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

Alpha-Lipoic Acid and Its Enantiomers Prevent Methemoglobin Formation and DNA Damage Induced by Dapsone Hydroxylamine: Molecular Mechanism and Antioxidant Action

Dapsone (DDS) therapy can frequently lead to hematological side effects, such as methemoglobinemia and DNA damage. In this study, we aim to evaluate the protective effect of racemic alpha lipoic acid (ALA) and its enantiomers on methemoglobin induction. The pre- and post-treatment of erythrocytes with ALA, ALA isomers, or MB (methylene blue), and treatment with DDS-NOH (apsone hydroxylamine) was performed to assess the protective and inhibiting effect on methemoglobin (MetHb) formation. Methemoglobin percentage and DNA damage caused by dapsone and its metabolites were also determined by the comet assay. We also evaluated oxidative parameters such as SOD, GSH, TEAC (Trolox equivalent antioxidant capacity) and MDA (malondialdehyde). In pretreatment, ALA showed the best protector effect in 2.5 µg/mL of DDS-NOH. ALA (1000 µM) was able to inhibit the induced MetHb formation even at the highest concentrations of DDS-NOH. All ALA tested concentrations (100 and 1000 µM) were able to inhibit ROS and CAT activity, and induced increases in GSH production. ALA also showed an effect on DNA damage induced by DDS-NOH (2.5 µg/mL). Both isomers were able to inhibit MetHb formation and the S-ALA was able to elevate GSH levels by stimulating the production of this antioxidant. In post-treatment with the R-ALA, this enantiomer inhibited MetHb formation and increased GSH levels. The pretreatment with R-ALA or S-ALA prevented the increase in SOD and decrease in TEAC, while R-ALA decreased the levels of MDA; and this pretreatment with R-ALA or S-ALA showed the effect of ALA enantiomers on DNA damage. These data show that ALA can be used in future therapies in patients who use dapsone chronically, including leprosy patients

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

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

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

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

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

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

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