A proposed in vitro cytotoxicity test battery to detect and predict hepatotoxicity via multiple mechanisms and pathways: a minireview.

The 21st-century toxicity testing program recommends the use of cytotoxicity data from human cells in culture for rapid in vitro screening focusing on biological pathways of potential toxicants to predict in vivo toxicity. Liver is the major organ for both endogenous and exogenous chemical metabolism of xenobiotics. Therefore, this review was undertaken to evaluate side by side five different currently used commercial cytotoxicity assay kits for purpose of rapid predictive screening of potential hepatotoxicants. The test compounds for this review were selected from the NIH LiverTox and FDA Liver Toxicity Knowledge Base (LTKB) databases. Human liver HepG2, HepaRG, and rat liver Clone 9 cell cultures were used as the in vitro liver models. Five commercial assay kits representing different biomarkers or pathways were selected for this review. These kits are Vita-Orange Cell Viability Assay Kit (Sigma-Aldrich), CellTiter-Glo Cell Viability Assay Kit (Promega), CytoTox-ONE Homogeneous Membrane Integrity Assay Kit (Promega), DNA Quantitation Fluorescence Assay Kit (Sigma-Aldrich), and Neutral Red Based In Vitro Toxicology Assay Kit (Sigma-Aldrich). This review found that these kits can all be used for rapid predictive cytotoxicity screening of potential hepatotoxicants in human liver HepG2 and rat liver Clone 9 cells in culture as in vitro liver models without compromising quality and accuracy of endpoint measurements as well as the length of toxicity screening time. Unraveling the structure-activity relationship of potential hepatotoxins would help to classify their hepatotoxic effects. Therefore, in addition to the current regulatory hepatotoxicity testing strategies, development and regulatory approval of hepatotoxins need to be discussed in order to identify potential gaps in the safety assessment. The overall results of our study support the hypothesis that a battery of rapid, simple, and reliable assays is an excellent tool for predicting in vivo effects of suspected liver toxins. The human liver HepaRG cells do not appear to be an ideal in vitro liver model for this purpose.

Contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by the cytokinesis-block micronucleus assay.

Extensive human exposure to food- and cosmetics-related consumer products containing nanosilver is of public concern because of the lack of information about their safety. Genotoxicity is an important endpoint for the safety and health hazard assessment of regulated products including nanomaterials. The in vitro cytokinesis-block micronucleus (CBMN) assay is a very useful test for predictive genotoxicity testing. Recently, we have reported the genotoxicity of 20 nm nanosilver in human liver HepG2 and colon Caco2 cells evaluated using the CBMN assay. The objective of our present study was three-fold: (i) to evaluate if HepG2 and Caco2 cells are valuable in vitro models for rapid genotoxicity screening of nanosilver; (ii) to test the hypothesis that the nanoparticle size and cell types are critical determinants of its genotoxicity; and (iii) to determine if ionic silver contributes to the nanosilver genotoxicity. With these objectives in mind, we evaluated the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge, obtained from the same commercial source, under the same experimental conditions and the same genotoxic CBMN endpoint used for the previously tested 20 nm silver. The ionic silver (silver acetate) was also evaluated under the same conditions. Results of our study show that up to the concentrations tested in these cell types, the smaller (20 nm) nanosilver induces micronucleus formation in both the cell types but the larger (50 nm) nanosilver and the ionic silver provide a much weaker response compared with controls under the same conditions.

Flow cytometric evaluation of the contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells.

Exposure to nanosilver found in food- and cosmetics-related consumer products is of public concern because of the lack of information about its safety. In this study, two widely used in vitro cell culture models, human liver HepG2 and colon Caco2 cells, and the flow cytometric micronucleus (FCMN) assay were evaluated as tools for rapid predictive screening of the potential genotoxicity of nanosilver. Recently, we reported the genotoxicity of 20 nm nanosilver using these systems. In the current study presented here, we tested the hypothesis that the nanoparticle size and cell types were critical determinants of its genotoxicity. To test this hypothesis, we used the FCMN assay to evaluate the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge and obtained from the same commercial source using the same experimental conditions and in vitro models (HepG2 and Caco2) as previously tested for the 20 nm silver. Results of our study show that up to the concentrations tested in these cultured cell test systems, the smaller (20 nm) nanoparticle is genotoxic to both the cell types by inducing micronucleus (MN). However, the larger (50 nm) nanosilver induces MN only in HepG2 cells, but not in Caco2 cells. Also in this study, we evaluated the contribution of ionic silver to the genotoxic potential of nanosilver using silver acetate as the representative ionic silver. The MN frequencies in HepG2 and Caco2 cells exposed to the ionic silver in the concentration range tested are not statistically significant from the control values except at the top concentrations for both the cell types. Therefore, our results indicate that the ionic silver may not contribute to the MN-forming ability of nanosilver in HepG2 and Caco2 cells. Also our results suggest that the HepG2 and Caco2 cell cultures and the FCMN assay are useful tools for rapid predictive screening of a genotoxic potential of food- and cosmetics-related chemicals including nanosilver.

Toxicogenomic responses of human liver HepG2 cells to silver nanoparticles.

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.

Comparative genotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by a flow cytometric in vitro micronucleus assay.

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.

Comparative cytotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells in culture.

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 of toxicity induced by nanosilver may be largely as a consequence of the type of cells used. This differential rather than universal response of different cell types exposed to nanoparticles may play an important role in the mechanism of their toxicity. In summary, the results of this study indicate that the widely used in vitro models, HepG2 and Caco2 cells in culture, are excellent systems for screening cytotoxicity of silver nanoparticles. These long established cell culture models and simple assays used in this study can provide useful toxicity and mechanistic information that can help to better inform safety assessments of food- and cosmetic-related silver nanoparticles.

Comparative genotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by fluorescent microscopy of cytochalasin B-blocked micronucleus formation.

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.

Interactive toxicity of usnic acid and lipopolysaccharides in human liver HepG2 cells.

Usnic acid (UA), 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 to evaluate the interactive toxicity, if any, of UA with lipopolysaccarides (LPS), a potential contaminant of food, at low non-toxic concentrations. The human hepatoblastoma HepG2 cells were treated with the vehicle control and test agents, separately and in a binary mixture, for 24 h at 37°C in 5% CO2. After the treatment period, the cells were evaluated by the traditional biochemical endpoints of toxicity in combination with the toxicogenomic endpoints that included cytotoxicity, oxidative stress, mitochondrial injury and changes in pathway-focused gene expression profiles. Compared with the controls, low non-toxic concentrations of UA and LPS separately showed no effect on the cells as determined by the biochemical endpoints. However, the simultaneous mixed exposure of the cells to their binary mixture resulted in increased cytotoxicity, oxidative stress and mitochondrial injury. The pathway-focused gene expression analysis resulted in the altered expression of several genes out of 84 genes examined. Most altered gene expressions induced by the binary mixture of UA and LPS were different from those induced by the individual constituents. The genes affected by the mixture were not modulated by either UA or LPS. The results of the present study suggest that the interactions of low nontoxic concentrations of UA and LPS produce toxicity in HepG2 cells.

Effects of usnic acid exposure on human hepatoblastoma HepG2 cells in culture.

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.

Comparative hepatotoxicity of deoxynivalenol in rat, mouse and human liver cells in culture.

The present study was undertaken to assess, in vitro, the hepatotoxic potential of the food-borne mycotoxin, deoxynivalenol (DON), using rat (Clone9 and MH1C1), mouse (NBL CL2) and human (WRL68 and HepG2) liver cells in culture. The cells were treated with DON for 24 h at 37 degrees C in 5% CO(2) at concentrations of 0-25 microg ml(-1). Following the treatment period, the cells were assayed for biochemical markers of hepatotoxicity that included three independent cytotoxicity assays, oxidative stress and mitochondrial dysfunction. Concentration-dependent cytotoxicity of DON was observed in each of the five different liver cells derived from three different species (rat, mouse and human) over the entire concentration range studied, beginning at 0.1 microg ml(-1). At these concentrations DON did not induce a biologically significant increase in oxidative stress in these liver cells, and showed a significant decrease in the mitochondrial function only in the rat liver MH1C1 cells compared with the control. The results of this in vitro study suggest that DON is a potential hepatotoxin for the rat, mouse and human liver cells in the concentration range tested in this study. The liver cells used in this study showed distinct endpoint-sensitivity to DON related to the species.

A synthetic polypeptide based on human E-cadherin inhibits invasion of human intestinal and liver cell lines by Listeria monocytogenes.

Internalin A is a surface protein of the facultative intracellular pathogen Listeria monocytogenes that interacts with the human host cell protein E-cadherin to facilitate invasion of epithelial cells. A single amino acid substitution at position 16 in mouse E-cadherin prevents this interaction. Synthetic polypeptides of 30 aa encompassing position 16 of human and mouse E-cadherin were tested for their ability to inhibit in vitro invasion of Caco-2, HepG2 and TIB73 cell lines by L. monocytogenes. Only the human-derived peptide was capable of inhibiting invasion in the human-origin Caco-2 and HepG2 cell lines. These findings demonstrate that small polypeptides can inhibit invasion of biologically relevant cell types by L. monocytogenes in vitro and may be potentially useful as therapeutic agents in vivo.

Prooxidant activity and toxicity of nordihydroguaiaretic acid in clone-9 rat hepatocyte cultures.

Nordihydroguaiaretic acid (NDGA) is a polyphenol. It is present at high concentrations in the leaves of the evergreen desert shrub, Larrea tridentate (Creosote bush), which has a long history of medicinal use traditionally by the native Americans and Mexicans. It is generally believed that the antioxidant properties of NDGA are responsible for the medicinal value of this desert shrub. The clone-9 rat hepatocyte cultures were used as an in vitro model to assess the hepatotoxic potential of NDGA and to determine whether it exhibits any prooxidant activity. The hepatocyte cultures were treated with NDGA for 2 h at 37 degrees C at concentrations of 0-100 microM. After the treatment period the cells, the culture supernatants and cell lysates were assayed for evaluation of prooxidant activity and toxicity of NDGA. Oxidative stress level and oxidative cell injury as measured by the peroxidation of membrane lipids and DNA double-strand breaks were used to index prooxidant activity. Cytotoxicity as measured by the leakage of the liver enzyme lactate dehydrogenase (LDH) into the culture medium, mitochondrial function and extent of cell proliferation were used as the endpoints of toxicity. Significant concentration-dependent differences were observed in these biomarkers over the concentration range examined demonstrating the prooxidant activity and toxicity of NDGA in clone-9 rat hepatocyte cultures.

Altered global gene expression profiles in human gastrointestinal epithelial Caco2 cells exposed to nanosilver.

Extensive consumer exposure to food- and cosmetics-related consumer products containing nanosilver is of public safety concern. Therefore, there is a need for suitable in vitro models and sensitive predictive rapid screening methods to assess their toxicity. Toxicogenomic profile showing subtle changes in gene expressions following nanosilver exposure is a sensitive toxicological endpoint for this purpose. We evaluated the Caco2 cells and global gene expression profiles as tools for predictive rapid toxicity screening of nanosilver. We evaluated and compared the gene expression profiles of Caco-2 cells exposed to 20 nm and 50 nm nanosilver at a concentration 2.5 µg/ml. The global gene expression analysis of Caco2 cells exposed to 20 nm nanosilver showed that a total of 93 genes were altered at 4 h exposure, out of which 90 genes were up-regulated and 3 genes were down-regulated. The 24 h exposure of 20 nm silver altered 15 genes in Caco2 cells, out of which 14 were up-regulated and one was down-regulated. The most pronounced changes in gene expression were detected at 4 h. The greater size (50 nm) nanosilver at 4 h exposure altered more genes by more different pathways than the smaller (20 nm) one. Metallothioneins and heat shock proteins were highly up-regulated as a result of exposure to both the nanosilvers. The cellular pathways affected by the nanosilver exposure is likely to lead to increased toxicity. The results of our study presented here suggest that the toxicogenomic characterization of Caco2 cells is a valuable in vitro tool for assessing toxicity of nanomaterials such as nanosilver.

Hepatotoxicity of androstenedione in pregnant rats.

Androstenedione, a naturally occurring steroid hormone, is a dietary supplement used to enhance athletic performance. Little is known, however, about the safety of its use by young adults including women of child bearing age. To test the possible hepatotoxic effects of androstenedione use, this study was undertaken using a rat model. Pregnant rats (six rats/dose) were exposed to androstenedione in corn oil by gastric intubation at 0, 5, 30 or 60 mg/kg body weight/day beginning 2 weeks before mating and continuing through gestation day 19. On gestation day 20, blood and livers were collected from the pregnant rats for analysis of hepatotoxicity endpoints: serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), glutathione (GSH) and glutathione S-transferase (GST), total microsomal P450, nuclear DNA damage and lipid peroxidation. Under these experimental conditions, no significant differences were observed in any of these biomarkers over the concentration range examined.

Hepatocyte culture as an in vitro model for evaluating the hepatotoxicity of food-borne toxicants and microbial pathogens: a review.

Hepatocyte culture is a well-established, well-characterized, and widely used in vitro tool for pharamacological and toxicological studies. The hepatocytes of a wide range of species, including humans, can be maintained in culture with a high metabolizing capacity for several days under closely controlled and easily manipulated conditions. Numerous studies have reported good correlation between in vitro hepatocytes and in vivo siutations. They have been widely used for studies of the metabolism and of the toxicity and mechanisms of action of chemicals and drugs, for the screening of mutagens, carcinogens, and micotoxins, for virulence assessment of microbial pathogens and viruses, for qualitative and quantitative interspecies comparison, and for genomics and proteiomics studies. Hepatocytes, especially human hepatocytes, are used for preclinical drug evaluation and screening as well as for studies of drug metabolism, toxicity, interactions, and structure-activity relationships. They can be used for evaluating the hepatotoxicity of herbal products, dietary supplements, food additives, food-borne toxicants, and microbial pathogens. The results obtained from such in vitro screenings can be used for in vivo studies to asses the safety of test materials of interest. At the present time, there is insufficient evidence for their use in quantitative risk assessment. However, they are suitable for use in qualitative hazard assessment, which may be used for quantitative risk analysis.