Alteration of the Binding Characteristics of a Recombinant scFv Anti-parathion Antibody - 2. Computer Modelling of Hapten Docking and Correlation with ELISA Binding

Recombinant antibody technology and the ability to manipulate antibody binding characteristics offers much potential to agri-food analysis, and in particular to the generation of antibodies against pesticides. As part of the development of a mutagenesis strategy aimed at changing antibody specificity, the sequence of an scFv recombinant antibody specific to ethyl-parathion has been used to generate a molecular model using ProMod software. Docking studies showed the involvement in the binding pocket of CDR H 3 , as expected, but also of CDR L 2 and of small areas of the framework region of the light chain, based on the Kabat definition of CDRs. The majority of the L 2 contacts do, however, fall within a recently proposed re-definition of L 2 . The validity of the model was supported by comparing docking studies of structurally related pesticides with cross-reaction data from an ELISA.     

Neurotoxic substances also posing a cancer risk: A warning

A large proportion of compounds studied for their neurotoxic potential are at the same time suspected or proven carcinogens. This is demonstrated using the International Neurotoxicology Association (INA) Professional Interest Directory and publications from the field of neurotoxicology as examples. In addition to listing these compounds, the classification scheme used by the International Agency for Research on Cancer (IARC), the Commission of the European Communities (EC), the U.S. Environmental Protection Agency (EPA), the German MAK-Commission of the Deutsche Forschungsgemeinschaft (DGF) and the U.S. National Institute of Occupational Safety and Health (NIOSH) to make qualitative risk assessments is explained. Finally, a short initiation to quantitative risk assessment as performed by the US EPA and the World Health Organization (WHO) is given in order to put the reader into a position as to assess the cancer risk incurred by his/her co-workers and by himself.     

Radioimmunoassay for 2,4-dichlorophenoxyacetic acid

Antisera to 2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, have been obtained from rabbits following immunization with various 2,4-D-protein conjugates. Employing [¹²⁵I] 2,4-D-tyramine as the radioligand for the antisera, very poor assay sensitivity was achieved because of a much higher affinity of the antibodies to the tracer. When using [6-³H] 2,4-D(specific radioactivity 465 GBq/mmol) a sensitive and specific radioimmunoassay (RIA) for 2,4-D could be developed, which allows determination directly in water, plasma and urine samples. Levels as low as approximately 100 pg (450 femtomoles) of 2,4-D can be detected. The antiserum is fairly specific for 2,4-D. Other related phenoxycarboxylic acids and dichlorophenol showed a cross-reactivity smaller than 10%. After a single administration of 2,4-D (0.91 mg/100 g body weight, orally) to rats, plasma and urine levels were determined at different times. Results correspond to those found in the literature, thus indicating the utility of the RIA. Further applications and limitations are discussed.     

Pesticides and their binary combinations as P-glycoprotein inhibitors in NIH 3T3/MDR1 cells

The purpose of the present study was to assess do selected pesticides as well as their binary combinations act as inhibitors of P-glycoprotein (P-gp) activity of NIH 3T3 mouse fibroblasts stably transfected with human MDR1 gene (NIH 3T3/MDR1). As a result of P-gp inhibition, the increase of intracellular accumulation of a model P-gp substrate fluorescent calcein acetoxymethyl ester was measured. Pesticide and verapamil individual dose–response data were scaled and expressed as percent of maximum effect. Results showed that out of 14 pure pesticides tested, endosulfan, phosalone and propiconazole were nearly as potent as model inhibitor verapamil (EC₅₀ = 1.5 μM), while diazinon showed a lower potency of inhibiting P-gp transport activity (EC₅₀ = 58.4 μM). Concentrations of pesticides that produced the same inhibiting effect (isoboles) were combined binary. Results calculated using the isobole method revealed that diazinon caused synergistic effect in inhibiting P-gp transport activity in all combinations.     

The acute oral toxicity of Malathion in relation to dietary protein

Summary The acute oral toxicity of Malathion Technical (95%) was determined in young, male albino rats which had been fed either laboratory chow (group I), a purified diet containing protein as casein in normal (26%) amounts (group II), or the same diet containing a deficient (3.5%) amount of casein (group III). The LD₅₀±S.E. was 1090±83 mg/kg body weight in group I, 1401±99 in II and 599±138 in III. The clinicopathologic syndrome of toxicity was essentially the same in all three groups. The clinical signs included ataxia, bradypnea, dacryorrhea, dyspnea, diarrhea, exophthalmos, hypothermia, hyporeflexia, listlessness, miosis pallor, retching movements, sialorrhea, teeth-chattering, thirst and tremors. Most deaths occurred at 1–15 hours, following respiratory failure. At autopsy there were found an acute enteritis, degenerative changes in the liver and kidneys, a stress reaction, and widespread capillary-venous vasodilatation, dehydration and loss of weight in body organs. In survivors there was generally a temporary anorexia, oligodipsia, loss of body weight, diuresis, glycosuria or proteinuria, aciduria or alkalinuria, abdominal tenderness, epistaxis, hemodacryorrhea, hind limb paralysis, prostration and thoracic rales lasting for a day or two. At 6 days survivors appeared normal and at 2 weeks and 1 month organ weights and water contents were within normal limits.This project was assisted by research grants from the World Health Organization and the Medical Research Council of Canada.     

Do pesticides cause childhood cancer?

Objectives: Epidemiological studies have reported associations between childhood cancer and either parental or child exposure to pesticides. Reviews have been published in 1997 and 1998, where the evidence was found suggestive but not conclusive. An update of the current state of knowledge is warranted. Methods: A literature search has been conducted to identify and evaluate new research results on this topic issued between 1998 and 2004. Results: Eighteen new studies have been identified for this review. Collectively, the studies suggest an increase in the risk of different cancer types associated with exposure to pesticides. However, the evidence is conflicting with regard to cancer types as well as to causative factors across studies. The major shortcomings concern exposure assessment, where, e.g., “farming” is treated equal to “exposure to pesticides,” disregarding other possible exposures, e.g., to biological or infectious agents, and hitherto unidentified lifestyle factors. Also, many exposure questionnaires used in case–control studies are based on broad and sometimes implausible categories. In most studies exposures were categorized as “ever vs. never,” with no regard for exposure intensity or duration. Conclusions: The available literature does not allow firm conclusions with regard to pesticides and any type of childhood cancer. Investing in the acquisition and critical review of exposure information appears to be the crucial step for causal assessment in future research. However, focusing on the presence of pesticides, and not asking the question why they were used, might mask relevant associations to other causative agents.Lecture at the XXXII Medichem Congress, September 1–3, 2004, Paris, France     

Pesticides exposure in Culturama, Brazil--knowledge, attitudes, and practices

In this study, the knowledge, attitudes, and practices associated with pesticide use and exposure were evaluated in the agricultural community of Culturama, in the state of Mato Grosso do Sul, Brazil. A standardized questionnaire was completed by 250 farm workers aged > or =18 years old. The average age of the studied population was 43.6 years and 17.6% had never been to school. Their farms were small (approximately 30ha) and family operated and did not utilize advanced farming technology. About 92% of the interviewees had worked directly with pesticides and 59.6% reported typical intoxication symptoms. Only 44.3%, however, believe that they had been intoxicated. A significant correlation was found between hand washing after pesticide application and reporting symptoms (P=0.014). Over 90% of the farmers reported using the organophosphorus insecticide methamidophos. A great majority (>90%) considered pesticides to be harmful to human health, but less than 20% used masks, impermeable clothes, or gloves during pesticide application. These results indicate that special educational programs, legislation promoting the use of safer pesticides, and implementation of personal protective measures are necessary to decrease the pesticide exposure of farmers in Culturama.     

Use of the dog as non-rodent test species in the safety testing schedule associated with the registration of crop and plant protection products (pesticides): present status

The results from a survey of the expert information that is publicly accessible on the use of the dog as test species during the regulatory evaluation of agricultural chemicals and pesticides are reported. Methods that are being used or considered in order to reduce the number of dogs used for this purpose are described. Regulatory evaluation aims at establishing threshold values for safe human exposure; it is based on no-observed-adverse-effect levels (NOELs) determined in animal studies. Current regulations require testing in two species, a rodent species (usually rat or mouse), and a non-rodent species (usually the dog). Subchronic (90-day) and chronic (12-month) repeated-dose feeding studies must be routinely conducted in dogs. This report first focuses on the results from a retrospective study analysing data on 216 pesticides kept on record by the Bundesinstitut für Risikobewertung, BfR (German Federal Institute for Risk Assessment), the competent regulatory authority in Germany. The study was sponsored and coordinated by SET, the German Foundation for the Promotion of Research on Replacement and Complementary Methods to Reduce Animal Testing (Stiftung zur Förderung der Erforschung von Ersatz-und Ergänzungsmethoden zur Einschränkung von Tierversuchen, Mainz) and conducted by the BfR. Since the data submitted for registration of a product is the property of the manufacturer, the study could only proceed with the collaboration of the German Association of Manufacturers of Agricultural Chemicals (Industrieverband Agrar, IVA). To ensure confidentiality, designated codes were used instead of the compounds proper names when the study was published. The results support two major conclusions. The use of the dog for the testing of pesticides is indeed necessary because the dog has proved to be the most sensitive species for about 15% of the compounds examined. However, chronic studies are only of limited value since they only provide essential information that cannot be obtained in sub-chronic studies in about 5% of cases. These conclusions are supported by several retrospective analyses using data on pharmaceutical drugs carried out in the context of the International Conference on Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH). Over 90% of drugs elicited no toxic symptoms in 12-month studies in dogs in addition to those that had been recorded previously in studies conducted for 90 or 180 days in dogs and rats. Another approach comparing the results from pre-clinical animal studies with clinical studies noted that animal studies predicted about 70% of the effects observed in volunteers, and in about 94% of cases the effects occurred in animal studies lasting not more than one month. Furthermore, the report summarises the current methods under consideration that could refine or reduce the use of dogs in toxicity testing: industrial data sharing and harmonisation of guidelines, in vitro methods, human studies, computational prediction models, and integrated testing approaches. The integrated Agricultural Chemicals Safety Assessment (ACSA) testing scheme, which is currently being developed in an international project initiated by the International Life Sciences Institute (ILSI, USA), is of particular relevance, since an ambitious attempt is being made to design a new comprehensive test framework incorporating modern scientific approaches and covering most aspects of current regulatory testing requirements. The ACSA project has access to the pesticide database of the US EPAs Office of Pesticide Programs (OPP). Preliminary results have confirmed the two major conclusions from the joint SET/BfR study conducted in Germany. Taking these results into account, it is recommended that the regulatory requirement for 12-month studies to be routinely carried out in dogs should be abandoned. While 90-day studies should be conducted in both rats and dogs, chronic studies should only take place in rats. If the dog is more sensitive than the rat in the 90-day study, an additional safety factor to the NOEL value obtained in the chronic rat study should be applied in order to set the threshold for safe human exposure, instead of conducting a 12-month study in dogs. This safety factor may be calculated from chronic NOEL data available in several pesticide databases. Chronic tests using dogs would then only be required if the test compound belongs to a new class of chemicals that has never been tested before. Thus, the report concludes that, according to current scientific knowledge, the routine 12-month studies in dogs are no longer required for agricultural chemicals and pesticides, and international regulations should be changed accordingly. Active international support of such measures is welcomed, from both an economical and an animal welfare perspective.Electronic Supplementary Material Supplementary material is available for this article at     

Changes in antioxidant enzymes in humans with long-term exposure to pesticides

Different pesticides, including organophosphates (OPs), have been reported to induce oxidative stress due to generation of free radicals and alteration in antioxidant defence mechanisms. In this study, a cohort of 81 intensive agriculture workers (pesticide sprayers) was assessed twice during the course of a spraying season for changes in erythrocyte antioxidant enzymes. Acetylcholinesterase (AChE) was used as a reference biomarker. Sprayers presented lower levels of superoxide dismutase (SOD) and glutathione reductase (GR) as compared to controls independently of age, BMI, smoking habit or alcohol consumption. A positive correlation between SOD and AChE was observed at the high exposure period. Those individuals with a decrease in AChE greater than 15% exhibited lower SOD and catalase (CAT) activities at the same period. Glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PDH) remained unaffected in the exposed population. Paraoxonase (PON1) polymorphism influenced erythrocyte CAT and GR, as subjects with the R allele presented lower CAT and higher GR levels. Whether or not the decreased enzyme activities found in this study are linked to the adverse health effects related to chronic pesticide toxicity (in which oxidative damage plays a pathophysiological role, such as cancer or neurodegenerative disorders) is an attractive hypothesis that warrants further investigation.     

Simplified models to analyse time- and dose-dependent responses of populations to toxicants

The basis of ecotoxicology lies currently in the dose–response of organisms to toxicants, as typically described by probit and logistic models. While recognising its merits, standard endpoints ignore the process of toxicity with time, and consequently our ability to predict direct toxic effects in environmental risk assessments is seriously curtailed. Although the response of toxicants with time has been studied before, its application in ecotoxicology remains underutilised. One reason is that no convincing mechanism has been proposed to explain the hyperbolic curves of such responses, whereas a variety of models have been used to describe them. The explanation of both time- and dose-dependent responses is found ultimately in the natural variability of receptor sites among individuals of populations exposed to a toxicant inhibitor with time. The process can be explained by the kinetics of inhibition, and is appropriately described by a simple mathematical expression like the Michaelis–Menten equation, though other asymptotic models (e.g. logistic model) can also be used. The advantage of the hyperbolic model is that median effect values (e.g. LC₅₀ for dose- and ET₅₀ for time-dependent responses) enable calculation of toxicity effects at any concentration level and/or time of exposure, thus making it especially attractive for risk assessment.