# Effects of Oxidized Pyrenes on the Biological Responses in the Human Bronchial Epithelial Cells
## 3 Results
### Effects of Pyrene and Its Quinone Forms on ROS Production by Bronchial Epithelial Cells  
BEAS-2B cells were treated with 1, 3, and 10 \(\upmu\)M pyrene, 4,5-PyQ, and 1,8-PyQ + 1,6-PyQ, and ROS production was examined. No significant difference in ROS production was observed when the cells were treated with 1 \(\upmu\)M pyrene or 1,8-PyQ + 1,6-PyQ compared with that in the control. However, ROS production increased after 4,5-PyQ exposure (\(p<0.01\) vs. control). At 3 and 10 \(\upmu\)M, no significant difference in ROS production was observed upon pyrene exposure compared with that in the control; nonetheless, ROS production increased after exposure to 4,5-PyQ or 1,8-PyQ + 1,6-PyQ (\(p<0.01\)).  
Figure 3: Effect of pyrene and its quinone forms on interleukin (IL)-6, IL-8, and soluble intercellular adhesion molecule (sICAM)-1 production from human bronchial epithelial cells. The protein levels of IL-6 (**A**), IL-8 (**B**), and sICAM-1 (**C**) in the culture supernatants, after exposure to pyrene and its quinone forms for 24 h, were measured using enzyme-linked immunosorbent assay (ELISA). The data are expressed as the mean \(\pm\) standard error of the mean (SEM) of four individual cultures. *\(p<0.05\), **\(p<0.01\)** vs. control, **#\(p<0.01\)** vs. pyrene at the same concentration, §§§§ \(p<0.01\)** vs. each other.  
Exposure to 1 \(\upmu\)M 4,5-PyQ increased ROS production compared to that after pyrene exposure (\(p<\) 0.01), whereas no significant difference was observed after 1,8-PyQ + 1,6-PyQ exposure. There were no significant differences in ROS production between 4,5-PyQ- and 1,8-PyQ + 1,6-PyQ-treated groups. At 3 and 10 \(\upmu\)M, increased ROS production was observed after 4,5-PyQ and 1,8-PyQ + 1,6-PyQ exposure compared to that after pyrene exposure (\(p<\) 0.01). When the effects of 4,5-PyQ and 1,8-PyQ + 1,6-PyQ exposure were compared, higher ROS levels were observed in the 4,5-PyQ group (\(p<\) 0.01) (Figure 4A).  
In the absence of cells, treatment with 1, 3, and 10 \(\upmu\)M of pyrene, 4,5-PyQ, and 1,8-PyQ + 1,6-PyQ showed no significant differences in ROS production after exposure to 1 \(\upmu\)M pyrene and 4,5-PyQ compared with that in the control, whereas increased ROS production was observed after 1,8-PyQ + 1,6-PyQ exposure at 1 \(\upmu\)M (\(p<\) 0.01 vs. control). Similar results were observed in groups treated with 3 and 10 \(\upmu\)M of pyrene quinones.  
In the mutual comparison at 1 \(\upmu\)M, ROS production was not significantly different after 4,5-PyQ exposure compared with that after pyrene exposure, but it increased after 1,8-PyQ + 1,6-PyQ treatment (\(p<\) 0.01). ROS levels increased after exposure to 1,8-PyQ + 1,6-PyQ compared to those after 4,5-PyQ exposure (\(p<\) 0.01) (Figure 4B). Similar results were observed in cells treated with 3 and 10 \(\upmu\)M of each quinone derivative (Figure 4B).  
Overall, significantly higher ROS production was induced in bronchial epithelial cells after treatment with oxidized pyrenes, following a dose-dependent pattern.  
Figure 4: Effects of pyrene and its quinone forms on reactive oxygen species (ROS) production by airway epithelial cells. The intracellular (with cell condition) (**A**) and extracellular (without cell conditions) (**B**) ROS production during 3 h exposure. The data are expressed as the mean \(\pm\) standard error of the mean (SEM) from four individual cultures. **\(p<\) 0.01 vs. control, #\(p<\) 0.01 vs. pyrene at the same concentration, §§§ \(p<\) 0.01 vs. each other.