Evaluation of oxidative stress and genotoxicity in organophosphorus insecticide formulators

The aim of this study was to evaluate genotoxicity and oxidative stress in workers who formulate organophosphorus (OP) pesticides. In this survey, blood leukocytes and erythrocytes of a group of 21 pesticide formulating workers and an equal number of control subjects were examined for genotoxicity and oxidative stress parameters. The mean comet tail length and mean comet length were used to measure DNA damage. Lipid peroxidation level, catalase, superoxide dismutase (SOD) and glutathione peroxidase activities in erythrocytes were analysed as biomarkers of oxidative stress. In addition, the acetylcholinesterase activity was measured as a biomarker of toxicity. The average duration of employment of workers in the factory was 97 months. Results indicated that chronic exposure (multiple-dose, greater than or equal to 6 months duration) to OP pesticides was associated with increased activities of catalase, SOD and glutathione peroxidase in erythrocytes. The level of lipid peroxidation and acetylcholinesterase activity did not show any significant differences between the two groups. The results also indicated that chronic exposure to OP pesticides was associated with increased DNA damage. It is concluded that human chronic exposure to OP pesticides may result in stimulated antioxidant enzymes and increased DNA damage in the absence of depressed acetylcholinesterase levels. Routine genotoxicity monitoring concomitant to acetylcholinesterase activity in workers occupationally exposed to OP insecticides is suggested.     

Delayed reduction of hippocampal synaptic transmission and spines following exposure to repeated subclinical doses of organophosphorus pesticide in adult mice

Agricultural and household organophosphorus (OP) pesticides inhibit acetylcholinesterase (AchE), resulting in increased acetylcholine (Ach) in the central nervous system. In adults, acute and prolonged exposure to high doses of AchE inhibitors causes severe, clinically apparent symptoms, followed by lasting memory impairments and cognitive dysfunction. The neurotoxicity of repeated environmental exposure to lower, subclinical doses of OP pesticides in adults is not as well studied. However, repeated exposure to acetylcholinesterase inhibitors, such as chlorpyrifos (CPF), pyridostigmine, and sarin nerve agent, has been epidemiologically linked to delayed onset symptoms in Gulf War Illness and may be relevant to environmental exposure in farm workers among others. We treated adult mice with a subclinical dose (5 mg/kg) of CPF for 5 consecutive days and investigated hippocampal synaptic transmission and spine density early (2-7 days) and late (3 months) after CPF administration. No signs of cholinergic toxicity were observed at any time during or after treatment. At 2-7 days after the last injection, we found increased synaptic transmission in the CA3-CA1 region of the hippocampus of CPF-treated mice compared with controls. In contrast, at 3 months after CPF administration, we observed a 50% reduction in synaptic transmission likely due to a corresponding 50% decrease in CA1 pyramidal neuron synaptic spine density. This study is the first to identify a biphasic progression of synaptic abnormalities following repeated OP exposure and suggests that even in the absence of acute cholinergic toxicity, repeated exposure to CPF causes delayed persistent damage to the adult brain in vivo.     

The effect of organophosphorus compounds on serum pseudocholinesterase levels in a group of industrial workers.

This paper presents the findings of a study of serum pseudocholinesterase activity in a group of 36 industrial workers chronically exposed to organophosphorus (OP) compounds. The mean pseudocholinesterase level of the workers was significantly lower than that of 36 other workers without a history of similar exposure. Although there was a high incidence of clinical features suggestive of OP compound toxicity in the exposed workers, no significant correlation between serum pseudocholinesterase levels and clinical symptoms and signs was found. Six exposed workers, found to have low serum pseudocholinesterase levels, were transferred for 6 months to work areas which did not involve OP exposure, whereupon their levels rose significantly back to the normal range.     

Physical exercise affects cholinesterases and organophosphate response

1. Cholinesterase activities in blood and tissues of control and exercising rats with and without organophosphate (OP) exposure were studied. 2. Physical exercise increased total cholinesterase and butyrylcholinesterase activities in rats without OP exposure in blood and diaphragm. In brain physical exercise had no effect on acetylcholinesterase activity. 3. Physical exercise diminished cholinesterase inhibition in blood and tissues after OP exposure.     

PON1Q192R genetic polymorphism modifies organophosphorous pesticide effects on semen quality and DNA integrity in agricultural workers from southern Mexico

Pesticide exposure, including organophosphorous (OP) insecticides, has been associated with poor semen quality, and paraoxonase (PON1), an enzyme involved in OP deactivation, may have a role on their susceptibility, due to PON1 polymorphisms. Our objective was to evaluate the role of PON1Q192R polymorphism on the susceptibility to OP toxicity on semen quality and DNA integrity in agricultural workers. A cross-sectional study was conducted in farmers with Mayan ascendancy from southeastern Mexico chronically exposed to pesticides; mostly OP. Fifty four agricultural workers (18-55 years old) were included, who provided semen and blood samples. Semen quality was evaluated according to WHO, sperm DNA damage by in situ-nick translation (NT-positive cells), PON1Q192R polymorphism by real-time PCR and serum PON1 activity by using phenylacetate and paraoxon. Two OP exposure indexes were created: at the month of sampling and during 3 months before sampling, representing the exposure to spermatids-spermatozoa and to cells at one spermatogenic cycle, respectively. PON1 192R and 192Q allele frequencies were 0.54 and 0.46, respectively. Significant associations were found between OP exposure at the month of sampling and NT-positive cells and sperm viability in homozygote 192RR subjects, and dose-effect relationships were observed between OP exposure during 3 months before sampling and sperm quality parameters and NT-positive cells in homozygote 192RR farmers. This suggests that cells at all stages of spermatogenesis are target of OP, and that there exists an interaction between OP exposure and PON1Q192R polymorphism on these effects; farmers featuring the 192RR genotype were more susceptible to develop reproductive toxic effects by OP exposure.     

Organophosphorous pesticides research in Mexico: epidemiological and experimental approaches

Non-persistent pesticides, such as organophosphorous (OP) insecticides have been extensively used in Mexico, and becoming a public health problem. This review presents data of OP use and related toxicity from epidemiological and experimental studies conducted in Mexico. Studies in agricultural workers from several regions of the country reported moderate to severe cholinergic symptoms, including decreased acetylcholinesterase (AChE) activity (the main acute OP toxic effect that causes an over accumulation of the neurotransmitter acetylcholine), revealing the potential risk of intoxication of Mexican farmers. OP exposure in occupational settings has been associated with decreased semen quality, sperm DNA damage and as endocrine disrupter, particularly in agricultural workers. Alterations in female reproductive function have also been observed, as well as adverse effects on embryo development by prenatal exposure in agricultural communities. This illustrates that OP exposure represents a risk for reproduction and offspring well-being in Mexico. The genotoxic effects of this group of pesticides in somatic and sperm cells are also documented. Lastly, we present data about gene-environmental interactions regarding OP metabolizing enzymes, such as paraoxonase-1 (PON1) and its role in modulating their toxicity, particularly on semen quality and sperm DNA integrity. In summary, readers will see the important health problems associated with OP exposure in Mexican populations, thereby the need of capacitation programs to communicate farmers the proper handling of agrochemicals to prevent their toxic effects and of more well designed human studies to support data of the current situation of workers and communities dedicated to agriculture activities.     

Organophosphorous pesticides research in Mexico: epidemiological and experimental approaches

Non-persistent pesticides, such as organophosphorous (OP) insecticides have been extensively used in Mexico, and becoming a public health problem. This review presents data of OP use and related toxicity from epidemiological and experimental studies conducted in Mexico. Studies in agricultural workers from several regions of the country reported moderate to severe cholinergic symptoms, including decreased acetylcholinesterase (AChE) activity (the main acute OP toxic effect that causes an over accumulation of the neurotransmitter acetylcholine), revealing the potential risk of intoxication of Mexican farmers. OP exposure in occupational settings has been associated with decreased semen quality, sperm DNA damage and as endocrine disrupter, particularly in agricultural workers. Alterations in female reproductive function have also been observed, as well as adverse effects on embryo development by prenatal exposure in agricultural communities. This illustrates that OP exposure represents a risk for reproduction and offspring well-being in Mexico. The genotoxic effects of this group of pesticides in somatic and sperm cells are also documented. Lastly, we present data about gene-environmental interactions regarding OP metabolizing enzymes, such as paraoxonase-1 (PON1) and its role in modulating their toxicity, particularly on semen quality and sperm DNA integrity. In summary, readers will see the important health problems associated with OP exposure in Mexican populations, thereby the need of capacitation programs to communicate farmers the proper handling of agrochemicals to prevent their toxic effects and of more well designed human studies to support data of the current situation of workers and communities dedicated to agriculture activities.     

Long-term functional neurotoxicity of paraoxon and chlorpyrifos: behavioural and pharmacological evidence

Organophosphate (OP) compounds are chemicals widely used in agriculture, industry and households and even as chemical weapons. The major mechanism of acute toxic action is the inhibition of acetylcholinesterase (AChE), which is responsible for the degradation of the neurotransmitter acetylcholine. A chronic OP-induced neuropsychiatric disorder (COPIND), which could result from both long-term exposure to subclinical doses of OP and after acute intoxication, has been proposed. These reports claim to develop animal models that could parallel behavioural and cognitive effects and that could later help to elucidate the mechanisms involved in this long-term affectation of the central nervous system. The present study uses a series of behavioural tests to discern the short- and long-term effects of acute intoxications with paraoxon (Px) or chlorpyrifos (CPF). Our results suggest that months after acute exposure to these OPs functional central nervous system alterations can be detected using a repeated acquisition spatial task in the water maze, for CPF, and in amphetamine-induced place preference paradigm, for both Px and CPF.     

Rates of spontaneous reactivation and aging of acetylcholinesterase in human erythrocytes after inhibition by organophosphorus pesticides

The in vitro rates of spontaneous reactivation and aging in human erythrocyte acetylcholinesterase were studied after inhibition by a dimethoxy (R1R2) and diethoxy substituted (R1R2) organophosphate pesticide (OP) of general structure R1R2P(O)X. These have been compared with data for human plasma cholinesterase previously reported using a similar methodology. A significantly slower rate of aging for erythrocyte acetylcholinesterase was found compared to plasma cholinesterase, whether inhibited by dimethoxy or diethoxy substituted OPs. For diethoxy OPs the rate of spontaneous reactivation of the inhibited plasma enzyme was significantly slower than for the inhibited red cell enzyme. This acetylcholinesterase, and previously published plasma cholinesterase, data suggest that in practise a blood sample taken 30-40 h after significant acute OP exposure will still show inhibition in either plasma or erythrocyte cholinesterase when analysed, but that any inhibited plasma enzyme is more likely to be in the aged form. In contrast a substantial proportion of the erythrocyte acetylcholinesterase is found unaged and therefore sensitive to reactivation by oximes. Samples from an occupational exposure where depressions in plasma or erythrocyte cholinesterase activity from baseline measurements were reactivated ex vivo using the oxime 2-PAM support this hypothesis. These data also confirm that the plasma enzyme is a more sensitive than erythrocyte acetylcholinesterase as an indicator of OP exposure and thus the potential value of ex vivo oxime reactivation of erythrocyte acetylcholinesterase in a blood sample to indicate subclinical OP exposure may be limited. However, this study is too small to draw conclusions on the sensitivity of ex vivo oxime reactivation of acetylcholinesterase as a novel biomarker of excessive OP absorption. Given that there is a better relationship between anticholinergic symptoms and red cell acetylcholinesterase inhibition, and that the slower resynthesis rate of any aged or inhibited red cell enzyme may be interpretatively useful when venepuncture is delayed, it is suggested that red cell acetylcholinesterase activity does have a place in monitoring potential OP exposure.     

Clinical and biochemical changes in chronically exposed organophosphate workers

Health effects of occupational organophosphate exposure were investigated by subjecting 22 workers chronically exposed to an organophosphate pesticide, fenthion (O,O-dimethyl-O-(4-methylmercapto-3-methylphenyl)-phosphorothioate) to clinical evaluation, estimation of serum cholinesterase, serum alkaline phosphatase (SAP), serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT). The mean age of the workers was 31 years and the mean duration of fenthion exposure 8.2 years. Headache (59%), giddiness (50%), ocular symptoms (27%) and paresthesia (18%) were the commonest symptoms. Serum acetylcholinesterase and butyrylcholinesterase levels were significantly lower than in controls. After withdrawing the workers from organophosphate exposure for 3 weeks, the follow-up study revealed absence of transient symptoms. There was no change in their neurological status, and serum acetylcholinesterase and butyrylcholinesterase levels were raised significantly, whereas the other serum enzymes showed no significant change. For monitoring of occupational organophosphate exposure, the importance of both clinical and biochemical parameters is emphasised.     

Peripheral cholinesterase inhibition by occupational chlorpyrifos exposure in Australian termiticide applicators.

Occupational exposure to organophosphorus insecticides (OPs), such as chlorpyrifos, may be monitored by the measurement of the activity of peripheral cholinesterase (ChE) enzymes, including erythrocyte acetylcholinesterase (EAChE) and serum cholinesterase (SChE). Lymphocyte neuropathy target esterase (NTE) is thought to have potential as a predictor of organophosphate-induced delayed neuropathy (OPIDN). This paper describes work performed in 39 Australian pest control operators (PCOs) exposed to a termiticide containing chlorpyrifos, and 34 unexposed control subjects. EAChE activities in PCOs did not differ from those of unexposed control workers. Mean NTE activity was slightly higher in PCOs than in controls and mean SChE was 52% of control activity. These results indicate that exposure of Australian PCOs to termiticides containing chlorpyrifos may be monitored using SChE but not EAChE or NTE, and that workers in this industry have sufficiently high OP exposure to significantly depress SChE activity. SChE inhibition of 70-80% may be associated with symptoms. Although no current symptoms were reported to be associated with occupational OP exposure, these workers may be at increased risk of acute effects following inadvertent spills or self-contamination due to their background level of exposure to OPs. While it is preferable to compare ChE enzyme activities between pre- and post-exposure periods to evaluate OP-related effects in individuals, in some cases there is an absence of pre-exposure data. The results of this study suggest that a screening value for SChE of 550 nmol/min/ml in a single blood sample may be useful to identify potentially OP-exposed individuals in the Australian population. Australian control subjects were similar with respect to EAChE, but displayed activities of NTE and SChE approximately 50 and 23% lower than an unexposed UK reference group. While these comparisons are presently speculative, they suggest that there may be differences in SChE and NTE activities in control populations of the two countries. The routine treatment of Australian homes with termiticides containing OPs, or differences in the availability and use of domestic OP-containing insecticides may explain these population differences. Further work is required to examine whether these differences are real, and if so their likely cause.     

Health surveillance and biological monitoring of pesticide formulators in India

Results of health survey and biological monitoring in pesticide formulators exposed to a combination of pesticides, an organophosphorus (OP) insecticide (phorate) and a persistent chlorinated insecticide (technical hexachlorocyclohexane; HCH; BHC) are reported. Exposure of 160 workers to a combination of pesticides (malathion, parathion, DDT and HCH) resulted in 73% of the workers showing toxic signs and symptoms. Formulators showed marked inhibition of whole blood, plasma and red blood cell cholinesterase (ChE) activity and slightly higher concentrations of DDT and HCH in serum. An interesting observation was that over 25% of the formulators showed ECG aberrations. The ECG changes were not related to whole blood ChE activity. Exposure to the chlorinated insecticide HCH in 19 workers engaged in the manufacture of technical HCH resulted in toxic signs and symptoms in over 90% of the subjects. The HCH concentrations in serum showed a ten-fold increase. Changes in the liver enzymes ornithine carbamyl transferase (OCT), gamma-glutamyl transpeptidase (GGTP), leucine aminopeptidase (LAP) and in immunoglobulin M(IgM) showed possible effects on liver and humoral immunity. ECG monitoring showed evidence of cardiac effects. Exposure of 40 formulators to a highly toxic OP insecticide (phorate) showed that over 60% of the workers suffered from toxic effects in spite of using a complete set of protective clothing. A marked and progressive inhibition in whole blood and plasma ChE activity was found during the two weeks of exposure to phorate. An appreciable recovery in ChE activity was observed 10 days after cessation of exposure. These surveys have established the need to practice and develop biological monitoring techniques to assess exposure and predict health risks in workers occupationally exposed to pesticides.     

Biological monitoring of combined exposure to organophosphates and pyrethroids

A total of 132 farmers were selected from two areas (J and S) for this study. In area J, the subgroups Ja (40 subjects) and Jb (36 subjects) sprayed on rice crops either the organophosphate (OP) insecticide methamidophos or the mixture methamidophos-deltamethrin (a pyrethroid, Pyr). In area S, the subgroups Sa (27 subjects) and Sb (29 subjects) sprayed on cottons either the OP methyl-parathion or the mixture methyl-parathion-deltamethrin. Groups Jc (22 subjects) and Sc (20 subjects) were non-exposed controls. Erythrocyte acetylcholinesterase (AChE) was measured by the Ellman's method before spraying, after 2-h exposure and 1 h later. Inhibition of AChE occurred after 2-h exposure to both single OP (Ja and Sa) and OP-containing mixtures (Jb and Sb) as compared with both pre-exposure and control values (Jc and Sc). A similar recovery was found in subgroups Ja, Jb, Sa and Sb 1 h after spraying, but all remained lower than the pre-exposure level. There was no difference in AChE inhibition and recovery between the single OP subgroups and the OP mixture subgroups. The inhibitory effect on AChE shown in subjects with combined OP-Pyr exposure was induced by the OP component of the insecticide mixture and to a similar extent as that induced by the single OP formulations. In addition to the fact that the OP is more potent than Pyrs, the above results might explain why the clinical manifestations of OP mixture poisonings are similar to those of single OP poisoning.     

Diurnal variations of extrapyramidal symptoms induced by haloperidol in schizophrenic subjects

Despite the fact that the circadian variations in the density of striatal dopaminergic D2 receptors are well documented, there are few reports concerning whether these variations may regulate the extrapyramidal symptoms produced by antipsychotic drugs. To test this hypothesis, we selected 18 male hospitalized schizophrenic subjects, who had been treated for 2 wk with 10 mg haloperidol three times a day. The extrapyramidal symptoms were evaluated 1 h after each haloperidol administration (e.g. 09:00, 14:00 and 19:00 hours). The diurnal profiles of haloperidol-induced extrapyramidal symptoms showed that akathisia and dystonia were milder in the morning and increased in severity during the night. However, Parkinsonian symptoms were more severe during the morning evaluations than during the evening tests. The difference in the diurnal pattern of the haloperidol-induced side-effects, suggests that receptor-related mechanisms with a different circadian profile contribute to the induction of the extrapyramidal symptoms.     

Cholinesterase activities determination in frozen blood samples: an improvement to the occupational monitoring in developing countries

1. Studies were carried out on rural workers in Brazil to determine the decrease in the activity of plasma butyrylcholinesterase (BChE), erythrocyte cholinesterase (AChE) associated with exposure to organophosphorus pesticides (OP). The goal of this work is to help prevent injury to these workers. 2. In developing countries the distance between area of pesticide use and reference laboratories is a drawback for analytical techniques, since cholinesterase activity determinations require fresh blood samples. Field methodologies can be a useful alternative to laboratory tests, however they are not as sensitive as those found in laboratories. 3. The modification of Ellman's Method presented in this paper allows blood samples to be frozen and maintain enzymatic stability: 7 days for AChE and 3 days for BChE. The proposed method is also more sensitive than Ellman's Method Modified by Magnotti (EMMM). 4. The results suggest that the Ellman Method Modified by Oliveira-Silva (EMMOS) is valid for monitoring procedures. This method represents an important contribution to the process of monitoring OP exposures, since the evaluations no longer have to be conducted near the site of OP use.     

Differential protein adduction by seven organophosphorus pesticides in both brain and thymus

There is a need for mechanistic understanding of the lasting ill health reported in several studies of workers exposed to organophosphorus (OP) pesticide. Although the acute toxicity is largely explicable by acetylcholinesterase inhibition and the lasting effects of frank poisoning by direct excitotoxicity or indirect consequences of the cholinergic syndrome, effects at lower levels of exposure would not be predicted from these mechanisms. Similarly, reversible interactions with nicotinic and muscarinic receptors in adults would not predict continuing ill health. Many OP pesticides produce protein adduction, and the lasting nature of this makes it a candidate mechanism for the production of continuing ill health. We found significant adduction of partially characterized protein targets in both rat brain and thymus by azamethiphos, chlorfenvinphos, chlorpyrifos-oxon, diazinon-oxon, dichlorvos and malaoxon, in vitro and pirimiphos-methyl in vivo. The diversity in the adduction pattern seen across these agents at low dose levels means that any longer term effects of adduction would be specific to specific organophosphates, rather than generic. This presents a challenge to epidemiology, as most exposures are to different agents over time. However, some adducted proteins are also expressed in blood, notably albumin, and so may provide exposure measures to increase the power of future epidemiological studies.     

Alcohol abuse in the psychiatric hospital population

A survey was conducted to assess the presence of alcohol consumption and its associated effects in the nonsubstance abuse population of a VA neuropsychiatric hospital. The findings indicated that patients with alcoholism-related psychiatric diagnoses were more likely to be illegally consuming alcohol than those without these diagnoses. On the other hand, the effects of drinking appeared to be attributable to patients' drinking patterns rather than to their alcoholism histories. Finally, the incidence of alcoholism problems approximated that of the nonpsychiatric population, suggesting that there is no unique, etiological basis for alcoholism in other psychiatric conditions.     

Long-term effects of an organophosphate upon the human electroencephalogram.

The brain electrical activity of workers exposed to the organophosphate compound (OP), sarin, was compared to that of control subjects. Exposed workers had a history of one or more documented accidental exposures to toxic levels of sarin. However, no exposed subject had exposure within 1 year of his examination. The comparison included standard clinical electroencephalograms (EEGs), computer-derived EEG spectral analysis, and standard overnight sleep EEGs. It was not possible to diagnose subjects individually by expert visual inspection of their EEGs. However, statistically significant between-group differences for both the visually inspected and computer-derived data were reported by both univariate and multivariate statistical methods. Different EEG changes revealed by visual inspection and computer-derived spectral analysis appear to reflect the differing sensitivites of these two analytic techniques. Statistically significant group differences included increased beta activity, increased delta and theta slowing, decreased alpha activity, and increased amounts of rapid eye movement sleep in the exposed population. It is suggested that the above findings represent an unexpected persistence of known short-term OP actions. It is also suggested that these results, when taken along with the reported long-term behavioral effects of OP exposure, provide parallel evidence that OP exposure can produce long-term changes in brain function.     

可通过血脑屏障的乙酰胆碱酯酶重活化剂的研究进展

神经性毒剂和有机磷农药是乙酰胆碱酯酶的不可逆抑制剂,对人类生命安全构成重大威胁。目前可用于临床预防和治疗的药物大都为季铵型乙酰胆碱酯酶重活化剂,对外周组织和血液中乙酰胆碱酯酶中毒具有较好的活化作用,但季铵盐结构限制了血脑屏障通过率,使这类重活化剂在中枢神经系统发挥作用有限。因此,开发可通过血脑屏障的重活化剂是当前主要发展趋势。本文对可通过血脑屏障的重活化剂的研究进展进行了综述。     

Organophosphorus pesticide exposure decreases sperm quality: association between sperm parameters and urinary pesticide levels

Several studies have suggested that human semen quality has declined over the past decades and some of them have associated it with occupational exposure to pesticides. However, most of these studies have not been associated with a reliable exposure level and have been designed mostly as cross-sectional studies. The present work evaluates, in a longitudinal follow-up study, the effect of organophosphate pesticides (OP) at three occupational exposure levels on semen quality. In addition, the study examined the association between OP urinary levels and sperm parameters in exposed and unexposed workers. A total of 139 semen samples from 52 volunteers were assessed. Urinary OP levels were measured by gas-liquid chromatography.The results revealed that the poorest semen quality was found among the subjects with the highest OP exposure and the highest urinary OP levels. Seasonal variations in sperm concentration and sperm count were registered.The results showed a significant decrease in total sperm count among subjects with the highest exposure to OP. Further studies assessing the effects of OP on male reproductive health should be controlled by the variability in human sperm parameters, sperm seasonality, spermatogenesis time and the changing OP exposure level in men highly exposed to OP.