Comparison of compound cytotoxicity in A549 and H292 lung epithelial cell lines in vitro

D.W. Jenkins, S. Ashra & J.F. Unitt. Lead Generation, Molecular Biology, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 5RH

Lung epithelial cells form a barrier between the host and environment and play important roles in the pathophysiology of several lung diseases, including asthma and chronic obstructive pulmonary disease. The present study used two immortalized human lung alveolar epithelial cell lines, A549, which have some features of alveolar type II cells, and NCI-H292 to evaluate the cytotoxicity of a panel of 22 potential toxicants of different mechanisms (Table 1). Gross compound cytotoxicity was measured by monitoring the reduction of alamar blue to resorufin (O’Brien et al., 2000). Briefly, cells were incubated in the presence or absence of compound for 4 or 24 h prior to assessment of cellular viability following the addition of 400 μM alamar blue and reading on a fluorescence platereader (SpectraMax Gemini; λEx 560 nm, λEx 590 nm). Release of cytokines from the cells (IL-1β , IL-12, IL-10, IL-2, GM-CSF, TNFα , IL-8, IL-4, IL-5 & IL-6) was measured via multi-spot ELISA from MesoScale Discovery ( Gaithersburg, MD, USA) according to manufacturers instructions. After 4 h, Brefeldin A caused concentration-dependent inhibition of alamar blue reduction in both cell types (A549: pIC50 7.8 ± 0.31, max inhibition 43 ± 3.9%; H292: pIC50 7.9 ± 0.35, max inhibition 36 ± 1.7%; n=5). After 24 h, significant cytotoxicity (pIC50, max inhibition) was observed in A549 cells following treatment with Brefeldin A (7.7 ± 0.082, 37 ± 7%), cycloheximide (6.6 ± 0.21, 43 ± 2%) and Ro-318220 (5.6 ± 0.073, 86 ± 8%) (n=3-5). The effects of cycloheximide and Ro-318220 were associated with increases in basal IL-6 and decreases in IL-8 release, respectively. In contrast, in H292 cells, cytotoxicity was caused by Brefeldin A (7.7 ± 0.12, 70 ± 4%), Actinomycin D (7.1 ± 0.10, 75 ± 5.3%), Cycloheximide (6.3 ± 0.38, 49 ± 3.7%), Doxorubicin (5.6 ± 0.061, 84 ± 2.8%), MG-132 (6.0 ± 0.22, 80 ± 1.7%), Staurosporine (6.3 ± 0.22, 87 ± 5.6%) and Ro 31-8220 (5.8 ± 0.14, 99 ± 0.45%) with a concomitant decrease in basal IL-6 release (n=3-6). In addition, cycloheximide and doxorubicin caused concomitant increases in TNF a and IL-1β release, respectively. Taken together, the data suggests that A549 cells may be more resistant to potentially cytotoxic agents than H292 cells and further demonstrates the utility of both cell types as model systems that can be used to investigate compound cytotoxicity in vitro.

O’Brien, J et al. (2000). Eur. J. Biochem., 267, 5421-5426.