You are here:
Biochemical Effects of Silver Nanomaterials in Human Hepatocellular Carcinoma (HepG2) Cells
Citation:
Kitchin, K., J. Richards, B. Robinette, K. Wallace, Najwa Haykal Coates, B. Castellon, E. Grulke, J. Kou, AND R. Varma. Biochemical Effects of Silver Nanomaterials in Human Hepatocellular Carcinoma (HepG2) Cells. Journal of Nanoscience and Nanotechnology. American Scientific Publishers , VALENCIA, CA, 20(9):5833-5858, (2020). https://doi.org/10.1166/jnn.2020.17858
Impact/Purpose:
It is difficult to evaluate nanomaterials to determine their type and degree of toxicity and to subsequently make science-based decisions. For nanomaterials, major determinants of their biological action may include their size (hydrodynamic and dry), surface area, shape, release of biologically active metal ions, band gap, surface properties, 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 oxidative stress mode of action has frequently been mentioned among the major possible modes of action of nanomaterials. A second major theory of nanomaterial toxicity is inflammation. Oxidative stress and inflammation can be closely related in many ways. A third major theory of nanomaterial toxicity is dissolution of the solid phase particle into soluble metal ions. These soluble metal ions are then the primary causal biological/toxic agents. The dissolution into toxic metal ions theory is particularly germane to exposures to copper and silver nanomaterials. In the nanotoxicology world, 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 can serve as 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. In this study parameters were 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. Nanomaterials composed of silver or copper are of high interest because of their biocidal nature. Nano copper has found uses in many different products including medical and biocidal applications (e.g. bactericide, algicide and fungicide), capacitor materials, catalysts, conductive coatings, conductive inks, conductive pastes, high thermal conductivity materials, lubricant additives and sintering additives in ceramic and diamond tool production. The three Cu containing treatment chemicals were nano Cu, nano CuO and CuCl2 (ions). Table 1 presents publicly available physical-chemical characteristics of these three materials (e. g. vendor, lot number, dry estimated size and purity) and also physical-chemical characterization performed by our research team. In the structure-activity and dose-response studies presented here, nano Cu, nano CuO and CuCl2 (exposures ranged between 0.1 and 30 ug/ml for three days) were examined for their ability to cause cellular toxicity and biological effects on the activities of 11 enzymes and 4 cellular constituents (total bilirubin, triglycerides, micro albumin and total protein) in HepG2 cells. The five major purposes of this study were (a) to perform dose-response studies, (b) to obtain information on structure-activity relationships, (c) to better connect the physical-chemical characterization information to their toxic biochemical effects, (d) to contrast different possible mode of toxicity theories of Cu containing nanomaterials and (e) to generate possible functional assays for use in adverse outcome pathway (AOP) based risk assessment.
Description:
In dose–response and structure–activity studies, human hepatic HepG2 cells were exposed to between 0.01 and 300 ug/ml of different silver nanomaterials and AgNO3 for 3 days. Treatment chemicals included a custom synthesized rod shaped nano Ag, a glutathione capped nano Ag, polyvinylpyrrolidone (PVP) capped nano Ag (75 nm) from Nanocomposix and AgNO3. Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function and oxidative stress. Few indications of cytotoxicity were observed between 0.1 ug/ml and 6 ug/ml of any nano Ag. At 10 ug/ml and above, Ag containing nanomaterials caused a moderate to severe degree of cytotoxicity in HepG2 cells. Lactate dehydrogenase and aspartate transaminase activity alterations were the most sensitive cytotoxicity parameters. Some biochemical parameters were altered by exposures to both nano Ag and AgNO3 (statistically significant increases in alkaline phosphatase, gamma glutamyltranspeptidase, glutathione peroxidase and triglycerides; in contrast both glutathione reductase and HepG2 protein concentration were both decreased). Three parameters were significantly altered by nano Ag but not by AgNO3 (decreases in glucose 6-phosphate dehydrogenase and thioredoxin reductase and increases in catalase). Cytotoxicity per se did not appear to fully explain the patterns of biological responses observed. Some of the observations with the three nano Ag (increases in alkaline phosphatase, catalase, gamma glutamyltranspeptidase, as well as decreases in glucose 6-phosphate dehydrogenase and glutathione reductase) are in the same direction as HepG2 responses to other nanomaterials composed of TiO2, CeO2, SiO2, CuO and Cu. Therefore, these biochemical responses may be due to micropinocytosis of nanomaterials, membrane damage, oxidative stress and/or cytotoxicity. 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.
URLs/Downloads:
DOI: Biochemical Effects of Silver Nanomaterials in Human Hepatocellular Carcinoma (HepG2) Cells