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Phagocytic uptake of oxidized heme polymer is highly cytotoxic to macrophages.

Deshmukh R, Trivedi V - PLoS ONE (2014)

Bottom Line: Toxicological potentiation of pro-oxidant molecules toward macrophages depends on generation of H2O2 and independent to release of free iron from pro-oxidant molecules.Methemoglobin oxidizes β-hematin to form oxidized β-hematin (βH*) through single electron transfer mechanism.Acridine orange/ethidium bromide staining and DNA fragmentation analysis indicate that macrophage follows an oxidative stress dependent apoptotic pathway to cause death.

View Article: PubMed Central - PubMed

Affiliation: Malaria Research Group, Department of Biotechnology, Indian Institute of Technology-Guwahati, Assam, India.

ABSTRACT
Apoptosis in macrophages is responsible for immune-depression and pathological effects during malaria. Phagocytosis of PRBC causes induction of apoptosis in macrophages through release of cytosolic factors from infected cells. Heme polymer or β-hematin causes dose-dependent death of macrophages with LC50 of 132 µg/ml and 182 µg/ml respectively. The toxicity of hemin or heme polymer was amplified several folds in the presence of non-toxic concentration of methemoglobin. β-hematin uptake in macrophage through phagocytosis is crucial for enhanced toxicological effects in the presence of methemoglobin. Higher accumulation of β-hematin is observed in macrophages treated with β-hematin along with methemoglobin. Light and scanning electron microscopic observations further confirm accumulation of β-hematin with cellular toxicity. Toxicological potentiation of pro-oxidant molecules toward macrophages depends on generation of H2O2 and independent to release of free iron from pro-oxidant molecules. Methemoglobin oxidizes β-hematin to form oxidized β-hematin (βH*) through single electron transfer mechanism. Pre-treatment of reaction mixture with spin-trap Phenyl-N-t-butyl-nitrone dose-dependently reverses the β-hematin toxicity, indicates crucial role of βH* generation with the toxicological potentiation. Acridine orange/ethidium bromide staining and DNA fragmentation analysis indicate that macrophage follows an oxidative stress dependent apoptotic pathway to cause death. In summary, current work highlights mutual co-operation between methemoglobin and different pro-oxidant molecules to enhance toxicity towards macrophages. Hence, methemoglobin peroxidase activity can be probed for subduing cellular toxicity of pro-oxidant molecules and it may in-turn make up for host immune response against the malaria parasite.

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MetHb and β-hematin interaction generate single electron containing species (β-hematin*) to exhibit cyto-toxicity towards macrophages.(A) Optical spectra of β-hematin oxidation by methemoglobin. Soret spectra were recorded in 100 mM Tris-HCl buffer, pH 7.4, in a total volume of 0.8 ml. Soret spectrum (a) of MetHb (1 µM); (b) a + H2O2 (100 µM); (c) b + β-hematin (10 µM). Equal concentration of β-hematin (10 µM) was added in the reference cuvette to correct the absorbance in soret region. (B) Scavenging single electron containing species (β-hematin*) restores cellular viability of macrophages. Cells were treated with different concentration of β-hematin (0–150 µg/ml)/methemoglobin (7.75 µM) mixture in the presence of different concentration of PBN (50–300 µM) or remained untreated. Cellular viability was determined by MTT assay as described in “material and methods”. Cells treated with incomplete media was considered as 100% viable. Data is the mean ± SD of three independent experiments (n = 3) with triplicate measurement. (C) Light microscopic observation of macrophages treated in (B) with 20x objective to detect cellular morphology at 0 hr and 6 hrs. (D) Binding of PBN to the oxidized βH. β-hematin was incubated with the different concentration of PBN (0–600 µM) in the presence of MetHb (7.75 µM), H2O2 and optical spectra were recorded.
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pone-0103706-g005: MetHb and β-hematin interaction generate single electron containing species (β-hematin*) to exhibit cyto-toxicity towards macrophages.(A) Optical spectra of β-hematin oxidation by methemoglobin. Soret spectra were recorded in 100 mM Tris-HCl buffer, pH 7.4, in a total volume of 0.8 ml. Soret spectrum (a) of MetHb (1 µM); (b) a + H2O2 (100 µM); (c) b + β-hematin (10 µM). Equal concentration of β-hematin (10 µM) was added in the reference cuvette to correct the absorbance in soret region. (B) Scavenging single electron containing species (β-hematin*) restores cellular viability of macrophages. Cells were treated with different concentration of β-hematin (0–150 µg/ml)/methemoglobin (7.75 µM) mixture in the presence of different concentration of PBN (50–300 µM) or remained untreated. Cellular viability was determined by MTT assay as described in “material and methods”. Cells treated with incomplete media was considered as 100% viable. Data is the mean ± SD of three independent experiments (n = 3) with triplicate measurement. (C) Light microscopic observation of macrophages treated in (B) with 20x objective to detect cellular morphology at 0 hr and 6 hrs. (D) Binding of PBN to the oxidized βH. β-hematin was incubated with the different concentration of PBN (0–600 µM) in the presence of MetHb (7.75 µM), H2O2 and optical spectra were recorded.

Mentions: Methemoglobin in the presence of H2O2 oxidizes a number of aromatic and halide substrates through single electron oxidation mechanism [37], [38], [39]. When H2O2 was added to native MetHb (Fe-III), a shift of soret peak from 406 nm (Figure 5A, spectrum a) to 417 nm (Figure 5A, spectrum b) was observed indicating the formation of an intermediate higher oxidation ferryl state (FeIV = O) complex, compound II. When β-hematin (10 µM) was added to this complex (417 nm), it gets reduced to the native FeIII state (403 nm) by one electron transfer process (Figure 5A, spectrum c). An equal amount of β-hematin was added to the reference cuvette to avoid artifacts of shift in soret peak. The UV-Visible spectral studies strongly support that the β-hematin probably been oxidized to single electron containing species βH* following a single electron oxidation mechanism.


Phagocytic uptake of oxidized heme polymer is highly cytotoxic to macrophages.

Deshmukh R, Trivedi V - PLoS ONE (2014)

MetHb and β-hematin interaction generate single electron containing species (β-hematin*) to exhibit cyto-toxicity towards macrophages.(A) Optical spectra of β-hematin oxidation by methemoglobin. Soret spectra were recorded in 100 mM Tris-HCl buffer, pH 7.4, in a total volume of 0.8 ml. Soret spectrum (a) of MetHb (1 µM); (b) a + H2O2 (100 µM); (c) b + β-hematin (10 µM). Equal concentration of β-hematin (10 µM) was added in the reference cuvette to correct the absorbance in soret region. (B) Scavenging single electron containing species (β-hematin*) restores cellular viability of macrophages. Cells were treated with different concentration of β-hematin (0–150 µg/ml)/methemoglobin (7.75 µM) mixture in the presence of different concentration of PBN (50–300 µM) or remained untreated. Cellular viability was determined by MTT assay as described in “material and methods”. Cells treated with incomplete media was considered as 100% viable. Data is the mean ± SD of three independent experiments (n = 3) with triplicate measurement. (C) Light microscopic observation of macrophages treated in (B) with 20x objective to detect cellular morphology at 0 hr and 6 hrs. (D) Binding of PBN to the oxidized βH. β-hematin was incubated with the different concentration of PBN (0–600 µM) in the presence of MetHb (7.75 µM), H2O2 and optical spectra were recorded.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4117526&req=5

pone-0103706-g005: MetHb and β-hematin interaction generate single electron containing species (β-hematin*) to exhibit cyto-toxicity towards macrophages.(A) Optical spectra of β-hematin oxidation by methemoglobin. Soret spectra were recorded in 100 mM Tris-HCl buffer, pH 7.4, in a total volume of 0.8 ml. Soret spectrum (a) of MetHb (1 µM); (b) a + H2O2 (100 µM); (c) b + β-hematin (10 µM). Equal concentration of β-hematin (10 µM) was added in the reference cuvette to correct the absorbance in soret region. (B) Scavenging single electron containing species (β-hematin*) restores cellular viability of macrophages. Cells were treated with different concentration of β-hematin (0–150 µg/ml)/methemoglobin (7.75 µM) mixture in the presence of different concentration of PBN (50–300 µM) or remained untreated. Cellular viability was determined by MTT assay as described in “material and methods”. Cells treated with incomplete media was considered as 100% viable. Data is the mean ± SD of three independent experiments (n = 3) with triplicate measurement. (C) Light microscopic observation of macrophages treated in (B) with 20x objective to detect cellular morphology at 0 hr and 6 hrs. (D) Binding of PBN to the oxidized βH. β-hematin was incubated with the different concentration of PBN (0–600 µM) in the presence of MetHb (7.75 µM), H2O2 and optical spectra were recorded.
Mentions: Methemoglobin in the presence of H2O2 oxidizes a number of aromatic and halide substrates through single electron oxidation mechanism [37], [38], [39]. When H2O2 was added to native MetHb (Fe-III), a shift of soret peak from 406 nm (Figure 5A, spectrum a) to 417 nm (Figure 5A, spectrum b) was observed indicating the formation of an intermediate higher oxidation ferryl state (FeIV = O) complex, compound II. When β-hematin (10 µM) was added to this complex (417 nm), it gets reduced to the native FeIII state (403 nm) by one electron transfer process (Figure 5A, spectrum c). An equal amount of β-hematin was added to the reference cuvette to avoid artifacts of shift in soret peak. The UV-Visible spectral studies strongly support that the β-hematin probably been oxidized to single electron containing species βH* following a single electron oxidation mechanism.

Bottom Line: Toxicological potentiation of pro-oxidant molecules toward macrophages depends on generation of H2O2 and independent to release of free iron from pro-oxidant molecules.Methemoglobin oxidizes β-hematin to form oxidized β-hematin (βH*) through single electron transfer mechanism.Acridine orange/ethidium bromide staining and DNA fragmentation analysis indicate that macrophage follows an oxidative stress dependent apoptotic pathway to cause death.

View Article: PubMed Central - PubMed

Affiliation: Malaria Research Group, Department of Biotechnology, Indian Institute of Technology-Guwahati, Assam, India.

ABSTRACT
Apoptosis in macrophages is responsible for immune-depression and pathological effects during malaria. Phagocytosis of PRBC causes induction of apoptosis in macrophages through release of cytosolic factors from infected cells. Heme polymer or β-hematin causes dose-dependent death of macrophages with LC50 of 132 µg/ml and 182 µg/ml respectively. The toxicity of hemin or heme polymer was amplified several folds in the presence of non-toxic concentration of methemoglobin. β-hematin uptake in macrophage through phagocytosis is crucial for enhanced toxicological effects in the presence of methemoglobin. Higher accumulation of β-hematin is observed in macrophages treated with β-hematin along with methemoglobin. Light and scanning electron microscopic observations further confirm accumulation of β-hematin with cellular toxicity. Toxicological potentiation of pro-oxidant molecules toward macrophages depends on generation of H2O2 and independent to release of free iron from pro-oxidant molecules. Methemoglobin oxidizes β-hematin to form oxidized β-hematin (βH*) through single electron transfer mechanism. Pre-treatment of reaction mixture with spin-trap Phenyl-N-t-butyl-nitrone dose-dependently reverses the β-hematin toxicity, indicates crucial role of βH* generation with the toxicological potentiation. Acridine orange/ethidium bromide staining and DNA fragmentation analysis indicate that macrophage follows an oxidative stress dependent apoptotic pathway to cause death. In summary, current work highlights mutual co-operation between methemoglobin and different pro-oxidant molecules to enhance toxicity towards macrophages. Hence, methemoglobin peroxidase activity can be probed for subduing cellular toxicity of pro-oxidant molecules and it may in-turn make up for host immune response against the malaria parasite.

Show MeSH
Related in: MedlinePlus