Adducts with protein, detection

Acrylonitrile forms adducts with proteins and glutathione. It also forms DNA adducts in vitro, but only after cytochrome P450 bioactivation, most likely through its epoxide metabolite (cyanoethylene oxide), which is also formed in vivo. Acrylonitrile-haemoglobin adducts have been detected in exposed workers. 

Elaboration of nerve agent toxicokinetics requires sophisticated analytical tools to detect and, if possible, to quantify the free toxicants as well as adducts with proteins and enzymes. Analysis of OP nerve agents has been performed by capillary electrophoresis (CE), biosensors, matrix-assisted laser desorption/ionization (MALDI) MS, desorption electrospray ionization MS (DESI MS), ion mobility time-of-flight MS (IM-TOF MS), nuclear magnetic resonance spectroscopy (NMR), LC-UV, gas chromatography (GC), and many more techniques (Hooijschuur et al, 2002 John et al, 2008). 

As discussed above, the majority of ultimate chemical mutagens and chemical carcinogens and also many cytotoxic agents possess electrophilic reactivity. It is generally believed that reaction between electrophilic centres in such toxicants with nucleophilic centres in informational or important structural or functional macromolecules is the key event in the toxicity of such compounds. Furthermore the determination of covalent adducts with proteins and nucleic acids may provide the basis of a valuable approach not only for the detection of cytotoxic and genotoxic activities but also in discriminating between these activities. 

LC-MS is a powerful method used to detect and quantify CWAs. The use of LC-MS for CWA and hydrolysis product detection has been reviewed [7,20,26]. LC-MS methods are often used to detect CWA hydrolysis/ degradation products instead of the active agents [27-28]. LC-MS serves as a bioanalytical method for CWA detection in living systems and its contributions have also been reviewed [7, 26, 29]. A LC-MS method using an on-line trypsin digestion is used to identify GB and sulfur mustard adducts with proteins and enzymes like human butyryl cholinesterase [30]. This technique, along with similar techniques, could be applied to confirm CWA exposure when illness is suspected from an unknown toxin. 

From these mechanistic studies, specific biomarkers of effective exposure were proposed the DNA adducts ethenodeoxyadenosine and ethenodeoxycytidine. These can be detected in liver biopsy and white blood cells and so can be used to monitor workers. Furthermore, more recently, a specific biomarker of response has also been detected. This is a mutant p21 ras protein, which results from the interaction with DNA and can be detected in the serum of workers exposed to vinyl chloride. The level of this protein detected in workers was found to have increased as exposure to vinyl chloride increased. Therefore, both these biomarkers can be used in risk assessment. 

Muldrew, K. L., James, L. P., Coop, L., McCullough, S. S., Hendrickson, H. P., Hinson, J. A., and Mayeux, P. R. (2002). Determination of acetaminophen-protein adducts in mouse liver and serum and human serum after hepatotoxic doses of acetaminophen using high-performance liquid chromatography with electrochemical detection. Drug Metab. Dispos. 30 446-451. 

Pentosidine is determined by HPLC with spectrofluorimetric detection (excitation and emission wavelengths of 335 and 385 nm, respectively) (S14), although immunochemical and ELISA assays for determination of various protein oxidative modification products have become increasingly popular (08). Protein-aldehyde adducts can be estimated using adduct-specific antibodies (U2, Wl). Another approach requires stabilization of adducts, producing derivatives resistant to conditions used in protein acid hydrolysis and quantification of hydrolysis products by gas chromatography-mass spectrometry (R7). 

Within cells, sulfur mustard forms adducts with DNA, primarily those described in Toxicity of sulfur mustard, above. Adducts can also be formed with nucleophilic sites of amino acids and proteins. Contrary to DNA adducts, there is no specific mechanism to reverse protein adduct formation. For this reason, there is a strong forensic interest in the detection of protein adducts of sulfur mustard as these may provide evidence of sulfur mustard exposure for prolonged periods after the incident. 

Once incorporated, unbound lewisite is quickly hydrolyzed. Its predominant metabolite is 2-chlorovinylarsonous acid, CVAA (Figure 50.8). Analytical methods to confinn lewisite exposure have, at least in the past, focused on the detection and quantification of CVAA. However, Noort et al. (2002) also pointed out that due to the high affinity of arsenic towards sulfhydryl groups, adducts of lewisite/ CVAA and cysteine residues of proteins are formed. In an in vitro study, incubating " C-labeled lewisite with human blood samples, 90% of lewisite was found in erythrocytes, whereas 25 to 50% of arsenic was bound to globin. From these protein adducts, CVAA can be released to form an adduct with the antidote British Anti-Lewisite (BAL) (Fidder et al, 2000). The authors were also able to identify a specific protein adduct of lewisite formed with the cysteine residues 93 and 112 of P-globin. See Detection of DNA and protein adducts of vesicants, below, for analytical... 

Incubation of lewisite-protein adducts with BAL is capable of transferring its metabolite 2-chlorovinylarsonous acid (CVAA) into a BAL-CVAA derivative. This derivative can be quantified using GC-MS. The method is able to detect a 1 nM lewisite exposure of human blood in vitro (Fidder et al, 2000). 

Uvr A, a damage recognition protein, detects helical distortion caused by DNA adducts such as thymine dimers (a). It then associates with Uvr B to form the A2B complex. After binding to the damaged segments, A2B forces DNA to bend. Uvr A then dissociates (b). The binding of the nuclease Uvr C to Uvr B (c) and the action of the helicase Uvr D (d) results in the excision of a 12-nucleotide DNA strand (12-mer). After Uvr B is released (e) the excision gap is repaired by pol I (f). 

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