Quinones represent an important class of endogenous compounds such as neurotransmitters and coenzyme Q10, electrophilic xenobiotics and environmental toxicants that have known reactivity based on their ability to redox cycle and generate oxidative stress, as well as to alkylate target proteins. 1,4-Benzoquinone (BQ) is a reactive quinone that we have used to help predict target residue covalent binding by such compounds. Hydroquinone glutathione conjugates (HQ-GSH) cause renal cell necrosis by producing reactive oxygen species (ROS) and by adducting proteins preferentially localized in the S3 segment of the renal proximal tubules. In vitro experimentation using model peptides and proteins have identified cysteine, lysine, arginine, and glutamic acid as amino acids targeted for quinone-thioether adduction. By mimicking a standard protein digestion protocol (100 mM ammonium bicarbonate pH 7.5, or 50 mM Tris-HCl pH 7.5), we demonstrated that cysteine-BQ adducts are unstable. Taken together, these results indicate that BQ-adduct formation on cysteine residues may be a transient interaction, where physiological conditions may play a role in adduct stability. In vivo experimentation following administration of 2-(glutathion-S-yl)HQ (MGHQ, 400 μmol/kg, iv, 2 hr) to Long Evans rats identified the specific site of quinone-thioether protein adduction on a number of proteins. Urinary proteins were isolated, and either trypsin digested en masse and analyzed by multi-dimensional protein identification technology (MuDPIT) or, following SDS-PAGE, single immunopositive bands were excised, trypsin digested and analysed by LC-MSMS. Following site-specific identification of adducts, 3-dimensional protein modeling of adducts on the protein was performed as a way to reveal the potential structural consequence of the modification on 3D structure. The outer stripe of the outer medulla (OSOM) is the target site of protein adduction caused by quinone-thioethers. Using a 2DGE-Western blot approach, in combination with an extensive knowledge of quinol-thioether chemistry, LC-MSMS, and the latest MSMS analysis software, we identified the specific amino acid site of adduction on 17 unique peptides from 34 target proteins within the OSOM. Of the 22 bands analyzed, adducted peptides were identified in 11 of them. Many of the target proteins identified have previously been identified as a target of other electrophiles, producing additional evidence that such protein adduction is selective rather than random. The site-specific identification of covalently adducted proteins is a prerequisite for understanding the biological significance of chemical-induced PTMs and the subsequent toxicological response.

Formation of advanced glycation endproducts (AGEs) on proteins has been linked to the pathogenesis of diabetic complications. Importantly, elevated levels of methylglyoxal (MG) occur in type 2 diabetes mellitus (T2DM), and the resulting site-specific formation of stable adducts on arginine residues can cause protein damage. Using MG, site-specific modifications on the plasma protein plasminogen (Pg) were determined following protein digestion into peptides and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis, and 30 arginine sites were identified on the protein. Investigation into three of the most highly modified sites, R504, R530, and R561, using molecular modeling, identified likely functional changes to the Pg cleavage and the lysine binding pocket as a result of adduct formation at these sites. Overall functional changes to Pg were examined using silver staining and kinetic assays to examine normal protein cleavage by activator enzymes streptokinase (STK), tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). MG-modified Pg exhibited decreased activation into plasmin (Pn), which is the active enzyme that forms via normal Pg cleavage, by all three activator enzymes. Activation into Pn by STK was significantly delayed by MG modification on plasminogen. Similar effects were observed with tPA and uPA. Efforts to identify the primary sites of MG adduction on Pg by two dimensional gel electrophoresis (2DGE) identified six sites, including R504 and R530, as the earliest modified sites. In order to probe MG site specific modification effects on lysine binding, MG-modified protein was run through a lysine-sepharose binding column and fractions were collected. The results indicated that MG-modified Pg bound more weakly to the column and eluted easier than unmodified Pg and LC-MS/MS using a LTQ Orbitrap Velos of the fraction indicated that R504 and R530 were the primary sites of MG adduction within the eluate. To assess MG-modification of Pg in humans, 12 plasma samples were immunodepleted of the top 14 abundant proteins and samples were analyzed by LC-MS/MS using a LTQ Orbitrap Velos. Nine of the 12 patient samples indicated the presence of MG-modified peptides. The antihyperglycemic drug metformin, a drug that scavenges MG and lowers formation of AGEs, was studied in order to better elucidate this scavenging mechanism. A novel reaction imidazolinone product, IMZ, was determined to be the primary product formed in the reaction between metformin and MG, confirmed unequivocally through x-ray diffraction analysis. In order to determine levels of IMZ in human patients on metformin therapy, multiple reaction monitoring (MRM) was employed to quantify the compound. Human urine samples from 92 patients on metformin treatment were analyzed. 66 of the 68 patients to exhibit high concentrations of metformin also indicated the presence of IMZ in their urine. The remaining samples either exhibited no metformin, or levels of metformin too low to detect the presence of IMZ. Importantly, IMZ was never identified in patients without a metformin signal, indicating the validity and quality of the assay. This dissertation builds upon the current knowledge of site-specific MG modifications, both in vitro, identifying for the first time Pg as a sensitive site-specific target of glycation, with functional effects, and importantly in humans, as this is the first identification of MG-modified Pg in vivo. The functional effects associated with this modification may provide a link between elevated MG in T2DM, and resulting cardiovascular complications. Additionally, the identification of the novel reaction product IMZ is important, as it helps to fully elucidate the role metformin plays in treating T2DM patients. The detection of IMZ in the urine of human patients on metformin therapy indicates that metformin plays a role in the reducing MG levels through scavenging in vivo, and the developed MRM method allows for future rapid, sensitive study of cohorts to better understand this mechanism and the role it plays in reducing AGEs and diabetic complications.

Defining the mechanics and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. In this dissertation I describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein using quantitative mass spectrometry. Adducts are formed by electrophiles at Cys-14 and Cys-47 on the metabolic enzyme glutathione-S-transferase P1-1 and accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples. This method was used to measure rate constants for adduction at both positions with two different model electrophiles, IAB and BMCC. The results indicate that Cys-47 was approximately 2-3-fold more reactive toward both electrophiles than was Cys-14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system. Quantitative analyses of protein modifications provide a means of determining the reactivity and selectivity of damaging protein modifications in chemical toxicity.Another area of study explored in this dissertation is looking at the effects of protein alkylation on activating cellular signaling pathways, specifically the JNK signaling pathway. Protein adduction has been shown to be selective between different alkylating agents. It would then be reasonable to think this selectivity of adduction translates to selectivity of downstream consequences or cellular events directly tied to specific adductions. My work will show how treatment of HEK293 cells with either IAB or BMCC leads to differences in activation of JNK signaling. In addition, I've been able to show a difference in selectivity of a number of adducted targets by each alkylating agent, which are directly involved in regulation of the JNK signaling pathway. These studies illustrate not only the significance of protein adduction, but the importance for continual research to better understand their behavior in living systems.

<p>The nasal passages are constantly exposed to both air- and bloodborne foreign compounds. In particular, the olfactory mucosa is demonstrated to be susceptible to a variety of drugs and chemicals. In this thesis, mechanisms involved in tissue-selective toxicity in the olfactory mucosa of rodents have been investigated using the olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO₂) as a model compound. Comparative studies were performed with the non-toxic 2,5-dichlorophenyl methylsulphone (2,5-diClPh-MeSO₂) and the reasons for the strikingly different toxicity were investigated. </p><p>A strong bioactivation and protein adduction of 2,6-diClPh-MeSO₂ in olfactory microsomes and S9-fractions of rodents was demonstrated. In contrast, no significant metabolic activation of 2,5-diClPh-MeSO₂ was observed and the bioactivation in the liver for both chlorinated isomers was negligible. <i>In vitro</i> studies with recombinant yeast cell microsomes expressing mouse cytochrome P450 2A5 (CYP2A5) demonstrated a metabolic activation of 2,6-diClPh-MeSO₂. The 2,6-diClPh-MeSO₂-induced lesions and CYP2A5 expression preferentially occurred in Bowman’s glands and sustentacular cells of the olfactory mucosa. A significant depletion of glutathione (GSH) in the olfactory mucosa was demonstrated <i>in vivo</i>, while no changes were observed in the liver. There was a rapid induction of the endoplasmic reticulum (ER)-specific chaperone Grp78, activation of the ER-specific caspase-12 and the downstream caspase-3 in the Bowman’s glands. Electron microscopy revealed swelling of ER and mitochondria and a lost integrity of the Bowman’s glands. </p><p>Based on these results, the proposed mechanism for 2,6-diClPh-MeSO₂-induced toxicity in the olfactory mucosa is bioactivation by CYP2A5 into a reactive intermediate causing protein adduction and GSH-depletion. This is initiating a sequence of downstream events of ER-stress, changes in ion homeostasis, ultrastructural organelle disruption and apoptotic signalling. In spite of the initial apoptotic signals, the terminal phase of apoptosis seemed to be blocked and necrotic features occurred. The predominant expression of CYP2A5 in the olfactory mucosa is proposed to play a key role for the tissue- and cell-specific toxicity induced by 2,6-diClPh-MeSO₂.</p>

The nasal passages are constantly exposed to both air- and bloodborne foreign compounds. In particular, the olfactory mucosa is demonstrated to be susceptible to a variety of drugs and chemicals. In this thesis, mechanisms involved in tissue-selective toxicity in the olfactory mucosa of rodents have been investigated using the olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO2) as a model compound. Comparative studies were performed with the non-toxic 2,5-dichlorophenyl methylsulphone (2,5-diClPh-MeSO2) and the reasons for the strikingly different toxicity were investigated. A strong bioactivation and protein adduction of 2,6-diClPh-MeSO2 in olfactory microsomes and S9-fractions of rodents was demonstrated. In contrast, no significant metabolic activation of 2,5-diClPh-MeSO2 was observed and the bioactivation in the liver for both chlorinated isomers was negligible. In vitro studies with recombinant yeast cell microsomes expressing mouse cytochrome P450 2A5 (CYP2A5) demonstrated a metabolic activation of 2,6-diClPh-MeSO2. The 2,6-diClPh-MeSO2-induced lesions and CYP2A5 expression preferentially occurred in Bowman’s glands and sustentacular cells of the olfactory mucosa. A significant depletion of glutathione (GSH) in the olfactory mucosa was demonstrated in vivo, while no changes were observed in the liver. There was a rapid induction of the endoplasmic reticulum (ER)-specific chaperone Grp78, activation of the ER-specific caspase-12 and the downstream caspase-3 in the Bowman’s glands. Electron microscopy revealed swelling of ER and mitochondria and a lost integrity of the Bowman’s glands. Based on these results, the proposed mechanism for 2,6-diClPh-MeSO2-induced toxicity in the olfactory mucosa is bioactivation by CYP2A5 into a reactive intermediate causing protein adduction and GSH-depletion. This is initiating a sequence of downstream events of ER-stress, changes in ion homeostasis, ultrastructural organelle disruption and apoptotic signalling. In spite of the initial apoptotic signals, the terminal phase of apoptosis seemed to be blocked and necrotic features occurred. The predominant expression of CYP2A5 in the olfactory mucosa is proposed to play a key role for the tissue- and cell-specific toxicity induced by 2,6-diClPh-MeSO2.