Investigation of toxicity effect of TiCN coated on 304 SS and 410 SS substrates in rat fibroblasts and B-lymphocytes

Abstract

In the present study, TiCN thin films were coated on AISI 304 and AISI 410 stainless steel (SS) substrates by Cathodic Arc Physical Vapor Deposition method. TiCN-coated substrates were confirmed by the XRD analysis results. Dense morphology and fine-grained surface of TiCN film were established by SEM images. Cellular toxicity of the coated 304 SS and 410 SS substrates was investigated in the fibroblasts and B-lymphocyte. In respect to that, we have shown coated substrates cytotoxicity, oxidative stress as well as cell viability, reactive oxygen species (ROS), lipid peroxidation (MDA), protein carbonyl, glutathione oxidase (GSSG), and glutathione reductase (GSH) assessment, releasing cytochrome c (Cytc), lysosomal membrane destabilization (AO) may lead to cell death signaling. Our results showed that the coated 304 SS and 410 SS substrates induced cells dysfunction via a significant increase in ROS production, MDA (P < 0.01 and P < 0.001), protein carbonyl (P < 0.05), and GSSG (P < 0.05 and P < 0.01) that correlated to cytochrome c release (P < 0.01). In addition, increased disturbance in oxidative phosphorylation was also shown by the decrease in cell viability (P < 0.001) and GSH (P < 0.01 and P < 0.001) in the coated 304 SS and 410 SS substrates-treated fibroblast and B-lymphocytes. The coated 304 SS and 410 SS substrates contacted cells and trafficked to the lysosomes and this is followed by lysosomal damage, leading to apoptosis/Necrosis. Our results indicated that these materials cause cellular dysfunction and subsequent oxidative stress leading to cognitive impairment in the rat fibroblasts and B-lymphocytes cells.

Keywords: TiCN coating; cathodic arc physical vapor deposition; cell viability; oxidative stress pathway.

Graphical Abstract

Fig. 1

XRD patterns of the uncoated and TiCN coated of: a) 304 SS and b) 410 SS samples.

Fig. 2

SEM microstructure images of TiCN coating deposited on a) 304 SS and b) 410 SS substrates.

Fig. 3

Cell viability was evaluated by MTT staining on fibroblast cell a) and B-lymphocytes b) (1 × 10⁴ cells/well) after exposure 304 SS, 410 SS substrates on 6, 12, and 24 h. The values are expressed as means ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 compared with control c.

Fig. 4

ROS detection by DCFH-DA was utilized to measure intracellular on fibroblast cell a, b) and B-lymphocytes c, d) (1 × 10⁶ cells/well) after exposure 304 SS, 410 SS substrates on 6 (red line), 12 (yellow line), and 24 h (green line) and control group (black line).

Fig. 5

Protein carbonyl assay on fibroblast cell a) and B-lymphocytes b) (1 × 10⁶ cells/well) after exposure 304 SS, 410 SS substrates on 6, 12, and 24 h. The values are expressed as means ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 compared with control c.

Fig. 6

Glutathione reductase content on fibroblast cell a) and B-lymphocytes b) and glutathione oxidase on fibroblast cell c) and B-lymphocytes d) (1 × 10⁶ cells/well) after exposure 304 SS, 410 SS substrates on 6, 12, and 24 h. The values are expressed as means ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 compared with control c.

Fig. 7

TBARS assay for quantification of the products of lipid peroxidation, which is malondialdehyde on fibroblast cell a) and B-lymphocytes b) (1 × 10⁶ cells/well) after exposure 304 SS, 410 SS substrates on 6, 12, and 24 h. The values are expressed as means ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 compared with control c.

Fig. 8

Release of cytochrome c on fibroblast cell a) and B-lymphocytes b) (1 × 10⁶ cells/well) after exposure 304 SS, 410 SS substrates on 6, 12, and 24 h. The values are expressed as means ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 compared with control c.

Fig. 9

Lysosome damage assay by AO on fibroblast cell a) and B-lymphocytes b) (1 × 10⁶ cells/well) after exposure 304 SS, 410 SS substrates on 6, 12, and 24 h. The values are expressed as means ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 compared with control c.