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. 2012 May 29;18(1):1045–1055. doi: 10.2119/molmed.2012.00154

Figure 7.

Figure 7

Role of heme, iron and oxidative stress in RKA182 and ARS toxicity. (A) HL-60 cells were pretreated or not for 1 h with the δ-aminolevulinate dehydratase inhibitor SA (1 mmol/L) and/or the heme precursors ALA (1 mmol/L) or PPIX (1 μmol/L), before a further 24-h exposure to ARS (2 μmol/L) or RKA182 (10 μmol/L). (B) HL-60 cells were pre-treated or not for 1 h with the iron chelator DFO (10 μmol/L) or the exogenous iron source HTF (10 μmol/L), before a further 24-h exposure to ARS (10 μmol/L) or RKA182 (15 μmol/L). (C) HL-60 cells were pretreated or not for 1 h with the superoxide scavenger tiron (1 mmol/L) or the thiol antioxidant NAC (10 mmol/L), before a further 24-h exposure to ARS (30 μmol/L) or RKA182 (20 μmol/L). (D–E) Cells were pretreated or not for 16 h with the glutathione synthesis inhibitor BSO (30 μmol/L), followed by a further 1-h exposure to NAC (10 mmol/L). (D) Total glutathione content was determined using the DTNB recycling assay, and is normalized to total protein content. (E) Cells were subsequently exposed to ARS (1 μmol/L) or RKA182 (2 μmol/L) for a further 24 h. In all cases, cytotoxicity was determined by quantification of cellular ATP content. Data are expressed relative to the ATP content of cells exposed only to vehicle (methanol, 0.5%). All data represent mean + SD, n = 3. One-way ANOVA, *P ≥ 0.05, **P ≥ 0.01, ***P ≥ 0.001 versus non-pretreated cells, or for the indicated comparison, N.S., no statistically significant change in ATP content with indicated pretreatment. (F) Proposed mechanisms underpinning cytotoxicity induced by artemisinins, trioxolanes and tetraoxanes.