In vivo imaging of ICAM-1 targeted nanoparticles in XPC and wild mice exposed to ambient air pollution in São Paulo, Brazil Nilmara Alves Post-doctoral researcher School of Medicine of University of São Paulo, Brazil Supervisor: Prof. Paulo Saldiva Good afternoon! I’m Nilmara Alves. I’m a post-doc researcher in the School of Medicine of University of São Paulo and my supervisor is Prof Paulo Saldiva. The title of my work is...

Increase in the incidence São Paulo Metropolitan Area ~ 20 million people ~ 8 million vehicles Increase in the incidence of lung adenoma in mice (Reymão et al., 1997) Alterations of alveolar structure in mice exposed to particulate matter (Mauad et al., 2008) The emissions from megacities in the world are strongly impacted by the transportation sector. São Paulo Metropolitan Area located in the southeast of Brazil has a population of nearly 20 million people and 8 million vehicles. In this context, many studies show the impacts of exposure to ambient levels in São Paulo. It is long known that exposure to air pollutants can cause various health effects such as increase in the incidence of lung adenoma in mice; alterations of alveolar structure and lung elastic properties in mice exposed to PM; increase of inflammatory response and DNA damage. Increase of airway inflammation in motorcyclists exposed to pollutants in São Paulo (Brant et al., 2014)

Urban Pollution in Real Time ? Urban Pollution in Real Time However, the effects of DNA damage caused by air pollutants in real time and the association with the mechanism of repair pathways are largely unknown. Adapted by Hoeijmakers, Nature 411, 2001.

X Nucleotide Excision Repair - NER Studies show that knockout mice in the XPC gene have an increase in lung tumors (Melis et al., 2008; 2013) X One important defense mechanisms against the accumulation of DNA damage is the nucleotide excision repair (NER), which includes the XPC protein. Different studies have shown that knockout mice in the XPC gene have an increased occurrence of lung tumors compared to wild animals.

Accumulated dose of PM2.5 : (World Health Organization) Harvard Ambient Particle Concentrator Accumulated dose of PM2.5 : 600 µg.m-3 in 1 hour Based on that, in this study, we used the Harvard Ambient Particle Concentrator to expose mice to Sao Paulo pollution in real time. Mice were exposed to an accumulated dose (concentration vs time of exposure in hours) of 600 µg.m-3 for 1 hour, which corresponds to an average (or mean) concentration of 25 µg.m-3 in a day, the limit established by the World Health Organization. PM2.5 = 25 µg.m-3 in 24-hour mean (World Health Organization)

Harvard Ambient Particle Concentrator - Particle size of Ambient Particle Concentrator using Scanning Mobility Particle Sizer (SMPS) We characterize the particle size of PM2,5 of our concentrator using SMPS equipment. The results show a fairly constant concentration factor on this size range, including particle size of tens of nanometers. The fairly constant concentration factor on this size range, including particle size of tens of nanometers.

Inhaled Lipopolysaccharide (LPS) – ( n=5 animals) Groups of animals C57-WT C57-XPC Filtered Air (n=5 animals) Inhaled Lipopolysaccharide (LPS) – ( n=5 animals) In this study, we used four groups with wild type wild and XPC mice: group with filtered air, polluted air group and the group which inhaled LPS as a positive control. Polluted Air 1 day of exposure (n=5 animals) Polluted Air 5 days of exposure (n=5 animals)

Methodology using Intercellular Adhesion Molecule-1 24 hours after the exposure 24 hours after the exposure, we injected intravenously Intercellular Adhesion Molecule-1(anti-ICAM-1) targeted nanoparticles and evaluated the inflammatory response using IVIS spectrum. anti-ICAM-1 targeted nanoparticles injected via intravenous Analysis using IVIS spectrum

control isotype antibody Experimental Control control isotype antibody anti-ICAM antibody PM PM PM ICAM PM To evaluate the inflammatory response we did a test using liposome with fluorochrome. This nanoparticle is associated with an anti-ICAM antibody. To have control of the particle, we used a control isotype antibody. Therefore, the greater the inflammation, the greater the response ICAM and the greater the interaction with the target particle. PM = DiD- lipophilic fluorochrome Unspecific or no interaction PM PM Slide by PhD Wesley Fotoran (ICB-USP)

Fluorescence Intensity Results Fluorescence Intensity - 1 DAY EXPOSURE Filtered Air Polluted Air Filtered Air Polluted Air 24 HOURS after 1 day exposure, we observed a significantly stronger fluorescent marker for anti-ICAM in the polluted group compared to the filtered air group. C57-XPC C57-XPC

Fluorescence Intensity Results Fluorescence Intensity - 1 DAY EXPOSURE Filtered Air Inhaled LPS Polluted Air Filtered Air LPS Polluted Air Here, we observe the expected effect of Inhaled LPS, which is a good positive control. C57-XPC C57-XPC C57-XPC

Fluorescence Intensity Results Fluorescence Intensity - 1 DAY EXPOSURE Polluted Air XPC Polluted Air WT Filtered Air Polluted Air - XPC Polluted Air - WT Filtered Air Here, in the pollutand group, we observed an increase of inflammatory response in both wild type and XPC animals. However, XPC mice are more sensitive for anti-ICAM marker. Furthermore, among the animals exposed to ambient pollution, XPC mice have shown a stronger inflammatory response compared to wild mice.

Results - 5 DAYS EXPOSURE Filtered Air Polluted Air C57-WT C57-WT Fluorescence Intensity - 5 DAYS EXPOSURE Filtered Air Polluted Air 5d Filtered Air Polluted Air After 5 days of exposure in wild type animals, we observed that the inflammatory response continues to ICAM marker. C57-WT C57-WT

Results - 5 DAYS EXPOSURE Filtered Air Polluted Air - WT Polluted Air - XPC However, when we compared this response between wild type and XPC mice, we observed that the result is different. Unlike the wild animals, the XPC mice did not show inflammatory response to ICAM during the five-days exposure C57-XPC C57-WT C57-XPC

Results Filtered Air Polluted Air 5 days Polluted Air 1 day C57-XPC Fluorescence Intensity Polluted Air 5d Filtered Air Polluted Air 1d Filtered Air Polluted Air 5 days Polluted Air 1 day Here, we observed a diference in the inflammattory response in XPC animals after 1 and 5 days exposure. C57-XPC C57-XPC C57-XPC

Conclusion Exposure to ambient air pollution in São Paulo promotes acute inflammatory responses in mice, including a single day exposure; XPC mice has shown higher inflammation response for a 1-day exposure relative to wild animals; Wild type animals still depict inflammation after 5 days of exposure. However, XPC animals do not.

Acknowledgments DNA Repair Laboratory (ICB-USP) Air Pollution Laboratory (FMUSP) - Atmospheric Pollution Laboratory (Institute of Physics – USP) Financial support: Email: nilmaraoalves@gmail.com or nilmara@usp.br