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• Biphasic (anatase-brookite) TiO 2 NPs were synthesized and tested. • Anatase-prevailing TiO 2 NPs induced more oxidative stress and DNA fragmentation. • Anatase-prevailing TiO 2 NPs deregulated proteome of benign epithelial cells. • Anatase crystalline phase contributes to overall cytotoxicity more than brookite. Nanoparticular form of titanium dioxide (TiO 2 NPs) belongs to important industrial material. Despite being widely used, serious contradictions regarding biosafety of TiO 2 NPs remain. We anticipate that such discrepancies could be due to a lack of understanding of a linkage between TiO 2 NPs phase composition and cytotoxicity. Therefore, we synthesized two types of biphasic TiO 2 NPs differing in an anatase-brookite phase composition. The study presents an array of in vitro data suggesting that TiO 2 NPs with a prevailing anatase phase composition possess higher cytotoxicity compared to TiO 2 NPs with an equal anatase-brookite crystallinity. This phenomenon was evidenced by significantly higher inhibition of metabolic activity and growth of epithelial and neuroblast-like cells. Moreover, anatase-prevailing TiO 2 NPs tend to produce higher amount of reactive oxygen species resulting in DNA fragmentation. Further insights into the molecular aspects of cytotoxicity of anatase-prevailing TiO 2 NPs were obtained by comparative proteomics delineating that TiO 2 NPs deregulate expression of a variety of proteins and associated pathways. This inevitably results in a decreased cellular ability to detoxify reactive oxygen species and respond to various stress conditions. The study provides novel data that add another piece to the jigsaw of the relation between structural features of NPs and biosafety. [ABSTRACT FROM AUTHOR]