ACCUMULATION OF IRON BY PRIMARY RAT HEPATOCYTES IN LONG-TERM CULTURE - CHANGES IN NUCLEAR SHAPE MEDIATED BY NON-TRANSFERRIN-BOUND FORMS OF IRON

We have preciously shown that hepatocytes in longterm dimethylsulfoxid e (DMSO) culture, fed a chemically defined medium, are highly differen tiated and an excellent in vitro model of adult liver. Hepatocytes in long-term DMSO culture can be iron loaded by exposure to non-transferr in-bound iron (NTBI) in the form of ferrous sulfate (FeSO4), ferric ni trilotriacetate, or trimethylhexanoyl (FeSO4)-ferrocene. Hole-transfer rin, at equivalent times and concentrations, was unable to load hepato cytes. Of the it on compounds tested, TMH-ferrocene most accurately si mulated the morphological features of iron-loaded hepatocytes in vivo. When exposed to 25 mu mol/L TMH-ferrocene, hepatocytes loaded increas ing amounts of iron for 2 months before the cells died. When exposed t o lower concentrations of TMH-ferrocene (as low as 2.5 mu mol/L), hepa tocytes continuously loaded iron and remained viable for more than 2 m onths. The cellular deposition of iron was different in hepatocytes ex posed to TMH-ferrocene compared with those exposed to FeSO4; exposure to TMH-ferrocene resulted in the presence of more ferritin cores withi n lysosomes than were seen with FeSO4. When the concentration of TMH-f errocene was increased, a greater number of ferritin cores were observ ed within the lysosome, and total cellular ferritin, as assessed by We stern blot, increased. The formation of hemosiderin was also observed. Furthermore, nuclear shape was distorted in iron-loaded hepatocytes. The extent of deviation from circularity in the nucleus correlated wit h increasing concentrations of TMH-ferrocene and was greater in hepato cytes exposed to FeSO4 than an equivalent concentration of TMH-ferroce ne. The deviation from circularity was smallest in hepatocytes that co ntained well formed ferritin cores and increased in hepatocytes that c ontained greater amounts of hemosiderin. Furthermore, in hepatocytes t reated with FeSO4, a large amount of cell-associated iron was detected but without a significant increase in the total amount of ferritin. T he deviation from circularity was the largest in FeSO4-treated hepatoc ytes, indicating that iron not properly incorporated into ferritin cau sed more cellular damage. We conclude that hen-loaded hepatocytes in l ong-term DMSO culture represent a flexible system for studying the eff ects of chronic iron loading on hepatocytes.