Antimutagenic components in <i>Glycyrrhiza</i> against <i>N</i>-methyl-<i>N</i>-nitrosourea in the Ames assay

<p>Antimutagenesis against <i>N</i>-nitroso compounds contribute to prevention of human cancer. We have found that <i>Glycyrrhiza aspera</i> ethanolic extract exhibits antimutagenic activity against <i>N</i>-methyl-<i>N</i>-nitrosourea (MNU) using the Ames assay with <i>Salmonella typhimurium</i> TA1535. In the present study, eight purified components from <i>Glycyrrhiza</i>, namely glabridin, glycyrrhetinic acid, glycyrrhizin, licochalcone A, licoricesaponin H2, licoricesaponin G2, liquiritigenin and liquiritin were evaluated for their antimutagenicity against MNU in the Ames assay with <i>S. typhimurium</i> TA1535. Glycyrrhetinic acid, glycyrrhizin, licoricesaponin G2, licoricesaponin H2 and liquiritin did not show the antimutagenicity against MNU in <i>S. typhimurium</i> TA1535. Glabridin, licochalcone A and liquiritigenin reduced revertant colonies derived from MNU in <i>S. typhimurium</i> TA1535 without showing cytotoxic effects, indicating that these compounds possess antimutagenic activity against MNU. The inhibitory activity of glabridin and licochalcone A was more effective than that of liquiritigenin. Thus, <i>Glycyrrhiza</i> contains antimutagenic components against DNA alkylating, direct-acting carcinogens.</p

Accelerated Recovery of Mitochondrial Membrane Potential by GSK-3β Inactivation Affords Cardiomyocytes Protection from Oxidant-Induced Necrosis

<div><p>Loss of mitochondrial membrane potential (ΔΨ_m) is known to be closely linked to cell death by various insults. However, whether acceleration of the ΔΨ_m recovery process prevents cell necrosis remains unclear. Here we examined the hypothesis that facilitated recovery of ΔΨ_m contributes to cytoprotection afforded by activation of the mitochondrial ATP-sensitive K⁺ (mK_ATP) channel or inactivation of glycogen synthase kinase-3β (GSK-3β). ΔΨ_m of H9c2 cells was determined by tetramethylrhodamine ethyl ester (TMRE) before or after 1-h exposure to antimycin A (AA), an inducer of reactive oxygen species (ROS) production at complex III. Opening of the mitochondrial permeability transition pore (mPTP) was determined by mitochondrial loading of calcein. AA reduced ΔΨ_m to 15±1% of the baseline and induced calcein leak from mitochondria. ΔΨ_m was recovered to 51±3% of the baseline and calcein-loadable mitochondria was 6±1% of the control at 1 h after washout of AA. mK_ATP channel openers improved the ΔΨ_m recovery and mitochondrial calcein to 73±2% and 30±7%, respectively, without change in ΔΨ_m during AA treatment. Activation of the mK_ATP channel induced inhibitory phosphorylation of GSK-3β and suppressed ROS production, LDH release and apoptosis after AA washout. Knockdown of GSK-3β and pharmacological inhibition of GSK-3β mimicked the effects of mK_ATP channel activation. ROS scavengers administered at the time of AA removal also improved recovery of ΔΨ_m. These results indicate that inactivation of GSK-3β directly or indirectly by mK_ATP channel activation facilitates recovery of ΔΨ_m by suppressing ROS production and mPTP opening, leading to cytoprotection from oxidant stress-induced cell death.</p></div

Effects of cyclosporine A on antimycin A-induced changes in ΔΨ_m and LDH release.

<p>A: Level of TMRE fluorescence was determined as an index of ΔΨ_m. Cyclosporine A (CsA, 0.5 µM) was added to the medium 60 min before antimycin A (AA) treatment. AA-induced reduction of TMRE fluorescence at 60 min after the onset of AA treatment was slightly attenuated in the CsA-treated group compared to that in the AA group, indicating partial suppression of ROS-induced mPTP opening. Recovery of TMRE fluorescence at 60 min after washout of AA was also slightly improved by CsA. N = 5 per group. B: LDH released after washout of AA was significantly reduced by CsA. *p<0.05 vs. Vehicle+AA. N = 8 per group.</p

Effects of mK_ATP channel openers on antimycin A-induced mPTP opening and its recovery in H9c2 cells.

<p>A and B: Calcein and MitoTracker images before (A) and after (B) antimycin A (AA) treatment. C and D: Levels of calcein-positive mitochondria 60 min after AA treatment (C) and 60 and 120 min after washout of AA (D). Level of calcein-positive mitochondria is expressed as the ratio of calcein-positive area to MitoTracker-positive area. *p<0.05 vs. Vehicle+AA, #p<0.05 vs. NC+AA.</p

Effects of inhibition of GSK-3β on antimycin A-induced changes in ΔΨ_m and its recovery.

<p>A: Western blotting for Ser9-phospho- and total GSK-3β in mitochondria, B: Effects of antimycin A (AA) on phospho-GSK-3β level. C: TMRE fluorescence after AA treatment in vehicle- and LiCl-pretreated cells. Western blotting for Ser9-phospho-GSK-3β, total GSK-3β, Ser641/645-phospho-glycogen synthase (GS), non-phospho-GS and β-actin (loading control) in total lysates of vehicle-treated and LiCl-treated cells. Treatments with 30 mM and 60 mM LiCl for 60 min induced phosphorylation of GSK-3β and dephosphorylation of GS. Increased phosphorylation of GSK-3β by LiCl reflects reduced activity of protein phosphatase 1, which is positively regulated by GSK-3β activity. D: TMRE fluorescence after AA treatment in control siRNA- and GSK-3β-siRNA-pretreated cells. NC =  nicorandil. Treatment  =  time after onset of treatment with AA, Washout  =  time after washout of AA. *p<0.05 vs. Vehicle or Control siRNA. N = 8.</p

Effects of ROS scavengers on time course of ΔΨ_m.

<p>A and B: Effects of MPG administered during AA treatment (A) and effects of treatment with MPG or NAC commenced at the time of AA washout (B) on TMRE fluoresence. MPG-Tx  =  treatment with mercaptopropionyl glycine (MPG) during AA treatment, MPG  =  MPG treatment commenced at the time of AA washout, NAC  =  N-acetylcysteine treatment commenced at the time of AA washout. Treatment  =  time after onset of treatment with AA, Washout  =  time after washout of AA. *p<0.05 vs. AA+Vehicle. N = 8.</p

Effects of mK_ATP channel openers and a glycolysis inhibitor on antimycin A-induced loss of ΔΨ_m and its recovery in H9c2 cells.

<p>A: TMRE images before and after antimycin A (AA) treatment (Images were from different cells). B and C: TMRE fluorescence in NC- (B) and DZ-pretreated cells (C). D: TMRE fluorescence in IAA-treated cells. TMRE fluorescence in NC =  nicorandil, DZ =  diazoxide, 5-HD =  5-hydoxydecanote, IAA  =  iodoacetate, Treatment  =  time after onset of treatment with AA, Washout  =  time after washout of AA. *p<0.05 vs. Vehicle+AA or AA, #p<0.05 vs. NC+AA. N = 8.</p

Necrosis and apoptosis after antimycin A treatment.

<p>A–C: Cell necrosis indicated by LDH release. LDH release at the end of AA treatment (A) and during a 2-h period after washout of AA (B, C) are shown. D and E: representative images of nuclear staining with Hoechst33342 (D) and apoptosis at 2 h after washout of AA (E). AA =  antimycin A, NC =  nicorandil, DZ =  diazoxide, 5-HD  =  5-hydroxydecanoate. *p<0.05 vs. Vehicle, †p<0.05 vs. Vehicle+AA. N = 8∼12.</p

Effects of an mK_ATP channel opener on antimycin A-induced loss of ΔΨ_m and its recovery in C2C12 cells.

<p>TMRE fluorescence in untreated and diazoxide-pretreated cells. AA =  antimycin A, DZ =  diazoxide, Treatment  =  time after onset of treatment with AA, Washout  =  time after washout of AA. *p<0.05 vs. Vehicle+AA. N = 8.</p

ROS generated by antimycin A.

<p>A: Representative DCF images. B: DCF fluorescence during and after antimycin A (AA) treatment in vehicle-, NC- and LiCl-pretreated cells. C: Effects of MPG administered during AA treatment on DCF. D: Effects of MPG or NAC treatment commenced at the time of AA washout on DCF. MPG-Tx  =  treatment with mercaptopropionyl glycine (MPG) during AA treatment, MPG =  MPG treatment commenced at the time of AA washout, NAC =  N-acetylcysteine treatment commenced at the time of AA washout. Treatment  =  time after onset of treatment with AA, Washout  =  time after washout of AA. *p<0.05 vs. Vehicle+AA or AA+Vehicle. N = 8.</p