RESUMO
Organophosphate insecticides (OPs) continue to be an important class of agrochemicals used in modern agriculture worldwide. Even though these pesticides persist in the environment for a relatively short time, they show a high acute toxicity that may represent a serious hazard for wildlife. Sub-lethal effects on non-target species are a focus in pest management programs and should be used as biomarkers. Cholinesterases (ChEs) are the most used biomarker of OP exposure in vertebrate and invertebrate species. However, the combined monitoring of ChE and carboxylesterase (CE) activities may provide a more useful indication of exposure and effect of the organisms. The objective of the present work was to find the most sensitive combination of enzyme, substrate, tissue and capacity to recovery of B-esterases in the freshwater gastropod Planorbarius corneus exposed to the OP azinphos-methyl. For this purpose, ChE and CE activities in different tissues of P. corneus (head-foot, pulmonary region, digestive gland, gonads and whole organism soft tissue) were studied. Measurements of ChE activity were performed using three substrates: acetylthiocholine, propionylthiocholine and butyrylthiocholine and CE activity using four different substrates: p-nitrophenyl acetate, p-nitrophenyl butyrate, 1-naphthyl acetate, and 2-naphthyl acetate in control and exposed organisms. Finally, the recovery rates of ChE and CE activities following 48h exposure to azinphos-methyl were analyzed. Our results show a preference for acetylthiocholine as substrate, a high inhibition with eserine (a selective ChE inhibitor) and inhibition with excess of substrate in all the analyzed tissues. The highest ChE and CE activity was found in the pulmonary region and in the digestive gland, respectively. The highest CE Vmax was obtained with 1 and 2-naphthyl acetate in all the tissues. CEs were more sensitive than ChE to azinphos-methyl exposure. The highest sensitivity was found using p-nitrophenyl acetate and butyrate as substrates. On the other hand, CEs of the digestive gland and the pulmonary region were more sensitive than CEs of the whole organism soft tissue. Regarding the recovery of enzyme activities after 48h exposure, ChE and CEs with p-nitrophenyl butyrate reached control values after 14days in the digestive gland and after 21days in the pulmonary region. Our results show marked differences in P. corneus basal ChE and CE activities depending on substrates and the tissue. Also, both tissue-dependent and substrate-dependent variations in sensitivity to azinphos-methyl exposure and recovery were obtained. CEs measured with p-nitrophenyl butyrate in the pulmonary region were the best combination to be used as biomarker of exposure to azinphos-methyl due to their sensitivity and low recovery capacity. Environmental concentrations of azinphos-methyl inhibited CE activity so they could be used as effective biomarkers of aquatic contamination.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Colinesterases/metabolismo , Inseticidas/toxicidade , Caramujos/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Azinfos-Metil/toxicidade , Biomarcadores/metabolismo , Butiratos/metabolismo , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Colinesterases/química , Concentração Inibidora 50 , Cinética , Nitrofenóis/metabolismo , Compostos Organofosforados/toxicidade , Caramujos/enzimologia , Caramujos/metabolismo , Especificidade por SubstratoRESUMO
Organophosphorus-induced delayed neuropathy (OPIDN) is a central and peripheral distal axonopathy characterized by ataxia and paralysis. Trichlorfon and acephate are two organophosphorus compounds (OPs) used worldwide as insecticide and which cause serious effects to non-target species. Despite that, the neuropathic potential of these OPs remains unclear. The present study addressed the neurotoxic effects and the neuropathic potential of trichlorfon and acephate in SH-SY5Y human neuroblastoma cells, by evaluating inhibition and aging of neuropathy target esterase (NTE), inhibition of acetylcholinesterase (AChE), neurite outgrowth, cytotoxicity and intracellular calcium. Additionally, the effects observed were compared to those of two well-studied OPs: mipafox (known as neuropathic) and paraoxon (known as non-neuropathic). Trichlorfon and mipafox presented the lowest percentage of reactivation of inhibited NTE and the lowest ratio IC50 NTE/IC50 AChE. Moreover, they caused inhibition and aging of at least 70% of the activity of NTE at sub-lethal concentrations. All these effects have been associated with induction of OPIDN. When assayed at these concentrations, trichlorfon and mipafox reduced neurite outgrowth and increased intracellular calcium, events implicated in the development of OPIDN. Acephate caused effects similar to those caused by paraoxon (non-neuropathic OP) and was only able to inhibit 70% of NTE activity at lethal concentrations. These findings suggest that trichlorfon is potentially neuropathic, whereas acephate is not.
Assuntos
Inseticidas/toxicidade , Compostos Organotiofosforados/toxicidade , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Fosforamidas/toxicidade , Triclorfon/toxicidade , Cálcio/metabolismo , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Caspase 3/metabolismo , Linhagem Celular , Inibidores da Colinesterase/toxicidade , Ativação Enzimática/efeitos dos fármacos , Humanos , Técnicas In Vitro , Neuritos/efeitos dos fármacosRESUMO
This work evaluated the potential of the isoforms of methamidophos to cause organophosphorus-induced delayed neuropathy (OPIDN) in hens. In addition to inhibition of neuropathy target esterase (NTE) and acetylcholinesterase (AChE), calpain activation, spinal cord lesions and clinical signs were assessed. The isoforms (+)-, (±)- and (-)-methamidophos were administered at 50mg/kg orally; tri-ortho-cresyl phosphate (TOCP) was administered (500mg/kg, po) as positive control for delayed neuropathy. The TOCP hens showed greater than 80% and approximately 20% inhibition of NTE and AChE in hen brain, respectively. Among the isoforms of methamidophos, only the (+)-methamidophos was capable of inhibiting NTE activity (approximately 60%) with statistically significant difference compared to the control group. Calpain activity in brain increased by 40% in TOCP hens compared to the control group when measured 24h after dosing and remained high (18% over control) 21 days after dosing. Hens that received (+)-methamidophos had calpain activity 12% greater than controls. The histopathological findings and clinical signs corroborated the biochemical results that indicated the potential of the (+)-methamidophos to be the isoform responsible for OPIDN induction. Protection against OPIDN was examined using a treatment of 2 doses of nimodipine (1mg/kg, i.m.) and one dose of calcium gluconate (5mg/kg, i.v.). The treatment decreased the effect of OPIDN-inducing TOCP and (+)-methamidophos on calpain activity, spinal cord lesions and clinical signs.
Assuntos
Inseticidas/toxicidade , Síndromes Neurotóxicas/etiologia , Compostos Organotiofosforados/toxicidade , Tritolil Fosfatos/toxicidade , Acetilcolinesterase/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Administração Oral , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Cálcio/metabolismo , Gluconato de Cálcio/farmacologia , Calpaína/efeitos dos fármacos , Calpaína/metabolismo , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/metabolismo , Galinhas , Feminino , Homeostase , Síndromes Neurotóxicas/prevenção & controle , Nimodipina/farmacologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Fatores de TempoRESUMO
Organophosphorus-induced delayed neuropathy (OPIDN) is a neurodegenerative disorder characterised by ataxia progressing to paralysis with concomitant central and peripheral distal axonopathy. Symptoms of OPIDN in people include tingling of the hands and feet. This tingling is followed by sensory loss, progressive muscle weakness and flaccidity of the distal skeletal muscles of the lower and upper extremities and ataxia, which appear about 8-14 days after exposure. Some organophosphorus compounds (OPs) that are still used in worldwide agriculture have potential to induce OPIDN, including methamidophos, trichlorfon, dichlorvos and chorpyrifos. This review summarizes experimental attempts to prevent and/or treat OPIDN and the different mechanisms involved in each approach. The initial mechanism associated with development of OPIDN is phosphorylation and inhibition of neuropathy target esterase (NTE). The phosphorylated enzyme undergoes a second reaction known as "aging" that results in the loss of one of the "R" groups bound to the phosphorus of the OP. A second mechanism involved in OPIDN is an imbalance in calcium homeostasis. This can lead to the activation of calcium-activated neutral protease and increases in calcium/calmodulin-dependent protein kinases. These events contribute to aberrant phosphorylation of cytoskeletal proteins and protein digestion in the terminal axon that can proceed similarly to Wallerian-type degeneration. Several experimental studies demonstrated alleviation of the signs and symptoms of OPIDN by restoring calcium balance. Other studies have used preadministration of NTE inhibitors, such as carbamates, thiocarbamates, sulfonyl fluorides and phosphinate to prevent OPIDN. Progress is being made, but there is yet no single specific treatment available for use in clinical practice to prevent or alleviate the severe effects of OPIDN.
Assuntos
Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/prevenção & controle , Compostos Organofosforados/toxicidade , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Síndromes Neurotóxicas/terapia , Fosforilação/efeitos dos fármacos , Degeneração Walleriana/induzido quimicamente , Degeneração Walleriana/metabolismoRESUMO
Organophosphorus (OP) used as pesticides and hydraulic fluids can produce acute poisoning known as OP-induced delayed neuropathy (OPIDN), whose effects take long time to recover. Thus a secure therapeutic strategy to prevent the most serious effects of this poisoning would be welcome. In this study, tri-o-cresyl phosphate (TOCP, 500 mg/kg p.o.) was given to hens, followed or not by nimodipine (1mg/kg i.m.) and calcium gluconate (Ca-glu 5mg/kg i.v.). Six hours after TOCP intoxication, neuropathy target esterase (NTE) activity inhibition was observed, peaking after 24h exceeding 80% inhibition. A fall in the plasmatic calcium levels was noted 12h after TOCP was given and, in the sciatic nerve, Ca(2+) fell 56.4% 24h later; at the same time calcium activated neutral protease (CANP) activity increased 308.7%, an effect that lasted 14 days. Any bird that received therapeutic treatment after TOCP intoxication presented significant signs of OPIDN. These results suggest that NTE may be implicated in the regulation of calcium entrance into cells being responsible for the maintenance of normal function of calcium channels, and that increasing CANP activity is responsible to triggering OPIDN. Thus, with one suitably adjusted dose of nimodipine as well as Ca-glu, we believe that this treatment strategy may be used in humans with acute poisoning by neuropathic OP.
Assuntos
Doenças do Sistema Nervoso/induzido quimicamente , Praguicidas/intoxicação , Tritolil Fosfatos/intoxicação , Animais , Cálcio/sangue , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Gluconato de Cálcio/uso terapêutico , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/metabolismo , Galinhas/metabolismo , Modelos Animais de Doenças , Homeostase/efeitos dos fármacos , Músculos/metabolismo , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/metabolismo , Nimodipina/uso terapêuticoRESUMO
To examine the efficacy of calcium gluconate (two doses of Ca-Glu 5 mg/kg i.v.) to alleviate the injurious effects of organophosphorus induced delayed neuropathy (OPIDN) in the presence or absence of phenylmethanesulfonyl fluoride (PMSF 90 mg/kg i.m.), 14 groups of four isabrown hens were used. To measure the lymphocyte neuropathy target esterase (LNTE)activity, groups receiving just distilled water (control), groups receiving just Tri-orto-cresyl phosphate (TOCP; 500 mg/kg p.o.) (Positive control), and other groups receiving TOCP and Ca-Glu or PMSF simultaneously or 12 hours later following intoxication by TOCP were used. They were sacrificed 12 and 24 hours after the administration of TOCP. To observe a 28-day time course of neurotoxicity scores and calcium plasma concentration, five groups were used. Regarding free Ca(2+)in the plasma, the positive control produced a characteristic profile time course up and down during 28 days, and some hens with maximum score of neurotoxicity in 28 days. The treatment, which prevented greater oscillation in free Ca(2+) in the plasma, presented a decrease in OPIDN in relation to the positive control. Twelve hours after the administration of TOCP, LNTE was 70-80% inhibited when compared with control, whereas the first decrease in the free Ca(2+) in the plasma was significantly different from the control only 24 hours after the administration of TOCP. In summary, the sooner the Ca-Glu is started, the less severe the neuropathy effects.
Assuntos
Gluconato de Cálcio/uso terapêutico , Fluoreto de Fenilmetilsulfonil/uso terapêutico , Tritolil Fosfatos/intoxicação , Doença Aguda , Animais , Cálcio/sangue , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Galinhas , Síndromes Neurotóxicas/tratamento farmacológicoRESUMO
This study evaluated the ability of the probiotic organism Lactobacillus plantarum to inhibit the pathogenic activity of Pseudomonas aeruginosa, both in vitro and in vivo, and investigated the mechanisms involved in such protection. L. plantarum whole cultures, culture filtrates (acid filtrate and neutralised acid filtrate) and isolated, washed cells were tested in vitro for their effects on the production of the P. aeruginosa quorum-sensing signal molecules, acyl-homoserine-lactones (AHLs), and two virulence factors controlled by these signal molecules, elastase and biofilm. All were inhibited by L. plantarum cultures and filtrates, but not by isolated, washed cells. The acid L. plantarum growth medium itself had some inhibitory activity, but the greatest activity was exerted by the whole culture. To test the in-vivo activity of L. plantarum, a burned-mouse model was used in which burns infected with P. aeruginosa were treated with L. plantarum at 3, 4, 5, 7 and 9 days post-infection. Samples from skin, liver and spleen taken after 5, 10 and 15 days demonstrated inhibition of P. aeruginosa colonisation by L. plantarum. There was also an improvement in tissue repair, enhanced phagocytosis of P. aeruginosa by tissue phagocytes, and a decrease in apoptosis at 10 days. These results indicate that L. plantarum and/or its by-products are potential therapeutic agents for the local treatment of P. aeruginosa burn infections.
Assuntos
Queimaduras/terapia , Lactobacillus/fisiologia , Probióticos/uso terapêutico , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/fisiologia , Animais , Antibiose , Biofilmes/crescimento & desenvolvimento , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Meios de Cultivo Condicionados , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Elastase Pancreática/antagonistas & inibidores , Fagocitose , Probióticos/administração & dosagem , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Fatores de Tempo , Infecção dos Ferimentos/terapiaRESUMO
The expression of sarcoplasmic esterases, lipases as well as the lipid content in the myofibers of the diaphragm of rats intoxicated with the organophosphate isofenphos was studied. Lipid accumulation was documented at light, electron microsopic and by morphometric studies. The distribution of these lipid droplets was irregular and abundant in myofibers with numerous mitochondria (predominantly oxidative fibers). Histochemical inhibition of sarcoplasmic esterases and lipases was observed in the intoxicated animals. This sarcoplasmic inhibition of esterases occurs roughly in parallel to the inhibition of plasma cholinesterase activity. The inhibition of sarcoplasmic lipases may explain, at least partially, the accumulation of lipids. This inhibition probably makes difficult the use of lipids as fuel, especially in the oxidative fibers. In contrast to the small amount of muscle necrosis, (1.30+/-0.745), metabolic muscle impairment was intense and extensive, i.e., decreased activities of esterases and lipases in the sarcoplasm, that should contribute to muscle weakness. Therefore, because segmental necrosis was most prominent in oxidative fibers (and these fibers use lipids as the principal fuel and contain the greater amount of lipases in the sarcoplasm), it is possible that inhibition of activity of lipases is responsible for the segmental necrosis. Although the exact role of these metabolic changes is not known, it is possible that they contribute not only to the induction and evolution of muscle cell necrosis but also to the muscle weakness and clinical impairment of animals and humans in the acute intoxication by these compounds.