The use of silver nanoparticles in food, food contact materials, dietary supplements and cosmetics has increased significantly owing to their antibacterial and antifungal properties. As a consequence, the need for validated rapid screening methods to assess their toxicity is necessary to ensure consumer safety. This study evaluated two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, as tools for assessing the potential cytotoxicity of food- and cosmetic-related nanoparticles. The two cell culture models were utilized to compare the potential cytotoxicity of 20-nm silver. The average size of the silver nanoparticle determined by our transmission electron microscopy (TEM) analysis was 20.4 nm. The dynamic light scattering (DLS) analysis showed no large agglomeration of the silver nanoparticles. The concentration of the 20-nm silver solution determined by our inductively coupled plasma-mass spectrometry (ICP-MS) analysis was 0.962 mg ml(-1) . Our ICP-MS and TEM analysis demonstrated the uptake of 20-nm silver by both HepG2 and Caco2 cells. Cytotoxicity, determined by the Alamar Blue reduction assay, was evaluated in the nanosilver concentration range of 0.1 to 20 µg ml(-1) . Significant concentration-dependent cytotoxicity of the nanosilver in HepG2 cells was observed in the concentration range of 1 to 20 µg ml(-1) and at a higher concentration range of 10 to 20 µg ml(-1) in Caco2 cells compared with the vehicle control. A concentration-dependent decrease in dsDNA content was observed in both cell types exposed to nanosilver but not controls, suggesting an increase in DNA damage. The DNA damage was observed in the concentration range of 1 to 20 µg ml(-1) . Nanosilver-exposed HepG2 and Caco2 cells showed no cellular oxidative stress, determined by the dichlorofluorescein assay, compared with the vehicle control in the concentration range used in this study. A concentration-dependent decrease in mitochondria membrane potential in both nanosilver exposed cell types suggested increased mitochondria injury compared with the vehicle control. The mitochondrial injury in HepG2 cells was significant in the concentration range of 1 to 20 µg ml(-1) , but in Caco2 cells it was significant at a higher concentration range of 10 to 20 µg ml(-1) . These results indicated that HepG2 cells were more sensitive to nanosilver exposure than Caco2 cells. It is generally believed that cellular oxidative stress induces cytotoxicity of nanoparticles. However, in this study we did not detect any nanosilver-induced oxidative stress in either cell type at the concentration range used in this study. Our results suggest that cellular oxidative stress did not play a major role in the observed cytotoxicity of nanosilver in HepG2 and Caco2 cells and that a different mechanism of nanosilver-induced mitochondrial injury leads to the cytotoxicity. The HepG2 and Caco2 cells used this study appear to be targets for silver nanoparticles. The results of this study suggest that the differences in the mechanisms...
Two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, and flow cytometry techniques were evaluated as tools for rapid screening of potential genotoxicity of food-related nanosilver. Comparative genotoxic potential of 20 nm silver was evaluated in HepG2 and Caco2 cell cultures by a flow cytometric-based in vitro micronucleus assay. The nanosilver, characterized by the dynamic light scattering, transmission electron microscopy and inductively coupled plasma-mass spectrometry analysis, showed no agglomeration of the silver nanoparticles. The inductively coupled plasma-mass spectrometry and transmission electron microscopy analysis demonstrated the uptake of 20 nm silver by both cell types. The 20 nm silver exposure of HepG2 cells increased the concentration-dependent micronucleus formation sevenfold at 10 µg ml(-1) concentration in attached cell conditions and 1.3-fold in cell suspension conditions compared to the vehicle controls. However, compared to the vehicle controls, the 20 nm silver exposure of Caco2 cells increased the micronucleus formation 1.2-fold at a concentration of 10 µg ml(-1) both in the attached cell conditions as well as in the cell suspension conditions. Our results of flow cytometric in vitro micronucleus assay appear to suggest that the HepG2 cells are more susceptible to the nanosilver-induced micronucleus formation than the Caco2 cells compared to the vehicle controls. However, our results also suggest that the widely used in vitro models, HepG2 and Caco2 cells and the flow cytometric in vitro micronucleus assay are valuable tools for the rapid screening of genotoxic potential of nanosilver and deserve more careful evaluation.
The increased use of silver nanoparticles (AgNPs) in foods and cosmetics has raised public safety concerns. However, only limited knowledge exists on the effect of AgNPs on the cellular transcriptome. This study evaluated global gene expression profiles of human liver HepG2 cells exposed to 20 and 50 nm AgNPs for 4 and 24 h at 2.5 µg ml(-1) . Exposure to 20 nm AgNPs resulted in 811 altered genes after 4 h, but much less after 24 h. Exposure to 50 nm AgNPs showed minimal altered genes at both exposure times. The HepG2 cells responded to the toxic insult of AgNPs by transiently upregulating stress response genes such as metallothioneins and heat shock proteins. Functional analysis of the altered genes showed more than 20 major biological processes were affected, of which metabolism, development, cell differentiation and cell death were the most dominant categories. Several cellular pathways were also impacted by AgNP exposure, including the p53 signaling pathway and the NRF2-mediated oxidative stress response pathway, which may lead to increased oxidative stress and DNA damage in the cell and potentially result in genotoxicity and carcinogenicity. Together, these results indicate that HepG2 cells underwent a multitude of cellular processes in response to the toxic insult of AgNP exposure, and suggest that toxicogenomic characterization of human HepG2 cells could serve as an alternative model for assessing toxicities of NPs.
As a consequence of the increased use of silver nanoparticles in food, food contact materials, dietary supplements and cosmetics to prevent fungal and bacterial growth, there is a need for validated rapid screening methods to assess the safety of nanoparticle exposure. This study evaluated two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, as tools for assessing the potential genotoxicity of 20-nm nanosilver. The average silver nanoparticle size as determined by transmission electron microscopy (TEM) was 20.4 nm. Dynamic light scattering (DLS) analysis showed no large agglomeration of the silver nanoparticles. The silver concentration in a 20-nm nanosilver solution determined by the inductively coupled plasma-mass spectrometry (ICP-MS) analysis was 0.962 mg ml(-1) . Analysis by ICP-MS and TEM demonstrated the uptake of 20-nm silver by both HepG2 and Caco2 cells. Genotoxicity was determined by the cytochalasin B-blocked micronucleus assay with acridine orange staining and fluorescence microscopy. Concentration- and time-dependent increases in the frequency of binucleated cells with micronuclei induced by the nanosilver was observed in the concentration range of 0.5 to 15 µg ml(-1) in both HepG2 and Caco2 cells compared with the control. Our results indicated that HepG2 cells were more sensitive than Caco2 cells in terms of micronuclei formation induced by nanosilver exposure. In summary, the results of this study indicate that the widely used in vitro models, HepG2 and Caco2 cells in culture, represent potential screening models for prediction of genotoxicity of silver nanoparticles by in vitro micronucleus assay.
Table 1. The table shows the US federal departments, independent agencies, and commissions that are participating in the National Nanotechnology Initiative (NNI). a Departments, independent agencies, and commissions with budgets for nanotechnology research and development Other federal departments, independent agencies, and commissions that are participating Consumer Product Safety Commission
The US National Nanotechnology Initiative (NNI) defines nanotechnology as "the understanding and control of matter at dimensions between approximately 1 and 100 nm, where unique phenomena enable novel applications." Recent scientific reports available in the literature clearly demonstrate the potential benefits of nanotechnology in consumer and industrial products. More and more nanomaterials are expected to be used in consumer products. This is expected to lead to increased human exposure to nanomaterials in their daily lives. Therefore, the effect of nanomaterials present in human environment is an area of increasing scientific interest. The information presented in this review is obtained from the current literature. It indicates that nanomaterials found in human environment may have potential for toxicological effects. However, the current literature on toxicological effects of nanomaterials is diverse. The current data are presented from studies without harmonization. These studies have used different in vitro and in vivo test models, different sources of test nanomaterials, different methods for nanomaterial characterization, and different experimental conditions. Therefore, these data are hard to interpret. More research on nanomaterial characterization, biological interaction, toxicity, and health effects is needed. The test methods need to be validated. Positive and negative controls for nanotoxicity need to be identified. Toxicity data harmonization needs to be done. Therefore, general information is not currently available for risk evaluation of certain nanomaterials that might be present in consumer products or that may enter into the market in future. Standardized and validated methods are necessary for toxicity assessment of nanomaterials. Therefore, in the absence of standardized validated methods any specific regulatory testing requirements for nanomaterials are currently premature. We conclude that the benefits of nanomaterials found currently in human environment are many, but their overall adverse effects on human health are limited.
Usnic acid, a natural botanical product, is a constituent of some dietary supplements used for weight loss. It has been associated with clinical hepatotoxicity leading to liver failure in humans. The present study was undertaken for metabolism and toxicity evaluations of usnic acid in human hepatoblastoma HepG2 cells in culture. The cells were treated with the vehicle control and usnic acid at concentrations of 0-100 µm for 24 h at 37 °C in 5% CO2 . Following the treatment period, the cells were evaluated by biochemical and toxicogenomic endpoints of toxicity that included cytochrome P450 activity, cytotoxicity, oxidative stress, mitochondrial dysfunction and changes in pathway focused gene expression profiles. Usnic acid exposure resulted in increased P450 activity, cytotoxicity, oxidative stress and mitochondrial dysfunction in HepG2 cells. The pathway-focused gene expression analysis resulted in significantly altered expression of six genes out of a total of 84 genes examined. Of the six altered genes, three genes were up-regulated and three genes down-regulated. A marked up-regulation of one gene CCL21 associated with inflammation, one gene CCNC associated with proliferation and carcinogenesis and one gene UGT1A4 associated with metabolism as well as DNA damage and repair were observed in the usnic acid-treated cells compared with the vehicle control. Also a marked down-regulation of one gene CSF2 associated with inflammation and two genes (CYP7A1 and CYP2E1) associated with oxidative metabolic stress were observed in the usnic acid-treated cells compared with the control. The biomarkers used in this study demonstrate the toxicity of usnic acid in human hepatoblastoma HepG2 cells, suggesting an oxidative mechanism of action.
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