Determining the parameters making it possible to assay intra-erythrocytic chromium: I - In vitro study

Hexavalent chromium is the most toxic form of chromium. Currently, no biological exposure marker exists that is specific to hexavalent chromium. A bibliographic study has shown that Cr(VI) is incorporated into erythrocytes while the other species of chromium are not or are not to any significant extent. That assumption therefore needs to be verified before this assay is used as a marker specific to exposure to Cr(VI).
The following points will be verified:
- that the erythrocytes incorporate hexavalent chromium exclusively or almost exclusively;
- whether a relationship exists between a quantity of hexavalent chromium added to a volume of blood and intra-erythrocytic chromium.
- the quantification limit for intra-erythrocytic chromium;
- any toxicity threshold for hexavalent chromium in such erythrocytes.

Hexavalent chromium and trivalent chromium will be added to tubes of blood. After incubation, the chromium will be analysed in the various compartments of the blood: erythrocytes, lymphocytes, plasma, and the total blood. The operating mode for conducting these experiments and the analytical techniques should be developed and optimised.
Increasing concentrations of chromium (VI) will be added to the tubes of blood in order to look for any relationship between the Cr assayed in the erythrocytes, in the lymphocytes, and in the supernatants, and the quantity of Cr(VI) (chromate and dichromate) deposited in the tubes of blood before incubation.
The literature indicates that Cr(VI) is toxic for erythrocytes; that toxicity threshold will be sought in our trials. A comet assay will be conducted on the lymphocytes in order to quantify any damage caused to the DNA.
The various forms of hexavalent chromium will be studied (chromate or dichromate, influence of the counterion, etc.) but the initial trials will be conducted with sodium dichromate.
The influence of incubation temperature and time will also be studied.

After this feasibility study and the subsequently scheduled long study have been conducted, the objective will be to establish a biological indicator value for exposure to hexavalent chromium.