Copper Oxide Nanoparticles Induced Mitochondria Mediated Apoptosis in Human Hepatocarcinoma Cells

<div><p>Copper oxide nanoparticles (CuO NPs) are heavily utilized in semiconductor devices, gas sensor, batteries, solar energy converter, microelectronics and heat transfer fluids. It has been reported that liver is one of the target organs for nanoparticles after they gain entry into the body through any of the possible routes. Recent studies have shown cytotoxic response of CuO NPs in liver cells. However, the underlying mechanism of apoptosis in liver cells due to CuO NPs exposure is largely lacking. We explored the possible mechanisms of apoptosis induced by CuO NPs in human hepatocellular carcinoma HepG2 cells. Prepared CuO NPs were spherical in shape with a smooth surface and had an average diameter of 22 nm. CuO NPs (concentration range 2–50 µg/ml) were found to induce cytotoxicity in HepG2 cells in dose-dependent manner, which was likely to be mediated through reactive oxygen species generation and oxidative stress. Tumor suppressor gene p53 and apoptotic gene caspase-3 were up-regulated due to CuO NPs exposure. Decrease in mitochondrial membrane potential with a concomitant increase in the gene expression of bax/bcl2 ratio suggested that mitochondria mediated pathway involved in CuO NPs induced apoptosis. This study has provided valuable insights into the possible mechanism of apoptosis caused by CuO NPs at <i>in vitro</i> level. Underlying mechanism(s) of apoptosis due to CuO NPs exposure should be further invested at <i>in vivo</i> level.</p></div

CuO NPs induced dose-dependent oxidative stress in HepG2 cells.

<p>(A) ROS level in HepG2 cells treated with CuO NPs in presence or absence of NAC, (B) lipid peroxidation and (C) glutathione level. Data represented are mean ± SD of three identical experiments made in three replicate. *Statistically significant difference as compared to the controls (p<0.05 for each).</p

CuO NPs induced apoptosis in HepG2 cells.

<p>(A) Quantitative real-time PCR analysis of mRNA levels of apoptotic genes in HepG2 cells exposed to CuO NPs. (I) p53, (II) bax, (III) bcl-2 and (IV) caspase-3. Data represented are mean ± SD of three identical experiments made in three replicate. *Statistically significant difference as compared to the controls (p<0.05 for each). (B) Western blot analysis of protein levels of apoptotic genes in HepG2 cells treated with CuO NPs. (I) Immunoblot images of p53, bax, bcl-2 and bax proteins. (II) Protein levels were also analyzed by desitometric analysis using AlphaEase TM FC StandAlone V.4.0.0 software. Results are expressed as a fold change over the control group. *Statistically significant difference as compared to the controls (p<0.05 for each).</p

Characterization of CuO NPs.

<p>(A) XRD pattern, (C) FETEM image (inset with higher magnification), (C) FESEM image and (D) EDS spectrum.</p

CuO NPs induced dose-dependent cytotoxicity in HepG2 cells.

<p>(A) MTT assay and (B) NRU assay. Data represented are mean ± SD of three identical experiments made in three replicate. *Statistically significant difference as compared to the controls (p<0.05 for each).</p

CuO NPs induced dose-dependent mitochondrial membrane potential in HepG2 cells.

<p>CuO NPs induced dose-dependent mitochondrial membrane potential in HepG2 cells.</p

Physicochemical properties of CuO NPs.

<p>Physicochemical properties of CuO NPs.</p

N-acetyl-cystein (NAC) significantly preserved the viability (MTT assay) of HepG2 cells caused by CuO NPs.

<p>Data represented are mean ± SD of three identical experiments made in three replicate. *Statistically significant difference in percentage of cells as compared to the controls (p<0.05). ^#Significant inhibitory effect of NAC on cell viability reduction (p<0.05).</p

Toxicogenomic Mechanisms of 6-HO-BDE-47, 6-MeO-BDE-47, and BDE-47 in <i>E. coli</i>

Cytotoxicity of 6-HO-BDE-47 and its two analogues, BDE-47 and 6-MeO-BDE-47, and the associated molecular mechanisms were assessed by use of a live cell reporter assay system which contains a library of 1820 modified green fluorescent protein (GFP) expressing promoter reporter vectors constructed from <i>E. coli</i> K12 strains. 6-HO-BDE-47 inhibited growth of <i>E. coli</i> with a 4 h median effect concentration (EC50) of 22.52 ± 2.20 mg/L, but neither BDE-47 nor 6-MeO-BDE-47 were cytotoxic. Thus, 6-HO-BDE-47 might serve as an antibiotic in some living organisms. Exposure to 6-HO-BDE-47 resulted in 65 (fold change >2) or 129 (fold change >1.5) genes being differentially expressed. The no observed transcriptional effect concentration (NOTEC) and median transcriptional effect concentration (TEC50) based on transcriptional end points, of 6-HO-BDE-47 were 0.0438 and 0.580 mg/L, respectively. The transcriptional responses were 514- and 39-fold more sensitive than the acute EC50 to inhibit cell growth. Most of the genes that were differentially expressed in response to 6-HO-BDE-47 were not modulated by BDE-47 or 6-MeO-BDE-47. These results suggest that cytotoxicity of 6-HO-BDE-47 to <i>E. coli</i> was via a mechanism that was different from that of either BDE-47 or 6-MeO-BDE-47. Gene expression associated with metabolic pathways was more responsive to 6-HO-BDE-47, which suggests that this pathway might be the primary target of this compound