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This investigation sought to establish the cellular expression and distribution of the alpha, pi, and mu classes of glutathione S-transferase (GST) enzymes in murine lung under control conditions and after treatment with tert-butyl-4-hydroxyanisole (BHA). Immunohistochemical and in situ hybridization studies were used to identify lung cells that were labeled for the GST subunits Yp, Ya, and Yb1. Immunoblotting of cytosolic proteins produced single bands of 28, 29, and 31 kD for Ya, Yp, and Yb1, respectively, in samples from untreated and BHA-treated mice. Treatment with BHA increased Ya and Yp reactivity, but this was not as marked for Yb1. Immunohistochemical staining for the Yp, Ya, and Yb1 subunits was localized in bronchioles and parenchyma of untreated and BHA-treated mice. Bronchiolar Clara and alveolar type II cells were stained to the greatest extent for all of the GST subunits. BHA treatment produced increased staining that was most pronounced in the bronchiolar epithelium. Ya and Yp were localized in the cytoplasm and nucleus, whereas Yb1 was found mainly in the cytoplasm. Immunoblots of extracted nuclear proteins revealed a band of 29 kD for Ya, with increased immunoreactivity in BHA-treated mice. In situ hybridization done with oligonucleotide probes showed abundant silver grains representing Ya, Yp, and Yb1 messenger RNA (mRNA) transcripts in the bronchioles. Grains were also localized in alveolar septa, and were most numerous in type II cells. Quantitative image analysis confirmed good agreement between relative levels of protein and mRNA transcripts. Quantities of mRNA transcripts for all subunits were increased in the parenchyma by BHA treatment, but the magnitudes of induction were most striking for Ya and Yp in the bronchioles. These results demonstrated that Ya, Yp, and Yb1 reside in specific lung areas and cells, and that in induced states, their increased expression is accompanied by increased mRNA.