How to Improve Detection of Mammary Toxicants 1.Similarity between human and rodent, so good model to continue with 2.Critical periods and need for treatment with test chemicals during that time 3.Methods that could be incorporated to improve over current – Whole mounts - what are they and how utilized – Longitudinal section evaluation 4.Where can these changes be made...? 5.PFOA as an example

Similar Developmental Events in Human and Rodent Mammary Tissue Developmental EventHumanRodent milk streak evidentEW4-6GD10-11 (mice) mammary epithelial bud formsEW10-13GD12-14 (mice), GD (rat) female nipple and areola formEW12-16GD18 (mice)/GD20 (rat) branching and canalization of epithelium EW20-32GD16 to birth (mice), GD 18 to birth (rat) secretion is possibleEW32-40 (ability lost postnatally) at birth, with hormonal stimuli isometric development of ductsbirth to puberty TEBs present (peri-pubertal)8-13 year old girls 23 to 60 days old (rodents) formation of lobular unitsEW32-40, or within 1-2 yr. of first menstrual cycle puberty and into adulthood TEB=terminal end bud, EW=embryonic week, GD=gestational day taken from S.E. Fenton, 2006 Endocrinology 147(Supplement):S18-S24. RODENT Terminal end buds (TEB) HUMAN Terminal ductal lobular unit (TDLU)

Puberty Gestation Altered growth and development; altered carcinogen susceptibility Adulthood Pregnancy/ lactation Breast cancer Early life Normal MG (rat) Fat pad and bud form Epithelium forms ductal tree Isometric epithelial growth Branching ducts and budding TEBs develop Exponential epithelial growth TEBs differentiate Epithelium is predominant Lobulo-alveolar development Altered hormone/growth factor levels and responsiveness; effects may be systemic or localized to MG Static resting state, changing with cyclicity Responsive to hormonal changes MG after early EDC exposure Lactational impairment Exposure during MG development

Mouse Mammary Gland Development BirthWeaning

RAT MAMMARY WHOLE MOUNTS Longitudinal Growth Branching & Budding

Inguinal mammary gland sampling Cross Section or Transverse Horizontal Section or Longitudinal From J. Latendresse, NCTR OLD METHOD BETTER METHOD

Where can these techniques be used? NTP – modified one-gen, repro studies, 2 yr bioassay OECD – EOGRTS modified one-gen repro screen Academic labs – targeted testing Pesticide testing EPA Tier I or II screening and testing.

Perfluorooctanoic Acid  What is PFOA? PFOA is a synthetic, fluorinated organic compound used in the production of weather-proof or grease-proof food, clothing, and household products and may result from the breakdown of broadly used polyfluorinated chemicals.  Why does PFOA pose a health concern? - Persistent chemical : half-life in humans 3.8 years; mice days - Present in 100% of human blood: mean in U.S. reported as ng/ml (or ppb) - Detected at ~3.6ppb in drinking water supplies near production plants -Rat studies: common tumor triad- hepatocellular, pancreatic acinar cell, and Leydig cell tumors -Female rats exhibit rapid elimination of PFOA compared to male rats, humans, and mice, thus are poor models for human developmental toxicity studies. -CD-1 mice are suitable model for human health translation purposes -Multiple developmental end point delays- eye opening, growth, full litter resorption and postnatal mortality (Lau et al., 2006).

Note. Mammary gland measurements from late gestation females at PND 21. Data presented as the mean ± SEM. Significant effects compared to controls, * p<0.05, **p<0.01, ***p< All images at the same 8X magnification. **Transient liver:body weight elevation at 1 mg/kg only Macon, M.B., Villanueva, L.R., Tatum-Gibbs, K., Zehr, R.D., Strynar, M.J., Stanko, J.P., White, S.S., Helfant, L., S.E. Fenton. Prenatal perfluorooctanoic acid exposure in CD-1 mice: low dose developmental effects and internal dosimetry. Toxicol Sci, Accepted March 22, 2011; doi: /toxsci/kfr076. Control0.01 mg/kg0.1 mg/kg1.0 mg/kg Low Dose PFOA Effects on PND 21 Mammary Gland Development Developmental Score Longitudinal Growth (μm) Lateral Growth (μm) Δ Longitudinal Growth (μm) Δ Lateral Growth (μm) Terminal End Buds (TEBs) Terminal Ends (TEs) Control (n)3.3 ± 0.3 (5)4321 ± 306 (5)5941 ± 280 (5)3394 ± 306 (5)4358 ± 280 (5)40 ± 4 (5)81 ± 12 (5) 0.01 mg/kg (n)2.2 ± 0.2* (4)3803 ± 386 (4)5420 ± 326 (4)3087 ± 386 (4)3899 ± 326 (4)33 ± 4 (4)61 ± 8 (4) 0.1 mg/kg (n)1.8 ± 0.3** (3)3615 ± 320 (3)4822 ± 672 (3)2370 ± 320 (3)3035 ± 672 (3)24 ± 4* (3)58 ± 4 (3) 1.0 mg/kg (n)1.6 ±0.1*** (5)2775 ± 285** (5)4822 ± 313 (5)1553 ± 301** (5)3382 ± 313 (5)15 ± 2***(5)47 ± 11 (5)

Serum PFOA concentration of female offspring - late gestation study PND 1 a (n)PND 7 (n)PND 14 (n)PND 21 b (n) Serum (ng/ml) Control22.6 ± 5.5 (4)7.8 ± 2.1 (5)7.8 ± 1.6 (8)4.1 ± 0.6 (7) 0.01 mg/kg284.5 ± 21.0* (3)150.7 ± 20.9* (7)80.2 ± 13.9* (8)16.5 ± 2.1* (10) 0.1 mg/kg ± 114.1* (2) ± 122.6* (8)645.4 ± 114.2* (7)131.7 ± 24.5* (7) 1.0 mg/kg ± 873.6* (7) ± * (11) ± 662.6* (11) ± 281.9* (11) Calculated Blood Burden (ng) Control1.3 ± 0.38 (4)1.5 ± 0.4 (5)2.7 ± 0.7 (8)1.2 ± 0.6 (7) 0.01 mg/kg15.2 ± 1.7* (3)27.3 ± 3.8* (7)27.0 ± 4.6* (8)7.8 ± 1.0* (10) 0.1 mg/kg114.3 ± 5.4* (2)221.7 ± 24.9* (8)218.5 ± 39.8* (7)65.5 ± 12.3* (7) 1.0 mg/kg926.0 ± 47.6* (7) ± 256.7* (11) ± 293.5* (11)977.6 ± 146.3* (11) Macon, M.B., Villanueva, L.R., Tatum-Gibbs, K., Zehr, R.D., Strynar, M.J., Stanko, J.P., White, S.S., Helfant, L., S.E. Fenton. Prenatal perfluorooctanoic acid exposure in CD-1 mice: low dose developmental effects and internal dosimetry. Toxicol Sci, Accepted March 22, 2011; doi: /toxsci/kfr076.

Control Control + 5 ppb PFOA 1 mg/kg 1 mg/kg + 5 ppb PFOA 5 mg/kg PND 22 PND 42PND 63 * * * * * * * * * * * * # F1 generation following gestational +/- chronic 5 ppb in water supply White, S.S., Stanko, J.P., Kato, K., Calafat, A.M., Hines, E.P. and S.E. Fenton. Developmental and chronic low-dose PFOA exposures decrease mammary gland growth and differentiation in three generations of CD-1 mice. Environ Health Perspect, Accepted April 18, 2011; doi: /ehp