109 Combined exposure effects of multi-walled carbon nanotubes and silver nanoparticles in <i>in vitro</i> models

Abstract Exposure to nanomaterials can occur via multiple routes, eg upon inhalation of polluted air, via ingestion following mucociliary clearance or with consumption of foods and beverages. To date, investigations of adverse effects in biological systems have focused on exposures to individual materials in relation to their dosimetry and influence of physicochemical characteristics like shape, size, and surface charge. Consequences of exposures to mixtures of nanomaterials remain understudied. In a two-tiered approach, human in vitro models (Caco-2 as monoculture and co-culture with HT29-MTX-E12, A549 and PMA-differentiated THP-1) were exposed to either multi-walled carbon nanotubes (MWCNT) or silver nanoparticles (AgNPs). Based on the cytotoxicity analysis (WST-1), no-/low-toxicity and medium-toxicity dosing combinations were selected. For co-exposures to MWCNT and AgNPs, either two separate dispersions were prepared and combined right before the exposure or both materials were combined and sonicated as mixture. After 24 h, cytotoxicity (WST-1 or LDH assay) and cytokine release (interleukin (IL)-8, IL-1β, tumour necrosis factor (TNF)-α) were quantified. In mono-exposures, both nanomaterials induced a dose-dependent decrease in cell viability. The magnitude differed between the individual cell systems. In co-exposures, both the cell system and the dispersion protocol impacted the outcomes. Extrapolating from the results of the mono-exposures failed to predict the effects of co-exposures in the different cell systems. The standardisation of dispersion protocols and exposure strategies has long been part of the hazard assessment of individual nanomaterials. However, the previously developed approaches may not be suitable to equally arrive at conclusions for the hazard assessment of mixtures.