Effects of anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene on human small airway epithelial cells and the protective effects of myo-inositol

Benzo[a]pyrene (B[a]P), a tobacco-derived carcinogen, induces lung tumors in rodents through its carcinogenic metabolite, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE). Tumorigenesis is inhibited by dietary myo-inositol in the post-initiation phase. However, little is known about how B[a]PDE and myo-inositol affect normal human lung cells. We addressed this question using untransformed human small airway epithelial (SAE) cells. SAE cell viability decreased < 50% in parallel to an increase of apoptotic cells (> 20%) 2 days after the cells were treated for 1 h with B[a]PDE (> 100 nM). In contrast, the cell number and viability were not altered in A549 human lung cancer cells by B[a]PDE treatment up to 10 μM with < 5% apoptotic cells and < 10 U/l LDH in the medium. SAE cells retain the features of basal cells in serum-free, low Ca²⁺ (4 nM) medium up to 4-5 passages, but in serum-supplemented or serum-free, high Ca²⁺ (1 mM) cultures, they differentiate into non-ciliated epithelial cells expressing Clara cell secretory protein (CCSP). A non-toxic, physiologically relevant dose of B[a]PDE (1 nM partially inhibited serum and Ca²⁺-induced SAE cell differentiation. This effect was abolished by wortmannin, a phosphatidylinositol-3 kinase (PI-3K) inhibitor, and PD98059, a mitogen activated protein kinase (MAPK) kinase-1 (MEK1) inhibitor, but not by SB202190, a p38 MAPK inhibitor, or melittin, a protein kinase C inhibitor. Myo-inositol (10-100 μM) did not alter growth or differentiation of untreated SAE or A549 cells, but reversed the inhibitory effect of B[a]PDE on serum and Ca²⁺-induced SAE cell differentiation when supplemented to the culture after B[a]PDE treatment. This myo-inositol action was not altered by PD98059, wortmannin or melittin, but was partially suppressed by SB202190. Collectively, these results indicate that B[a]PDE inhibits serum-induced SAE cell differentiation, possibly involving activating signals through a PI-3K/MEK1 mediated MAPK pathway, whereas myo-inositol protects SAE cells against this inhibitory effect of B[a]PDE perhaps through both PI-3K/MEK1 and p38 MAPK pathways.