Alicia Ávalos

Cytotoxicity and ROS production of manufactured silver nanoparticles of different sizes in hepatoma and leukemia cells

Silver nanoparticles (AgNPs), which have well‐known antimicrobial properties, are extensively used in various medical and general applications. In spite of the widespread use of AgNPs, relatively few studies have been undertaken to determine the cytotoxic effects of AgNPs. The aim of this study was investigate how AgNPs of different sizes (4.7 and 42 nm) interact with two different tumoral human cell lines (hepatoma [HepG2] and leukemia [HL‐60]). In addition, glutathione depletion, inhibition of superoxide dismutase (SOD) and reactive oxygen species (ROS) generation were used to evaluate feasible mechanisms by which AgNPs exerted its toxicity. AgNPs of 4.7 nm and 42 nm exhibited a dramatic difference in cytotoxicity. Small AgNPs were much more cytotoxic than large AgNPs. A difference in the cellular response to AgNPs was found. HepG2 cells showed a higher sensitivity to the AgNPs than HL‐60. However, the cytotoxicity induced by AgNPs was efficiently prevented by NAC treatment, which suggests that oxidative stress is primarily responsible for the cytotoxicity of AgNPs. Furthermore, cellular antioxidant status was disturbed: AgNPs exposure caused ROS production, glutathione depletion and slight, but not statistically significant inactivation of SOD. Copyright

Oxidative stress contributes to gold nanoparticle-induced cytotoxicity in human tumor cells

Abstract Due to their exceptional properties, gold nanoparticles (AuNPs) have shown promising medical and technological applications in the treatment of cancer and the development of antimicrobial packaging and time–temperature indicators in the food sector. However, little is known about their cytotoxicity when they come into contact with biological systems. The aim of this work was to compare the effects of three commercially available AuNPs of different sizes (30, 50 and 90 nm) on human leukemia (HL-60) and hepatoma (HepG2) cell lines. AuNP-induced cytotoxicity was dose and time-dependent, with IC50 values higher than 15 μg/mL. Nanoparticle (NP) size and cell line slightly influenced on the cytotoxicity of AuNPs, although HL-60 cells proved to be more sensitive to the cytotoxic response than HepG2. N-Acetyl-l-cysteine (NAC) protected HL-60 and HepG2 cells only against treatment with 30 nm AuNPs. In both cell types, glutathione (GSH) content was drastically depleted after 72 h of incubation with the three AuNPs (less than 30% in all cases), while the reduction of superoxide dismutase activity (SOD) activity depended on cell line. HepG2, but not HL-60 cells, exhibited a decrease of SOD activity (∼45% of activity). The three AuNPs also caused a two-fold elevation of reactive oxygen species (ROS) production in both cell lines. Thus, protective effect of NAC, depletion of GSH and increase of ROS appear to be determined by NP size and indicate that oxidative stress contributes to cytotoxicity of AuNPs.

Interactions of manufactured silver nanoparticles of different sizes with normal human dermal fibroblasts

Silver compounds have been used for their medicinal properties for centuries. At present, silver nanoparticles (AgNPs) are reemerging as a viable topical treatment option for infections encountered in burns, open wounds and chronic ulcers. This study evaluated the in vitro mechanisms of two different sizes of AgNPs (4·7 and 42 nm) toxicity in normal human dermal fibroblasts. The toxicity was evaluated by observing cell viability and oxidative stress parameters. In all toxicity endpoints studied (MTT and lactate dehydrogenase assays), AgNPs of 4·7 nm were much more toxic than the large AgNPs (42 nm). The cytotoxicity of both AgNPs was greatly decreased by pre‐treatment with the antioxidant N‐acetyl‐L‐cysteine. The oxidative stress parameters showed significant increase in reactive oxygen species levels, depletion of glutathione level and slight, but not statistically significant inactivation of superoxide dismutase, suggesting generation of oxidative stress. Thus, AgNPs should be used with caution for the topical treatment of burns and wounds, medical devices etc, because their toxicity depends on the size, the smaller NPs being much more cytotoxic than the large.

Effects of silver and gold nanoparticles of different sizes in human pulmonary fibroblasts.

Abstract Silver and gold nanoparticles (Ag–AuNPs) are currently some of the most manufactured nanomaterials. Accordingly, the hazards associated with human exposure to Ag–AuNPs should be investigated to facilitate the risk assessment process. In particular, because pulmonary exposure to Ag–AuNPs occurs during handling of these nanoparticles, it is necessary to evaluate the toxic response in pulmonary cells. The aim of this study was to evaluate the in vitro mechanisms of toxicity of different sizes of silver (4.7 and 42 nm) and gold nanoparticles (30, 50 and 90 nm) in human pulmonary fibroblasts (HPF). The toxicity was evaluated by observing cell viability and oxidative stress parameters. Data showed that AgNPs-induced cytotoxicity was size-dependent, whereas the AuNPs of the three sizes showed similar cytotoxicity. Silver nanoparticles of 4.7 nm were much more toxic than the large silver nanoparticles and the AuNPs. However, the pre-treatment with the antioxidant, N-acetyl-l-cysteine, protected HPF cells against treatment with Ag–AuNPs. The oxidative stress parameters revealed significant increase in reactive oxygen species levels, depletion of glutathione level and slight, but not statistically significant inactivation of superoxide dismutase, suggesting generation of oxidative stress. Hence, care has to be taken while processing and formulating the Ag–AuNPs till their final finished product.

Comparative cytotoxicity evaluation of different size gold nanoparticles in human dermal fibroblasts

The aim of this work was to compare the effects of three commercially available gold nanoparticles (AuNPs) of different sizes (30, 50 and 90 nm) on the viability of normal human dermal fibroblasts (NHDF). In addition, we evaluated protective effect of N-Acetyl-L-cysteine (NAC), total glutathione content (GSH/GSSG), superoxide dismutase (SOD) activity and reactive oxygen species (ROS) production to investigate if oxidative stress was involved in the cytotoxic response of these AuNPs. Although AuNP-induced cytotoxicity was dose and time dependent, nanoparticle size slightly influenced the cytotoxic response of AuNPs assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase. Regarding oxidative parameters, NAC produced no significant protection of NHDF cells against treatment with any of the three AuNPs. Independently on nanoparticle size, GSH/GSSG content was drastically depleted after 24 h of incubation with the three AuNPs (less than 15% in all cases), while no statistically significant changes on SOD activity were reported (∼90% of activity). The three AuNPs also caused a notable increase in the ROS production of NHDF cells. In conclusion, our data suggest that AuNP-induced cytotoxicity in NHDF is mediated by oxidative stress and it is independent of nanoparticle size.

Validation of coffee silverskin extract as a food ingredient by the analysis of cytotoxicity and genotoxicity

The aim of the present study was to validate the food safety of CSE, by studying its effect on cytotoxicity (100-20000μg/ml) and genotoxicity (10, 100 and 1000μg/ml) and also to investigate its preventive potential (1, 10 and 100μg/ml) against B(a)P induced DNA damage. Prior to analyses, the antioxidant capacity and the microbiological quality of CSE were tested. DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or comet assay. HepG2 cells were pre-treated with CSE (1, 10 and 100μg/ml) for 24h followed by the addition of 100μM B(a)P in presence of CSE for other 24h. Detection of oxidized purines and pyrimidines was carried out using Formamidopyrimidine DNA glycosylase or Endonuclease III enzymes, respectively. Chlorogenic acid (CGA), the major antioxidant present in coffee, was used as a control. Treatment with 100 μM B(a)P significantly increased (p<0.05) levels of DNA strand breaks and oxidized purine and pyrimidine bases. Treatment of HepG2 cells with CSE did not induce either cytotoxicity or genotoxicity. CSE significantly inhibited (p<0.05) genotoxicity induced by B(a)P and the observed effect may be associated to its antioxidant capacity. CGA alone at the concentration present in CSE was effective against B(a)P. Thus, CGA seems to be a contributor to the preventive effect of CSE against B(a)P induced DNA damage in HepG2 cells. In conclusion, CSE presents potential as a natural sustainable chemoprotective agent against the chemical carcinogen B(a)P.

In vitro and in vivo genotoxicity assessment of gold nanoparticles of different sizes by comet and SMART assays

Due to the increasing use of gold nanoparticles (AuNPs) in different areas such as medicine, biotechnology or food sector, human exposure to them has grown significantly and its toxicity evaluation has become essential. Therefore, the purpose of this study was to compare the potential genotoxic effects of 30, 50 and 90 nm AuNPs, using in vitro comet assay with the in vivo mutagenic and recombinogenic activity (SMART Test) in Drosophila. The results indicated that in both cell lines, 30, 50 and 90 nm (1-10 μg ml-1) AuNPs increased DNA strand breaks following 24 h treatment. This damage was not dose and size-dependent. Moreover, a modified comet assay using endonuclease III and formamidopyrimidine-DNA glycosylase restriction enzymes showed that in both cell lines, pyrimidines and purines were oxidatively damaged by all AuNPs, being 90 nm AuNPs slightly more genotoxic. However, the data obtained with SMART showed that 30 nm AuNPs did not modify the spontaneous frequencies of spots indicating lack of mutagenic and recombinogenic activity. Therefore, further experiments must be carried out to gain a better understanding of the mechanism of action of AuNPs to ensure their safe use.