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

Abstract

Background: It 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.

Methods: In 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.

Results: We 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.

Conclusions: While 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.

Figure 1

IL6 (black bars) and IL8 (grey bars) production by HK-2 cells in the presence of the panel of engineered nanomaterials. The cells were exposed to the sub-lethal concentrations of the NMs for 24 hr with cytokine secretion measured utilising the FACSArray. Values represent mean ± SEM (n = 3), significance indicated by * = p < 0.05 and ** = p < 0.005, when material treatments are compared to the control. A) NM 110 B) NM 111 C) NRCWE 402 D) NM 400 E) NRCWE 002 F) NRCWE 003 G) NRCWE 001 H) NRCWE 004 I) NM 300 J) NM 101. The Figure has been arranged according to the biological effect of individual NMs.

Figure 2

Intracellular ROS production following exposure of the HK-2 cells to the panel of engineered nanomaterials. The cells were exposed to the NM for 4 hr with oxidative stress measured via HE oxidation by flow cytometry. Values represent mean ± SEM (n = 6) A) Ag - NM 300 B) ZnO - NM 110 and NM 111 C) TiO₂ - NM 101, NRCWE 001, 002, 003 and 004 D) MWCNT - NM 400 and NM 402.

Figure 3

DNA damage expressed as percent of tail DNA following exposure of the HK-2 cells to the ENPRA panel of engineered nanomaterials. The cells were exposed to cell medium (control), 60 μM H₂O₂ and NMs for 4 hr. Values represent mean ± SEM (n = 3), significance indicated by * = p < 0.05 and ** = p < 0.005, when material treatments are compared to the control. A) NM 300 B) NRCWE 002 C) NM 400 D) NRCWE 004 E) NM 402 F) NM 101 G) NM 110 H) NRCWE 003 I) NM 111 J) NRCWE 001. The LC₂₀ ± one serial dilution has been used for the majority of NMs (NM 110, NM 111, NM 300, NM 400, NM 402, NRCWE 003 and NRCWE 004). For NMs in which an LC₂₀ was not reached the three highest concentrations were utilised.