“The cold aqueous extract of Baccharis articulata stimulates apoptosis process in human lymphocytes”.

CARIDDI LAURA NOELIA; ESCOBAR FRANCO MATÍAS; SABINI MARÍA CAROLA; ZAMORA FERNANDA; TORRES CRISTINA VANESA; REINOSO ELINA; SABINI LILIANA INÉS

PUERTO MADRYN

Congreso; “XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica”; 2010

THE COLD AQUEOUS EXTRACT OF Baccharis articulata STIMULATES APOPTOSIS PROCESS IN HUMAN LYMPHOCYTESBaccharis articulata STIMULATES APOPTOSIS PROCESS IN HUMAN LYMPHOCYTES Cariddi L, Escobar F, Sabini M, Zamora F, Torres C, Reinoso E,Sabini L Facultad de Ciencias Exactas Fco-Qcas y Naturales. Universidad Nacional de Río Cuarto. E-mail: lcariddi@exa.unrc.edu.ar The cytotoxic action of Baccharis articulata (Asteraceae) has not been studied. The aim was to determine the toxic effects of cold aqueous extract of B. articulata (Ba-CAE) on human lymphocytes. Lymphocytes separated from peripheral blood of healthy volunteers by density gradient were exposed to Ba-CAE (10, 20, 40, 80, 160, 320, 640 and 1280 ìg/mL) for 18-24 h. Cell viability was determined by staining of trypan blue exclusion and MTT reduction. Apoptosis was determined by Hoechst 33258 staining, TUNEL and DNA fragmentation analysis by agarose gel electrophoresis. A dose-dependent toxicity of Ba-CAE was demonstrated by staining of trypan blue exclusion (CC50=150 ìg/mL). By MTT assay could not quantify this effect, since Ba-CAE was able to reduce tetrazolium salt in absence of cells. Hoechst staining, showed apoptotic figures (fragmented nuclei, kidney shaped nuclei and small nuclear blebs) in cells treated with Ba-CAE (80 to 1280 ìg/mL). The percentage of TUNEL-positive per 400 cells was: cell with media alone: 7+1%, cells treated with Ba-CAE: 10ìg/mL (8+1%), 20ìg/mL (9+3%), 40ìg/mL (43+11%)*, 80ìg/mL (58+18%)*, 160ìg/mL (62+13%)* 320ìg/mL (68+20%)**, 640ìg/mL (73+10%)**, 1280ìg/mL (88+13%)**; *p<0.01; **p<0.001. Agarose gel electrophoresis showed typical DNA laddering in cells treated with Ba-CAE (40 to 1280 ìg/mL). Ba-CAE stimulated apoptosis in human lymphocytes. Baccharis articulata (Asteraceae) has not been studied. The aim was to determine the toxic effects of cold aqueous extract of B. articulata (Ba-CAE) on human lymphocytes. Lymphocytes separated from peripheral blood of healthy volunteers by density gradient were exposed to Ba-CAE (10, 20, 40, 80, 160, 320, 640 and 1280 ìg/mL) for 18-24 h. Cell viability was determined by staining of trypan blue exclusion and MTT reduction. Apoptosis was determined by Hoechst 33258 staining, TUNEL and DNA fragmentation analysis by agarose gel electrophoresis. A dose-dependent toxicity of Ba-CAE was demonstrated by staining of trypan blue exclusion (CC50=150 ìg/mL). By MTT assay could not quantify this effect, since Ba-CAE was able to reduce tetrazolium salt in absence of cells. Hoechst staining, showed apoptotic figures (fragmented nuclei, kidney shaped nuclei and small nuclear blebs) in cells treated with Ba-CAE (80 to 1280 ìg/mL). The percentage of TUNEL-positive per 400 cells was: cell with media alone: 7+1%, cells treated with Ba-CAE: 10ìg/mL (8+1%), 20ìg/mL (9+3%), 40ìg/mL (43+11%)*, 80ìg/mL (58+18%)*, 160ìg/mL (62+13%)* 320ìg/mL (68+20%)**, 640ìg/mL (73+10%)**, 1280ìg/mL (88+13%)**; *p<0.01; **p<0.001. Agarose gel electrophoresis showed typical DNA laddering in cells treated with Ba-CAE (40 to 1280 ìg/mL). Ba-CAE stimulated apoptosis in human lymphocytes. B. articulata (Ba-CAE) on human lymphocytes. Lymphocytes separated from peripheral blood of healthy volunteers by density gradient were exposed to Ba-CAE (10, 20, 40, 80, 160, 320, 640 and 1280 ìg/mL) for 18-24 h. Cell viability was determined by staining of trypan blue exclusion and MTT reduction. Apoptosis was determined by Hoechst 33258 staining, TUNEL and DNA fragmentation analysis by agarose gel electrophoresis. A dose-dependent toxicity of Ba-CAE was demonstrated by staining of trypan blue exclusion (CC50=150 ìg/mL). By MTT assay could not quantify this effect, since Ba-CAE was able to reduce tetrazolium salt in absence of cells. Hoechst staining, showed apoptotic figures (fragmented nuclei, kidney shaped nuclei and small nuclear blebs) in cells treated with Ba-CAE (80 to 1280 ìg/mL). The percentage of TUNEL-positive per 400 cells was: cell with media alone: 7+1%, cells treated with Ba-CAE: 10ìg/mL (8+1%), 20ìg/mL (9+3%), 40ìg/mL (43+11%)*, 80ìg/mL (58+18%)*, 160ìg/mL (62+13%)* 320ìg/mL (68+20%)**, 640ìg/mL (73+10%)**, 1280ìg/mL (88+13%)**; *p<0.01; **p<0.001. Agarose gel electrophoresis showed typical DNA laddering in cells treated with Ba-CAE (40 to 1280 ìg/mL). Ba-CAE stimulated apoptosis in human lymphocytes. ìg/mL) for 18-24 h. Cell viability was determined by staining of trypan blue exclusion and MTT reduction. Apoptosis was determined by Hoechst 33258 staining, TUNEL and DNA fragmentation analysis by agarose gel electrophoresis. A dose-dependent toxicity of Ba-CAE was demonstrated by staining of trypan blue exclusion (CC50=150 ìg/mL). By MTT assay could not quantify this effect, since Ba-CAE was able to reduce tetrazolium salt in absence of cells. Hoechst staining, showed apoptotic figures (fragmented nuclei, kidney shaped nuclei and small nuclear blebs) in cells treated with Ba-CAE (80 to 1280 ìg/mL). The percentage of TUNEL-positive per 400 cells was: cell with media alone: 7+1%, cells treated with Ba-CAE: 10ìg/mL (8+1%), 20ìg/mL (9+3%), 40ìg/mL (43+11%)*, 80ìg/mL (58+18%)*, 160ìg/mL (62+13%)* 320ìg/mL (68+20%)**, 640ìg/mL (73+10%)**, 1280ìg/mL (88+13%)**; *p<0.01; **p<0.001. Agarose gel electrophoresis showed typical DNA laddering in cells treated with Ba-CAE (40 to 1280 ìg/mL). Ba-CAE stimulated apoptosis in human lymphocytes.