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HepG2 cells under silver nanoparticle-induced stress - comet assay investigation

Scientists in Poland have been investigating the adaptation of various cells to silver nanoparticle-induced stress.  The researchers reported that silver nanoparticles are one of the most widely used nanomaterials due to their antibacterial properties.  They also commented that due to the recent boost in the usage of silver nanoparticle-containing products, human exposure to silver nanoparticles is increasing.  Understandably, the scientists believe this highlights the need for careful evaluation of silver nanoparticle toxicity to humans.

The investigators used two cellular models:
hepatic HepG2
epithelial A549 cell lines
to study the mechanism of silver nanoparticle-induced toxicity at the cellular level.

A variety of assays were used in this investigation; including real-time PCR, the alkaline comet assay and the neutral red assay.

The comet assay (single cell gel electrophoresis) was performed to measure DNA damage.  For positive controls, nanoparticle-untreated cells were X-irradiated with the dose of 3 Gy at a dose rate of 1.14 Gy/min.  Image analysis of the data was performed by the Comet Assay IV Image Analysis System (Perceptive Instruments, UK). Fifty randomly selected comets per slide were analysed, two slides per experimental point. In this study, the percentage of DNA in the comet’s tail was used as a measure of DNA breakage.

The researchers concluded that these two cell lines differ significantly in their response to silver nanoparticle treatment.

A549 cells -
A minor decrease in viability and increase in the extent of DNA breakage were observed.

HepG2 cells -
A markedly different response to silver nanoparticles was observed in HepG2 cells. In the short term, a massive induction of DNA breakage was observed, suggesting that the basal activity of antioxidant defence in these cells was not sufficient to effectively protect them from the nanoparticle-induced oxidative stress.

After prolonged exposure, the extent of DNA breakage decreased to the level observed in the control cells suggesting that a successful adaptation to the new conditions had taken place. The investigators believe that the cells that were unable to adapt must have died, as revealed by the Neutral Red assay that indicated less than half viable cells after 24-h treatment with 100 µg/ml of 20nm silver nanoparticles.

The gene expression analysis revealed that the observed adaptation was underlain by a pro-proliferative, anti-apoptotic signal leading to up-regulation of the genes promoting proliferation and inflammatory response (EGR1, FOS, JUN, HK2, IL4, MMP10, VEGFA, WISP1, CEBPB, IL8, SELPLG), genes coding the anti-apoptotic proteins (BCL2A1, CCL2) and factors involved in the response to stress (HSPB1, GADD45A).

Conclusions and novel findings

According to the authors, the main novelty of the presented study is the correlation of the gene expression data, DNA breakage measurements and viability assay leading to the hypothesis that some type of cells can adapt to the silver nanoparticle-induced stress through activation of the pro-proliferative and anti-apoptotic transcriptional program.

The investigators believe that further research is needed to investigate, if the observed changes in HepG2 cells adapted to the nanoparticles are permanent or transient and whether the cells return to the initial physiological state, when the silver nanoparticle-induced stress is stopped.

The findings presented here could potentially effect the medical applications of nanoparticles, since the sustained proliferative signalling and resistance to cell death are hallmarks of cancer, acquired by the cells in the process of carcinogenesis.

Case study based on:

Adaptation of HepG2 cells to silver nanoparticles-induced stress is based on the pro-proliferative and anti-apoptotic changes in gene expression
Brzóska K, Męczyńska-Wielgosz S, Stępkowski TM, Kruszewski M.
Mutagenesis. 2015 Feb 13.