Transgenerational (In Utero/Lactational) Exposure to Investigate the Effects of Endocrine Disrupting Compounds (EDCs) in Rats

Joseph S. Ostby1, L. Earl Gray1

1 United States Environmental Protection Agency, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 16.8
DOI:  10.1002/0471140856.tx1608s19
Online Posting Date:  May, 2004
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Abstract

This protocol is designed to evaluate the effects of endocrine disrupting compounds (EDCs) through fetal (transplacental) and/or neonatal (via the dam's milk) exposure during the critical periods of reproductive organogenesis in the rat. Continued direct exposure to the F1 pups after weaning is an option that can also be included. Reproductive indices, growth, and viability are monitored throughout the life of the F1 generation and an extensive necropsy is conducted after sexual maturity is attained. The endpoints evaluated are designed to detect compounds such as androgen receptor antagonists or steroid hormone synthesis inhibitors which inhibit normal development of the male reproductive system and growth stimulating hormones which masculinize females and facilitate development of male reproductive tissues. Data collected from these studies can be used to determine the no and lowest observed effects levels and predict potential adverse consequences of EDCs in human and wildlife populations. The information also provides a foundation for future studies designed to identify mechanism(s) of action, specific genes and or proteins involved, and susceptible populations these compounds might target.

Keywords: Endocrine Disrupting Compounds (EDCs); (Anti)Androgen; (Anti)Estrogen

     
 
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Table of Contents

  • Basic Protocol 1: Study Design
  • Basic Protocol 2: Measuring Anogenital Distance
  • Basic Protocol 3: Assessing Areolae/Nipple Development in Infant Male and Female Rats
  • Basic Protocol 4: Detecting Puberty—Vaginal Opening (VO) and Preputial Separation (PPS)
  • Basic Protocol 5: Necropsy
  • Support Protocol 1: Data Interpretation and Summary
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Study Design

  Materials
  • Individually housed timed‐pregnant rats
  • Test compounds
  • Laboratory‐grade corn oil (Sigma)
  • Balance, accurate to 0.1 g, that integrates the weight over several measurements (e.g., Sartorius IP65)
  • 1‐ml glass tuberculin syringe (BD)
  • 1.5‐in. × 18‐ or 20‐G curved gavage needle (Popper and Sons)
  • 25‐G × 5/8‐in. or 1/2‐in. sterile needle (BD)
  • Saturated picric acid solution and envelope moistener or cotton swabs to apply picric acid to dams for treatment identification
  • Software such as Microsoft Excel or SAS (PC SAS or IBM Host on Demand) for calculating means and standard errors by treatment
  • FTP95 for transferring SAS output data sets to WordPerfect or Microsoft Word

Basic Protocol 2: Measuring Anogenital Distance

  Materials
  • Sprague‐Dawley or Long Evans pups at birth or on PND 2 and as adults
  • Dissecting microscope, 0.63× to 4× and 10‐mm (0.1‐mm divisions) ocular reticle (e.g., Leica MZ6)
  • 1‐mm stage micrometer (0.01‐mm divisions)
  • Vernier caliper (0 to 150 mm with 0.1‐mm divisions)
  • Two‐place balance (e.g., Mettler PM2000)

Basic Protocol 3: Assessing Areolae/Nipple Development in Infant Male and Female Rats

  Materials
  • Infant male and female rats (e.g., Sprague‐Dawley)
  • Bouins's fixative (see recipe)
  • Balance (e.g., Mettler PM2000)
  • Small animal clippers with size no. 40 clipper blade (Oster)

Basic Protocol 4: Detecting Puberty—Vaginal Opening (VO) and Preputial Separation (PPS)

  Materials
  • Rats from litters of dams in treatment groups, approaching puberty
  • Balance for animal weight recorded to the nearest 0.01 g (e.g., Sartorius IP65) with integration capability to accurately weigh moving animals

Basic Protocol 5: Necropsy

  Materials
  • Male and female rats, 90 to 120 days old
  • CO 2
  • Bouin's fixative (see recipe)
  • Balance accurate to 0.1 g with integration capability for weighing rats (e.g., Sartorius IP65)
  • Rodent restraint cones (Harvard Apparatus)
  • Rodent guillotine
  • Vernier caliper (0 to 150 mm with 0.1‐mm divisions)
  • Small animal clippers with size # 40 clipper blade (Oster) for shaving ventral surface to check areolae/nipples
  • Surgical instruments (Roboz): micro dissecting forceps (RS‐5230 for grasping delicate tissues such as ventral prostate and RS‐5236 for holding tougher tissue such as skin), micro dissecting scissors (RS‐5852 for delicate dissections and RS‐6814 for cutting tougher tissue such as skin) and hemostats (RS‐7172 for clamping seminal vesicles)
  • Balance accurate to 0.0001 g for weighing tissues (e.g., Sartorius BP 121 S)
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Figures

Videos

Literature Cited

Literature Cited
   Bentvelsen, F.M., Brinkmann, A.O., van der Schoot, P., van der Linden, J.E., van der Kwast, T.H., Boersma, W.J., Schroder, F.H., and Nijman, J.M. 1995. Developmental pattern and regulation by androgens of androgen receptor expression in the urogential tract of the rat. Mol. Cell Endocrinol. 113:245‐253
   Clark, R.L., Antonello, J.M., Grossman, S.J., Wise, L.D., Anderson, C., Bagdon, W.J., Prahalada, S., MacDonald, J.S., and Robertson, R.T. 1990. External genitalia abnormalities in male rats exposed in utero to finasteride, a 5 alpha‐reductase inhibitor. Teratology 42:91‐100.
   Clark, R.L., Anderson, C.A., Prahalada, S., Robertson, R.T., Lochry, E.A., Leonard, Y.M., Stevens, J.L., and Hoberman, A.M. 1993. Critical developmental periods for effects on male rat genitalia induced by finasteride, a 5 alpha–reductase inhibitor. Toxicol. Appl. Pharmacol. 119:34‐40.
   Gray, L.E. Jr., Ostby, J.S., and Kelce, W.R. 1994. Developmental effects of an environmental antiandrogen: The fungicide vinclozolin alters sex differentiation of the male rat. Toxicol. Appl. Pharmacol. 129:46‐52.
   Gray, L.E. Jr., Ostby, J.S., and Kelce, W.R. 1997a. A dose‐response analysis of the reproductive effects of a single gestational dose of 2,3,7,8‐tetreachlorodibenzo‐p‐dioxin in male Long Evans hooded rat offspring. Toxicol. Appl. Pharmacol. 146:11‐20.
   Gray, L.E. Jr., Ostby, J.S., and Kelce, W.R. 1997b. In utero exposure to low doses of 2,3,7,8‐tetreachlorodibenzo‐p‐dioxin alters reproductive development of female Long Evans hooded rat offspring. Toxicol. Appl. Pharmacol. 146:237‐244.
   Gray, L.E. Jr., Wolf, C., Lambright, C., Mann, P., Price, M., Cooper, R.L., and Ostby, J. 1999. Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p′‐DDE, and ketoconazole) and toxic substances (dibutyl‐ and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol. Ind. Health 15:94‐118.
   Gray, L.E. Jr., Ostby, J., Furr, J., Price, M., Veeramachaneni, D.N., and Parks, L. 2000. Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat. Toxicol. Sci. 58:350‐365.
   Gray, L.E. Jr., Ostby, J., Wilson, V., Lambright, C., Bobseine, K., Hartig, P., Hotchkiss, A., Wolf, C., Furr, J., Price, M., Parks, L., Cooper, R.L., Stoker, T.E., Laws, S.C., Degitz, S.J., Jensen, K.M., Kahl, M.D., Korte, J.J., Makynen, E.A., Tietge, J.E., and Ankley, G.T. 2002. Xenoendocrine disrupters_tiered screening and testing: Filling key data gaps. Toxicology 182:371‐382.
   Imperato‐McGinley, J., Sanchez, R.S., Spencer, J.R., Yee, B., and Vaughan, E.D. 1992. Comparison of the effects of the 5 alpha‐reductase inhibitor finasteride and the antiandrogen flutamide on prostate and genital differentiation: Dose‐response studies. Endocrinology 131:1149‐1156.
   Korenbrot, C.C., Huhtaniemi, I.T., and Weiner, R.I. 1977. Preputial separation as an external sign of pubertal development in the male rat. Biol. Reprod. 17:298‐303.
   Kubota, Y., Nef, S., Farmer, P.J., Temelcos, C., Parada, L.F., and Hutson, J.M. 2001. Leydig insulin‐like hormone, gubernacular development and testicular descent. J. Urol. 165:1673‐1675.
   Lambright, C., Ostby, J., Bobseine, K., Wilson, V., Hotchkiss, A.K., Mann, P.C., and Gray, L.E. Jr. 2000. Cellular and molecular mechanisms of action of linuron: An antiandrogenic herbicide that produces reproductive malformations in male rats. Toxicol. Sci. 56:389‐399.
   McIntyre, B.S., Barlow, N.J., and Foster, P.M. 2002. Male rats exposed to linuron in utero exhibit permanent changes in anogenital distance, nipple retention, and epididymal malformations that result in subsequent testicular atrophy. Toxicol. Sci. 65:62‐70.
   Ojeda, S.R., Wheaton, J.E., Jameson, H.E., and McCann, S.M. 1976. The onset of puberty in the female rat: Changes in plasma prolactin, gonadotropins, luteinizing hormone‐releasing hormone (LHRH), and hypothalamic LHRH content. Endocrinology 98:630‐638.
   Parks, L.G., Ostby, J.S., Lambright, C.R., Abbott, B.D., Klinefelter, G.R., Barlow, N.J., and Gray, L.E. Jr. 2000. The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat. Toxicol. Sci. 58:339‐349.
   Wolf, C.J., Hotchkiss, A., Ostby, J.S., LeBlanc, G.A., and Gray, L.E. Jr. 2002. Effects of prenatal testosterone propionate on the sexual development of male and female rats: A dose‐response study. Toxicol. Sci. 65:71‐86.
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