Characterization of Fibrotic Changes in the Lungs of Mice Following Oral Administration of 4NQO

Abstract

IntroductionWe have shown that oral administration of 4‐nitroquinoline‐1‐oxide (4NQO), a carcinogen used to induce oral cancer in a mouse model, can cause inflammatory changes in the lung as manifested by a significant increase in the presence of macrophages, neutrophils, and T lymphocytes in the lung parenchyma. The inflammatory changes in the lung were affected by the sex of animals, since the number of macrophages and neutrophils was significantly higher in male than female mice. In addition, dietary content of saturated fat also played a role, since number of neutrophils was significantly higher in 4NQO‐treated males on high fat diet. Accumulation of inflammatory cells and their products in the lung may result in tissue injury and lung fibrosis. For example, activated macrophages can produce TGF‐β, which promotes collagen synthesis and formation of scar tissue. In this study we examined the effects of 4NQO administration on fibrotic changes in the mouse lungs. We hypothesized that treatment with 4NQO promotes development of fibrosis, and that sex and dietary fat content would affect severity of fibrotic changes.Materials and methods5‐week‐old male and female C57Bl/6 mice (n=36 each gender), were divided into a low fat (10kcal% fat; LF) and high fat (60 kcal% fat; HF) diet groups. After one week mice in the experimental groups began a treatment with 50μg/mL of 4NQO in 1.25% propylene glycol (PG) in water. Mice in control groups were provided with water or PG in water. After 17 weeks of treatment all mice were given water alone for 6 weeks prior to euthanasia. Lung tissues from all treatment groups were harvested, fixed in formalin, and processed for histological evaluation. Lung tissue sections were stained using Masson’s Trichrome method. Severity of pulmonary fibrosis was assessed using modified Ashcroft method with a scale 0–3, where ‘0’ characterized normal lung, while ‘3’ indicated complete obliteration of normal lung architecture.Results4NQO administration resulted in significant fibrotic changes (control 0.13 vs. 4NQO 0.67, p<0.001). Among 4NQO‐treated mice, males showed significantly higher fibrotic changes than females (0.87 vs. 0.47, p=0.0015). The presence of fibrotic changes was associated with an increase in lymphoid cell numbers. In addition, recent reports implicate propylene glycol (PG), present in e‐cigarettes, in lung pathology due to vaping. The presence of PG had no effect on the fibrotic changes and lung injury in our study. Also, dietary fat content did not affect fibrotic changes in the lung. Neither low nor high fat diets contributed to fibrotic changes in the control animals. In addition, fat content did not affect 4NQO‐induced fibrosis.ConclusionOral administration of 4NQO resulted in fibrotic changes in the lungs. A degree of pulmonary fibrosis correlated with an increase in the numbers of lymphoid cells in the lungs. This mouse model of oral cancer that was developed to mimic the effects of smoking may be extended to serve as a model to study the effects of tobacco on lung pathology.Support or Funding InformationMidwestern University