The alkaline comet assay in vitro with primary rat alveolar macrophages

Inhalation of respirable crystalline silica (RCS) may lead to lung diseases, e.g. chronic inflammation, silicosis and cancer. In 1997 RCS has been classified as human carcinogen by the IARC, but the multifactorial aspects of RCS toxicity were acknowledged and industry and science were encouraged to characterise the properties of RCS-containing dusts from various industrial sectors by physicochemical methods and to discriminate their toxic potential by appropriate in vitro/in vivo tests. The SILICERAM project has thus been initiated as a European Collective Research Project to provide those, responsible for setting legislation, with data that will ensure definition of appropriate RCS air limits for the ceramic industry. Within the SILICERAM project, the suitability of the in vitro alkaline comet assay (CA) for screening of the genotoxic potential of ceramic dusts was investigated. Primary rat alveolar macrophages, pre-cultured for 24h, were used as a model system. Mid-size DQ12 quartz was chosen as a positive control, demonstrating an almost linear, concentration-dependent (25-200µg/cm2) induction of DNA strand breaks (SB) after 2h of incubation. SB formation was inhibited by co-treatment with 10µM aluminium lactate, indicating a quartz-specific, surface-dependent clastogenic effect. In addition, DQ12 induced PGE2 liberation and LDH release. TiO2 Bayertitan T (rutile) was investigated as a potential negative control, but was shown to be inappropriate. Unexpectedly, TiO2 induced a significant increase in SB formation, not counteracted by aluminium lactate. Unlike TiO2, Al2O3 could subsequently be identified as an appropriate negative control for the CA. Contrived samples (representative of mineral mixtures employed in ceramic production) composed of different amounts of DQ12, China clay, and feldspar were investigated and could be ranked by their RCS content. Using the optimised CA, different ceramic dust samples collected in the tile, table- and sanitary ware, refractory and brick sector, will be screened and ranked concerning their genotoxic potential. Aluminium lactate will be used to differentiate quartz-dependent and -independent effects.