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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, AND E. Grulke. Biochemical effects of some CeO2, SiO2, and TiO2 nanomaterials in HepG2 cells. CELL BIOLOGY AND TOXICOLOGY. Springer, New York, NY, 2(35):129-145, (2019). https://doi.org/10.1007/s10565-018-9445-x
Impact/Purpose:
Introduction: This biochemical study is part of a large, coordinated US Environmental Protection Agency study of metal oxide nanomaterials composed of CeO2, SiO2 and TiO2 for systemic toxicity in several organs including the liver. Because study parameters have been selected to evaluate cell growth, cytotoxicity, hepatic function and oxidative stress, comparisons can be made to determine which parameters respond to low exposure concentrations, which parameters are the most responsive and to what degree the observed effects are driven by cytotoxicity. Other completed studies in this series include in vitro immuno spin-trapping effects (oxidative stress) (Kitchin, Prasad et al. 2011), proteomics effects, (Ge, Bruno et al. 2011) genomics studies in HepG2 cells (Thai, Wallace et al. 2015, Thai, Wallace et al. 2016) and metabolomics (Kitchin 2014). The central purpose of this study was to further investigate the potential hepatotoxicity of CeO2 containing nanoparticles. Thus nano CeO2 particles W4, X5, Y6 and Z7 were selected. Nano SiO2 particles K1 and N2 were selected in an attempt to study thin coatings of nano CeO2 on a SiO2 base particle (J0)). CeO2 Q was selected as a non-nano, larger CeO2 particle which had been well studied by an European group (Geraets, Oomen et al. 2012). Finally, TiO2 T8141 was included as an additional control in the sense of (a) not CeO2 and (b) not nano sized but still a metal oxide particle. In a more detailed view, the four major purposes of this present study were (a) dose-response, (b) structure-activity, (c) better connecting the physical-chemical characterization information to their toxic biochemical effects and (d) contribute a nanomaterial-toxicity data set useful for structure-activity and dose-response modelers. Many of the study parameters were related to oxidative stress (e. g. superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GRD), glucose 6-phosphate dehydrogenase (G6PDH), gamma glutamyltranspeptidase (GGT), reduced glutathione concentration (GSH) and thioredoxin reductase (THRR). Two other parameters were related to cell growth (microalbulmin (MIA) and protein concentration). Cytotoxicity related parameters were done by a variety of methods (cytotoxicity by dyes and by visual criteria using a microscope), released enzymes subsequent to membrane damage and toxicity (percentage of total lactate dehydrogenase (%LDH), alanine aminotransferase (%ALT) and aspartate transaminase (%AST). Hepatic function was assessed by alkaline phosphatase (ALP), total bilirubin (T BIL) and triglycerides (TRIG). In respect to structure-activity issues, we tried to determine if the studied CeO2, SiO2 and TiO2 nanomaterials are similar toxicologically or if they have quite different biological properties. Specifically, we studied the differences in biochemical effects altered by these 9 metal oxide nanomaterials ranging in dry primary particle size of 8 to 214 nm. These 9 particles (Table 1) also differed in other physical-chemical characteristics (e. g. specific surface area/porosity, primary and agglomerated particle size and particle shape, as well as oxygen, electron and metal vacancies or excesses on the surfaces). These biochemical parameters were chosen to (a) evaluate the type and degree of possible cellular toxicity and to (b) evaluate the oxidative stress theory of nanomaterial-induced toxicity. The resulting data is interpreted in terms of possible mode of action (free radical attack, glutathione depletion and oxidative stress), dose-response and structure-activity relationship.
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.
