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Biochemical effects of some CeO2, SiO2, and TiO2 nanomaterials in HepG2 cells.
Citation:
Kitchin, K., J. Richards, B. Robinette, K. Wallace, N. Coates, B. Castellon, E. Grulke, AND J. Kou. Biochemical effects of some CeO2, SiO2, and TiO2 nanomaterials in HepG2 cells. CELL BIOLOGY AND TOXICOLOGY. Springer, New York, NY, 35(2):129-145, (2019). https://doi.org/10.1007/s10565-018-9445-x
Impact/Purpose:
It is difficult to evaluate nanomaterials to determine their type and degree of toxicity and to subsequently make science-based decisions. For major determinants of their biological action may include their size (hydrodynamic and dry), shape, release of biologically active metal ions, band gap, surface area and surface properties, particularly the ability to donate or accept electrons, to release or absorb oxygen and to generate free radicals such as reactive oxygen species (ROS). Thus, the major theories of nanomaterial action are a) oxidative stress, b) inflammation and c) dissolution of the solid phase particle into toxic, soluble metal ions. Nanomaterials composed of silver or copper are of high interest because of their biocidal nature. Thus, nanosilver is used in hospital equipment including catheters, stents, bandages, wound dressings, as well as on surfaces including wheelchair seats and door handles; other nanosilver uses include clothing, paints, bed and bath linens, keyboards, stuffed animals, cosmetics, baby bottles, and food containers. Functional assays have recently been proposed as a way to better predict and connect the physical-chemical properties of nanomaterials and their potential adverse health outcomes. Biochemical parameters offer many good possible functional assays as intermediates in the long causal chain between physical-chemical properties of nanomaterials and eventual toxicity. This biochemical study is part of a large US Environmental Protection Agency coordinated study of many nanomaterials composed of Ag and Cu for systemic toxicity in several organs including the liver. Study parameters have been selected to evaluate cell growth, cytotoxicity, hepatic function and oxidative stress. Parameter comparisons can be made to determine which parameters respond to low exposure concentrations, which biological parameters are the most responsive to various nanomaterials and to what degree the observed effects are associated with or driven by cytotoxicity. These biochemical parameters were chosen to (a) on dose-response, (b) on structure-activity, (c) to better connect the physical-chemical characterization information to their toxic biochemical effects and (d) to contrast different possible mode of toxicity theories of Ag containing nanomaterials and (e) to generate possible functional assays. The resulting data are interpreted in terms of possible mode of action (e. g. free radical attack, glutathione related effects, oxidative stress and release of metal ions), dose-response, structure-activity relationship and adverse outcome pathway. Major conclusions and summary The major findings of this experimental study in HepG2 cells are: A) Lactic dehydrogenase and aspartate transaminase were the most sensitive cytotoxicity and biochemical parameters to the four Ag exposures, B) Decreased G6PDH (by all three nano Ag forms) and GRD activity (only nano Ag R did not cause decreases) support and are consistent with the oxidative stress theory of Ag nanomaterial action, C) Not all forms of Ag produced the same pattern of biochemical effects. For example, AgNO3 did not decrease G6PDH, nano Ag S and nano Ag R did not increase GPX, nano Ag R did not decrease GRD and only nano Ag PVP decreased THRR D) Use of as many as 10 cytotoxicity and growth parameters (3 released enzymes, MTT, MTS, alamar blue, ATP, microscope examination of the cells, protein, MIA content) provided a much fuller complex view of cytotoxic and biological response to the stressor nanomaterials than the use of just one or two cytotoxicity parameters would have provided and E) cytotoxicity per se does not appear to fully explain the patterns of biological responses observed.
Description:
The potential mammalian hepatotoxicity of nanomaterials was explored in dose-response and structure-activity studies in human hepatic HepG2 cells exposed to between 10 and 1000 μg/ml of five different CeO2, three SiO2, and one TiO2-based particles for 3 days. Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function, and oxidative stress. Few indications of cytotoxicity were observed between 10 and 30 μg/ml. In the 100 to 300 μg/ml exposure range, a moderate degree of cytotoxicity was often observed. At 1000 μg/ml exposures, all but TiO2 showed a high degree of cytotoxicity. Cytotoxicity per se did not seem to fully explain the observed patterns of biochemical parameters. Four nanomaterials (all three SiO2) decreased glucose 6-phosphate dehydrogenase activity with some significant decreases observed at 30 μg/ml. In the range of 100 to 1000 μg/ml, the activities of glutathione reductase (by all three SiO2) and glutathione peroxidase were decreased by some nanomaterials. Decreased glutathione concentration was also found after exposure to four nanomaterials (all three nano SiO2 particles). In this study, the more responsive and informative assays were glucose 6-phosphate dehydrogenase, glutathione reductase, superoxide dismutase, lactate dehydrogenase, and aspartate transaminase. In this study, there were six factors that contribute to oxidative stress observed in nanomaterials exposed to hepatocytes (decreased glutathione content, reduced glucose 6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, superoxide dismutase, and increased catalase activities). With respect to structure-activity, nanomaterials of SiO2 were more effective than CeO2 in reducing glutathione content, glucose 6-phosphate dehydrogenase, glutathione reductase, and superoxide dismutase activities
