Evaluation of Gastric Ulcerogenic and Healing Impairment Effects of Bisphosphonates: Adverse Gastric Reactions of Bisphosphonate

Koji Takeuchi1, Kikuko Amagase1

1 Department of Pharmacology and Experimental Therapeutics, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto, Japan
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 21.10
DOI:  10.1002/0471140856.tx2110s53
Online Posting Date:  August, 2012
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Abstract

Bisphosphonates (BPPs) were developed as antiresorptive drugs capable of treating diseases related to bone remodeling; however, they have untoward effects including ulceration in the upper gastrointestinal tract and worsen the healing‐impairment action of nonsteroidal anti‐inflammatory drugs, prescribed in patients with arthritis or osteoporosis. We produced ulcers in the antrum by administering BPPs to fasted rats, followed by refeeding, and confirmed their healing‐impairment action on pre‐existing gastric ulcers; the ulcerogenic effect is due to direct mucosal irritation and decrease in the mucosal anti‐oxidative system, while the latter effect is due to dysregulation of growth factor expression, such as vascular endothelial growth factor and basic fibroblast growth factor, and angiogenesis in the ulcerated mucosa. In this article, we describe these two animal models for investigating BPP‐related adverse reactions, including methods for the induction of antral ulcers and healing impairment of gastric ulcers, as well as measurement of pathogenic functional and biochemical changes. Curr. Protoc. Toxicol. 53:21.10.1‐21.10.29. © 2012 by John Wiley & Sons, Inc.

Keywords: bisphosphonate; gastric adverse effect; antral ulceration; healing‐impairment action

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Antral Ulcers
  • Basic Protocol 2: Healing‐Impairment Effect on Chronic Gastric Ulcers
  • Support Protocol 1: Quantification of Microvascular Permeability by Evans Blue Staining
  • Support Protocol 2: Measuring Myeloperoxidase (MPO) Activity
  • Support Protocol 3: Assaying Lipid Peroxidation via TBARS
  • Support Protocol 4: Evaluation of SOD Activity
  • Support Protocol 5: Quantification of GSH Content Using the DNTB Assay
  • Support Protocol 6: Quantification of Acid Secretion
  • Support Protocol 7: Assessment of Mucosal PGE2 Content
  • Support Protocol 8: Quantification of COX‐2 mRNA Expression
  • Support Protocol 9: Analysis of VEGF and bFGF Protein Expression
  • Support Protocol 10: Evaluation of Angiogenesis by Immunostaining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of Antral Ulcers

  Materials
  • Male Sprague‐Dawley rats (6‐ to 8‐weeks old; 200 to 260 g; Charles River Laboratories)
  • Alendronate (see recipe) or a similar bisphosphonate
  • Compounds for assessing prophylactic efficacy (optional):
    • Rebamipide (see recipe)
    • Omeprazole (see recipe)
    • Indomethacin (see recipe)
    • Allopurinol (Sigma; dissolve in 0.9% NaCl)
    • Superoxide dismutase (SOD, Nacalai Tesque, http://www.nacalai.co.jp/global/; dissolve in 0.9% NaCl)
  • Anesthetic agent suitable for survival surgery (diethyl ether recommended; Wako)
  • 2% (v/v) formalin in normal saline
  • Hematoxylin and eosin (e.g., Fisher, Sigma)
  • Gavage needles appropriate for rats
  • Dissecting instruments
    • Scalpel or blunt‐end scissors
    • Forceps
  • Dissecting microscope (e.g., Olympus) with 1‐mm square‐grid eyepiece (10×)
  • Additional reagents and equipment for paraffin embedding and sectioning (Hofman, )
NOTE: If performing assays described in the support protocols, please refer to the respective support protocol for additional required materials.

Basic Protocol 2: Healing‐Impairment Effect on Chronic Gastric Ulcers

  Materials
  • Male Sprague Dawley rats (6‐ to 8‐weeks old; 200 to 260 g; Charles River Laboratories)
  • Anesthetic agent suitable for survival surgery (diethyl ether recommended; Wako)
  • Alendronate (see recipe) or indomethacin (see recipe)
  • Dissecting instruments
    • Scalpel or blunt‐end scissors
    • Forceps
  • Electric thermal cauterization probe (Fuchigami, Kyoto, Japan)
  • Dissecting microscope (e.g., Olympus) with 1‐mm square‐grid eyepiece (10×)
  • Gavage needle appropriate for rats
NOTE: If performing assays described in the support protocols, please refer to the respective support protocol for additional required materials.

Support Protocol 1: Quantification of Microvascular Permeability by Evans Blue Staining

  • 1% (w/w) Evans blue dye (Sigma)
  • Diethyl ether (Wako)
  • Normal saline (0.9% NaCl)
  • 3.5 M potassium hydroxide (KOH)
  • 4 M phosphoric acid (H 3PO 4)
  • Acetone
  • Dissecting instruments
  • Glass tubes with stoppers
  • Centrifuge
  • Spectrophotometer (U‐2000; Hitachi, http://www.hitachi.com/)
  • Additional reagents and equipment for parenteral injection of the rat (Donovan and Brown, )

Support Protocol 2: Measuring Myeloperoxidase (MPO) Activity

  • Normal saline (0.9% NaCl)
  • MPO homogenization buffer (see recipe)
  • MPO reaction buffer (see recipe)
  • Horseradish peroxidase (as standard)
  • Compatible protein assay kit (Pierce)
  • Polytron tissue homogenizer (Ika, http://www.ika.com/)
  • Centrifuge
  • Microplate reader (Thermo Max; Molecular Devices)

Support Protocol 3: Assaying Lipid Peroxidation via TBARS

  • 1.15% (w/v) KCl
  • 8.1% (w/v) sodium dodecyl sulfate (SDS)
  • 20% (v/v) acetic acid
  • 0.8% (w/v) butylhydroxytoluene (BHT)
  • 0.8% (w/v) thiobarbituic acid
  • n‐butanol
  • Pyridine
  • TBA malondialdehyde standard solution (supplied with the TBARS Assay Kit, Cayman Chemical Company)
  • Polytron tissue homogenizer (Ika)
  • Spectrophotometer (U‐2000; Hitachi) or Spectrofluorimeter with 530‐540/590 excitation/emission filters

Support Protocol 4: Evaluation of SOD Activity

  • Sucrose buffer (see recipe)
  • SOD Assay Kit‐WST (Dojindo Laboratories, http://www.dojindo.com/)
  • Polytron tissue homogenizer (Ika, http://www.ika.com/)
  • Ultracentrifuge
  • Microplate reader (Thermo Max; Molecular Devices)

Support Protocol 5: Quantification of GSH Content Using the DNTB Assay

  • GSH phosphate buffer (see recipe), 4°C
  • 25% (w/v) trichloroacetic acid
  • DNTB GSH assay kit (e.g., Cayman Chemical, Sigma Aldrich)
  • Polytron tissue homogenizer (Ika, http://www.ika.com/)
  • Centrifuge
  • Microplate reader (Thermo Max; Molecular Devices)

Support Protocol 6: Quantification of Acid Secretion

  • Scalpel or blunt‐end scissors
  • Forceps, fine
  • Needle holder
  • Suture (size No. 5)
  • Centrifuge (5010; Kubota, http://www.centrifuge.jp/)
  • Automatic titrator (COM‐555; Hiranuma, http://www.hiranuma.com/english/)

Support Protocol 7: Assessment of Mucosal PGE2 Content

  • Methanol
  • PGE 2 enzyme immunoassay (EIA) kit (Cayman Chemical Company)
  • Polytron tissue homogenizer (Ika, http://www.ika.com/)
  • Centrifuge

Support Protocol 8: Quantification of COX‐2 mRNA Expression

  • Liquid N 2
  • Additional reagents and equipment for extraction of total RNA (Tanaka et al., ), synthesis of cDNA from RNA (Feng et al., ), agarose gel electrophoresis (Voytas, ), and quantitative RT‐PCR (unit 4.38 in this manual); also see appendix 3A in this manual for additional cross‐references to Current Protocols in Molecular Biology

Support Protocol 9: Analysis of VEGF and bFGF Protein Expression

  • Western blot homogenization buffer (see recipe)
  • Bicinchoninic acid (BCA) protein assay kit (Pierce) and albumin standard
  • 15% SDS‐PAGE gel ( appendix 3F)
  • Transfer buffer (see recipe)
  • 2% skim milk in PBST [PBS ( appendix 2A) containing 0.1% (v/v) Tween 20]
  • Anti‐VEGF and anti‐bFGF antibodies (Santa Cruz Biotechnology)
  • Horseradish peroxidase‐conjugated anti‐rabbit IgG (Santa Cruz Biotechnology)
  • Enhanced chemiluminescence detection system: Versa Doc (BioRad) or Western Blot Chemiluminescence Reagent Plus (PerkinElmer)
  • Centrifuge (Sigma Labs., Harz, Germany)
  • Polytron tissue homogenizer (Ika, http://www.ika.com/)
  • Nitrocellulose membranes (Protran; Schleicher & Schuell)
  • Additional reagents and equipment for parenteral injection of the rat (Donovan and Brown, ), SDS‐PAGE ( appendix 3F), and western blotting (immunoblotting; Gallagher et al., )

Support Protocol 10: Evaluation of Angiogenesis by Immunostaining

  • 0.3% H 2O 2
  • Antibody against von Willebrand factor (DAKO)
  • Vectastain ABC‐peroxidase kit (Vector Laboratories)
  • Hematoxylin stain (e.g., Fisher, Sigma)
  • Microscope (Olympus, Japan)
  • Additional reagents and equipment for parenteral injection of the rat (Donovan and Brown, ) and preparation of frozen sections (Hofman, )
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Figures

Videos

Literature Cited

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