# Effects of Oxidized Pyrenes on the Biological Responses in the Human Bronchial Epithelial Cells
## 5 Conclusions  
In this study, we investigated the respiratory toxicity of pyrene, a type of PAH, and its quinone forms in vitro by assessing the effects of varying concentrations of pyrene, 4,5- PyQ, and 1,8-PyQ + 1,6-PyQ, on human lung epithelial (BEAS-2B) cells. We evaluated cell viability, apoptosis, and the production of IL-6, IL-8, sICAM-1, and ROS. At 1 \(\upmu\)M, increased cellular activity was observed after exposure to 4,5-PyQ or 1,8-PyQ + 1,6-PyQ. At 3 \(\upmu\)M, 4,5-PyQ increased the number of late apoptotic and/or necrotic cells compared with the control, whereas 1,8-PyQ + 1,6-PyQ exposure increased the number of dead cells. IL-6 production was decreased after exposure to 10 \(\upmu\)M 4,5-PyQ and IL-8 production was decreased after exposure to 3 and 10 \(\upmu\)M of both 4,5-PyQ and 1,8-PyQ + 1,6-PyQ. sICAM-1 production was higher after 1,8-PyQ + 1,6-PyQ exposure at 10 \(\upmu\)M. In the presence of cells, 4,5-PyQ and 1,8-PyQ + 1,6-PyQ increased ROS production significantly in a concentration-dependent manner, whereas similar results were observed with 1,8-PyQ + 1,6-PyQ under cell-free conditions.  
This study has the following limitations: (1) it only examined in vitro effects; thus, the effects of these pyrene quinones on respiratory pathophysiology such as asthma, pneumonia, and COPD and (2) the contribution of ROS and/or other molecular mechanisms in these epithelial cell toxicities, including cell death patterns induced by pyrene quinones, remain unclear. Therefore, additional in vivo studies and molecular assessments are needed to address these limitations.  
Overall, these results show that the oxidized quinone forms of pyrene exert more harmful cytotoxic effects on respiratory organs than those of pyrene, resulting in exacerbation of respiratory diseases. Thus, future toxicological studies on the effects of PM should focus more on the oxidized forms of PAHs.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12199664/s1](https://www.mdpi.com/article/10.3390/app12199664/s1)]([https://www.mdpi.com/article/10.3390/app12199664/s1](https://www.mdpi.com/article/10.3390/app12199664/s1)), Figure S1: Chemical structural formula of pyrene and its quinone forms.  
Conceptualization, T.K. and H.T.; Investigation, A.H. and S.T.; Resources, T.K.; Data curation, S.T.; Writing--original draft preparation, A.H. and K.-i.L.; Writing--reviewand editing, A.H., K.-i.I., T.K., K.U. and H.T.; Supervision, K.-i.I., T.K., K.U. and H.T.; Project administration, A.H.; Funding acquisition, T.K. and H.T. All authors have read and agreed to the published version of the manuscript.  
This work was supported by CREST, JST (JPMJCR19H3: to [PERSON]), and the Japan Society for the Promotion of Science Grants in Aid for Scientific Research (B: 17H01906: to [PERSON]).  
Not applicable.  
The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to privacy.  
We thank [PERSON] for their technical assistance.  
The authors declare no conflict of interest.