The effects of biomass burning from Amazon region in human lung cells Nilmara Alves Post-doctoral researcher School of Medicine of University of São Paulo, Brazil nilmara@usp.br Good afternoon, I’m Nilmara Alves. I’m a pos-doctor in the School of Medicine of University of São Paulo. This work is the result of a collaboration between Federal University of Rio Grande do Norte and my university. The title is The effects of biomass burning from Amazon region in human lung cells. First, I’ll provide a brief overview of the studied region.

Amazon region The Amazon region occupies more than half of the Brazilian territory. This vast region includes both clear area and human impacted areas. The Brazilian Amazon has been affected by deforestation and biomass burning and these cause important impacts on the climate and with adverse effects on human health.

Effects on human health Amazon region Biomass Burning The Amazon region occupies more than half of the Brazilian territory. This vast region includes both clear area and human impacted areas. The Brazilian Amazon has been affected by deforestation and biomass burning and these cause important impacts on the climate and with adverse effects on human health. Effects on human health

The majority of forest fire hotspots in the Brazilian Amazon take place in the deforestation arc with a population of over 10 million people Amazon region The Amazon region occupies more than half of the Brazilian territory. This vast region includes both clear area and human impacted areas. The Brazilian Amazon has been affected by deforestation and biomass burning and these cause important impacts on the climate and with adverse effects on human health.

The majority of forest fire hotspots in the Brazilian Amazon take place in the deforestation arc with a population of over 10 million people Brazilian Amazon Amazon region The Amazon region occupies more than half of the Brazilian territory. This vast region includes both clear area and human impacted areas. The Brazilian Amazon has been affected by deforestation and biomass burning and these cause important impacts on the climate and with adverse effects on human health.

The majority of forest fire hotspots in the Brazilian Amazon take place in the deforestation arc with a population of over 10 million people Brazilian Amazon Amazon region The Amazon region occupies more than half of the Brazilian territory. This vast region includes both clear area and human impacted areas. The Brazilian Amazon has been affected by deforestation and biomass burning and these cause important impacts on the climate and with adverse effects on human health. Deforestation arc

It is important to note that the World Health Organization classified outdoor air pollution as carcinogenic to humans. Despite the effort from epidemiological studies to investigate the effects of the biomass burning on human health, there is not single definitive mechanism of action that explains those results.

Particulate matter smaller than 10 µm This is the first work that investigates the mechanism of action of PM emitted by biomass burning in the Amazon region. Sampling site To collect the samples we used a high volume sampler and we collected the particulate matter smaller than 10 microns because these are the inhalable particles. There are few works that study the mechanisms of action of atmospheric pollutants. As far as we know this is the first work that aims to investigate the pathways of action of organic particulate in the Amazon region. So, the organic matter was extracted and before investigating the mechanisms of action, we identified the compounds in this complex mixture. High Volume Sampler Particulate matter smaller than 10 µm

Polycyclic Aromatic Hydrocarbons (PAH) After collecting, we identified many compounds such as polycyclic aromatic hydrocarbons. Most of the identified compounds are mutagenic and carcinogenic. For example, benzo-a-pirene. Most of the identified PAH are mutagenic and carcinogenic. De Oliveira Alves et al., 2015 Atmospheric Environment

Viability Test – XTT Kit - Human lung cells (A549 cell line) * ** After the chemical analysis, we performed the viability test using A549 cells under different concentrations. We observed that the organic particulate matter altered the cell viability after 72 h of exposure at higher concentration. DC: DMSO 0.1% control PAHs: Polycyclic Aromatic Hydrocarbons (30 ng/mL) *p <0.05 and ** p <0.01 Statistically significant compared to negative control according to Dunnett's test.

Comet assay - Human lung cells after 24h exposure ** ** ** * * Negative Control ** * * Once the concentrations were defined, we analyzed the DNA damage. To all concentrations, we observed that PM organic cause genotoxic effects. Organic PM10 (400 µg.mL-1) NC: negative control DC: DMSO 0.1% control PAH: Polycyclic Aromatic Hydrocarbons (30 ng/mL) *p <0.05 and ** p <0.01 Statistically significant compared to negative control according to Dunnett's test.

Cytokinesis -block micronucleus assay (CBMN) - Human lung cells after 24h exposure * ** Also, using the micronucleus test, we observed that organic PM cause mutagenic effects in human lung cells *p <0.05 and ** p <0.01 Statistically significant compared to negative control according to Dunnett's test. De Oliveira Alves et al., 2014. Environmental Research

G1 arrest is followed by induction of p21 and p53 Cell cycle Analysis - Cell cycle after 24h exposure G1: 47.32% S: 13.71 G2: 38.97% G1: 67.13% S: 7.64% G2: 25.23% Negative control PM10 400 µg/mL Kmowing of the genotoxic effects, we analyzed the cell cycle after 24 hours exposure. At the highest concentration, we observed a cell cycle arrest in G1 folowed by induction of p21 and p53. G1 arrest is followed by induction of p21 and p53

Nuclei fragmentation (sub-G1 Analysis) - Human lung cells after 72h exposure Negative Control 200 µg/mL 400 µg/mL ** **p<0.01 according to Dunnett’ test To study cell death, we analyzed after 72 hours of exposure to organic particulate matter. We observed a statistically significant increase in sub​​-G1 cells at higher concentrations, indicating apoptosis.

This data is the result of DNA fragmentation during apoptosis H2AX and apoptosis Double marking of H2AX and caspase-3 after 72h exposure Negative control 400 µg/mL H2AX Caspase – 3 Here, we did the double marking of gamma-H2ax and caspase-3. We observed an increased gama-H2AX staining at higher concentrations and this result of DNA fragmentation during apoptosis. This data is the result of DNA fragmentation during apoptosis

Cell Death - Organic PM exposed in A549 cells during 72 h ** ** ** Normal cell Early apoptosis Late apoptosis Necrosis ** ** ** Therefore, in this test to differentiate apoptosis and necrosis, the cells continue showed signs of apoptosis in 20 % of the cells and a clear increase in the necrosis occurrence. NC: negative control ** p <0.01 Statistically significant compared to negative control according to Dunnett's test.

Biomass Burning in the Amazon region Organic PM10 Human lung cells G1-phase arrest Cell Death Apoptosis Necrosis DNA damage p53 p21 H2AX Genotoxic Mutagenic Genetic Instabiliy Cell Proliferation Lung Cancer Biomass Burning in the Amazon region

Conclusions - This work shows an important advance in understanding the cellular effects due to exposure of particulate matter from biomass burning; - One key step of this study is based on the identification of organic compounds that are known to affect the human health. Ler acima

Acknowledgments - Laboratory of Environmental Mutagenesis – UFRN - Laboratory of DNA Repair – USP - Laboratory of Atmospheric Chemistry (Institute of Chemistry – USP) - Laboratory of Atmospheric Pollution (Institute of Physics – USP) - National School of Public Health at Oswaldo Cruz Foundation – RJ Supervisors: Silvia Batistuzzo, Carlos Menck and Pérola Vasconcellos Email: nilmaraoalves@gmail.com or nilmara@usp.br Financial support: