405
The Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.40, No.3, 405-411, 2015
Original Article
A Biotin-PEAC5-maleimide labeling assay to detect electrophiles Yumi Abiko1, Nho Cong Luong2 and Yoshito Kumagai1,2
2
1Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan Master’s Program of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
(Received March 13, 2015; Accepted March 30, 2015)
ABSTRACT — 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. Key words: Electrophile, Thiol, Cysteine, S-arylation, Biotin-PEAC5-maleimide INTRODUCTION Electrophiles readily bind to nucleophiles such as protein thiol groups, resulting in functional changes of modified proteins. Electrophilic compounds ubiquitously exist in the environment and are taken up during daily living. For example, 1,2- and 1,4-naphthoquinones (1,2-NQ and 1,4-NQ) and 1,4-benzoquinone (1,4-BQ) are reaction products that contaminate the atmosphere during photooxidation of aromatic hydrocarbons such as naphthalene and benzene, and are also found as electrophilic metabolites of these chemicals in the body (Batterman et al., 2012; Cho et al., 2005; Eiguren-Fernandez et al., 2010; Jia and Batterman, 2010; Kautzman et al., 2010). 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 (Kumagai et al., 2012, 2013; Endo et al., 2007, 2011; Kobayashi 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. Thus, a simple and convenient assay to determine protein modification should be established. We have developed a biotin-PEAC 5 -maleimide (BPM)-labeling assay using Western blotting with a horseradish peroxidase (HRP)-linked anti-biotin antibody to detect MeHg-induced S-modification of proteins (Toyama et al., 2013) (see Fig. 1). However, application of this method to organic electrophiles remains to be determined. Because it was reported that maleimide,
Correspondence: Yoshito Kumagai (E-mail: [email protected]) Vol. 40 No. 3
406 Y. Abiko et al.
E Protein
S
Protein
E, electrophiles
BPM
O
O
O
Protein
S
E
BPM
O
N N
S
Biotin
CH3
N
Biotin
N N
CH3
N
O
H3C
Protein
O
S
E
Detected by avidin-HRP Western blotting ELISA Fig. 1.
Schematic strategy of the BPM-labeling assay for detecting quinones bound to cellular proteins.
which has an α,β-unsaturated carbonyl, covalently modifies cysteine residues in proteins (Lind et al., 2002; Muthuramalingam et al., 2013), we hypothesized that covalent modification of proteins by organic electrophiles may be detected using BPM. In this study, we analyzed covalent modification of various quinones to proteins using the BPM labeling assay as shown in Fig. 1. We also examined whether this assay was applicable for monitoring air samples containing environmental electrophiles such as 1,4-BQ, 1,2-NQ, and 1,4-NQ (Cho et al., 2004; Eiguren-Fernandez et al., 2008; Jakober et al., 2007). MATERIALS AND METHODS Materials 1,2-NQ (97.4% purity determined by high-performance liquid chromatography (HPLC)), 1,4-NQ (98% purity determined by gas chromatography (GC)), 5,8-dihydroxy1,4-NQ (79.8% purity determined by HPLC), 1,4-BQ (98% purity determined by iodometric titration), tertbutyl-1,4-benzoquinone (TBQ, 98% purity determined by GC), 2-hydroxy-1,4-NQ (99.9% purity determined by HPLC), and APAP were purchased from Tokyo Chemical Industry (Tokyo, Japan). 2-Methyl-1,4-NQ (98% purity determined by HPLC) was obtained from Nacalai (Kyoto, Japan). 5-Hydroxy-1,4-NQ (95% purity determined by HPLC), NAPQI, BPM, and the HRP-linked anti-biotin antibody were purchased from Sigma-Aldrich Vol. 40 No. 3
(St. Louis, MO, USA), Invitrogen (Carlsbad, CA, USA) and Cell Signaling Technology (Beverly, MA, USA), respectively. Vapor phase samples were collected in the mid-town area of Los Angeles near the University of Southern California during June (from 17th to 22th, VP1) and July (from 1st to 7th, VP2) 2008 and extracted as reported previously (Eiguren-Fernandez et al., 2008, 2010). All other reagents were of the highest grade available. Cell culture Human epithelial carcinoma A431 cells were obtained from the RIKEN Cell Bank (Tsukuba, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium (Wako, Osaka, Japan) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-alanyl-L-glutamine in an incubator supplemented with 5% CO2 at 37°C. For extracting the total protein for the BPM-labeling assay, 8 × 105 cells were seeded in 35-mm dishes and incubated for 24 hr, and then pre-incubated in serum-free medium for 24 hr before treatment with quinones for 1 hr or before collection of the cell lysate. BPM-labeling assay Cell lysates, which were collected into a lysis buffer (50 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (HCl), pH 8.0; 150 mM sodium chloride (NaCl); 0.1% sodium dodecyl sulfate (SDS); 0.5% deoxychol-
407 BPM-labeling ELISA is a convenient method to detect electrophiles
ic acid; 1% Nonidet P-40) containing 1% protease inhibitor cocktail, were incubated with the quinones in 20 mM Tris-HCl (pH 8.0) at 37°C for 1 hr, then an aliquot (20 μg of protein) was reacted with 2 nmol of BPM in 20 mM Tris-HCl (pH 8.0) at 37°C for 30 min, and was subjected to Western blot analysis. Western blot analysis Each sample was mixed with a half-volume of SDSpolyacrylamide gel electrophoresis (PAGE) loading buffer (62.5 mM Tris-HCl, pH 6.8; 6% SDS; 24% glycerol; 50 mM tris(2-carboxyethyl)phosphine; and 0.015% bromophenol blue) and incubated at 95°C for 5 min. The cellular proteins were then separated using SDSPAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% skim milk in TTBS (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; and 0.1% Tween 20) and then incubated with a primary antibody in TTBS. To detect immunoreactive proteins, HRP-conjugated anti-rabbit immunoglobulin G or an HRP-conjugated anti-biotin antibody and an enhanced chemiluminescence system (Chemi-Lumi One; Nacalai) were employed. Enzyme-linked immunosorbent assay (ELISA) Adult C57BL/6J mice livers were homogenized in three volumes of 3 M potassium chloride and centrifuged at 9,000 × g for 10 min. The protein concentration of the liver lysate was determined by the bicinchoninic acid assay. Samples were store at -80°C prior to use. Nunc MaxiSoap 96 well plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 50 μL of liver lysate (200 μg/mL) in sodium carbonate buffer (pH 9.5) at 4°C for overnight. The coat proteins were reduced by 10 mM dithiothreitol at 37°C for 30 min and washed with phosphate-buffered saline (PBS), and then reacted with quinones at 37°C for 30 min. After the wells were washed with PBS, 50 μL of 2 μM BPM in 50 mM Tris-HCl (pH 7.5) was added into the well and incubated at 37°C for 30 min, then the wells were blocked with 1% bovine serum albumin in PBS. To detect BPM-labeled proteins, an HRP-conjugated anti-biotin antibody and an ABTS peroxidase substrate system (KPL, Gaithersburg, MD, USA) were used according to the manufacturer’s instructions. All experiments were done in duplicate determinations. RESULTS AND DISCUSSION We previously prepared specific antibodies against 1,2-NQ and 1,4-NQ (Miura and Kumagai, 2010; Hirose
et al., 2012). With these antibodies, we detected covalent modifications of the cellular proteins during exposure of A431 cells to 1,2-NQ and 1,4-NQ. As shown in Figs. 2A and 2C, blot intensities were enhanced in a concentration-dependent manner following exposure to these quinones. By contrast, blot intensities as detected by the BPM-labeling assay were decreased in a concentration-dependent manner in either case (Figs. 2B and 2D). Interestingly, there was an inverse relationship of blot intensity between those obtained using specific antibodies against 1,2-NQ and 1,4-NQ and the BPM-labeling assay (Figs. 2E and 2F). These results indicated that covalent modification of quinones to proteins could also be detected by the BPM-labeling assay or Western blotting (Fig. 1). We next detected 1,2-NQ and 1,4-NQ protein modification using ELISA. As expected, antibodies against these quinones detected their binding to proteins, whereas 1,2-NQ and 1,4-NQ blocked BPM-binding to proteins (Fig. 3). While the sensitivity of BPM-labeling ELISA was lower than established ELISA with the antibodies, BPM-labeling ELISA appears to be a simple and convenient assay to detect organic electrophiles. To confirm this hypothesis, we also tested the covalent modification of electrophilic quinones such as 5-hydroxy-1,4-NQ, 5,8-dihydroxy-1,4-NQ, 1,4-BQ, and TBQ to proteins by the BPM-labeling ELISA. As shown in Fig. 4, these quinones blocked binding of BPM to proteins, whereas 2-hydroxy-1,4-NQ, 2-methyl-1,4-NQ, 9,10-phenanthraquinone, and anthraquinone, which have little capability for covalent modification to proteins (Kumagai et al., 1998; Ollinger and Brunmark, 1991), did not (Fig. 5). APAP is metabolized by CYPs to form NAPQI, resulting in liver toxicity through electrophilic modification of hepato-proteins (Hinson et al., 2010). As shown in Fig. 6, NAPQI, but not APAP, disrupted BPM modification of protein (Fig. 6), suggesting that BPM-labeling ELISA is useful to detect electrophilic products derived from parent drugs (e.g., tamoxifen, diclofenac) through CYP-dependent metabolic activation (Evans et al., 2004). We found that PM2.5 samples collected in Riverside, California are contaminated with 1,2-NQ and 1,4-NQ, presumably through photooxidation of naphthalene (Cho et al., 2004). We have shown by MALDI-TOF/MS that such ambient samples certainly contain electrophiles (Iwamoto et al., 2010). It was also shown that vaporphase samples collected in the midtown area of Los Angeles activate the Keap1/Nrf2 pathway, supporting the existence of electrophilic chemicals in the ambient samples. In the present study, we evaluated whether or not the atmospheric samples are contaminated with electrophiles by using BPM-labeling ELISA. Modification of proteins Vol. 40 No. 3
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Fig. 2.
Western blot analyses of 1,2-NQ and 1,4-NQ dependent S-arylation of cellular proteins using specific antibodies against these quinones (A and C) and by the BPM-labeling assay (B and D). A431 cells were treated with 1,2-NQ (A, B, E) and 1,4-NQ (C, D, F) (0, 25, 50, or 100 μM) for 1 hr and lysed with a lysis buffer, and then the cell lysates were analyzed. The bands were quantified using ImageJ software (E and F).
B 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
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Fig. 3.
antibody
1 2 3 4 1,2-NQ [log10 (nM)]
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Evaluation of 1,2-NQ- and 1,4-NQ-mediated S-arylation of proteins in mice liver by established ELISA and BPM-labeling ELISA. The binding of 1,2-NQ (A) or 1,4-NQ (B) to proteins from mice liver were detected by ELISA with the indicated antibodies and BPM-labeling ELISA.
Vol. 40 No. 3
409 BPM-labeling ELISA is a convenient method to detect electrophiles
0.5
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0.4 0.3 OH
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0.1 0 0 1 2 3 4 5 5-hydroxy-1,4-NQ [log10 (nM)]
Absorbance at 405 nm
C
OH
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OH
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D 0.5
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S-Arylation of mice liver proteins by electrophilic quinones. A, 5-hydroxy-1,4-NQ; B, 5,8-dihydroxy-1,4-NQ; C, 1,4-BQ; D, TBQ. Binding of quinones to proteins from mice liver was detected by BPM-labeling ELISA.
B 0.8 0.6 O
0.4 0.2
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0 1 2 3 4 5 2-methyl-1,4-NQ [log10 (nM)]
C Absorbance at 405 nm
Absorbance at 405 nm
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A
Fig. 5.
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D 0.8 0.6 O
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0.2 0 0 1 2 3 4 5 9,10-phenanthraquinone [log10 (nM)]
Absorbance at 405 nm
Fig. 4.
Absorbance at 405 nm
B
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Absorbance at 405 nm
A
0.8 0.6 O
0.4 0.2 O
0 0
1 2 3 4 5 anthraquinone [log10 (nM)]
S-Arylation of mice liver proteins by less electrophilic quinones. A, 2-methyl-1,4-NQ; B, 2-hydroxy-1,4-NQ; C, 9,10-phenanthraquinone; D, anthraquinone. Binding of quinones to proteins from mice liver was detected by BPM-labeling ELISA. Vol. 40 No. 3
410 Y. Abiko et al.
0.8 0.6 H 3COC
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0 1 2 3 4 5 acetaminophen [log10 (nM)]
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Acetaminophen and its electrophilic metabolite mediated S-arylation of mice liver proteins by BPM-labeling ELISA. Binding of acetoaminophen (A) or NAPQI (B) to proteins from mice liver was detected by BPM-labeling ELISA.
Absorbance at 405 nm
A
Fig. 7.
0.8
B 0.8 0.6 0.4 0.2 0 0
1 2 3 3 VP1 [log10 (m /L)]
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Absorbance at 405 nm
Fig. 6.
B Absorbance at 405 nm
Absorbance at 405 nm
A
0.6 0.4 0.2 0 0
1 2 3 3 VP2 [log10 (m /L)]
4
Detection of electrophiles in ambient vapor phase samples determined by BPM-labeling ELISA. Binding of electrophiles in VP1 (A) or VP2 (B) to proteins from mice liver was detected by BPM-labeling ELISA.
with electrophiles in the VP1 and VP2 air samples was detected using this assay (Fig. 7), suggesting it is also suitable for the determination of electrophiles in a variety of environmental samples. In addition to cellular toxicity, electrophiles activate electrophilic signaling pathways such as PTP1B/EGFR signaling, and the Keap1/Nrf2 pathway through covalent attachment of highly reactive thiols in redox sensor proteins (Kumagai et al., 2012; Jacobs and Marnett, 2010). Thus, we postulated that BPM-labeling ELISA would be an efficient assay to identify electrophiles covalently bound to the redox sensor proteins, which regulate electrophilic signal transduction pathways.
Vol. 40 No. 3
ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid (#25220103 to Y.K.) for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Conflict of interest---- The authors declare that there is no conflict of interest. REFERENCES Abiko, Y., Miura, T., Phuc, B.H., Shinkai, Y. and Kumagai, Y. (2011): Participation of covalent modification of Keap1 in the activation of Nrf2 by tert-butylbenzoquinone, an electrophilic metabolite of butylated hydroxyanisole. Toxicol. Appl.
411 BPM-labeling ELISA is a convenient method to detect electrophiles Pharmacol., 255, 32-39. Batterman, S., Chin, J.Y., Jia, C., Godwin, C., Parker, E., Robins, T., Max, P. and Lewis, T. (2012): Sources, concentrations, and risks of naphthalene in indoor and outdoor air. Indoor Air, 22, 266-278. Cho, A.K., Sioutas, C., Miguel, A.H., Kumagai, Y., Schmitz, D.A., Singh, M., Eiguren-Fernandez, A. and Froines, J.R. (2005): Redox activity of airborne particulate matter at different sites in the Los Angeles Basin. Environ. Res., 99, 40-47. Cho, A.K., Stefano, E.D., You, Y., Rodriguez, C.E., Schmitz, D.A., Kumagai, Y., Miguel, A.H., Eiguren-Fernandez, A., Kobayashi, T., Avol, E. and Froines, J.R. (2004): Determination of four quinones in diesel exhoust particles, SRM 1649a, and atmospheric PM2.5. Aerosol. Sci. Tech., 38, 68-81. Eiguren-Fernandez, A., Miguel, A.H., Stefano, E.D., Schmit, D.A., Cho, A.K., Thurairatnam, S., Avol, E.L. and Froines, J.R. (2008): Atmospheric distribution of gas- and particle-phase quinones in Southern California. Aerosol Sci. Tech., 42, 854-861. Eiguren-Fernandez, A., Shinyashiki, M., Schmitz, D.A., DiStefano, E., Hinds, W., Kumagai, Y., Cho, A.K. and Froines, J.R. (2010): Redox and electrophilic properties of vapor- and particle-phase components of ambient aerosols. Environ. Res., 110, 207-212. Endo, A., Sumi, D., Iwamoto, N. and Kumagai, Y. (2011): Inhibition of DNA binding activity of cAMP response element-binding protein by 1,2-naphthoquinone through chemical modification of Cys-286. Chem. Biol. Interact., 192, 272-277. Endo, A., Sumi, D. and Kumagai, Y. (2007): 1,2-Naphthoquinone disrupts the function of cAMP response element-binding protein through covalent modification. Biochem. Biophys. Res. Commun., 361, 243-248. Evans, D.C., Watt, A.P., Nicoll-Griffith, D.A. and Baillie, T.A. (2004): Drug-protein adducts: an industry perspective on minimizing the potential for drug bioactivation in drug discovery and development. Chem. Res. Toxicol., 17, 3-16. Hinson, J.A., Roberts, D.W. and James, L.P. (2010): Mechanisms of acetaminophen-induced liver necrosis. Handb. Exp. Pharmacol., 196, 369-405. Hirose, R., Miura, T., Sha, R., Shinkai, Y., Tanaka-Kagawa, T. and Kumagai, Y. (2012): A method for detecting covalent modification of sensor proteins associated with 1,4-naphthoquinone-induced activation of electrophilic signal transduction pathways. J. Toxicol. Sci., 37, 891-898. Iwamoto, N., Nishiyama, A., Eiguren-Fernandez, A., Hinds, W., Kumagai, Y., Froines, J.R., Cho, A.K. and Shinyashiki, M. (2010): Biochemical and cellular effects of electrophiles present in ambient air samples. Atmos. Environ., 44, 1483-1489. Iwamoto, N., Sumi, D., Ishii, T., Uchida, K., Cho, A.K., Froines, J.R. and Kumagai, Y. (2007): Chemical knockdown of proteintyrosine phosphatase 1B by 1,2-naphthoquinone through covalent modification causes persistent transactivation of epidermal growth factor receptor. J. Biol. Chem., 282, 33396-33404. Jacobs, A.T. and Marnett, L.J. (2010): Systems analysis of protein modification and cellular responses induced by electrophile
stress. Acc. Chem. Res., 43, 673-683. Jakober, C.A., Riddle, S.G., Robert, M.A., Destaillats, H., Charles, M.J., Green, P.G. and Kleeman, M.J. (2007): Quinone emissions from gasoline and diesel motor vehicles. Environ. Sci. Technol., 41, 4548-4554. Jia, C. and Batterman, S. (2010): A critical review of naphthalene sources and exposures relevant to indoor and outdoor air. Int. J. Environ. Res. Public Health, 7, 2903-2939. Kautzman, K.E., Surratt, J.D., Chan, M.N., Chan, A.W., Hersey, S.P., Chhabra, P.S., Dalleska, N.F., Wennberg, P.O., Flagan, R.C. and Seinfeld, J.H. (2010): Chemical composition of gas- and aerosol-phase products from the photooxidation of naphthalene. J. Phys. Chem. A, 114, 913-934. Kobayashi, M., Li, L., Iwamoto, N., Nakajima-Takagi, Y., Kaneko, H., Nakayama, Y., Eguchi, M., Wada, Y., Kumagai, Y. and Yamamoto, M. (2009): The antioxidant defense system Keap1Nrf2 comprises a multiple sensing mechanism for responding to a wide range of chemical compounds. Mol. Cell. Biol., 29, 493502. Kumagai, Y., Kanda, H., Shinkai, Y. and Toyama, T. (2013): The role of the Keap1/Nrf2 pathway in the cellular response to methylmercury. Oxid. Med. Cell. Longev., 2013, 848279. Kumagai, Y., Nakajima, H., Midorikawa, K., Homma-Takeda, S. and Shimojo, N. (1998): Inhibition of nitric oxide formation by neuronal nitric oxide synthase by quinones: nitric oxide synthase as a quinone reductase. Chem. Res. Toxicol., 11, 608-613. Kumagai, Y., Shinkai, Y., Miura, T. and Cho, A.K. (2012): The chemical biology of naphthoquinones and its environmental implications. Annu. Rev. Pharmacol. Toxicol., 52, 221-247. Lind, C., Gerdes, R., Hamnell, Y., Schuppe-Koistinen, I., von Löwenhielm, H.B., Holmgren, A. and Cotgreave, I.A. (2002): Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis. Arch. Biochem. Biophys., 406, 229-240. Miura, T. and Kumagai, Y. (2010): Immunochemical method to detect proteins that undergo selective modification by 1,2-naphthoquinone derived from naphthalene through metabolic activation. J. Toxicol. Sci., 35, 843-852. Miura, T., Shinkai, Y., Jiang, H.Y., Iwamoto, N., Sumi, D., Taguchi, K., Yamamoto, M., Jinno, H., Tanaka-Kagawa, T., Cho, A.K. and Kumagai, Y. (2011): Initial response and cellular protection through the Keap1/Nrf2 system during the exposure of primary mouse hepatocytes to 1,2-naphthoquinone. Chem. Res. Toxicol., 24, 559-567. Muthuramalingam, M., Matros, A., Scheibe, R., Mock, H.P. and Dietz, K.J. (2013): The hydrogen peroxide-sensitive proteome of the chloroplast in vitro and in vivo. Front. Plant Sci., 4, 54. Ollinger, K. and Brunmark, A. (1991): Effect of hydroxy substituent position on 1,4-naphthoquinone toxicity to rat hepatocytes. J. Biol. Chem., 266, 21496-21503. Toyama, T., Shinkai, Y., Kaji, T. and Kumagai, Y. (2013): Convenient method to assess chemical modification of protein thiols by electrophilic metals. J. Toxicol. Sci., 38, 477-484.
Vol. 40 No. 3
The Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.40, No.3, 405-411, 2015
Original Article
A Biotin-PEAC5-maleimide labeling assay to detect electrophiles Yumi Abiko1, Nho Cong Luong2 and Yoshito Kumagai1,2
2
1Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan Master’s Program of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
(Received March 13, 2015; Accepted March 30, 2015)
ABSTRACT — 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. Key words: Electrophile, Thiol, Cysteine, S-arylation, Biotin-PEAC5-maleimide INTRODUCTION Electrophiles readily bind to nucleophiles such as protein thiol groups, resulting in functional changes of modified proteins. Electrophilic compounds ubiquitously exist in the environment and are taken up during daily living. For example, 1,2- and 1,4-naphthoquinones (1,2-NQ and 1,4-NQ) and 1,4-benzoquinone (1,4-BQ) are reaction products that contaminate the atmosphere during photooxidation of aromatic hydrocarbons such as naphthalene and benzene, and are also found as electrophilic metabolites of these chemicals in the body (Batterman et al., 2012; Cho et al., 2005; Eiguren-Fernandez et al., 2010; Jia and Batterman, 2010; Kautzman et al., 2010). 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 (Kumagai et al., 2012, 2013; Endo et al., 2007, 2011; Kobayashi 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. Thus, a simple and convenient assay to determine protein modification should be established. We have developed a biotin-PEAC 5 -maleimide (BPM)-labeling assay using Western blotting with a horseradish peroxidase (HRP)-linked anti-biotin antibody to detect MeHg-induced S-modification of proteins (Toyama et al., 2013) (see Fig. 1). However, application of this method to organic electrophiles remains to be determined. Because it was reported that maleimide,
Correspondence: Yoshito Kumagai (E-mail: [email protected]) Vol. 40 No. 3
406 Y. Abiko et al.
E Protein
S
Protein
E, electrophiles
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O
O
Protein
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E
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Biotin
CH3
N
Biotin
N N
CH3
N
O
H3C
Protein
O
S
E
Detected by avidin-HRP Western blotting ELISA Fig. 1.
Schematic strategy of the BPM-labeling assay for detecting quinones bound to cellular proteins.
which has an α,β-unsaturated carbonyl, covalently modifies cysteine residues in proteins (Lind et al., 2002; Muthuramalingam et al., 2013), we hypothesized that covalent modification of proteins by organic electrophiles may be detected using BPM. In this study, we analyzed covalent modification of various quinones to proteins using the BPM labeling assay as shown in Fig. 1. We also examined whether this assay was applicable for monitoring air samples containing environmental electrophiles such as 1,4-BQ, 1,2-NQ, and 1,4-NQ (Cho et al., 2004; Eiguren-Fernandez et al., 2008; Jakober et al., 2007). MATERIALS AND METHODS Materials 1,2-NQ (97.4% purity determined by high-performance liquid chromatography (HPLC)), 1,4-NQ (98% purity determined by gas chromatography (GC)), 5,8-dihydroxy1,4-NQ (79.8% purity determined by HPLC), 1,4-BQ (98% purity determined by iodometric titration), tertbutyl-1,4-benzoquinone (TBQ, 98% purity determined by GC), 2-hydroxy-1,4-NQ (99.9% purity determined by HPLC), and APAP were purchased from Tokyo Chemical Industry (Tokyo, Japan). 2-Methyl-1,4-NQ (98% purity determined by HPLC) was obtained from Nacalai (Kyoto, Japan). 5-Hydroxy-1,4-NQ (95% purity determined by HPLC), NAPQI, BPM, and the HRP-linked anti-biotin antibody were purchased from Sigma-Aldrich Vol. 40 No. 3
(St. Louis, MO, USA), Invitrogen (Carlsbad, CA, USA) and Cell Signaling Technology (Beverly, MA, USA), respectively. Vapor phase samples were collected in the mid-town area of Los Angeles near the University of Southern California during June (from 17th to 22th, VP1) and July (from 1st to 7th, VP2) 2008 and extracted as reported previously (Eiguren-Fernandez et al., 2008, 2010). All other reagents were of the highest grade available. Cell culture Human epithelial carcinoma A431 cells were obtained from the RIKEN Cell Bank (Tsukuba, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium (Wako, Osaka, Japan) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-alanyl-L-glutamine in an incubator supplemented with 5% CO2 at 37°C. For extracting the total protein for the BPM-labeling assay, 8 × 105 cells were seeded in 35-mm dishes and incubated for 24 hr, and then pre-incubated in serum-free medium for 24 hr before treatment with quinones for 1 hr or before collection of the cell lysate. BPM-labeling assay Cell lysates, which were collected into a lysis buffer (50 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (HCl), pH 8.0; 150 mM sodium chloride (NaCl); 0.1% sodium dodecyl sulfate (SDS); 0.5% deoxychol-
407 BPM-labeling ELISA is a convenient method to detect electrophiles
ic acid; 1% Nonidet P-40) containing 1% protease inhibitor cocktail, were incubated with the quinones in 20 mM Tris-HCl (pH 8.0) at 37°C for 1 hr, then an aliquot (20 μg of protein) was reacted with 2 nmol of BPM in 20 mM Tris-HCl (pH 8.0) at 37°C for 30 min, and was subjected to Western blot analysis. Western blot analysis Each sample was mixed with a half-volume of SDSpolyacrylamide gel electrophoresis (PAGE) loading buffer (62.5 mM Tris-HCl, pH 6.8; 6% SDS; 24% glycerol; 50 mM tris(2-carboxyethyl)phosphine; and 0.015% bromophenol blue) and incubated at 95°C for 5 min. The cellular proteins were then separated using SDSPAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% skim milk in TTBS (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; and 0.1% Tween 20) and then incubated with a primary antibody in TTBS. To detect immunoreactive proteins, HRP-conjugated anti-rabbit immunoglobulin G or an HRP-conjugated anti-biotin antibody and an enhanced chemiluminescence system (Chemi-Lumi One; Nacalai) were employed. Enzyme-linked immunosorbent assay (ELISA) Adult C57BL/6J mice livers were homogenized in three volumes of 3 M potassium chloride and centrifuged at 9,000 × g for 10 min. The protein concentration of the liver lysate was determined by the bicinchoninic acid assay. Samples were store at -80°C prior to use. Nunc MaxiSoap 96 well plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 50 μL of liver lysate (200 μg/mL) in sodium carbonate buffer (pH 9.5) at 4°C for overnight. The coat proteins were reduced by 10 mM dithiothreitol at 37°C for 30 min and washed with phosphate-buffered saline (PBS), and then reacted with quinones at 37°C for 30 min. After the wells were washed with PBS, 50 μL of 2 μM BPM in 50 mM Tris-HCl (pH 7.5) was added into the well and incubated at 37°C for 30 min, then the wells were blocked with 1% bovine serum albumin in PBS. To detect BPM-labeled proteins, an HRP-conjugated anti-biotin antibody and an ABTS peroxidase substrate system (KPL, Gaithersburg, MD, USA) were used according to the manufacturer’s instructions. All experiments were done in duplicate determinations. RESULTS AND DISCUSSION We previously prepared specific antibodies against 1,2-NQ and 1,4-NQ (Miura and Kumagai, 2010; Hirose
et al., 2012). With these antibodies, we detected covalent modifications of the cellular proteins during exposure of A431 cells to 1,2-NQ and 1,4-NQ. As shown in Figs. 2A and 2C, blot intensities were enhanced in a concentration-dependent manner following exposure to these quinones. By contrast, blot intensities as detected by the BPM-labeling assay were decreased in a concentration-dependent manner in either case (Figs. 2B and 2D). Interestingly, there was an inverse relationship of blot intensity between those obtained using specific antibodies against 1,2-NQ and 1,4-NQ and the BPM-labeling assay (Figs. 2E and 2F). These results indicated that covalent modification of quinones to proteins could also be detected by the BPM-labeling assay or Western blotting (Fig. 1). We next detected 1,2-NQ and 1,4-NQ protein modification using ELISA. As expected, antibodies against these quinones detected their binding to proteins, whereas 1,2-NQ and 1,4-NQ blocked BPM-binding to proteins (Fig. 3). While the sensitivity of BPM-labeling ELISA was lower than established ELISA with the antibodies, BPM-labeling ELISA appears to be a simple and convenient assay to detect organic electrophiles. To confirm this hypothesis, we also tested the covalent modification of electrophilic quinones such as 5-hydroxy-1,4-NQ, 5,8-dihydroxy-1,4-NQ, 1,4-BQ, and TBQ to proteins by the BPM-labeling ELISA. As shown in Fig. 4, these quinones blocked binding of BPM to proteins, whereas 2-hydroxy-1,4-NQ, 2-methyl-1,4-NQ, 9,10-phenanthraquinone, and anthraquinone, which have little capability for covalent modification to proteins (Kumagai et al., 1998; Ollinger and Brunmark, 1991), did not (Fig. 5). APAP is metabolized by CYPs to form NAPQI, resulting in liver toxicity through electrophilic modification of hepato-proteins (Hinson et al., 2010). As shown in Fig. 6, NAPQI, but not APAP, disrupted BPM modification of protein (Fig. 6), suggesting that BPM-labeling ELISA is useful to detect electrophilic products derived from parent drugs (e.g., tamoxifen, diclofenac) through CYP-dependent metabolic activation (Evans et al., 2004). We found that PM2.5 samples collected in Riverside, California are contaminated with 1,2-NQ and 1,4-NQ, presumably through photooxidation of naphthalene (Cho et al., 2004). We have shown by MALDI-TOF/MS that such ambient samples certainly contain electrophiles (Iwamoto et al., 2010). It was also shown that vaporphase samples collected in the midtown area of Los Angeles activate the Keap1/Nrf2 pathway, supporting the existence of electrophilic chemicals in the ambient samples. In the present study, we evaluated whether or not the atmospheric samples are contaminated with electrophiles by using BPM-labeling ELISA. Modification of proteins Vol. 40 No. 3
408 Y. Abiko et al.
Fig. 2.
Western blot analyses of 1,2-NQ and 1,4-NQ dependent S-arylation of cellular proteins using specific antibodies against these quinones (A and C) and by the BPM-labeling assay (B and D). A431 cells were treated with 1,2-NQ (A, B, E) and 1,4-NQ (C, D, F) (0, 25, 50, or 100 μM) for 1 hr and lysed with a lysis buffer, and then the cell lysates were analyzed. The bands were quantified using ImageJ software (E and F).
B 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
BPM
0
Fig. 3.
antibody
1 2 3 4 1,2-NQ [log10 (nM)]
Absorbance at 405 nm
Absorbance at 405 nm
A
5
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
BPM
0
antibody
1 2 3 4 1,4-NQ [log10 (nM)]
5
Evaluation of 1,2-NQ- and 1,4-NQ-mediated S-arylation of proteins in mice liver by established ELISA and BPM-labeling ELISA. The binding of 1,2-NQ (A) or 1,4-NQ (B) to proteins from mice liver were detected by ELISA with the indicated antibodies and BPM-labeling ELISA.
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409 BPM-labeling ELISA is a convenient method to detect electrophiles
0.5
O
0.4 0.3 OH
0.2
O
0.1 0 0 1 2 3 4 5 5-hydroxy-1,4-NQ [log10 (nM)]
Absorbance at 405 nm
C
OH
O
OH
O
0.4 0.2 0 0 1 2 3 4 5 5,8-dihydroxy-1,4-NQ [log10 (nM)]
D 0.5
O
0.4 0.3 0.2
O
0.1 0 0
1 2 3 4 1,4-BQ [log10 (nM)]
5
0.5
O
0.4 0.3 0.2
O
0.1 0 0
1 2 3 4 TBQ [log10 (nM)]
5
S-Arylation of mice liver proteins by electrophilic quinones. A, 5-hydroxy-1,4-NQ; B, 5,8-dihydroxy-1,4-NQ; C, 1,4-BQ; D, TBQ. Binding of quinones to proteins from mice liver was detected by BPM-labeling ELISA.
B 0.8 0.6 O
0.4 0.2
O
0
0 1 2 3 4 5 2-methyl-1,4-NQ [log10 (nM)]
C Absorbance at 405 nm
Absorbance at 405 nm
Absorbance at 405 nm
A
Fig. 5.
0.6
0.8 0.6 O
0.4
OH
0.2 O
0
0 1 2 3 4 5 2-hydroxy-1,4-NQ [log10 (nM)]
D 0.8 0.6 O
0.4
O
0.2 0 0 1 2 3 4 5 9,10-phenanthraquinone [log10 (nM)]
Absorbance at 405 nm
Fig. 4.
Absorbance at 405 nm
B
Absorbance at 405 nm
Absorbance at 405 nm
A
0.8 0.6 O
0.4 0.2 O
0 0
1 2 3 4 5 anthraquinone [log10 (nM)]
S-Arylation of mice liver proteins by less electrophilic quinones. A, 2-methyl-1,4-NQ; B, 2-hydroxy-1,4-NQ; C, 9,10-phenanthraquinone; D, anthraquinone. Binding of quinones to proteins from mice liver was detected by BPM-labeling ELISA. Vol. 40 No. 3
410 Y. Abiko et al.
0.8 0.6 H 3COC
0.4
N
H
0.2 OH
0
0 1 2 3 4 5 acetaminophen [log10 (nM)]
H 3COC
0.6
N
0.4 0.2
O
0 0
1 2 3 4 NAPQI [log10 (nM)]
5
Acetaminophen and its electrophilic metabolite mediated S-arylation of mice liver proteins by BPM-labeling ELISA. Binding of acetoaminophen (A) or NAPQI (B) to proteins from mice liver was detected by BPM-labeling ELISA.
Absorbance at 405 nm
A
Fig. 7.
0.8
B 0.8 0.6 0.4 0.2 0 0
1 2 3 3 VP1 [log10 (m /L)]
4
Absorbance at 405 nm
Fig. 6.
B Absorbance at 405 nm
Absorbance at 405 nm
A
0.6 0.4 0.2 0 0
1 2 3 3 VP2 [log10 (m /L)]
4
Detection of electrophiles in ambient vapor phase samples determined by BPM-labeling ELISA. Binding of electrophiles in VP1 (A) or VP2 (B) to proteins from mice liver was detected by BPM-labeling ELISA.
with electrophiles in the VP1 and VP2 air samples was detected using this assay (Fig. 7), suggesting it is also suitable for the determination of electrophiles in a variety of environmental samples. In addition to cellular toxicity, electrophiles activate electrophilic signaling pathways such as PTP1B/EGFR signaling, and the Keap1/Nrf2 pathway through covalent attachment of highly reactive thiols in redox sensor proteins (Kumagai et al., 2012; Jacobs and Marnett, 2010). Thus, we postulated that BPM-labeling ELISA would be an efficient assay to identify electrophiles covalently bound to the redox sensor proteins, which regulate electrophilic signal transduction pathways.
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ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid (#25220103 to Y.K.) for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Conflict of interest---- The authors declare that there is no conflict of interest. REFERENCES Abiko, Y., Miura, T., Phuc, B.H., Shinkai, Y. and Kumagai, Y. (2011): Participation of covalent modification of Keap1 in the activation of Nrf2 by tert-butylbenzoquinone, an electrophilic metabolite of butylated hydroxyanisole. Toxicol. Appl.
411 BPM-labeling ELISA is a convenient method to detect electrophiles Pharmacol., 255, 32-39. Batterman, S., Chin, J.Y., Jia, C., Godwin, C., Parker, E., Robins, T., Max, P. and Lewis, T. (2012): Sources, concentrations, and risks of naphthalene in indoor and outdoor air. Indoor Air, 22, 266-278. Cho, A.K., Sioutas, C., Miguel, A.H., Kumagai, Y., Schmitz, D.A., Singh, M., Eiguren-Fernandez, A. and Froines, J.R. (2005): Redox activity of airborne particulate matter at different sites in the Los Angeles Basin. Environ. Res., 99, 40-47. Cho, A.K., Stefano, E.D., You, Y., Rodriguez, C.E., Schmitz, D.A., Kumagai, Y., Miguel, A.H., Eiguren-Fernandez, A., Kobayashi, T., Avol, E. and Froines, J.R. (2004): Determination of four quinones in diesel exhoust particles, SRM 1649a, and atmospheric PM2.5. Aerosol. Sci. Tech., 38, 68-81. Eiguren-Fernandez, A., Miguel, A.H., Stefano, E.D., Schmit, D.A., Cho, A.K., Thurairatnam, S., Avol, E.L. and Froines, J.R. (2008): Atmospheric distribution of gas- and particle-phase quinones in Southern California. Aerosol Sci. Tech., 42, 854-861. Eiguren-Fernandez, A., Shinyashiki, M., Schmitz, D.A., DiStefano, E., Hinds, W., Kumagai, Y., Cho, A.K. and Froines, J.R. (2010): Redox and electrophilic properties of vapor- and particle-phase components of ambient aerosols. Environ. Res., 110, 207-212. Endo, A., Sumi, D., Iwamoto, N. and Kumagai, Y. (2011): Inhibition of DNA binding activity of cAMP response element-binding protein by 1,2-naphthoquinone through chemical modification of Cys-286. Chem. Biol. Interact., 192, 272-277. Endo, A., Sumi, D. and Kumagai, Y. (2007): 1,2-Naphthoquinone disrupts the function of cAMP response element-binding protein through covalent modification. Biochem. Biophys. Res. Commun., 361, 243-248. Evans, D.C., Watt, A.P., Nicoll-Griffith, D.A. and Baillie, T.A. (2004): Drug-protein adducts: an industry perspective on minimizing the potential for drug bioactivation in drug discovery and development. Chem. Res. Toxicol., 17, 3-16. Hinson, J.A., Roberts, D.W. and James, L.P. (2010): Mechanisms of acetaminophen-induced liver necrosis. Handb. Exp. Pharmacol., 196, 369-405. Hirose, R., Miura, T., Sha, R., Shinkai, Y., Tanaka-Kagawa, T. and Kumagai, Y. (2012): A method for detecting covalent modification of sensor proteins associated with 1,4-naphthoquinone-induced activation of electrophilic signal transduction pathways. J. Toxicol. Sci., 37, 891-898. Iwamoto, N., Nishiyama, A., Eiguren-Fernandez, A., Hinds, W., Kumagai, Y., Froines, J.R., Cho, A.K. and Shinyashiki, M. (2010): Biochemical and cellular effects of electrophiles present in ambient air samples. Atmos. Environ., 44, 1483-1489. Iwamoto, N., Sumi, D., Ishii, T., Uchida, K., Cho, A.K., Froines, J.R. and Kumagai, Y. (2007): Chemical knockdown of proteintyrosine phosphatase 1B by 1,2-naphthoquinone through covalent modification causes persistent transactivation of epidermal growth factor receptor. J. Biol. Chem., 282, 33396-33404. Jacobs, A.T. and Marnett, L.J. (2010): Systems analysis of protein modification and cellular responses induced by electrophile
stress. Acc. Chem. Res., 43, 673-683. Jakober, C.A., Riddle, S.G., Robert, M.A., Destaillats, H., Charles, M.J., Green, P.G. and Kleeman, M.J. (2007): Quinone emissions from gasoline and diesel motor vehicles. Environ. Sci. Technol., 41, 4548-4554. Jia, C. and Batterman, S. (2010): A critical review of naphthalene sources and exposures relevant to indoor and outdoor air. Int. J. Environ. Res. Public Health, 7, 2903-2939. Kautzman, K.E., Surratt, J.D., Chan, M.N., Chan, A.W., Hersey, S.P., Chhabra, P.S., Dalleska, N.F., Wennberg, P.O., Flagan, R.C. and Seinfeld, J.H. (2010): Chemical composition of gas- and aerosol-phase products from the photooxidation of naphthalene. J. Phys. Chem. A, 114, 913-934. Kobayashi, M., Li, L., Iwamoto, N., Nakajima-Takagi, Y., Kaneko, H., Nakayama, Y., Eguchi, M., Wada, Y., Kumagai, Y. and Yamamoto, M. (2009): The antioxidant defense system Keap1Nrf2 comprises a multiple sensing mechanism for responding to a wide range of chemical compounds. Mol. Cell. Biol., 29, 493502. Kumagai, Y., Kanda, H., Shinkai, Y. and Toyama, T. (2013): The role of the Keap1/Nrf2 pathway in the cellular response to methylmercury. Oxid. Med. Cell. Longev., 2013, 848279. Kumagai, Y., Nakajima, H., Midorikawa, K., Homma-Takeda, S. and Shimojo, N. (1998): Inhibition of nitric oxide formation by neuronal nitric oxide synthase by quinones: nitric oxide synthase as a quinone reductase. Chem. Res. Toxicol., 11, 608-613. Kumagai, Y., Shinkai, Y., Miura, T. and Cho, A.K. (2012): The chemical biology of naphthoquinones and its environmental implications. Annu. Rev. Pharmacol. Toxicol., 52, 221-247. Lind, C., Gerdes, R., Hamnell, Y., Schuppe-Koistinen, I., von Löwenhielm, H.B., Holmgren, A. and Cotgreave, I.A. (2002): Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis. Arch. Biochem. Biophys., 406, 229-240. Miura, T. and Kumagai, Y. (2010): Immunochemical method to detect proteins that undergo selective modification by 1,2-naphthoquinone derived from naphthalene through metabolic activation. J. Toxicol. Sci., 35, 843-852. Miura, T., Shinkai, Y., Jiang, H.Y., Iwamoto, N., Sumi, D., Taguchi, K., Yamamoto, M., Jinno, H., Tanaka-Kagawa, T., Cho, A.K. and Kumagai, Y. (2011): Initial response and cellular protection through the Keap1/Nrf2 system during the exposure of primary mouse hepatocytes to 1,2-naphthoquinone. Chem. Res. Toxicol., 24, 559-567. Muthuramalingam, M., Matros, A., Scheibe, R., Mock, H.P. and Dietz, K.J. (2013): The hydrogen peroxide-sensitive proteome of the chloroplast in vitro and in vivo. Front. Plant Sci., 4, 54. Ollinger, K. and Brunmark, A. (1991): Effect of hydroxy substituent position on 1,4-naphthoquinone toxicity to rat hepatocytes. J. Biol. Chem., 266, 21496-21503. Toyama, T., Shinkai, Y., Kaji, T. and Kumagai, Y. (2013): Convenient method to assess chemical modification of protein thiols by electrophilic metals. J. Toxicol. Sci., 38, 477-484.
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