Expression of Cyp2B1 in Freshly Isolated and Proliferating Cultures of Epithelial Rat Lung Cells

Abstract

Bronchiolar Clara cells and alveolar type 2 cells of the lung are known to express relatively high levels of P450 enzymes compared to other pulmonary cells. Populations of enriched type 2 cells and Clara cells were isolated from rat lung by a procedure including lung perfusion, protease digestion, centrifugal elutriation, and differential attachment. Alveolar macrophages were removed by lavage. The purity of the type 2 cell-enriched population was approximately 90%, and the purity of the Clara cell-enriched population was 40-50%. Both type 2 cells and the cells of the Clara cell-enriched population proliferated in culture. CYP2B1 mRNA was expressed approximately to the same level in type 2 cells and the Clara cell-enriched population. The mRNA levels remained roughly constant for both cell types throughout the culture period, except for an early transient reduction. The apoenzyme level of CYP2B1 was 2-3 times higher in freshly isolated cells of the Clara cell-enriched population than in the type 2 cells. Both epithelial cell types showed decreased level of CYP2B1 apoenzyme in culture. The differences in the CYP2B1 mRNA and apoenzyme expression levels in freshly isolated cells and cultured cells suggest the existence of a post-transcriptional regulatory mechanism for CYP2B1 expression in lung cells. The characterization of specific functions of lung cells in culture, such as P450 gene expression, provides necessary information for the use of the cells in in vitro pulmonary toxicology.

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“…The animals weighed 200–250 g at the time of sacrifice, and were given Ewos standard pelleted laboratory chow from Astra Ewos AB, Södertälje, Sweden and water ad libitum . Alveolar macrophages were obtained by lung lavage [ 43 ]. The macrophages were suspended in RPMI medium with ampicillin (0.1 mg/ml), penicillin (0.1 mg/ml), streptomycin (0.1 mg/ml), fungizone (0.25 μg/ml) and 5% heat-inactivated fetal bovine serum (FBS) at a cell density of 1.5 × 10 6 cells/ml and added to 35-mm Costar wells (1 ml/well).…”

The Organophosphorus Pesticide Chlorpyrifos Induces Sex-Specific Airway Hyperreactivity in Adult Rats

“…CYP2B6 is also one of the most highly polymorphic CYPs with 9 major variants identified in the Caucasian population alone, which may also explain differences in individual susceptibility to CPF intoxication (Crane et al, 2012). The rat homolog of CYP2B6 is CYP2B1, which is highly expressed in type II alveolar cells (Lå g et al, 1996), which could explain why the whole lung tissue, which is largely comprised of alveoli, was the only tissue in which significant inhibition of AChE activity by CPF was observed.…”