Iron mobilization, cytoprotection, and inhibition of cell proliferation in normal and transformed rat hepatocyte cultures by the hydroxypyridinone CP411, compared to CP20: a biological and physicochemical study

Abstract

The present study analyzes the iron mobilization, the cytoprotective, and the antiproliferative effects of the lipophilic hydroxypyridinone CP411, in comparison with the hydrophilic chelator CP20 or deferiprone used in the treatment of iron overload. Primary rat hepatocyte cultures and the rat hepatoma cell line Fao were used. Chelator cell uptake was evaluated by mass spectrometry in the two models. This method was also used to investigate the stability of the chelators in an acellular system as well as their scavenging and chelating effects against the hydroxyl radical generated by the Fenton reaction. The iron mobilization and the cytoprotective effects of the chelators were evaluated in primary cultures by measuring respectively 55 Fe and lactate dehydrogenase release in the culture medium. The antiproliferative effect of the chelators was studied using the Fao cell line and measuring DNA synthesis by thymidine incorporation and DNA content by flow cytometry. We observed that CP411 entered the hepatocytes and the Fao cells respectively 4 and 13 times more than CP20. CP411 was 2.5 times more effective than CP20 to mobilize iron from preloaded hepatocytes. Pretreatment of the hepatocytes with CP20 or CP411 decreased the toxic effect of iron and CP411 was 1.6 times more effective than CP20. A dose-dependent decrease of DNA synthesis, correlated to an accumulation of cells in S phase, was observed in the Fao cell line in the presence of CP411, while CP20 was without effect. CP411 effect was inhibited by addition of iron simultaneously with the chelator, the addition of Zn or Cu was without effect. The inhibitory effect of CP411 was reversible since, 24 hr after removal of the chelator, DNA replication reached the control level. The results show that CP411 is more efficient to protect the hepatocyte from the toxic effect of iron load and to inhibit tumor cell proliferation. Its higher efficiency may result from its better cell uptake since equimolar solutions of the two chelators in an acellular system exhibit the same ability to inhibit the Fenton reaction.