Crystalline silica particles cause rapid NLRP3-dependent mitochondrial depolarization and DNA damage in airway epithelial cells
Authors: Rongrong Wu
Mikael Adner
Patricia Ramos-Ramírez
Johan Högberg
Huiyuan Zheng
Ulla Stenius
Journal: Particle and Fibre Toxicology (2020)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: Autotaxin (ATX); CC10; Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP); Double strand brakes (DSB); GPRC5A
Affiliations: Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-17177, Stockholm, Sweden., Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-17177, Stockholm, Sweden. .

Abstract:  Background: Respirable crystalline silica causes lung carcinomas and many thousand future cancer cases are expected in e.g. Europe. Critical questions are how silica causes genotoxicity in the respiratory epithelium and if new cases can be avoided by lowered permissible exposure levels. In this study we investigate early DNA damaging effects of low doses of silica particles in respiratory epithelial cells in vitro and in vivo in an effort to understand low-dose carcinogenic effects of silica particles. Results: We find DNA damage accumulation already after 5-10 min exposure to low doses (5 μg/cm2) of silica particles (Min-U-Sil 5) in vitro. DNA damage was documented as increased levels of γH2AX, pCHK2, by Comet assay, AIM2 induction, and by increased DNA repair (non-homologous end joining) signaling. The DNA damage response (DDR) was not related to increased ROS levels, but to a NLRP3-dependent mitochondrial depolarization. Particles in contact with the plasma membrane elicited a Ser198 phosphorylation of NLRP3, co-localization of NLRP3 to mitochondria and depolarization. FCCP, a mitochondrial uncoupler, as well as overexpressed NLRP3 mimicked the silica-induced depolarization and the DNA damage response. A single inhalation of 25 μg silica particles gave a similar rapid DDR in mouse lung. Biomarkers (CC10 and GPRC5A) indicated an involvement of respiratory epithelial cells. Conclusions: Our findings demonstrate a novel mode of action (MOA) for silica-induced DNA damage and mutagenic double strand breaks in airway epithelial cells. This MOA seems independent of particle uptake and of an involvement of macrophages. Our study might help defining models for estimating exposure levels without DNA damaging effects. 

Retracted: No

PubPeer comments:
 Control flow cytometry sample is reused between Figures 1E and 2A. The control condition is matching between the two panels, so probably it does not affect the conclusions, but I could not find a clear indication in the text that the data from the same experiment were plotted. CDK2 blots in Figure 1A and Figure 3A: the shapes of the bands are very similar, but the brightness of the images is not. In case the CDK2 blots are the same sample, were NLRP3, CDK2 (Fig. 1A), gammaH2AX and pCHK2 (Fig. 3A) also all probed on the same membrane? , 
 Control sample is reused between Figures 1E and 2A (please see image below): ![file] Loading control may have been reused between Figures 1A and 3A (please see image below - original sizes and resized panel 3A for comparison): ![file] , 
 I think the authors mentioned that they want to use FCCP to mimic silica induced MMP drop, so figure 1 e and figure 2 a maybe the same experiment put into different figures?  FCCP is an uncoupler of the electron transport chain inducing oxidative phosphorylation, which decreases the mitochondrial membrane potential, and also the plasma membrane potential [21]. We found that 500u2009nM FCCP induced a similar mitochondrial depolarization as silica (Fig. 2a). So maybe that s why the control is the same. ,