Controlled chamber exposure of healthy human subjects to BD100 exhaust triggered airway inflammatory events, in addition to the cardiovascular consequences recently shown in a companion study, employing a similar exposure [19]. These respiratory and cardiovascular effects were seen, despite the fact that the use of the RME-based biodiesel fuel resulted in roughly half of the PM mass concentration in the chamber, as compared to petrodiesel. Results were based on the engine running at the same load and speed, according to a standardised urban traffic running cycle, during all diesel exposures. The present study indicates that renewable biodiesel fuels may not have a proficient potential to reduce the well known adverse health effects associated with petrodiesel-related traffic air pollution [1–4].
Previous petrodiesel exhaust exposure studies in humans have commonly shown a neutrophilic response in the bronchial mucosa, epithelium and bronchial wash, which was clearly confirmed also in the present study after exposure to BD100 exhaust (see Table 4 for comparison). The pathways demonstrated for petrodiesel-induced neutrophil recruitment, including EGFR tyr 1173 phosphorylation, activation of AhR, CYP1A1, p65 NFкB, and MAPkinase,as well as IL-8 and GRO-alpha release, may also be involved after biodiesel exhaust exposure[14, 22–24]. The increased expression of vascular adhesion molecules, providing rolling and firm adhesion for the recruitment of neutrophils and monocytes from the blood stream to the bronchial mucosa after BD100 exhaust exposure, was also in line with previous petrodiesel exhaust exposure studies [8, 14, 15].
Table 4
Comparison of airway inflammatory effects between biodiesel and petrodiesel exhaust exposure
| BD100 vs. air Present study | Petrodiesel vs. air Salvi et al. AmJRCCM 1999 (8) | Petrodiesel vs. air Friberg et al. PFT 2023 (15) |
BRONCHIAL BIOPSY | | | |
P-selectin subm | ++ | ns | ND |
ICAM-1 subm | (+) | ++ | ND |
Neutrophil subm | ++ | ++ | ++ |
Neutrophil epi | + | ++ | ND |
Mast cells subm | + | + | + |
CD68 + Macrophage subm | + | ND | ND |
Lymphocytes subm | ns | + | + |
BW | | | |
Neutrophil | ++ | ++ | ++ |
Macrophage | + | ns | ns |
+ and ++ = significant increase at p < 0.05 and p < 0.01 respectively |
(+) = non-significant trend p < 0.07 |
ns = non-significant |
ND = no data |
Inflammatory effects in the lungs in the current BD100 exhaust exposure study and two previous petrodiesel studies. Similar exposure protocol used with bronchoscopies performed 6 hours after controlled diesel exhaust vs. filtered air exposures. Findings in bronchial biopsy submucosa (subm) and epithelium (epi), as well as bronchial wash (BW). |
A novel finding was the recruitment of CD68 + macrophages into the bronchial mucosa together with an increase in macrophages in BW after BD100 exhaust exposure, not previously reported after petrodiesel exhaust exposure (Table 4). The early appearance of monocytic cells in the bronchial mucosa, appears mediated by upregulated vascular adhesion expression. These cells with phagocytic properties, expressing the scavenger receptor CD68+, are expected to be of importance for the T-cell interaction after biodiesel particles exposure. At the investigated time point, T-cells were not yet elevated in the bronchial mucosa, but they have been common compatriots in the bronchial inflammation following petrodiesel exposure [8, 14]. When it comes to alveolar macrophages in the peripheral airspaces, sampled by BAL, we did not find any increase at the 6-hour time point. This finding is in line with previous petrodiesel research, showing alveolar macrophages to appear en masse at a later phase, i.e. 24 hours after exposure in human subjects, due to demand for clearance of exhaust particles [7]. While the macrophage recruitment enhances the clearance capacity, we reported petrodiesel exhaust particles to adversely impair the phagocytic capacity of the individual macrophages [7]. This will be addressed in future biodiesel studies, when the later phase outcomes will be nvestigated.
The influx of macrophages and neutrophils into the airways reflected by the BW, was not accompanied by any increase in the levels of the neutrophil peroxidase MPO or metalloproteases and inhibitors, mainly secreted by neutrophils and macrophages, at the investigated 6-hour time point. Forthcoming research will address these components at a later time frame, as indicated by a previous study [25].
In a companion paper [21], it was shown that biodiesel exposure significantly increased BAL-fluid levels of 12, 13-dihydroxyoctadecenoic acid (12,13-DiHOME), a product from linoleic acid, through the CYP pathway and downstream epoxide hydrolase oxidation. This lipokine, released by activated leukocytes, has been associated with the recruitment of neutrophils, lymphocytes and monocytes. BD100 exposure also increased BAL levels of the lipid mediators 13-hydroxyoctadecadienoic acid (13(S)-HODE) and 12,13-dihydroxy-9Z-octadecenoic acid (13-HODE). 13(S)-HODE may be produced during oxidative stress and has been associated with airway epithelial injury, by several pathways including binding to phospholipids in mitochondrial membranes, leading to increased permeability and functional impairment [26]. Interestingly, 13(S)-HODE activates the transient receptor potential cation channel subfamily V 1 (TRPV1), which contributes to autonomic nervous dysfunction and adverse cardiovascular effects, such as impaired vasomotor function, myocardial dysfunction and ST-segment depression on ECG, as previously reported following petrodiesel exposure [10, 16]. The elevated BAL 13(S)-HODE levels may thus suggest similar vascular effects by exposure to BD100 exhaust. Furthermore, we reported a BAL fluid increase in prostaglandin E2 (PGE2) after BD100 exhaust exposure in human subjects, as well as in vitro using a multicell model [27]. PGE2 may have several effects related to petrodiesel- and biodiesel exhaust exposure, including activation of TRPV1, but also impairment of phagocytosis, as previously identified after petrodiesel exposure [7].
So far, few studies have addressed effects of biodiesel exhaust exposure in human subjects. Mehus et al investigated exposures to diesel exhaust from soy methyl ester (SME) (75% blend in 25% petrodiesel fuel) in 48 subjects in an open heavy load-haul-dump (LHD), during mucking operations underground in a mining environment [25]. PM10 concentrations were 336 µg/m3 for petrodiesel and 268 µg/m3 for SME75 biodiesel during exposure for 200 minutes, thus a much higher exposure dose than in the present study. Both petrodiesel and SME75 biodiesel reduced FEV1 and FVC 6 hours after exposure, not considered to be a common finding after experimental petrodiesel studies using lower exposure burdens. The reductions in FEV1 and FVC were in the range of 100–200 ml and were marginally less for SME75. Induced sputum analyses showed increases in neutrophils, macrophages, MMP-9 and MPO across the exposures, both for bio- and petrodiesel. As induced sputum collects an airway secretion induced by a strong provocation with hypertonic saline, it does not always reflect the unprovoked state of the lung tissue, as reflected in biopsies and bronchoalveolar lavages. This could together with the probably higher total exposure dose (PM concentration x time) contribute to differences in effects between the findings by Mehus et al and the present study. Neither can it be excluded that RME and SME may act differentially in the airways, due to different physico-chemical factors. Toxicological effects of exhaust from various biodiesel and petrodiesel fuels have been investigated using animal, cell culture and bacterial models. Biodiesel fuels, despite being described as “green”, renewable and CO2-neutral, do not necessarily provide less toxic or proinflammatory potential as compared to petrodiesel, when a variety of endpoints are taken into account [27–30].
For comparisons of environmental and health impacts between different fuels, not at least biodiesel and petrodiesel, it has been highlighted that it may not be sufficient to rely only on the engine exhaust PM mass equivalence emission factors, e.g. given as g PM per kWh. Since the actual emissions to the ambient air depend on a combination of the PM mass equivalence emission factors per energy (fuel) used and the fuel consumption, an assessment of the emissions per distance for the specific vehicle when using different fuels, could be a more relevant measure to assess real life environmental health impacts [31, 32]. Biodiesel fuels, which consist of fatty acid methyl esters, have higher cetane number than petrodiesel, resulting in faster ignition and combustion. Moreover, an increased ratio of oxygen within the fuel may affect the combustion efficiency, which together with less aromatics and sulphur, contributes to reduced soot formation and particle generation. This has been taken into account when it comes to the design of the present and our preceding studies, in which biodiesel PM concentrations often have been lower compared with corresponding petrodiesel fuel exhaust emissions, given the same engine load and running conditions [19, 21, 25, 29, 30, 32]. In addition, we have shown that the reduction in biodiesel PM mass in the present engine set-up was associated with a higher fraction of organic matter, considerable less PAHs but a relatively higher fraction of oxygenated PAHs (oxy-PAHs), as well as higher numbers of small nanoparticles [18, 19].
Strengths and limitations
Strengths include investigations in human subjects, which provide species validation as compared to other research approaches. Each subject was investigated twice, thereby serving as her or his own control. The random order of exposures was blinded to the subjects and the investigators, but known to the technical staff providing the exposure atmosphere, with codes broken only after completion of statistical analyses. The same engine set-up was used with comparable running conditions as in a series of earlier studies [15, 19, 33, 34].
Limitations include a single exposure, which only presents information regarding acute effects, whereas long-term repeated exposures are difficult to perform in human subjects under controlled conditions. It is recognised that information of health effect parameters in elderly individuals and subjects with respiratory and cardiovascular diseases is highly relevant from health care perspectives. We have previously undertaken exposure studies in elderly as well as individuals with allergy, asthma, COPD and cardiovascular diseases [10, 20, 35–37], which would be of importance to study in regard to health effects of exhaust from novel fuels.
Complementary panels of markers of inflammation, oxidative burden and other mechanisms are motivated, and remain in focus for forthcoming reports.
Investigations with invasive procedures, such as bronchoscopies in humans, are limited to the number of subjects that can practically be investigated, depending on staff and financial resources. It is recognised that a larger number of subjects could have provided additional statistical strength. Furthermore, the time point chosen for bronchoscopy investigation is a critical issue to identify effects and, while multiple time points for bronchoscopies would be ideal, this would demand more than two investigations within the same individual, which is difficult, as well as an ethical consideration. On the other hand, including additional subjects with sampling at different time points is possible, however, will make the study substantially larger and much more resource-demanding. Complementary future studies are needed to address several of these issues.
Similarities and differences when it comes to the physical and chemical properties of exhaust from biodiesel and petrodiesel fuels deserve further exploration, in order to determine whether future diesel, or diesel-like fuels, fuels could be designed to avoid the toxicological properties that lead to adverse respiratory and cardiovascular responses.