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

Medical Research Council Applied Neuroscience Group, School of Biomedical Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom.
Human &amp Experimental Toxicology (Impact Factor: 1.41). 05/2007; 26(4):347-53. DOI: 10.1177/0960327107074617
Source: PubMed

ABSTRACT 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.

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    • "The most extensive data are for OP labeling of albumin. Several laboratories agree that albumin is covalently modified by OP both in vitro and in vivo (Adams et al., 2004;Peeples et al., 2005;Carter et al., 2007;Williams et al., 2007;Ding et al., 2008;Tarhoni et al., 2008). "
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    ABSTRACT: Acute toxicity of organophosphorus poisons (OP) is explained by inhibition of acetylcholinesterase in nerve synapses. Low-dose effects are hypothesized to result from modification of other proteins, whose identity is not yet established. The goal of the present work was to obtain information that would make it possible to identify tubulin as a target of OP exposure. Tubulin was selected for study because live mice injected with a nontoxic dose of a biotinylated organophosphorus agent appeared to have OP-labeled tubulin in brain as determined by binding to avidin beads and mass spectrometry. The experiments with live mice were not conclusive because binding to avidin beads could be nonspecific. To be convincing, it is necessary to find and characterize the OP-labeled tubulin peptide. The search for OP-labeled tubulin peptides was begun by identifying residues capable of making a covalent bond with OP. Pure bovine tubulin (0.012 mM) was treated with 0.01-0.5 mM chlorpyrifos oxon for 24 h at 37 degrees C in pH 8.3 buffer. The identity of labeled amino acids and percent labeling was determined by mass spectrometry. Chlorpyrifos oxon bound covalently to tyrosines 83, 103, 108, 161, 224, 262, 272, 357, and 399 in bovine alpha tubulin, and to tyrosines 50, 51, 59, 106, 159, 281, 310, and 340 in bovine beta tubulin. The most reactive were tyrosine 83 in alpha and tyrosine 281 in beta tubulin. In the presence of 1 mM GTP, percent labeling increased 2-fold. Based on the crystal structure of the tubulin heterodimer (PDB 1jff) tyrosines 83 and 281 are well exposed to solvent. In conclusion seventeen tyrosines in tubulin have the potential to covalently bind chlorpyrifos oxon. These results will be useful when searching for OP-labeled tubulin in live animals.
    Toxicology and Applied Pharmacology 08/2009; 240(2):149-58. DOI:10.1016/j.taap.2009.07.020 · 3.63 Impact Factor
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    • "In addition to its pharmacological application in humans, DDVP is widely used as an insecticide for domestic purposes (Choudhary et al., 2001). There exists a large body of experimental evidence suggesting that inhibitors of AChE used to potentiate cognition exert their effect through other targets (Richards et al., 2000, Xie et al., 2000, Duysen et al., 2001, Ray and Richards, 2001, Carter et al., 2007). For example, it has been described that low doses of DDVP given to rats can potentiate cognitive abilities without causing a significant inhibition of brain AChE (Van der Staay et al., 1996). "
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    ABSTRACT: Dichlorvos is the active molecule of the pro-drug metrifonate used to revert the cognitive deficits associated with Alzheimer's disease. A few years ago it was reported that dichlorvos inhibits the enzyme acylpeptide hydrolase at lower doses than those necessary to inhibit acetylcholinesterase to the same extent. Therefore, the aim of our investigation was to test the hypothesis that dichlorvos can enhance synaptic efficacy through a mechanism that involves acylpeptide hydrolase instead of acetylcholinesterase inhibition. We used long-term potentiation induced in rat hippocampal slices as a model of synaptic plasticity. Our results indicate that short-term exposures (20 min) to 50 microM dichlorvos enhance long-term potentiation in about 200% compared to the control condition. This effect is correlated with approximately 60% inhibition of acylpeptide hydrolase activity, whereas acetylcholinesterase activity remains unaffected. Paired-pulse facilitation and inhibition experiments indicate that dichlorvos does not have any presynaptic effect in the CA3-->CA1 pathway nor affect gabaergic interneurons. Interestingly, the application of 100 nM methyllicaconitine, an alpha(7) nicotinic receptor antagonist, blocked the enhancing effect of dichlorvos on long-term potentiation. These results indicate that under the exposure conditions described above, dichlorvos enhances long-term potentiation through a postsynaptic mechanism that involves (a) the inhibition of the enzyme acylpeptide hydrolase and (b) the modulation of alpha(7) nicotinic receptors.
    Toxicology and Applied Pharmacology 05/2009; 238(1):37-46. DOI:10.1016/j.taap.2009.04.011 · 3.63 Impact Factor
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    • "Rat plasma was diluted to 320 mg ml (1 and preincubated with pesticide-oxon or solvent for 20 min in vitro at concentration/times confirmed to produce approximately 30% inhibition of thymus tissue AChE activity (Carter et al. 2007). The actual concentrations used were: azamethiphos (oxon) 0.12 mM, chlorfenvinphos (oxon) 1.5 mM, chlorpyrifos-oxon 0.03 mM, diazinon-oxon 0.04 mM and malaoxon 0.1 mM. "
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    ABSTRACT: We have evaluated the potential of plasma albumin to provide a sensitive biomarker of exposure to commonly used organophosphorus pesticides in order to complement the widely used measure of acetylcholinesterase (AChE) inhibition. Rat or human plasma albumin binding by tritiated-diisopropylfluorophosphate ((3)H-DFP) was quantified by retention of albumin on glass microfibre filters. Preincubation with unlabelled pesticide in vitro or dosing of F344 rats with pesticide in vivo resulted in a reduction in subsequent albumin radiolabelling with (3)H-DFP, the decrease in which was used to quantify pesticide binding. At pesticide exposures producing approximately 30% inhibition of AChE, rat plasma albumin binding in vitro by azamethiphos (oxon), chlorfenvinphos (oxon), chlorpyrifos-oxon, diazinon-oxon and malaoxon was reduced from controls by 9+/-1%, 67+/-2%, 56+/-2%, 54+/-2% and 8+/-1%, respectively. After 1 h of incubation with 19 microM (3)H-DFP alone, the level of binding to rat or human plasma albumins reached 0.011 or 0.039 moles of DFP per mole of albumin, respectively. This level of binding could be further increased by raising the concentration of (3)H-DFP, increasing the (3)H-DFP incubation time, or by substitution of commercial albumins for native albumin. Pesticide binding to albumin was presumed covalent since it survived 24 h dialysis. After dosing rats with pirimiphos-methyl (dimethoxy) or chlorfenvinphos (oxon) (diethoxy) pesticides, the resultant albumin binding were still significant 7 days after dosing. As in vitro, dosing of rats with malathion did not result in significant albumin binding in vivo. Our results suggest albumin may be a useful additional biomonitor for moderately low-level exposures to several widely used pesticides, and that this binding differs markedly between pesticides.
    Biomarkers 07/2008; 13(4):343-63. DOI:10.1080/13547500801973563 · 2.52 Impact Factor
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