In vitro protective effect and antioxidant mechanism of Resveratrol induced by Dapsone Hydroxylamine in human cells

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDSNHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10-1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy

Clinical applications and methemoglobinemia induced by dapsone

Dapsone is a synthetic sulfone that is used as an antibiotic in humans and animals to prevent and treat diseases including leprosy, tuberculosis, malaria, and Pneumocystis carinii pneumonia and Toxoplasma gondii encephalitis in acquired immune deficiency syndrome (AIDS) patients as well as in anti-inflammatory conditions, such as dermatitis herpetiformis. However, this drug is also associated with several adverse effects, including dose-related hemolysis, methemoglobinemia, psychosis, peripheral neuropathy, agranulocytosis, aplastic anemia, hypersensitivity syndrome, sulfone syndrome, and others. Of these effects, methemoglobinemia is the most common side effect of dapsone, which leads to functional anemia and cellular hypoxia with symptoms of cyanosis, headache, fatigue, tachycardia, weakness, and dizziness. Thus, this review summarizes relevant information on the structure, mechanism of action, clinical indication, and adverse reactions of dapsone

In Vitro Protective Effect and Antioxidant Mechanism of Resveratrol Induced by Dapsone Hydroxylamine in Human Cells.

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDS-NHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10-1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy

Redox mechanism.

<p>Ionization potential and stabilization energy of dapsone hydroxylamine (DDS-NHOH), resveratrol (RSV), and methylene blue (MET) on antioxidant and methemoglobinemia reversion.</p

Proposal for a possible action mechanism of resveratrol (RSV) in inhibiting methemoglobin formation and DNA damage induced by DDS hydroxylamine (DDS-NHOH) <i>in vitro</i> model.

<p>Proposal for a possible action mechanism of resveratrol (RSV) in inhibiting methemoglobin formation and DNA damage induced by DDS hydroxylamine (DDS-NHOH) <i>in vitro</i> model.</p

CAT and SOD activity.

<p>Erythrocytes were pretreated with resveratrol (RSV, 100 μM and 1000 μM) for 1 h at 37°C and incubated for 30 min with DDS-NHOH (2.5 μg/ml) or T-BHP (200 μM). Results are expressed as mean ± S.E.M. *P < 0.05 compared to methanol group.</p

Effect of treatment with resveratrol on DNA damage induced by DDS-NHOH.

<p>Tail Length (μm—<b>A</b>), DNA in tail (%—<b>B</b>) Tail Moment (TM—<b>C</b>) and Olive Moment (OM—<b>D</b>) were used as a marker of DNA damage in lymphocyte using Comet assay. As positive control was used H₂O₂ (200 μM). All values are depicted as mean ± S.E.M.</p

Comparative effect of post-treatment with resveratrol (RSV) or methylene blue (MET) on methemoglobin formation induced by DDS-NHOH.

<p>Erythrocytes were incubated for 1 h with DDS-NHOH (2.5 μg/mL), then these cells were incubated with RSV (100μM) for 1 h or MET(40 nM). Data are reported as mean ± S.E.M. *P < 0.05 compared to methanol group. ^#P < 0.05 compared to DDS-NHOH group.</p

Effect of the DDS-NHOH on methemoglobin formation in human erythrocytes.

<p>Erythrocytes were incubated with different concentrations of DDS-NHOH (2.5; 5.0 and 7.5 μg/mL) for 1 h at 37°C. Data are reported as means ± S.E.M from three independent experiments done in triplicate. *P < 0.05 compared to methanol group.</p

HOMO surface.

<p>Structure for HOMO of the dapsone hydroxylamine (DDS-NHOH), resveratrol (RSV), and methylene blue (MET). All nodal patterns related to individual group contributions are presented by blue or yellow for negative or positive wave function, respectively.</p