Periodic Reporting for period 1 - Chenitar (Impact of chemical form of nickel ions on its molecular targets in some human cells) Reporting period: 2015-07-01 to 2017-06-30 Summary of the context and overall objectives of the project Nickel is classified under Regulation (EC) No. 1272/2008 as carcinogenic category 2. Besides, nickel allergy is very common worldwide, being the most common cause of contact dermatitis of the skin. Even though nickel allergy and nickel carcinogenicity are well-known health effects related to human exposure to nickel (either during production of nickel-containing products or by direct contact with the final item), the responsible molecular mechanisms events are still unknown, and require additional research. Information about the uptake of nickel to the cell and on the molecular targets of nickel is critical to resolve these health issues related to nickel. In fact, the molecular targets which determine carcinogenicity are important issues to EU according to the Scientific Committee of the European Commission. In this context, the main goal of this study is the identification of the molecular targets of different chemical forms of nickel in human skin cells as well to establish correlation between the toxicity and uptake with time of exposure, concentration and chemical form of nickel. Conclusions of the actionOxidic nickel (NiO) was found to be the most toxic nickel form towards human keratynocites among the different chemical forms of nickel tested. The determination of the nickel uptake by cells showed that the highest amount of nickel was taken up by cells treated with NiO. However, a direct relation between toxicity, chemical form and uptake by cells could not be obtained. By using a non-denaturing separation strategy, a nickel-binding protein produced by cells under stress in the presence of nickel was detected in cytosols from cells treated with different chemical forms of nickel. Finally, the development of analytical technique based on separation techniques and high-resolution electrospray mass spectrometry allowed the identification of the nickel-binding protein involved in nickel toxicity: tumor protein p63-regulated gene 1. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far 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 resultsThe 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. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) For the first time, the toxicity of a wide range of nickel towards human skin cells was studied, and this information further correlated with the information obtained about the uptake of nickel by cells. An analytical strategy based on separation strategies and last generation high resolution mass spectrometry was developed and optimized for the first time in order to identify a nickel-binding protein related with nickel toxicity.The results of the project related with nickel toxicity will help to implement new EU directives regarding the safety and control of nickel content and nickel release in different consumer items. Besides, the finding that NiNPs can release nickel ions that are taken up by cells will be important from the point of view of the risk assessments of new nanomaterials, which is starting to be a major concern in EU. On the other hand, the knowledge generated by the identification of a protein produce by human skin cells involved in the regulation of nickel toxicity is of great importance for a better comprehension of the molecular mechanisms related to nickel allergy and can be further used in other studies. In addition, the analytical methodology used for the identification of the nickel-binding protein can be further adapted for the study of other metal-binding proteins.
