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Reactive intermediates: molecular and MS-based approaches to assess the functional significance of chemical-protein adducts.

ABSTRACT: Biologically reactive intermediates formed as endogenous products of various metabolic processes are considered important factors in a variety of human diseases, including Parkinson's disease and other neurological disorders, diabetes and complications thereof, and other inflammatory-associated diseases. Chemical-induced toxicities are also frequently mediated via the bioactivation of relatively stable organic molecules to reactive electrophilic metabolites. Indeed, chemical-induced toxicities have long been known to be associated with the ability of electrophilic metabolites to react with a variety of targets within the cell, including their covalent adduction to nucleophilic residues in proteins, and nucleotides within DNA. Although we possess considerable knowledge of the various biochemical mechanisms by which chemicals undergo metabolic bioactivation, we understand far less about the processes that couple bioactivation to toxicity. Identifying specific sites within a protein, which are targets for adduction, can provide the initial information necessary to determine whether such adventitious posttranslational modifications significantly alter either protein structure and/or function. To address this problem, we have developed mass spectrometry (MS)-based approaches to identify specific amino acid targets of electrophile adduction (electrophile-binding motifs), coupled with molecular modeling of such adducts, to determine the potential structural and functional consequences. Where appropriate, functional assays are subsequently conducted to assess protein function.

SUBMITTER: Monks TJ

PROVIDER: S-EPMC4007760 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Reactive intermediates: molecular and MS-based approaches to assess the functional significance of chemical-protein adducts.

Monks Terrence J TJ Lau Serrine S SS

Toxicologic pathology 20121206 2

Biologically reactive intermediates formed as endogenous products of various metabolic processes are considered important factors in a variety of human diseases, including Parkinson's disease and other neurological disorders, diabetes and complications thereof, and other inflammatory-associated diseases. Chemical-induced toxicities are also frequently mediated via the bioactivation of relatively stable organic molecules to reactive electrophilic metabolites. Indeed, chemical-induced toxicities h ...[more]

PMID: 23222993

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Project description:DNA alkylation or adduct formation occurs at nucleophilic sites in DNA, mainly the N7-position of guanine. Ever since identification of the first N7-guanine adduct, several hundred studies on DNA adducts have been reported. Major issues addressed include the relationships between N7-guanine adducts and exposure, mutagenesis, and other biological endpoints. It became quickly apparent that N7-guanine adducts are frequently formed, but may have minimal biological relevance, since they are chemically unstable and do not participate in Watson Crick base pairing. However, N7-guanine adducts have been shown to be excellent biomarkers for internal exposure to direct acting and metabolically activated carcinogens. Questions arise, however, regarding the biological significance of N7-guanine adducts that are readily formed, do not persist, and are not likely to be mutagenic. Thus, we set out to review the current literature to evaluate their formation and the mechanistic evidence for the involvement of N7-guanine adducts in mutagenesis or other biological processes. It was concluded that there is insufficient evidence that N7-guanine adducts can be used beyond confirmation of exposure to the target tissue and demonstration of the molecular dose. There is little to no evidence that N7-guanine adducts or their depurination product, apurinic sites, are the cause of mutations in cells and tissues, since increases in AP sites have not been shown unless toxicity is extant. However, more research is needed to define the extent of chemical depurination versus removal by DNA repair proteins. Interestingly, N7-guanine adducts are clearly present as endogenous background adducts and the endogenous background amounts appear to increase with age. Furthermore, the N7-guanine adducts have been shown to convert to ring opened lesions (FAPy), which are much more persistent and have higher mutagenic potency. Studies in humans are limited in sample size and differences between controls and study groups are small. Future investigations should involve human studies with larger numbers of individuals and analysis should include the corresponding ring opened FAPy derivatives.

Project description:Acetaminophen (APAP) is metabolized in the liver to N-acetyl-p-benzoquinone imine (NAPQI), an electrophilic metabolite known to bind liver proteins resulting in hepatotoxicity. Mammalian thioredoxin reductase (TrxR) is a cellular antioxidant containing selenocysteine (Sec) in its C-terminal redox center, a highly accessible target for electrophilic modification. In the present study, we determined if NAPQI targets TrxR. Hepatotoxicity induced by APAP treatment of mice (300 mg/kg, i.p.) was associated with a marked inhibition of both cytosolic TrxR1 and mitochondrial TrxR2 activity. Maximal inhibition was detected at 1 and 6 h post-APAP for TrxR1 and TrxR2, respectively. In purified rat liver TrxR1, enzyme inactivation was correlated with the metabolic activation of APAP by cytochrome P450, indicating that enzyme inhibition was due to APAP-reactive metabolites. NAPQI was also found to inhibit TrxR1. NADPH-reduced TrxR1 was significantly more sensitive to NAPQI (IC50 = 0.023 μM) than the oxidized enzyme (IC50 = 1.0 μM) or a human TrxR1 Sec498Cys mutant enzyme (IC50 = 17 μM), indicating that cysteine and selenocysteine residues in the redox motifs of TrxR are critical for enzyme inactivation. This is supported by our findings that alkylation of reduced TrxR with biotin-conjugated iodoacetamide, which selectively reacts with selenol or thiol groups on proteins, was inhibited by NAPQI. LC-MS/MS analysis confirmed that NAPQI modified cysteine 59, cysteine 497, and selenocysteine 498 residues in the redox centers of TrxR, resulting in enzyme inhibition. In addition to disulfide reduction, TrxR is also known to mediate chemical redox cycling. We found that menadione redox cycling by TrxR was markedly less sensitive to NAPQI than disulfide reduction, suggesting that TrxR mediates these reactions via distinct mechanisms. These data demonstrate that APAP-reactive metabolites target TrxR, suggesting an additional mechanism by which APAP induces oxidative stress and hepatotoxicity.

Project description:IntroductionFlavor aldehydes in e-cigarettes, including vanillin, ethyl vanillin (vanilla), and benzaldehyde (berry/fruit), rapidly undergo chemical reactions with the e-liquid solvents, propylene glycol, and vegetable glycerol (PG/VG), to form chemical adducts named flavor aldehyde PG/VG acetals that can efficiently transfer to e-cigarette aerosol. The objective of this study was to compare the cytotoxic and metabolic toxic effects of acetals and their parent aldehydes in respiratory epithelial cells.Aims and methodsCell metabolic assays were carried out in bronchial (BEAS-2B) and alveolar (A549) epithelial cells assessing the effects of benzaldehyde, vanillin, ethyl vanillin, and their corresponding PG acetals on key bioenergetic parameters of mitochondrial function. The potential cytotoxic effects of benzaldehyde and vanillin and their corresponding PG acetals were analyzed using the LIVE/DEAD cell assay in BEAS-2B cells and primary human nasal epithelial cells (HNEpC). Cytostatic effects of vanillin and vanillin PG acetal were compared using Click-iT EDU cell proliferation assay in BEAS-2B cells.ResultsCompared with their parent aldehydes, PG acetals diminished key parameters of cellular energy metabolic functions, including basal respiration, adenosine triphosphate production, and spare respiratory capacity. Benzaldehyde PG acetal (1-10 mM) increased cell mortality in BEAS-2B and HNEpC, compared with benzaldehyde. Vanillin PG acetal was more cytotoxic than vanillin at the highest concentration tested while both diminished cellular proliferation in a concentration-dependent manner.ConclusionsReaction products formed in e-liquids between flavor aldehydes and solvent chemicals have differential toxicological properties from their parent flavor aldehydes and may contribute to the health effects of e-cigarette aerosol in the respiratory system of e-cigarette users.ImplicationsWith no inhalation toxicity studies available for acetals, data from this study will provide a basis for further toxicological studies using in vitro and in vivo models. This study suggests that manufacturers' disclosure of e-liquid ingredients at time of production may be insufficient to inform a comprehensive risk assessment of e-liquids and electronic nicotine delivery systems use, due to the chemical instability of e-liquids over time and the formation of new compounds.

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Reactive intermediates: molecular and MS-based approaches to assess the functional significance of chemical-protein adducts.

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Reactive intermediates: molecular and MS-based approaches to assess the functional significance of chemical-protein adducts.

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