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Cytotoxicity by MTT assay. After 24 h of incubation, the human lymphocytes were counted and 1 *104 cells, seeded into the 96-well culture plates with different concentration of Co3O4, Fe2O3, Silicon dioxide, and Al2O3 NPs(10, 25, 50, 75, and 100 mg/ml) and incubated at 37C for 24 h. After incubation, cells were treated with 5 mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 37C for 4 h in 5% CO2 incubator. Then 50 ml of dimethyl sulfoxide was added to solubilize the formed formazan. The number of viable cells was read by a Microplate Absorbance Reader (BioTek, Winooski, Vermont,USA) at 532 nm. 

The human lymphocytes not treated with NPs were taken as a control. Lactate dehydrogenase leakage assay. About 0.1 ml of Co3O4,Fe2O3, Silicon dioxide, and Al2O3 NPs (50, 75, and 100 mg/ml) were added with 0.2 mM tris(hydroxymethyl)aminomethane(Tris)–hydrochloric acid, 30 mM sodium pyruvate,and 6.6 mM nicotinamide adenine dinucleotide. The absorbance was measured at 340 nm, and the LDH release expressed in terms of percentage.The lactate dehydrogenase leakage (LDH) activity was an alternative assay to determine the cytotoxicity of NPs. The membrane integrity damage of cells was estimated using LDH by measuring the amount of enzyme released by the human lymphocytes.

Oxidative stress in human lymphocytes. Oxidative stress in human lymphocytes was estimated by the reactive oxygen species (ROS) generation. In 96-well culture plates, Silicon dioxide coetent was also estimated.1*10cells/ml were seeded and incubated for 24 h at 37C. After 24 h, different concentration of NPs (50, 75, and 100 mg/ml) was added and incubated for 24 h. Then the cells were incubated at 37C for 30 min with dichlorodihydrofluoresceindiacetate (10 mM). The intensities of fluorescence were detected with wavelength excitation at 485 nm and wavelength emission at 528 nm using a fluorescence spectrophotometer (SL174, ELICO, India).



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