Study of oncogene expression in co-culture of BEAS-2B cells and type 1 or type 2 macrophages exposed to anthracose and particulate matter from atmospheric pollution (PM2.5)

Author(s)

Daniela Perroni Frias

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2024-01-23

Examining board members

Mariangela Macchione; Fernanda Degobbi Tenorio Quirino dos Santos Lopes; Thais Mauad; Sergio Daishi Sasaki

• Advisor: Mariangela Macchione

Abstract

Anthracosis is a term used to describe the black pigmentation of the lungs and tracheobronchial tree caused by the deposition of inhaled carbon particles. Recent evidence indicates that particulate matter from air pollution plays an important role in the establishment of anthracosis and possibly in the development of cancer in the respiratory system, due to carcinogenic substances from pollution that remain deposited in the lung. The aim of this work was to verify the reactivity of anthracosis particulate matter (MPA), evaluating the expression of oncogenes in normal human bronchial cells (BEAS-2B) or lung adenocarcinoma (A549) in co-culture with M1 or M2 macrophages exposed to MPA. Environmental particulate matter (DEP) was collected from diesel exhaust engine and MPA was collected from at the USP Capital Death Verification Service (SVO). Both were chemically characterized for Polycyclic Aromatic Hydrocarbons (PAH) by gas chromatography coupled to mass spectrometry. After exposure to different concentrations of each particulate, MTT and LDH viability tests were performed on monocultures of BEAS-2B and A549. After 24 hours of exposure to MPA or DEP at 50 g/mL in co-cultures and monocultures, the expressions of genes involved in the xenobiotic response CYP1A1 and CYP1B1 and cancer markers TP53, AGR2 (DNA repair mechanism and cell cycle), PTEN, PIK3CA and EGFR (cell growth) were evaluated by RT-PCR. Immunophenotyping of macrophages exposed to MPA was performed by Flow Cytometry and the cytokines IL-1, TNF-, IL-6, IL-8, IL-10 and INF- were measured in the supernatant of mono and co-cultures. The results showed PAHs with 2 rings for MPA and 4 or 5 rings for DEP, also a higher amount of PAH in g/g in DEP compared to MPA. MTT and LDH showed low cytotoxicity of MPA and DEP after 24 hours of exposure. Immunophenotyping showed heterogeneity in macrophages, displaying M1 and M2 markers both in mono and co-cultures, while the cytokines IL-1, TNF-, IL-6, IL-8 increased after exposure to MPA, but not DEP. Oncogenes remained unchanged in mono- and co-cultures, while CYP1A1 and CYP1B1 genes increased expression after exposure to DEP, but not MPA. The findings of this work indicate that MPA is not an inert particulate, but induced a different response in pulmonary cells and co-cultures compared to DEP, probably related to differences in their compositions (AU)

• FAPESP's process: 19/03586-2 - Study of the expression of oncogenes in co-culture of BEAS-2B cells and macrophage type 1 or type 2 exposed to anthracosis and atmospheric particulate matter (PM2.5)
• Grantee: Daniela Perroni Frias
• Support Opportunities: Scholarships in Brazil - Doctorate