Accumulation of iron by primary rat hepatocytes in long-term culture: Changes in nuclear shape mediated by non-transferrin-bound forms of iron

Edward E. Cable, James Connor, Harriet C. Isom

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

We have previously shown that hepatocytes in longterm dimethylsulfoxide (DMSO) culture, fed a chemically defined medium, are highly differentiated and an excellent in vitro model of adult liver. Hepatocytes in long-term DMSO culture can be iron loaded by exposure to non-transferrin-bound iron (NTBD in the form of ferrous sulfate (FeSO4), ferric nitrilotriacetate, or trimethylhexanoyl (TMH)-ferrocene. Holotransferrin, at equivalent times and concentrations, was unable to load hepatocytes. Of the iron compounds tested, TMH-ferrocene most accurately simulated the morphological features of iron- loaded hepatocytes in vivo. When exposed to 25 μmol/L TMH-ferrocene, hepatocytes loaded increasing amounts of iron for 2 months before the cells died. When exposed to lower concentrations of TMH-ferrocene (as low as 2.5 μmol/L), hepatocytes continuously loaded iron and remained viable for more than 2 months. The cellular deposition of iron was different in hepatocytes exposed to TMH-ferrocene compared with those exposed to FeSO4; exposure to TMH-ferrocene resulted in the presence of more ferritin cores within lysosomes than were seen with FeSO4. When the concentration of TMH-ferrocene was increased, a greater number of ferritin cores were observed within the lysosome, and total cellular ferritin, as assessed by Western 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 with increasing concentrations of TMH-ferrocene and was greater in hepatocytes exposed to FeSO4 than an equivalent concentration of TMH-ferrocene. The deviation from circularity was smallest in hepatocytes that contained well formed ferritin cores and increased in hepatocytes that contained greater amounts of hemosiderin. Furthermore, in hepatocytes treated with FeSO4, a large amount of cell-associated iron was detected but without a significant increase in the total amount of ferritin. The deviation from circularity was the largest in FeSO4-treated hepatocytes, indicating that iron not properly incorporated into ferritin caused more cellular damage. We conclude that iron-loaded hepatocytes in long-term DMSO culture represent a flexible system for studying the effects of chronic iron loading on hepatocytes.

Original languageEnglish (US)
Pages (from-to)781-792
Number of pages12
JournalAmerican Journal of Pathology
Volume152
Issue number3
StatePublished - Mar 1 1998

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Hepatocytes
Iron
Ferritins
Dimethyl Sulfoxide
Hemosiderin
ferrous sulfate
Lysosomes
Iron Compounds
ferrocene
Western Blotting

All Science Journal Classification (ASJC) codes

  • Pathology and Forensic Medicine

Cite this

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title = "Accumulation of iron by primary rat hepatocytes in long-term culture: Changes in nuclear shape mediated by non-transferrin-bound forms of iron",
abstract = "We have previously shown that hepatocytes in longterm dimethylsulfoxide (DMSO) culture, fed a chemically defined medium, are highly differentiated and an excellent in vitro model of adult liver. Hepatocytes in long-term DMSO culture can be iron loaded by exposure to non-transferrin-bound iron (NTBD in the form of ferrous sulfate (FeSO4), ferric nitrilotriacetate, or trimethylhexanoyl (TMH)-ferrocene. Holotransferrin, at equivalent times and concentrations, was unable to load hepatocytes. Of the iron compounds tested, TMH-ferrocene most accurately simulated the morphological features of iron- loaded hepatocytes in vivo. When exposed to 25 μmol/L TMH-ferrocene, hepatocytes loaded increasing amounts of iron for 2 months before the cells died. When exposed to lower concentrations of TMH-ferrocene (as low as 2.5 μmol/L), hepatocytes continuously loaded iron and remained viable for more than 2 months. The cellular deposition of iron was different in hepatocytes exposed to TMH-ferrocene compared with those exposed to FeSO4; exposure to TMH-ferrocene resulted in the presence of more ferritin cores within lysosomes than were seen with FeSO4. When the concentration of TMH-ferrocene was increased, a greater number of ferritin cores were observed within the lysosome, and total cellular ferritin, as assessed by Western 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 with increasing concentrations of TMH-ferrocene and was greater in hepatocytes exposed to FeSO4 than an equivalent concentration of TMH-ferrocene. The deviation from circularity was smallest in hepatocytes that contained well formed ferritin cores and increased in hepatocytes that contained greater amounts of hemosiderin. Furthermore, in hepatocytes treated with FeSO4, a large amount of cell-associated iron was detected but without a significant increase in the total amount of ferritin. The deviation from circularity was the largest in FeSO4-treated hepatocytes, indicating that iron not properly incorporated into ferritin caused more cellular damage. We conclude that iron-loaded hepatocytes in long-term DMSO culture represent a flexible system for studying the effects of chronic iron loading on hepatocytes.",
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Accumulation of iron by primary rat hepatocytes in long-term culture : Changes in nuclear shape mediated by non-transferrin-bound forms of iron. / Cable, Edward E.; Connor, James; Isom, Harriet C.

In: American Journal of Pathology, Vol. 152, No. 3, 01.03.1998, p. 781-792.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Accumulation of iron by primary rat hepatocytes in long-term culture

T2 - Changes in nuclear shape mediated by non-transferrin-bound forms of iron

AU - Cable, Edward E.

AU - Connor, James

AU - Isom, Harriet C.

PY - 1998/3/1

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N2 - We have previously shown that hepatocytes in longterm dimethylsulfoxide (DMSO) culture, fed a chemically defined medium, are highly differentiated and an excellent in vitro model of adult liver. Hepatocytes in long-term DMSO culture can be iron loaded by exposure to non-transferrin-bound iron (NTBD in the form of ferrous sulfate (FeSO4), ferric nitrilotriacetate, or trimethylhexanoyl (TMH)-ferrocene. Holotransferrin, at equivalent times and concentrations, was unable to load hepatocytes. Of the iron compounds tested, TMH-ferrocene most accurately simulated the morphological features of iron- loaded hepatocytes in vivo. When exposed to 25 μmol/L TMH-ferrocene, hepatocytes loaded increasing amounts of iron for 2 months before the cells died. When exposed to lower concentrations of TMH-ferrocene (as low as 2.5 μmol/L), hepatocytes continuously loaded iron and remained viable for more than 2 months. The cellular deposition of iron was different in hepatocytes exposed to TMH-ferrocene compared with those exposed to FeSO4; exposure to TMH-ferrocene resulted in the presence of more ferritin cores within lysosomes than were seen with FeSO4. When the concentration of TMH-ferrocene was increased, a greater number of ferritin cores were observed within the lysosome, and total cellular ferritin, as assessed by Western 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 with increasing concentrations of TMH-ferrocene and was greater in hepatocytes exposed to FeSO4 than an equivalent concentration of TMH-ferrocene. The deviation from circularity was smallest in hepatocytes that contained well formed ferritin cores and increased in hepatocytes that contained greater amounts of hemosiderin. Furthermore, in hepatocytes treated with FeSO4, a large amount of cell-associated iron was detected but without a significant increase in the total amount of ferritin. The deviation from circularity was the largest in FeSO4-treated hepatocytes, indicating that iron not properly incorporated into ferritin caused more cellular damage. We conclude that iron-loaded hepatocytes in long-term DMSO culture represent a flexible system for studying the effects of chronic iron loading on hepatocytes.

AB - We have previously shown that hepatocytes in longterm dimethylsulfoxide (DMSO) culture, fed a chemically defined medium, are highly differentiated and an excellent in vitro model of adult liver. Hepatocytes in long-term DMSO culture can be iron loaded by exposure to non-transferrin-bound iron (NTBD in the form of ferrous sulfate (FeSO4), ferric nitrilotriacetate, or trimethylhexanoyl (TMH)-ferrocene. Holotransferrin, at equivalent times and concentrations, was unable to load hepatocytes. Of the iron compounds tested, TMH-ferrocene most accurately simulated the morphological features of iron- loaded hepatocytes in vivo. When exposed to 25 μmol/L TMH-ferrocene, hepatocytes loaded increasing amounts of iron for 2 months before the cells died. When exposed to lower concentrations of TMH-ferrocene (as low as 2.5 μmol/L), hepatocytes continuously loaded iron and remained viable for more than 2 months. The cellular deposition of iron was different in hepatocytes exposed to TMH-ferrocene compared with those exposed to FeSO4; exposure to TMH-ferrocene resulted in the presence of more ferritin cores within lysosomes than were seen with FeSO4. When the concentration of TMH-ferrocene was increased, a greater number of ferritin cores were observed within the lysosome, and total cellular ferritin, as assessed by Western 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 with increasing concentrations of TMH-ferrocene and was greater in hepatocytes exposed to FeSO4 than an equivalent concentration of TMH-ferrocene. The deviation from circularity was smallest in hepatocytes that contained well formed ferritin cores and increased in hepatocytes that contained greater amounts of hemosiderin. Furthermore, in hepatocytes treated with FeSO4, a large amount of cell-associated iron was detected but without a significant increase in the total amount of ferritin. The deviation from circularity was the largest in FeSO4-treated hepatocytes, indicating that iron not properly incorporated into ferritin caused more cellular damage. We conclude that iron-loaded hepatocytes in long-term DMSO culture represent a flexible system for studying the effects of chronic iron loading on hepatocytes.

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