Effect of iron chelators on methemoglobin and thrombin preconditioning

Abstract

Cell loss immediately adjacent to an intracerebral hemorrhage may be mediated in part by the toxicities of extracellular hemoglobin (Hb) and thrombin. However, at low concentrations, these proteins induce tolerance to hemin and iron that may limit further peri-hematomal injury as erythrocyte lysis progresses. The mechanisms mediating these preconditioning effects have not been completely defined, but increased expression of both heme oxygenase (HO)-1 and iron binding proteins likely contributes. In the present study, we hypothesized that iron chelator therapy would attenuate this protective response. Pretreatment of cortical glial cultures (> 90 % GFAP+) with 3 μM methemoglobin (metHb) or 5 units/ml thrombin for 24 h was nontoxic per se, and increased HO-1 and ferritin expression. When challenged with a toxic concentration of hemin, the increase in cellular redox-active iron was attenuated in preconditioned cultures and cell survival was increased. However, if cultures were pretreated with metHb or thrombin plus deferoxamine or 2,2'-bipyridyl, ferritin induction was prevented and cellular redox-active iron increased with hemin treatment. Preconditioning-mediated cytoprotection was consistently reduced by deferoxamine, while 2,2'-bipyridyl had a variable effect. Neither chelator altered HO-1 expression. A cytoprotective response was preserved when chelator therapy was limited to 11 hours of the 24 h preconditioning interval. These results suggest a potentially deleterious effect of continuous iron chelator therapy after ICH. Intermittent therapy may remove peri-hematomal iron without negating the benefits of exposure to low concentrations of Hb or thrombin.

Keywords: Heme; Intracerebral hemorrhage; Iron; Ischemia; Stroke; Subarachnoid hemorrhage.

Fig. 1

Iron chelators reduce ferritin induction by methemoglobin. Bars represent mean HO-1 (a) or ferritin (b) band densities in lysates of cultures (5/condition) treated with 3 μM methemoglobin (MetHb) for 24 hours, alone or with 100 μM of either deferoxamine (DFO) or 2,2′-bipyridyl (BP), expressed as a multiple of the mean value in control cultures subjected to sham medium changes only (= 1). Lane order of representative immuno-blots is same as bar order; actin is gel-loading control. ***P<0.001 v. sham controls, #P<0.05, ##P<0.01 v. metHb alone condition

Fig. 2

Iron chelators prevent ferritin induction by thrombin. Cultures (5/condition) were treated with 5 units/ml thrombin for 24 hours alone or with 100 μM deferoxamine (DFO) or 2,2′-bipyridyl (BP). Bars represent mean HO-1 (a) or ferritin (b) band densities, scaled to the mean value in control cultures subjected to sham medium exchange only (= 1). Lane order of representative immunoblots is same as bar order; actin is gel-loading control. *P<0.05, **P<0.01, ***P<0.001 v. sham controls, ##P<0.01 v. thrombin alone condition

Fig. 3

Methemoglobin and thrombin preconditioning reduce the cytosolic labile iron increase after hemin treatment; chelators reverse this effect. Phase contrast (a,c) and fluorescence (b, d-h) photomicrographs of cortical glial cultures stained with calcein AM after: a,b) control medium exchanges only; culture monolayer is intact with bright calcein fluorescence; c,d) sham pretreatment (MEM10) for 24 h followed by hemin 30 μM for 3 h, showing intact culture monolayer but calcein fluorescence quenching, indicating increased cytosolic labile iron; e) same culture as in c, d, after adding cell-permeable iron chelator 1,10-phenanthroline, which reverses calcein fluorescence quenching, demonstrating membrane integrity and ability to retain calcein; f) culture pretreated with 3 μM methemoglobin for 24 h, then 30 μM hemin for 3 hours; g,h) cultures pretreated with 3 μM methemoglobin plus 100 μM deferoxamine (g) or 100 μM 2,2′-bipyridyl (h) for 24 h, then hemin 30 μM for 3 hours; preconditioning effect is reduced. Scale bar = 100 μm. Bars represent mean calcein fluorescence intensity in cultures pretreated for 24 hours with MEM10 medium only, 3 μM methemoglobin (Met, i), or 5 units/ml thrombin (Thr, j), alone or with 100 μM deferoxamine (DFO) or 2,2′-bipyridyl (BP), then washed and treated with 30 μM hemin without chelators for 3 hours. Calcein fluorescence values are normalized to that in control cultures exposed to MEM10 medium only (= 100). ***P<0.001 v. fluorescence in cultures pretreated with methemoglobin or thrombin alone and then treated with hemin, ###P<0.001 v. that in control cultures treated with MEM10 medium only

Fig 4

Protective effect of methemoglobin and thrombin preconditioning is attenuated by iron chelators. Phase contrast photomicrographs of glial cultures treated with: a) medium exchanges only; b-d) hemin 30 μM for 8 h, after pretreatment for 24 hours with MEM10 medium only (b), metHb 3 μM (c), or metHb 3 μM plus 2,2′-bipyridyl 100 μM (d). Scale bar=100 μm. Bars represent mean medium LDH in cultures pretreated for 24 h with 3 μM methemoglobin (Met, e) or 5 units/ml thrombin (Thr, f) alone or with 100 μM deferoxamine (DFO) or 100 μM 2,2′-bipyridyl (BP); other cultures were incubated with MEM10 medium only. After medium exchange, cultures were then treated with 30 μM hemin for 8 or 24 hours, without chelators. LDH values are scaled to the mean value in cultures from the same plating treated with 0.1 % Triton X-100 (= 100), which lyses all cells. The weak signal in control cultures subjected to medium exchange only was subtracted from all values to yield the LDH activity associated with neurotoxicity. **P<0.01, ***P<0.001 v. mean value in cultures pretreated with methemoglobin or thrombin alone, #P<0.05, ###P<0.001 v. value in cultures pretreated with MEM10 medium alone, n=14–18/condition

Fig. 5

Decreasing the duration of chelator therapy preserves the cyto-protective effect of metHb and thrombin preconditioning. Cultures were pretreated for 24 h with 3 μM methemoglobin (Met, a) or 5 units/ml thrombin (Thr, b) as in Fig. 4, but 100 μM DFO was present only for the first 11 hours of the preconditioning interval. After medium exchange, cultures were then treated with 30 μM hemin for 8 or 24 hours, without DFO. Medium LDH values are scaled to the mean value in cultures treated with 0.1 % Triton X-100 (= 100), which lyses all cells. The weak signal in control cultures subjected to medium exchange only was subtracted from all values to yield the LDH activity associated with neurotoxicity. ###P<0.001 v. value in control cultures incubated in MEM10 medium alone, n=14–18/condition