An in vitroassessment of panel of engineered nanomaterials using a human renal cell line: cytotoxicity, pro-inflammatory response, oxidative stress and genotoxicity

Abstract

BackgroundIt has been shown that nanomaterials (NMs) are able to translocate to secondary tissues one of the important being the kidneys. Oxidative stress has been implicated as a possible mechanism for NM toxicity, hence effects on the human renal proximal tubule epithelial cells (HK-2) treated with a panel of engineered nanomaterials (NMs) consisting of two zinc oxide particles (ZnO - coated - NM 110 and uncoated - NM 111), two multi walled carbon nanotubes (MWCNT) (NM 400 and NM 402), one silver (NM 300) and five TiO2 NMs (NM 101, NRCWE 001, 002, 003 and 004) were evaluated.MethodsIn order to assess the toxicological impact of the engineered NMs on HK-2 cells - WST-1 cytotoxicity assay, FACSArray, HE oxidation and the comet assays were utilised. For statistical analysis, the experimental values were compared to their corresponding controls using an ANOVA with Tukey’s multiple comparison.ResultsWe found the two ZnO NMs (24 hr LC50 – 2.5 μg/cm2) and silver NM (24 hr LC50 – 10 μg/cm2) were highly cytotoxic to the cells. The LC50 was not attained in the presence of any of the other engineered nanomaterials (up to 80 μg/cm2). All nanomaterials significantly increased IL8 and IL6 production. Meanwhile no significant change in TNF-α or MCP-1 was detectable. The most notable increase in ROS was noted following treatment with the Ag and the two ZnO NMs. Finally, genotoxicity was measured at sub-lethal concentrations. We found a small but significant increase in DNA damage following exposure to seven of the ten NMs investigated (NM 111, NRCWE 001 and NRCWE 003 being the exception) with this increase being most visible following exposure to Ag and the positively charged TiO2.ConclusionsWhile the NMs could be categorised as low and highly cytotoxic, sub-lethal effects such as cytokine production and genotoxicity were observed with some of the low toxicity materials.

Silver nanoparticles induce cytotoxicity, but not cell transformation or genotoxicity on Balb3T3 mouse fibroblasts

“…The IC50 we found for NM-300 after 24 h of exposure is much lower than the one observed by Kermanizadeh and coauthors testing the same material on human renal (HK-2) cells by the EST-1 colorimetric assay [43]. In fact, in the same exposure conditions, they calculated IC50 of 31.25 µg/mL and 14.06 µg/mL in two different cell culture media [43]. Our result confirms the higher sensitivity of the Balb3T3 and the CFE assay in comparison with other cell lines and the colorimetric assay.…”

“…After 24 h of treatment, only NM-300 showed an identifiable IC50 value at 8 µM (0.86 µg of Ag/mL), in the concentration range tested. The IC50 we found for NM-300 after 24 h of exposure is much lower than the one observed by Kermanizadeh and coauthors testing the same material on human renal (HK-2) cells by the EST-1 colorimetric assay [43]. In fact, in the same exposure conditions, they calculated IC50 of 31.25 µg/mL and 14.06 µg/mL in two different cell culture media [43].…”

“…For the NM-300 tested on human renal cells (HK-2) exposed for 4 h, it was observed dose-dependent DNA damage at concentrations of 3.9, 7.8 and 15 µg/mL, by Comet assay [43]. The chromosome aberration test can be performed in vitro or in vivo and is able to highlights alterations both of number and structure of the chromosomes in metaphase arrested cells.…”

A 3D co-culture of three human cell lines to model the inflamed intestinal mucosa for safety testing of nanomaterials

“…This might be explained by the fact that the inflamed co-culture is already stressed by the inflammation. High release of IL-8 was also observed in other studies that addressed the toxic potential of Ag NPs in human renal proximal tubule epithelial cells (HK-2) (Kermanizadeh et al, 2013b), C3A cells (Kermanizadeh et al, 2013a), and also in mice (Gaiser et al, 2012). It is not clear why the Ag NPs show such a high toxicity to the cells.…”

Genotoxicity of nanomaterials in vitro: treasure or trash?

“…Having a look at published genotoxicity studies of nanomaterials, indeed hardly any study has used a rather large set of nanomaterials. One of the rare exceptions is the work published by Kermanizadeh and coworkers, where the cytotoxicity and genotoxicity of ten different nanomaterials were assessed in human HK-2 cells (Kermanizadeh et al 2013). Due to contradicting data on the in vitro genotoxicity of nanomaterials in different studies, and given that no benchmark or reference materials exist, it appears highly advisable to test a large panel of nanomaterials in one and the same study though.…”