XII Congresso Brasileiro de Ecotoxicologia
25 a 28 de setembro de 2012
Porto de Galinhas – PE
MAPKs PATHWAYS, CITOTOXICITY ASSAY AND CYTOSKELETON INTEGRITY OF
AIRWAY EPITHEIAL CELLS EXPOSED TO DIESEL EXHAUST PARTICLES
Robson Seriani1; Mara S. Junqueira1; Cláudia E. C. Sousa2; Regina P. Markus2; Ana L. Garippo2;
Elnara M. Negri1; Thaís Mauad1; Paulo H. N. Saldiva1; Mariangela Macchione1.
[email protected] (Faculdade de Medicina – USP, SP, SP)
[email protected] (Faculdade de Medicina – USP, SP, SP
2
[email protected] (Instituto de Biociências, USP, SP,SP)
2
[email protected] (Instituto de Biociências, USP, SP, SP).
3
[email protected] (Instituto do Coração, InCor, SP, SP)
1
[email protected] (Faculdade de Medicina – USP, SP, SP)
1
[email protected] (Faculdade de Medicina – USP, SP, SP)
1
[email protected] (Faculdade de Medicina – USP, SP, SP)
1
[email protected] (Faculdade de Medicina – USP, SP, SP)
1
1
Particulate matter from diesel exhaust represents a problem for public health, mainly due to the presence of
toxic compounds that can damage the respiratory system. This study evaluated activation of MAPK (pERK,
pJNK and pP38), mitochondrial activity (MTT assay), lactate dehydrogenase (LDH) assay as a biomarker of
cell death and cytoskeleton integrity, in bronchial epithelial airways (BEAS-2B) exposed to diesel exhaust
particles (DEP). BEAS-2B were maintained in plates with LHC-9 medium supplemented 48h before
experiment, at 37°C and 5% CO2. Experimental groups were stablished as follows: 15, 30 and 60 minutes of
exposition to 100µg/mL of DEP and Graphite (inert material) respectively and 200µM of Vanadium
(Na3VO4 – positive control). LHC-9 medium supplemented cells were used as control. The MAPKs pathway
was identified by western blot and protein densitometry, MTT and LDH assays was done according standard
protocol and cytoskeleton integrity was studied using AlexaFluor488-phalloidin labelling and confocal laser
scanning microscopy. To compare differences between control and treatments groups ANOVA were used.
The significance level was set at 5%. pERK in DEP expression was enhanced at 15 minutes and reduced
gradually overdose time. In Graphite group pERK expression was time-depedent. In Vanadium group the
enhanced expression oscillated between 15 and 60 minutes. pJNK in DEP enhanced expression in 15
minutes, decreased in 30 and 60 minutes, in Graphite enhance in 15 minutes with a decrease in 30 and 60
minutes, in Vanadium group constant decrease was observed. The pP38 was not expressed in all samples.
MTT and LDH assays showed differences between treatments mainly in 15 minutes, for DEP and Graphite
that had significant decrease in mitochondrial activity and overflowed LDH (p<0,01). About cytoskeleton
integrity measured by fluorescence, we did not observe differences on times of treatments, but comparing
between groups in 30 minutes significant difference (p<0,05) was observed in DEP compared to control and
Vanadium. Coincidently, this had relationship with low expression of MAPKs. In summary, DEP, Graphite
and Vanadium cause metabolic changes (decrease) in mitochondrial activity, LDH overflow and MAPKs
pathways activation (pERK and pJNK) at short time of exposition.
Keywords: diesel exhaust particles, toxicity, epithelial cells
Sociedade Brasileira de Ecotoxicologia (SBE)
Universidade Federal de Pernambuco (UFPE)
633