Biochemical effects of copper nanomaterials in human hepatocellular carcinoma (HepG2) cells.

In dose-response and structure-activity studies, human hepatic HepG2 cells were exposed for 3 days to nano Cu, nano CuO or CuCl2 (ions) at doses between 0.1 and 30 ug/ml (approximately the no observable adverse effect level to a high degree of cytotoxicity). Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function, and oxidative stress. With nano Cu and nano CuO, few indications of cytotoxicity were observed between 0.1 and 3 ug/ml. In respect to dose, lactate dehydrogenase and aspartate transaminase were the most sensitive cytotoxicity parameters. The next most responsive parameters were alanine aminotransferase, glutathione reductase, glucose 6-phosphate dehydrogenase, and protein concentration. The medium responsive parameters were superoxide dismutase, gamma glutamyltranspeptidase, total bilirubin, and microalbumin. The parameters glutathione peroxidase, glutathione reductase, and protein were all altered by nano Cu and nano CuO but not by CuCl2 exposures. Our chief observations were (1) significant decreases in glucose 6-phosphate dehydrogenase and glutathione reductase was observed at doses below the doses that show high cytotoxicity, (2) even high cytotoxicity did not induce large changes in some study parameters (e.g., alkaline phosphatase, catalase, microalbumin, total bilirubin, thioredoxin reductase, and triglycerides), (3) even though many significant biochemical effects happen only at doses showing varying degrees of cytotoxicity, it was not clear that cytotoxicity alone caused all of the observed significant biochemical effects, and (4) the decreased glucose 6-phosphate dehydrogenase and glutathione reductase support the view that oxidative stress is a main toxicity pathway of CuCl2 and Cu-containing nanomaterials.

Biochemical effects of some CeO2, SiO2, and TiO2 nanomaterials in HepG2 cells.

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.

Metabolomic effects of CeO2, SiO2 and CuO metal oxide nanomaterials on HepG2 cells.

BACKGROUND: To better assess potential hepatotoxicity of nanomaterials, human liver HepG2 cells were exposed for 3 days to five different CeO2 (either 30 or 100 µg/ml), 3 SiO2 based (30 µg/ml) or 1 CuO (3 µg/ml) nanomaterials with dry primary particle sizes ranging from 15 to 213 nm. Metabolomic assessment of exposed cells was then performed using four mass spectroscopy dependent platforms (LC and GC), finding 344 biochemicals. RESULTS: Four CeO2, 1 SiO2 and 1 CuO nanomaterials increased hepatocyte concentrations of many lipids, particularly free fatty acids and monoacylglycerols but only CuO elevated lysolipids and sphingolipids. In respect to structure-activity, we now know that five out of six tested CeO2, and both SiO2 and CuO, but zero out of four TiO2 nanomaterials have caused this elevated lipids effect in HepG2 cells. Observed decreases in UDP-glucuronate (by CeO2) and S-adenosylmethionine (by CeO2 and CuO) and increased S-adenosylhomocysteine (by CuO and some CeO2) suggest that a nanomaterial exposure increases transmethylation reactions and depletes hepatic methylation and glucuronidation capacity. Our metabolomics data suggests increased free radical attack on nucleotides. There was a clear pattern of nanomaterial-induced decreased nucleotide concentrations coupled with increased concentrations of nucleic acid degradation products. Purine and pyrimidine alterations included concentration increases for hypoxanthine, xanthine, allantoin, urate, inosine, adenosine 3',5'-diphosphate, cytidine and thymidine while decreases were seen for uridine 5'-diphosphate, UDP-glucuronate, uridine 5'-monophosphate, adenosine 5'-diphosphate, adenosine 5'-monophophate, cytidine 5'-monophosphate and cytidine 3'-monophosphate. Observed depletions of both 6-phosphogluconate, NADPH and NADH (all by CeO2) suggest that the HepG2 cells may be deficient in reducing equivalents and thus in a state of oxidative stress. CONCLUSIONS: Metal oxide nanomaterial exposure may compromise the methylation, glucuronidation and reduced glutathione conjugation systems; thus Phase II conjugational capacity of hepatocytes may be decreased. This metabolomics study of the effects of nine different nanomaterials has not only confirmed some observations of the prior 2014 study (lipid elevations caused by one CeO2 nanomaterial) but also found some entirely new effects (both SiO2 and CuO nanomaterials also increased the concentrations of several lipid classes, nanomaterial induced decreases in S-adenosylmethionine, UDP-glucuronate, dipeptides, 6-phosphogluconate, NADPH and NADH).

Differential genomic effects of four nano-sized and one micro-sized CeO2 particles on HepG2 cells.

The objective of this research was to perform a genomics study of five cerium oxide particles, 4 nano and one micrometer-sized particles which have been studied previously by our group with respect to cytotoxicity, biochemistry and metabolomics. Human liver carcinoma HepG2 cells were exposed to between 0.3 to 300 ug/ml of CeO2 particles for 72 hours and then total RNA was harvested. Fatty acid accumulation was observed with W4, X5, Z7 and less with Q but not Y6. The gene expression changes in the fatty acid metabolism genes correlated the fatty acid accumulation we detected in the prior metabolomics study for the CeO2 particles named W4, Y6, Z7 and Q, but not for X5. In particular, the observed genomics effects on fatty acid uptake and fatty acid oxidation offer a possible explanation of why many CeO2 particles increase cellular free fatty acid concentrations in HepG2 cells. The major genomic changes observed in this study were sirtuin, ubiquitination signaling pathways, NRF2-mediated stress response and mitochondrial dysfunction. The sirtuin pathway was affected by many CeO2 particle treatments. Sirtuin signaling itself is sensitive to oxidative stress state of the cells and may be an important contributor in CeO2 particle induced fatty acid accumulation. Ubiquitination pathway regulates many protein functions in the cells, including sirtuin signaling, NRF2 mediated stress, and mitochondrial dysfunction pathways. NRF2-mediated stress response and mitochondrial were reported to be altered in many nanoparticles treated cells. All these pathways may contribute to the fatty acid accumulation in the CeO2 particle treated cells.

Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth.

There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural cultures were more sensitive to neurite outgrowth inhibitors, they also had a lower dynamic range for detecting chemical-induced neurite outgrowth inhibition and greater variability from culture-to-culture as compared to rat primary cortical cultures.

Effects of Silver Nanoparticles and Silver Nitrate on mRNA and microRNA Expression in Human Hepatocellular Carcinoma Cells (HepG2).

In order to understand toxicity of nano silver, human hepatocellular carcinoma (HepG2) cells were treated either with silver nitrate (AgNO3) or with nano silver capped with glutathione (Ag-S) at various concentration. Differentially expressed genelists for mRNA and microRNA were obtained through Illumina RNA sequencing and DEseq data analyses. Both treatments showed non-linear dose response relationships for mRNA and microRNA. Gene expression analysis showed signaling pathways common to both nano Ag-S and AgNO3, such as cell cycle regulation, DNA damage response and cancer related pathways. But, nano Ag-S caused signaling pathway changes that were not altered by AgNO3 such as NRF2-mediated oxidative stress response inflammation, cell membrane signaling, and cell proliferation. Nano Ag-S also affected p53 signaling, survival, apoptosis, tissue repair, lipid synthesis, angiogenesis, liver fibrosis and tumor development. Several of the pathways affected by nano Ag-S are hypothesized as major contributors to nanotoxicity. MicroRNA target filter analysis revealed additional affected pathways that were not reflected in the mRNA expression response alone, including DNA damage signaling, genomic stability, ROS, cell cycle, ubiquitination, DNA methylation, cell proliferation and fibrosis for AgNO3; and cell cycle regulation, P53 signaling, cell proliferation, survival, apoptosis, tissue repair and so on for nano Ag-S. These pathways may be mediated by microRNA repression of protein translation.Our study clearly showed that the addition of microRNA profiling increased the numbers of signaling pathways discovered that affected by the treatments on HepG2 cells and gave US a better picture of the effects of these reagents in the cells.

Effects of Copper Nanoparticles on mRNA and Small RNA Expression in Human Hepatocellular Carcinoma (HepG2) Cells.

With the advancement of nanotechnology, nanoparticles are widely used in many different industrial processes and consumer products. Copper nanoparticles (Cu NPs) are among the most toxic nanomaterials. We investigated Cu NPs toxicity in Human Hepatocellular carcinoma (HepG2) cells by examining signaling pathways, and microRNA/mRNA interactions. We compared the effects of exposures to Cu NPs at various concentrations and CuCl2 was used as a control. The number of differentially expressed mRNA did not follow a linear dose-response relationship for either Cu NPs or CuCl2 treatments. The most significantly altered genes and pathways by Cu NPs exposure were NRF2 (nuclear factor erythroid 2 related factor 2)-mediated oxidative stress response, protein ubiquitination, Tumor protein p53 (p53), phase I and II metabolizing enzymes, antioxidant proteins and phase III detoxifying gene pathways.Messenger RNA-microRNA interaction from MicroRNA Target Filter Analyses revealed more signaling pathways altered in Cu NPs treated samples than transcriptomics alone, including cell proliferation, DNA methylation, endoplasmic reticulum (ER) stress, apoptosis, autophagy, reactive oxygen species, inflammation, tumorigenesis, extracellular matrix/angiogenesis and protein synthesis. In contrast, in the control (CuCl2) treated samples showed mostly changes in inflammation mainly through regulation of the Nuclear Factor Kappa-light-chain-enhancer of Activated B-cells (NFκB). Further, some RNA based parameters that showed promise as biomarkers of Cu NPs exposure including both well and lesser known genes: heme oxygenase 1 (HMOX1), heat shock protein, c-Fos proto-oncogene, DNA methyltransferases, and glutamate-cysteine ligase modifier subunit (GCLM, part of the glutathione synthesis pathway). The differences in signaling pathways altered by the Cu NPs and CuCl2 treatments suggest that the effects of the Cu NPs were not the results of nanomaterial dissolution to soluble copper ions.

Biochemical Effects of Silver Nanomaterials in Human Hepatocellular Carcinoma (HepG2) Cells.

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.

Media formulation influences chemical effects on neuronal growth and morphology.

Screening for developmental neurotoxicity using in vitro, cell-based systems has been proposed as an efficient alternative to performing in vivo studies. One tool currently used for developmental neurotoxicity screening is automated high-content imaging of neuronal morphology. While high-content imaging (HCI) has been demonstrated to be useful in detection of potential developmental neurotoxicants, comparison of results between laboratories or assays can be complicated due to methodological differences. In order to determine whether high-content imaging-based developmental neurotoxicity assays can be affected by differences in media formulation, a systematic comparison of serum-supplemented (Dulbecco's modified Eagle's media (DMEM) + 10% serum) and serum-free (Neurobasal A + B27) culture media on neuronal morphology was performed using primary rat cortical neurons. Concentration-response assays for neuritogenesis, axon and dendrite outgrowth, and synaptogenesis were performed in each media type using chemicals with previously demonstrated effects. Marked qualitative and quantitative differences in the characteristics of neurons cultured in the two media types were observed, with increased neuronal growth and less basal cell death in Neurobasal A + B27. Media formulation also affected assay sensitivity and selectivity. Increases in assay sensitivity were observed in Neurobasal A + B27 media as compared to serum-supplemented DMEM. In some instances, a greater difference between effective concentrations for cell death and neurodevelopmental-specific endpoints was also observed in Neurobasal A + B27 media as compared to serum-supplemented DMEM. These data show that media formulation must be considered when comparing data for similar endpoints between studies. Neuronal culture maintained in Neurobasal A + B27 media had several features advantageous for HCI applications including less basal cell death, less cell clustering and neurite fasciculation, and a tendency towards increased sensitivity and selectivity in chemical concentration-response studies.

Use of high content image analyses to detect chemical-mediated effects on neurite sub-populations in primary rat cortical neurons.

Traditional developmental neurotoxicity tests performed in vivo are costly, time-consuming and utilize a large number of animals. In order to address these inefficiencies, in vitro models of neuronal development have been used in a first tier screening approach for developmental neurotoxicity hazard identification. One commonly used endpoint for assessing developmental neurotoxicity in vitro is measurement of neurite outgrowth. This biological process is amenable to high-throughput measurement using high content imaging (HCI) based methodologies. To date, a majority of HCI studies of neurite outgrowth have focused on measurements of total neurite outgrowth without examining whether stereotypic neuronal growth patterns are disrupted or whether specific sub-populations of neurites (i.e. axons or dendrites) are selectively affected. The present study describes the development and implementation of two HCI based analysis methods for assessing chemical effects on neuronal maturation. These methods utilize the stereotypical growth pattern of primary rat cortical neurons in culture (i.e. the Staging Method), as well as the differential cytoplasmic distribution of ß(III)-tubulin and MAP2 (i.e. the Subtraction Method), to quantify inhibition of neurite initiation, axon outgrowth and secondary neurite (or dendrite) outgrowth in response to chemical exposure. Results demonstrate that these distinct maturational processes are differentially affected by pharmacological compounds (K252a, Na(3)VO(4), Bis-1) known to inhibit neurite outgrowth. Furthermore, a group of known developmental neurotoxicants also differentially affected the growth of axons and secondary neurites in primary cortical culture. This work improves upon previous HCI methods by providing a means in which to rapidly and specifically quantify chemical effects on the growth of axons and dendrites in vitro.

Use of high content image analysis to detect chemical-induced changes in synaptogenesis in vitro.

Synaptogenesis is a critical process in nervous system development whereby neurons establish specialized contact sites which facilitate neurotransmission. Early life exposure to chemicals can result in persistent deficits in nervous system function at later life stages. These effects are often the result of abnormal development of synapses. Given the large number of chemicals in commerce with unknown potential to result in developmental neurotoxicity (DNT), the need exists for assays that can efficiently characterize and quantify chemical effects on brain development including synaptogenesis. The present study describes the application of automated high content image analysis (HCA) technology for examining synapse formation in rodent primary mixed cortical cultures. During the first 15 days in vitro (DIV) cortical neurons developed a network of polarized neurites (i.e., axons and dendrites) and expression of the pre-synaptic protein synapsin increased over time. The localization of punctate synapsin protein in close apposition to dendrites also increased, indicating an increase in synapse formation. Results demonstrated that: (1) punctate synapsin protein with a spatial orientation consistent with synaptic contact sites could be selectively measured, (2) the critical period for synaptogenesis in cortical cultures was consistent with previous reports, (3) chemicals known to inhibit synapse formation decreased automated measurements of synapse number and (4) parallel evaluation of neuron density, dendrite length and synapse number could distinguish frank cytotoxicity from specific effects on synapse formation or neuronal morphology. Collectively, these data demonstrate that automated image analysis can be used to efficiently assess synapse formation in primary cultures and that the resultant data is comparable to results obtained using lower throughput methods.

In vitro assessment of developmental neurotoxicity: use of microelectrode arrays to measure functional changes in neuronal network ontogeny.

Because the Developmental Neurotoxicity Testing Guidelines require large numbers of animals and is expensive, development of in vitro approaches to screen chemicals for potential developmental neurotoxicity is a high priority. Many proposed approaches for screening are biochemical or morphological, and do not assess function of neuronal networks. In this study, microelectrode arrays (MEAs) were used to determine if chemical-induced changes in function could be detected by assessing the development of spontaneous network activity. MEAs record individual action potential spikes as well as groups of spikes (bursts) in neuronal networks, and activity can be assessed repeatedly over days in vitro (DIV). Primary cultures of rat cortical neurons were prepared on MEAs and spontaneous activity was assessed on DIV 2, 6, 9, 13, and 20 to determine the in vitro developmental profile of spontaneous spiking and bursting in cortical networks. In addition, 5 µM of the protein kinase C inhibitor bisindolylmaleamide-1 (Bis-1) was added to MEAs (n = 9-18) on DIV 5 to determine if changes in spontaneous activity could be detected in response to inhibition of neurite outgrowth. A clear profile of in vitro activity development occurred in control MEAs, with the number of active channels increasing from 0/MEA on DIV 2 to 37 ± 5/MEA by DIV 13; the rate of increase was most rapid between DIV 6 and 9, and activity declined by DIV 20. A similar pattern was observed for the number of bursting channels, as well as the total number of bursts. Bis-1 decreased the number of active channels/MEA and the number of bursting channels/MEA. Burst characteristics, such as burst duration and the number of spikes in a burst, were unchanged by Bis-1. These results demonstrate that MEAs can be used to assess the development of functional neuronal networks in vitro, as well as chemical-induced dysfunction.

Comparison of PC12 and cerebellar granule cell cultures for evaluating neurite outgrowth using high content analysis.

Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process underlying nervous system development that can be quantified using automated microscopy and image analysis (high content analysis). The present study characterized and compared the PC-12 cell line (NS-1) and primary cultures of cerebellar granular cells (CGC), as models for assessing chemical effects on neurite outgrowth. High content analysis of neurite outgrowth was performed using the Cellomics ArrayScan V(Ti) automated epifluorescent imaging system to acquire and analyze images of beta-tubulin immunostained cells in 96-well plates. Cell viability was assessed using the CellTiter-Glo assay. Culture of NS-1 or CGC in nerve growth factor or serum respectively, rapidly induced neurite outgrowth that increased over four days in vitro. Seven compounds previously shown to affect neurite outgrowth in vitro were tested in both models for changes in total neurite length and cell viability. In NS-1 cells, four chemicals (PKC inhibitor Bis-I, MEK inhibitor U0126, trans-Retinoic acid, methylmercury) inhibited neurite outgrowth, while lead, amphetamine and valproic acid had no effect. In CGC, five chemicals inhibited neurite outgrowth (Bis-I, U0126, lead, methylmercury, and amphetamine), while trans-Retinoic acid decreased cell viability but not neurite outgrowth. Valproic acid was without effect. The sensitivity of the two models was chemical specific: NS-1 cells were more sensitive to Bis-I, methylmercury and trans-Retinoic acid, while CGC were more sensitive to U0126, lead, and amphetamine. For every chemical (except trans-Retinoic acid), neurite outgrowth was equal to or more sensitive than cell viability. In comparison, out of seven chemicals without prior evidence for effects on neurite outgrowth, only one decreased neurite outgrowth (diphenhydramine in CGC). These findings demonstrate that the effects of chemicals on neurite outgrowth may be cell type specific.