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A2222 - Oropharyngeal Bleomycin Upregulates Drug Efflux Transporters and Reduces Lung Exposure to Substrate Compounds: A Pitfall for Pharmacological Studies on Pulmonary Fibrosis
Author Block: J. Park1, N. J. Coffey1, S. Bodine2, W. A. Gahl2, M. V. Malicdan2, B. R. Gochuico2, G. Kunos1, R. Cinar1; 1Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD, United States, 2Medical Genetics Branch, National Human Genome Research Institute, Bethesda, MD, United States.
Rationale: Lung epithelium is exposed to airborne toxins and pathogens as a natural consequence of respiratory gas exchange. An evolutionary protective mechanism involves ATP-binding cassette (ABC) transporters, which mediate cellular detoxification by exporting xenobiotic compounds. These transporters, however, can also extrude various pharmaceutical agents from the lung, limiting drug accumulation and reducing treatment efficacy. The resulting “multidrug resistant” phenotype is an active area of study in the context of lung cancer as it is a major challenge to clinical oncology. To date, however, the role of ABC transporters in the progression and treatment of idiopathic pulmonary fibrosis (IPF) has not been described.
Methods: To determine whether ABC transporters are upregulated in IPF, we used immunohistochemistry to assess the expression of three canonical ABC transporters, p-glycoprotein (MDR1), breast cancer resistance protein (BCRP), and multi drug resistance protein 1 (MRP1), in lung tissue from IPF patients and experimental mouse models of IPF induced by bleomycin, either via a single oropharyngeal aspiration (1.5 U/kg) or systemic exposure by subcutaneous osmotic minipump (100 U/kg over 7 days). Additionally, we used fluorescence in situ hybridization to localize mRNA transcripts to CD68+ macrophages, SPC+ alveolar type II (ATII) cells, and PECAM1+ endothelial cells. Separately, cell lines of interest (RLE-6TN ATII cells and NR8383 macrophages) were incubated with vehicle or bleomycin (5 mU/ml) for a maximum of five days to explore cell type-specific changes in gene expression of ABC transporters in response to bleomycin treatment.
Results: Patient-derived IPF lung tissue expressed baseline levels of the ABC transporters studied, comparable to non-fibrotic lung tissue from healthy controls. Surprisingly, however, whole murine lung tissue from the oropharyngeal bleomycin model expressed 2-fold increased levels of MDR1 and BCRP. This upregulation was predominantly localized to alveolar macrophages and ATII cells, as suggested by a significant and sustained increase in gene expression for MDR1 and BCRP (30- to 40-fold increase, ≥5 days) in response to bleomycin challenge in vitro. Meanwhile, the systemic bleomycin administration was not associated with such upregulation, consistent with findings in human IPF.
Conclusion: We report that the oropharyngeal bleomycin upregulates two ABC transporters associated with drug efflux, which is a deviation from the disease behavior of human IPF. Therefore, we identify a deficiency in the most widely-used model of pulmonary fibrosis that may be confounding pharmacological studies developing new therapeutic compounds for IPF.