1. The Intact 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
&
The American Chemistry Council

2. Outline Background Overview of the 15-day intact male assay
Study rationale
Study design considerations
Case study with flutamide, ketoconazole, & finasteride
Future considerations and final thoughts
Point out that we are not making a direct comparison to the other tier I screening assays. We are illustrating the utility of the intact male assay for detecting EACs.

3. Comparison of the EDSTAC-Recommended and Alternative Tier I Screening Batteries for Identifying EACs

4. Desirable Attributes of a Screen
Reliable (identified known EACs)
Predictive (known EACs are identified for their mode of action)
Sensitive (low false-negatives)
Quick (i.e., short-term)
Cost effective
Minimize animal usage

5. Proposed Tier I Screening Battery
Uterotrophic
Assay
ER Agonists
ER Antagonists
Receptor Binding/
Transactivation
Agonist/Antagonist
(ER, AR)
Highlight that male is more sensitive than female for detecting thyroid modulators.
Thyroid Effects
Steroid Biosynthesis
ER/AR Agonists
ER/AR Antagonists
Intact Male
Assay

6. 15-Day Intact Male Assay Model: Required Endpoints:
10-Week old intact male rats
n = 15/group
Control + 3 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, estradiol, prolactin, LH, FSH, T3, T4, TSH
Biochemical - preparation of hepatic microsomes
Optional Endpoints (if warranted by other findings)
Histopathology – liver
Hormonal assessment – DHT
Biochemical Assessment
Hepatic UDP-glucuronyltransferase activity
Hepatic aromatase activity
Focus discussion on the study design and required endpoints.

7. Mechanisms that Modify Hormone Action Positive/Negative Influences
Hormone receptor agonist/antagonist
Alter hormone synthesis (e.g., steroid hormones)
Alter hormone storage or release (e.g., peptide hormones)
Alter hormone metabolism
Alter hormone transport
Alter bioavailability by displacing hormone bound in serum or by altering enterohepatic recirulation
Alter post-receptor activation
Alter endocrine axis centrally (neuroendocrine)

8. Hypothalamic-Pituitary-Testis Axis
CNS
Hypothalamus
Anterior Pituitary
GnRH
(+)
FSH (+)
Sertoli Cell Leydig Cell
Inhibin
Testosterone
Aromatase
Estradiol
(-)
Testis
LH (+)
DHT
5-Reductase
Target Peripheral Tissues X
Dopamine Agonist
(Muselergine)
Antiandrogens
(Flutamide)
Steroid Inhibitors
(Ketoconazole)
Aromatase Inhibitors
(Aminoglutethemide)
5a-Reductase Inhibitors
(Finasteride)

9. Hypothalamic-Pituitary-Ovary Axis
CNS
Hypothalamus
Anterior Pituitary
Pulsatile GnRH
(+)
LH (+)
Theca Cell Granulosa Cell
Androgens
Progesterone +
Estradiol
Aromatase
(-)
Ovary
FSH (+)
Inhibin X
Target Peripheral Tissues
Dopamine Agonist
(Muselergine)
Antiestrogens
(ICI-182,780)
Steroid Inhibitors
(Ketoconazole)
Aromatase Inhibitors
(Aminoglutethemide)

10. Intact Male Assay: “Expected” Profile for Unknowns

11. Intact 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: )
Adult vs. immature animals
Immature are more sensitive to organ weight changes
Mature are more sensitive to hormonal changes
Duration – 2-week

12. Effect of Diet Restriction on Organ Weights in Sprague-Dawley Rats

13. Effect of Diet Restriction on Serum Hormones in Sprague-Dawley Rats
One important aspect in the design of endocrine studies is the effect of body weight on circulating hormone concentrations. In the experiment summarized above [O’Connor et al., 2000; Toxicol. Sci. 54, ], male rats were maintained on various levels of dietary restriction in order to produce a range of body weight decrements. The male rats were necropsied after 15 days of dietary restriction and a series of parameters evaluated including reproductive organ weights, reproductive hormone concentrations, and thyroid parameters. When interpreting changes in hormone concentrations, as well as other endpoints, it is important that the effect of body weight loss on the endpoints is evaluated in order to eliminate/minimize potential confounding due to overt toxicity.

14. Effect of Diet Restriction on Thyroid Hormones in Sprague-Dawley Rats

15. Immature vs. Mature Rats

16. Immature vs. Mature Rats

17. Intact Male Assay: Thyroid Timecourse

18. Intact Male Assay: Thyroid Timecourse

19. Case Study With Flutamide, Ketoconazole, & Finasteride

20. Hypothalamic-Pituitary-Testis Axis
CNS
Hypothalamus
Anterior Pituitary
GnRH
(+)
FSH (+)
Sertoli Cell Leydig Cell
Inhibin
Testosterone
Aromatase
Estradiol
(-)
Testis
LH (+)
DHT
5-Reductase
Target Peripheral Tissues
Antiandrogens
(Flutamide) X
Steroid Inhibitors
(Ketoconazole)
5a-Reductase Inhibitors
(Finasteride)

21. 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

22. Case Study: Organ Weights

23. Case Study: Serum Hormones

24. 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)
Pregnenolone
Cholesterol
Progesterone
17a-Hydroxyprogesterone
Androstenedione
Testosterone 1a 1b 1c ② ③
④ Ketoconazole ⑤ ⑥
5a-Dihydroxytestosterone ⑦ ⑧
Estrone
17b-Estradiol
Leydig Cell X
Finasteride
Target peripheral tissues
Flutamide

25. Case Study: Flutamide, Ketoconazole, & Finasteride Comparison of Organ Weight Data
Cannot differentiate mode of action based on organ weight changes

26. Case Study: Flutamide, Ketoconazole, & Finasteride Comparison of Serum Hormone Data
Mode of action can be determined based on hormonal changes

27. Hypothalamic-Pituitary-Testis Axis
CNS
Hypothalamus
Anterior Pituitary
GnRH
(+)
FSH (+)
Sertoli Cell Leydig Cell
Inhibin
Testosterone
Aromatase
Estradiol
(-)
Testis
LH (+)
DHT
5-Reductase
Target Peripheral Tissues
Antiandrogens
(Flutamide) X
Steroid Inhibitors
(Ketoconazole)
5a-Reductase Inhibitors
(Finasteride)
Regulation of the hypothalamic-pituitary-testis axis in mammals. Abbreviations: central nervous system (CNS); gonadotropin releasing hormone (GnRH); follicle stimulating hormone (FSH); luteinizing hormone (LH); dihydrotestosterone (DHT). Potential sites of endocrine disruption include: ① Dopamine agonists would act on the CNS to affect GnRH release; ② Steroid biosynthesis inhibitors would inhibit testosterone production, reducing the amount of testosterone and perhaps DHT or estradiol; ③Aromatase inhibitors would inhibit the conversion of testosterone to estradiol; ④ 5α-reductase inhibitors would inhibit the conversion of testosterone to DHT; ⑤Androgen receptor blockers would interfere with the normal androgen feedback to the pituitary and brain, as well as decreasing androgen action peripherally; ⑥Estrogen receptor blockers would interfere with the normal estrogen feedback to the pituitary and brain, as well as decreasing estrogen action peripherally. Inhibin has not yet been rigorously evaluated in environmental toxiciology.

28. EACs Examined in the 15-Day Intact Male Assay
Mention studies were performed under GLPs.

29. EACs Examined in the Pubertal Assays
Point out that for the pubertal assays there is no agreement on how to set and MTD
Point out that we did not come prepared to make definitive comparisons between assays. Goal is to return at a later date to discuss differences between the assays.

30. Profile of Selected EACs Examined in the Intact Male Assay

31. Profile of Selected EACs Examined in the Pubertal Male Assay

32. Profile of Selected EACs Examined in the Hershberger Assay

33. Detection of p,p’-DDE in the Intact 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, )
Will discuss if time allows.

34. Effect of p,p’-DDE on Organ Weights in the Intact Male Assay

35. Effect of p,p’-DDE on Serum Hormone Levels in the Intact Male Assay

36. Effect of p,p’-DDE on Thyroid Hormone Levels in the Intact Male Assay

37. Detection of Di-n-Butyl Phthalate (DBP) in the Intact Male Assay
Antiandrogen-like mode of action
Inhibition of steroidogenesis?
Mylchreest et al., 2002 (Reprod. Toxicol. 16, 19-28)
Shultz et al., 2001 (Toxicol. Sci. 64, )
Results from intact male assay are consistent with steroid biosynthesis inhibition as the mode of action of DBP

38. Effect of DBP on Organ Weights & Histopathology in the Intact Male Assay

39. Effect of DBP on Organ Weights in the Intact Male Assay

40. Comparison of the EDSTAC-Recommended and Alternative Tier I Screening Batteries for Identifying EACs

41. Advantages of Tier I Using Intact 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
Intact endocrine system
Design allows integration of new endpoints if desired
Consider value of using Intact male in Tier 1
Need a more in-depth analysis – side by side comparison (apples to apples) of Tier 1 in vivo assays (Hershberger/pubertals/intact male)
Specificity and sensitivity of the alternative approaches should be directly assessed with common set of substances across different modes of action
O’Connor et al., (2002). Evaluation of the Tier I screening options for detecting endocrine-active compounds (EACs). Critical Reviews in Toxicology, 32:

42. Inter-Laboratory Studies Using the Intact Male Assay
EPA
Linuron
Methoxychlor
3 to 4 more in 2004?
CTL
Genistein
Dow
Flutamide
Bayer
Nonylphenol
WIL
Methyltestosterone
Exxon-Mobil
p,p’-DDE (2004)

43. Identification of EACs
Tier I
Uterotrophic
Assay
ER Agonists
ER Antagonists
Receptor Binding/
Transactivation
Agonist/Antagonist
(ER, AR)
Thyroid Effects
Steroid Biosynthesis
ER/AR Agonists
ER/AR Antagonists
Intact Male
Assay

44. Publications: 15-Day Intact Male Assay
O’Connor, J.C., Cook, J.C., Marty, M.S., Davis, L.G., Kaplan, A.M., and Carney, E.W. (2002). Evaluation of the Tier I screening options for detecting endocrine-active compounds (EACs). Critical Reviews in Toxicology, 32:
O’Connor, J.C., Frame, S.R., and Ladics, G.S. (2002). Evaluation of a 15-day screening assay using intact male rats for identifying antiandrogens. Toxicological Sciences, 69:
O’Connor, J.C., Frame, S.R., and Ladics, G.S. (2002). Evaluation of a 15-day screening assay using intact male rats for identifying steroid biosynthesis inhibitors and thyroid modulators. Toxicological Sciences, 69:
O’Connor, J.C., Davis, L.G., Frame, S.R., and Cook, J.C. (2000). Detection of dopaminergic modulators in a Tier I screening battery for identifying endocrine-active compounds (EACs). Reproductive Toxicology, 14:
O’Connor, J.C., Davis, L.G., Frame, S.R., and Cook, J.C. (2000). Evaluation of a Tier I screening battery for detecting endocrine-active compounds (EACs) using the positive controls testosterone, coumestrol, progesterone, and RU486. Toxicological Sciences, 54:
O’Connor, J.C., Cook, J.C., Frame, S.R., and Davis, L.G. (1999). Detection of the environmental antiandrogen p,p’-DDE in Sprague-Dawley and Long-Evans rats using a Tier I screening battery and a Hershberger Assay. Toxicological Sciences, 51:
O’Connor, J.C., Frame, S.R., and Cook, J.C. (1999). Detection of thyroid toxicants in a Tier I screening battery and alterations in thyroid endpoints over 28 days of exposure. Toxicological Sciences, 51:
O’Connor, J.C., Cook, J.C., Slone, T.W., Frame, S.R., and Davis, L.G. (1998). An ongoing validation of a Tier I screening battery for detecting endocrine-active compounds (EACs). Toxicological Sciences, 46:
O’Connor, J.C., Frame, S.R., Biegel, L.B., Cook, J.C., and Davis, L.G. (1998). Sensitivity of a tier I screening battery compared to an in utero exposure for detecting the estrogen receptor agonist 17-estradiol. Toxicological Sciences 44:
Cook, J.C., Kaplan, A.M., Davis, L.G., and O’Connor, J.C. (1997). Development of a tier I screening battery for detecting endocrine active compounds (EACs). Regulatory Toxicology and Pharmacology 26: