Toxicity of combustion condensates on human cells

Abstract

Combustion exhausts emitted from traffic and industry sources represent the main contributors to air pollution in urban areas,and as such they have been associated with increased mortality and cardiorespiratory morbidity. Despite intense research, the mechanism of these correlations is still not fully understood. Most of the studies published so far have focused their research on only soot particles or their surrogates (e.g. carbon black), thereby neglecting other combustion by-products present in exhausts. The “cool trap” method, applied in this work, collects particulate and gaseous components of the combustion exhaust. Utilisation of such heterogeneous mixtures can give a better insight in the toxicity of combustion exhausts. <br /> The main aim of this work was to study the toxicity of combustion condensates on human alveolar (A-549) and human monocyte-like (U-937) cell lines. Pre-screening of toxicity was performed by using bacterial SWITCH test which utilises the recombinant <i>S. typhimurium</i> TA1535 pSWITCH strain. The combustion condensates originated from premixed ethylene/oxygen laboratory flames or from diesel fuel combustion in a diesel power generator. <br /> Physical characterisation of the combustion condensates, performed via HRTEM and spectrophotometry, has verified the presence of combustion generated particles: soot and nanoparticles. Total organic carbon (TOC) content proved to be a suitable dose measure for the toxicity assessment of combustion condensates. Combustion condensates’ toxic effects (genotoxic and cytotoxic) were observed with the SWITCH test. Thereby, ethylene combustion condensates generated at different combustion conditions (C/O ratios ranging from 0.63 to 0.93) and diesel combustion condensates (generated during 4kW-load or no-load engine operating modes) induced 50% cell death (LC₅₀) and maximum genotoxic effect in the concentration range between 19 to 46 ppm of TOC. TOC content-dependant cytotoxicity of combustion condensates was demonstrated on both human cell lines. While U-937 cells died predominately via apoptosis, no apoptosis induction was observed in A-549 cells. The NF-κB activation in U-937 cells was clearly observed after 24 h incubation with ethylene combustion condensates containing up to 4.8 ppm of TOC. The NF-κB activation in A-549 cells was investigated by examining the expressions of two NF-κB target genes: IL-6 and NFκBIA. Short-term exposure (2 h and 6 h) to ethylene- or diesel combustion condensates containing 10 ppm of TOC had no impact on the IL-6 gene expression. Longer incubation (24 h) with ethylene combustion condensate significantly increased the level of IL-6 and NFκBIA mRNAs, indicating NF-κB activation and a pro-inflammatory effect. <br /> Data presented in this work clearly show that the toxicity of combustion condensates is proportional to their TOC content and that it is not soot-mediated. Moreover, they show that toxicologically relevant constituent(s) of combustion condensates can neither be retained with a 20 nm pore-size filter nor precipitated by 14 h centrifugation at 116 000 x g.