Electrophilic Metabolites Research Articles

Identification of Novel Metabolic Pathways of Sitagliptin (STG) by LC/MS and LC/MS2 after Incubations with Rat Hepatocytes

Sitagliptin (STG), a drug for treating Type Ii Diabetes Mellitus (T2DM), has been associated with severe joint pain in some patients. In this paper the metabolic profile of the drug has been investigated in order to determine metabolism and formation of reactive compounds which may contribute to this adverse effect. Metabolism of STG was investigated in vitro by incubation with freshly isolated Sprague-Dawley rat hepatocytes, to characterize Phase I and II metabolites, and the reaction mixture analysed on a zwitter ionic hydrophilic interaction (ZIC®-HILIC) column using LC-MS and LC-MS2 utilising electrospray ionization (ESI) in the positive ion mode. STG was metabolised to yield eleven metabolites, but in total only 3.1% of the parent drug was metabolised over 2 hrs incubation. These metabolites were structurally characterized on the basis of accurate mass analyses and the major metabolic routes for STG determined to be via aromatic oxidation (0.86%) and desaturation of N-C and C-C of the piperazine (0.44%). Novel metabolites of STG detected using these methods included STG N-glucuronide (M6) and a di-ketone metabolite (M4), hydroxylation of both the amine group and aromatic ring followed by formation of glucuronide metabolites (M5, M5’), oxidative desaturation of NH2 and di-hydroxylation of metabolites followed by loss of HF. Also, observed was an N-sulfate metabolite (0.07%) and acetylation followed by glucuronide conjugation was also found in trace amounts (<0.01%). MS2 fragment ions provide additional structural confirmation providing a possible structure for most metabolites such as by fragment ion loss of the glucuronide group (176 Da) from metabolite M5 and loss of the phenolic sulfate (80 Da) of N-Sulfate metabolite (M7). Reduction reaction of piperazine ring probably generates highly electrophilic metabolite of STG, which may be susceptible to produce adverse effects. Furthermore, N-oxidation reaction forming reactive intermediates metabolic to give a hydroxylamine metabolite that may undergo further reactions to yield electrophilic intermediate metabolites.

The role of glutathione transferases in renal cell carcinoma

Mounting evidence suggest that members of the subfamily of cytosolic glutathione S-transferases (GSTs) possess roles far beyond the classical glutathione-dependent enzymatic conjugation of electrophilic metabolites and xenobiotics. Namely, monomeric forms of certain GSTs are capable of forming protein: protein interactions with protein kinases and regulate cell apoptotic pathways. Due to this dual functionality of cytosolic GSTs, they might be implicated in both the development and the progression of renal cell carcinoma (RCC). Prominent genetic heterogeneity, resulting from the gene deletions, as well as from SNPs in the coding and non-coding regions of GST genes, might affect GST isoenzyme profiles in renal parenchyma and therefore serve as a valuable indicator for predicting the risk of cancer development. Namely, GSTs are involved in the biotransformation of several compounds recognized as risk factors for RCC. The most potent carcinogen of polycyclic aromatic hydrocarbon diol epoxides, present in cigarette smoke, is of benzo(a)pyrene (BPDE), detoxified by GSTs. So far, the relationship between GST genotype and BPDE-DNA adduct formation, in determining the risk for RCC, has not been evaluated in patients with RCC. Although the association between certain individual and combined GST genotypes and RCC risk has been debated in a the literature, the data on the prognostic value of GST polymorphism in patients with RCC are scarce, probably due to the fact that the molecular mechanism supporting the role of GSTs in RCC progression has not been clarified as yet.

Second primary malignancies of cancer patients: treatment-related carcinogenesis

Cytostatic drugs used in chemotherapy cause secondary tumors in some patients cured of the antecedent neoplasm. Thus the incidence of second neoplasia for cancer survivors compared with the general population represents a 4-fold to 6-fold increased risk.The review presents recent data on the mechanisms of malignant transformation of cells by cytostatics, on the characteristics of high- risk groups associated with congenital polymorphisms of xenobiotic metabolizing enzymes and DNA repair systems. Increased risk of drug-related cancer was observed in patients with high activity of P450 isoforms, transforming cytotoxic prodrugs into the active electrophilic metabolites and low level of detoxifying enzymes. All things being equal the risk of therapy related neoplasia is higher in patients with low activity of DNA repair enzymes. It is evaluated the possibility of experimental study of new targeted drugs carcinogenic potential in order to minimize the future risk of secondary malignancies.

Formation of sulfur adducts of N-acetyl-p-benzoquinoneimine, an electrophilic metabolite of acetaminophen in vivo: participation of reactive persulfides.

While N-acetyl-p-benzoquinoneimine (NAPQI), an electrophilic metabolite of acetaminophen (APAP), has been found to undergo GSH conjugation associated with its detoxification, interaction of NAPQI with nucleophilic per- and polysulfides produced by cystathionine γ-lyase (CSE), cystathionine β-synthase, and/or other enzymes is not known. In the present study, we found that sulfur adducts such as the NAPQIH2-SSSCys adduct and the NAPQIH2-SSG adduct are produced in biological samples of mice upon APAP exposure. Our in vitro experiments indicated that the formation of these novel APAP metabolites is, at least in part, attributable to the interaction of CysSSnSH produced by CSE and GSH persulfide with APAP-derived NAPQI.

Physiological roles of bacillithiol in intracellular metal processing.

Glutathione (GSH) is an abundantly produced low-molecular-weight (LMW) thiol in many organisms. However, a number of Gram-positive bacteria do not produce GSH, but instead produce bacillithiol (BSH) as one of the major LMW thiols. Similar to GSH, studies have found that BSH has various roles in the cell, including protection against hydrogen peroxide, hypochlorite and disulfide stress. BSH also participates in the detoxification of thiol-reactive antibiotics and the electrophilic metabolite methylglyoxal. Recently, a number of studies have highlighted additional roles for BSH in the processing of intracellular metals. Herein, we examine the potential functions of BSH in the biogenesis of Fe-S clusters, cytosolic metal buffering and the prevention of metal intoxication.

Sustained NRF2 activation in hereditary leiomyomatosis and renal cell cancer (HLRCC) and in hereditary tyrosinemia type 1 (HT1).

The nuclear erythroid 2-like 2 transcription factor (NRF2), is a major regulator of cellular redox balance. Although NRF2 activation is generally regarded as beneficial to human health, recent studies have identified that sustained NRF2 activation is over-represented in many cancers. This raises the question regarding the role of NRF2 activation in the development and progression of those cancers. This review focuses on the mechanisms and the effects of NRF2 activation in two hereditary cancer predisposition syndromes: hereditary leiomyomatosis and renal cell cancer (HLRCC) and hereditary tyrosinemia type 1 (HT1). Because the cancer initiating mutations in these hereditary syndromes are well defined, they offer a unique opportunity to explore the roles of NRF2 activation in the early stages of carcinogenesis. Over the years, a variety of approaches have been utilized to study the biology of HLRCC and HT1. In HLRCC, in vitro studies have demonstrated the importance of NRF2 activation in sustaining cancer cell proliferation. In the mouse model of HT1 however, NRF2 activation seems to protect cells from malignant transformation. In both HT1 and HLRCC, NRF2 activation promotes the clearance of electrophilic metabolites, enabling cells to survive cancer-initiating mutations. Biological insights gained from the hereditary syndromes' studies may shed light on to the roles of NRF2 activation in sporadic tumours.

Modeling Epoxidation of Drug-like Molecules with a Deep Machine Learning Network.

Drug toxicity is frequently caused by electrophilic reactive metabolites that covalently bind to proteins. Epoxides comprise a large class of three-membered cyclic ethers. These molecules are electrophilic and typically highly reactive due to ring tension and polarized carbon–oxygen bonds. Epoxides are metabolites often formed by cytochromes P450 acting on aromatic or double bonds. The specific location on a molecule that undergoes epoxidation is its site of epoxidation (SOE). Identifying a molecule’s SOE can aid in interpreting adverse events related to reactive metabolites and direct modification to prevent epoxidation for safer drugs. This study utilized a database of 702 epoxidation reactions to build a model that accurately predicted sites of epoxidation. The foundation for this model was an algorithm originally designed to model sites of cytochromes P450 metabolism (called XenoSite) that was recently applied to model the intrinsic reactivity of diverse molecules with glutathione. This modeling algorithm systematically and quantitatively summarizes the knowledge from hundreds of epoxidation reactions with a deep convolution network. This network makes predictions at both an atom and molecule level. The final epoxidation model constructed with this approach identified SOEs with 94.9% area under the curve (AUC) performance and separated epoxidized and non-epoxidized molecules with 79.3% AUC. Moreover, within epoxidized molecules, the model separated aromatic or double bond SOEs from all other aromatic or double bonds with AUCs of 92.5% and 95.1%, respectively. Finally, the model separated SOEs from sites of sp2 hydroxylation with 83.2% AUC. Our model is the first of its kind and may be useful for the development of safer drugs. The epoxidation model is available at http://swami.wustl.edu/xenosite.

2-Amino-9H-pyrido[2,3-b]indole (AαC) Adducts and Thiol Oxidation of Serum Albumin as Potential Biomarkers of Tobacco Smoke

2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine formed during the combustion of tobacco. AαC undergoes bioactivation to form electrophilic N-oxidized metabolites that react with DNA to form adducts, which can lead to mutations. Many genotoxicants and toxic electrophiles react with human serum albumin (albumin); however, the chemistry of reactivity of AαC with proteins has not been studied. The genotoxic metabolites, 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC), 2-nitroso-9H-pyrido[2,3-b]indole (NO-AαC), N-acetyloxy-2-amino-9H-pyrido[2,3-b]indole (N-acetoxy-AαC), and their [(13)C6]AαC-labeled homologues were reacted with albumin. Sites of adduction of AαC to albumin were identified by data-dependent scanning and targeted bottom-up proteomics approaches employing ion trap and Orbitrap MS. AαC-albumin adducts were formed at Cys(34), Tyr(140), and Tyr(150) residues when albumin was reacted with HONH-AαC or NO-AαC. Sulfenamide, sulfinamide, and sulfonamide adduct formation occurred at Cys(34) (AαC-Cys(34)). N-Acetoxy-AαC also formed an adduct at Tyr(332). Albumin-AαC adducts were characterized in human plasma treated with N-oxidized metabolites of AαC and human hepatocytes exposed to AαC. High levels of N-(deoxyguanosin-8-yl)-AαC (dG-C8-AαC) DNA adducts were formed in hepatocytes. The Cys(34) was the sole amino acid of albumin to form adducts with AαC. Albumin also served as an antioxidant and scavenged reactive oxygen species generated by metabolites of AαC in hepatocytes; there was a strong decrease in reduced Cys(34), whereas the levels of Cys(34) sulfinic acid (Cys-SO2H), Cys(34)-sulfonic acid (Cys-SO3H), and Met(329) sulfoxide were greatly increased. Cys(34) adduction products and Cys-SO2H, Cys-SO3H, and Met(329) sulfoxide may be potential biomarkers to assess exposure and oxidative stress associated with AαC and other arylamine toxicants present in tobacco smoke.

Use of nuclear magnetic resonance-based metabolomics to characterize the biochemical effects of naphthalene on various organs of tolerant mice.

Naphthalene, the most common polycyclic aromatic hydrocarbon, causes airway epithelium injury in mice. Repeated exposure of mice to naphthalene induces airway epithelia that are resistant to further injury. Previous studies revealed that alterations in bioactivation enzymes and increased levels of gamma-glutamylcysteine synthase in the bronchioles protect tolerant mice from naphthalene and its reactive metabolites. In our current study, tolerance was induced in male ICR mice using a total of 7 daily intraperitoneal injections of naphthalene (200 mg/kg). Both naphthalene-tolerant and non-tolerant mice were challenged with a dose of 300 mg/kg naphthalene on day 8 to investigate metabolite differences. The lungs, liver, and kidneys were collected for histopathology 24 h after the challenge dose. Bronchial alveolar lavage fluid (BALF) and both hydrophilic and hydrophobic extracts from each organ were analyzed using nuclear magnetic resonance (NMR)-based metabolomics. The histological results showed no observable injuries to the airway epithelium of naphthalene-tolerant mice when compared with the control. In contrast, airway injuries were observed in mice given a single challenge dose (injury mice). The metabolomics analysis revealed that the energy metabolism in the lungs of tolerant and injury mice was significantly perturbed. However, antioxidant metabolites, such as glutathione and succinate, were significantly increased in the lungs of tolerant mice, suggesting a role for these compounds in the protection of organs from naphthalene-induced electrophilic metabolites and free radicals. Damage to the airway cellular membrane, as shown by histopathological results and increased acetone in the BALF and perturbation of hydrophobic lung extracts, including cholesterol, phosphorylcholine-containing lipids, and fatty acyl chains, were observed in injury mice. Consistent with our histopathological results, fewer metabolic effects were observed in the liver and kidney of mice after naphthalene treatments. In conclusion, NMR-based metabolomics reveals possible mechanisms of naphthalene tolerance and naphthalene-induced toxicity in the respiratory system of mice.

Site of reactivity models predict molecular reactivity of diverse chemicals with glutathione.

Drug toxicity is often caused by electrophilic reactive metabolites that covalently bind to proteins. Consequently, the quantitative strength of a molecule's reactivity with glutathione (GSH) is a frequently used indicator of its toxicity. Through cysteine, GSH (and proteins) scavenges reactive molecules to form conjugates in the body. GSH conjugates to specific atoms in reactive molecules: their sites of reactivity. The value of knowing a molecule's sites of reactivity is unexplored in the literature. This study tests the value of site of reactivity data that identifies the atoms within 1213 reactive molecules that conjugate to GSH and builds models to predict molecular reactivity with glutathione. An algorithm originally written to model sites of cytochrome P450 metabolism (called XenoSite) finds clear patterns in molecular structure that identify sites of reactivity within reactive molecules with 90.8% accuracy and separate reactive and unreactive molecules with 80.6% accuracy. Furthermore, the model output strongly correlates with quantitative GSH reactivity data in chemically diverse, external data sets. Site of reactivity data is nearly unstudied in the literature prior to our efforts, yet it contains a strong signal for reactivity that can be utilized to more accurately predict molecule reactivity and, eventually, toxicity.

High throughput HPLC–ESI−-MS/MS methodology for mercapturic acid metabolites of 1,3-butadiene: Biomarkers of exposure and bioactivation

1,3-Butadiene (BD) is an important industrial and environmental carcinogen present in cigarette smoke, automobile exhaust, and urban air. The major urinary metabolites of BD in humans are 2-(N-acetyl-l-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-l-cystein-S-yl)-2-hydroxybut-3-ene (MHBMA), 4-(N-acetyl-l-cystein-S-yl)-1,2-dihydroxybutane (DHBMA), and 4-(N-acetyl-l-cystein-S-yl)-1,2,3-trihydroxybutyl mercapturic acid (THBMA), which are formed from the electrophilic metabolites of BD, 3,4-epoxy-1-butene (EB), hydroxymethyl vinyl ketone (HMVK), and 3,4-epoxy-1,2-diol (EBD), respectively. In the present work, a sensitive high-throughput HPLC–ESI–-MS/MS method was developed for simultaneous quantification of MHBMA and DHBMA in small volumes of human urine (200μl). The method employs a 96 well Oasis HLB SPE enrichment step, followed by isotope dilution HPLC–ESI−-MS/MS analysis on a triple quadrupole mass spectrometer. The validated method was used to quantify MHBMA and DHBMA in urine of workers from a BD monomer and styrene-butadiene rubber production facility (40 controls and 32 occupationally exposed to BD). Urinary THBMA concentrations were also determined in the same samples. The concentrations of all three BD-mercapturic acids and the metabolic ratio (MHBMA/(MHBMA+DHBMA+THBMA)) were significantly higher in the occupationally exposed group as compared to controls and correlated with BD exposure, with each other, and with BD-hemoglobin biomarkers. This improved high throughput methodology for MHBMA and DHBMA will be useful for future epidemiological studies in smokers and occupationally exposed workers.

15-Hydroxyprostaglandin Dehydrogenase Generation of Electrophilic Lipid Signaling Mediators from Hydroxy Ω-3 Fatty Acids

15-Hydroxyprostaglandin dehydrogenase (15PGDH) is the primary enzyme catalyzing the conversion of hydroxylated arachidonic acid species to their corresponding oxidized metabolites. The oxidation of hydroxylated fatty acids, such as the conversion of prostaglandin (PG) E2 to 15-ketoPGE2, by 15PGDH is viewed to inactivate signaling responses. In contrast, the typically electrophilic products can also induce anti-inflammatory and anti-proliferative responses. This study determined that hydroxylated docosahexaenoic acid metabolites (HDoHEs) are substrates for 15PGDH. Examination of 15PGDH substrate specificity was conducted in cell culture (A549 and primary human airway epithelia and alveolar macrophages) using chemical inhibition and shRNA knockdown of 15PGDH. Substrate specificity is broad and relies on the carbon position of the acyl chain hydroxyl group. 14-HDoHE was determined to be the optimal DHA substrate for 15PGDH, resulting in the formation of its electrophilic metabolite, 14-oxoDHA. Consistent with this, 14-HDoHE was detected in bronchoalveolar lavage cells of mild to moderate asthmatics, and the exogenous addition of 14-oxoDHA to primary alveolar macrophages inhibited LPS-induced proinflammatory cytokine mRNA expression. These data reveal that 15PGDH-derived DHA metabolites are biologically active and can contribute to the salutary signaling actions of Ω-3 fatty acids.

Mechanisms of keratinocyte death in Stevens-Johnsonsyndrome/toxic epidermal necrolysis

The apoptosis of keratinocytes involves drug metabolism and immune mechanism. The former mainly refers to the induction of apoptosis by active electrophilic drug metabolites through the mitochondrial pathway. Comparatively, the immune mechanism is more complex with the involvement of many immune cells including CD8+ cytotoxic T cells, natural killer cells, monocytes and regulatory T cells. Some cytokines, such as tumor necrosis factor α(TNF-α), soluble FasL, granulysin, TNF-related apoptosis-inducing ligand(TRAIL)and TNF-like weak inducer of apoptosis(TWEAK), contribute to the amplification of apoptosis. The necrosis of keratinocytes is considered to be associated with the alarm family members S100A, α-defensins and high mobility group protein B1. Key words: Stevens-Johnson syndrome; Epidermal necrolysis, toxic; Keratinocytes; Apoptosis

Induction of GST and related events by dietary phytochemicals: sources, chemistry, and possible contribution to chemoprevention.

This review provides an overview of the chemical constituents of regularly consumed plants that increase the activity or induce expression of glutathione S-transferases (GSTs), a major family of detoxification/ cytoprotective enzymes of ubiquitous occurrence in the body. Since induction of phase II (cytoprotective) enzymes, essentially GSTs, is a principal strategy in deactivation of potential carcinogens, it is reasonable to conclude that phytochemicals that enhance the activity/expression of GST isoforms/isoenzymes may play a role in cancer prevention. In this respect, classes of natural products that exhibit this ability are presented. In addition, their possible contribution to chemoprevention is discussed. GSTs constitute a large family of detoxification enzymes in nature. GSTs has been long known to deactivate electrophilic xenobiotics or metabolites, reactive oxygen species as well as certain endogenous substrates. However, there is a growing appreciation that GSTs may have an even wider relevance to cancer, in that they can directly modulate the activity of a number of protein targets, including other enzymes in redox pathways and in signaling networks of cell division and cell cycle control. The following aspects will be treated herein: botanical sources, phytochemical classes, chemical structures of these natural products, bioactivity relevant to chemoprevention, and their influence on induction of GST in vitro and in animal models. A hint on the SAR of organosulfur compounds, isothiocyanates, and limonoids as GST inducers, is added. The few clinical and/or epidemiological studies that associate GST induction with prevention of carcinogenesis are also reviewed.

A Biotin-PEAC5-maleimide labeling assay to detect electrophiles.

Recently, we established a biotin-PEAC5-maleimide (BPM)-labeling assay, which can be used to determine the modification of electrophilic metals to proteins (Toyama et al., J. Toxicol. Sci., 38, 477-484, 2013). In the present study, we applied a BPM-labeling assay to detect protein S-modification by environmental organic electrophiles. After exposing A431 cells to 1,2-naphthoquinone (1,2-NQ) and 1,4-naphthoquinone (1,4-NQ), there was an inverse correlation between Western blot analysis with specific antibody against these electrophiles and that with BPM on the blot intensity to detect protein modification. Similar results were also observed using enzyme-linked immunosorbent assay (ELISA) with BPM. Modification of proteins in mouse liver cytosol by 5-hydroxy-1,4-NQ, 5,8-dihydroxy-1,4-NQ, 1,4-benzoquinone (1,4-BQ), tert-butyl-1,4-BQ, and N-acetyl-p-benzoquinoneimine, an electrophilic metabolite of acetaminophen, was detected using ELISA, but not non-electrophilic quinones or hydroquinone. We also tested whether ELISA could be used to detect electrophiles contained in the vapor phase of ambient air samples collected in the midtown area of Los Angeles. Taken together, the results suggested that the ELISA, developed in this study, can detect the existence of electrophilic quinones that covalently modify cellular proteins, resulting in modulation of redox-signal transduction pathways or cell damage.

Protective effects of hydrogen sulfide anions against acetaminophen-induced hepatotoxicity in mice.

The key mechanism for hepatotoxicity resulting from acetaminophen (APAP) overdose is cytochrome P450-dependent formation of N-acetyl-p-benzoquinone imine (NAPQI), a potent electrophilic metabolite that forms protein adducts. The fundamental roles of glutathione in the effective conjugation/clearance of NAPQI have been established, giving a molecular basis for the clinical use of N-acetylcysteine as a sole antidote. Recent evidence from in vitro experiments suggested that sulfide anions (S(2-)) to yield hydrogen sulfide anions (HS(-)) under physiological pH could effectively react with NAPQI. This study evaluated the protective roles of HS(-) against APAP-induced hepatotoxicity in mice. We utilized cystathionine γ-lyase-deficient (Cth(-/-)) mice that are highly sensitive to acetaminophen toxicity. Intraperitoneal injection of acetaminophen (150 mg/kg) into Cth(-/-) mice resulted in highly elevated levels of serum alanine/aspartate aminotransferases and lactate dehydrogenase associated with marked increases in oncotic hepatocytes; all of which were significantly inhibited by intraperitoneal preadministration of sodium hydrosulfide (NaHS). NaHS preadministration significantly suppressed APAP-induced serum malondialdehyde level increases without abrogating APAP-induced rapid depletion of hepatic glutathione. These results suggest that exogenous HS(-) protects hepatocytes by directly scavenging reactive NAPQI rather than by increasing cystine uptake and thereby elevating intracellular glutathione levels, which provides a novel therapeutic approach against acute APAP poisoning.

Safrole and the Versatility of a Natural Biophore

Safrole (1), obtained from essential oils from different plant species, has wide application in the chemical industry as a synthetic precursor of piperonyl butoxide, piperonal and drugs such as tadalafil, cinoxacin and levodopa. From the toxicological point of view, it is considered a hepatotoxin through metabolic bioactivation, leading to the formation of electrophilic metabolites, and has been described as an inhibitor of different CYP450 isoenzymes. The versatility of its structure, allowing various chemical transformations, and the biophoric nature of its benzodioxole or methylenedioxy subunit, give it unique features and make it an attractive starting material for the synthesis of compounds with different pharmacological activities. Selected examples of natural and synthetic bioactive compounds containing the benzodioxole system will be discussed, including those from specific contribution of LASSBio®.

Should the incorporation of structural alerts be restricted in drug design? An analysis of structure-toxicity trends with aniline-based drugs.

Certain idiosyncratic adverse drug reactions (IADRs) can be triggered by electrophilic protein-reactive metabolites that are formed in the process of drug metabolism. While methodologies (e.g., structural alert concept in drug design, glutathione (GSH) trapping, and protein covalent binding) for examining reactive metabolite (RM) formation are available, predicting the IADR potential applying these parameters remains a significant challenge. The present work examines toxicity trends associated with the aniline structural alert in the top 200 prescribed drugs of 2011 and recently approved (2009-2013) small molecule drugs, in relation with 30 aniline-based drugs withdrawn from commercial use or associated with a black box warning for IADRs. The aniline sub-structure was found in several drugs from the toxic, most prescribed, and recently approved category. RMs resulting from the bioactivation of the aniline alert was also noted in the three categories chosen for comparison. A major discriminator between the toxic drugs and the majority of drugs in the most-prescribed list, however, was the daily dose--drugs most frequented associated with IADRs were the ones with higher daily doses (exceeding hundreds of milligrams). A greater tolerance for IADRs was also noted with certain drugs intended to treat rare, unmet medical needs (e.g., cancer). Overall, the analysis suggests that optimization of pharmacologic potency and pharmacokinetics that would lead to a lower daily dose, and therefore, a lower body burden of parent drug/metabolites, should be taken into consideration in drug discovery.

LC-MS/MS screening strategy for unknown adducts to N-terminal valine in hemoglobin applied to smokers and nonsmokers.

Electrophilically reactive compounds have the ability to form adducts with nucleophilic sites in DNA and proteins, constituting a risk for toxic effects. Mass spectrometric detection of adducts to N-terminal valine in hemoglobin (Hb) after detachment by modified Edman degradation procedures is one approach for in vivo monitoring of exposure to electrophilic compounds/metabolites. So far, applications have been limited to one or a few selected reactive species, such as acrylamide and its metabolite glycidamide. This article presents a novel screening strategy for unknown Hb adducts to be used as a basis for an adductomic approach. The method is based on a modified Edman procedure, FIRE, specifically developed for LC-MS/MS analysis of N-terminal valine adducts in Hb detached as fluorescein thiohydantoin (FTH) derivatives. The aim is to detect and identify a priori unknown Hb adducts in human blood samples. Screening of valine adducts was performed by stepwise scanning of precursor ions in small mass increments, monitoring four fragments common for the FTH derivative of valine with different N-substitutions in the multiple-reaction mode, covering a mass range of 135 Da (m/z 503-638). Samples from six smokers and six nonsmokers were analyzed. Control experiments were performed to compare these results with known adducts and to check for artifactual formation of adducts. In all samples of smokers and nonsmokers, seven adducts were identified, of which six have previously been studied. Nineteen unknown adducts were observed, and 14 of those exhibited fragmentation patterns similar to earlier studied FTH derivatives of adducts to valine. Identification of the unknown adducts will be the focus of future work. The presented methodology is a promising screening tool using Hb adducts to indicate exposure to potentially toxic electrophilic compounds and metabolites.

Protective roles of aldo-keto reductase 1B10 and autophagy against toxicity induced by p-quinone metabolites of tert-butylhydroquinone in lung cancer A549 cells

tert-Butylhydroquinone (BHQ), an antioxidant used as a food additive, exhibits an anticancer effect at low doses, but is carcinogenic in rodents at high doses. BHQ is metabolized into cytotoxic tert-butylquinone (TBQ), which is further converted to 6-tert-butyl-2,3-epoxy-4-hydroxy-5-cyclohexen-1-one (TBEH) through 6-tert-butyl-2,3-epoxy-4-benzoquinone (TBE). Both TBQ and TBE are cytotoxic, but their toxic mechanisms have not been fully characterized. In this study, we have investigated the toxic mechanisms of TBQ and TBE, and the defense system against the two p-quinones using lung cancer A549 cells. TBQ and TBE, but not BHQ and TBEH, showed cytotoxicity to A549 cells. Neither caspase-3 activation nor an increase in the expression of endoplasmic reticulum stress-associating target genes was observed. TBQ and TBE reacted with reduced glutathione, and significantly decreased the glutathione level in A549 cells, suggesting that the cytotoxicity of the p-quinones is caused by their high electrophilicity reacting with biomolecules. The A549 cells treated with the p-quinones also showed increased levels of autophagic vacuoles and LC3-II protein, which are specific autophagy markers. An autophagy inhibitor, 3-methyladenine (3MA), decreased the LC3-II production by the p-quinones, but enhanced the cytotoxicity induced by TBQ and TBE, suggesting that autophagy contributes to alleviating the p-quinone-triggered cytotoxicity. In addition, the TBE-induced cytotoxicity and autophagy activation in the cells were significantly suppressed by overexpression of aldo-keto reductase (AKR)1B10 that efficiently reduces TBE into TBEH, and were augmented by pretreatment with a potent AKR1B10 inhibitor, C1. The effects of 3MA and C1 on the TBE-induced cytotoxicity were additive. The data provides evidence for the first time that autophagy and AKR1B10 contribute to the defense system against the cytotoxicity caused by the electrophilic p-quinone metabolites of BHQ.