Development of Polyclonal Antibodies for Detection of Diosbulbin B

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Article Cite This: Chem. Res. Toxicol. 2018, 31, 231−237

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Development of Polyclonal Antibodies for Detection of Diosbulbin B‑Derived cis-Enedial Protein Adducts Zixia Hu,† Shenzhi Zhou,† Na Zhang,† Weiwei Li,‡ Dongju Lin,† Ying Peng,*,† and Jiang Zheng*,†,‡ †

Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou 550025, P. R. China

‡

S Supporting Information *

ABSTRACT: Diosbulbin B (DSB), a major component of herbal medicine Dioscorea bulbifera L. (DB), can be metabolized to an electrophilic intermediate, DSB-derived cis-enedial (DDE). DDE was suggested to contribute to the hepatotoxicity observed in experimental animals and humans after their exposure to DSB. Our previous work found that DDE reacted with primary amino and/or sulfhydryl groups of hepatic protein. The objective of the study was to develop polyclonal antibodies that can recognize DDE-derived protein adducts. Immunogens synthesized from DDE and keyhole limpet hemocyanin were employed to raise polyclonal antibodies in rabbits. An enzyme-linked immunosorbent assay (ELISA) demonstrated high titers of antisera obtained from immunized rabbits. Immunoblot analysis showed that DDE-modified bovine serum albumin (BSA) was recognized by the obtained polyclonal antibodies in a concentration-dependent manner and without cross-reaction to native BSA. Competitive ELISA and competitive immunoblot analyses defined the specificity of the antibodies to recognize BSA modified by DDE. Immunoblot analysis also detected a multitude of chemiluminescent bands with a variety of molecular weights in liver homogenates that were harvested from mice treated with DSB. In summary, we have successfully raised polyclonal antibodies to detect protein adducts derived from DDE.

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alone.22 Recently, we succeeded in detection of protein adductions derived from DDE in liver of animals given DSB, using the LC−MS/MS technique.23 We hypothesize that modification of vital cellular proteins by DDE triggers the development of hepatotoxicity of DSB. Immunochemical approaches have been successfully undertaken to selectively recognize protein modified by reactive metabolites or xenobiotics, such as styrene,24 1,2-naphthoquinone,25 1-aminohydantoin,26 flucloxacillin,27 and menthofuran.28 Additionally, antibodies can be applied to monitor the toxicological importance of protein modification in exposed humans and experimental animals. We aimed to raise polyclonal antibodies against protein adducts derived from DDE. The antibodies obtained from sera of rabbits immunized with DDE-derived keyhole limpet hemocyanin adducts demonstrated specific recognition of the DSB moiety of protein adducts formed in vitro and in vivo. It is anticipated that these antibody-based immunoassays facilitate our investigation of the biochemical mechanism of DSB-induced toxicity.

INTRODUCTION Dioscorea bulbifera L. (DB), a Chinese medicinal herb that belongs to the yam family Dioscoreaceae, has been widely employed for the treatment of breast lumps, goiters, carbuncles, and lung abscesses.1,2 In addition, studies demonstrated that DB displays a variety of pharmacological properties, such as antitumor,3 anti-inflammatory,4 antisalmonellal,5 and antifeedant6 activities. However, its clinical use carries the risk of serious liver injury.7−9 Hepatitis induced after chronic exposure to DB has been reported in patients, and the clinical parameters are consistent with those observed in animals.10−13 Diosbulbin B (DSB), a furan-containing diterpenoid lactone, is a major component of DB.14,15 Our previous studies showed that DSB was metabolized by P450 enzymes to the corresponding cis-enedial [diosbulbin B-derived cis-enedial (DDE)], an electrophilic intermediate.16 The cis-enedial is required for DSB-induced hepatotoxicities.17 The primary amino groups of lysine (Lys), N-acetyl lysine (NAL), and glutathione (GSH) and/or the sulfhydryl of cysteine (Cys), Nacetyl cysteine (NAC), and GSH can react with the electrophilic species to form chemically stable pyrrole or pyrroline derivatives.18−21 Our previous study employed NAL and NAC or GSH to trap DDE in both rat and human liver microsomal incubation systems supplemented with DSB, and pyrrole derivatives were detected in the incubation systems in the presence of GSH and NAL.16 In addition, a pyrroline was formed when the cis-enedial was reacted with NAL or GSH © 2018 American Chemical Society

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MATERIALS AND METHODS

Chemicals and Materials. Dry rhizomes of DB were purchased from Tong-Ren-Tang Pharmacy (Shenyang, China). DSB was isolated and purified from DB rhizomes according to a reported protocol,29 Received: October 31, 2017 Published: March 23, 2018 231

DOI: 10.1021/acs.chemrestox.7b00299 Chem. Res. Toxicol. 2018, 31, 231−237

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Chemical Research in Toxicology and its structure identity was characterized by mass spectrometry and NMR. The purity of DSB was >98% as determined by HPLC with a diode array detector. Water-soluble keyhole limpet hemocyanin (KLH), 3,3′,5,5′-tetramethylbenzidine, 2,5-dimethylfuran (DMF), Nacetyl cysteine (NAC), N-acetyl lysine (NAL), glutathione (GSH), Freund’s complete adjuvant, and Freund’s incomplete adjuvant were purchased from Sigma-Aldrich (St. Louis, MO). Horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Rockland (Limerick, PA). Bovine serum albumin (BSA) and ovalbumin (OVA) were purchased from Salarbio (Beijing, China). The BCA protein assay kit and the BeyoECL Star detection kit were purchased from Beyotime (Shanghai, China). All of the other reagents or solvents were of analytical grade. Greiner Bio-One (Frickenhausen, Germany) 96-well microtiter plates were used in enzyme-linked immunosorbent assays (ELISAs). A Thermo Scientific Varioskan Flash Spectral Scanning Multimode Reader was applied to measure the ELISA absorbance. A Bio-Rad basic electrophoresis system was employed for SDS−PAGE. Membrane transfer was accomplished with a Bio-Rad Tans-Blot SD Semi-Dry Transfer Cell. Synthesis. Immunogen. DSB (5 mg) was dissolved in 200 μL of acetone and cooled in a dry ice bath. To the resulting solution that was being stirred was added dropwise dimethyldioxirane (DMDO, 1.0 mL), pre-prepared according to the published method.30 The stirring continued for 1 h at room temperature, followed by condensation with a gentle flow of nitrogen gas. The residue was reconstituted with 1.0 mL of PBS containing KLH (3 mg/mL), and the resultant mixture was incubated for 1 h at 37 °C while being stirred and then frozen and kept at −20 °C after dialysis. Competitors. Competitive agents were synthesized via published protocols with slight modifications.16,22 Two duplicates of DSB (5 mg) and two duplicates of 2,5-dimethylfuran (DMF, 1.4 mg) were individually oxidized by DMDO as described before and dissolved in 1.0 mL of PBS containing NAC (0.71 mg) and/or NAL (0.82 mg) to obtain the corresponding competitors, including the DDE−pyrrole derivative (1), the DDE−pyrroline derivative (2), the DMF−pyrrole derivative (3), and the DMF−pyrroline derivative (4) (Scheme 2). The reaction mixtures were stirred for 1 h at 37 °C and concentrated under a gentle flow of nitrogen gas. Purification of products was accomplished by a semipreparative HPLC system. BSA−DDE and BSA−DDE−GSH Adducts. Protein adducts, namely, bovine serum albumin−diosbulbin B-derived cis-enedial intermediate (BSA−DDE) and bovine serum albumin−diosbulbin B-derived cisenedial intermediate−glutathione (BSA−DDE−GSH), were synthesized as positive controls in an immunoblot, and BSA−DDE was also used as a coating antigen in ELISAs. All procedures were undertaken as described in Immunogen except KLH was replaced with BSA (1.0 mg) or BSA (1.0 mg) combined with GSH (1.3 mg). Animals. Japanese white rabbits (female) weighing 2.0−2.5 kg and Kunming mice (male) weighing 18−20 g were used for immunization. They were both provided by the Animal Center of Shenyang Pharmaceutical University (Shenyang, China). Animals were placed in a controlled environment (temperature of 25 °C and 12 h dark/light cycle) and provided ad libitum access to food and water. All animal studies were conducted in accordance with procedures approved by the Ethics Review Committee for Animal Experimentation of Shenyang Pharmaceutical University. Immunization of Rabbits. Japanese white rabbits were immunized for preparation of antisera. The rabbits were immunized with 20 μL of immunogen emulsion. The immunogen (50 μg) was dissolved in 0.5 mL of PBS (pH 7.4), followed by emulsification with 0.5 mL of Freund’s complete adjuvant. The rabbits were treated subcutaneously with the emulsion (1.0 mL/rabbit) at 10 sites along the back. Booster injections with a 2 week interval were performed by the same procedure except that Freund’s complete adjuvant was replaced with Freund’s incomplete adjuvant. Peripheral blood was collected from the ear vein before each boosting injection, and titers were examined by an ELISA. Booster injections were finished until no further elevation in antibody titer was observed. Assessment of Antisera Titer. An ELISA was employed for the assessment of antiserum titers by measuring the binding of serial

dilutions (1:400 to 1:819200) of antisera to microtiter plates coated with coating antigen BSA−DDE or native BSA. The coating antigen solution (100 μL) in PBS (200 mM phosphate-buffered saline solution at pH 7.4) containing BSA−DDE or native BSA (20 μg/mL) was incubated overnight in 96-well microtiter plates at 4 °C. The following day, the coating antigen solution was discarded, and the plates were washed three times with PBST (200 mM phosphate-buffered saline solution containing 0.02% Tween 20 at pH 7.4). After being washed, the wells were blocked with 150 μL of 5% nonfat milk in PBST buffer and incubated at room temperature for 1.5 h, and then the plates were washed as described above. Aliquots (100 μL) of the antisera in PBST buffer at various dilutions were added to the wells, followed by 2 h incubation at room temperature and three washes in the same manner. A solution (100 μL) of HRP-conjugated secondary antibody (1:10000) in PBST buffer was added to each well and incubated at room temperature for 1 h, and the wells were washed again as described previously. Finally, the bound secondary antibody was measured by adding 100 μL of a freshly prepared substrate solution (0.3 mM tetramethylbenzidine and 0.4 mM H2O2 in 0.1 M sodium acetate buffer at pH 5.5). After incubation at room temperature for 30 min, 50 μL of a 4 N H2SO4 solution was added to quench the colorimetric development. The absorbance at dual wavelengths (450− 650 nm) was evaluated; data processing was performed using SigmaPlot, and curves were fitted with a four-parameter logistic equation. Competitive ELISA. A competitive ELISA was undertaken to characterize the hapten selectivity of the antibodies. A 96-well microtiter plate, whose wells were filled with 100 μL of PBS containing coating antigen BSA−DDE adducts (20 μg/mL), was incubated at 4 °C overnight. The four competitors (1−4, 0.121−250 μM) were serially diluted in 5% nonfat milk PBST buffer that included primary antiserum (1:17000), followed by incubation at 4 °C overnight. The following day, the same procedure described in the titer analysis was performed. The absorbance at dual wavelengths (450−650 nm) was read. The equation control (%) = A/A0 × 100% was employed to convert the absorbance values. The value in the presence of competitor is represented by A, and that without a competitor is represented by A0. IC50 values, the concentration that can produce 50% inhibition, could be obtained from the curve. Immunoblot Analysis. Protein bands were separated by SDS− PAGE with 5% stacking and 12% resolving gels and then electrophoretically transferred to polyvinylidene fluoride membranes by a semidry method at 15 V for 30 min. A protein assay was performed to make sure that an equal amount of protein was analyzed in advance of loading and a total of 2.0 μg of protein was loaded. The membranes were blocked with 5% nonfat milk in Tris-buffered saline with Tween 20 [TBST, 10 mM Tris, 0.15 M NaCl, and 0.05% Tween 20 (pH 7.5)] at room temperature for 1 h, followed by incubation with primary rabbit antiserum (1:17000) in TBST buffer containing 5% nonfat milk at 4 °C overnight. The next day, the membranes were washed in TBST buffer (5 × 5 min) and subsequently incubated with HRP-conjugated secondary antibody (1:10000) in TBST buffer containing 5% nonfat milk for 1 h at room temperature. The immunoblots were washed again in TBST buffer as described above. Bound antibodies were visualized by chemiluminescence with a Beyo ECL Star detection kit. Competitive immunoblot analysis was undertaken in parallel except that the primary rabbit antiserum was preincubated with serially diluted competitors (100, 10, and 1 μM) in TBST buffer containing 5% nonfat milk at 4 °C overnight. In Vivo Study. Kunming mice were intragastrically treated with DSB (0, 75, 150, or 200 mg/kg) dissolved in corn oil. Liver was harvested 12 h after administration. The freshly harvested liver tissues (0.2 g) were washed in a saline solution to remove blood and homogenized in 2.0 mL of cold lysis buffer (1 mM phenylmethanesulfonyl fluoride, 5 mM sodium fluoride, and 1 mM sodium orthovanadate), followed by centrifugation at 10000g for 20 min at 4 °C to discard tissue debris. The resulting supernatants were collected and diluted with PBS to determine protein concentration, using the BCA protein assay kit, followed by SDS−PAGE. 232

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Chemical Research in Toxicology Scheme 1. Chemical Synthesis Routes of DDE-Derived Protein Adducts

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RESULTS AND DISCUSSION There is abundant evidence of the formation of electrophilic cisenedial intermediates, namely the reactive metabolite of furan or furan-containing compounds, with high reactivity to form conjugates with Lys, GSH, Cys, NAC, or NAL.18−21 It is proposed that those interactions also occur with protein to allow disruption of protein function and even to elicit toxicities. The furan moiety of DSB can be bioactivated and metabolized into cis-enedial in vitro and in vivo by P450 enzymes,16 and hepatotoxicity was induced as a consequence of excess DDE exposure.17 Conjugates formed by reaction of DDE with nucleophilic functional groups (such as the amino group of NAL and the sulfhydryl group of GSH or NAC) were detected.16 In view of the information mentioned above, it is important for us to elucidate the relationship between DDEmodified cellular protein and DSB-induced hepatotoxicity. Immunochemical techniques have been extensively employed to determine target protein by detecting the hapten-modified portion that is formed after exposure to electrophilic intermediates.24−27 Previous study in our laboratory has demonstrated that DDE reacted with sulfhydryl- and/or amino-containing compounds to generate pyrrole or pyrroline derivatives.16 Hence, we hypothesize that DSB-derived DDE would react with lysine or cysteine residues of the protein. If such reactions occur, the pyrrole and pyrroline structures would be formed during the reaction of the DDE with KLH as the carrier protein. We reasoned that polyclonal antibodies to be raised would recognize the pyrrole and/or pyrroline moieties of DDE-modified protein. Immunogen and coating antigen were prepared by reaction of KLH or BSA with DDE as designed in Scheme 1. Briefly, DSB is oxidized by DMDO to generate DDE with a structure of chemically reactive α,β-unsaturated dialdehyde, a soft electrophile, that preferably reacts with soft nucleophiles by Michael addition, such as the sulfhydryl group of KLH or BSA, and then reacts with the amino group to form a Schiff base, followed by intramolecular cyclization and dehydration to

produce the immunogen with pyrrole. The immunogen with pyrroline was formed by a direct condensation of the cis-endial with primary amines. Eight rabbits were immunized with the immunogen, and polyclonal antibodies with the ability to recognize the corresponding pyrrole or pyrroline moieties were raised. An ELISA was performed to assess the titers of collected sera by detecting the serially diluted antisera (1:400 to 1:819200) binding to the 96-well plates coated with BSA− DDE or native BSA. The absorbance was read at dual wavelengths (450−650 nm), and data were converted into curves. The titration of antisera against the hapten−protein conjugate is shown in Figure 1. An appreciable immune response presented in Figure 1 indicates that we successfully

Figure 1. Titer analysis of antisera obtained from rabbits immunized with the immunogen. The 96-well microtiter plates were coated with BSA−DDE or native BSA and blocked with 5% nonfat milk. After incubation with diluted antisera and the secondary antibody, the substrate was added sequentially and the absorbance was read at dual wavelengths (450−650 nm). The absorbance from triplicate determinations is presented as the mean ± SD. 233

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Chemical Research in Toxicology Scheme 2. Structures of DDE−Pyrrole 1, DDE−Pyrroline 2, DMF−Pyrrole 3, and DMF−Pyrroline 4

Figure 2. Inhibition of binding of the antiserum to coating antigen BSA−DDE was investigated by a competitive ELISA. The 96-well microtiter plates were coated with BSA−DDE and blocked with 5% nonfat milk. After incubation with the antiserum preincubated with serially diluted DDE− pyrrole 1, DDE−pyrroline 2, DMF−pyrrole 3, and DMF−pyrroline 4 (0.121−250 μM), the secondary antibody and substrate were added sequentially and the absorbance was read at dual wavelengths (450−650 nm). The absorbance from triplicate determinations is presented as the mean ± SD.

showed inhibitory effects on the binding of the antiserum to the coating antigen in a concentration-dependent manner (Figure 2), which demonstrated that the high absorbance was caused by specific binding of polyclonal antibodies to the hapten moiety. Interestingly, we found that both IC50 values of DDE−pyrrole 1 and DDE−pyrroline 2 obtained from the curve were similar. It suggests that the pyrrole and pyrroline moieties elicited similar immunogenicity in rabbits immunized with the immunogen. The specificity of the antibodies was evaluated by the same technique. As expected, no such inhibition was observed when DMF−pyrrole 3 and DMF−pyrroline 4 were employed as competitors (Figure 2). This indicates the antibodies were highly selective with respect to the designed haptens. We further investigated whether the hapten loading in denatured BSA−DDE adducts could be recognized by antibodies, and an immunoblot was performed to identify whether the connection exists in denatured BSA−DDE adducts and antibodies. The adducts were synthesized as described in Scheme 1 and diluted to a series of concentration ratios (0:1, 1:9, 1:1, 9:1, and 1:0) with native BSA, followed by loading 2.0 μg of prepared samples on 10% Tris-glycine gels for electrophoresis. After separation and electrotransfer of protein, immunodetection was employed with an incubation-blotted membrane with the antiserum and then anti-rabbit IgG conjugated with horseradish peroxidase. The ECL chemiluminescence kit was used to monitor the bands on the final

immunized rabbits with immunogen and raised antisera against coating antigen. The recognition ability was evaluated by titration of antisera and determination of the degree of crossreaction to native BSA. Results showed that immunized rabbits produced very high titers (approximately 1:17000) of antibodies against the antigen, and cross-reaction between antiserum and native BSA can almost be neglected. A similar observation was made when the ovalbumin−DDE adduct (OVA−DDE) was employed as the coating antigen (Figure S1). To clarify whether the appreciable immune response in the titration tests was caused by the formation of pyrrole or pyrroline, resulting from the reaction of DDE with the amino group of the lysine residues and/or the sulfhydryl group of the cysteine residues, competitive ELISAs were performed to characterize the selectivity of the antiserum against hapten. Our previous work detected pyrroles and pyrrolines in protein adducts obtained from liver tissues of mice given DSB.16,22 On the basis of the findings, we rationally designed and synthesized four competitors (DDE−pyrrole 1, DDE−pyrroline 2, DMF− pyrrole 3, and DMF−pyrroline 4) as illustrated in Scheme 2. Before incubation with the coating antigen, the antiserum was individually preincubated with serially diluted competitors. The dilution rate (1:17000) of the antiserum was adopted in the study, according to the titer analysis. The absorbance measured at dual wavelengths (450−650 nm) was fitted against the logarithm of the different molarity of the competitors. As anticipated, both DDE−pyrrole 1 and DDE−pyrroline 2 234

DOI: 10.1021/acs.chemrestox.7b00299 Chem. Res. Toxicol. 2018, 31, 231−237

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Figure 3. Immunoblot analysis of BSA−DDE adducts or BSA−DDE−GSH adducts with the antiserum obtained from a rabbit immunized with the immunogen. (a) BSA−DDE adducts were diluted with native BSA at ratios of 0:1 (lane 1), 1:9 (lane 2), 1:1 (lane 3), 9:1 (lane 4), and 1:0 (lane 5). (b) BSA−DDE adducts or BSA−DDE−GSH adducts. Protein samples (2.0 μg) were loaded and analyzed.

To verify the formation of DDE-derived protein adducts in liver and validate the ability of the antiserum raised from rabbits to detect such protein adducts, liver homogenates, obtained from mice treated with DSB at different doses (the dose was selected on the basis of our previous study),17 were prepared for immunoblot analysis. After protein separation and blotting, the membrane was treated with the antiserum (1:17000) and consecutively incubated with the second antibody. As shown in Figure 5, multiple protein bands were detected in the lanes

antibody-incubated membrane. As expected, a chemiluminescent band of approximately 66 kDa (the molecular weight of BSA was 66 kDa) was presented in the lane loaded with BSA− DDE adducts, and the intensity of chemiluminescent bands was accumulatively increased with the higher ratio of BSA−DDE adducts in samples (Figure 3a). It suggests that the antibodies were able to detect not only the protein adducts in denatured form but also protein adduction in quantity. Our previous study showed that DDE reacted with amino groups of protein and the sulfhydryl group of GSH to form GSH-derived protein adducts.31 To determine whether the antibodies could recognize such protein adducts, BSA−DDE−GSH was synthesized and subjected to immunoblot analysis. A chemiluminescent band was observed at the molecular weight of BSA (Figure 3b). Additionally, no chemiluminescent band was observed in the lane loaded with native bovine albumin. This indicates that the antibodies were able to detect not only the pyrroline moiety but also the pyrrole structure of the protein adducts. A competitive immunoblot was conducted to further confirm the selectivity of the antibodies. The membrane blotted with BSA−DDE was divided into three pieces and incubated with the antiserum preincubated with a serial dilution of DDE− pyrrole 1 and DDE−pyrroline 2 at concentrations of 100, 10, and 1 μM, followed by incubation with the secondary antibody and detection of the chemiluminescent band with the ECL kit. As presented in Figure 4, the intensity of the chemiluminescent

Figure 5. Immunoblot analysis of DDE-modified protein samples prepared from liver of Kunming mice. Mice were treated with DSB at a dose of 75 mg/kg (lane 1), 150 mg/kg (lane 2), 200 mg/kg (lane 3), or 0 mg/kg (lane 4). Protein samples (1.0 μg) were loaded and analyzed.

loaded with protein samples obtained from animals treated with DSB, particularly for the mice given a 200 mg/kg dose. Additionally, the number and the intensity of chemiluminescence bands were increased and enhanced with the increases in the dose of DSB. Interestingly, much more intense protein bands were observed in the lane loaded with samples obtained from mice treated with DSB at 200 mg/kg than in those with samples obtained from animals given a 150 mg/kg dose of DSB, which is consistent with the pattern of the dosedependent hepatotoxicity found in our previous work.17 The observed correlation indicates the importance of protein covalent binding in DSB liver toxicity. However, a protein band at 70 kDa with weak intensity was observed in the control lane, which may result from cross-reaction. Apparently, the minor cross-reaction revealed limited interference upon detection of protein adduction resulting from metabolic activation of DSB. It appears that DSB was metabolized to an electrophilic intermediate in situ, followed by modification of cysteine and lysine residues of cellular protein in liver.

Figure 4. Competitive immunoblot of BSA−DDE adducts. Polyvinylidene fluoride membranes blotted with BSA−DDE were incubated with the antiserum preincubated with DDE−pyrrole 1 or DDE−pyrroline 2 at a concentration of 100, 10, or 1 μM. Protein samples (2.0 μg) were loaded and analyzed.

band decreased with an increase in the applied competitor molarity. Furthermore, the band responsible for BSA−DDE completely disappeared when the incubation was undertaken in the presence of the competitors at a concentration of 100 μM. This result further demonstrated that the cysteine or lysine residues of protein modified by DDE are responsible for the immunorecognition of the polyclonal antibodies to protein adducts. 235

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(4) Demetzos, C., Dimas, K., Hatziantoniou, S., Anastasaki, T., and Angelopoulou, D. (2001) Cytotoxic and anti-inflammatory activity of labdane and cis-clerodane type diterpenes. Planta Med. 67, 614−618. (5) Teponno, R. B., Tapondjou, A. L., Gatsing, D., Djoukeng, J. D., Abou-Mansour, E., Tabacchi, R., Tane, P., Stoekli-Evans, H., and Lontsi, D. (2006) Bafoudiosbulbins A, and B, two anti-salmonellal clerodane diterpenoids from Dioscorea bulbifera L. var. Phytochemistry 67, 1957−1963. (6) Cifuente, D. A., Borkowski, E. J., Sosa, M. E., Gianello, J. C., Giordano, O. S., and Tonn, C. E. (2002) Clerodane diterpenes from Baccharis sagittalis:insect antifeedant activity. Phytochemistry 61, 899− 905. (7) Murray, R. D. H., Jorge, Z. D., Khan, N. H., Shahjahan, M., and Quaisuddin, M. (1984) Diosbulbin D and 8-epidiosbulbin E acetate, norclerodane diterpenoids from Dioscorea bulbifera tubers. Phytochemistry 23, 623−625. (8) Yang, H., Li, J., Cui, X., Yang, C., Li, L., and Liu, J. (2006) Clinical use and adverse drug reaction of compound prescription of Dioscorea bulbifera L. in clinical trial. Linchuang Wuzhen Wuzhi 19, 85−87. (9) Liu, J. R. (2002) Two cases of toxic hepatitis caused by Dioscorea Bulbifera L. Adverse Drug React. 2, 129−130. (10) Wang, J., Liang, Q., Ji, L., Liu, H., Wang, C., and Wang, Z. (2011) Genderrelated difference in liver injury induced by Dioscorea bulbifera L. rhizome in mice. Hum. Exp. Toxicol. 30, 1333−1341. (11) Ma, Y., Niu, C., Wang, J., Ji, L., and Wang, Z. (2014) Diosbulbin B-induced liver injury in mice and its mechanism. Hum. Exp. Toxicol. 33, 729−736. (12) Niu, Z. M., and Chen, A. Y. (1994) 16 cases report of toxic hepatitis caused by Dioscorea bulbifera. Zhongxiyi Jiehe Ganbing Zazhi 4, 55−56. (13) Wang, J., Ji, L., Liu, H., and Wang, Z. (2010) Study of the hepatotoxicity induced by Dioscorea bulbifera L. rhizome in mice. BioSci. Trends 4, 79−85. (14) Liu, H., Chou, G., Guo, Y., Ji, L., Wang, J., and Wang, Z. (2010) Norclerodane diterpenoids from the rhizomes of Dioscorea bulbifera. Phytochemistry 71, 1174−1180. (15) Gao, H., Kuroyanagi, M., Wu, L., Kawahara, N., Yasuno, T., and Nakamura, Y. (2002) Antitumor-promoting constituents from Dioscorea bulbifera L. in JB6 mouse epidermal cells. Biol. Pharm. Bull. 25, 1241−1243. (16) Lin, D., Li, C., Peng, Y., Gao, H., and Zheng, J. (2014) Cytochrome P450-mediated metabolic activation of Diosbulbin B. Drug Metab. Dispos. 42, 1727−1736. (17) Li, W. W., Lin, D. J., Gao, H. Y., Xu, Y. J., Meng, D. Y., Smith, C. V., Peng, Y., and Zheng, J. (2016) Metabolic activation of furan moiety makes diosbulbin B hepatotoxic. Arch. Toxicol. 90, 863. (18) Grill, A. E., Schmitt, T., Gates, L. A., Lu, D., Bandyopadhyay, D., Yuan, J. M., Murphy, S. E., and Peterson, L. A. (2015) Abundant Rodent Furan-Derived Urinary Metabolites Are Associated with Tobacco Smoke Exposure in Humans. Chem. Res. Toxicol. 28, 1508−1516. (19) Lu, D., and Peterson, L. A. (2010) Identification of Furan Metabolites Derived from Cysteine-cis-2-Butene-1,4-dial-Lysine CrossLinks. Chem. Res. Toxicol. 23, 142−151. (20) Li, C., Lin, D., Gao, H., Hua, H., Peng, Y., and Zheng, J. (2015) N-Acetyl lysine/glutathione-derived pyrroles as potential Ex Vivo biomarkers of bioactivated furan-containing compounds. Chem. Res. Toxicol. 28, 384−393. (21) Yang, B., Liu, W., Chen, K., Wang, Z., and Wang, C. (2014) Metabolism of diosbulbin B in vitro and in vivo in rats: formation of reactive metabolites and human enzymes involved. Drug Metab. Dispos. 42 (10), 1737−1750. (22) Lin, D. J., Guo, X. C., Gao, H. Y., Cheng, L., Cheng, M. S., Song, S. J., Peng, Y., and Zheng, J. (2015) In Vitro and in Vivo Studies of the Metabolic Activation of 8-Epidiosbulbin E Acetate. Chem. Res. Toxicol. 28, 1737−1746. (23) Wang, K., Lin, D. J., Guo, X. C., Huang, W. L., Zheng, J., and Peng, Y. J. (2017) Chemical Identity of Interaction of Protein with

Characterization of the modified proteins is critical for understanding of the mechanisms of toxic action of DSB, and efforts will be made to define the insight into the mechanisms of toxic action of DSB. In conclusion, polyclonal antibodies have been successfully raised to detect DDE-derived protein adducts with modifications at lysine and/or cysteine residues. The antibodies revealed high titers with neglected cross-reaction toward native proteins. This would provide a powerful tool for investigating the biochemical mechanism of toxicities induced by DDE.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.7b00299.

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Figure S1 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Wuya College of Innovation, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, Liaoning 110016, P. R. China, and State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University, 9 Beijing Rd., Guiyang, Guizhou 550025, P. R. China. E-mail: [email protected]. Telephone: +86-2423986361. Fax: +86-24-23986510. The two corresponding units contributed equally to this work. *Wuya College of Innovation, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, Liaoning 110016, P. R. China. E-mail: [email protected]. Telephone: +86-24-23986361. Fax: +86-24-23986510. ORCID

Jiang Zheng: 0000-0002-0340-0275 Funding

This work was supported in part by the National Natural Science Foundation of China (Grants 81373471, 81430086, and 81773813). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS DSB, diosbulbin B; DB, D. bulbifera L.; DDE, DSB-derived cisenedial; NAL, N-acetyl lysine; NAC, N-acetyl cysteine; GSH, glutathione; KLH, thiolated keyhole limpet hemocyanin; DMDO, dimethyldioxirane; BSA, bovine serum albumin; OVA, ovalbumin; DMF, 2,5-dimethylfuran

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REFERENCES

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