air or hyperoxia exposed WT mice (with corn-oil or BNF treatment) stained for macrophages are shown in Figures 5D. Similarly, neutrophils were increased in the lungs of corn-oil treated WT mice exposed to hyperoxia compared to room air controls of either sex and no significant increase was seen in Cyp1a2−/− mice (Figure 5E). Among BNF treated mice, WT males had decreased pulmonary neutrophils after hyperoxia treatment (Figure 5F). When both sexes were combined, analysis showed that BNF treatment significantly decreased neutrophil recruitment in the lung of hyperoxia-exposed WT mice compared to room-air controls (Figure 5G). Representative lung sections immunostained for neutrophils from WT mice are shown in Figure 5H.

Figure 5. Effect of hyperoxia on pulmonary neutrophil and macrophage infiltration.

Figure 5

Open in a new tab

Representative lung sections (40x magnification) immunostained for macrophages (Figure 5D) and neutrophils (Figure 5H) in lungs of WT and Cyp1a2−/− neonatal mice. Quantitation of macrophages (Figure 5A–C) and neutrophils (5E–G) in the different experimental groups is also shown. Figure 5A: Macrophages/high power field (hpf) in male and female mice (WT and Cyp1a2−/−) in the corn-oil group. Figure 5B: Macrophages/hpf in male and female mice (WT and Cyp1a2−/−) that received treatment with BNF. Figure 5C: Effect of BNF treatment on macrophages/hpf under hyperoxia and room air conditions compared to corn-oil treated controls. Figure 5E: Neutrophils/high power field (hpf) in male and female mice (WT and Cyp1a2−/−) in the corn-oil group. Figure 5F: Neutrophils/hpf in male and female mice (WT and Cyp1a2−/−) that received treatment with BNF. Figure 5G: Effect of BNF treatment on neutrophils/hpf under hyperoxia and room air conditions compared to corn-oil treated controls. Values are means ± SEM from at least 3 individual animals of each sex and genotype. Significant differences between room air and hyperoxia exposed mice are indicated by *, p<0.05. Differences between corn-oil and BNF treated are indicated by #, p<0.05 and ###, p<0.001.

Pulmonary and hepatic expression of Cyp1a1 and Cyp1a2 after hyperoxia exposure in WT and Cyp1a2−/− mice

Upon exposure to hyperoxia, there was a significant induction of Cyp1a1 in WT mice administered corn-oil (Figure 6A). A similar induction was noted in BNF treated WT mice exposed to hyperoxia and the increase was greater compared to corn-oil treated controls. In Cyp1a2−/− mice, there was no induction in Cyp1a1 expression under hyperoxia and no increase was seen with BNF treatment. Thus, genotype had a significant effect on pulmonary Cyp1a1 expression and absence of the Cyp1a2 gene abrogated the induction in Cyp1a1 mRNA expression in the lung under hyperoxic conditions in Cyp1a2−/− mice. Western blot analysis for CYP1A1 protein expression (Figure 6B) showed an induction in WT mice treated with BNF under room air. The significant induction in gene expression under hyperoxia was not observed in the expression of the corresponding protein. This difference between mRNA and protein levels may be attributed to alteration in post-transcriptional regulation or due to increased protein catabolism, including the ubiquitin-proteosome system as shown previously (Chambellan et al., 2006). Similar to mRNA levels, Cyp1a2−/− mice showed no induction of CYP1A1 either under hyperoxic conditions or with BNF treatment. In the liver, BNF administration lead to a significant increase in CYP1A1 expression in normoxic mice compared to corn-oil treated controls. Hyperoxia exposure significantly decreased hepatic Cyp1a1 expression in BNF treated WT mice. Cyp1a2−/− mice did not show a significant increase in hepatic Cyp1a1 expression despite BNF treatment. This was similar to the absence of induction in Cyp1a1 gene expression noted in the lung in Cyp1a2−/− mice. Liver western blot (Figure 6D) showed induction of CYP1A1 in BNF treated WT mice as shown in Figure 6C. A CYP1A1 protein band was not detected in any of the other experimental groups neither in WT mice nor in Cyp1a2−/− mice.

Figure 6. Effect of hyperoxia on pulmonary and hepatic Cyp1a1 mRNA and CYP1A1 protein expression in WT and Cyp1a2−/− mice.

Figure 6

Open in a new tab

Figure 6A: Pulmonary Cyp1a1 mRNA expression in WT and Cyp1a2−/− mice exposed to hyperoxia or room air that received corn-oil or BNF treatment. Figure 6B: Representative western blot image and densitometric analysis of lung CYP1A1 protein expression in WT and Cyp1a2−/− mice exposed to hyperoxia or room air that received corn-oil or BNF treatment. The positive control (labeled “PC”) was 0.5 μg of liver microsomes from mice treated with 3-MC. Figure 6C: Hepatic Cyp1a1 mRNA expression in WT and Cyp1a2−/− mice exposed to hyperoxia or room air that received corn-oil or BNF treatment. Figure 6D: Representative western blot image of liver CYP1A1 protein expression in WT and Cyp1a2−/− mice exposed to hyperoxia or room air that received corn-oil or BNF treatment. “PC” refers to positive control as described above. Values are means ± SEM from at least 3 individual animals of each genotype. Significant differences between room air and hyperoxia exposed mice are indicated by **, p<0.01 and ***, p<0.001. Differences between corn-oil and BNF treated are indicated by ###, p<0.001. Differences between WT and Cyp1a2−/− mice are indicated by δδδ, p <0.001.

CYP1A1 (EROD) and CYP1A2 (MROD) activity in in WT and Cyp1a2−/− mice upon hyperoxia exposure

Liver MROD assay, which measures CYP1A2 activity, showed a significant increase in BNF treated WT mice under room air conditions, and a significant decrease with hyperoxia exposure. As expected, MROD activity in Cyp1a2−/− mice was very low (Figure 7A). Liver EROD assay measures the CYP1A1 activity. There was a significant induction in EROD activity with BNF administration in WT mice, which was decreased upon hyperoxia exposure. Similar to mRNA levels, there was no induction in EROD (CYP1A1) activity in Cyp1a2−/− mice either with hyperoxia exposure or BNF administration (Figure 7B). Lung EROD activity increased with BNF administration in WT mice and decreased following hyperoxia exposure (Figure 7C). This was similar to the trend observed with pulmonary CYP1A1 protein levels as shown in Figure 6B. Cyp1a2−/− mice did not show any induction in lung EROD under hyperoxia or with BNF treatment.

Figure 7. Effect of hyperoxia on pulmonary and hepatic EROD (CYP1A1) and MROD (CYP1A2) activity in WT and Cyp1a2−/− mice.

Figure 7

Open in a new tab

Liver MROD (Figure 7A) and EROD (Figure 7B) and Lung EROD (Figure 7C) activity in WT and Cyp1a2−/− mice exposed to hyperoxia or room air that received corn-oil or BNF treatment. Values are means ± SEM from at least 3 individual animals of each genotype. Significant differences between room air and hyperoxia exposed mice are indicated by *, p<0.05 and ***, p<0.001. Differences between corn-oil and BNF treated are indicated by #, p<0.05 and ###, p<0.001. Differences between WT and Cyp1a2−/− mice are indicated by δδδ, p <0.001.

Pulmonary and hepatic expression Nqo1 after hyperoxia exposure in WT and Cyp1a2−/− mice

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a major antioxidant/phase II gene in the lung. We measured Nqo1 mRNA and protein expression in WT and Cyp1a2−/− mice under the different experimental conditions. In WT mice, changes in hepatic Nqo1 mRNA expression were not statistically significant. Interestingly, corn-oil treated Cyp1a2−/− mice showed increased hepatic Nqo1 mRNA expression under hyperoxic conditions. Compared to corn-oil treated controls, BNF treatment led to increase in Nqo1 mRNA expression in Cyp1a2−/− mice. NQO1 protein levels increase with BNF treatment in the livers of WT mice in room air with a decrease following hyperoxia exposure (Figure 8A). Cyp1a2−/− mice also showed a similar pattern in liver NQO1 protein expression (Figure 8B).

Figure 8. Effect of hyperoxia on pulmonary and hepatic Nqo1mRNA and NQO1 protein expression in WT and Cyp1a2−/− mice.

Figure 8

Open in a new tab

Figure 8A: Hepatic Nqo1 mRNA expression in WT and Cyp1a2−/− mice exposed to hyperoxia or room air that received corn-oil or BNF treatment. Figure 8B: Representative western blot image and densitometric analysis of liver NQO1 protein expression in WT and Cyp1a2−/− mice exposed to hyperoxia or room air that received corn-oil or BNF treatment. Figure 8C: Pulmonary Nqo1 mRNA expression in WT and Cyp1a2−/− mice exposed to hyperoxia or room air that received corn-oil or BNF treatment. Figure 8D: Representative western blot image of lung NQO1 protein expression in WT and Cyp1a2−/− mice exposed to hyperoxia or room air that received corn-oil or BNF treatment. Values are means ± SEM from at least 3 individual animals of each genotype. Significant differences between room air and hyperoxia exposed mice are indicated by *, p<0.05, and **, p<0.01. Differences between corn-oil and BNF treated are indicated by #, p<0.05, ##, p<0.01 and ###, p<0.001. Differences between WT and Cyp1a2−/− mice are indicated by δ, p <0.05.

In the lung, BNF treatment did not significantly increase Nqo1 mRNA expression in WT mice compared to corn-oil treated controls. Hyperoxia exposure induced expression in both corn-oil and BNF treated WT and Cyp1a2−/− mice. BNF treatment significantly increased Nqo1 mRNA expression in Cyp1a2−/− mice both in room air and hyperoxic conditions (Figure 8C). This induction was significantly different in Cyp1a2−/− mice compared to similarly treated WT mice in room air. Lung NQO1 protein levels were increased with hyperoxia exposure in WT mice both in corn-oil and BNF treated mice and this was greater in BNF-treated mice compared to corn-oil treated controls (Figure 8D). Corn-oil treated Cyp1a2−/− mice showed increased showed increased NQO1 protein expression in the lung after hyperoxia exposure.

DISCUSSION

The role of the CYP1A subfamily of enzymes in hyperoxic lung injury have been reported in many previous publications (Lingappan et al., 2014; Okamoto et al., 1993; Sinha et al., 2005; Wang et al., 2015). Induction of CYP1A is protective against, while inhibition increases hyperoxic lung injury in adult mice (Moorthy et al., 2000). In neonatal mice, we have shown that prenatal administration of CYP1A inducer (BNF) attenuates lung injury and preserves lung development (Couroucli et al., 2011). Cyp1a1−/− mice have greater alveolar simplification and impairment in angiogenesis compared to WT mice following postnatal hyperoxia exposure (Maturu et al., 2017). Cyp1a1−/− mice displayed a greater induction of hepatic CYP1A2 and pulmonary NQO1 enzymes and the protective effect of BNF was speculated to be mediated through these mechanisms.

The primary objective of this investigation was to determine the role of hepatic CYP1A2 in neonatal hyperoxic lung injury using mice lacking the gene for CYP1A2. CYP1A2 is expressed in the liver, and is absent in the lung. Cyp1a2−/− adult mice exposed to hyperoxia display increased lung injury, pulmonary edema and inflammation compared to similarly exposed WT mice (Wang et al., 2015). This led us to speculate that liver CYP1A2 may be imparting a protective role in the setting of hyperoxic lung injury. However, in neonatal mice, absence of Cyp1a2 did not increase susceptibility to hyperoxic lung injury and did not attenuate the protective effect of BNF. Lung alveolarization as measured by morphometric indices such as MLI and RAC, though adversely impacted by hyperoxia, were not different between WT and Cyp1a2−/− mice. The interesting finding in this manuscript is the sharp contrast between the adult (previously published;(Wang et al., 2015) and neonatal lung phenotype in Cyp1a2−/− mice in the setting of hyperoxic lung injury and in the expression of Cyp1a1 in Cyp1a2−/− mice. In the Cyp1a2−/− neonatal mice, Cyp1a1 expression was low at baseline and no induction was seen with hyperoxia or with BNF treatment. In contrast, in adult Cyp1a2−/− mice, there was a marked increase in pulmonary Cyp1a1 expression. Also, the increased expression of NQO1 in Cyp1a2−/− neonatal mice after BNF treatment has not been reported before and this may attenuate the effect of hyperoxia on the developing lung in these animals. BNF administration ameliorated the adverse impact of hyperoxia in both WT and Cyp1a2−/− mice by improving alveolarization, angiogenesis, Nqo1 expression and decreasing inflammation.

Since, the Cyp1a2−/− mice weighed less than WT mice at PND15, we did not use lung weight/body weight ratios as a marker of lung injury in the present manuscript. Instead we reported body weight and lung weight separately. BNF had a strong effect in preventing weight loss in hyperoxia exposed Cyp1a2−/− neonatal mouse pups. Several differences can be highlighted based on the findings in the current study and previously published results on the role of CYP1A2 in adult and neonatal lungs in the setting of hyperoxia exposure. Cyp1a2−/− adult mice showed increased expression of Cyp1a1 in the liver and lung (Wang et al., 2015). Endogenous AHR ligands, which may be metabolized by CYP1A2, could accumulate in Cyp1a2−/− adult mice, thereby leading to CYP1A1 induction even in room air. This was not observed in neonatal mice and CYP1A1 was not inducible in these mice even after BNF administration. This suggests that absence of CYP1A2 leads to CYP1A1 induction in adult mice but not in neonatal mice. Cyp1a2−/− adult mice showed increased pulmonary neutrophil infiltration upon exposure to hyperoxia. In the neonates, in the present study, there was no increase in pulmonary or macrophage recruitment in the lung in Cyp1a2−/− neonatal mice, while WT mice showed increase in both. Developmental differences in key biological pathways modulating hyperoxic lung injury have been reported by other investigators (Wright et al., 2009); (Yang et al., 2004); (Yang et al., 2000).

Cyp1a2 is not expressed in the neonatal liver in the first 2 weeks, with a slow increase in expression noted thereafter (Selwyn et al., 2015). In a study by Hart et al, describing the ontogeny of different cytochrome P450 genes in the maturing mouse liver, Cyp1a2 expression gradually increased after birth till PND20 and remained constant until PND45 (Hart et al., 2009). Similar findings were reported in neonatal rats with immunodetectable CYP1A2 at PND3 (at approximately 25% of adult levels) with gradual increase reaching 50% of adult levels by PND42 and adult levels by 6 to 9 weeks of life (Elbarbry et al., 2007). In human livers, a similar delay in the ontogeny of CYP1A2 expression has been reported (Sonnier and Cresteil, 1998). CYP1A2 is not detectable in the immediate perinatal period with levels slowly increasing beyond infancy. However, CYP1A2 is inducible in the neonatal murine liver with prenatal (Couroucli et al., 2011) or neonatal administration of BNF (CYP1A inducer) (Maturu et al., 2017), resulting in increased expression of mRNA and protein levels of CYP1A2 and attenuated hyperoxic lung injury. Based on these data and the increased lung injury noted in Cyp1a2−/− adult mice led to the hypothesis that absence of the inducible CYP1A2 in Cyp1a2−/− neonatal mice would partially decrease the beneficial effect of BNF in the model of postnatal hyperoxia exposure. However, results from this study show that BNF has beneficial effects in Cyp1a2−/− neonatal mice to the same extent as WT mice. This shows that BNF treatment attenuates hyperoxic lung injury independent of CYP1A2 induction probably through mechanisms including upregulation of other antioxidant genes such as Nqo1 or due its anti-inflammatory and pro-angiogenic effect.

Despite the well-established sex-specific differences in the incidence of BPD and impaired lung function in males, the molecular mechanism(s) behind this are not completely understood (Stevenson et al., 2000). There are sex-related differences in the constitutive expression of CYP1A in vivo (Asaoka et al., 2010; Eber and Zach, 2001; Kojima et al., 2010; 2008; Rasmussen et al., 2011) and differential modulation of CYP1A by testosterone and estradiol in vitro (Lee et al., 1998; Monostory et al., 2009; Spink et al., 2003). We have shown that exposure to hyperoxia leads to a greater induction in the expression and activity of CYP1A in female WT adult mice in a model of acute hyperoxic lung injury (Lingappan et al., 2013). BNF treated Cyp1a2−/− male mice showed greater reduction in radial alveolar count (a marker of alveolarization; Figure 3E) and angiogenesis (Figure 4J) than WT male mice. Cyp1a2−/− female mice had better preservation of pulmonary angiogenesis at PND15 compared to similarly exposed males (Figure 4I).

NF-E2-related factor-2 (Nrf2) activates the antioxidant response elements (ARE) pathway which comprises of many genes which regulate the cellular response to oxidative stress (Cho et al., 2002; Cho and Kleeberger, 2015). One of these genes is NAD(P)H:quinone oxidoreductase 1 (NQO1), which plays a role as a major antioxidant/phase II gene in the lung. By catalyzing two-electron reduction of a variety of quinone compounds, it prevents generation of free radicals and protects cells from oxidative damage. Single nucleotide polymorphisms in the region of the NQO1 gene were found to be associated with increased risk for the development of BPD in human patients(Sampath et al., 2015). Induction of NQO1 in the lung by hyperoxia exposure has been reported in both adult and neonatal lungs (Cho et al., 2002; Maturu et al., 2017). Mice deficient in Nqo1 were more susceptible to hyperoxic lung injury (Das et al., 2006). Based on the previously established evidence, we measured Nqo1 expression in WT and Cyp1a2−/− mice after BNF expression if this could be in part responsible for the protective effect of BNF in this model. In this study, we show significant induction in pulmonary Nqo1 expression by hyperoxia in both WT and Cyp1a2−/− mice. The response to BNF treatment was greater in Cyp1a2−/− mice. This may have provided some protection against oxidative stress in the setting of postnatal hyperoxia exposure.

In conclusion, absence of Cyp1a2 gene in the neonatal mouse does not increase hyperoxia-mediated arrest in alveolarization and pulmonary angiogenesis. Treatment with BNF imparts protection to both WT and Cyp1a2−/− mice with preservation of lung alveolar and vascular development and attenuation of inflammation in WT mice. Several differences between the neonatal and adult phenotype based on previously published findings were also highlighted. Cyp1a2−/− mice showed a robust induction in Nqo1 expression with hyperoxia and BNF treatment, which could in part play a protective role in this model of postnatal hyperoxia exposure. Further studies are needed to understand the mechanistic role of CYP1A2 in neonatal lung injury, as well as the protective effects of BNF. These studies could lead to additional translational studies that could benefit preterm infants undergoing supplemental oxygen therapy.

Highlights.

  • BNF administration ameliorates lung injury in both wild type and Cyp1a2−/− mice.

  • Cyp1a2−/− mice show increased expression of Nqo1 mRNA after BNF treatment.

  • NQO1 can contribute to the protective effects of BNF

Acknowledgments

This work was supported in part by the National Institutes of Health [K08-HL-127103 to K.L. and R01 grants ES-019689, ES-001932, HL-129794, and HL-112516 to B.M, and HL088343 to XC], the American Lung Association grant RG-418067 to KL. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Asaoka Y, Sakai H, Sasaki J, Goryo M, Yanai T, Masegi T, Okada K. Changes in the gene expression and enzyme activity of hepatic cytochrome P450 in juvenile Sprague-Dawley rats. J Vet Med Sci. 2010;72:471–479. doi: 10.1292/jvms.09-0397. [DOI] [PubMed] [Google Scholar]
  2. Baker CD, Abman SH. Impaired pulmonary vascular development in bronchopulmonary dysplasia. Neonatology. 2015;107:344–351. doi: 10.1159/000381129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berger J, Bhandari V. ANIMAL MODELS OF BRONCHOPULMONARY DYSPLASIA. I: THE TERM MOUSE MODELS. AJP: Lung Cellular and Molecular Physiology. 2014 doi: 10.1152/ajplung.00159.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bhakta KY, Jiang W, Couroucli XI, Fazili IS, Muthiah K, Moorthy B. Regulation of cytochrome P4501A1 expression by hyperoxia in human lung cell lines: Implications for hyperoxic lung injury. Toxicol Appl Pharmacol. 2008;233:169–178. doi: 10.1016/j.taap.2008.08.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bhandari V. Hyperoxia-derived lung damage in preterm infants. Semin Fetal Neonatal Med. 2010;15:223–229. doi: 10.1016/j.siny.2010.03.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chambellan A, Cruickshank PJ, McKenzie P, Cannady SB, Szabo K, Comhair SAA, Erzurum SC. Gene expression profile of human airway epithelium induced by hyperoxia in vivo. American Journal of Respiratory Cell and Molecular Biology. 2006;35:424–435. doi: 10.1165/rcmb.2005-0251OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cho HY, Jedlicka AE, Reddy SPM, Kensler TW, Yamamoto M, Zhang LY, Kleeberger SR. Role of NRF2 in protection against hyperoxic lung injury in mice. American Journal of Respiratory Cell and Molecular Biology. 2002;26:175–182. doi: 10.1165/ajrcmb.26.2.4501. [DOI] [PubMed] [Google Scholar]
  8. Cho HY, Kleeberger SR. Association of Nrf2 with airway pathogenesis: lessons learned from genetic mouse models. Archives of Toxicology. 2015;89:1931–1957. doi: 10.1007/s00204-015-1557-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cooney TP, Thurlbeck WM. The radial alveolar count method of Emery and Mithal: a reappraisal 1--postnatal lung growth. Thorax. 1982;37:572–579. doi: 10.1136/thx.37.8.572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Couroucli XI, Liang YHW, Jiang W, Wang L, Barrios R, Yang P, Moorthy B. Prenatal administration of the cytochrome P4501A inducer, B-naphthoflavone (BNF), attenuates hyperoxic lung injury in newborn mice: implications for bronchopulmonary dysplasia (BPD) in premature infants. Toxicol Appl Pharmacol. 2011;256:83–94. doi: 10.1016/j.taap.2011.06.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Couroucli XI, Welty SE, Geske RS, Moorthy B. Regulation of pulmonary and hepatic cytochrome P4501A expression in the rat by hyperoxia: implications for hyperoxic lung injury. Mol Pharmacol. 2002;61:507–515. doi: 10.1124/mol.61.3.507. [DOI] [PubMed] [Google Scholar]
  12. Das A, Kole L, Wang L, Barrios R, Moorthy B, Jaiswal AK. BALT development and augmentation of hyperoxic lung injury in mice deficient in NQO1 and NQO2. Free Radic Biol Med. 2006;40:1843–1856. doi: 10.1016/j.freeradbiomed.2006.01.025. [DOI] [PubMed] [Google Scholar]
  13. Eber E, Zach MS. Long term sequelae of bronchopulmonary dysplasia (chronic lung disease of infancy) Thorax. 2001;56:317–323. doi: 10.1136/thorax.56.4.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Elbarbry FA, McNamara PJ, Alcorn J. Ontogeny of hepatic CYP1A2 and CYP2E1 expression in rat. J Biochem Mol Toxicol. 2007;21:41–50. doi: 10.1002/jbt.20156. [DOI] [PubMed] [Google Scholar]
  15. Hart SN, Cui Y, Klaassen CD, Zhong XB. Three patterns of cytochrome P450 gene expression during liver maturation in mice. Drug Metab Dispos. 2009;37:116–121. doi: 10.1124/dmd.108.023812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hilgendorff A, Reiss I, Ehrhardt H, Eickelberg O, Alvira CM. Chronic Lung Disease in the Preterm Infant: Lessons Learned From Animal Models. American Journal of Respiratory Cell and Molecular Biology. 2013 doi: 10.1165/rcmb.2013-0014TR. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jiang W, Welty SE, Couroucli XI, Barrios R, Kondraganti SR, Muthiah K, Yu L, Avery SE, Moorthy B. Disruption of the Ah receptor gene alters the susceptibility of mice to oxygen-mediated regulation of pulmonary and hepatic cytochromes P4501A expression and exacerbates hyperoxic lung injury. J Pharmacol Exp Ther. 2004;310:512–519. doi: 10.1124/jpet.103.059766. [DOI] [PubMed] [Google Scholar]
  18. Jobe AH. The new bronchopulmonary dysplasia. Curr Opin Pediatr. 2011;23:167–172. doi: 10.1097/MOP.0b013e3283423e6b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kojima M, Sekimoto M, Degawa M. Androgen-mediated down-regulation of CYP1A subfamily genes in the pig liver. Journal of Endocrinology. 2010;207:203–211. doi: 10.1677/JOE-10-0160. [DOI] [PubMed] [Google Scholar]
  20. Kojima M, Sekimoto M, Degawa M. A novel gender-related difference in the constitutive expression of hepatic cytochrome P4501A subfamily enzymes in Meishan pigs. Biochem Pharmacol. 2008;75:1076–1082. doi: 10.1016/j.bcp.2007.10.030. [DOI] [PubMed] [Google Scholar]
  21. Lee SS, Jeong HG, Yang KH. Effects of estradiol and progesterone on cytochrome P4501A1 expression in Hepa 1c1c7 cells. Biochem Mol Biol Int. 1998;45:775–781. doi: 10.1080/15216549800203192. [DOI] [PubMed] [Google Scholar]
  22. Lingappan K, Jiang W, Wang L, Couroucli XI, Barrios R, Moorthy B. Sex-specific differences in hyperoxic lung injury in mice: implications for acute and chronic lung disease in humans. Toxicol Appl Pharmacol. 2013;272:281–290. doi: 10.1016/j.taap.2013.06.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lingappan K, Jiang W, Wang L, Couroucli XI, Moorthy B. Sex-specific differences in hyperoxic lung injury in mice: Role of cytochrome P450 (CYP)1A. Toxicology. 2015;331:14–23. doi: 10.1016/j.tox.2015.01.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lingappan K, Jiang W, Wang L, Moorthy B. Sex-specific differences in neonatal hyperoxic lung injury. AJP: Lung Cellular and Molecular Physiology. 2016 doi: 10.1152/ajplung.00047.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lingappan K, Jiang W, Wang L, Wang G, Couroucli XI, Shivanna B, Welty SE, Barrios R, Khan MF, Nebert DW, Roberts LJ, Moorthy B. Mice Deficient in the Gene for Cytochrome P450 (CYP)1A1 are More Susceptible than Wild Type to Hyperoxic Lung Injury: Evidence for Protective Role of CYP1A1 Against Oxidative Stress. Toxicol Sci. 2014 doi: 10.1093/toxsci/kfu106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lingappan K, Maity S, Jiang W, Wang L, Couroucli X, Veith A, Zhou G, Coarfa C, Moorthy B. Role of Cytochrome P450 (CYP)1A in Hyperoxic Lung Injury: Analysis of the Transcriptome and Proteome. Sci Rep. 2017;7:642. doi: 10.1038/s41598-017-00516-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maturu P, Wei-Liang Y, Jiang W, Wang L, Lingappan K, Barrios R, Liang Y, Moorthy B, Couroucli XI. Newborn mice lacking the gene for Cyp1a1 are more susceptible to oxygen-mediated lung injury, and are rescued by postnatal β-napthoflavone (BNF) administration: Implications for bronchopulmonary dysplasia (BPD) in premature infants. Toxicol Sci. 2017;157:260–271. doi: 10.1093/toxsci/kfx036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McGowan S, Jackson SK, Jenkins-Moore M, Dai HH, Chambon P, Snyder JM. Mice bearing deletions of retinoic acid receptors demonstrate reduced lung elastin and alveolar numbers. American Journal of Respiratory Cell and Molecular Biology. 2000;23:162–167. doi: 10.1165/ajrcmb.23.2.3904. [DOI] [PubMed] [Google Scholar]
  29. Monostory K, Pascussi JM, Kóbori L, Dvorak Z. Hormonal regulation of CYP1A expression. Drug Metab Rev. 2009;41:547–572. doi: 10.1080/03602530903112284. [DOI] [PubMed] [Google Scholar]
  30. Moorthy B, Nguyen UT, Gupta S, Stewart KD, Welty SE, Smith CV. Induction and decline of hepatic cytochromes P4501A1 and 1A2 in rats exposed to hyperoxia are not paralleled by changes in glutathione S-transferase-alpha. Toxicol Lett. 1997;90:67–75. doi: 10.1016/s0378-4274(96)03832-5. [DOI] [PubMed] [Google Scholar]
  31. Moorthy B, Parker KM, Smith CV, Bend JR, Welty SE. Potentiation of oxygen-induced lung injury in rats by the mechanism-based cytochrome P-450 inhibitor, 1-aminobenzotriazole. J Pharmacol Exp Ther. 2000;292:553–560. [PubMed] [Google Scholar]
  32. Nicola T, Hagood JS, James ML, Macewen MW, Williams TA, Hewitt MM, Schwiebert L, Bulger A, Oparil S, Chen YF, Ambalavanan N. Loss of Thy-1 inhibits alveolar development in the newborn mouse lung. Am J Physiol Lung Cell Mol Physiol. 2009;296:L738–50. doi: 10.1152/ajplung.90603.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Okamoto T, Mitsuhashi M, Fujita I, Sindhu RK, Kikkawa Y. Induction of cytochrome P450 1A1 and 1A2 by hyperoxia. Biochem Biophys Res Commun. 1993;197:878–885. doi: 10.1006/bbrc.1993.2561. [DOI] [PubMed] [Google Scholar]
  34. Rasmussen MK, Zamaratskaia G, Ekstrand B. Gender-related differences in cytochrome P450 in porcine liver--implication for activity, expression and inhibition by testicular steroids. Reprod Domest Anim. 2011;46:616–623. doi: 10.1111/j.1439-0531.2010.1714.x. [DOI] [PubMed] [Google Scholar]
  35. Sampath V, Garland JS, Helbling D, Dimmock D, Mulrooney NP, Simpson PM, Murray JC, Dagle JM. Antioxidant response genes sequence variants and BPD susceptibility in VLBW infants. Pediatr Res. 2015;77:477–483. doi: 10.1038/pr.2014.200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Selwyn FP, Cheng SL, Bammler TK, Prasad B, Vrana M, Klaassen C, Cui JY. Developmental Regulation of Drug-Processing Genes in Livers of Germ-Free Mice. Toxicol Sci. 2015;147:84–103. doi: 10.1093/toxsci/kfv110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shertzer HG, Clay CD, Genter MB, Schneider SN, Nebert DW, Dalton TP. Cyp1a2 protects against reactive oxygen production in mouse liver microsomes. Free Radic Biol Med. 2004;36:605–617. doi: 10.1016/j.freeradbiomed.2003.11.013. [DOI] [PubMed] [Google Scholar]
  38. Shim SY, Cho SJ, Kong KA, Park EA. Gestational age-specific sex difference in mortality and morbidities of preterm infants: A nationwide study. Sci Rep. 2017;7:6161. doi: 10.1038/s41598-017-06490-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sindhu RK, Sakai H, Kikkawa Y. Effect of hyperoxia on rat pulmonary and hepatic cytochrome P450 monooxygenases. Archives of Toxicology. 2000;73:540–546. doi: 10.1007/s002040050006. [DOI] [PubMed] [Google Scholar]
  40. Sinha A, Muthiah K, Jiang W, Couroucli X, Barrios R, Moorthy B. Attenuation of hyperoxic lung injury by the CYP1A inducer beta-naphthoflavone. Toxicol Sci. 2005;87:204–212. doi: 10.1093/toxsci/kfi226. [DOI] [PubMed] [Google Scholar]
  41. Sonnier M, Cresteil T. Delayed ontogenesis of CYP1A2 in the human liver. Eur J Biochem. 1998;251:893–898. doi: 10.1046/j.1432-1327.1998.2510893.x. [DOI] [PubMed] [Google Scholar]
  42. Spink DC, Katz BH, Hussain MM, Pentecost BT, Cao Z, Spink BC. Estrogen regulates Ah responsiveness in MCF-7 breast cancer cells. Carcinogenesis. 2003;24:1941–1950. doi: 10.1093/carcin/bgg162. [DOI] [PubMed] [Google Scholar]
  43. Stenmark KR, Abman SH. Lung vascular development: implications for the pathogenesis of bronchopulmonary dysplasia. Annu Rev Physiol. 2005;67:623–661. doi: 10.1146/annurev.physiol.67.040403.102229. [DOI] [PubMed] [Google Scholar]
  44. Stevenson DK, Verter J, Fanaroff AA, Oh W, Ehrenkranz RA, Shankaran S, Donovan EF, Wright LL, Lemons JA, Tyson JE, Korones SB, Bauer CR, Stoll BJ, Papile LA. Sex differences in outcomes of very low birthweight infants: the newborn male disadvantage. Arch Dis Child Fetal Neonatal Ed. 2000;83:F182–5. doi: 10.1136/fn.83.3.F182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Taylor SC, Berkelman T, Yadav G, Hammond M. A defined methodology for reliable quantification of Western blot data. Mol Biotechnol. 2013;55:217–226. doi: 10.1007/s12033-013-9672-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wang L, Lingappan K, Jiang W, Couroucli XI, Welty SE, Shivanna B, Barrios R, Wang G, Khan MF, Gonzalez FJ, Roberts LJ, Moorthy B. Disruption of Cytochrome P4501A2 in mice leads to increased susceptibility to hyperoxic lung injury. Free Radic Biol Med. 2015 doi: 10.1016/j.freeradbiomed.2015.01.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wright CJ, Zhuang T, La P, Yang G, Dennery PA. Hyperoxia-induced NF-kappaB activation occurs via a maturationally sensitive atypical pathway. Am J Physiol Lung Cell Mol Physiol. 2009;296:L296–306. doi: 10.1152/ajplung.90499.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yang G, Abate A, George AG, Weng YH, Dennery PA. Maturational differences in lung NF-kappaB activation and their role in tolerance to hyperoxia. J Clin Invest. 2004;114:669–678. doi: 10.1172/JCI19300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yang G, Madan A, Dennery PA. Maturational differences in hyperoxic AP-1 activation in rat lung. Am J Physiol Lung Cell Mol Physiol. 2000;278:L393–8. doi: 10.1152/ajplung.2000.278.2.L393. [DOI] [PubMed] [Google Scholar]

RESOURCES