Sex-specific differences in hyperoxic lung injury in mice: role of cytochrome P450 (CYP)1A

Abstract

Sex-specific differences in pulmonary morbidity in adults and preterm infants are well documented. Hyperoxia contributes to lung injury in experimental animals and humans. Cytochrome P450 (CYP) 1A enzymes have been shown to play a mechanistic role in hyperoxic lung injury (HLI) in animal models. Whether CYP1A enzymes contribute to gender-specific differences in relation to HLI is unknown. In this investigation, we tested the hypothesis that mice will display gender-specific differences in HLI, and that this phenomenon will be altered in mice lacking the genes for Cyp1a1 or 1a2. Eight week-old male and female wild type (WT) (C57BL/6J) mice, Cyp1a1-/-, and Cyp1a2-/- mice were exposed to 72h of hyperoxia (FiO2>0.95). Lung injury and inflammation were assessed and pulmonary and hepatic CYP1A1 and CYP1A2 levels were quantified at the enzyme activity, protein and mRNA level. Upon exposure to hyperoxia, liver and lung microsomal proteins showed higher pulmonary CYP1A1 (apoprotein level and activity) in WT females compared to WT males and a greater induction in hepatic CYP1A2 mRNA levels and activity in WT females after hyperoxia exposure. The gender based female advantage was lost or reversed in Cyp1a1-/- and Cyp1a2-/- mice. These findings suggest an important role for CYP1A enzymes in the gender-specific modulation of hyperoxic lung injury.

Keywords: CYP1A; Hyperoxia; Lung injury; Sex-specific.

Figure 1

Effect of hyperoxia (72 h) on lung weight and body weight: 1A: Effect of hyperoxia on body weights of WT, Cyp1a1−/− and Cyp1a2−/− mice. Values are means ± SEM from at least 5 individual animals. 1B: Fold change in lung weight following hyperoxia exposure in male and female WT, Cyp1a1−/− and Cyp1a2−/− mice. Values are means ± SEM from at least 3 individual animals. Significant differences between male and female mice are indicated by δ, p <0.05, between WT and knock-out (Cyp1a1−/− or Cyp1a2−/−) mice within the same sex are indicated by #, p <0.05 and ###, p<0.001, and between room air and hyperoxia exposed mice are indicated by *, p<0.05 and ***, p<0.001.

Figure 2. Effect of hyperoxia on lung histopathology

Representative hematoxylin eosin stained images from the lungs of WT and Cyp1a1−/− and Cyp1a2−/− mice (n=5 mice per group). WT, Cyp1a1−/− and Cyp1a2−/− mice were exposed to hyperoxia (72 h) at 4 x (2A) and 10x (2B) magnification of the lung fields. WT males show more perivascular, bronchiolar edema and alveolar hemorrhage when compared to female mice. Cyp1a2−/− females show greater signs of lung injury than males. Thick arrows point to areas of alveolar edema. Thin arrows point to regions showing perivascular edema.

Figure 3. Lung injury in WT, Cyp1a1−/− and Cyp1a2−/− male and female mice

Lung injury scores in 20 random high-power fields (400× total magnification, n = 3 mice per group) in hematoxylin and eosin stained lung sections obtained from male and female mice of all three genotypes after exposure to hyperoxia (72 h). Significant differences between male and female mice are indicated by δ, p <0.05, between WT and knock-out (Cyp1a1−/− or Cyp1a2−/−) mice within the same sex are indicated by #, p <0.05 and ###, p<0.001

Figure 4

Representative immunostained images for lung neutrophils. Hyperoxia-induced neutrophil recruitment was determined by immunohistochemistry with anti-neutrophil antibodies in WT, Cyp1a1−/− and Cyp1a2−/− mice (n=4 mice per group) at room air and after 72 h of hyperoxia. Arrow points to brown-staining neutrophils.

Figure 5

Representative quantitative analysis of neutrophil infiltration in the lungs at room air and after exposure to 72 h of hyperoxia (FiO2>95%). Neutrophil count per high power (40x magnification) was done as described under materials and methods. Data represent means ± SEM from at least 3 individual animals in each group. Significant differences between male and female mice are indicated by δ, p <0.05. Significant differences between WT and knock-out (Cyp1a1−/− or Cyp1a2−/− mice) within the same sex are indicated by #, p <0.05 and ###, p<0.001.

Figure 6

Real time PCR analysis showing the fold increase in lung TNF- α mRNA expression in over baseline room air levels after 72 h of hyperoxia exposure. Values are means ± SEM from at least 3 individual animals. Figures 6A, 6B and 6C show the changes in male and female mice in WT, Cyp1a1−/− and Cyp1a2−/− mice. Significant differences between male and female mice are indicated by δ, p <0.05. Significant differences form room air controls are indicated by *, p <0.05; **, p<0.01 and ***, p<0.001.

Figure 7

Hepatic CYP1A activity and expression 7A: MROD (CYP1A2) activity in the liver as measured by the MROD assay at room air (Air) and after 72 h of hyperoxia exposure (O₂). Data represent means±SEM from at least 4 individual animals in each group. 7B: Representative western blot assays showing hepatic CYP1A2 (apoprotein) expression in liver microsomes isolated from WT and Cyp1a1−/− mice at room air (Air) and after 72 h of hyperoxia exposure (O₂). GRP-78 (glucose regulated protein; 78kDa) was used as the loading control. The positive control (labeled “PC”) was 0.5 μg of liver microsomes from mice treated with 3-methylcholanthrene. 7C: Densitometric analysis of hepatic CYP1A2 immunoblots. 7D: Real-time RT-PCR analysis for CYP1A2 mRNA (Liver) in WT and Cyp1a1−/− mice. Values are means±SEM from at least 3 individual animals. Liver CYP1A2 mRNA expression at 24 h of hyperoxia exposure expressed as fold change over expression level in WT males at room air. Significant differences between male and female mice are indicated by δ, p <0.05 and δδ, p <0.01. Significant differences between WT and knock-out (Cyp1a1−/− mice) within the same sex are indicated by #, p <0.05 and ###, p<0.001. Significant differences form room air controls are indicated by *, p <0.05; **, p<0.01 and ***, p<0.001.

Figure 8

Pulmonary CYP1A1 activity and western blot assay 8A: CYP1A1 activity in the lung as measured by the EROD assay at room air (Air) and after 72 h of hyperoxia exposure (O₂). Data represent means±SEM from at least 4 individual animals in each group. Significant differences between male and female mice are indicated by δ, p <0.05 and δδ, p <0.01. 8B: Whole lung proteins (20μg) were subjected to Western blotting using monoclonal antibodies raised against CYP1A1, as described in “Materials and methods”. Whole lung proteins were isolated from WT and Cyp1a2−/− mice at room air (Air) and after 48 h of hyperoxia exposure (O₂). Under each sample lane is the corresponding β-actin blot to assess for protein loading. The positive control (labeled “PC”) was 0.5 μg of liver microsomes from mice treated with 3-MC. 8C: Densitometric analysis of pulmonary CYP1A1 immunoblots at room air and after 48 h of hyperoxia exposure (O₂). . Significant differences between male and female mice are indicated by δ, p <0.05.