Detection of Adduct on Tyrosine 411 of Albumin in Humans Poisoned by Dichlorvos

Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska 68198-5950, USA.
Toxicological Sciences (Impact Factor: 3.85). 07/2010; 116(1):23-31. DOI: 10.1093/toxsci/kfq117
Source: PubMed


Studies in mice and guinea pigs have shown that albumin is a new biomarker of organophosphorus toxicant (OP) and nerve agent exposure. Our goal was to determine whether OP-labeled albumin could be detected in the blood of humans exposed to OP. Blood from four OP-exposed patients was prepared for mass spectrometry analysis by digesting 0.010 ml of serum with pepsin and purifying the labeled albumin peptide by offline high performance liquid chromatography. Dimethoxyphosphate-labeled tyrosine 411 was identified in albumin peptides VRY411TKKVPQVSTPTL and LVRY411TKKVPQVSTPTL from two patients who had attempted suicide with dichlorvos. The butyrylcholinesterase activity in these serum samples was inhibited 80%. A third patient whose serum BChE activity was inhibited 8% by accidental inhalation of dichlorvos had undetectable levels of adduct on albumin. A fourth patient whose BChE activity was inhibited 60% by exposure to chlorpyrifos had no detectable adduct on albumin. This is the first report to demonstrate the presence of OP-labeled albumin in human patients. It is concluded that tyrosine 411 of human albumin is covalently modified in the serum of humans poisoned by dichlorvos and that the modification is detectable by mass spectrometry. The special reactivity of tyrosine 411 with OP suggests that other proteins may also be modified on tyrosine. Identification of other OP-modified proteins may lead to an understanding of neurotoxic symptoms that appear long after the initial OP exposure. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected]
/* */

Download full-text


Available from: Florian Nachon
  • Source
    • "The literature contains only two previous publications on OP adducts on protein tyrosine in humans (Li et al., 2010d; van der Schans et al., 2013). The subjects had poisoned themselves with dichlorvos or chlorpyrifos (Li et al., 2010d; van der Schans et al., 2012). Two dichlorvos-poisoned subjects had dimethoxyphosphorylated albumin and monomethoxyphosphorylated butyrylcholinesterase in their plasma. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos.
    Full-text · Article · Apr 2013 · Toxicology and Applied Pharmacology
  • Source
    • "Based on the instability of phosphonylated adducts to AChE and BChE, the detection of HSA-OP adducts represent a more feasible strategy to detect in vivo biomarkers of OP exposure because the abundance of serum albumin is 10,000 times more than either AChE or BChE and the Tyr 411 adduct is extremely stable (Ding et al., 2008). Tyr 411 OP adducts of HSA have been reported in a number of cases of OP exposure (Peeples et al., 2005; Li et al., 2010; Lockridge and Schopfer, 2010; Marsillach et al., 2011). Protein sequence alignment of serum albumin indicates that across different species, including humans, rats, and guinea pigs, the region surrounding Tyr 411 is highly conserved (Fig. 1). "
    [Show abstract] [Hide abstract]
    ABSTRACT: A major challenge in organophosphate (OP) research has been the identification and utilization of reliable biomarkers for the rapid, sensitive and efficient detection of OP exposure. Although tyrosine (Tyr) 411 OP adducts to human serum albumin (HSA) have been suggested to be one of the most robust biomarkers in the detection of OP exposure, the analysis of HSA-OP adduct detection has been limited to techniques using mass spectrometry. Herein, we describe the procurement of two monoclonal antibodies (mAb-HSA-GD and mAb-HSA-VX) that recognized the HSA Tyr 411 adduct of soman (GD) or VX, respectively, but did not recognize non-phosphonylated HSA. We showed that mAb-HSA-GD was able to detect the HSA Tyr 411 OP adduct at a low level (i.e., human blood plasma treated with 180 nM soman) that could not be detected by mass spectrometry. mAb-HSA-GD and mAb-HSA-VX showed an extremely low level detection of GD-adducted to HSA (on the order of picograms or 10-12 g). mAb-HSA-GD could also detect serum albumin OP adducts in blood plasma samples from different animals administered GD including rat, guinea pig and monkey. The ability of the two antibodies to selectively recognize nerve agents adducted to serum albumin suggests that these antibodies could be used to identify biomarkers of OP exposure and provide a new biological approach to detect OP exposure in animals.
    Preview · Article · Nov 2012 · Journal of Pharmacology and Experimental Therapeutics
  • Source
    • "To date only one study has examined both BChE and albumin adducts in the plasma of human subjects exposed to OP (Li et al., 2010). BChE reacts more readily than albumin with OP. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity is an established biomarker of exposure to organophosphorus poisons (OP). Inhibition of activity is due to covalent binding of the OP to the active site serine. Mass spectrometry has made it possible to monitor OP exposure by analyzing OP adducts on tyrosine in proteins that have no active site serine. Our goal was to test the hypothesis that OP-tyrosine may serve as a biomarker of OP exposure in mice. A MALDI-TOF mass spectrometry strategy to analyze diethoxyphosphate-tyrosine of m/z 318 was developed. The adduct was synthesized by incubating l-tyrosine with chlorpyrifos oxon at pH 8.1. The adduct eluted from a reverse phase HPLC column with 22-23% acetonitrile. The fragmentation spectrum of the m/z 318 precursor ion confirmed its identity as diethoxyphosphate-tyrosine. Diethoxyphosphate-tyrosine was isolated from chlorpyrifos oxon treated mouse albumin after digesting the protein with pronase. Mice (n=3 per group) were treated with a nontoxic dose of chlorpyrifos oxon (3 mg/kg) and a toxic dose (10 mg/kg transdermally). The pronase digested plasma yielded diethoxyphosphate-tyrosine up to 120 h after treatment with 3 mg/kg chlorpyrifos oxon and up to 144 h after 10 mg/kg. In contrast plasma AChE activity returned to normal after 24-72 h. In conclusion MALDI-TOF mass spectrometry can be used to diagnose exposure to chlorpyrifos oxon days after AChE inhibition assays are uninformative.
    Full-text · Article · Mar 2012 · Toxicology
Show more