15 research outputs found
Comparative Analysis of Toxic Responses of Organic Extracts from Diesel and Selected Alternative Fuels Engine Emissions in Human Lung BEAS-2B Cells
This study used toxicogenomics to identify the complex biological response of human lung BEAS-2B cells treated with organic components of particulate matter in the exhaust of a diesel engine. First, we characterized particles from standard diesel (B0), biodiesel (methylesters of rapeseed oil) in its neat form (B100) and 30% by volume blend with diesel fuel (B30), and neat hydrotreated vegetable oil (NEXBTL100). The concentration of polycyclic aromatic hydrocarbons (PAHs) and their derivatives in organic extracts was the lowest for NEXBTL100 and higher for biodiesel. We further analyzed global gene expression changes in BEAS-2B cells following 4 h and 24 h treatment with extracts. The concentrations of 50 µg extract/mL induced a similar molecular response. The common processes induced after 4 h treatment included antioxidant defense, metabolism of xenobiotics and lipids, suppression of pro-apoptotic stimuli, or induction of plasminogen activating cascade; 24 h treatment affected fewer processes, particularly those involved in detoxification of xenobiotics, including PAHs. The majority of distinctively deregulated genes detected after both 4 h and 24 h treatment were induced by NEXBTL100; the deregulated genes included, e.g., those involved in antioxidant defense and cell cycle regulation and proliferation. B100 extract, with the highest PAH concentrations, additionally affected several cell cycle regulatory genes and p38 signaling
Nucleotide excision repair is not induced in human embryonic lung fibroblasts treated with environmental pollutants.
The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells) and tested their response to treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 µm (PM2.5) collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs) including the levels of bulky DNA adducts and the nucleotide excision repair (NER) response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS)]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9) and absence (-S9) of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (-S9); the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced
Western blotting analyses of the levels of XPE, XPC and XPA proteins.
<p>Protein expression was measured after the 24 h treatment of HEL12469 cells with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) in the absence (–S9) and presence (+S9) of the microsomal S9 fraction. A representative result of two independent experiments is shown. Amido Black-stained proteins were used as a loading control.</p
PAH content in extractable organic matter (EOM) from individual samplings.
*<p>Significant differences (p<0.001) in PAH concentrations between EOMs from Prague-summer vs. Ostrava-summer and Prague-winter vs. Ostrava-winter.</p
Comparative Analysis of Toxic Responses of Organic Extracts from Diesel and Selected Alternative Fuels Engine Emissions in Human Lung BEAS-2B Cells
Relative levels of XPE, XPC and XPA proteins in lysates of HEL12469 cells.
<p>The cells were treated with B[a]P and EOMs for 24 h. The data represent mean protein levels relative to the control sample from two independent experiments. P–W = Prague-winter, O–W = Ostrava-winter, P–S = Prague-summer, O–S = Ostrava-summer.</p
Relative levels of unscheduled DNA synthesis (UDS).
<p>UDS in HEL12469 cells was studied after 24 h treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOMs) in the absence (–S9) and presence (+S9) of the S9 microsomal fraction. Mean ± SD UDS values relative to the DMSO-treated control are shown, asterisks denote a significant (p<0.05) increase/decrease in the activity of UDS. The baseline levels of UDS in cells treated with DMSO is represented by a bold horizontal line. Each mean UDS value is based on the analysis of 700–2500 cells.</p
In vitro transformation of human bronchial epithelial cells by diesel exhaust particles. Gene expression profiling and early toxic responses
Final concentration of benzo[a]pyrene (B[a]P) in culture media after treatment of the cells with various concentrations of EOM.
<p>Final concentration of benzo[a]pyrene (B[a]P) in culture media after treatment of the cells with various concentrations of EOM.</p
Bulky DNA adduct levels/10<sup>8</sup> nucleotides detected in DNA extracted from HEL12469 cells.
<p>The cells were treated for 24 h with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) in the absence (–S9) and presence (+S9) of the microsomal S9 fraction. P–W = Prague-winter, O–W = Ostrava-winter, P–S = Prague-summer, O–S = Ostrava-summer, N.D. – not detectable.</p