GSH adducts

FIGURE 8.5 Simultaneous detection and identification of clozapine-GSH adduct in one single injection ... 

A) mass spectrum from negative precursor ion scan of 272 detected the clozapine-GSH adduct at m/z 630 ... 

However, the temptation to link glutathione (GSH) adducts to MBI must be avoided since the identification of metabolite adducts with GSH does not confirm MBI, albeit it may be an indicator of other adverse drug reactions (Walgren et al., 2005). By definition, exogenous nucleophiles such as GSH should offer no protection from MBI [161], and in principle, even when GSH adducts are formed from the same chemical entity responsible for MBI, the exogenous adducts themselves may not be related to the enzyme-inactivating species [174]. 

This reaction is mediated by an enzyme called a glutathione transferase, which facilitates the encounter of GSH and the compound it is attacking (Mannervik, 1985 Vuilleumier, 1997). Formation of the GSH adduct (i.e., the compound formed when the two reactants are attached to one another) permits the attack of water on the previously chlorinated carbon and since the resulting intermediate is not particularly stable in this case, it decomposes, releasing formaldehyde and regenerating glutathione in a reaction much like the dehydration of gem-diols ... 

Figure 4.21. Nominal mass isobaric interferences for the GSH adduct of nefazodone when tandem quadmpole mass spectrometry is used.

Figure 4.23. Low and high energy acquisitions denoting GSH adducts of nefazodone which correspond to the loss of the pyroglutamic acid and further confirmed in the low energy trace with exact mass.

Figure 4.24. LC-MS (bottom panel) and MS/MS (top panel) spectra of GSH adduct of nefazo-done obtained using negative ion mode ESI-MS.

Ma, L., Zhang, H., Humphreys, G., Sanders, M., and Zhu, M. (2005). Selective and sensitive detection of GSH adducts in complex biological samples using high resolution LC/FIMS with mass defect filtering. In Proceedings of the 53rd ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, TX. 

Prostaglandin synthetase, peroxidase or lipid peroxidation have been shown to oxidise arylamine xenobiotics to reactive species that bind extensively to DNA. The binding could be an initial event in the toxic or carcinogenic process. Evidence is presented that cation radicals are involved in the formation of the various oxidation products and DNA adduct formation with the carcinogen aminofluorene. Furthermore methylaminoazobenzene (butter yellow) was found to form the same major GSH adduct as is formed in vivo. 

In the presence of glutathione, HCHO and AB formation were inhibited and water-soluble GSH adducts were formed if the peroxidase... 

At pH < 6 or at higher peroxide concentrations, other GSH adducts were also formed but have not yet been identified. Presumably they are formed by further oxidation of GS-CH2-AB and/or GSH adduct formation with other MAB oxidation products. Under optimal conditions in the H202 peroxidase system, 83% of the MAB could be recovered as GS-CH2 AB as calculated from a molar extinction coefficient for GS-CH2 AB of 22,600 M at 395 nm in methanol water 1 1. 

Another consequence of these findings is that the same adduct can be formed by a free radical mediated pathway from MAB following a one electron oxidation by peroxides as that formed from methylol or me thimine by a two electron oxidation catalyzed by cytochrome P450. Clearly identification of the GSH adducts of carcinogens in vivo may not distinguish both metabolic activation systems. It is also still not clear whether cytochrome P450 and peroxidases form common intermediates during N-demethylation reactions (22-24). 

Biotransformation studies involving incubations in human plasma, exposure to tumor cells and dosing to mice have shown that AMD473 is detectable up to 6 h post administration (i.p. or oral) to mice and is mainly biotransformed to aquated activation products [55], Notably, and in contrast to results obtained for JM216 [29], only AMD473 itself was detectable within human ovarian carcinoma cells no GSH adduct was formed [55]. 

Depletion of cellular GSH has been widely studied with hundreds of chemicals including APAP and bromobenzene. These studies demonstrated very clearly that bioactivation followed by GSH adduct formation causes depletion of cytosolic glutathione and oxidative stress as indicated by indicators including enhanced levels of GSSG, lipid peroxidation, and loss of membrane integrity. 

GSTs also catalyze a number of reactions in which an S-substituted GSH adduct is not formed or in which this adduct is oxidized glutathione. Examples of these reactions include the release of nitrate from nitrate esters and the release of cyanide from thiocyanates. Some GSTs also have glutathione peroxidase activity. 

Reactive metabolite GSH adduct formation GSH depletion (e.g., monochlorobimane) [47, 48]... 

DDT dehydrochiorinase has been highly purified from DDT-resis-tant houseflies (47) and has been characterized as a soluble lipoprotein with a molecular weight of about 120,000 and consisting of four equal subunits. Formation of the tetramer reportedly occurs only in the presence of DDT, and GSH is required for dehydrochlorination ( ). In contrast to most GSH-S-transferases there is no evidence for the formation of a DDT-GSH adduct and no evidence that GSH is depleted during the course of the reaction. 

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