1. IntroductionEpidemiological and experimental studies have reported compelling evidence that pollution by airborne particulate matter (PM) is an important cause of health outcomes associated with respiratory and cardiovascular diseases as well as cancer mortality [1,2,3,4]. In urban areas, the traffic sector is a major contributor to PM emissions as well as other pollutants, including carbon monoxide (CO), volatile organic compounds (VOCs), nitrogen oxides (NOX), polycyclic aromatic hydrocarbons (PAHs), and PM [5,6]. Moreover, diesel exhaust has been classified as carcinogenic (Group 1) and gasoline exhaust as possibly carcinogenic (Group 2B) to humans by the International Agency for Research on Cancer (IARC) [7].Today’s transports depend mainly on fossil fuels, and alternatives are needed against increasingly stringent emission regulations, rising oil prices, and a finite supply of fossil fuels. Biofuels are renewable, sustainable, efficient, and cost-effective energy sources [8] and represent an attractive option to replace fossil fuels. Biofuels blended with gasoline have been gradually introduced to the global market; an example is E10, constituted of 10% ethanol and 90% gasoline. Among the diversity of biofuels, second-generation biofuels (B2G) are derived from lignocellulosic biomass. The special feature of the B2G is that “nonfood biomass” such as agricultural waste, straw, grass, and wood, is used as raw materials for their production [9,10,11]. Compared to the abundant literature on diesel and gasoline emissions, relatively few studies focus on B2G and their health effects. Several studies have shown that adding ethanol to gasoline reduces carbonaceous pollutants, such as carbon monoxide (CO), total hydrocarbons (THC), and particulate matter. However, some studies also reported the formation of more complex compounds, such as acetaldehydes, that could lead to greater health effects [12,13,14].The mechanisms emphasizing the health outcomes of PM are considered to involve mainly oxidative stress and inflammation. Oxidative stress results from an imbalance between the generation of reactive oxygen species (ROS) and the antioxidant defense. ROS are indispensable for regulating critical signaling pathways involved in cell growth, proliferation, differentiation, and survival. However, an excess of ROS can lead to oxidative stress [15]. Hence, the measurement of the oxidative potential (OP) of PM could be used as an indicator of particle toxicity. The OP can be defined as the PM capacity to oxidize target molecules and induce the formation of oxidizing species in the lung [16]. This evaluation can be performed by various acellular methods, such as the DTT assay (OPDTT) or the antioxidant depletion assay (OPAO), which are simpler and quicker to achieve than cellular assays. Moreover, studies have established a link between OP and biological responses to particle exposure, making it a relevant toxicological metric [17,18,19].Besides chemical composition, size is also an essential factor for PM assessment. Fine particles (FP) and ultrafine particles (UFPs) have attracted more attention as they can enter deeper into the respiratory tract [20]. UFPs are also associated with lower mass concentrations than coarser fractions; they are more numerous and have a higher specific area, which may significantly impact their consequences on human health [4,21]. The health effects of traffic emissions from diesel and gasoline engines have been extensively studied in vitro; nevertheless, there are few studies concerning new fuel emissions, and in most of the studies, particle suspensions were used for submerged exposures [22,23,24,25]. Therefore, new approaches to assessing health effects from particles, especially UFPs, are needed to support biofuel development, but generating realistic aerosols and performing in vitro exposures at relevant particle-doses is challenging.

The aim of this study was to compare the physicochemical and toxicological characteristics of UFPs emitted during the combustion of gasoline fuel and biofuel Surrogates under controlled combustion and exposure conditions. This study focused on one variable, the fuel: propane as a reference fuel, a gasoline surrogate, and two biofuel surrogates consisting of a Surrogate blended with anisole to mimic a B2G or ethanol to mimic an E10. These Surrogates were combusted under identical operating conditions, and the physicochemical characterization was performed in order to investigate how the composition of these four types of UFP affects their biological effects in vitro.

4. Discussion

The use of biofuels results in multiple technical, economic, environmental, and health challenges. In terms of health, the main interest is the reduction of harmful emissions. The aim of this study was to compare the physicochemical profile and the toxicological effects of gasoline and B2G fuels.

Gas analysis showed that for carbon dioxide, the highest levels were by the CAST1 condition, and the addition of anisole to the Surrogate slightly increased the amount while the ethanol slightly reduced it. Regarding the chemical composition of PM, PAHs were identified as major contributors, especially from liquid fuels, compared to CAST1 propane. The addition of anisole to the Surrogate is the condition showing the higher PAHs amounts, mainly acenaphthylene and naphthalene. This agrees with several studies showing that biofuel combustion, especially from ethanol blends, reduces carbon dioxide, hydrocarbons, PM, and PAHs emissions while increasing NOx and aldehydes [41,42,43,44]. The particle size measurements showed that the addition of an oxygenated additive (ethanol or anisole) decreased the geometric diameter of the primary spherules constituting soot aggregates, as previously observed by Verma et al., 2021, from biodiesels. However, the CAST1 primary particles presented the smallest size [45].The smaller-sized particles could result in higher oxidation reactivity given their larger specific surface area. This can be detected by measures of oxidative potential (OP), an indicator of the biological reactivity of particles. Two types of acellular tests were used in this work, using increasing concentrations of particles. Results demonstrated that both DTT and ascorbic acid were more depleted by CAST1 particles compared to the other particles. This observation agrees with the hypothesis that size may contribute to OP, given a larger specific area. However, for the smallest concentration, OPDTT results demonstrated that particles from liquid fuels depleted more DTT than CAST1 particles, probably due to PAHs, which are known to correlate strongly with DTT [46,47]. However, this depletion remained modest, contrary to the results obtained with the CAST1 particles. The OPAA results were less consistent, probably due to the absence of metals (i.e., Mn, Fe, Cu, Cr, and Zn) known to drive the AA response [48]. Indeed, important contents of Fe, Cu, and Zn are found in emissions from commercial diesel and gasoline fuels [17,47,49]. Hence, further analyses are needed to elucidate the complexity of metal content in fuel emissions and their involvement in the oxidative potential of emitted UFPs.As inhaled UFPs deposition may occur at the bronchial level [20], the bronchial epithelial BEAS-2B cells were used in this study. BEAS-2B cells were exposed to freshly generated aerosols from the miniCAST, and biological endpoints were performed after 3 or 24 h of incubation with particles. Each aerosol was diluted to 50 mg/m3 for a comparable estimated depositional dose of particles (370 ng/cm2) in order to investigate an early and a late response. The cytotoxicity performed by the MTT assay was reported to be 50]. This ratio is an indicator of the energetic cellular state, which may reflect an increase in the hydrolysis of ATP and, therefore, its consumption or a decrease in its production. Although these effects on energetic metabolism were modest, it can be assumed that they could have cellular consequences if prolonged or repeated exposures occurred. Indeed, the reduction of ATP affects many essential cellular systems, such as motor protein functions, including ciliary motility and intracellular transport. Although further studies are needed to explain the origin of the alterations observed here and their potential consequences, these results are consistent with previous studies, highlighting impaired energy homeostasis after particle exposure. A previous study has shown that airborne particles may disrupt the alveolar/endothelial barrier function, which is tightly regulated by intracellular ATP [51]. Jin et al. have also shown an alteration of ATP production and energy metabolism in the lungs of rats after sub-chronic PM2.5 exposures [52]. These observations outline the use of ATP assessments as an indicative biomarker of PM exposures.Our results on NQO1 and HO-1 mRNA expression did not show important effects regarding oxidative stress. NQO1 reported a slight up-regulation by the S+10A condition at 3 h and by Surrogate at 24 h. NQO1 is induced by Nrf2 and exerts cytoprotective, antioxidant, and anti-inflammatory effects in the lungs [53] against particles, which leads to its induction [22,54]. Concerning HO-1, Surrogate and S+10A induced an important up-regulation 3 h after exposure, and after 24 h, expression was restored to normal values. This kinetic of HO-1 expression response is similar to that observed in a previous study with organic ultrafine particles [29], underlining the cellular adaptation to transient production of reactive oxygen species through the induction of antioxidant response. Interestingly, this result agrees with the results of the OP at the lowest dose, which represents the same order of magnitude as that estimated in culture (more precisely 1.5 times). Inflammation response and, more specifically, IL-8 and IL-6 mRNA expression were also studied by qRT-PCR. IL-8 was slightly downregulated at 3 h, but the opposite response was observed at 24 h. IL-6 showed similar results. Studies on PFs and UFPs mainly reported the induction of inflammation genes at higher exposure doses [55,56] as well as cytokine release after longer incubation times [57,58]. In addition, immunosuppressive effects have been seen to be related to PAHs amount [59]. Our results suggest that a down-regulation of IL-8 and IL-6 may be associated with an early inflammation response, probably due to the organic content. Further research should consider measuring cytokine release to validate these conclusions. The xenobiotic metabolism was also considerably disturbed. CYP1A1 and CYP1B1 mRNA were highly upregulated by liquid fuels at both time conditions. These two cytochrome P450 (CYP) enzymes may be induced by PAHs presented in particles. According to the above-reported results, CAST1 does not significantly impact the gene expression of the studied markers, while Surrogate and S+10A or S+10E do.

Despite the interesting approach proposed in this study, there are a few limitations. Although using a soot generator and Surrogate fuels allows the generation of reproducible aerosol emissions, in real-world conditions, fuel emissions encounter physicochemical processes such as oxidation and interaction with other pollutants that can modify PM characteristics, resulting in different effects compared to the laboratory conditions. Indeed, the composition of the exhaust particles can vary according to parameters such as the type and age of the engine, the drive cycle, the presence of additives in fuels, etc. Another limitation of our work concerns the in vitro model based on a cell line. The BEAS-2B cell model is widely used in particle toxicology; nevertheless, primary cell models are more representative of the pulmonary epithelium.