Differential effects of the particle core and organic extract of diesel exhaust particles
Highlights
► Native DEP and methanol DEP-extract induced marked expression of COX-2, IL-6 and IL-8, as well as cytotoxicity. ► Residual DEPs did not induce the inflammatory mediators, whereas CYP1A1 expression was markedly increased. ► In spite of the CYP1A1-expression by residual DEP the majority of the PAHs were in the extractable fraction. ► However, certain PAH-derivatives, probably their carboxylic isomers, tended to be retained on the residual DEPs.
Introduction
Exposure to diesel engine exhaust particles (DEPs) has been associated with several adverse health outcomes in which pulmonary inflammation seems to play a key role. Inflammation involves cellular release of a range of inflammatory mediators, including interleukin (IL)-6 and IL-8. A major challenge is to identify specific components or characteristics of the complex and variable mixture that DEPs represent, which may be responsible for such effects. Typically, DEPs consist of a carbonaceous nuclei and a vast number of inorganic and organic compounds, including polycyclic aromatic hydrocarbons (PAHs) and PAH-derivatives, such as quinones, carboxyl- and nitro-PAHs (Cho et al., 2004, Jakober et al., 2007, Schuetzle et al., 1981). The metabolism of PAHs typically involves binding to the aryl hydrocarbon receptor (AhR) and subsequent activation of CYP1A1-enzymes (Ma and Lu, 2007), and PAHs as well as DEPs may induce pro-inflammatory cellular responses, including cytotoxicity and expression and release of inflammatory mediators (Øvrevik et al., 2010, Steerenberg et al., 1998, Totlandsdal et al., 2010).
Studies on effects of native DEPs vs. extracted components and the residual carbonaceous nuclei indicate that extractable organic species are more important to DEP-induced effects than the residual nuclei (Bonvallot et al., 2001, Takano et al., 2007, Yang et al., 1997). These findings are further supported by studies on effects of single DEP-associated organic compounds (Baulig et al., 2003, Hiyoshi et al., 2005, Inoue et al., 2007, Øvrevik et al., 2009), as well as studies of effects induced by different DEP-extracts (Kawasaki et al., 2001) or particles coated with common DEP-associated PAHs (Goulaouic et al., 2008). However, as previously reviewed elsewhere (Ma and Ma, 2002), the particulate component of DEPs is also known to induce effects, which may differ from those associated with organics.
We have recently shown that CYP1A1 expression was induced at very low DEP-concentrations in bronchial epithelial cells, and that this induction appeared to be suppressed at higher concentrations promoting pro-inflammatory responses (Totlandsdal et al., 2010). Nevertheless, basal CYP-activity seemed essential in facilitating DEP-induced expression of various pro-inflammatory mediators such as cytokines (Totlandsdal et al., 2010). The present study was initiated to further investigate the role of organic constituents of DEPs in CYP1A1 vs. cytokine/chemokine responses. We therefore compared the potency of native DEPs and corresponding methanol DEP-extract and residual DEPs to induce cytotoxicity and expression of multiple genes, including CYP1A1, in human bronchial epithelial cells.
Section snippets
Collection and preparation of particle samples
As previously described (Totlandsdal et al., 2010), the DEPs have been chemically characterised and were generated by an unloaded diesel engine (Deutz, 4 cylinder, 2.2 l, 500 rpm) using gas oil. Methanol DEP-extracts and corresponding residual DEP-particles were prepared as previously described (Kocbach et al., 2008), by two-stage sonication in methanol, subsequent separation of particles from the extract by centrifugation, and drying under nitrogen gas, before storage at −20 °C until use. When
Cytotoxicity
As indicated in Fig. 1, the DEP-extract, but not the residual DEPs induced necrotic cell death. The DEP-extract tended to be more toxic than the native DEPs, although the difference was not statistically significant. A slight increase in the percentage of apoptotic cells was noted in response to native DEPs, but remained below 2.5% (data not shown).
Multiple gene expression analysis
Native DEPs, corresponding methanol-DEP-extract and residual DEPs were examined for their effects on the expression of 20 different
Discussion
Gaining more knowledge about the potential of different components of diesel engine exhaust to induce biological responses, is of key importance for the development of more targeted strategies to protect against the adverse health effects associated with DEP-exposure. The present study indicated that residual components of methanol-extracted DEPs accounted for the DEP-induced expression of CYP1A1, whereas extracted organic components primarily were responsible for the DEP-induced cytotoxicity
Funding
This work was funded by the Research Council of Norway through the “Environment, Genetics and Health” program (grant no. 185620/V40).
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
At the Department of Air Pollution and Noise at the Norwegian Institute of Public Health (NIPH), we would like to thank Tonje Skuland and Edel Lilleaas for assistance with the mRNA screening array and culture and maintenance of the BEAS-2B cells. Furthermore, F.R. Cassee at the National Institute for Public Health and the Environment in the Netherlands is thanked for providing us with the DEPs. NEXAFS spectra were recorded by Qianli Chen (Empa and ETH Zürich). The ALS is supported by the U.S.
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