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.

Differential Genomic Effects of Six Different TiO2 Nanomaterials on Human Liver HepG2 Cells.

Human HepG2 cells were exposed to six TiO2 nanomaterials (with dry primary particle sizes ranging from 22 to 214 nm, either 0.3, 3, or 30 µg/mL) for 3 days. Some of these canonical pathways changed by nano-TiO2 in vitro treatments have been already reported in the literature, such as NRF2-mediated stress response, fatty acid metabolism, cell cycle and apoptosis, immune response, cholesterol biosynthesis, and glycolysis. But this genomic study also revealed some novel effects such as protein synthesis, protein ubiquitination, hepatic fibrosis, and cancer-related signaling pathways. More importantly, this genomic analysis of nano-TiO2 treated HepG2 cells linked some of the in vitro canonical pathways to in vivo adverse outcomes: NRF2-mediated response pathways to oxidative stress, acute phase response to inflammation, cholesterol biosynthesis to steroid hormones alteration, fatty acid metabolism changes to lipid homeostasis alteration, G2/M cell checkpoint regulation to apoptosis, and hepatic fibrosis/stellate cell activation to liver fibrosis.

Signaling Pathways and MicroRNA Changes in Nano-TiO2 Treated Human Lung Epithelial (BEAS-2B) Cells.

The effect of titanium dioxide nanoparticles (nano-TiO2 Degussa p25) treatment of human lung epithelial cells (BEAS-2B) was examined by analyzing changes in messenger [mRNA] and microRNA [miRNA]. BEAS-2B cells were treated with 0, 3, 10, 30 or 100 µg/ml nano-TiO2 for 1 day (for mRNA analysis) or 3 days (for miRNA analysis). Differentially expressed mRNA and miRNA were analyzed using Affymetrix microarrays and Affymetrix miRNA microarrays, respectively. Although, the tested doses were not cytotoxic, there were alterations in both mRNA and miRNA expression. The expression of mRNA/miRNA changes were examined in MetaCore (GeneGo) and IPA (Ingenuity Pathway Analysis) to delineate associated canonical/signaling pathways. Canonical/signaling pathways altered by nano-TiO2 treatments included: cell cycle regulation, apoptosis, calcium signaling, translation, NRF2-mediated oxidative response, IGF1 signaling, RAS signaling, PI3K/AKT signaling, cytoskeleton remodeling, cell adhesion, BMP signaling, and inflammatory response. Many of the genes in these pathways are known to be regulated by the miRNAs whose expressions were altered by the nano-TiO2 treatment. The miRNA 17-92 cluster and let-7 miRNA family that are involved in lung cancer formation were altered by nano-TiO2 treatment. The miR-17-92 cluster, an oncogenic microRNA cluster, is induced while the tumor suppressor microRNA, let-7 family, is suppressed. The changes of let-7/KRAS signaling pathway was observed in all the doses treated. The observed changes in miRNA expression introduces an additional mechanistic dimension that supports the significance of the observed mRNA expression changes, and demonstrated that the nano-TiO2 in vitro treatment in human lung cells can cause diverse but coordinated pathway alterations associated with changes in in vivo response to tumorigenes.

Differential Genomic Effects on Signaling Pathways by Two Different CeO2 Nanoparticles in HepG2 Cells.

To investigate genomic effects, human liver hepatocellular carcinoma (HepG2) cells were exposed for three days to two different forms of nanoparticles both composed of CeO2 (0.3, 3 and 30 µg/mL). The two CeO2 nanoparticles had dry primary particle sizes of 8 nanometers {(M) made by NanoAmor} and 58 nanometers {(L) made by Alfa Aesar} and differ in various other physical-chemical properties as well. The smaller particle has stronger antioxidant properties, probably because it has higher Ce3+ levels on the particle surface, as well as more surface area per unit weight. Nanoparticle M showed a normal dose-response pattern with 363, 633 and 1273 differentially expressed genes (DEGs) at 0.3, 3 and 30 µg/mL, respectively. In contrast, nanoparticle L showed a puzzling dose-response pattern with the most DEGs found in the lowest exposure group with 1049, 303 and 323 DEGs at 0.3, 3 and 30 µg/mL, respectively. This systems biological genomic study showed that the major altered pathways by these two nano cerium oxides were protein synthesis, stress response, proliferation/cell cycle, cytoskeleton remodeling/actin polymerization and cellular metabolism. Some of the canonical pathways affected were mTOR signaling, EIF2 signaling, fatty acid activation, G2/M DNA damage checkpoint regulation, glycolysis and protein ubiquitination. These two CeO2 nanoparticles differed considerably in their genomic effects. M is more active than L in respect to altering the pathways of mitochondrial dysfunction, acute phase response, apoptosis, 14-3-3 mediated signaling, remodeling of epithelial adherens junction signaling, actin nucleation by ARP-WASP complex, altered TCA cycle and elevated fatty acid concentrations by metabolomics. However, L is more active than M in respect to the pathways of NRF2-mediated stress response and hepatic fibrosis/hepatic stellate cell activation. One major difference in the cell response to nano M and L is that nano M caused the Warburg effect while nano L did not.

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.

Catalase has a key role in protecting cells from the genotoxic effects of monomethylarsonous acid: a highly active metabolite of arsenic.

Although it is widely known that arsenic-contaminated drinking water causes many diseases, arsenic's exact mode of action (MOA) is not fully understood. Induction of oxidative stress has been proposed as an important key event in the toxic MOA of arsenic. The authors' studies are centered on identifying a reactive species involved in the genotoxicity of arsenic using a catalase (CAT) knockout mouse model that is impaired in its ability to breakdown hydrogen peroxide (H2 O2 ). The authors assessed the induction of DNA damage using the Comet assay following exposure of mouse Cat(+/) (+) and Cat(-) (/) (-) primary splenic lymphocytes to monomethylarsonous acid (MMA(III) ) to identify the potential role of H2 O2 in mediating cellular effects of this metalloid. The results showed that the Cat(-) (/) (-) lymphocytes are more susceptible to MMA(III) than the Cat(+/) (+) lymphocytes by a small (1.5-fold) but statistically significant difference. CAT activity assays demonstrated that liver tissue has approximately three times more CAT activity than lymphocytes. Therefore, Comet assays were performed on primary Cat(+/) (+) , Cat(+/) (-) , and Cat(-) (/) (-) hepatocytes to determine if the Cat(-) (/) (-) cells were more susceptible to MMA(III) than lymphocytes. The results showed that the Cat(-) (/) (-) hepatocytes exhibit higher levels of DNA strand breakage than the Cat(+/) (+) (approximately fivefold) and Cat(+/) (-) (approximately twofold) hepatocytes exposed to MMA(III) . Electron spin resonance using 5,5-dimethyl-1-pyrroline-N-oxide as the spin-trap agent detected the generation of ·OH via MMA(III) when H2 O2 was present. These experiments suggest that CAT is involved in protecting cells against the genotoxic effects of the ·OH generated by MMA(III) .