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The 15-Day Intact Adult Male Assay As An Alternative Tier I Screening Assay For Detecting Endocrine-Active Compounds. John C. O’Connor DuPont Haskell Laboratory for Health and Environmental Sciences. Outline. Background Overview of the 15-day intact adult male assay Study rationale
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The 15-Day Intact Adult Male Assay As An Alternative Tier I Screening Assay For Detecting Endocrine-Active Compounds John C. O’Connor DuPont Haskell Laboratory for Health and Environmental Sciences
Outline • Background • Overview of the 15-day intact adult male assay • Study rationale • Study design considerations • Case study with flutamide, ketoconazole, & finasteride • Path Forward
Comparison of the EDSTAC-Recommended and Alternative Tier I Screening Batteries for Identifying EACs
Desirable Attributes of a Screen • Reliable (identifies known EACs for EAT activity) • Predictive (known EACs are identified for their mode of action) • Sensitive (low false-negatives) • Quick (i.e., short-term) • Cost effective • Minimize animal usage
Study Design: 15-Day Intact Adult Male Assay • Model: • 10-Week old intact male rats • n = 15/group • Control + 4 dose groups • 15-Day test (oral) • Required Endpoints: • Organ weights - liver, testes, thyroid, epididymides, prostate, seminal ves., ASG unit • Histopathology - testis, epididymides, thyroid • Hormonal battery - testosterone, DHT, estradiol, prolactin, LH, FSH, T3, T4, TSH • Biochemical - preparation of hepatic microsomes • Optional Endpoints (if warranted by other findings) • Histopathology – liver • Biochemical Assessment • Hepatic UDP-glucuronyltransferase activity • Hepatic aromatase activity • Others?
CNS X Hypothalamus (-) (-) (+) GnRH X (-) (-) Anterior Pituitary FSH (+) LH (+) Sertoli Cell Leydig Cell Target Peripheral Tissues Testis X Inhibin X Testosterone X X Aromatase 5-Reductase Estradiol DHT Hypothalamic-Pituitary-Testis Axis
Adult Male Assay: Study Design Issues • Oral dosing – most relevant route • Dose level selection • Based on range-finder studies • Target ≤ 10% final body weight • Based on dietary restriction studies • O’Connor et al., 2000 (Toxicol. Sci. 54: 338-354) • Adult vs. immature animals • Immature are more sensitive to organ weight changes • Mature are more sensitive to hormonal changes • Mature have greater blood volume for hormonal assessment • Duration – 2-week • Strain sensitivity differences
Effect of Diet Restriction on Organ Weights in Sprague-Dawley Rats
Effect of Diet Restriction on Serum Hormones in Sprague-Dawley Rats
Effect of Diet Restriction on Thyroid Hormones in Sprague-Dawley Rats
Data Interpretation • High dose level should not exceed MTD, defined as <10% difference in final body weight when compared to the control group • Organ Weights • Absolute • Testes, epididymis • Relative (% final body weight) • Liver, thyroid, prostate, seminal vesicles, ASG unit • Histopathology - testis, epididymides, thyroid • Control + high dose unless effects are observed • Hormonal battery - testosterone, DHT, estradiol, prolactin, LH, FSH, T3, T4, TSH • Evaluate trends across dose response and overall pattern of hormonal effects
Immature vs. Mature Rats Mature Immature
Immature vs. Mature Rats Mature Immature
Immature vs. Mature Comparison: Study Design • Goal: Compare responses of immature and mature rats to 2 positive controls using identical study designs, and evaluate the intrinsic variability of serum hormone and organ weight measurements in mature and immature rats. • Model: • Immature intact male rats (age 53 days at necropsy) • n = 15/group • 15-Day test (oral) • Control + 4 dose groups • Vinclozolin (75 and 150 mg/kg/day) • Phenobarbital (5 and 25 mg/kg/day) • Included feed restriction group (targeted to 10% body weight decrement from con. • Included two additional “control” groups (age 49 and 57 days at necropsy) • Second experiment currently being conducted with PTU, flutamide, linuron, & DBP
Organ Weights in Immature and Mature Rats Gavaged With Vinclozolin for 15 Days Age – 84 Days Age – 53 Days Note: Organ weight alterations were more pronounced at 300 mg/kg/day in mature rats, and included microscopic alterations of the testes at 150 and 300 mg/kg/day.
Serum Hormone Concentrations in Immature and Mature Rats Gavaged With Vinclozolin for 15 Days Age – 84 Days Age – 53 Days Note: Hormonal alterations were more pronounced at 300 mg/kg/day in mature rats.
Serum Hormone Concentrations in Immature and Mature Rats Gavaged With Phenobarbital for 15 Days Age – 84 Days Age – 53 Days Note: Thyroid weight was significantly increased at both dose levels in mature rats but was NOT affected in immature rats.
Correlation Between Age and Variability in Serum Hormone Concentrations in Rats
Correlation Between Age and Variability in Serum Hormone Concentrations in Rats
Correlation Between Age and Variability in Serum Hormone Concentrations in Rats
Detection of p,p’-DDE in the Adult Male Assay • Weak AR antagonist • Strain differences observed in 15-day intact male assay • O’Connor et al., 1999 (Toxicol. Sci. 51: 44-53) • CD rats – not clearly identified as an AR antagonist • LE rats – identified as an AR antagonist • Consistent with strain differences observed in studies of You et al., 1998 (Toxicol. Sci. 45, 162-173)
Effect of p,p’-DDE on Serum Hormone Levels in the Adult Male Assay
Effect of p,p’-DDE on Thyroid Hormone Levels in the Adult Male Assay
CNS X Hypothalamus (-) (-) (+) GnRH X (-) (-) Anterior Pituitary FSH (+) LH (+) Sertoli Cell Leydig Cell Target Peripheral Tissues Testis X Inhibin X Testosterone X X Aromatase 5-Reductase Estradiol DHT Hypothalamic-Pituitary-Testis Axis
Case Study: Study Design • Model: • 10-Week old intact male rats • n = 15/group • 15-Day test (oral) • Control + 4 dose groups • Dose levels selected based on range-finder studies • Flutamide (10 mg/kg/day; high dose) • Ketoconazole (100 mg/kg/day; high dose) • Finasteride (25 mg/kg/day; high dose) • Measured Endpoints: • Organ weights - liver, testes, thyroid, epididymides, prostate, seminal ves., ASG unit • Histopathology - testis, epididymides, thyroid • Hormonal battery - testosterone, DHT, estradiol, prolactin, LH, FSH, T3, T4, TSH
Effect Of Ketoconazole On Testosterone Biosynthesis From Isolated Rat Leydig Cells
1a 1b 1c Testosterone Biosynthesis (4 Pathway) ① Cholesterol SCC enzyme (CP-450) a. 20-hydroxylase b. 22-hydroxylase c. 20,22-lyase ② 3b-Hydroxysteroid Dehydrogenase ③ 4,5-Ketosteroid Isomerase ④ 17-Hydroxylase (CP-450) ⑤ C-17,20-Lyase (CP-450) ⑥ 17b-Hydroxysteroid Dehydrogenase ⑦ 5a-Reductase ⑧ Aromatase (CP-450) Cholesterol Leydig Cell Pregnenolone ② ③ Progesterone X ④ Ketoconazole 17a-Hydroxyprogesterone ⑤ ⑧ Androstenedione Estrone ⑥ ⑧ Testosterone 17b-Estradiol X ⑦ Finasteride X 5a-Dihydroxytestosterone Target peripheral tissues Flutamide
Case Study: Flutamide, Ketoconazole, & FinasterideComparison of Organ Weight Data Cannot differentiate mode of action based on organ weight changes
Case Study: Flutamide, Ketoconazole, & FinasterideComparison of Serum Hormone Data Mode of action can be determined based on hormonal changes
CNS Hypothalamus (-) (-) X Antiandrogens (Flutamide) (+) GnRH (-) (-) Anterior Pituitary FSH (+) LH (+) Sertoli Cell Leydig Cell Target Peripheral Tissues Steroid Inhibitors (Ketoconazole) Testis X Inhibin X Testosterone Antiandrogens (Flutamide) X 5-Reductase Aromatase 5a-Reductase Inhibitors (Finasteride) Estradiol DHT Hypothalamic-Pituitary-Testis Axis
Proposed Tier I Screening Battery Tier I ER Agonists ER Antagonists Uterotrophic Assay Receptor Binding/ Transactivation Agonist/Antagonist (ER, AR) Thyroid Effects Steroid Biosynthesis ER/AR Agonists ER/AR Antagonists Intact Adult Male Assay
Advantages of Tier I Using Adult Male Assay • Comprehensive mode-of-action screen • Capable of evaluating several different modes of action in a single assay -- by measuring mechanistic endpoints (androgen, estrogen and thyroid agonists/antagonists; steroid hormone synthesis (aromatase & steroidogenesis) • Tier I with intact male provides mode of action “profile” to focus direction of any further testing • Reduces the number of animals needed for Tier I • Intact endocrine system • Design allows integration of new endpoints if desired • O’Connor et al., (2002). Evaluation of the Tier I screening options for detecting endocrine-active compounds (EACs). Critical Reviews in Toxicology, 32: 521-549.
Completed Inter-Laboratory Studies Using the 15-Day Intact Adult Male Rat Assay Protocol *Data available though the EPA (1) or ACC (2)
Proposed Inter-Laboratory Studies Using the 15-Day Intact Male Rat Assay Protocol
Rationale for Compound Selection • The mode-of-action of the compounds focus on EAT • Linuron – weak anti-androgen • Phenobarbital – weak thyroid-active agent • Ketoconazole – steroid biosynthesis inhibitor • All compounds have been previously run in the 15-day intact male assay • Provide a base-line for expected results • Will facilitate inter-laboratory comparisons of the transferability of the 15-day intact male assay • All compounds have been previously evaluated in other potential Tier I screening assays (Hershberger, pubertal male and/or pubertal female assays) • Will facilitate future comparisons among assays
Issues/Questions for the EDMVAC • Is the protocol ready for inter-laboratory validation? • If not, what additional data is needed? • Does the EDMVAC agree with the test material selection for inter-laboratory validation studies?