RESUMEN
Glyphosate, the world's most widely used herbicide, has a low toxicity rating despite substantial evidence of adverse health effects. Furthermore, glyphosate-based formulations (GBFs) contain several other chemicals, some of which are known to be harmful. Additionally, chronic, and acute exposure to GBFs among rural workers may lead to health impairments, such as neurodegenerative diseases and cancer. P53 is known as a tumor suppressor protein, acting as a key regulator of the cellular response to stress and DNA damage. Therefore, mutations in the TP53 gene, which encodes p53, are common genetic alterations found in various types of cancer. Therefore, this study aimed to evaluate the cytotoxicity and genotoxicity of GBF in two glioblastoma cell lines: U87MG (TP53-proficient) and U251MG (TP53-mutant). Additionally, the study aimed to identify the main proteins involved in the response to GBF exposure using Systems Biology in a network containing p53 and another network without p53. The MTT assay was used to study the toxicity of GBF in the cell lines, the clonogenic assay was used to investigate cell survival, and the Comet Assay was used for genotoxicity evaluation. For data analysis, bioinformatics tools such as String 12.0 and Stitch 5.0 were applied, serving as a basis for designing binary networks in the Cytoscape 3.10.1 program. From the in vitro test analyses, it was observed a decrease in cell viability at doses starting from 10â¯ppm. Comet Assay at concentrations of 10â¯ppm and 30â¯ppm for the U251MG and U87MG cell lines, respectively observed DNA damage. The network generated with systems biology showed that the presence of p53 is important for the regulation of biological processes involved in genetic stability and neurotoxicity, processes that did not appear in the TP53-mutant network.
RESUMEN
Callingcard Vine (Entada polystachya (L.) DC. var. polystachya - Fabaceae) is a common plant in coastal thickets from western Mexico through Central America to Colombia and Brazil, especially in Amazon biome. It has been popularly used as a urinary burning reliever and diuretic. However, the plant chemical constituents are poorly understood and Entada spp. genotoxic potential have not been previously investigated. In the present study we determined the chemical composition of the aqueous E. polystachya crude seed extract (EPCSE) and evaluated the cytotoxic, genotoxic and mutagenic properties of EPCSE in Salmonella typhimurium and Chinese hamster ï¬broblast (V79) cells. Cytotoxic activity was also evaluated in tumor cell lines (HT29, MCF7 and U87) and non-malignant cells (MRC5). The chemical analysis by High Resolution Mass Spectrometry (HRMS) of EPCSE indicated the presence of saponin and chalcone. The results of the MTT and clonal survival assays suggest that EPCSE is cytotoxic to V79 cells. Survival analysis showed higher IC50 in non-tumor compared with tumor cell lines. EPCSE showed induction of DNA strand breaks as revealed by the alkaline comet assay and micronucleus test. Using the modified comet assay, it was possible to detect the induction of oxidative DNA base damage by EPCSE in V79 cells. Consistently, the extract induced increase lipid peroxidation (TBARS), superoxide dismutase (SOD) and catalase (CAT) activities in V79 cells. In addition, EPCSE induced mutations in S. typhimurium TA98 and TA100 strains, confirming a mutagenic potential. Taken together, our results suggest that EPCSE is cytotoxic and genotoxic to V79 cells and mutagenic to S. typhimurium. These properties can be related to the pro-oxidant ability of the extract and induction of DNA lesions. Additionally, EPCSE could inhibit the growth of tumor cells, especially human colorectal adenocarcinoma (HT29) cell line, and can constitute a possible source of antitumor natural agents.
Asunto(s)
Antineoplásicos , Fabaceae , Cricetinae , Animales , Humanos , Mutágenos/toxicidad , Daño del ADN , Cricetulus , Ensayo Cometa , Línea Celular Tumoral , Extractos Vegetales/toxicidad , ADNRESUMEN
Diphenyl ditelluride (DPDT) is an organotellurium (OT) compound with pharmacological properties, including antioxidant, antigenotoxic and antimutagenic activities when applied at low concentrations. However, DPDT as well as other OT compounds also show cytotoxicity against mammalian cells when treatments occur at higher drug concentrations. Considering that the underlying mechanisms of toxicity of DPDT against tumor cells have been poorly explored, the objective of our study was to investigate the effects of DPDT against both human cancer and non-tumorigenic cells. As a model, we used the colonic HCT116 cancer cells and the MRC5 fibroblasts. Our results showed that DPDT preferentially targets HCT116 cancer cells when compared to MRC5 cells with IC50 values of 2.4 and 10.1 µM, respectively. This effect was accompanied by the induction of apoptosis and a pronounced G2/M cell cycle arrest in HCT116 cells. Furthermore, DPDT induces DNA strand breaks at concentrations below 5 µM in HCT116 cells and promotes the occurrence of DNA double strand breaks mostly during S-phase as measured by γ-H2AX/EdU double staining. Finally, DPDT forms covalent complexes with DNA topoisomerase I, as observed by the TARDIS assay, with a more prominent effect observed in HCT116 than in MRC5 cells. Taken together, our results show that DPDT preferentially targets HCT116 colon cancer cells likely through DNA topoisomerase I poisoning. This makes DPDT an interesting molecule for further development as an anti-proliferative compound in the context of cancer.
Asunto(s)
Neoplasias del Colon , ADN-Topoisomerasas de Tipo I , Animales , Humanos , Células HCT116 , ADN-Topoisomerasas de Tipo I/metabolismo , Apoptosis , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , ADN , Mamíferos/metabolismoRESUMEN
The sodium valproate has been largely used as an anti-epilepsy drug and, recently, as a putative drug in cancer therapy. However, the treatment with sodium valproate has some adverse effects. In this sense, more effective and secure complexes than sodium valproate should be explored in searching for new active drugs. This study aims to evaluate the cytotoxicity of sodium valproate, mixed ternary mononuclear Cu(II) complexes based on valproic acid (VA) with 1,10-phenanthroline (Phen) or 2,2'- bipyridine (Bipy) ligands - [Cu2(Valp)4], [Cu(Valp)2Phen] and [Cu(Valp)2Bipy] - in yeast Saccharomyces cerevisiae, proficient or deficient in different repair pathways, such as base excision repair (BER), nucleotide excision repair (NER), translesion synthesis (TLS), DNA postreplication repair (PRR), homologous recombination (HR) and non-homologous end-joining (NHEJ). The results indicated that the Cu(II) complexes have higher cytotoxicity than sodium valproate in the following order: [Cu(Valp)2Phen] > [Cu(Valp)2Bipy] > [Cu2(Valp)4] > sodium valproate. The treatment with Cu(II) complexes and sodium valproate induced mutations in S. cerevisiae. The data indicated that yeast strains deï¬cient in BER (Ogg1p), NER (complex Rad1p-Rad10p) or TLS (Rev1p, Rev3p and Rad30p) proteins are associated with increased sensitivity to sodium valproate. The BER mutants (ogg1Δ, apn1Δ, rad27Δ, ntg1Δ and ntg2Δ) showed increased sensitivity to Cu(II) complexes. DNA damage induced by the complexes requires proteins from NER (Rad1p and Rad10p), TLS (Rev1p, Rev3p and Rad30p), PRR (Rad6 and Rad18p) and HR (Rad52p and Rad50p) for efficient repair. Therefore, Cu(II) complexes display enhanced cytotoxicity when compared to the sodium valproate and induce distinct DNA lesions, indicating a potential application as cytotoxic agents.
Asunto(s)
Cobre/farmacología , Reparación del ADN/efectos de los fármacos , Preparaciones Farmacéuticas/administración & dosificación , Fenantrolinas/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Ácido Valproico/farmacología , ADN/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Ligandos , Mutación/efectos de los fármacos , Recombinación Genética/efectos de los fármacosRESUMEN
Cell-based therapy provides a novel strategy to restore lost neurons or modulate the degenerating microenvironment in amyotrophic lateral sclerosis (ALS). This study verified the therapeutic potential of bone marrow mononuclear cells (BMMCs) in SOD1G93A mice. BMMCs were obtained from enhanced green fluorescent protein (EGFP) transgenic C57BL/6 mice (EGFPBMMCs) or from SOD1G93A transgenic mice (mSOD1BMMCs) and given to mice at the pre-symptomatic or late symptomatic stage. Survival, body weight and motor performance data were recorded. DNA integrity was evaluated using the alkaline comet assay. The spinal cords were collected to assess motoneuron preservation and cell migration. EGFPBMMCs and mSOD1BMMCs transplantation to pre-symptomatic SOD1G93A mice prolonged survival and delayed disease progression. The effects were more significant for the EGFPBMMC-transplanted mice. In late symptomatic mice, EGFPBMMCs promoted a discrete increase in survival, without other clinical improvements. DNA from EGFPBMMCs and mSOD1BMMCs was found in the spinal cords of transplanted animals. DNA damage was not modified by BMMCs in any of the studied groups. Despite positive behavioral effects observed in our study, the limited results we observed for late transplanted mice call for caution before clinical application of BMMCs in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/terapia , Trasplante de Médula Ósea , Neuronas/patología , Esclerosis Amiotrófica Lateral/mortalidad , Esclerosis Amiotrófica Lateral/patología , Animales , Muerte Celular , Supervivencia Celular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones Transgénicos , Neuronas Motoras/patología , Superóxido Dismutasa/genética , TransgenesRESUMEN
The organoselenium compound, dicholesteroyl diselenide (DCDS) is a structural analogue of diphenyl diselenide (DPDS) and may be considered as a promising antioxidant drug in vivo. Nevertheless, little is known about the toxicological properties of DCDS. In the present study we evaluated the cytotoxic, genotoxic and mutagenic properties of DCDS in Chinese hamster lung fibroblasts (V79) and in strains of the yeast Saccharomyces cerevisiae, proficient and deficient in several DNA-repair pathways. The results with V79 cells show that DCDS induced cytotoxicity, GSH depletion and elevation of lipid peroxidation at lower concentrations than did DPDS. DCDS also generated single- and double-strand DNA breaks in V79 cells, both in the presence and in the absence of metabolic activation, as revealed by alkaline and neutral comet assays. Moreover, the induction of oxidative DNA base-damage was demonstrated by means of a modified comet assay with formamidopyrimidine-DNA glycosylase and endonuclease III. Treatment with DCDS also induced micronucleus formation in V79 cells as well as point and frame-shift mutations in a haploid wild-type strain of S. cerevisiae. Yeast mutants defective in base excision-repair proteins were the most sensitive to DCDS. Pre-incubation with N-acetylcysteine reduced DCDS's oxidative, genotoxic and mutagenic effects in yeast and in V79 cells. Our findings indicate that the presence of cholesteroyl substituents in DCDS results in elevation of its cytotoxic and genotoxic potential compared with that of DPDS in yeast and in V79 cells. However, due to dose-dependent contrasting behaviour of organoselenium compounds and differences in their toxicity in in vitro and in vivo systems, further studies are needed in order to establish the non-toxic concentration range for treatment in mammals.
Asunto(s)
Colesterol/análogos & derivados , Daño del ADN , Micronúcleos con Defecto Cromosómico/inducido químicamente , Mutágenos/toxicidad , Compuestos de Organoselenio/toxicidad , Saccharomyces cerevisiae/efectos de los fármacos , Animales , Biomarcadores/análisis , Línea Celular , Supervivencia Celular/efectos de los fármacos , Colesterol/toxicidad , Ensayo Cometa , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Mutación del Sistema de Lectura/efectos de los fármacos , Humanos , Peroxidación de Lípido/efectos de los fármacos , Pruebas de Micronúcleos , Estrés Oxidativo/efectos de los fármacos , Saccharomyces cerevisiae/genética , Pruebas de Toxicidad/métodosRESUMEN
MutY is a glycosylase known for its role in DNA base excision repair (BER). It is critically important in the prevention of DNA mutations derived from 7,8-dihydro-8-oxoguanine (8-oxoG), which are the major lesions resulting from guanine oxidation. MutY has been described as a DNA repair enzyme in the GO system responsible for removing adenine residues misincorporated in 8-oxoG:A mispairs, avoiding G:C to T:A mutations. Further studies have shown that this enzyme binds to other mispairs, interacts with several enzymes, avoids different transversions/transitions in DNA, and is involved in different repair pathways. Additional activities have been reported for MutY, such as the repair of replication errors in newly synthesized DNA strands through its glycosylase activity. Moreover, MutY is a highly conserved enzyme present in several prokaryotic and eukaryotic organisms. MutY defects are associated with a hereditary colorectal cancer syndrome termed MUTYH-associated polyposis (MAP). Here, we have reviewed the roles of MutY in the repair of mispaired bases in DNA as well as its activities beyond the GO system.
Asunto(s)
ADN Glicosilasas/fisiología , Guanosina/análogos & derivados , Mutagénesis/genética , Animales , Reparación del ADN/genética , Guanosina/metabolismo , HumanosRESUMEN
The clinical manifestations of Lonomia obliqua caterpillar envenomation are systemic hemorrhage and acute kidney injury. In an effort to better understand the physiopathological mechanisms of envenomation, a rat model was established to study systemic tissue damage during L. obliqua envenomation. An array of acute venom effects was characterized, including biochemical, hematological, histopathological, myotoxic and genotoxic alterations. Rapid increases in serum alanine and aspartate transaminases, γ-glutamyl transferase, lactate dehydrogenase, hemoglobin, bilirubin, creatinine, urea and uric acid were observed, indicating that intravascular hemolysis and liver and kidney damage had occurred. Treatment with a specific antivenom (antilonomic serum) for up to 2 h post-venom injection neutralized the biochemical alterations. However, treatment after 6 h post-venom injection failed to normalize all biochemical parameters, despite its efficacy in reversing coagulation dysfunction. The hematological findings were consistent with hemolytic anemia and neutrophilic leukocytosis. The histopathological alterations were mainly related to hemorrhage and inflammation in the subcutaneous tissue, lung, heart and kidneys. Signs of congestion and hemosiderosis were evident in the spleen, and hemoglobin and/or myoglobin casts were also detected in the renal tubules. Increased levels of creatine kinase and creatine kinase-MB were correlated with the myocardial necrosis observed in vivo and confirmed the myotoxicity detected in vitro in isolated extensor digitorum longus muscles. Significant DNA damage was observed in the kidneys, heart, lung, liver and lymphocytes. The majority of the DNA lesions in the kidney were due to oxidative damage. The results presented here will aid in understanding the pathology underlying Lonomia's envenomation.
Asunto(s)
Venenos de Artrópodos/toxicidad , Mordeduras y Picaduras de Insectos/fisiopatología , Mariposas Nocturnas/química , Animales , Antivenenos/farmacología , Venenos de Artrópodos/química , Coagulación Sanguínea/efectos de los fármacos , Cardiotoxinas/química , Cardiotoxinas/toxicidad , Daño del ADN/efectos de los fármacos , Hemorragia/tratamiento farmacológico , Hemorragia/fisiopatología , Mordeduras y Picaduras de Insectos/tratamiento farmacológico , Riñón/efectos de los fármacos , Riñón/patología , Larva/química , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Ratas , Ratas WistarRESUMEN
Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5-10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Inestabilidad Genómica/efectos de los fármacos , Micronutrientes/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo/farmacología , HumanosRESUMEN
Patulin, a known mycotoxin, is considered a significant contaminant in apples, apple-derived products and feeds. This study investigated the genotoxic effects of patulin in multiple organs (brain, kidney, liver and urinary bladder) of mice using an in vivo comet assay. We assessed the mechanism underlying this genotoxicity by measuring the GSH content and the thiobarbituric acid-reactive species (TBARS) level. Male CF-1 mice were given 1.0-3.75 mg/kg patulin intraperitoneally. The effect of patulin was dose-dependent and the highest patulin dose induced DNA strand breaks in the brain (damage index, DI, in hippocampus increased from 36.2 in control animals to 127.5), liver (44.3-138.4) and kidneys (31.5-99); decreased levels of GSH (hippocampus--from 46.9 to 18.4 nmol/mg protein); and an increase in lipid peroxidation (hippocampus--from 5.8 to 20.3 MDA equivalents/mg protein). This finding establishes an interrelationship between the pro-oxidant and genotoxic effects of patulin. Pre-treatment administration of N-acetyl-cysteine reduced patulin-induced DNA damage (hippocampus--DI from 127.5 to 39.8) and lipid peroxidation (hippocampus--20.3 to 12.8 MDA equivalents/mg protein) by restoring cellular GSH levels, reinforcing the positive relationship between patulin-induced GSH depletion and DNA damage caused by systemic administration of this mycotoxin.
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Daño del ADN/efectos de los fármacos , Patulina/toxicidad , Acetilcisteína/farmacología , Animales , Ensayo Cometa , Relación Dosis-Respuesta a Droga , Glutatión/metabolismo , Hipocampo/efectos de los fármacos , Riñón/efectos de los fármacos , Masculino , Ratones , Estructura Molecular , Estrés Oxidativo , Patulina/administración & dosificación , Patulina/químicaRESUMEN
Diphenyl ditelluride (DPDT) is a potential prototype for the development of novel biologically active molecules. Thus, it is important to evaluate the toxic effects of this compound. In the present study, we evaluated the cytotoxic, genotoxic and mutagenic properties of DPDT in Chinese hamster fibroblast (V79) cells, in strains of the yeast Saccharomyces cerevisiae both proficient and deficient in several DNA repair pathways and in Salmonella typhimurium. DPDT induced frameshift mutations in both S.typhimurium and a haploid wild-type strain of S.cerevisiae. Mutants of S.cerevisiae defective in base excision repair and recombinational repair were more sensitive to DPDT. The results of a lactate dehydrogenase leakage assay suggest that DPDT is cytotoxic to V79 cells. At cytotoxic concentrations, this compound increased thiobarbituric reactive species levels and decreased the glutathione:GSSH ratio in yeast and V79 cells. DPDT generated single- and double-strand DNA breaks in V79 cells, both with and without metabolic activation, as revealed by alkaline and neutral comet assays. Moreover, an induction of oxidative DNA base damage was indicated by a modified comet assay using formamidopyrimidine DNA glycosylase and endonuclease III. Treatment with DPDT also induced micronucleus formation in V79 cells. Pre-incubation with N-acetylcysteine reduced DPDT's oxidative, genotoxic and mutagenic effects in yeast and V79 cells. Our results suggest that the toxic and mutagenic properties of DPDT may stem from its ability to disturb the redox balance of the cell, which leads to oxidative stress and the induction of DNA damage.
Asunto(s)
Derivados del Benceno/toxicidad , Modelos Biológicos , Mutágenos/toxicidad , Compuestos Organometálicos/toxicidad , Animales , Derivados del Benceno/química , Biomarcadores/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Cricetinae , Cricetulus , Daño del ADN , L-Lactato Deshidrogenasa/metabolismo , Viabilidad Microbiana/efectos de los fármacos , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Pruebas de Mutagenicidad , Mutágenos/química , Compuestos Organometálicos/química , Estrés Oxidativo/efectos de los fármacos , Mutación Puntual/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Salmonella/citología , Salmonella/efectos de los fármacos , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
The nitroreductase family is comprised of a group of FMN- or FAD-dependent enzymes that are able to metabolize nitrosubstituted compounds using the reducing power of NAD(P)H. These nitroreductases can be found in bacterial species and, to a lesser extent, in eukaryotes. There is little information on the biochemical functions of nitroreductases. Some studies suggest their possible involvement in the oxidative stress response. In the yeast Saccharomyces cerevisiae, two nitroreductase proteins, Frm2p and Hbn1p, have been described. While Frm2p appears to act in the lipid signalling pathway, the function of Hbn1p is completely unknown. In order to elucidate the functions of Frm2p and Hbn1p, we evaluated the sensitivity of yeast strains, proficient and deficient in both oxidative stress proteins, for respiratory competence, antioxidant-enzyme activities, intracellular reactive oxygen species (ROS) production and lipid peroxidation. We found reduced basal activity of superoxide dismutase (SOD), ROS production, lipid peroxidation and petite induction and higher sensitivity to 4-nitroquinoline-oxide (4-NQO) and N-nitrosodiethylamine (NDEA), as well as higher basal activity of catalase (CAT) and glutathione peroxidase (GPx) and reduced glutathione (GSH) content in the single and double mutant strains frm2Delta and frm2Delta hbn1Delta. These strains exhibited less ROS accumulation and lipid peroxidation when exposed to peroxides, H(2)O(2) and t-BOOH. In summary, the Frm1p and Hbn1p nitroreductases influence the response to oxidative stress in S. cerevisae yeast by modulating the GSH contents and antioxidant enzymatic activities, such as SOD, CAT and GPx.
Asunto(s)
Nitrorreductasas/metabolismo , Estrés Oxidativo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Antioxidantes/metabolismo , Catalasa/metabolismo , Dietilnitrosamina/metabolismo , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Peroxidación de Lípido , Mutación , Nitrorreductasas/genética , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Superóxido Dismutasa/metabolismoRESUMEN
The nitroreductase family comprises a group of FMN- or FAD-dependent and NAD(P)H-dependent enzymes able to metabolize nitrosubstituted compounds. The nitroreductases are found within bacterial and some eukaryotic species. In eukaryotes, there is little information concerning the phylogenetic position and biochemical functions of nitroreductases. The yeast Saccharomyces cerevisiae has two nitroreductase proteins: Frm2p and Hbn1p. While Frm2p acts in lipid signaling pathway, the function of Hbn1p is unknown. In order to elucidate the function of Frm2p/Hbn1p and the presence of homologous sequences in other prokaryotic and eukaryotic species, we performed an in-depth phylogenetic analysis of these proteins. The results showed that bacterial cells have Frm2p/Hbn1p-like sequences (termed NrlAp) forming a distinct clade within the fungal Frm2p/Hbn1p family. Hydrophobic cluster analysis and three-dimensional protein modeling allowed us to compare conserved regions among NrlAp and Frm2/Hbn1p proteins. In addition, the possible functions of bacterial NrlAp and fungal Frm2p/Hbn1p are discussed.
Asunto(s)
Proteínas Bacterianas/clasificación , Proteínas Bacterianas/metabolismo , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/metabolismo , Nitrorreductasas/clasificación , Nitrorreductasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Fenómenos Químicos , Química Física , Biología Computacional , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Modelos Moleculares , Nitrorreductasas/química , Nitrorreductasas/genética , Filogenia , Estructura Terciaria de Proteína , Homología Estructural de ProteínaRESUMEN
The Guaíba Basin is a source of drinking water for Porto Alegre (RS, Brazil). The water from this basin receives industrial, urban, and rural waste from many sources. The mussel species Limnoperna fortunei was chosen based on population data, distribution, and sensitivity. Previous tests with comet assay and micronuclei frequency in this freshwater mussel have shown to be successful in biomonitoring studies. The aim of this study was to evaluate the genotoxic contamination of the Guaíba Lake Hydrographic Region, through the determination of damage by the micronuclei and comet assays in L. fortunei (golden mussel). Nine sampling sites were evaluated in three different seasons: five sites in the mouths of the main rivers that flow into Guaíba lake; one site at the mouth of a stream; one major site of sewage discharge; two sites at Guaíba lake, near a sewage discharge; and the control site in a preservation area. DNA damage was detected by the single cell gel assay, as well as the frequency of micronuclei in hemocytes of mussels exposed under laboratory conditions for 7 days to water and sediment samples. Significant results were found in different seasons in almost all sampling sites (P<0.05, ANOVA Dunnet's test). Most of the positive results were found in samples affected mainly by urban effluents. It was possible to observe that there was a weak relation between mutagenic and genotoxic responses and mussels inorganic elements contents. Seasonal variation was observed at different sampling sites, but always indicating a huge contamination near urban sewage discharge. These results are consistent with previous studies, allowing us to infer that urban contamination is the biggest problem in this region. It is also possible to infer that L. fortunei is a good sentinel organism for the Guaíba Basin.
Asunto(s)
Ensayo Cometa/métodos , Daño del ADN , Pruebas de Micronúcleos/métodos , Animales , Bivalvos , Brasil , Agua Dulce/análisis , Sustancias Peligrosas/toxicidad , Estaciones del Año , Contaminación Química del Agua/análisisRESUMEN
The development of methodologies for biomonitoring freshwater ecosystems is of particular relevance in view of the serious problem of aquatic environmental pollution. The mussel species Limnoperna fortunei (golden mussel) was chosen to be tested as a biomonitor organism based on its population data and distribution. L. fortunei individuals were exposed to UV radiation in vitro, and in vivo to pentachlorophenol (PCP) and copper sulphate (CuSO(4)), with the aim of standardizing comet assay and micronucleus test methodologies and evaluating the potential of this organism as a biomonitor. Haemolymph cells immobilized in agarose on slides exposed to UV radiation showed a dose-response relationship with maximum damage at 4.2 J/m(2). For the chemical tests, individuals were exposed for 2h for the comet assay and 24 and 48 h for the micronucleus test. A dose-response relationship was observed for both chemicals. 3x10(-5) M CuSO(4) induced high genotoxicity, also producing some toxicity after 48 h of exposure. PCP induced maximum damage in both assays at 150 µg/L. Individuals exposed to PCP showed 100% repair 2 h after the exposure period, as assessed by the comet assay. Exposure to an environmental sample over 7 days confirmed the mussel sensitivity to water contaminants, detected both by the comet assay and the micronucleus test. The results allow us to suggest the golden mussel as a potential biomonitor organism.