The toxicity of different chemical forms of nickel (NiO, NiCl2, NiSO4, Ni2S3, NiNPs) towards commercially available human skin cells (Human Epidermal Keratinocytes, adult (HEKa); provided by Thermo Fischer Scientific) was determined. The uptake of nickel by cells incubated with the same chemical forms of nickel at different incubation times and with different nickel concentrations was determined by inductively coupled plasma mass spectrometry (ICPMS). Besides, the physicochemical form of the nickel found in cytosols from experiments with NiNPs was determined by single particle ICPMS. An analytical methodology based on non-denaturing separation strategies with multidimensional chromatography was developed. Intact nickel species present in cell cytosols were separated by size exclusion chromatography (SEC) according to their size and detected by ICPMS. Immobilized Metal Affinity Chromatography (IMAC) was used to compare the results with cells incubated under the same conditions of the samples but without the presence of nickel. In order to isolate the peak of interest for further identification, size exclusion columns with different size ranges were tested. By using the SEC column that produced a better resolution, the fraction corresponding to the nickel species produced by cells was collected and a second separation step was performed by using hydrophilic interaction liquid chromatography (HILIC) coupled to ICPMS.
An analytical strategy was developed in order to obtain information about the molecular targets of nickel involved in nickel toxicity. The developed strategy consisted of the coupling of a ZIC-HILIC column in parallel with ICPMS and a high-resolution electrospray mass spectrometer (ESI-FT-MS/MS) with post-column acidification. The HILIC-ESI-FT-MS/MS method was optimized by using a commercially available metallothionein standard. The sample collected from SEC was injected into HILIC coupled to ESI-FT-MS/MS, and a fraction was collected at the retention time of the nickel specie, estimated previously by ICPMS. This fraction was injected in infusion mode and analyzed directly by ESI-FT-MS/MS under the optimized conditions. A signal corresponding to a protein was obtained by MS and later fragmented, obtaining the corresponding fragments by MS2. The data obtained by MS and MS2 were treated with a software for top-down proteomics.
Overview of the results
The order of toxicity of different chemical forms towards human epidermal cells was: NiO > NiSO NiCl2 > Ni2S3. A toxicity value for NiNPs could not be obtained. The presence of nickel in cytosols increased when cells were treated with those nickel levels that produced medium cell mortality compared to cells treated with nickel levels that produced low cell mortality. The highest amount of nickel was found in the cell treatment with the most toxic nickel compound: NiO. The nickel present in cytosols of cells treated with NiNPs was found as dissolved form (nickel ions) and not as nanoparticles. The analysis by SEC-ICPMS showed the presence of new nickel specie(s) in cytosols from cells treated with NiCl2, NiSO4, NiNPs and NiO, only in the experiments where the highest concentration of nickel was used. However, this nickel specie(s) was not present in cytosols treated with low nickel concentrations, cytosols from cells treated with the least toxic compkund (NisS3), or cytosols from control cells spiked with nickel. Therefore, this nickel-binding protein(s) was produced by cells under stress in the presence of nickel and hence is involved in the molecular mechanisms of nickel toxicity. Through the development and optimization of a novel analytical strategy, this protein expressed by human epidermal cells was identified: tumor protein p63-regulated gene 1.
The results about the project have been disseminated to the scientific community through a poster communication and two oral communications at international meetings (one of them the most important in the field of Bioinorganic Analytical Chemistry) and an oral communication in a seminar. Moreover, two publications are being currently produced and will be submitted to high ranked journals as Open Access.
