ABSTRACT
PHARMACOLOGY RELEVANCE: Baccharis trinervis (Lam, Persoon) leaves are used in the traditional medicine for the treatment of high fevers, edema, inflammation, sores and muscle cramps, snakebites and as antiseptic. AIM OF THE STUDY: To investigate the cytotoxic, genotoxic, and mutagenic effects of extracts and fractions of B. trinervis from Brazil and Colombia in Chinese Hamster Ovary (CHO) cells, and to examine the mutagenic activity in Salmonella typhimurium. MATERIAL AND METHODS: Aqueous extracts (AE) of aerial parts of B. trinervis from Brazil (B) and Colombia (C) were fractioned in ethyl acetate fraction (EAF), butanol extract (BF), and aqueous residue fraction (ARF). Qualitative chemical screening and determination of total flavonoid content were made. Identification of chemical constituents was performed by High Performance Liquid Chromatography (HPLC) and High Resolution Mass Spectrometry (HRMS). For the in vitro tests, CHO cells were treated for 3h with extracts and fractions. The cytotoxic activity was evaluated by clonal survival and 3-(4.5-dimethylthiazole-2-yl)-2.5-biphenyl tetrazolium bromide reduction assay (MTT). Genotoxic and mutagenic effects were evaluated by the alkaline comet assay and Cytokinesis-blockage micronucleus test (CBMN), respectively. Additionally, Salmonella/microsome assay was carried out to determinate the mutagenic effects in EAF from Brazil and Colombia. RESULTS: Phytochemical analyses indicated the presence of saponins and flavonoids. AE and EAF were the samples with the highest quantity of total flavonoids. HPLC showed the presence of luteolin only in AEC, and caffeic acid, ellagic acid, rosmarinic acid, and rutin were identified in AEB and AEC (AEC>AEB). The HRMS in positive mode of EAFB and EAFC showed presence of two carboxylic acids, coumarin, and two terpenoids. In addition, were identified one terpenoid and two carboxylic acids in AE, BF and ARF of B. trinervis from both countries in negative mode. Dose-dependent cytotoxic effects were observed in CHO cells treated with B. trinervis extracts and fractions by using clonal survival and MTT at concentrations higher than 0.05mg/mL. All the extracts and fractions induced DNA strand breaks in CHO cells with dose-dependent response, mostly EAFB and EAFC. The EAF from Brazil and Colombia showed mutagenic effect at 0.5mg/mL, while the other fractions did not show a significant difference in relation to the control. No mutagenic effects were found in EAF from both countries by the Salmonella/microsome assay. CONCLUSIONS: Cytotoxic and genotoxic effects were demonstrated in all extracts and fractions used, although only EAF showed mutagenic effects by CBMN, but not by Salmonella/microsome assay. Our results suggest that flavonoids, phenylpropanoids, coumarins, and diterpenes may be responsible for the cytotoxic, genotoxic and mutagenic effects observed.
Subject(s)
Baccharis/chemistry , Cell Survival/drug effects , DNA Damage/drug effects , Flavonoids/analysis , Mutagens/pharmacology , Plant Extracts/toxicity , Animals , Brazil , Colombia , Comet Assay , Cricetulus , Dose-Response Relationship, Drug , Micronucleus Tests , Microsomes/drug effects , Plant Leaves/chemistryABSTRACT
Increasing evidence suggest that occupational exposure to open-cast coal mining residues like dust particles, heavy metals and Polycyclic Aromatic Hydrocarbons (PAHs) may cause a wide range of DNA damage and genomic instability that could be associated to initial steps in cancer development and other work-related diseases. The aim of our study was to evaluate if key polymorphisms in metabolism genes CYP1A1Msp1, GSTM1Null, GSTT1Null and DNA repair genes XRCC1Arg194Trp and hOGG1Ser326Cys could modify individual susceptibility to adverse coal exposure effects, considering the DNA damage (Comet assay) and micronucleus formation in lymphocytes (CBMN) and buccal mucosa cells (BMNCyt) as endpoints for genotoxicity. The study population is comprised of 200 healthy male subjects, 100 open-cast coal-mining workers from "El Cerrejón" (world's largest open-cast coal mine located in Guajira - Colombia) and 100 non-exposed referents from general population. The data revealed a significant increase of CBMN frequency in peripheral lymphocytes of occupationally exposed workers carrying the wild-type variant of GSTT1 (+) gene. Exposed subjects carrying GSTT1null polymorphism showed a lower micronucleus frequency compared with their positive counterparts (FR: 0.83; P=0.04), while BMNCyt, frequency and Comet assay parameters in lymphocytes: Damage Index (DI) and percentage of DNA in the tail (Tail % DNA) were significantly higher in exposed workers with the GSTM1Null polymorphism. Other exfoliated buccal mucosa abnormalities related to cell death (Karyorrhexis and Karyolysis) were increased in GSTT/M1Null carriers. Nuclear buds were significantly higher in workers carrying the CYP1A1Msp1 (m1/m2, m2/m2) allele. Moreover, BMNCyt frequency and Comet assay parameters were significantly lower in exposed carriers of XRCC1Arg194Trp (Arg/Trp, Trp/Trp) and hOGG1Ser326Cys (Ser/Cys, Cys/Cys), thereby providing new data to the increasing evidence about the protective role of these polymorphisms. This modulation may involve specific and differentiated pathways in different tissues that also may cause a differential sensitivity related to differential induction of some enzymes.
Subject(s)
Coal Mining/methods , DNA Repair/genetics , Occupational Exposure/adverse effects , Polymorphism, Single Nucleotide , Adult , Case-Control Studies , Colombia , Cross-Sectional Studies , Cytochrome P-450 CYP1A1/genetics , DNA Damage , DNA Glycosylases/genetics , DNA-Binding Proteins/genetics , Genetic Association Studies , Glutathione Transferase/genetics , Heterozygote , Humans , Inactivation, Metabolic/genetics , Male , Middle Aged , X-ray Repair Cross Complementing Protein 1 , Young AdultABSTRACT
RESUMEN Introducción: La exposición ocupacional a residuos de minería de carbón puede generar un amplio rango de lesiones en el ADN potencialmente asociadas a procesos carcinogénicos y a otras enfermedades laborales. Objetivo: Evaluar el efecto genotóxico en el ADN de individuos ocupacionalmente expuestos a residuos de minería de carbón mediante la determinación de la frecuencia de micronúcleos (MN) en linfocitos y el posible efecto modulador del polimorfismo de reparo de DNA hOGG1Ser326Cys (rs.1052133). Metodología: Fueron estudiados 74 trabajadores expuestos (GE) y 74 individuos no expuestos de la población general como grupo control (GC), a los cuales se le realizaron las técnicas moleculares test de MN y genotipificación. Resultados: El valor promedio de la frecuencia de MN para el GE fue 8.8±4.9, mientras que el valor promedio para el GC fue de 2.9±4.0. En relación al tiempo de exposición y la frecuencia de MN, individuos con más de 19 años de exposición presentaron una frecuencia de MN mayor (13 - 20 MN) que los individuos entre 2 y 18 años de exposición (2-12 MN). La frecuencia de MN por áreas de trabajo, reveló que los individuos involucrados en actividades de minería presentaron una mayor frecuencia (11.3±3.4), seguidos de los involucrados en embarque (9.0±5.3) y trabajadores del área de acarreo (8.3±5.3). La actividad moduladora del polimorfismo hOGG1Ser326Cys sobre la frecuencia de MN en individuos del GE, evidenció una menor frecuencia (8.32± 4.70) en individuos portadores del polimorfismo Ser/Cys, Cys/Cys con relación a individuos portadores del genotipo Ser/ Ser (9.06± 4.95). Estos hallazgos sobre la posible actividad protectora de hOGG1Ser326Cys en poblaciones expuestas proveen nuevos datos sobre el posible efecto protector de este polimorfismo. Conclusiones: Los datos muestran que la exposición a residuos de minería de carbón genera efectos genotóxicos, y que estos daños son modulados por variantes genotípicas de los genes de reparación involucrados en la remoción de daño oxidativo.
ABSTRACT Introduction: Occupational exposure to coal mining residues can generate a wide range of DNA lesions potentially associated with carcinogenic processes and other work related diseases. Objetive: To evaluate the genotoxic effects in the DNA of individuals occupationally exposed to coal mining residues considering micronucleus formation in lymphocytes (MN) as endpoints for genotoxicity and the possible modulating effect of DNA repair polymorphism hOGG1Ser326Cys (rs. 1052133). Methodology: The studied population was comprised by 74 exposed workers (GE) and 74 office non-exposed referents from general population as a control group (CG). The mean frequency of MN for GE was 8.8 ± 4.9, while for GC was 2.9 ± 4.0. In regard to time of exposure and MN frequency, individuals over 19 years of exposure presented a higher frequency of MN (13-20 MN) than individuals between 2 and 18 years of exposure (2-12 MN). Results: Frequency of MN discriminated by working areas, revealed that the individuals involved in mining activities had a higher MN frequency (11.3 ± 3.4), followed by those involved in embarking (9.0 ± 5.3) and coal carrying activities (8.3 ± 5.3). Modulatory activity of the hOGG1Ser326Cys polymorphism on MN frequency in GE individuals, showed a lower frequency (8.32 ± 4.70) in individuals carrying the polymorphism Ser / Cys, Cys / Cys compared to Ser / Ser (9.06 ± 4.95) carriers. These findings on the possible protective activity of hOGG1Ser326Cys in exposed populations provide new data to the increasing evidence about the protective role of this polymorphism. Conclusions: Data obtained showed that exposure to coal mining residues generates genotoxic effects that could be modulated by genetic variants of repair genes involved in removal of oxidative damage.