Pro-oxidant effects of nano-TiO2 on Chlamydomonas reinhardtii during short-term exposure

This study sheds light on the short-term dynamics of pro-oxidant processes related to the exposure of C. reinhardtii microalgae to nano-TiO2 using (a) conventional fluorescence probes for cellular pro-oxidant process and (b) a recently developed cytochrome c biosensor for the continuous quantification of extracellular H2O2. The main aims are to investigate nano-TiO2 toxicity and the modifying factors thereof based on the paradigm of oxidative stress and to explore the utility of extracellular H2O2 as a potential biomarker of the observed cellular responses. This is the first study to provide continuous quantitative data on abiotic and biotic nano-TiO2-driven H2O2 generation to systematically investigate the link between extracellular and cellular pro-oxidant responses. Acute exposures of 1 h were performed in two different exposure media (MOPS and lake water), with nominal particle concentrations from 10 mg L-1 to 200 mg L-1, with and without UV pre-illumination. Abiotic and biotic extracellular H2O2 were continuously measured with the biosensor and complemented with endpoints for abiotic ROS (H2DCF-DA), oxidative stress (CellROX (R) Green) and damage (propidium iodide) measured by flow cytometry at the beginning and end of exposure. Results showed that nano-TiO2 suspensions generated ROS under UV light (abiotic origin) and promoted ROS accumulation in C. reinhardtii (biotic origin). However, extracellular and intracellular pro-oxidant processes differed. Hence, extracellular H2O2 cannot per se serve as a predictor of cellular oxidative stress or damage. The main predictors best describing the cellular responses included ``exposure medium'', ``exposure time'', ``UV treatment'' as well as ``exposure concentration''.