Electrophilic Chemicals Research Articles

The glycolytic metabolite methylglyoxal covalently inactivates the NLRP3 inflammasome

The NLRP3 inflammasome promotes inflammation in disease, yet the full repertoire of mechanisms regulating its activity is not well delineated. Among established regulatory mechanisms, covalent modification of NLRP3 has emerged as a common route for the pharmacological inactivation of this protein. Here, we show that inhibition of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) results in the accumulation of methylglyoxal, a reactive metabolite whose increased levels decrease NLRP3 assembly and inflammatory signaling in cells. We find that methylglyoxal inactivates NLRP3 via a non-enzymatic, covalent-crosslinking-based mechanism, promoting inter- and intraprotein MICA (methyl imidazole crosslink between cysteine and arginine) posttranslational linkages within NLRP3. This work establishes NLRP3 as capable of sensing a host of electrophilic chemicals, both exogenous small molecules and endogenous reactive metabolites, and suggests a mechanism by which glycolytic flux can moderate the activation status of a central inflammatory signaling pathway.

Protein Haptenation and Its Role in Allergy.

Humans are exposed to numerous electrophilic chemicals either as medicines, in the workplace, in nature, or through use of many common cosmetic and household products. Covalent modification of human proteins by such chemicals, or protein haptenation, is a common occurrence in cells and may result in generation of antigenic species, leading to development of hypersensitivity reactions. Ranging in severity of symptoms from local cutaneous reactions and rhinitis to potentially life-threatening anaphylaxis and severe hypersensitivity reactions such as Stephen-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), all these reactions have the same Molecular Initiating Event (MIE), i.e. haptenation. However, not all individuals who are exposed to electrophilic chemicals develop symptoms of hypersensitivity. In the present review, we examine common chemistry behind the haptenation reactions leading to formation of neoantigens. We explore simple reactions involving single molecule additions to a nucleophilic side chain of proteins and complex reactions involving multiple electrophilic centers on a single molecule or involving more than one electrophilic molecule as well as the generation of reactive molecules from the interaction with cellular detoxification mechanisms. Besides generation of antigenic species and enabling activation of the immune system, we explore additional events which result directly from the presence of electrophilic chemicals in cells, including activation of key defense mechanisms and immediate consequences of those reactions, and explore their potential effects. We discuss the factors that work in concert with haptenation leading to the development of hypersensitivity reactions and those that may act to prevent it from developing. We also review the potential harnessing of the specificity of haptenation in the design of potent covalent therapeutic inhibitors.

Reactivity of Acrylamides Causes Cytotoxicity and Activates Oxidative Stress Response.

Acrylamides are widely used industrial chemicals that cause adverse effects in humans or animals, such as carcinogenicity or neurotoxicity. The excess toxicity of these reactive electrophilic chemicals is especially interesting, as it is mostly triggered by covalent reactions with biological nucleophiles, such as DNA bases, proteins, or peptides. The cytotoxicity and activation of oxidative stress response of 10 (meth)acrylamides measured in three reporter gene cell lines occurred at similar concentrations. Most acrylamides exhibited high excess toxicity, while methacrylamides acted as baseline toxicants. The (meth)acrylamides showed no reactivity toward the hard biological nucleophile 2-deoxyguanosine (2DG) within 24 h, and only acrylamides reacted with the soft nucleophile glutathione (GSH). Second-order degradation rate constants (kGSH) were measured for all acrylamides with N,N'-methylenebis(acrylamide) (NMBA) showing the highest kGSH (134.800 M-1 h-1) and N,N-diethylacrylamide (NDA) the lowest kGSH (2.574 M-1 h-1). Liquid chromatography coupled to high-resolution mass spectrometry was used to confirm the GSH conjugates of the acrylamides with a double conjugate formed for NMBA. The differences in reactivity between acrylamides and methacrylamides could be explained by the charge density of the carbon atoms because the electron-donating inductive effect of the methyl group of the methacrylamides lowered their electrophilicity and thus their reactivity. The differences in reactivity within the group of acrylamides could be explained by the energy of the lowest unoccupied molecular orbital and steric hindrance. Cytotoxicity and activation of oxidative stress response were linearly correlated with the second-order reaction rate constants of the acrylamides with GSH. The reaction of the acrylamides with GSH is hence not only a detoxification mechanism but also leads to disturbances of the redox balance, making the cells more vulnerable to reactive oxygen species. The reactivity of acrylamides explained the oxidative stress response and cytotoxicity in the cells, and the lack of reactivity of the methacrylamides led to baseline toxicity.

Quantification and Mapping of Alkylation in the Human Genome Reveal Single Nucleotide Resolution Precursors of Mutational Signatures.

Chemical modifications to DNA bases, including DNA adducts arising from reactions with electrophilic chemicals, are well-known to impact cell growth, miscode during replication, and influence disease etiology. However, knowledge of how genomic sequences and structures influence the accumulation of alkylated DNA bases is not broadly characterized with high resolution, nor have these patterns been linked with overall quantities of modified bases in the genome. For benzo(a) pyrene (BaP), a ubiquitous environmental carcinogen, we developed a single-nucleotide resolution damage sequencing method to map in a human lung cell line the main mutagenic adduct arising from BaP. Furthermore, we combined this analysis with quantitative mass spectrometry to evaluate the dose-response profile of adduct formation. By comparing damage abundance with DNase hypersensitive sites, transcription levels, and other genome annotation data, we found that although overall adduct levels rose with increasing chemical exposure concentration, genomic distribution patterns consistently correlated with chromatin state and transcriptional status. Moreover, due to the single nucleotide resolution characteristics of this DNA damage map, we could determine preferred DNA triad sequence contexts for alkylation accumulation, revealing a characteristic DNA damage signature. This new BaP damage signature had a profile highly similar to mutational signatures identified previously in lung cancer genomes from smokers. Thus, these data provide insight on how genomic features shape the accumulation of alkylation products in the genome and predictive strategies for linking single-nucleotide resolution in vitro damage maps with human cancer mutations.

Comparison of urinary mercapturic acid excretions in users of various tobacco/nicotine products.

Urinary mercapturic acids (MAs) are detoxification products for electrophiles occurring in the human body. They are suitable biomarkers of exposure to directly acting electrophilic chemicals or to chemicals which generate the electrophile during its metabolism. We determined the urinary excretion of 19 MAs in habitual users of combustible cigarettes (CCs), electronic cigarettes (ECs), heated tobacco products (HTPs), oral tobacco (OT), and nicotine replacement therapy (NRT) products, and nonusers (NUs) of any tobacco/nicotine products. The 19 MAs are assumed to be physiologically formed primarily from 15 toxicants with three of them belonging to IARC Group 1 (human carcinogen), seven to Group 2A (probable human carcinogen), four to Group 2B (possible human carcinogen), and one to Group 3 (not classifiable as carcinogen). Smoking (CC) was found to be associated with significantly elevated exposure to ethylene oxide (or ethylene), 1,3-butadiene, benzene, dimethylformamide, acrolein, acrylamide, styrene, propylene oxide, acrylonitrile, crotonaldehyde, and isoprene compared with the other user groups and NU. Users of HTPs revealed slight elevation in the MAs related to acrolein, acrylamide, and crotonaldehyde compared with the other non-CC groups. Vaping (EC) was not found to be associated with any of the MAs studied. In conclusion, the determination of urinary MAs is a useful tool for assessing the exposure to toxicants (mainly potential carcinogens) in users of various tobacco/nicotine products. Our data also give cause to clarify the role of vaping (EC) in urinary excretion of DHPMA (precursor: glycidol).

Mapping the dynamics of Nrf2 antioxidant and NFκB inflammatory responses by soft electrophilic chemicals in human liver cells defines the transition from adaptive to adverse responses

A comprehensive understanding of the dynamic activation and crosstalk between different cellular stress response pathways that drive cell adversity is crucial in chemical safety assessment. Various chemicals have electrophilic properties that drive cell injury responses in particular oxidative stress signaling and inflammatory signaling. Here we used bacterial artificial chromosome-based GFP cellular stress reporters with live cell confocal imaging, to systematically monitor the differential modulation of the dynamics of stress pathway activation by six different soft electrophiles: sulforaphane, andrographolide, diethyl maleate, CDDO-Me, ethacrynic acid and tert-butyl hydroquinone. The various soft electrophiles showed differential potency and dynamics of Nrf2 activation and nuclear translocation. These differences in Nrf2 dynamics correlated with distinct activation pattern of Nrf2 downstream targets SRNX1 and HMOX1. All soft electrophiles caused a strong dose dependent suppression of a cytokine-induced NFĸB response represented by suppression of NFĸB nuclear oscillation and inhibition of the downstream target gene activation A20 and ICAM1, which followed the potency of Nrf2 modulation but occurred at higher concentration close to saturation of Nrf2 activation. RNAi-based depletion of RelA resulted in a prolonged presence of Nrf2 in the nucleus after soft electrophile treatment; depletion of Nrf2 caused the induction of NFĸB signaling and activation of its downstream targets A20 and ICAM1. A systematic transcriptome analysis confirmed these effects by soft electrophiles on Nrf2 and NFκB signaling crosstalk in human induced-pluripotent stem cell-derived hepatocyte-like cells. Altogether our data indicate that modulation of Nrf2 by soft electrophiles may have consequences for efficient inflammatory signaling.

HPLC-ESI-HRMS2 Determination of N-(2-Hydroxyethyl)-l-valyl-l-leucine in Human Urine: Method Validation.

Biomonitoring of human exposure to reactive electrophilic chemicals such as ethylene oxide (EO) has been commonly based on the determination of adducts with N-terminal valine in blood protein globin, but a systematic search has also been undertaken to find surrogate markers enabling non-invasive sampling. Recently, N-(2-hydroxyethyl)-L-valyl-L-leucine (HEVL) has been identified as an ultimate cleavage product of EO-adducted globin in the urine of occupationally exposed workers. Herein, full validation of the analytical procedure consisting of solid-phase extraction of HEVL from urine samples (2 mL) followed by high-performance liquid chromatography-electrospray ionization-high-resolution mass spectrometry determination using deuterium-labeled HEVL as an internal standard (IS) is described. Method limit of quantitation is 0.25 ng/mL, and its selectivity is excellent as demonstrated by the invariable ratio of the qualifier and quantifier ion intensities across diverse urine samples and synthetic standard. The linear calibration model was applicable over the whole concentration range tested (0.25-10 ng/mL). The method accuracy assessed as a recovery of HEVL using a spiking experiment was 98-100%. Within-day precision of the method ranged from 1.8% to 3.0%, while the results from consecutive analytical runs conducted within 1 week or within 10-150 weeks differed in the range of 2.2-9.7%. The stability study on urine samples (-20°C up to 3 years, freeze-and-thaw up to 10 cycles) as well as on aqueous solutions (5°C up to 4 months) indicated no relevant changes in HEVL concentration (≤4%) over the time tested. Analytical responses of both HEVL and IS correlated with urinary creatinine as an index of matrix composition, but this matrix effect was mostly eliminated using the HEVL/IS peak area ratio, attaining the IS-normalized relative matrix effect <3%. In conclusion, the method complied successfully with the bioanalytical method validation criteria, making it a reliable tool for HEVL determination in human biomonitoring.

Electrophilic and Drug-Induced Stimulation of NOTCH3 N-terminal Fragment Oligomerization in Cerebrovascular Pathology.

Small vessel disease is a prevalent age-related condition linked to increased risk of dementia and stroke. We investigate the most commonly inherited form, CADASIL, caused by cysteine-involving mutations in NOTCH3. Recent studies highlight accumulation of NOTCH3 N-terminal fragmentation product (NTF) in disease. In vitro, NTF is capable of both spontaneous and catecholamine-enhanced cysteine-mediated oligomerization. Despite well-characterized genetic influence on CADASIL, environmental effects, including medication usage, on disease remain unclear. We studied effects of assorted electrophilic compounds and drugs on NTF oligomerization by SDS-PAGE and dynamic light scattering. We then examined direct proton pump inhibitor-NTF binding with antibodies designed against proton pump inhibitor-labeled proteins and mass spectrometry. Finally, we used monoclonal NTF antibodies with Proximity Ligation Assay to identify NTF oligomers in 3 CADASIL and 2 age-matched control brains. We identified enhancement of NTF oligomerization by two electrophilic cysteine-modifying compounds, N-ethylmaleimide and iodoacetamide, and an electrophilic compound capable of oxidizing cysteines, ferric chloride. Electrophilic clinical drugs (fenoldopam, omeprazole, tenatoprazole, lansoprazole, and rabeprazole) also promoted oligomerization, and we identified direct omeprazole-NTF and tenatoprazole-NTF complexes. Additionally, we provide novel evidence of NTF multimers in human CADASIL brains. A broad array of electrophilic chemicals, including clinically relevant drugs, influences oligomerization of a pathological CADASIL protein, providing mechanistic insight into disease protein oligomerization. We posit that environmental influences, which may include usage of electrophilic drugs, may affect CADASIL presentations.

Glucocorticoid receptor signaling represses the antioxidant response by inhibiting histone acetylation mediated by the transcriptional activator NRF2

NRF2 (nuclear factor erythroid 2-related factor 2) is a key transcriptional activator that mediates the inducible expression of antioxidant genes. NRF2 is normally ubiquitinated by KEAP1 (Kelch-like ECH-associated protein 1) and subsequently degraded by proteasomes. Inactivation of KEAP1 by oxidative stress or electrophilic chemicals allows NRF2 to activate transcription through binding to antioxidant response elements (AREs) and recruiting histone acetyltransferase CBP (CREB-binding protein). Whereas KEAP1-dependent regulation is a major determinant of NRF2 activity, NRF2-mediated transcriptional activation varies from context to context, suggesting that other intracellular signaling cascades may impact NRF2 function. To identify a signaling pathway that modifies NRF2 activity, we immunoprecipitated endogenous NRF2 and its interacting proteins from mouse liver and identified glucocorticoid receptor (GR) as a novel NRF2-binding partner. We found that glucocorticoids, dexamethasone and betamethasone, antagonize diethyl maleate-induced activation of NRF2 target genes in a GR-dependent manner. Dexamethasone treatment enhanced GR recruitment to AREs without affecting chromatin binding of NRF2, resulting in the inhibition of CBP recruitment and histone acetylation at AREs. This repressive effect was canceled by the addition of histone deacetylase inhibitors. Thus, GR signaling decreases NRF2 transcriptional activation through reducing the NRF2-dependent histone acetylation. Consistent with these observations, GR signaling blocked NRF2-mediated cytoprotection from oxidative stress. This study suggests that an impaired antioxidant response by NRF2 and a resulting decrease in cellular antioxidant capacity account for the side effects of glucocorticoids, providing a novel viewpoint for the pathogenesis of hypercorticosteroidism.

Diacetyl and related flavorant α-Diketones: Biotransformation, cellular interactions, and respiratory-tract toxicity.

Exposure to diacetyl and related α-diketones causes respiratory-tract damage in humans and experimental animals. Chemical toxicity is often associated with covalent modification of cellular nucleophiles by electrophilic chemicals. Electrophilic α-diketones may covalently modify nucleophilic arginine residues in critical proteins and, thereby, produce the observed respiratory-tract pathology. The major pathway for the biotransformation of α-diketones is reduction to α-hydroxyketones (acyloins), which is catalyzed by NAD(P)H-dependent enzymes of the short-chain dehydrogenase/reductase (SDR) and the aldo-keto reductase (AKR) superfamilies. Reduction of α-diketones to the less electrophilic acyloins is a detoxication pathway for α-diketones. The pyruvate dehydrogenase complex may play a significant role in the biotransformation of diacetyl to CO2. The interaction of toxic electrophilic chemicals with cellular nucleophiles can be predicted by the hard and soft, acids and bases (HSAB) principle. Application of the HSAB principle to the interactions of electrophilic α-diketones with cellular nucleophiles shows that α-diketones react preferentially with arginine residues. Furthermore, the respiratory-tract toxicity and the quantum-chemical reactivity parameters of diacetyl and replacement flavorant α-diketones are similar. Hence, the identified replacement flavorant α-diketones may pose a risk of flavorant-induced respiratory-tract toxicity. The calculated indices for the reaction of α-diketones with arginine support the hypothesis that modification of protein-bound arginine residues is a critical event in α-diketone-induced respiratory-tract toxicity.

Plasma nitrogenation of polymer surfaces with a new type of combinatorial plasma‐printing reactor

A new method for combinatorial area‐selective polymer modification or deposition using DBD‐type plasmas at atmospheric pressure (“plasma‐printing”) is presented. Thereby a gradient of two gases perpendicular to the flow direction is established by triangular‐shaped overlapping gas inlets. Design of this reactor allows generation of spot arrays with controlled gradients of physicochemical surface properties which is first applied by investigating the influence of H2 admixtures to N2‐plasma‐treatment of polymers. Formation of functional groups is quantitatively characterized by ATR FT‐IR with preceding chemical derivatization using nucleophilic and electrophilic chemicals. It is shown that treatments using high gas flow velocities are more efficient than treatments with low gas speed. Also it is shown that direct plasma treatment leads to polymer surfaces with nucleophilic and electrophilic properties and that H2 content influences formation of both types of functionalities.

Pulmonary effects by diacetyl exposure

Exposure to diacetyl and related α-diketones causes respiratory-tract damage in humans and experimental animals. Chemical toxicity is often associated with covalent modification of cellular nucleophiles by electrophilic chemicals. Electrophilic α-diketones may covalently modify nucleophilic arginine residues in critical proteins and, thereby, produce the observed respiratory-tract pathology. The major pathway for the biotransformation of α-diketones is reduction to α-hydroxyketones (acyloins), which is catalyzed by NAD(P)H-dependent enzymes of the short-chain dehydrogenase/reductase (SDR) and the aldo-keto reductase (AKR) superfamilies. Reduction of α-diketones to the less electrophilic acyloins is a detoxication pathway for α-diketones. The pyruvate dehydrogenase complex may play a significant role in the biotransformation of diacetyl to CO2. The interaction of toxic electrophilic chemicals with cellular nucleophiles can be predicted by the hard and soft, acids and bases (HSAB) principle. Application of the HSAB principle to the interactions of electrophilic α-diketones with cellular nucleophiles shows that α-diketones react preferentially with arginine residues. Furthermore, the respiratory-tract toxicity and the quantum-chemical reactivity parameters of diacetyl and replacement flavorant α-diketones are similar. Hence, the identified replacement flavorant α-diketones may pose a risk of flavorant-induced respiratory-tract toxicity. The calculated indices for the reaction of α-diketones with arginine support the hypothesis that modification of protein-bound arginine residues is a critical event in α-diketone-induced respiratory-tract toxicity.

In vivo and in vitro inhibition of rat liver glutathione transferases activity by extracts from Combretum zeyheri (Combretaceae) and Parinari curatellifolia (Chrysobalanaceae).

BackgroundParinari curatellifolia and Combretum zeyheri are medicinal plants used in Zimbabwe and other Southern African countries for stomach ailments, fever, body aches, wound healing, cancer and tuberculosis. Glutathione transferases (GSTs) are mammalian enzymes that play a significant role in the detoxification and metabolism of many xenobiotic and endogenous compounds and as such can interact with many exogenous compounds including herbal medicines.The effects of Parinari curatellifolia and Combretum zeyheri leaf extracts on glutathione transferases of male Sprague–Dawley rats were investigated in vivo and in vitro after oral administration of either leaf ethanol or water extracts of each plant.MethodsFor Parinari curatellifolia, 18 male Sprague-Dawley rats were administered with 0, 500 and 1000 mg/kg body weight of the leaf extracts in corn oil or saline. Animals were sacrificed after 96 h and the kidney and liver samples were removed and used to prepare the cytosolic fractions. GST activity was determined using 1-chloro-2, 4-dinitrobezene. For Combretum zeyheri, twenty four male Sprague–Dawley rats were randomly divided into two groups. These two groups were further divided into three groups of four animals each. They were given either the aqueous or ethanol extract at doses of C. zeyheri at 0, 50 mg/kg body weight and 200 mg/kg body weight. The extracts were administered orally by oral gavage for four consecutive days and the rats were sacrificed by cervical dislocation on the fifth day.ResultsIn animals administered with C. zeyheri, GST activity was significantly increased by the 200 mg/kg aqueous extract in the kidneys and livers in vivo whilst the ethanolic extract at 200 mg/kg decreased enzyme activity significantly both organs. Both the ethanol and aqueous extracts inhibited GST activity in vitro with the ethanol extract being more potent inhibitor than ethacrynic acid, a standard GST inhibitor. The increased GST activity in vivo and versus inhibition in vitro suggests that metabolites may be responsible for the effects observed in vivo. For P. curatellifolia, a dose-dependent decrease in GST activity was observed in vivo for the animals given the aqueous extract but no changes were observed with the ethanol extract. There was a concentration-dependent inhibition of cytosolic GSTs when P. curatellifolia leaf extracts in vitro. The ethanol extract of P. curatellifolia exhibited GST-inhibitory activity in the liver with an IC50 value of 12 μg/mL and for ethacrynic acid, the IC50 was found to be 10 μg/mL. This showed that this extract was a potent inhibitor of GSTs in vitro.ConclusionsC. zeyheri had an inductive effect on GST activity when administered in aqueous solution but inhibited GST in vitro whilst P. curatellifolia inhibited GST activity in vivo. Induction of GSTs would be cytoprotective against the toxic effects electrophilic chemicals. Since GSTs are responsible for the synthesis of prostaglandins, the inhibition of GST activity of by these two plants in vivo maybe one of the reasons that makes the plants important for use in the treatment pain and fever in ethnopharmacology.

Diet-related DNA adduct formation in relation to carcinogenesis.

The human diet contributes significantly to the initiation and promotion of carcinogenesis. It has become clear that the human diet contains several groups of natural foodborne chemicals that are at least in part responsible for the genotoxic, mutagenic, and carcinogenic potential of certain foodstuffs. Electrophilic chemicals are prone to attack nucleophilic sites in DNA, resulting in the formation of altered nucleobases, also known as DNA adducts. Since DNA adduct formation is believed to signal the onset of chemically induced carcinogenesis, the DNA adduct-inducing potential of certain foodstuffs has been investigated to gain more insight into diet-related pathways of carcinogenesis. Many studies have investigated diet-related DNA adduct formation. This review summarizes work on known or suspected dietary carcinogens and the role of DNA adduct formation in hypothesized carcinogenesis pathways.

Using novel in vitro methods to predict of the skin sensitization potential of botanical mixtures

Determination of skin sensitization is a critical toxicological endpoint in the safety evaluation of botanical ingredients. With the European Union regulatory ban of animal testing for cosmetic ingredients, and many companies proactively choosing to eliminate animal testing due to ethical considerations, non-animal methods are increasingly being used for safety assessments. The KeratinoSens assay is a cell-based reporter gene assay which may be used to assess the potential of chemicals to induce skin sensitization in humans. A feature all chemical allergens have in common is their intrinsic electrophilicity, or their potential to be transformed to electrophilic chemicals. The Nrf-2-electrophile sensing pathway comprised of the repressor protein Keap1, the transcription factor Nrf2 and the antioxidant response element (ARE), is capable of detecting skin sensitizers. In the KeratinoSens assay, the induction of a luciferase gene under the control of the antioxidant response element derived from the human gene AKR1C2 gene is quantified. Botanical mixtures were tested in the KeratinoSens assay and the resulting data compared to available historical animal data or clinical data. Since the botanical extract portion may only be a small fraction of the complete mixture, as a proof of concept low levels of known sensitizers were spiked into botanical mixtures with different excipient solvent systems and luciferase induction and cytotoxicity measured. Even at levels as low as 1%, the assay was capable of detecting the spiked sensitizers. These data suggest that the KeratinoSens assay has the potential to identify electrophile allergens within a botanical extract mixture.

HDAC inhibitors increase NRF2-signaling in tumour cells and blunt the efficacy of co-adminstered cytotoxic agents.

The NRF2 signalling cascade provides a primary response against electrophilic chemicals and oxidative stress. The activation of NRF2-signaling is anticipated to have adverse clinical consequences; NRF2 is activated in a number of cancers and, additionally, its pharmacological activation by one compound can reduce the toxicity or efficiency of a second agent administered concomitantly. In this work, we have analysed systematically the ability of 152 research, pre-clinical or clinically used drugs to induce an NRF2 response using the MCF7-AREc32 NRF2 reporter. Ten percent of the tested drugs induced an NRF2 response. The NRF2 activators were not restricted to classical cytotoxic alkylating agents but also included a number of emerging anticancer drugs, including an IGF1-R inhibitor (NVP-AEW541), a PIM-1 kinase inhibitor (Pim1 inhibitor 2), a PLK1 inhibitor (BI 2536) and most strikingly seven of nine tested HDAC inhibitors. These findings were further confirmed by demonstrating NRF2-dependent induction of endogenous AKR genes, biomarkers of NRF2 activity. The ability of HDAC inhibitors to stimulate NRF2-signalling did not diminish their own potency as antitumour agents. However, when used to pre-treat cells, they did reduce the efficacy of acrolein. Taken together, our data suggest that the ability of drugs to stimulate NRF2 activity is common and should be investigated as part of the drug-development process.

Chapter Four - TRPA1 Channels: Chemical and Temperature Sensitivity

Transient receptor potential ankyrin 1 (TRPA1) is a polymodal excitatory ion channel found in sensory neurons of different organisms, ranging from worms to humans. Since its discovery as an uncharacterized transmembrane protein in human fibroblasts, TRPA1 has become one of the most intensively studied ion channels. Its function has been linked to regulation of heat and cold perception, mechanosensitivity, hearing, inflammation, pain, circadian rhythms, chemoreception, and other processes. Some of these proposed functions remain controversial, while others have gathered considerable experimental support. A truly polymodal ion channel, TRPA1 is activated by various stimuli, including electrophilic chemicals, oxygen, temperature, and mechanical force, yet the molecular mechanism of TRPA1 gating remains obscure. In this review, we discuss recent advances in the understanding of TRPA1 physiology, pharmacology, and molecular function.

Protease-activated receptor-2 activates NQO-1 via Nrf2 stabilization in keratinocytes

BackgroundProtease-activated receptor-2 (PAR-2) mediates inflammation and immune responses by serine proteinases. NF-E2-related factor 2 (Nrf2) confers protection against tissue injury through antioxidant responses to oxidative stress induced by a variety of factors, including electrophilic chemicals, hydrogen peroxide, and ultraviolet irradiation. ObjectiveIn this study, we investigated if PAR-2 activation can stimulate Nrf2 signaling to preserve homeostasis in keratinocytes. MethodsWe performed western blotting, real-time reverse transcription polymerase chain reaction, and immunocytochemistry of keratinocyte cultures, as well as immunohistochemical labeling of human skin samples. Short interfering RNA (siRNA) was employed to confirm the effects of PAR-2 activation. ResultsPAR-2 activation with a selective PAR-2 agonist peptide increased the nuclear level of Nrf2 protein and subsequently induced phase II enzyme expression. Nrf2 signaling via PAR-2 activation was confirmed with experiments using PAR-2-siRNA-treated keratinocytes. The activation of an Nrf2-targeted gene by PAR-2 activation was not induced by new production of Nrf2 but by prolonged stabilization of Nrf2. Lesional skin samples from vitiligo patients showed significantly lower expression of PAR-2 and Nrf2 than control skin samples. ConclusionCollectively, PAR-2 activation enhanced nuclear Nrf2 translocation, and PAR-2-mediated Nrf2 activation was attributable to existing Nrf2 stabilization rather than de novo production. Our findings suggest that in addition to induction of inflammation, PAR-2 can play a chemopreventative role via Nrf2 stabilization in keratinocytes.

Allergic Skin Inflammation Induced by Chemical Sensitizers Is Controlled by the Transcription Factor Nrf2

Allergic contact dermatitis (ACD) is induced by low-molecular weight electrophilic chemicals and metal ions. Chemical contact sensitizers trigger reactive oxygen species production and provoke electrophilic stress, leading to the accumulation of the transcription factor nuclear-related factor 2 (Nrf2) in innate immune cell types. The objective of this work was to identify the role of Nrf2 in the regulation of ACD. We used the local lymph node assay (LLNA) and the mouse ear swelling test (MEST) to study the role of Nrf2 in both the sensitization and elicitation phase in nrf2 knockout (nrf2(-/-)) and wild-type (nrf2(+/+)) mice. Five chemicals were used: two compounds known to react with cysteine residues, 2,4-dinitrochlorobenzene (DNCB) and cinnamaldehyde (CinA); one sensitizer known to exhibit mixed reactivity to cysteine and lysine residues, isophorone diisocyanate; and one reacting specifically with lysine residues, trimellitic anhydride and croton oil, a well-known irritant. In the MEST assay, DNCB (1 and 2%) induced a significant increase in ear thickness in nrf2(-/-) compared with nrf2(+/+) mice, suggesting a role for Nrf2 in the control of the inflammatory process. When DNCB was used at 0.25 and 0.5% or when mice were treated with CinA, inflammation was found only in nrf2(-/-) mice. In the LLNA, all chemical sensitizers induced an increase of lymphocyte proliferation in nrf2(-/-) compared with nrf2(+/+) mice for the same chemical concentration. These results reveal an important role for Nrf2 in controlling ACD and lymphocyte proliferation in response to sensitizers.

Regulation of Phase I and Phase II Enzyme Gene Expressions and their activities by caffeic acid, major compound of perilla frutescens.

Caffeic acid (CA) is a natural polyphenol containing carboxylic acid found in many herbs, and known as an anti‐inflammatory, anti‐tumorigenic and anti‐cancer effect in vitro and in vivo. And cytochrome P450 1A1/2 (CYP1A1/2) are members of the cytochrome P450 superfamily of enzymes involved in xenobiotic and metabolism. These enzymes play a major role in bioactivation of polycyclic aromatic hydrocarbons (PAHs) and other environmental pro‐carcinogens into their carcinogenic forms. Because of this, the accumulations of CYP1A1/2 remain a focus of interest in cancer research and food toxicology. Phase II enzymes such as heme oxygenase‐1 (HO‐1) and quinone reductase (QR) catalyze conjugation by sulfation, glucuronidation, and neutralize electrophilic chemicals to facilitate elimination of activated chemical compound by CYP1A1/2. In this study, we investigated the potential ability of CA to induce CYP1A enzymes in HepG2 human hepatoma cells. Our results showed that CA increases CYP1A1/2 mRNA expression dose‐dependently. Also gene expressions of HO‐1 and QR (phase II enzymes), were significantly up‐regulated with CA treatment. Thus, these data suggest that CA has potentials of enhancing capacity on CYP1A1/2 expressions (phase I enzymes) and also increasing HO‐1 and QR enzymes (pahse II enzymes), and may prove to be an effective chemopreventive agent.