Development of Polyclonal Antibodies for Detection of Diosbulbin B-Derived <i>cis</i>-Enedial Protein Adducts

Hu, Zixia; Zhou, Shenzhi; Zhang, Na; Li, Weiwei; Lin, Dongju; Peng, Ying; Zheng, Jiang

doi:10.1021/acs.chemrestox.7b00299

Cited by 12 publications

(4 citation statements)

References 25 publications

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“…Facilitating the recognition of the DDE-derived protein adduction, we further successfully raised the polyclonal antibodies with high titers in rabbits immunized with the antigen prepared by reaction of KLH with DDE. Immunoblot analysis of the liver homogenates obtained from mice given different doses of DSB showed that much more intense protein bands were observed in the samples from 200 mg/kg DSB-treated mice than that from mice administered with 150 mg/kg DSB, similar to the dose-13 dependent hepatotoxicity observed above (Hu et al, 2018).…”

Section: Diosbulbin Bsupporting

confidence: 64%

Metabolic Activation and Hepatotoxicity of Furan-Containing Compounds

Tian

Peng

Zheng

2022

Drug Metabolism and Disposition

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Furan-containing compounds are abundant in nature, of which many but not all have been found to be hepatotoxic and carcinogenic. The furan ring present in the chemical structures may be one of the domineering factors to bring about the toxic response resulting from the generation of reactive epoxide or cis-enedial intermediates which are of the potential to react with biomacromolecules. This review sets out to explore the relationship between the metabolic activation and hepatotoxicity of furan-containing compounds on the strength of scientific reports on several typical alkylated furans, synthetic pharmaceuticals and components extracted from herbal medicines. The pharmacological activities as well as concrete evidence of their liver injuries are described, and the potential toxic mechanisms were discussed partly based on our previous work. Efforts were made to understand the development of liver injury and seek solutions to prevention and interference of the adverse effects.

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Section: Diosbulbin Bsupporting

confidence: 64%

Metabolic Activation and Hepatotoxicity of Furan-Containing Compounds

Tian

Peng

Zheng

2022

Drug Metabolism and Disposition

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“…DSB, the major component in traditional Chinese medicine DBL, was found to be hepatotoxic in our early studies, and its cis -enedial metabolite was responsible for the reported toxicity. , The reactive intermediate was found to react with GSH, protein, and DNA. ,,− The present study aimed at the interaction of intermediate 2 (Scheme ) with polyamines/biogenic amines/amino acids, along with its potential correlation with DSB-induced cell death. We employed BBM to trap toxic cis -enedial intermediate 2 resulting from the bioactivation of the furan-ring moiety of DSB.…”

Section: Discussionmentioning

confidence: 97%

Evidence for Polyamine, Biogenic Amine, and Amino Acid Adduction Resulting from Metabolic Activation of Diosbulbin B

Zhang

et al. 2020

Chem. Res. Toxicol.

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Dioscorea bulbifera L. (DBL), a traditional Chinese medicine, is a well-known herb with hepatotoxicity, and the biochemical mechanisms of the toxic action remain unknown. Diosbulbin B (DSB), a major component of DBL, can induce severer liver injury which requires cytochrome P450-catalyzed oxidation of the furan ring. It is reported that a cis-enedial reactive intermediate resulting from metabolic activation of DSB can react with thiols and amines to form pyrrole or pyrroline derivatives. In this study, we investigated the interaction of the reactive intermediate with polyamines, biogenic amines, and amino acids involved in the polyamine metabolic pathway, including putrescine, spermidine, spermine, histamine, arginine, ornithine, lysine, glutamine, and asparagine. Seven DSB-derived amine adducts were detected in microsomal incubations supplemented with DSB and individual amines. Six adducts were observed in cultured rat primary hepatocytes after exposure to DSB. DSB was found to induce apoptosis and cell death in time-and concentration-dependent manners. Apparently, the observed apoptosis was associated with the detected amine adduction. The findings facilitate the understanding of the mechanisms of toxic action of DSB.

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“…The regression curves of the competitors were fitted Western Blot. Protein (1.0 μg/well) obtained from in vitro and in vivo studies were separated by SDS-PAGE as we previously described, 32 and then the proteins resolved were transferred to polyvinylidene fluoride (PVDF) membranes by the semidry technique at 10 V for 15 min. After transferring, the PVDF membranes with protein bands were blocked with 5% nonfat milk in TBST buffer (0.05% Tween 20, 0.15 M NaCl, and 10 mM Tris at pH 7.5) for 2 h at room temperature.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Immunochemical Detection of Protein Modification Derived from Metabolic Activation of 8-Epidiosbulbin E Acetate

Zhou

Zhang

et al. 2020

Chem. Res. Toxicol.

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Furanoid 8-epidiosbulbin E acetate (EEA) is one of the most abundant diterpenoid lactones in herbal medicine Dioscorea bulbifera L. (DB). Our early work proved that EEA could be metabolized to EEA-derived cis-enedial (EDE), a reactive intermediate, which is required for the hepatotoxicity observed in experimental animals exposed to EEA. Also, we found that EDE could modify hepatic protein by reaction with thiol groups and/or primary amines of protein. The present study was inclined to develop polyclonal antibodies to detect protein modified by EDE. An immunogen was prepared by reaction of EDE with keyhole limpet hemocyanin (KLH), and polyclonal antibodies were raised in rabbits immunized with the immunogen. Antisera collected from the immunized rabbits demonstrated high titers evaluated by enzyme-linked immunosorbent assays (ELISAs). Immunoblot analysis showed that the polyclonal antibodies recognized EDEmodified bovine serum albumin (BSA) in a hapten load-dependent manner but did not cross-react with native BSA. Competitive inhibition experiments elicited high selectivity of the antibodies toward EDE-modified BSA. The antibodies allowed us to detect and enrich EDE-modified protein in liver homogenates obtained from EEA-treated mice. The developed immunoprecipitation technique, along with mass spectrometry, enabled us to succeed in identifying multiple hepatic proteins of animals given EEA. We have successfully developed polyclonal antibodies with the ability to recognize EDE-derived protein adducts, which is a unique tool for us to define the mechanisms of toxic action of EEA.

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Development of Polyclonal Antibodies for Detection of Diosbulbin B-Derived cis-Enedial Protein Adducts

Cited by 12 publications

References 25 publications

Metabolic Activation and Hepatotoxicity of Furan-Containing Compounds

Metabolic Activation and Hepatotoxicity of Furan-Containing Compounds

Evidence for Polyamine, Biogenic Amine, and Amino Acid Adduction Resulting from Metabolic Activation of Diosbulbin B

Immunochemical Detection of Protein Modification Derived from Metabolic Activation of 8-Epidiosbulbin E Acetate

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