Macrophages follow oxidative stress mediated apoptosis to exhibit death.

<p>(<b>A</b>) Macrophages either remained untreated or treated with MetHb (7.75 µM), β-Hematin (60 µg/ml), or combination of β-Hematin (60 µg/ml)/MetHb (7.75 µM) for 6 hrs at 37°C in incomplete media. Cells were stained with acridine orange/Et-Br and analyzed in flow cytometry to characterize healthy, early or late apoptotic, and necrotic cell population. (<b>B</b>) DNA fragmentation analysis of macrophages treated with methemoglobin (7.75 µM), different concentration of β-Hematin (40 or 60 µg/ml) or combination of β-hematin (60 µg/ml)/methemoglobin (7.75 µM) in incomplete media for 6 hrs at 37°C. DNA fragmentation analysis was performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103706#s2" target="_blank">material and methods</a>”. (<b>C</b>) Oxidative stress is essential for induction of apoptosis during treatment of macrophages with combination of β-hematin/methemoglobin. DNA fragmentation analysis of macrophages either remains untreated or treated with combination of β-hematin (60 µg/ml)/methemoglobin (7.75 µM) for 6 hrs at 37°C in the absence or presence of different antioxidant molecules (NAC/mannitol). DNA fragmentation analysis was performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103706#s2" target="_blank">material and methods</a>”. (<b>D</b>) Methemoglobin mediated generation of βH* in the presence of extracellular H₂O₂ is responsible for macrophage apoptosis. DNA fragmentation analysis of macrophage remains untreated or treated with combination of β-hematin (60 µg/ml)/methemoglobin (7.75 µM) for 6 hrs at 37°C in absence or presence of catalase (200 units) or PBN (300 µM) respectively. DNA fragmentation analysis was performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103706#s2" target="_blank">material and methods</a>”. (<b>E</b>) Schematic Diagram of methemoglobin mediated β-hematin toxicological potentiation towards macrophages. A details description is given in the text.