Hazard identification of exhausts from gasoline-ethanol fuel blends using a multi-cellular human lung model

Ethanol can be produced from biomass and as such is renewable, unlike petroleum-based fuel. Almost all gasoline cars can drive with fuel containing 10% ethanol (E10), flex-fuel cars can even use 85% ethanol (E85). Brazil and the USA already include 10-27% ethanol in their standard fuel by law. Most health effect studies on car emissions are however performed with diesel exhausts, and only few data exists for other fuels. In this work we investigated possible toxic effects of exhaust aerosols from ethanol-gasoline blends using a multi-cellular model of the human lung. A flex-fuel passenger car was driven on a chassis dynamometer and fueled with E10, E85, or pure gasoline (E0). Exhausts obtained from a steady state cycle were directly applied for 6h at a dilution of 1:10 onto a multi-cellular human lung model mimicking the bronchial compartment composed of human bronchial cells (16HBE14o-), supplemented with human monocyte-derived dendritic cells and monocyte-derived macrophages, cultured at the air-liquid interface. Biological endpoints were assessed after 6h post incubation and included cytotoxicity, pro-inflammation, oxidative stress, and DNA damage. Filtered air was applied to control cells in parallel to the different exhausts; for comparison an exposure to diesel exhaust was also included in the study. No differences were measured for the volatile compounds, i.e. CO, NO_x, and T.HC for the different ethanol supplemented exhausts. Average particle number were 6×10² #/cm³ (E0), 1×10⁵ #/cm³ (E10), 3×10³ #/cm³ (E85), and 2.8×10⁶ #/cm³ (diesel). In ethanol-gasoline exposure conditions no cytotoxicity and no morphological changes were observed in the lung cell cultures, in addition no oxidative stress - as analyzed with the glutathione assay - was measured. Gene expression analysis also shows no induction in any of the tested genes, including mRNA levels of genes related to oxidative stress and pro-inflammation, as well as indoleamine 2,3-dioxygenase 1 (IDO-1), transcription factor NFE2-related factor 2 (NFE2L2), and NAD(P)H dehydrogenase [quinone] 1 (NQO1). Finally, no DNA damage was observed with the OxyDNA assay. On the other hand, cell death, oxidative stress, as well as an increase in pro-inflammatory cytokines was observed for cells exposed to diesel exhaust, confirming the results of other studies and the applicability of our exposure system. In conclusion, the tested exhausts from a flex-fuel gasoline vehicle using different ethanol-gasoline blends did not induce adverse cell responses in this acute exposure. So far ethanol-gasoline blends can promptly be used, though further studies, e.g. chronic and in vivo studies, are needed.