Hershberger Assay to Investigate the Effects of Endocrine‐Disrupting Compounds with Androgenic or Antiandrogenic Activity in Castrate‐Immature Male Rats

L. Earl Gray1, Johnathan Furr1, Joseph S. Ostby1

1 United States Environmental Protection Agency, NHEERL, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 16.9
DOI:  10.1002/0471140856.tx1609s26
Online Posting Date:  December, 2005
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Abstract

The protocol described in this unit is designed to evaluate the effects of androgenic and antiandrogenic endocrine‐disrupting compounds (EDCs) in the castrate‐immature male rat. Continuous 10‐day exposure of the prepubertal male to chemicals is used to identify androgenic or antiandrogenic activities based on the weights of several androgen‐dependent tissues on the day after treatment is ended. Androgen‐dependent organ weights and growth, along with kidney, liver, and adrenal weights are measured at necropsy. The androgen‐dependent tissues include the ventral prostate, seminal vesicles (with fluid plus coagulating glands), levator ani plus bulbocavernosus muscles, Cowper's glands, and glans penis. Optional measures, which facilitate interpretation of the data when positive effects are detected, include serum testosterone and luteinizing hormone levels. Androgenic chemicals cause increases in one or all of the androgen‐dependent tissue weights after being administered by oral gavage or subcutaneously (s.c.) to castrate‐immature male rats. Antiandrogenic chemicals cause reductions in androgen‐dependent tissue weights after being coadministered with testosterone propionate (s.c.).

Keywords: Endocrine Disrupting Compounds (EDCs); Antiandrogens; Androgens

     
 
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Table of Contents

  • Basic Protocol 1: Study Design and Exposure to Assess Test Compounds for Androgenic or Antiandrogenic Effects
  • Support Protocol 1: Preparation and Administration of Dosing Solutions
  • Basic Protocol 2: Necropsy of Hershberger Assay Animals
  • Support Protocol 2: Assessing Androgenic Effects of Testosterone Propionate on Glans Penis Maturation
  • Basic Protocol 3: Statistical Analysis of Hershberger Assay Data
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Study Design and Exposure to Assess Test Compounds for Androgenic or Antiandrogenic Effects

  Materials
  • Immature, male Sprague‐Dawley rats
  • Saturated picric acid solution (Sigma or equivalent)
  • Dosing solutions (see protocol 2)
  • Vehicle: laboratory‐grade corn oil (Sigma or equivalent)
  • Testosterone propionate (TP)
  • Housing cages
  • Balance accurate to 0.1 g, which integrates the weight over several measurements (e.g., Sartorius IP65)
  • Temporary cages
  • Racks for cages
  • Envelope moistener or cotton swab
  • Numbered ear tags (optional)
  • 18‐ or 20‐G × 1.5‐in. curved animal feeding needle (Popper and Sons)
  • 1‐cc glasspack tuberculin syringe, sterile (BD)
  • 25‐G × 5/8‐in. needle (BD) or 27‐G × 1/2‐in. needle (BD), sterile

Support Protocol 1: Preparation and Administration of Dosing Solutions

  Materials
  • Test compounds (xenobiotic)
  • Vehicle (e.g., laboratory‐grade corn oil; Sigma‐Aldrich)
  • Testosterone propionate (TP)
  • Test animals
  • Dosing solution vials
  • Balance accurate to at least 0.01 mg
  • 1‐cc glasspack tuberculin syringe, sterile (BD or equivalent)
  • 18‐ or 20‐G × 1.5‐in. curved animal feeding needle (Popper and Sons)
  • 25‐G × 5/8‐in. needle or 27‐G × 1/2‐in. needle, sterile (BD, or equivalent)

Basic Protocol 2: Necropsy of Hershberger Assay Animals

  Materials
  • Rats from the study
  • Balance accurate to 0.1 g with integration capability for weighing rats (e.g., Sartorius IP65)
  • 5‐ml syringe and 25‐G × 5/8 in. needle (optional)
  • Dissection boards
  • Balance accurate to 0.0001 g for weighing tissues (e.g., Sartorius BP 121 S)
  • Surgical equipment (eg., Roboz):
    • Microdissecting forceps: 12 cm, straight pointed style, 1 mm wide, blunt tip (to prevent tissue puncture and fluid leakage), serrated
    • Dissecting scissors: 6 inches, straight pointed style, sharp tip, not serrated
    • Microdissecting scissors: 4 inches, straight pointed style, sharp tip, not serrated
    • Hemostats: 14 cm, straight pointed style, serrated tip
  • Weigh boats
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Figures

Videos

Literature Cited

Literature Cited
   Dorfman, R.I. 1962. Standard Methods Adopted by Official Organization. Academic Press, New York.
   Freyberger, A., Hartmann, E., and Krotlinger, F. 2005. Evaluation of the rodent Hershberger bioassay using three reference (anti)androgens. Arh. Hig. Rad. Toksikol. 56:131‐139.
   Hershberger, L.G., Shipley, E.G., and Meyer, R.K. 1953. Myotrophic activity of 19‐nortestosterone and other steroids determined by modified levator ani muscle method. Proc. Soc. Exp. Biol. Med. 83:175‐180.
   Kennel, P.F., Pallen, C.T., and Bars, R.G. 2004. Evaluation of the rodent Hershberger assay using three reference endocrine disrupters (androgen and antiandrogens). Reprod. Toxicol. 18:63‐73.
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