Determination of lethal dose (LD50) of chromium (Cr), cobalt (Co) and nickel (Ni) in HepaRG cells. Comparison to concentrations found in liver from autopsied prosthesis-bearing patients

Objective Many publications have highlighted the release of metal particles used in the composition of metal-metal prostheses, such as Cr, Co and Ni. They are stored in the liver and can accumulate there. Few data are available on their toxicity in this tissue when present. The cytotoxicity of these 3 heavy metals was investigated in cultures of human hepatocytes cell lines, in order to determine LD50s and to compare to the concentrations found in the liver from 10 autopsied prosthesis-bearing patients. Methods HepaRG is a human hepatoma cell line commonly used as a model for toxicity studies. Cells were cultured in William's E medium with glutamine supplemented with 10% fetal bovine serum, human insulin, hydrocortisone hemisuccinate and penicillin/streptomycin. Differentiation was induced by adding 2% DMSO to nutrient medium. After 14 days, the cells were placed in a 24-well plate in a concentration of 500,000 cells/well. Solutions with ten increasing concentrations of NiCl 2 or CoCl 2 or Na 2 Cr 2 O 7 were incubated with cells for 48 hours at 37 °C. These concentrations were established based on literature data for other hepatocyte models [1] , [2] . Cell viability was evaluated by LDH release (Pierce LDH Cytotoxicity Assay Kit, ThermoFisher) and MTT assay (Vybrant ® MTT cell proliferation assay kit). Concentration response profiles were realized with GraphPad Prism 2.0. Measurements of the heavy metals in liver from autopsied patients were carried out using ICP-HRMS (Element XR, ThermoFisher) after mineralization by microwave (Ultrawave ® , ThermoFisher) [3] . Results Expected LD50 according to literature data were around 25, 500 and 1000 μM for Cr, Co and Ni, respectively. For Cr, testing concentrations were 0.5, 5, 10, 50, 100, 500, 1000, 5000, 10,000, 50,000 μM. For Co and Ni, they were 10, 50, 100, 250, 500, 1000, 2500, 5000, 10,000, 50,000 μM. The first cytotoxicity assay performed was the LDH release. It measures quantitatively the LDH released in the medium when cells are damaged. But William's E medium is pink and had altered the reading of absorbance. So, results could not be exploited. The MTT assay is carried out on the cell pellet and thus, makes it possible to overcome the culture medium. The dose/response curves for each metal could be realized. Experimental LD50s are 2 μM for Cr, 940 μM for Co, 1380 μM for Ni. The average Cr, Co, Ni concentrations found in the livers of the 10 dead patients carrying prostheses were 0.05, 0.03 and 0.035 μM, respectively. As expected, concentrations found in the liver of autopsied patients are much lower than the experimental LD50. Conclusion The LD50s were determined for Cr, Co and Ni in HepaRG cells, much higher than concentrations found in the liver from 10 autopsied prosthesis-bearing patients. However, the impact of these elements on hepatocyte metabolism can be reasonably considered at low concentrations. Consequently, further researches using metabolomics approach will be performed.