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Graphene and its derivatives have become promising materials for biomedical applications in the last decade. Before their widespread application, however, evaluating their toxicity and mechanisms underlying interactions with cellular components is imperative. Aims: Assessment of the effect of two graphene derivatives, pristine graphene oxide (GO) and ammonia-modified GO (GO-NH2) particles, on the ATPase activity of rat liver mitochondria and ROS production. Methods: Liver mitochondria were isolated from male albino rats and treated with different concentrations of GO and GO-NH2 particles (4, 10, 25, and 50 μg/ml). ATPase activity of both, intact and uncoupled by freezing/thawing mitochondria was determined by the measurement of inorganic phosphate (Pi) released from ATP. The generation of hydrogen peroxide (H2O2) after exposure of mitochondria to GO and GO-NH2 particles was determined by a DCFH-D assay. Results: GO and GO-NH2 particles applied at concentrations of 4 and 50 μg/ml did not affect the ATPase activity of intact mitochondria. In contrast, in uncoupled mitochondria, they demonstrated a stimulating effect on ATPase activity. The impact of GO-NH2 was more substantial and concentration-dependent. ROS production was also higher in GO-NH2-treated mitochondria. Conclusion: The present study demonstrated that GO and GO-NH2 particles can exert a cytotoxic effect on mitochondria even after a short-time of exposure to both types of particles. [ABSTRACT FROM AUTHOR]