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In Vitro and In Vivo Cytogenotoxic Effects of Hot Aqueous Extract of Achyrocline satureioides (Lam.) DC.

Cariddi LN, Sabini MC, Escobar FM, Bacchetti R, Montironi I, Merckis C, Reinoso EB, Núñez Montoya S, Zanon SM, Comini LR, Sabini LI - Biomed Res Int (2015)

Bottom Line: The in vivo micronucleus test showed that As-HAE exerted cytogenotoxic effects on bone marrow of mice, contrary to what was observed with cold extract.The chemical study of As-HAE allowed identifying the flavonoids found in cold extract: luteolin, quercetin, and 3-O-methylquercetin, but at higher concentrations.We suggest that toxic effects induced by As-HAE could be due to high concentrations of these flavonoids.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología e Inmunología, Universidad Nacional de Río Cuarto, Ruta 36, Km 601, Río Cuarto, C5800 Córdoba, Argentina ; Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Rivadavia 1917, C1033AAJ Buenos Aires, Argentina.

ABSTRACT
In this work we extend the toxicological studies of hot aqueous extract of A. satureioides (As-HAE) evaluating cytotoxic and apoptotic effects on human peripheral blood mononuclear cells (PBMCs). We also determine genotoxic action of this extract in vivo. In addition, the extract was chemically characterized. Finally, we established a comparison with previous data of cold aqueous extract. The As-HAE induced cytotoxicity on PBMCs determined by trypan blue dye exclusion (CC50 = 653 μg/mL) and MTT (CC50 = 588 μg/mL) assays being more toxic than cold extract. However, As-HAE as well as cold extract did not induce apoptosis measured by Hoechst 33258 staining, TUNEL assay, and DNA fragmentation analysis. The in vivo micronucleus test showed that As-HAE exerted cytogenotoxic effects on bone marrow of mice, contrary to what was observed with cold extract. The chemical study of As-HAE allowed identifying the flavonoids found in cold extract: luteolin, quercetin, and 3-O-methylquercetin, but at higher concentrations. We suggest that toxic effects induced by As-HAE could be due to high concentrations of these flavonoids. Given that As-HAE is the most used in folkloric medicine, its administration should be controlled in order to prevent potential cell damage.

No MeSH data available.


Related in: MedlinePlus

(a) Quantification of apoptotic human PBMCs by TUNEL assay after treatment with hot aqueous extract of Achyrocline satureioides (10, 600, and 1000 μg/mL). (b) Photomicrographs of human PBMCs treated with hot aqueous extract of Achyrocline satureioides (As-HAE) and stained with TUNEL (100x) using the commercial kit ApopTag Plus Peroxidase In Situ Apoptosis (Chemicon International, USA). (A) Medium alone (control), (B) cells treated with hydrogen peroxide (1 mmol/L), (C) As-HAE (10 μg/mL), (D) As-HAE (600 μg/mL), and (E) As-HAE (1000 μg/mL). Apoptotic cells are shown in brown.
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fig3: (a) Quantification of apoptotic human PBMCs by TUNEL assay after treatment with hot aqueous extract of Achyrocline satureioides (10, 600, and 1000 μg/mL). (b) Photomicrographs of human PBMCs treated with hot aqueous extract of Achyrocline satureioides (As-HAE) and stained with TUNEL (100x) using the commercial kit ApopTag Plus Peroxidase In Situ Apoptosis (Chemicon International, USA). (A) Medium alone (control), (B) cells treated with hydrogen peroxide (1 mmol/L), (C) As-HAE (10 μg/mL), (D) As-HAE (600 μg/mL), and (E) As-HAE (1000 μg/mL). Apoptotic cells are shown in brown.

Mentions: In order to determine whether the cytotoxic effect of As-HAE was due to apoptosis, DNA fragmentation induced by the extract was analyzed. PBMCs morphology was evaluated followed by Hoechst 33258 DNA staining. The nuclei of cells cultured in medium alone were uniformly blue (Figure 2). Fluorescence microscope showed that cells treated with all As-HAE concentrations showed to be similar to the control. However, only some nuclei of the PBMCs treated with the highest concentration of As-HAE (1000 μg/mL) contained small bright blue dots representing chromatin condensation and/or nuclear fragmentation. Other few apoptotic figures were observed such as the formation of membrane blebs and apoptotic bodies (Figure 2). We corroborated these results by TUNEL staining. The percentage of TUNEL-positive cells per 400 cells in cells cultured in medium alone was 7.89 + 0.75% TUNEL + PBMCs. Cells treated with all As-HAE concentrations did not show statistical difference with the negative control (Figures 3(a) and 3(b)). Similarly, any As-HAE concentration assayed showed the typical DNA laddering in agarose gels electrophoresis (data not shown). These results indicate that As-HAE did not cause apoptosis in human PBMCs.


In Vitro and In Vivo Cytogenotoxic Effects of Hot Aqueous Extract of Achyrocline satureioides (Lam.) DC.

Cariddi LN, Sabini MC, Escobar FM, Bacchetti R, Montironi I, Merckis C, Reinoso EB, Núñez Montoya S, Zanon SM, Comini LR, Sabini LI - Biomed Res Int (2015)

(a) Quantification of apoptotic human PBMCs by TUNEL assay after treatment with hot aqueous extract of Achyrocline satureioides (10, 600, and 1000 μg/mL). (b) Photomicrographs of human PBMCs treated with hot aqueous extract of Achyrocline satureioides (As-HAE) and stained with TUNEL (100x) using the commercial kit ApopTag Plus Peroxidase In Situ Apoptosis (Chemicon International, USA). (A) Medium alone (control), (B) cells treated with hydrogen peroxide (1 mmol/L), (C) As-HAE (10 μg/mL), (D) As-HAE (600 μg/mL), and (E) As-HAE (1000 μg/mL). Apoptotic cells are shown in brown.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4442415&req=5

fig3: (a) Quantification of apoptotic human PBMCs by TUNEL assay after treatment with hot aqueous extract of Achyrocline satureioides (10, 600, and 1000 μg/mL). (b) Photomicrographs of human PBMCs treated with hot aqueous extract of Achyrocline satureioides (As-HAE) and stained with TUNEL (100x) using the commercial kit ApopTag Plus Peroxidase In Situ Apoptosis (Chemicon International, USA). (A) Medium alone (control), (B) cells treated with hydrogen peroxide (1 mmol/L), (C) As-HAE (10 μg/mL), (D) As-HAE (600 μg/mL), and (E) As-HAE (1000 μg/mL). Apoptotic cells are shown in brown.
Mentions: In order to determine whether the cytotoxic effect of As-HAE was due to apoptosis, DNA fragmentation induced by the extract was analyzed. PBMCs morphology was evaluated followed by Hoechst 33258 DNA staining. The nuclei of cells cultured in medium alone were uniformly blue (Figure 2). Fluorescence microscope showed that cells treated with all As-HAE concentrations showed to be similar to the control. However, only some nuclei of the PBMCs treated with the highest concentration of As-HAE (1000 μg/mL) contained small bright blue dots representing chromatin condensation and/or nuclear fragmentation. Other few apoptotic figures were observed such as the formation of membrane blebs and apoptotic bodies (Figure 2). We corroborated these results by TUNEL staining. The percentage of TUNEL-positive cells per 400 cells in cells cultured in medium alone was 7.89 + 0.75% TUNEL + PBMCs. Cells treated with all As-HAE concentrations did not show statistical difference with the negative control (Figures 3(a) and 3(b)). Similarly, any As-HAE concentration assayed showed the typical DNA laddering in agarose gels electrophoresis (data not shown). These results indicate that As-HAE did not cause apoptosis in human PBMCs.

Bottom Line: The in vivo micronucleus test showed that As-HAE exerted cytogenotoxic effects on bone marrow of mice, contrary to what was observed with cold extract.The chemical study of As-HAE allowed identifying the flavonoids found in cold extract: luteolin, quercetin, and 3-O-methylquercetin, but at higher concentrations.We suggest that toxic effects induced by As-HAE could be due to high concentrations of these flavonoids.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología e Inmunología, Universidad Nacional de Río Cuarto, Ruta 36, Km 601, Río Cuarto, C5800 Córdoba, Argentina ; Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Rivadavia 1917, C1033AAJ Buenos Aires, Argentina.

ABSTRACT
In this work we extend the toxicological studies of hot aqueous extract of A. satureioides (As-HAE) evaluating cytotoxic and apoptotic effects on human peripheral blood mononuclear cells (PBMCs). We also determine genotoxic action of this extract in vivo. In addition, the extract was chemically characterized. Finally, we established a comparison with previous data of cold aqueous extract. The As-HAE induced cytotoxicity on PBMCs determined by trypan blue dye exclusion (CC50 = 653 μg/mL) and MTT (CC50 = 588 μg/mL) assays being more toxic than cold extract. However, As-HAE as well as cold extract did not induce apoptosis measured by Hoechst 33258 staining, TUNEL assay, and DNA fragmentation analysis. The in vivo micronucleus test showed that As-HAE exerted cytogenotoxic effects on bone marrow of mice, contrary to what was observed with cold extract. The chemical study of As-HAE allowed identifying the flavonoids found in cold extract: luteolin, quercetin, and 3-O-methylquercetin, but at higher concentrations. We suggest that toxic effects induced by As-HAE could be due to high concentrations of these flavonoids. Given that As-HAE is the most used in folkloric medicine, its administration should be controlled in order to prevent potential cell damage.

No MeSH data available.


Related in: MedlinePlus