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, 2002)

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

 Additional Materials (also see Basic Protocol 1)
  • 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 (H3PO4)
  • 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, 2006)

Support Protocol 2: Measuring Myeloperoxidase (MPO) Activity

 Additional Materials (also see Basic Protocol 1)
  • 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

 Additional Materials (also see Basic Protocol 1)
  • 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

 Additional Materials (also see Basic Protocol 1)
  • 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

 Additional Materials (also see Basic Protocol 1)
  • 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

 Additional Materials (also see Basic Protocol 1)
  • 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

 Additional Materials (also see Basic Protocol 1)
  • Methanol
  • PGE2 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

 Additional Materials (also see Basic Protocol 1)
  • Liquid N2
  • Additional reagents and equipment for extraction of total RNA (Tanaka et al., 2002), synthesis of cDNA from RNA (Feng et al., 1993), agarose gel electrophoresis (Voytas, 2000), 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

 Additional Materials (also see Basic Protocol 1)
  • 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, 2006), SDS-PAGE (appendix 3F), and western blotting (immunoblotting; Gallagher et al., 2008)

Support Protocol 10: Evaluation of Angiogenesis by Immunostaining

 Additional Materials (also see Basic Protocol 1)
  • 0.3% H2O2
  • 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, 2006) and preparation of frozen sections (Hofman, 2002)
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Figures

  •  FigureFigure 21.10.1 Chemical structures of BPPs (alendronate, risedronate, and minodronate).
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  •  FigureFigure 21.10.2 Development of antral ulcers induced by alendronate in rat stomachs. Animals fasted for 24 hr were given alendronate (100 to 600 mg/kg, p.o.), then refed, and killed 4 days later. (A) Gross appearance of antral ulcers on day 4 after the administration of alendronate (100 to 600 mg/kg). (B) Lesion score and the extravasated amount of Evans blue in the antral mucosa. Data are presented as the mean ± SE of 4 to 6 rats. Asterisk (*) means significant difference from normal, at p < 0.05. (C) Relationship between the lesion score and the extravasated amount of dye. There is a highly significant relationship between these two factors, the reciprocal coefficient (r) being 0.826. Data adapted after modification of Ohashi et al. (2009).
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  •  FigureFigure 21.10.3 Histological observations (HE staining, ×40) of the antral mucosa before (normal) and 1 or 3 days after the administration of alendronate (300 mg/kg, p.o.). Animals fasted for 18 hr were given alendronate (300 mg/kg, p.o.), refed, and killed on 1 and 3 days after administration.
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  •  FigureFigure 21.10.4 The effects of allopurinol, SOD, rebamipide, omeprazole, or indomethacin on antral ulcers induced by alendronate in the rat stomach. The animals fasted for 24 hr were given alendronate (300 mg/kg, p.o.), refed, and killed 4 days later. Allopurinol (100 mg/kg), SOD (30,000 units/kg) or rebamipide (30 mg/kg) was given i.p. or p.o., respectively, 30 min before and 10 hr after alendronate on the first day and twice daily for 2 days. Omeprazole (30 mg/kg, p.o.) or indomethacin (2 mg/kg) was given 30 min before alendronate and once daily for 2 days thereafter. Data are presented as the mean ± SE of 4 to 6 rats. Significant difference at p < 0.05, * from vehicle. Data adopted after modification of Ohashi et al. (2009).
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  •  FigureFigure 21.10.5 Schematic illustration of the method to produce chronic gastric ulcer by thermal cauterization. An electrically probe (8 mm in a diameter) is placed on the corpus mucosa of the stomach and heated electrically at 70°C for 30 sec. Three days later, a well-defined ulcer develops in the corpus of the stomach.
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  •  FigureFigure 21.10.6 Effects of alendronate and indomethacin on the healing of chronic gastric ulcers in rats. Gastric ulcers were induced by thermal cauterization (70°C for 30 sec). Alendronate (30 and 60 mg/kg) or indomethacin (2 mg/kg) was administered p.o. once daily for 7 days, starting 3 days after ulceration. (A) Data are presented as the means ± SE of 6 rats. *Significant difference from control, at p < 0.05. (B,C) Macroscopic and microscopic observations of gastric ulcers on day 10; a: control; b: alendronate (30 mg/kg); c: indomethacin (2 mg/kg). Data adapted after modification of Amagase et al. (2007).
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  •  FigureFigure 21.10.7 The effect of rebamipide on changes in SOD activity (A) and GSH content (B) induced in the antral mucosa by alendronate in rats. The animals fasted for 24 hr were given alendronate (300 mg/kg, p.o.), refed, and killed 4 days later. Rebamipide (30 mg/kg) was given p.o. 30 min before and 10 hr after alendronate on the first day and twice daily for 2 days. Data are presented as the means±SE for 4 to 6 rats. Significant difference at p < 0.05, *from normal, # from vehicle. Data adapted after modification of Ohashi et al. (2009).
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  •  FigureFigure 21.10.8 The effect of alendronate on acid secretion in pylorus-ligated rats. The animals fasted for 24 hr were given alendronate (300 mg/kg, p.o.), and refed thereafter. Acid secretion was measured in pylorus-ligated stomachs for 4 hr on day 1, 2, and 3 after the administration of alendronate. In cases of acid measurement on day 1, 2, and 3 after alendronate treatment, the animals were fasted for 18 hr before pylorus ligation. Data are presented as the mean ± SE of 4 to 6 rats. *Significant difference from control, at p < 0.05. Figures show: (A) gastric fluid volume; (B) acidity; (C) acid output. Data adapted after modification of Ohashi et al. (2009).
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  •  FigureFigure 21.10.9 Effects of alendronate and indomethacin on PGE2 contents (A) and COX-2 expression (B) in the ulcerated area in rat stomachs. Gastric ulcers were induced by thermal cauterization (70°C for 30 sec), and the PGE2 content was measured on day 10 after ulceration. The expression of COX-2 was examined on day 7 after ulceration. Alendronate (60 mg/kg, p.o.) or indomethacin (2 mg/kg, s.c.) was administered once daily for 4 days or 7 days, starting 3 days after ulceration. Data are presented as the means ± SE of 5 to 6 rats. Significantly different at p < 0.05; * from normal; # from control. Data adapted after modification of Amagase et al. (2007).
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  •  FigureFigure 21.10.10 Effects of alendronate and indomethacin on the expression of VEGF and bFGF protein in the ulcerated rat stomach. Gastric ulcers were induced by thermal cauterization (70°C for 30 sec), and rats were killed on day 10 after ulceration. Alendronate (60 mg/kg, p.o.) or indomethacin (2 mg/kg, s.c.) was administered once daily for 7 days, starting 3 days after ulceration. (A) Expression of VEGF and bFGF was determined by conventional immunoblotting; (B) densitometric quantification was determined by Quantity One software and the results are expressed as % of control (10 days after ulceration). Data adapted after modification of Amagase et al. (2007, 2011)
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  •  FigureFigure 21.10.11 Microscopic observation of Factor VIII–positive cells in the base of gastric ulcers. Gastric ulcers were induced by thermal cauterization (70°C for 30 sec), and the rats were killed on day 10 after ulceration. Alendronate (60 mg/kg, p.o.) or indomethacin (2 mg/kg, s.c.) was administered once daily for 7 days, starting 3 days after ulceration. Frozen sections were prepared, and immunostaining with anti–Factor VIII antibody was performed. Factor VIII–positive cells represent newly formed microvasculature. Figures show: (A) Control; (B) alendronate; (C) indomethacin. Data adapted after modification of Amagase et al. (2007).
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  •  FigureFigure 21.10.12 Possible pathogenic mechanisms of BPP-induced antral ulceration in the stomach. The development of BPP-induced antral ulcers is essentially due to the direct action of these drugs, followed by increased microvascular permeability and inflammation, resulting in the occurrence of ulceration. In addition, impairment of the mucosal antioxidative system, such as decreased SOD activity and GSH content, contributes to the pathogenesis of ulceration. Antioxidative drugs, such as SOD or allopurinol, as well as rebamipide (a mucosal protective drug), are effective to reduce the severity of BPP-induced antral ulceration, while antisecretory drugs, such as omeprazole (a proton pump inhibitor), have no effect.
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Literature Cited

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