Development of Polyclonal Antibodies for ... - ACS Publications

Subscriber access provided by - Access paid by the | UCSB Libraries

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 Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00299 • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 25, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Title page

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

Running title: Immunochemical Detection of Diosbulbin B Derived Protein Adducts

Corresponding Authors: Jiang Zheng, PhD Wuya College of Innovation, Shenyang Pharmaceutical University, No. 103, Wenhua Road, 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, 9 Beijing Road, Guiyang, Guizhou, 550025, P.R. China Email: [email protected] Tel: +86-24-23986361; Fax: +86-24-23986510 †, §: The two corresponding units contributed equally to this work. Ying Peng, PhD Wuya College of Innovation, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, Liaoning, 110016, P. R. China Email: [email protected] Tel: +86-24-23986361; Fax: +86-24-23986510

ACS Paragon Plus Environment

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 30

Abstract Diosbulbin B (DSB), a major component found in 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 human after 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 which can

recognize DDE-derived protein adducts.

Immunogens synthesized from DDE and

keyhole limpet hemocyanin (KLH) were employed to raise polyclonal antibodies in rabbits.

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 also detected a multitude of

chemiluminescent bands with a variety of molecular weight in liver homogenates which were harvested from mice treated with DSB.

In summary, we have

successfully raised polyclonal antibodies to detect protein adducts derived from DDE.


Page 3 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Dioscorea bulbifera L. (DB), a Chinese medicinal herb which belongs to the yam family Dioscoreaceae, has been widely employed for treatment of breast lumps, goiters, carbuncles, and lung abscesses.1,2 displays

a

variety

of

In addition, studies demonstrated that DB

pharmacological

properties,

such

anti-inflammatory,4 antisalmonellal,5 and antifeedant6 activities. clinical use carries the risk of serious liver injury.7-9

as

antitumor,3 However, its

Hepatitis induced after chronic

exposure to DB has been reported in patients and the clinical parameters are accordant with those observed in animals.10-13 Diosbulbin B (DSB), a furan-containing diterpenoid lactone, is a major component in 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 hepatotoxicities.17

The cis-enedial is required for DSB-induced

The primary amino groups of lysine (Lys), N-acetyl lysine (NAL)

and glutathione (GSH) and/or the sulfhydryl of cysteine (Cys), N-acetyl 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 both in 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 alone.22

Recently, we

succeeded in detection of protein adductions derived from DDE in liver of animals



Page 4 of 30

given DSB, using 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.

The present

study 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 on the biochemical mechanism of DSB-induced toxicity.


Page 5 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 and its structure identity was characterized by mass spectrometry and NMR.

The purity of DSB was > 98%

determined by HPLC with a diode array detector. hemocyanin

(KLH),

Water-soluble keyhole limpet

3,3',5,5'-tetramethylbenzidine,

2,5-dimethylfuran

(DMF),

N-acetyl 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) was purchased from Salarbio (Beijing, China). BCA protein assay kit and BeyoECL Star detection kit were purchased from Beyotime (Shanghai, China). grade.

All of other reagents or solvents were of analytical

Greiner Bio-One (Frickenhausen, Germany) 96-well microtiter plates were

used in ELISA.

Thermo Scientific Varioskan Flash Spectral Scanning Multimode

Reader was applied to measure the ELISA absorbance. electrophoresis system was employed for SDS-PAGE.

Bio-Rad basic

Membrane transfer was

accomplished with Bio-Rad Tans-Blot SD Semi-Dry Transfer Cell.

Synthesis Immunogen. ice bath.

DSB (5 mg) was dissolved in 200 µL of acetone and cooled in a dry To the resulting solution, dimethyldioxirane (DMDO, 1.0 mL),



Page 6 of 30

pre-prepared according to the published method,30 was dropwise added with stirring. The stirring continued for 1 h at room temperature, followed by condensing with a gentle flow of nitrogen gas.

The residue was reconstituted with 1.0 mL of PBS

buffer containing KLH (3 mg/mL), and the resultant mixture was incubated for 1 h at 37 °C with stirring, and then frozen and kept in -20 °C after dialysis.

Competitors.

Competitive agents were synthesized as published protocol 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 buffer containing NAC (0.71 mg) and/or NAL (0.82 mg) to obtain the corresponding competitors, including DDE-pyrrole derivative (1), DDE-pyrroline derivative (2), DMF-pyrrole derivative (3), and 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 semi-preparative 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

cis-enedial

intermediate─glutathione

(BSA-DDE-GSH), were synthesized as positive control in immunoblot, and BSA-DDE was also used for coating antigen in ELISA.

All procedure was

undertaken as described in “Immunogen” except KLH was replaced by BSA (1.0 mg) or BSA (1.0 mg) combination with GSH (1.3 mg).


Page 7 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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

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. of antisera.

Japanese white rabbits were immunized for preparation

The rabbits were immunized with 20 µL of immunogen emulsion.

The

immunogen (50 µg) was dissolved in 0.5 mL of PBS buffer (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 the Freund’s complete adjuvant was replaced by Freund’s incomplete adjuvant.

Peripheral blood was collected from the ear vein before each boosting

injection, titers were examined by ELISA.

Booster injections were finished until no

further elevation in antibody titer was observed.

Assessment of Antisera Titer.

ELISA was employed for the assessment of antisera

titers by measuring the binding of serial dilutions (1/400 to 1/819,200) of antisera to microtiter plates coated with coating antigens BSA-DDE or native BSA.

Coating

antigen solution (100 µL) in PBS buffer (200 mM phosphate-buffered saline solution



Page 8 of 30

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

wash procedure, 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 before.

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 washing three times in the same manner.

A solution (100 µL) of HRP conjugated secondary

antibody (1:10,000) 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 freshly prepared substrate solution (0.3 mM tetramethylbenzidine, 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 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 by four-parameter logistic equation.

Competitive ELISA.

Competitive ELISA was undertaken to characterize the

hapten-selectivity of the antibodies.

A 96-well microtier plate, whose wells were

filled with 100 µL of PBS buffer containing coating antigen BSA-DDE adducts (20 µg/mL), was incubated at 4 °C overnight.

The four competitors (1-4, 0.121 to 250


Page 9 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


µM ) were serially diluted in 5% nonfat milk-PBST buffer included primary antiserum (1:17,000) respectively, followed by incubation at 4 °C overnight.

The following

day, the same procedure described in the titer analysis was carried out. absorbance at dual wavelengths (450-650 nm) was read.

The

The equation: control (%)

= A/A0×100% was employed to convert the absorbance values.

The value in the

presence of competitor was represented by A, and without a competitor was represented by A0.

IC50 values, namely the concentration that can produced 50%

inhibition could be obtained from the curve.

Immunoblot Analysis.

Protein bands were separated by SDS-polyacrylamide gel

electrophoresis with 5% stacking and 12% resolving gels, then electrophoretically transferred to polyvinylidene fluoride membranes by semi-dry method at 15 V for 30 min.

A protein assay was carried out to make sure that an equal amount of protein

was analyzed in advance of loading and 2.0 µg protein was loaded totally.

The

membranes were blocked with 5% nonfat milk in Tris-buffered saline with Tween-20 (TBST, 10 mM Tris, 0.15 M NaCl, 0.05% Tween-20, pH 7.5) at room temperature for 1 h, followed by incubation with primary rabbit antiserum (1:17,000) 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 (1:10,000) in TBST buffer containing 5% nonfat milk for 1 h at room temperature.

The immunoblots were washed again in TBST buffer as before.

Bound antibodies were visualized by chemiluminescence with a Beyo ECL Star detection kit.

Competitive immunoblot analysis was undertaken in parallel except



Page 10 of 30

that the primary rabbit antiserum were 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. administration.

Liver was harvested at 12 h following the

The freshly harvested liver tissues (0.2 g) were washed in saline

solution to remove blood and homogenized in 2.0 mL cold Lysis buffer (1 mM phenylmethanesulfonyl

fluoride,

5

mM

sodium

fluoride,

1

mM

sodium

orthovanadate), followed by centrifugation at 10,000 g for 20 min at 4 °C to discard tissue debris.

The resulting supernatants were collected and diluted with PBS buffer

to determine protein concentration, using BCA protein assay kit, followed by SDS-polyacrylamide gel electrophoresis.


Page 11 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Results and Discussion There are abundant evidences for the formation of electrophilic cis-enedial intermediates, namely 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 to disrupt 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 amino group of NAL and sulfhydryl group of GSH or NAC) were detected.16

In view of these

information above, it is important for us to elucidate the relationship between DDE modified cellular protein and DSB-induced hepatotoxicity.

Immunochemical

techniques have been extensively employed to determine target protein by detecting the hapten-modified portion which 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 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.

Page 12 of 30

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 sulfhydryl group of KLH or BSA, and then reacts with amino group to form a Schiff’s base, followed by intramolecular cyclization and dehydration to produce the immunogen with pyrrole.

While 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 recognition ability towards the corresponding pyrrole or pyrroline moieties were raised.

ELISA

was carried out to assess the titers of collected sera by detecting the serially diluted antisera (1/400 to 1/819,200) binding to the 96-well plates coated with BSA-DDE or native BSA.

Absorbance was read at dual wavelengths (450-650 nm) and data were

converted into curves. The titration of antisera against hapten-protein conjugate is shown in Figure 1.

An appreciable immune response presented in Figure 1 indicates

that we successfully 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 cross-reaction to native BSA.

Results showed that

immunized rabbits produced very high titers (approximately 1:17,000) of antibodies against the antigen, and cross reaction between antiserum and native BSA can be almost neglected.

Similar observation was obtained when ovalbumin-DDE

(OVA-DDE) was employed as coating antigen (Figure S1). In order to clarify whether the appreciable immune response in the titration


Page 13 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tests was caused by the formation of pyrrole or pyrroline, resulting from the reaction of DDE with amino group of the lysine residues and/or sulfhydryl group of the cysteine residues, competitive ELISAs were carried out 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 Based on 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 coating antigen, antiserum was preincubated with a serially diluted competitors individually.

Dilution rate (1:17,000) 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 different molarity of the competitors.

As anticipated, both DDE-pyrrole 1 and DDE-pyrroline 2

showed inhibitory effects on the binding of 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 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 imminogenicity in rabbits immunized with the imminogen. 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 to the designed haptens.



Page 14 of 30

It would be of interest to further investigate whether the hapten loading in denatured BSA-DDE adducts could be recognized by antibodies, and immunoblot was carried out 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 serial of concentration ratios (0:1, 1:9, 1:1, 9:1, 1:0) with native BSA, followed by loading 2.0 µg of prepared samples on 10% Tris-glycine gels for electrophortesis.

After separation and electrotransfer of protein, immunodetection

was employed by incubation blotted membrane with antiserum and then anti-rabbit IgG conjugated with horseradish peroxidase.

ECL chemiluminescence kit was used

to monitor the bands on the final 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 the protein adduction in quantity.

Our previous study showed that DDE reacted with amino groups of

protein and the sulhydryl group of GSH to form GSH derived protein adducts.31 To determine whether the antibodies to recognize such protein adducts, BSA-DDE-GSH was synthesized and subjected to immunoblot.

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


Page 15 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


also the pyrrole structure of the protein adducts. 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 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 secondary antibody and detection of the chemiluminescent band with ECL kit. As presented in Figure 4, the intensity of chemiluminescent band decreased with the increase of competitor molarity applied.

Furthermore, the band responsible for

BSA-DDE completely disappeared when the incubation was undertaken in the presence of 100 µM of the competitors.

This result further demonstrated that the

cysteine or lysine residues of protein modified by DDE responsible for the immunorecognition of the polyclonal antibodies to protein adducts. 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 administered of DSB at different dosage (the dosage was selected on the base of our previous study),17 were prepared for immunoblot.

After protein separation and blotting, the membrane was treated with

the antiserum (1:17,000) and consecutively incubated with the second antibody.

As

shown in Figure 5, multiple protein bands were detected in the lanes loaded with protein samples obtained from animals treated with DSB, particularly for the mice dosed at 200 mg/kg.

Additionally, the number and the intensity of

chemiluminescence bands were increased and enhanced with the increases of the dose



of DSB.

Page 16 of 30

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 that of animals given 150 mg/kg DSB, which is consistent with the pattern of the dose-dependent hepatotoxicity study found in our previous work.17

The observed

correlation just 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 on the detection of protein adduction resulting from metabolic activation of DSB.

It appears that DSB was metabolized to

electrophilic intermediate in situ, followed by modification of cysteine and lysine residues of cellular protein in liver. Characterization of the modified proteins is critical for the 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 the 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 the investigation of biochemical

mechanism of toxicities induced by DDE.


Page 17 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Funding sources This work was supported in part by the National Natural Science Foundation of China [Grant 81373471, 81430086, 81773813].



Abbreviations: DSB, diosbulbin B; DB, Dioscorea bulbifera L.; DDE, DSB-derived cis-enedial; NAL, N-acetyl lysine; NAC, N-acetyl cysteine; GSH, glutathione; Lys, lysine; Cys, cysteine; KLH, thiolated keyhole limpet hemocyanin; DMDO, dimethyldioxirane; BSA, bovine serum albumin; OVA, ovalbumin; DMF, 2,5-dimethylfuran.


Page 18 of 30

Page 19 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


References (1) Li, S., Iliya, I. A., Deng, J., Zhao, S. (2000) Flavonoids and anthraquinone from Dioscorea bulbifera L. Chin. J. Chin. Mater. Med. 25, 159–160. (2) Gao, H. Y., Shui, A. L., Chen, Y. H., Zhang, X. Y., Wu, L. J. (2003) The chemical compositions of Dioscorea bulbifera L. J. Shenyang Pharm. Univ. 20, 178–180. (3) Grynberg, N. F., Echevarria, A., Lima, J. E., Pamplona, S. S., Pinto, A. C., Maciel, M. A. (1999) Anti-tumour activity of two 19-nor-clerodane diterpenes, trans-dehydrocrotonin and trans-crotonin, from Croton cajucara. Planta. Med. 65, 687–689. (4) Demetzos, C., Dimasm, K., Hatziantoniou, S., Anastasaki, T., 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., Lontsi, D. (2006) Bafoudiosbulbins A, and B, two anti-salmonellal clerodane diterpenoids from Dioscorea bulbifera L. var. sativa. Phytochemistry 67, 1957–1963. (6) Cifuente, D. A., Borkowski, E. J., Sosa, M. E., Gianello, J. C., Giordano, O. S., 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., 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., Liu, J. (2006) Clinical use and adverse drug reaction of compound prescription of Dioscorea bulbifera L. in clinical trial. Clin. Misdiagn. Misther. 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., Wang, Z. (2010) 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., Wang, Z. (2013) 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. Chin. J. Integr. Tradit. Western Liver Dis. 4, 55−56. (13) Wang, J., Ji, L., Liu, H., 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., Wang, Z. (2010) Norclerodane diterpenoids from the rhizomes of Dioscorea bulbifera. Phytochemistry. 71, 1174–1180. (15) Gao, H., Kuroyanaqi, M., Wu, L., Kawahara, N., Yasuno, T., 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., Zheng, J. (2015) Metabolic activation of furan moiety makes diosbulbin B hepatotoxic. Arch. Toxicol. doi: 10.1007. (18) Grill, A. E., Schmitt, T., Gates, L. A., Lu, D., Bandyopadhyay, D., Yuan, J. M., Murphy, S. E., 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., Peterson, L. A. (2010) Identification of Furan Metabolites Derived from Cysteine-cis-2-Butene-1,4-dial-Lysine Cross-Links. Chem. Res. Toxicol. 23, 142–151.


Page 20 of 30

Page 21 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(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. (2015) 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., Zheng, J. (2015) In Vitro and in Vivo Studies of the Metabolic Activation of 8‑Epidiosbulbin E Acetate. Chem. Res. Toxicol. doi: 10.1021. (23) Wang, K., Lin, D. J., Guo, X. C., Huang, W. L., Zheng J., Peng Y., J. (2017) Chemical Identity of Interaction of Protein with Reactive Metabolite of Diosbulbin B In Vitro and In Vivo. Toxins. doi:10.3390. (24) Yuan, W., Chung, J. K., Gee, S., Hammock, B. D., Zheng, J. (2007) Development of Polyclonal Antibodies for the Detection of Styrene Oxide Modified Proteins. Chem. Res. Toxicol. 20, 316–321. (25) Zheng, J., Hammock, B. D. (1996) Development of Polyclonal Antibodies for Detection of Protein Modification by 1,2-Naphthoquinone. Chem. Res. Toxicol. 9, 904–909. (26) Liu, W., Zhao, C. B., Zhang, Y. L., Lu, S. X., Liu, J. T., Xi, R. M. (2007) Preparation of Polyclonal Antibodies to a Derivative of 1-Aminohydantoin (AHD) and Development of an Indirect Competitive ELISA for the Detection of Nitrofurantoin. Residue. J. Agric. Food Chem. 55, 6829–6834. (27) Carey, M. A., Pelt, F. N. A. M. (2005) Immunochemical detection of flucloxacillin adduct formation in livers of treated rats. Toxicology. 216, 41–48. (28) Khojasteh, S. C., Hartley, D. P., Ford, K. A., Uppal, H., Oishi, S., Nelson, S. D. (2012) Characterization of Rat Liver Proteins Adducted by Reactive Metabolites of Menthofuran. Chem. Res. Toxicol. 25, 2301–2309. (29) Kawasaki, T., Komori, T., and Setoguchi, S. (1968) Furanoid norditerpenes from Dioscoreacae plants. 1. Diosbulins A, B, and C from Dioscorea bulbifera form a spontanea. Chem Pharm Bull (Tokyo) 16, 2430–2435. ACS Paragon Plus Environment


(30) Taber, D. F., DeMatteo, P. W., and Hassan, R. A. (2013) Simplified Preparation of Dimethyldioxirane (DMDO). Org. Synth. 90, 350–357. (31) Lin, D. J., Wang, K., Guo, X. C., Gao, H. Y., Peng, Y., Zheng, J. (2016) Lysine- and cysteine-based protein adductions derived from toxic metabolites of 8-epidiosbulbin E acetate. Toxicology Letters. 264, 20–28.


Page 22 of 30

Page 23 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC.graph 155x53mm (150 x 150 DPI)



Figure 1. Titer analysis of antisera obtained from rabbits immunized with immunogen. 96-Well microtiter plates were coated with BSA-DDE or native BSA and blocked with 5% nonfat milk. Followed by incubation with diluted antisera and secondary antibody, substrate was added sequentially and the absorbance was read at dual wavelength (450-650 nm). The absorbance from triplicate determinations is presented in the mean ± SD. 96x79mm (220 x 220 DPI)


Page 24 of 30

Page 25 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Inhibition of binding of antiserum to coating antigen BSA-DDE was investigated by competitive ELISA. 96-Well microtiter plates were coated with BSA-DDE and blocked with 5% nonfat milk. Followed by incubation with antiserum preincubated with serially diluted DDE-pyrrole 1, DDE-pyrroline 2, DMF-pyrrole 3 and DMF-pyrroline 4 (0.121 to 250 µM). Secondary antibody and substrate were added sequentially and the absorbance was read at dual wavelength (450-650 nm). The absorbance from triplicate determinations is presented in the mean ± SD. 165x65mm (220 x 220 DPI)



Figure 3. Immunoblot analysis of BSA-DDE adducts or BSA-DDE-GSH adducts by antiserum obtained from a rabbit immunized with immunogen. a: BSA-DDE adducts were diluted with native BSA at the ratio of 0:1 (lane 1), 1:9 (lane 2), 1:1 (lane 3), 9:1 (lane 4), 1:0 (lane 5). b: BSA-DDE adducts or BSA-DDE-GSH adducts. Protein samples (2.0 µg) were loaded and analyzed. 495x167mm (150 x 150 DPI)


Page 26 of 30

Page 27 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 concentrations of 100, 10, or 1 µM. Protein samples (2.0 µg) were loaded and analyzed. 56x24mm (150 x 150 DPI)



Figure 5. Immunoblot analysis of DDE-modified protein samples prepared from liver of Kunming mice. Mice were treated with DSB at a dosage 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. 56x71mm (150 x 150 DPI)


Page 28 of 30

Page 29 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. Chemical synthesis routes of DDE-derived protein adducts. 583x377mm (150 x 150 DPI)



Scheme 2. Structure of DDE-pyrrole 1, DDE-pyrroline 2, DMF-pyrrole 3, DMF-pyrroline 4. 484x126mm (150 x 150 DPI)


Page 30 of 30

Development of Polyclonal Antibodies for ... - ACS Publications

Recommend Documents