1. 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
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.

2. 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.

3. 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

4. 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.

5. 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.

6. 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

7. 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.

8. 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

9. 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

10. 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.

11. 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.

12. 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., Environmental Research

13. 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
PM 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

14. 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.

15. 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

16. 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.

17. 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

18. 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

19. 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 or
Financial support: