Project title: Association between Exposure to Diesel Exhaust Particles and Glutathione in Epithelial Lining Fluid of the Lung in Glutathione-Deficient Mice
Completed in: 2014
Inhalation of diesel exhaust particles (DEPs) has been associated with adverse health effects. An important and abundant antioxidant in the lungs is glutathione (GSH), which can attenuate oxidative stress induced by DEP exposure. Glutamate cysteine ligase (GCL) carries out the rate-determining step in GSH synthesis. GCL consists of modifier (Gclm) and catalytic (Gclc) subunits. In a previous study investigating the effects of DEP on lung inflammation, it was show that Gclm knock-out (KO) (-/-) mice were not more sensitive to DEP exposure that Gclm wild type (WT) (+/+) mice. Thus, it was hypothesized that Gclm KO (-/-) mice have likely adapted by up-regulating other genes involved in protecting the lungs from DEP-induced oxidative stress. While the exact mechanisms are not known, one possible explanation is that GSH transporters might be exporting GSH into the epithelial lining fluid (ELF). Four candidate GSH transporters include ATP-binding cassette (ABC) subfamily C protein 2 (ABCC2, also known as multidrug-associated resistance protein), ABCC12, ABC subfamily G protein (ABCG2), also known as breast cancer-related protein), and ABCC7 (also known as cystic fibrosis transmembrane regulator). This study was aimed to measure ELF GSH levels in mice with different GCLM genotypes (Gclm wild-type (+/+), Gclm heterozygous (+/-), and Gclm knock-out (-/-) mice) following DEP exposure and to measure candidate GSH transporter mRNA expression levels in these mice. The mice were exposed to DEPs via intranasal instillation and sacrificed after 6 hours. Total GSH levels in bronchoalveolar lavage fluid (BALF) were measured using a plate assay in which GSH reacts with naphthalene-2,3-dicarboxaldehyde (NDA), forming a fluorescent derivative compound. The mRNA levels of the candidate GSH transporter genes were analyzed using quantitative real –time polymerase chain reaction (qRT-PCR). The results showed that the total ELF GSH found in BALF increased in response to DEP exposure in Gclm-/- mice. It was observed that ABCC2 mRNA and ABCC12 mRNA in the lungs were statistically significantly upregulated following exposure to DEP in Gclm+/+ and Gclm+/- mice, but they had low expression. Furthermore, it was shown that among PBS-treated tontrols, CFTR expression was higher in Gclm-/- mice than in in Gclm+/+ and Gclm+/- mice, suggesting that CFTR is likely responsible for maintaining ELF GSH levels in the lungs of Gclm-/- mice. In summary, exposure to DEP can increase levels of ELF GSH in Gclm-/- mice and this may be the basis for adaptive response that can oppose oxidative stress and inflammation.