In vitro neurotoxicity of particles from diesel and biodiesel fueled engines following direct and simulated inhalation exposure

February 5, 2024

Lora-Sophie Gerber a b, Dirk C.A. de Leijer a, Andrea Rujas Arranz a, Jonas M.M.L. Lehmann a b, Meike E. Verheul a, Flemming R. Cassee a b, Remco H.S. Westerink a
a Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands
b National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands


  • Particles emitted from diesel or biodiesel combustion reduced neuronal activity.
  • Diesel particles exhibit higher direct neurotoxic potency than biodiesel particles.
  • Effects are caused by adsorbed chemicals and not by the clean carbon core.
  • Simulated inhalation exposure only had limited effects on neuronal activity.
  • Toxic effects were absent in the lung model used to simulate inhalation exposure.

Combustion-derived particulate matter (PM) is a major source of air pollution. Efforts to reduce diesel engine emission include the application of biodiesel. However, while urban PM exposure has been linked to adverse brain effects, little is known about the direct effects of PM from regular fossil diesel (PMDEP) and biodiesel (PMBIO) on neuronal function. Furthermore, it is unknown to what extent the PM-induced effects in the lung (e.g., inflammation) affect the brain. This in vitro study investigates direct and indirect toxicity of PMDEP and PMBIO on the lung and brain and compared it with effects of clean carbon particles (CP).

PM were generated using a common rail diesel engine. CP was sampled from a spark generator. First, effects of 48 h exposure to PM and CP (1.2–3.9 µg/cm2) were assessed in an in vitro lung model (air–liquid interface co-culture of Calu-3 and THP1 cells) by measuring cell viability, cytotoxicity, barrier function, inflammation, and oxidative and cell stress. None of the exposures caused clear adverse effects and only minor changes in gene expression were observed.

Next, the basal medium was collected for subsequent simulated inhalation exposure of rat primary cortical cells. Neuronal activity, recorded using microelectrode arrays (MEA), was increased after acute (0.5 h) simulated inhalation exposure. In contrast, direct exposure to PMDEP and PMBIO (1–100 µg/mL; 1.2–119 µg/cm2) reduced neuronal activity after 24 h with lowest observed effect levels of respectively 10 µg/mL and 30 µg/mL, indicating higher neurotoxic potency of PMDEP, whereas neuronal activity remained unaffected following CP exposure.

These findings indicate that combustion-derived PM potently inhibit neuronal function following direct exposure, while the lung serves as a protective barrier. Furthermore, PMDEP exhibit a higher direct neurotoxic potency than PMBIO, and the data suggest that the neurotoxic effects is caused by adsorbed chemicals rather than the pure carbon core.

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