3,5,5-TRIMETHYLHEXANOYLFERROCENE INDUCTION OF HEME OXYGENASE ACTIVITYIN NORMAL HEPATOCYTES

Recent work showed that the combination of 50 mu M glutethimide plus 5 0 mu M ferric nitrilotriacetate (FeNTA) synergistically induces heme o xygenase (HO) activity in cultured chick embryo liver cells (Cable et al., Biochem Biophys Res Commun 168: 176-181, 1990). This synergistic induction is due to increased heme synthesis, which then acts to incre ase HO gene transcription. The aim of the current studies was to chara cterize the effects on hepatic heme metabolism of (3,5,5-trimethylhexa noyl)ferrocene (TMH-ferrocene), which causes hepatic iron-loading in r ats. Unlike FeNTA, TMH-ferrocene alone maximally induced HO activity a t 5-10 mu M TMH-ferrocene. At higher concentrations, HO activities dec lined, as did total cellular protein synthesis. Induction of HO was ma ximal after a 12-hr exposure to TMH-ferrocene, similar to induction by glutethimide plus FeNTA. The effect of TMH-ferrocene on HO could not be ascribed to greater cellular uptake of iron, since cell-associated iron levels were higher after FeNTA. than after TMH-ferrocene treatmen t. TMH-ferrocene (up to 20 mu M) did not induce delta-aminolevulinic a cid synthase activity. Uroporphyrin accumulation in cells treated with TMH-ferrocene was minimal, but the combination of TMH-ferrocene and g lutethimide caused a synergistic increase in uroporphyrin accumulation , similar to treatment with glutethimide plus FeNTA. 4,6-Dioxoheptanoi c acid, an inhibitor of heme synthesis, blocked the induction of HO ca used by glutethimide and FeNTA, but did not decrease the induction of HO by TMH-ferrocene. TMH-ferrocene-mediated induction of HO does not a ppear to be due to lipid peroxidation, since malondialdehyde formation was greater for ferrocene (a structural analog of TMH-ferrocene that does not induce HO) than for TMH-ferrocene. Furthermore, the anti-oxid ant, butylated hydroxyanisole, which prevented lipid peroxidation, dec reased HO induced by glutethimide plus FeNTA, but butylated hydroxyani sole did not affect HO induced by TMH-ferrocene. We conclude that, unl ike the combination of glutethimide plus FeNTA, TMH-ferrocene induces HO activity by a mechanism that is independent of cellular heme synthe sis.