Initial Response and Cellular Protection through the Keap1/Nrf2 System during the Exposure of Primary Mouse Hepatocytes to 1,2-Naphthoquinone

Miura, Takashi; Shinkai, Yasuhiro; Jiang, Hai Yan; Iwamoto, Noriko; Sumi, Daigo; Taguchi, Keiko; Yamamoto, Masayuki; Jinno, Hideto; Tanaka-Kagawa, Toshiko; Cho, Arthur K.; Kumagai, Yoshito

doi:10.1021/tx100427p

Cited by 55 publications

(61 citation statements)

References 42 publications

(76 reference statements)

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“…As shown in Scheme 1B, there is little doubt that activation of both AhR and Nrf2 mediated by NQs derived from naphthalene causes increased levels of phase-I enzymes (e.g., CYP1A1), phase-II enzymes (e.g., aldo-keto reductases and UDPglucuronosyltransferases) and phase-III transporters (e.g., multi-drug resistance associated proteins). We therefore speculate that NQs would be unique metabolites that facilitate eventually the metabolism of its parent compound (i.e., naphthalene) associated with detoxification and excretion into extracellular space (Abiko et al, 2011;Kumagai et al, 2012;Miura et al, 2011) (Scheme 1B).…”

Section: Discussionmentioning

confidence: 99%

Covalent binding of quinones activates the Ah receptor in Hepa1c1c7 cells

2015

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-Highly reactive quinone species produced by photooxidation and/or metabolic activation of mono-or bi-aromatic hydrocarbons modulate cellular homeostasis and electrophilic signal transduction pathways through the covalent modification of proteins. Polycyclic aromatic hydrocarbons, but not mono-or bi-aromatic hydrocarbons, are well recognized as ligands for the aryl hydrocarbon receptor (AhR). However, quinone species produced from mono-and bi-aromatic hydrocarbons could potentially cause AhR activation. To clarify the AhR response to mono-and bi-aromatic hydrocarbon quinones, we studied Cyp1a1 (cytochrome P450 1A1) induction and AhR activation by these quinones. We detected Cyp1a1 induction during treatment with quinones in Hepa1c1c7 cells, but not their parent compounds. Nine of the twelve quinones with covalent binding capability for proteins induced Cyp1a1. Cyp1a1 induction mediated by 1,2-naphthoquinone (1,2-NQ), 1,4-NQ, 1,4-benzoquinone (1,4-BQ) and tert-butyl-1,4-BQ was suppressed by a specific AhR inhibitor and was not observed in c35 cells, which do not have a functional AhR. These quinones stimulated AhR nuclear translocation and interaction with the AhR nuclear translocator. Interestingly, 1,2-NQ covalently modified AhR, which was detected by an immunoprecipitation assay using a specific antibody against 1,2-NQ, resulting in enhancement of xenobiotic responsive element (XRE)-derived luciferase activity and binding of AhR to the Cyp1a1 promoter region. While mono-and bi-aromatic hydrocarbons are generally believed to be poor ligands for AhR and hence unable to induce Cyp1a1, our study suggests that the quinones of these molecules are able to modify AhR and activate the AhR/XRE pathway, thereby inducing Cyp1a1. Since we previously reported that 1,2-NQ and tert-butyl-1,4-BQ also activate NF-E2-related factor 2, it seems likely that some of quinones are bi-functional inducers for phase-I and phase-II reaction of xenobiotics.

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Section: Discussionmentioning

confidence: 99%

Covalent binding of quinones activates the Ah receptor in Hepa1c1c7 cells

2015

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“…The modification of protein thiols by electrophiles may cause loss of protein function involved in toxicity (Lopachin and Decaprio, 2005). Our recent findings indicate that thiol-modification of cellular proteins triggers activation of electrophilic signal transduction pathways such as Keap1/Nrf2 (Miura et al, 2011;Toyama et al, 2007) and PTP1B/EGFR (Iwamoto et al, 2007), leading to cell survival. Therefore, detection of S-modification of cellular proteins and identification of sensor proteins modified by electrophiles is a key to elucidating molecular mechanisms of not only intoxication but also detoxification of the electrophiles.…”

Section: Introductionmentioning

confidence: 99%

A convenient method to assess chemical modification of protein thiols by electrophilic metals

et al. 2013

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-Although covalent modification of protein thiols by electrophilic metals is implicated in disruption of protein functions associated with toxicity, there are limited methods available to detect such modifications. In the present study, we established a convenient method to assess modification of protein thiols by electrophiles, referred to as a biotin-PEAC 5 -maleimide (BPM)-labeling assay. In this assay, protein S-modification by electrophiles can be estimated by a decrease in protein modification by BPM, a thiol reactive probe. Using methylmercury (MeHg) as a model electrophilic metal, thiol modification of cellular proteins was detected by the BPM-labeling assay in SH-SY5Y cell lysates and primary mouse hepatocytes. The sensitivity and reliability of the assay was confirmed by atomic absorption spectrometry with recombinant Keap1 as a model thiol protein. This assay was applied to not only MeHg but also to other metals such as cadmium and lead. We also established a BPM-precipitation assay with avidin-agarose beads to separate BPM-modified cellular proteins followed by detection with the individual antibodies. This assay was available for detecting MeHg-induced S-modification of cellular Keap1 in SH-SY5Y cells. Taken together, we have developed reliable simple methods to estimate protein S-modification by electrophilic metals.

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“…Acetaminophen (APAP) is a convenient cold remedy that undergoes metabolic activation by cytochrome P-450 isozymes (CYPs) to yield N-acetyl-p-benzoquinoneimine (NAPQI), which readily reacts with hepatic protein through thiol groups, resulting in a severe liver injury (Hinson et al, 2010). Our previous studies indicate that covalent modification of proteins by exogenous electrophiles such as 1,2-NQ, tert-butyl-1,4-BQ (TBQ), and methylmercury (MeHg) activates several redox signal transduction pathways such as the Keap1/Nrf2 pathway, PTP1B/EGFR signaling, and Akt/CREB signaling, leading to cell survival, proliferation, or cell death, respectively Endo et al, 2007Endo et al, , 2011Kobayashi et al, 2009;Iwamoto et al, 2007;Abiko et al, 2011;Miura et al, 2011). To detect covalent modification of cellular proteins by these electrophiles on immune blot analysis, antibodies against them are required.…”

Section: Introductionmentioning

confidence: 99%

A Biotin-PEAC<sub>5</sub>-maleimide labeling assay to detect electrophiles

2015

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-Recently, we established a biotin-PEAC 5 -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 ELI-SA, 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.

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Initial Response and Cellular Protection through the Keap1/Nrf2 System during the Exposure of Primary Mouse Hepatocytes to 1,2-Naphthoquinone

Cited by 55 publications

References 42 publications

Covalent binding of quinones activates the Ah receptor in Hepa1c1c7 cells

Covalent binding of quinones activates the Ah receptor in Hepa1c1c7 cells

A convenient method to assess chemical modification of protein thiols by electrophilic metals

A Biotin-PEAC<sub>5</sub>-maleimide labeling assay to detect electrophiles

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