RESUMEN
Phosphonates (PHTs), organic compounds with a stable C-P bond, are widely distributed in nature. Glyphosate (GP), a synthetic PHT, is extensively used in agriculture and has been linked to various human health issues and environmental damage. Given the prevalence of GP, developing cost-effective, on-site methods for GP detection is key for assessing pollution and reducing exposure risks. We adopted Agrobacterium tumefaciens CHLDO, a natural GP degrader, as a host and the source of genetic parts for constructing PHT biosensors. In this bacterial species, the phn gene cluster, encoding the C-P lyase pathway, is regulated by the PhnF transcriptional repressor. We selected the phnG promoter, which displays a dose-dependent response to GP, to build a set of whole-cell biosensors. Through stepwise genetic optimization of the transcriptional cascade, we created a whole-cell biosensor capable of detecting GP in the 0.25-50 µM range in various samples, including soil and water.
Asunto(s)
Agrobacterium tumefaciens , Técnicas Biosensibles , Glicina , Glifosato , Organofosfonatos , Agrobacterium tumefaciens/genética , Técnicas Biosensibles/métodos , Glicina/análogos & derivados , Glicina/farmacología , Glicina/metabolismo , Organofosfonatos/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Familia de Multigenes , LiasasRESUMEN
Phosphonates are organic phosphorous (P) compounds frequently detected in the environment due to a very stable CP bond that render them relatively recalcitrant. Glyphosate [N-phosphonomethyl glycine] is the most widely used and best-known synthetic phosphonate, and one of the most concerning herbicides in the world today. Microbial degradation of glyphosate and organophosphonates in general, is the main dissipation mechanism operating in most environments. One microbial metabolic pathway in this process is the CP lyase pathway, entailing an enzymatic complex encoded by about 14 genes (the Phn operon). Our goal was to develop a quantitative polymerase chain reaction (qPCR) assay for a key enzyme, the CP lyase that breaks down the CP bond, via quantification of the codifying phnJ gene. The primers designed in this study fulfill the requirements for a successful qPCR assay, with high efficiency and sensitivity, as well as specific detection of the target sequence in a wide range of taxonomic groups. This is, to our knowledge, the first report of primers designed to target phnJ in both pure cultures and metagenomic DNA from different environmental sources. Direct quantification of phnJ may be a cost-effective proxy to determine glyphosate degradation potential in different matrixes.
Asunto(s)
Biodegradación Ambiental , Cartilla de ADN/genética , Glicina/análogos & derivados , Herbicidas/metabolismo , Liasas/genética , Organofosfonatos/metabolismo , Secuencia de Bases , Glicina/metabolismo , Liasas/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Análisis de Secuencia de ADN , GlifosatoRESUMEN
Glyphosate [N-phosphono-methylglycine (PMG)] is the most used herbicide worldwide, particularly since the development of transgenic glyphosate-resistant (GR) crops. Aminomethylphosphonic acid (AMPA) is the main glyphosate metabolite, and it may be responsible for GR crop damage upon PMG application. PMG degradation into AMPA has hitherto been reckoned mainly as a biological process, produced by soil microorganisms (bacteria and fungi) and plants. In this work, we use density functional calculations to identify the vibrational bands of PMG and AMPA in surface-enhanced Raman spectroscopy (SERS) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra experiments. SERS shows the presence of AMPA after glyphosate is deposited from aqueous solution on different metallic surfaces. AMPA is also detected in ATR-FTIR experiments when PMG interacts with metallic ions in aqueous solution. These results reveal an abiotic degradation process of glyphosate into AMPA, where metals play a crucial role.
Asunto(s)
Glicina/análogos & derivados , Metales/química , Organofosfonatos/química , Contaminantes del Suelo/química , Biodegradación Ambiental , Glicina/química , Glicina/metabolismo , Herbicidas/química , Herbicidas/metabolismo , Isoxazoles , Metales/metabolismo , Organofosfonatos/metabolismo , Contaminantes del Suelo/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría Raman , Tetrazoles , GlifosatoRESUMEN
Digitaria insularis biotypes resistant to glyphosate have been detected in Brazil. Studies were carried out in controlled conditions to determine the role of absorption, translocation, metabolism, and gene mutation as mechanisms of glyphosate resistance in D. insularis. The susceptible biotype absorbed at least 12% more (14)C-glyphosate up to 48 h after treatment (HAT) than resistant biotypes. High differential (14)C-glyphosate translocation was observed at 12 HAT, so that >70% of the absorbed herbicide remained in the treated leaf in resistant biotypes, whereas 42% remained in the susceptible biotype at 96 HAT. Glyphosate was degraded to aminomethylphosphonic acid (AMPA), glyoxylate, and sarcosine by >90% in resistant biotypes, whereas a small amount of herbicide (up to 11%) was degraded by the susceptible biotype up to 168 HAT. Two amino acid changes were found at positions 182 and 310 in EPSPS, consisting of a proline to threonine and a tyrosine to cysteine substitution, respectively, in resistant biotypes. Therefore, absorption, translocation, metabolism, and gene mutation play an important role in the D. insularis glyphosate resistance.
Asunto(s)
Digitaria/efectos de los fármacos , Digitaria/fisiología , Glicina/análogos & derivados , Resistencia a los Herbicidas , 3-Fosfoshikimato 1-Carboxiviniltransferasa/genética , 3-Fosfoshikimato 1-Carboxiviniltransferasa/metabolismo , Brasil , Glicina/farmacocinética , Glicina/farmacología , Glioxilatos/metabolismo , Herbicidas/farmacología , Isoxazoles , Mutación , Organofosfonatos/metabolismo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Sarcosina/metabolismo , Ácido Shikímico/análisis , Ácido Shikímico/metabolismo , Tetrazoles , GlifosatoRESUMEN
Acetaminophen (APAP) hepatotoxicity has been related with several cases of cirrhosis, hepatitis and suicides attempts. Notably, oxidative stress plays a central role in the hepatic damage caused by APAP and antioxidants have been tested as alternative treatment against APAP toxicity. In the present study, we observed the hepatoprotector activity of the diethyl-2-phenyl-2-tellurophenyl vinylphosphonate (DPTVP), an organotellurium compound with low toxicity and high antioxidant potential. When the dose of 200 mg/kg of APAP was used, we observed that all used doses of DPTVP were able to restore the -SH levels that were depleted by APAP. Furthermore, the increase in thiobarbituric acid reactive substances levels and in the seric alanine aminotransferase (ALT) activity and the histopathological alterations caused by APAP were restored to control levels by DPTVP (30, 50 and 100 µmol/kg). On the other hand, when the 300 mg/kg dose of APAP was used, DPTVP restored the non-proteic -SH levels and repaired the normal liver morphology of the intoxicated mice only at 50 µmol/kg. Our in vitro results point out to a scavenging activity of DPTVP against several reactive species, action that is attributed to its chemical structure. Taken together, our results demonstrate that the pharmacological action of DPTVP as a hepatoprotector is probably due to its scavenging activity related to its chemical structure.
Asunto(s)
Acetaminofén/efectos adversos , Citoprotección/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Hígado/efectos de los fármacos , Compuestos Organometálicos/farmacología , Organofosfonatos/farmacología , Animales , Relación Dosis-Respuesta a Droga , Depuradores de Radicales Libres/metabolismo , Radicales Libres/metabolismo , Hígado/patología , Masculino , Ratones , Compuestos Organometálicos/metabolismo , Organofosfonatos/metabolismoRESUMEN
The present study addressed the question whether ExoU, a Pseudomonas aeruginosa toxin with phospholipase A2 (PLA2) activity, may induce airway epithelial cells to overexpress tissue factor (TF) and exhibit a procoagulant phenotype. Cells from the human bronchial epithelial BEAS-2B line were infected with an ExoU-producing P. aeruginosa strain, pre-treated or not with the cytosolic PLA2 inhibitor methylarachidonyl fluorophosphate (MAFP), or with two ExoU-deficient mutants. Control noninfected and infected cells were assessed for the expression of: 1) TF mRNA by RT-PCR; 2) cell-associated TF by enzyme immunoassay and flow cytometry; 3) procoagulant activity by a colorimetric assay; and 4) microparticle-associated TF by flow cytometry. An enzyme immunoassay was also used to assess cell-associated TF in lung extracts from mice infected intratracheally with ExoU-producing and -deficient bacteria. Cells infected with the wild-type bacteria had higher levels of TF mRNA, cell-associated TF expression, procoagulant activity and released microparticle-associated TF than cells infected with the mutants. Bacterial treatment with MAFP significantly reduced the expression of TF by infected cells. Lung samples from mice infected with the wild-type bacteria exhibited higher levels of cell-associated TF and procoagulant activity. The present results demonstrate that ExoU may contribute to the pathogenesis of lung injury by inducing a tissue factor-dependent procoagulant activity in airway epithelial cells.
Asunto(s)
Proteínas Bacterianas/fisiología , Bronquios/microbiología , Coagulantes/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Proteínas Bacterianas/metabolismo , Bronquios/citología , Citosol/metabolismo , Femenino , Humanos , Ratones , Modelos Biológicos , Mutación , Organofosfonatos/metabolismo , Fosfolipasas A2/metabolismo , Infecciones por Pseudomonas/diagnóstico , Tromboplastina/metabolismoRESUMEN
The glutamatergic system is an important target in many neurodegenerative diseases and for several neurotoxic drugs. Organotellurium compounds are often very good free radical scavengers' agents. Recently, we reported that diethyl-2-phenyl-2-tellurophenyl vinylphosphonate is a compound with low toxicity in vitro and in vivo, as well as also possesses antioxidant activity against iron-induced lipid peroxidation. The aim of this study was to evaluate in vitro the antioxidant and mitochondrial protective effect of this organotellurium compound against quinolinic acid (QA) and sodium nitroprusside (SNP), and to evaluate the in vitro actions of this organotellurium compound in the glutamatergic system in brain of rats. We observed that the telluro vinylphosphonate possess an antioxidant activity against QA and SNP at micromolar concentrations. When tested at antioxidant concentrations (from 2 to 10muM), the compound does not affect the mitochondrial viability and [3H]glutamate uptake in slices from cerebral cortex, hippocampus and striatum, [3H]glutamate release from synaptosomal preparations and [3H]glutamate binding in membrane preparation. Our data suggest that the telluro vinylphosphonate act as an antioxidant in the central nervous system in vitro with no effects on the glutamatergic system; nevertheless more studies in different models of brain injury must be performed in order to corroborate our findings.
Asunto(s)
Antioxidantes , Encéfalo/metabolismo , Neurotoxinas/metabolismo , Organofosfonatos , Telurio , Compuestos de Vinilo , Animales , Antioxidantes/química , Antioxidantes/metabolismo , Encéfalo/anatomía & histología , Ácido Glutámico/metabolismo , Peroxidación de Lípido , Mitocondrias/metabolismo , Donantes de Óxido Nítrico/metabolismo , Nitroprusiato/metabolismo , Organofosfonatos/química , Organofosfonatos/metabolismo , Ácido Quinolínico/metabolismo , Ratas , Ratas Wistar , Sinaptosomas/metabolismo , Telurio/química , Telurio/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Compuestos de Vinilo/química , Compuestos de Vinilo/metabolismoRESUMEN
The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans is of great importance in biomining operations. During the bioleaching of ores, microorganisms are subjected to a variety of environmental stresses and to the limitations of some nutrients, such as inorganic phosphate (P(i)), which is an essential component for all living cells. Although the primary source of phosphorus for microorganisms is P(i), some bacteria are also able to metabolize P(i) esters (with a C-O-P bond) and phosphonates (with a very inert C-P bond). By using bioinformatic analysis of genomic sequences of the type strain of A. ferrooxidans (ATCC 23270), we found that as part of a Pho regulon, this bacterium has a complete gene cluster encoding C-P lyase, which is the main bacterial enzyme involved in phosphonate (Pn) degradation in other microorganisms. A. ferrooxidans was able to grow in the presence of methyl-Pn or ethyl-Pn as an alternative phosphorus source. Under these growth conditions, a great reduction in inorganic polyphosphate (polyP) levels was seen compared with the level for cells grown in the presence of P(i). By means of reverse transcription-PCR (RT-PCR), DNA macroarrays, and real-time RT-PCR experiments, it was found that A. ferrooxidans phn genes were cotranscribed and their expression was induced when the microorganism was grown in methyl-Pn as the only phosphorus source. This is the first report of phosphonate utilization in a chemolithoautotrophic microorganism. The existence of a functional C-P lyase system is a clear advantage for the survival under P(i) limitation, a condition that may greatly affect the bioleaching of ores.
Asunto(s)
Acidithiobacillus/genética , Liasas/genética , Operón , Organofosfonatos/metabolismo , Fosfatos/metabolismo , Acidithiobacillus/crecimiento & desarrollo , Acidithiobacillus/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Liasas/metabolismo , Viabilidad Microbiana/genética , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Regulón/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The availability of Roundup Ready (RR) varieties of soybean has increased the use of glyphosate for weed control in Argentina. Glyphosate [(N-phosphonomethyl)glycine] is employed for the eradication of previous crop vegetation and for weed control during the soybean growing cycle. Its action is effective, and low environmental impact has been reported so far. No residues have been observed in soil or water, either of glyphosate or its metabolite, AMPA (aminomethylphosphonic acid). The objective of this work was to monitor glyphosate and AMPA residues in soybean plants and grains in field crops in Santa Fe Province, Argentina. Five sites were monitored in 1997, 1998 and 1999. Individual soybean plants were sampled from emergence to harvest, dried and ground. Analysis consisted in residue extraction with organic solvents and buffers, agitation, centrifugation, clean-up and HPLC with UV detection. In soybean leaves and stems, glyphosate residues ranged from 1.9 to 4.4 mg kg(-1) and from 0.1 to 1.8 mg kg(-1) in grains. Higher concentrations were detected when glyphosate was sprayed several times during the crop cycle, and when treatments approached the flowering stage. AMPA residues were also detected in leaves and in grains, indicating metabolism of the herbicide.