Morphological analysis of the vestibular system of guinea pigs poisoned by organophosphate / Análise morfológica do sistema vestibular de cobaias intoxicadas por organofosforado

ABSTRACT INTRODUCTION: The vestibular system is responsible for body balance. There are substances that damage it, causing dizziness; these are termed vestibulotoxic substances. Agrochemicals have been investigated for ototoxicity because of studies that identified dizziness as a recurrent symptom among rural workers' complaints. OBJECTIVE: To histopathologically evaluate the vestibular system in guinea pigs exposed to an organophosphate, and to identify the drug's effects on this system. METHODS: Experimental clinical study. Eighteen guinea pigs were used; six of them poisoned with the organophosphate chlorpyrifos at doses of 0.5 mg/kg/day and seven of them at 1 mg/kg/day; and a control group of five guinea pigs was exposed to distilled water, all for 10 consecutive days. Later, ciliary tufts of saccule and utricle maculae were counted by scanning electron microscopy. RESULTS: Comparing the groups, a one-way ANOVA test for the variable "saccule" ( p = 0.0569) and a Kruskal-Wallis test for the variable "utricle" ( p = 0.8958) were performed, revealing no difference among groups in both variables. CONCLUSION: The histopathologic analysis of the vestibular system of guinea pigs exposed to an organophosphate showed no difference in the amount of ciliary tufts of saccule and utricle maculae at the doses tested, although the result for the variable "saccule" was considered borderline, showing a trend for significance.

RESUMO INTRODUÇÃO: O sistema vestibular é responsável pelo equilíbrio corporal. Existem substâncias que o danificam, causando tontura; são chamadas vestibulotóxicas. Agrotóxicos tornaram-se objeto de investigação da ototoxicidade a partir de pesquisas que apontaram tontura como sintoma recorrente entre as queixas de trabalhadores rurais. OBJETIVO: Constitui-se em avaliar a histopatologia do sistema vestibular de cobaias expostas a organofosforados, identificando os efeitos nesse sistema. MÉTODO: É um estudo clínico experimental, que utilizou 18 cobaias, sendo seis intoxicadas com organofosforadoclorpirifós na dose de 0,5 mg/kg/dia; sete na dose de 1 mg/kg/dia, e grupo controle com cinco cobaias expostas a água destilada, durante 10 dias consecutivos. Posteriormente realizou-se a contagem dos tufos ciliares nas máculas dos sáculos e utrículos através microscopia eletrônica de varredura. RESULTADOS: Na comparação intergrupos, para a variável sáculo realizou-se o teste ANOVA one-way (p = 0,0569); para a variável utrículo, utilizou-se o teste Kruskal-Wallis (p = 0,8958), revelando não haver diferença entre os grupos em ambas as variáveis. CONCLUSÃO: Análise histopatológica do sistema vestibular de cobaias expostas a organofosforado não demonstrou diferença na quantidade de tufos ciliares nas máculas dos sáculos e utrículos nas doses testadas, apesar do resultado para a variável sáculo ser considerado limítrofe mostrando uma tendência a significância.

Otoproteção em cobaias expostas a agrotóxico e ginkgo biloba / Otoprotection in guinea pigs exposed to pesticides and ginkgo biloba

Os agrotóxicos são amplamente utilizados na agricultura e, atualmente, fazem parte do grupo de agentes químicos que podem levar à perda auditiva. A identificação de drogas que, associadas aos ototóxicos, possam atuar como otoprotetores é objeto de estudo. OBJETIVO: Analisar a existência de efeito otoprotetor do extrato de Ginkgo biloba aos possíveis danos cocleares causados pelo agrotóxico do grupo dos organofosforados - metamidofós, avaliando-se as alterações anatômicas por meio da microscopia eletrônica de superfície. MATERIAL E MÉTODO: Estudo experimental prospectivo utilizando 21 cobaias, que sofreram ação da administração de soro fisiológico, agrotóxico e ginkgo biloba isoladamente e associadas, durante sete dias consecutivos. Após, as cócleas foram removidas e avaliadas anatomicamente pela microscopia eletrônica de varredura. RESULTADOS: As cobaias submetidas ao agrotóxico apresentaram alterações morfológicas cocleares, com lesões nas três espiras analisadas na microscopia eletrônica, intensificadas de acordo com a dosagem recebida do agente. As cobaias tratadas com agrotóxico e Ginkgo biloba apresentaram uma manutenção da arquitetura ciliar nas células ciliadas externas em todas as espiras da cóclea. CONCLUSÃO: O extrato de Ginkgo biloba, por sua ação antioxidante, atuou como fator otoprotetor à ototoxicidade pelo agrotóxico em cobaias.

Pesticides are widely used in agriculture, despite the risk of hearing loss related to the exposure to their chemical components. This study looks into protective drugs to counteract the ototoxicity of pesticides. OBJECTIVE: This study aims to analyze the effect ginkgo biloba extract may have in protecting against possible cochlear damage caused by organophosphate pesticides (methamidophos). Anatomic changes are assessed through surface and electron microscopy. MATERIALS AND METHODS: This is a prospective experimental study. Twenty-one guinea pigs were given saline solution, pesticide, and ginkgo biloba alone or combined for seven consecutive days. Then their cochleas were removed and examined in a scanning electron microscope. RESULTS: Pesticide-exposed guinea pigs had morphological alterations in their cochleas and injuries in the three turns analyzed through electron microscopy. Injury intensity varied according to the dosages of the agents given to the test subjects. Guinea pigs treated with pesticide and ginkgo biloba maintained the architecture of their outer hair cells in all cochlear turns. CONCLUSION: The antioxidant properties found in the ginkgo biloba extract protected guinea pigs from pesticide ototoxicity.

Acute toxicity and cholinesterase inhibition of the nematicide ethoprophos in larvae of gar Atractosteus tropicus (Semionotiformes: Lepisosteidae)

Biomarkers are a widely applied approach in environmental studies. Analyses of cholinesterase (ChE), glutathione S-transferase (GST) and lipid peroxidation (LPO) are biomarkers that can provide information regarding early effects of pollutants at different biochemical levels on an organism. The aim of this study was to evaluate the biomarker approach on a Costa Rican native and relevant species. For this, larvae of gar (Atractosteus tropicus) were exposed to the organophosphorus nematicide, ethoprophos. Acute (96hr) exposure was conducted with pesticide concentrations ranging from 0.1μg/L to 1 500μg/L. The 96hr LC50 calculated was 859.7μg/L. After exposure, three biomarkers (ChE, GST and LPO) were analyzed in fish that survived the acute test. The lowest observed effect concentration (LOEC) regarding ChE activity inhibition was 50μg/L. This concentration produced a significant inhibition (p<0.05) of the enzyme by 20%. The highest concentration tested without showing any effect on ChE activity and therefore considered as no observed effect concentration (NOEC) was 10μg/L. Ethoprophos concentration of 400μg/L caused a ChE inhibition by 79%. In this study, no significant variations (p>0.05) in GST activity and LPO were observed in A. tropicus larvae after exposure to ethoprophos.

El proceso de reproducción inducida de Atractosteus tropicus es útil para la acuicultura y la reintroducción en zonas donde las poblaciones silvestres se han reducido considerablemente. En larvas de esta especie se evaluó la toxicidad aguda, así como la respuesta de tres biomarcadores: actividad colinesterasa (ChE), actividad de Glutation S-transferasa (GST) y peroxidación de lípidos (LPO). Asimismo, se realizaron exposiciones agudas (96hr) a etoprofos (nematicida organofosforado), en donde se utilizaron concentraciones entre 0.1μg/L y 1 500μg/L del nematicida. La concentración letal 50 (LC50) calculada fue de 859.7μg/L; la máxima concentración sin efecto en los organismos (NOEC) 10μg/L y la concentración más baja en la cual se observó algún efecto (LOEC) 50μg/L. A esa concentración, el efecto observado fue una reducción significativa (p<0.05) en la actividad de la ChE. Una concetración de etoprofos de 400μg/L causó una inhibición del 79% en la actividad ChE. La actividad GST y la LPO no mostraron una respuesta significativa (p>0.05) luego de la exposición de los organismos a etoprofos.

Ototoxicidade por organofosforados: descrição dos aspectos ultraestruturais do sistema vestibulococlear de cobaias / Organophosphate-related ototoxicity: Description of the vestibulocochlear system ultrastructural aspects of guinea pigs

Os agrotóxicos organofosforados são amplamente utilizados na agricultura, e atualmente fazem parte do grupo de agentes químicos que podem levar à perda auditiva, no qual já estavam incluídos os solventes, os metais e os asfixiantes. OBJETIVO: Analisar a ação ototóxica aguda de um agrotóxico do grupo dos organofosforados na citoarquitetura do sistema vestibulococlear. Trata-se de um estudo experimental prospectivo. MATERIAL E MÉTODO: Foram utilizadas cobaias albinas machos, divididas em três grupos, nos quais se administrou água destilada (grupo 1 - controle), agrotóxico - 0,3mg/Kg/dia (grupo 2), agrotóxico - 3 mg/Kg/dia (grupo 3), durante sete dias consecutivos. O agrotóxico utilizado foi Tamaron BR (metamidofós). A avaliação anatômica da cóclea, sáculo e utrículo foi realizada através da microscopia eletrônica de varredura, após o período de aplicação do agrotóxico. RESULTADOS: As cobaias submetidas ao organofosforado apresentaram alterações morfológicas cocleares, com lesões nas três espiras analisadas, bem como alterações ciliares de sáculo e utrículo, intensificadas de acordo com a dosagem recebida do agente. CONCLUSÃO: As alterações morfológicas observadas nas células ciliadas nos grupos expostos a doses diárias de organofosforado promovem evidências de um efeito agudo degradante dos agrotóxicos no sistema vestibulococlear.

Organophosphate toxic agents are used in agriculture and are currently part of the group of toxic agents which can lead to hearing loss, in which we have solvents, metals and asphyxiation agents. AIM: to analyze the acute ototoxic action of a group of organophosphate agents in the vestibulo-cochlear system. This is a prospective experimental study. MATERIALS AND METHODS: we used male albino guinea pigs, broken down into three groups, to which we provided distilled water (group 1 - control), agrotoxic agent - 0.3mg/Kg/day (group 2), agrotoxic - 3 mg/Kg/day (group 3), during 7 seven consecutive days. The most used agrotoxic agent was Tamaron BR (metamidophos). The anatomical evaluation of the cochlea, saccule and utricle was carried out by means of electronic scanning microscopy after the use of the agrotoxic agent. RESULTS: the guinea pigs submitted to the organophosphate presented cochlear morphological alterations with lesions on the three turns analyzed, as well as cilia alterations in the saccule and utricle, intensified according to the agent dosage. CONCLUSION: the morphological alterations seen in the hair cells exposed to daily doses of organophosphate promote evidences of an acute deleterious effect of agrotoxic agents on the vestibulo-cochlear system.

Patterns of organophosphorous poisoning attending a teaching hospital.

The objective of this was to study the pattern of patients of organophosphorous (OP) poisoning attending Kathmandu Medical College Teaching Hospital (KMCTH). This is a retrospective study of OP poisoning from hospital records were analyzed meticulously and data extracted. Data collected were: patient's demographics, motive for poisoning, type of OP poison, arrival time, time since ingestion, patient's vitals, predisposing factors, serum cholinesterase levels, treatment given, duration of hospital stay and mortality. A total of 47 patients of OP poisoning attended KMCTH from Aug 2003 to July 2005. 22(46.8%) cases were male and 25(53.2%) were female. The maximum number of patients were between the age of 20-40 (33-70.2%). Married patients outnumbered the unmarried (35/74.5% vs 12/25.5%). The most common motive for poisoning was suicidal, 41 cases (87.2%). Metacid (methyl-parathion) was the most commonly used OP compounds in 32 (68%) patients. Interpersonal marital relationship seemed to be the commonest predisposing factor, 23 cases (48.9%).The commonest time of presentation was between 6 pm-12 midnight. 7(57.4%) cases presented within 2 hours of ingestion of the poison. Serum cholinesterase level measured after full atropinisation was >50% of normal level in 17 cases (36.2%). 10 cases (21.3%) required respiratory support. Intermediate syndrome (IMS) was observed in 3 cases. Overall mortality occurred in 3 cases (6.4%). This study highlights the problem of OP poisoning in our region. Establishment of strict policies against the sale and availability of insecticides and pesticides which are freely available in the market is an effective way to control OP poisoning.

Alpha lipoic acid ameliorate changes occur in neurotransmitters and antioxidant enzymes that influenced by profenofos insecticide

This study was designed to determine whether alpha lipoic acid [ALA] which has been shown to have substantial antioxidant properties would ameliorate some of profenofos insecticide toxic effects. ALA administered [60mg/kg b.w.] to adult female rats for 14 days 1 hour after administration of 1/10 LD50 [45 mg/kg b.w.] and 1/20 LD50 [22.5 mg/kg b.w.] for profenofos insecticide which act as free radical inducer. Neurotransmitters [Dopamine [DA], Norepinephrin [NE], Serotonine [5-HT] and 5-Hydroxy indol acetic acid [5-HIAA]] were estimated in plasma. While malondialdehyde [MDA], reduced glutathione [GSH] level, glutathione-S-transferase [GST] and superoxide dismutase [SOD] activities were determined in liver, kidney and brain. The results revealed an increase in plasma serotonine [5-HT] levels in group of rats intoxicated with low dose of profenofos. A significant increase in MDA level [an indicator for lipid peroxidation] in liver of rats intoxicated with both doses of profenofos was recorded, concurrent with a significant reduction in GSH level and GST and SOD activities in most tested tissues of rats intoxicated with both doses of profenofos. Supplementation with alpha lipoic acid [60 mg/kg b.wt] 1 h after profenofos administration induced some but not complete improvement in all parameters whereas, it induced significant increase in plasma DA and 5-HT while it reduced lipid peroxidation in each of the examined tissues. These results accompanied with improvement in GSH level especially in liver, in addition to GST and SOD activities in some organs. Its effect differ from tissue to another. In conclusion ALA supplementation to profenofos intoxicated rats induced improvement in lipid peroxidation, total glutathione level and glutathione-S- transferase activity

Cytogenetic and genotoxic effects of the insecticides, imidacloprid and methamidophos

We examined the cytogenetic and genotoxic effects of the neonicotinoid insecticide imidacloprid and the organophosphate insecticide methamidophos, when administered alone or in combination. These insecticides were tested with the bone marrow chromosome aberration assay and micronucleus test in rats and by the bacterial mutation assay (Salmonella/microsome mutagenicity assay). Wistar albino rats were orally fed daily with laboratory chow treated with various concentrations of insecticides, 50 and 100 mg/kg imidacloprid, 2.5 and 5 mg/kg methamidophos, and 2.5 and 5 mg/kg imidacloprid plus methamidophos, respectively, for 90 days. Numerical and structural chromosomal aberrations were evaluated. Significant differences were detected between all the insecticide-administered groups versus the control group and between the two concentrations of the pesticide-treated groups. Both concentrations of the insecticides induced a dose-related increase in the micronucleus frequency (P < 0.05). Dose-related increases in the number of revertants were observed with the two Salmonella strains (TA98 and TA100). All tested doses of the insecticides demonstrated mutagenic activity in the presence of S9 mix. These results lead us to the conclusion that the synergistic effect of methamidophos and imidacloprid causes an increase in potential damage to non-target organisms.

Evaluation of pirimiphos-methyl (50% EC) against the immatures of Anopheles stephensi/An. culicifacies (malaria vectors) and Culex quinquefasciatus (vector of bancroftian filariasis).

BACKGROUND & OBJECTIVES: In India, temephos and fenthion are used as larvicides in fresh and polluted waters. Since use of same insecticide may precipitate resistant, as an alternative bioefficacy of pirimiphos-methyl--an organophosphorus insecticide was evaluated against immatures of Anopheles and Culex species in different breeding habitats in District Ghaziabad (U.P.) and Goa. METHODS: Laboratory bioassays were carried out using standard WHO procedure. LC50 and LC90 were calculated using Probit analysis. Abbott's formula was used to calculate per cent corrected mortality in laboratory exposures while, Mulla's formula was applied for calculating per cent reduction in mosquito immatures under field conditions. RESULTS: Pirimiphos-methyl was found to be most effective against larvae of An. stephensi followed by An. culicifacies and Cx. quinquefasciatus. The LC50 and LC90 values against three species were 0.023, 0.032 and 0.04 ppm; and 0.045, 0.057 and 0.114 ppm respectively. In field, pirimiphos-methyl @ 200 g a.i./ha against Anopheles species and @ 300 g a.i./ha against Culex species can produce > 80% reduction in density of immatures up to one week in different habitats. Pirimiphos-methyl < 0.25 ppm is not toxic to fish. INTERPRETATION & CONCLUSION: Pirimiphos-methyl is more effective against anophelines in clean water than culicines in polluted water. This larvicide should not be used at a concentration above 0.25 ppm in habitats harbouring the larvivorous fish.

Acephate induced oxidative damage in erythrocytes.

The effect of oral administration of acephate (360 mg/kg body weight), for 15 days, daily, was investigated on the erythrocytes of male rats. Activities of acetyl cholinesterase and glucose-6-phosphate dehydrogenase decreased, while those of glutathione-s-transferase and glutathione reductase increased. Decreased glutathione content and increased lipid peroxidation suggest that there was increased oxidative stress in the erythrocytes of treated animals. Increased cholesterol/phospholipid ratio in the erythrocyte membranes and morphological changes in RBCs (scanning electron microscopy studies) were observed in acephate treated animals. The results clearly suggest that acephate induced oxidative stress in erythrocytes leads to morphological changes.

Neurobehavioural study of subchronic administration of oxydemeton-methyl (insecticide and acaricide) in rats.

Oxydemeton-methyl, an organophosphate insecticide and acaricide produced decrease in the exploratory behaviour and prolongation of barbitone sodium induced hypnosis in rats after intermittent aerosol spray inhalational exposure, for 1/2 hour daily for 7 consecutive days, compared to the saline control group. Further, ED50 +/- SEM value for haloperidol induced catalepsy, CD50 +/- SEM value for pentylenetetrazole induced seizure and CI50 +/- SEM value for electroshock (i.e. the dose of haloperidol, PTZ and intensity of electroshock producing catalepsy or positive seizure response in 50% of rats) were significantly decreased after 7 days exposure to oxydemeton-methyl compared to that of saline control group. The study has established the central nervous system depressant effect, extrapyramidal effect and proconvulsant potential of oxydemeton-methyl which is widely used by the agricultural workers in the form of field spray.

Antidotal efficacy of pyridinium oximes and cholineacetyltransferase inhibitors against organophosphorus intoxication in rodents.

In an attempt to develop effective antidote against organophosphorus intoxication, some new imidazole-pyridinium mono-oximes, long chain pyridinium mono-oximes and cholineacetyltransferase inhibitors were synthesised. These compounds were evaluated for their in vivo therapeutic protection and neuromuscular function studies in rodents. The results indicate that SPK-series oximes may be useful against sarin poisoning without any beneficial effect against VX (O-Ethyl S-2-NN-diisopropylaminoethyl methylphosphonofluoridate) intoxication. The cholineacetyltransferase (ChAT) inhibitors may not be of any help against any of the OP compounds studied in this study.

An observational study on neurobehavioural effects of acute oxydemeton-methyl (insecticide and acaricide) exposure in rats.

Oxydemeton-methyl, an organophosphate insecticide and acaricide produced decrease in the exploratory behaviour and prolongation of barbitone sodium-induced hypnosis after intermittent aerosol spray inhalational exposure for 1 h in rats compared to the saline control group. Further, CD50 +/- S.E.M. value for pentylenetetrazole (PTZ) and CI50 +/- S.E.M. value for electroshock (i.e. the dose of PTZ and intensity of electroshock producing positive seizure response in 50% of rats) were significantly decreased by acute exposure to oxydemeton-methyl compared to that of saline control group. The study has established the central nervous system depressant effect and proconvulsant potential of oxydemeton-methyl which is widely used by the agricultural workers in the form of field spray.

Relationship between acetylcholinesterase and monoamine oxidase in brain regions of Oreochromis mossambicus exposed to phosalone.

The level of acetylcholinesterase (AChE) in brain regions of O. mossambicus at different intervals showed the extent of phosalone toxicity. Significant inhibition of AChE at the end of 96 hr in the brain regions was observed. In contrast to AChE inhibition, the monoamine oxidase (MAO) activity showed significant increase in the regions of cerebral hemispheres, dien/mesencephalon, cerebellum and medulla oblongata. The increase of MAO activity in the brain regions under phosalone toxicity is considered to be one of the mechanisms to maintain the amines level in O. mossambicus.

Mutagenicity of systemic organophosphate pesticides metasystox and rogor.

Mutagenicity of metasystox and rogor could not be detected on the basis of tests employing Ames Salmonella/microsome assay even in the presence of wheat seedling or rat liver microsomal activation systems.