Evaluation of a 15-Day Screening Assay Using Intact Male Rats for Identifying Steroid Biosynthesis Inhibitors and Thyroid Modulators

John C. O’Connor1, Steven R. Frame and Gregory S. Ladics

DuPont Haskell Laboratory for Health and Environmental Sciences, P.O. Box 50, Newark, Delaware 19714

Received March 18, 2002; accepted June 7, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An in vivo screening assay using intact adult male rats has been evaluated for its ability to detect four endocrine-active compounds (EACs) via oral (gavage) administration. The test compounds included the aromatase inhibitor fadrozole (FAD), the testosterone biosynthesis inhibitor ketoconazole (KETO), and the thyroid modulators phenobarbital (PB) and propylthiouracil (PTU). Three of the test compounds (KETO, PB, and PTU) have been previously evaluated in the 15-day intact male assay with compound administration via intraperitoneal injection (ip). For the current studies, male rats were dosed for 15 days via oral gavage and euthanized on the morning of test day 15. The endpoints evaluated included final body and organ weights (liver, thyroid gland, testes, epididymides, prostate, seminal vesicles with fluid, accessory sex gland unit [ASG]), serum hormone concentrations (testosterone [T], estradiol [E2], dihydrotestosterone [DHT], luteinizing hormone [LH,] follicle stimulating hormone [FSH], prolactin [PRL], T3, T4, thyroid stimulating hormone [TSH]), and histopathology of the testis, epididymis, and thyroid gland; positive results for each endpoint are described below. In addition, an evaluation of immune system endpoints (humoral immune function, spleen and thymus weights, and spleen cell number) was conducted on a subset of animals dosed with either KETO or PB. FAD and KETO decreased the weights for the androgen-dependent tissues and caused similar patterns of hormonal alterations (decreased serum T and DHT; increased serum FSH and/or LH). In addition, KETO caused spermatid retention. For FAD and KETO, effects on thyroid parameters were not indicative of thyroid toxicity. PB and PTU caused thyroid effects consistent with thyroid modulators (increased thyroid weight, decreased serum T3 and T4, increased serum TSH, thyroid follicular cell hypertrophy/hyperplasia, and colloid depletion). In addition, PB increased relative liver weight and altered reproductive hormone concentrations (decreased serum DHT, PRL, LH; increased serum E2). Orally administered KETO and PB did not alter the primary humoral immune response to sheep red blood cells (SRBC), although spleen weights were increased at the highest doses for both compounds. In the current study, all four test substances were identified as endocrine-active. The effects that were observed in the current study via oral (gavage) compound administration were similar to the responses that were observed by the ip route in previous studies for KETO, PB, and PTU. Overall, the sensitivity (i.e., the dose required to elicit similar magnitude responses) between the ip and oral routes of administration were similar for the three EACs that were examined by both routes of administration. This article, in addition to the > 20 compounds that have already been examined using the 15-day intact male assay, supports this assay as a viable screening assay for detecting EACs, and also illustrates that the ability to identify EACs using the intact male assay will be equivalent regardless of the route of compound administration.

Key Words: screening; Tier I battery; rat; immunotoxicity; endocrine-active compounds; fadrozole; ketoconazole; phenobarbital; propylthiouracil.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Responding to concerns that endocrine-active compounds (EACs) may impact endocrine function in humans and wildlife, Congress passed amendments to the Food Quality Protection Act of 1996 and the Safe Drinking Water Act of 1996 requiring the U.S. Environmental Protection Agency (EPA) to implement screening/testing strategies for EACs. In response to the 1996 legislation, EPA convened the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) to advise the agency on a strategy to screen and test xenobiotics for endocrine disruption. EDSTAC completed their charter in 1998 by recommending a tiered screening and testing scheme to evaluate compounds for their potential to act as agonists or antagonists to the estrogen receptors (ER) or androgen receptors (AR), steroid biosynthesis inhibitors, or their ability to alter thyroid function (EDSTAC, 1998Go). For Tier I, the EDSTAC-recommended screening battery comprised 8 different assays, but EDSTAC also proposed two alternative batteries that were deemed worthy of further evaluation (Table 1Go). The current report examines the utility of one of the alternate assays, the 15-day intact male assay, for its ability to identify EACs.


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TABLE 1 Comparison of the EDSTAC-Recommended and Alternative Tier I Screening Batteries for Identifying Endocrine-Active Compounds
 
Over the last 7 years, DuPont has been evaluating the 15-day intact male assay as one component of a Tier I screening strategy for identifying EACs (Cook et al., 1997Go; O’Connor et al., 1998aGo,bGo; 1999aGo, bGo; 2000aGo, bGo; 2001Go). The rationale for the study design (Cook et al., 1997Go) and the pros and cons of this approach have been previously discussed (O’Connor et al., in pressGo). Briefly, adult male rats (15/group) are dosed daily for 15 days with the test compound and euthanized on the morning of test day 15, approximately 2 h after the last administered dose. At the terminal euthanasia, the liver, thyroid gland, and reproductive organs (testes, epididymides, prostate, seminal vesicles with fluid, accessory sex gland unit [ASG]) are weighed, and the testes, epididymides, and thyroid gland are saved for histopathological evaluation. Blood is collected and serum is prepared for hormonal evaluation. The original study design for this Tier I battery used the intraperitoneal route (ip) of compound administration. The ip route was selected to enhance the sensitivity of the assay (i.e., reduce false negative responses) and to facilitate potency comparisons by eliminating absorption as a variable (Cook et al., 1997Go). Because there will be several routes of potential exposure of humans to EACs (e.g., dermal, oral, or inhalation) and because of the unique exposure routes of wildlife (e.g., gills, eggs), the ip route was selected to maximize sensitivity realizing that subsequent studies (i.e., Tier II studies) would be required to determine whether sufficient exposure could occur via the most relevant route. The current studies examined compound administration via oral gavage.

Additionally, in evaluating currently available data on EACs, the Risk Assessment Forum of the EPA reported that the principal health effects following exposure to EACs included carcinogenisis, reproductive toxicity, neurotoxicity, and immunotoxicity (Crisp et al., 1997Go). A highly complex bidirectional interrelationship between the immune and neuroendocrine systems has been reported (reviewed in Besedovsky and del Rey, 1996Go; Chryssikopoulos, 1997Go; Fuchs and Sanders, 1994Go; Tomaszewska and Przekop, 1997Go). Products of the immune system (i.e., cytokines) have been reported to affect neuroendocrine functions (Koenig, 1991Go; Rivest and Laflamme, 1995Go; Roy, 1994Go), while various hormone receptors have been found on immune cells and a number of hormones have been reported to enhance (e.g., growth hormone, TSH, and prolactin), attenuate (e.g., gonadal steroids and endogenous opioids), or suppress (e.g., glucocorticoids and ACTH) responses of the immune system (Gaillard, 1995Go; Weigent and Blalock, 1987Go).

In the current study, four EACs have been examined using the 15-day intact male assay with compound administration via the oral (gavage) route. The readers are also referred to the accompanying article (O’Connor et al., 2002Go), which presents data from 6 additional EACs (antiandrogens) that were evaluated via oral compound administration in the 15-day intact male assay. These studies will provide a basis for comparing differences in sensitivity between the ip and oral routes of compound administration for the 15-day intact male assay, as well as increasing the number of compounds examined using the intact male assay. In addition to the endocrine endpoints, evaluation of immune system endpoints were conducted on two of the test compounds using a separate subset of animals. Spleen and thymus weights, spleen cell number, and the primary IgM humoral immune response to sheep red blood cells (SRBC) were evaluated. In the current report, the highly specific aromatase inhibitor fadrozole (FAD; Nunez et al., 1996Go) and the testosterone biosynthesis inhibitor ketoconazole (KETO; Feldman, 1986Go) were used to characterize the ability of the 15-day intact male assay to detect steroid biosynthesis inhibitors. Two thyroid modulators, phenobarbital (PB; a hepatic enzyme inducer that enhances the clearance of thyroid hormones) and propylthiouracil (PTU; a thyroperoxidase inhibitor) were used to characterize the ability of the assay to detect weak and potent thyroid modulators, respectively (reviewed in Capen, 1996Go, 1997Go). In addition, the data from the current report for KETO, PB, and PTU were compared to the data for each compound that were generated when the compounds were administered via ip injection. Finally, the data from the current report were compared to data from the pubertal male and female assays, two additional screening assays proposed by EDTAC, in order to facilitate comparisons of the three screening assays.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Test materials.
PB, PTU, and cyclophosphamide (CY) were obtained from the Sigma Chemical Company (St. Louis, MO). All other materials were obtained from the following manufacturers: Certified Rodent Diet #5002®, PMI Feeds, Inc. (St. Louis, MO); methylcellulose, Fisher Scientific (Springfield, NJ); KETO, ICN Pharmaceuticals, Inc. (Costa Mesa, CA); sterile sheep red blood cell (SRBC) in Alsevers solution, Rockland (Gilbertsville, PA); Hanks Balanced Salt Solution (HBSS) and 1.0 M HEPES, GIBCO Laboratories (Grand Island, NY); luteinizing hormone (LH; catalog #RPA.552), prolactin (PRL; catalog #RPA.553), thyroid stimulating hormone (TSH; catalog #RPA.554), and follicle stimulating hormone (FSH; catalog #RPA.550) radioimmunoassay (RIA) kits, Amersham Corp. (Arlington Heights, IL); testosterone (T; catalog #TKTT5), estradiol (E2; catalog #KE2D5), tri-iodothyronine (T3; catalog #TKT35), and thyroxine (T4; catalog #TKT45) RIA kits, Diagnostic Products Corp. (Los Angeles, CA); rT3 RIA kits (catalog #10834), Polymedco Inc. (Cortlandt Manor, NY); and, dihydrotestosterone (DHT; catalog #DSL-9600) RIA kit, Diagnostic Systems Laboratories (Webster, TX). FAD was generously donated by Novartis Pharmaceuticals Corporation (Summit, NJ).

Test species.
Male Sprague-Dawley (Crl:CD®(SD)IGS BR) rats were acquired from Charles River Laboratories, Inc. (Raleigh, NC). Male rats were approximately 63 days old upon arrival. Rats were housed in stainless steel, wire-mesh cages suspended above cage boards and were fed PMI Feeds, Inc., Certified Rodent Diet #5002 and provided with tap water (United Water Delaware) ad libitum. Rats were clinically normal and free of antibody titers to pathogenic murine viruses and mycoplasma and free of pathogenic endo- and ectoparasites and bacteria. Animal rooms were maintained on a 12-h light/dark cycle (fluorescent light), a temperature of 23 ± 2°C, and a relatively humidity of 50% ± 10%. After a quarantine period of approximately one week, rats that displayed adequate weight gain and freedom from clinical signs were divided by computerized, stratified randomization into 5 treatment groups so that there were no statistically significant differences among group body weight means. For KETO and PB, each treatment group contained 25 male rats; 15 were designated for endocrine analyses and 10 were designated for immune system analyses. For FAD and PTU, each treatment group contained 15 male rats designated for endocrine analyses; immune system analyses were not conducted.

Study design.
Each of the four test compounds were evaluated with their own concurrent control group in individual studies over a period of two years. All rats were weighed daily and cage-side examinations were performed to detect moribund or dead rats. At each weighing, rats were individually handled and examined for abnormal behavior or appearance. No mortality was observed for the four test substances except as noted below for KETO. All test compounds were prepared in 0.25% methylcellulose vehicle and administered by oral gavage at approximately 0900 h daily for 15 days. The dose volume was 5.0 ml/kg body weight. On the morning of test day +15, rats were anesthetized using CO2 and euthanized by exsanguination. The following concentrations of test compound were used: FAD (1, 5, 10, and 25 mg/kg/day), KETO (10, 25, 75, and 125 mg/kg/day), PB (5, 25, 50, and 100 mg/kg/day), and PTU (0.1, 1, 10, and 20 mg/kg/day). Due to unexpected deaths as a result of excessive toxicity in the 125 mg/kg/day KETO group, the dose level was reduced to 100 mg/kg/day on test day 6. Doses were selected in order to obtain the maximal pharmacologic effect for each compound and/or not exceed the maximum tolerated dose (MTD), defined as a 10% difference in final body weight from the ad libitum control group, as determined in range-finder studies.

Pathological evaluations.
For the endocrine subset (15/group) all rats were administered the final dose of test compound approximately 2 h prior to euthanization. Euthanization was performed between 0700 and 1000 h on the morning of test day +15, and necropsy was performed across the treatment groups in order to control for potential variation due to "time-of-day" effects. At necropsy, blood was collected from the descending vena cava, allowed to clot at 4°C for 1 h, and centrifuged for 20 min at 1500 x g (4°C) for preparation of serum for hormonal analyses. The liver, testes, epididymides, prostate, seminal vesicles, and ASG unit (composed of the prostate, seminal vesicles with fluid, and coagulating glands) were weighed and relative (to body weight) organ weights were calculated. The thyroid glands and surrounding tissue were removed and placed into formalin fixative for at least 48 h prior to trimming and weighing. Following fixation, final dissection of the thyroid gland was performed under a dissecting microscope by one individual in order to reduce the variability of the dissection procedure and hence, reduce the variability of the thyroid weights. Weights for the testes, epididymides, and seminal vesicles were paired weights. After weighing, the epididymides were placed in formalin fixative, the testes were placed in Bouin’s fixative, and both were examined microscopically. The formalin-fixed thyroid glands were also examined microscopically. The epididymides were not collected from the KETO experiment.

Immune system assessment.
For the immune system subset, rats were housed and dosed under the same conditions as the main study animals. Six days prior to study termination, rats dosed with KETO and PB that were designated for assessment of the primary humoral immune response (10/dose group) were injected into a tail vein with 0.5 ml of 4 x 108 SRBC in saline. This dose of SRBC, in conjunction with the timing of immunization relative to measurement of response, was found to elicit an optimal primary IgM response in this rat strain (data not shown). All rats were administered the final dose of test compound approximately 2 h prior to euthanization on the morning of test day +15. Between 0800 and 1100 h, rats were euthanized, blood collected, and the spleen and thymus were removed, weighed, and relative (to body weight) organ weights calculated. Sera were obtained and stored frozen (0°C) until analyzed. A single-cell suspension was prepared from half of the spleen in HBSS containing 1.0 M HEPES (GIBCO) by first cutting the spleens into several pieces and then placing the pieces in a Stomacher® Lab Blender (Seward Medical Limited, London, UK). Spleen cell numbers were determined using a Serono Baker 9000® hematology analyzer (Allentown, PA) and multiplied by the total spleen weight/weight of the spleen section to obtain cell number/total spleen. Individual serum samples were analyzed for SRBC-specific IgM antibody using an ELISA as previously described (Temple et al., 1993Go). Data were acquired using an MR 5000 96-well microplate reader (Dynatech Laboratories, Chantilly, VA) and analyzed using the Revelation Software (Version 2.0, Dynatech Laboratories). Sera pooled from male rats injected with SRBC and dosed with the known immunosuppressive agent CY (20 mg/kg/day, ip) for 6 days were evaluated with the study samples as a positive control. The data were expressed as the highest dilution (i.e., titer) to give an absorbance value of 0.5 (e.g., 1:500 = 500). The SRBC-specific serum IgM antibody titers were reported as log2 to normalize the data.

Hormonal measurements.
Blood was collected at the time of euthanization from all animals. Serum was prepared and stored at –80°C until analyzed for serum hormone concentrations. Serum T, E2, DHT, LH, FSH, PRL, TSH, T3, and T4 concentrations were measured by commercially available RIA kits. Details on standard curve concentrations for each RIA kit are available from the manufacturer. When hormone concentrations were below the limit of detection, a zero was used as the value for calculations. When hormone concentrations were above the highest standard, the sample was diluted using kit-specific assay "zero calibrator" and reanalyzed.

Statistical analyses.
Mean final body weights and organ weights were analyzed by a one-way ANOVA. When the corresponding F test for differences among test group means was significant, pairwise comparisons between test and control groups were made with Dunnett’s test (Dunnett, 1955Go). Bartlett’s test for homogeneity of variances was performed and, when significant (p < 0.005), was followed by nonparametric procedures (Dunn’s test; Dunn, 1964Go). Serum hormone concentrations and SRBC-specific IgM levels were analyzed using Jonckheere’s test for trend in a stepdown manner (Hochberg and Tamhane, 1987Go; Marcus et al., 1976Go). If a significant dose-response trend was detected, data from the top dose group was excluded and the test repeated until no significant trend was detected. Except for Bartlett’s test, all other significance was judged at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Final body and liver weights (Table 2Go).
Mean final body weights were significantly decreased at 10 and 20 mg/kg/day in rats treated with PTU (93 and 92% of control, respectively), and were unchanged in rats treated with FAD, KETO, or PB. Relative liver weights were significantly increased in rats treated with KETO and PB, with the greatest increases at the highest dosages (123 and 132% of control, respectively), and were significantly decreased in rats treated with 10 mg/kg/day PTU (90% of control). Relative liver weights were not affected by FAD treatment at the dose levels tested.


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TABLE 2 Intact Male Assay: Final Body Weights and Organ Weights
 
Reproductive organ weights and histopathology (Table 2Go).
FAD decreased relative ASG unit weights at 5, 10, and 25 mg/kg/day (88% of control at all dosages). Relative seminal vesicle weights were decreased at 5, 10, and 25 mg/kg/day (82, 83, and 85% of control, respectively). Absolute testis and epididymis weights and relative prostate weights were not affected by treatment with FAD at the dose levels tested. Microscopically, slight spermatid retention (primarily in stage IX/X tubules) was present in the testes of one rat administered 25 mg/kg/day. Since slight spermatid retention may be seen in control animals, the single occurrence in only one of 15 rats may represent a spurious finding. No microscopic changes were observed in the epididymides.

KETO decreased organ weights for all androgen-dependent tissues at the highest dosage (91, 82, 87, and 72% of control for epididymis, ASG unit, seminal vesicle, and prostate weights, respectively). Absolute testis weights were not affected by treatment with KETO at the dose levels tested. Microscopically, retention of mature (elongate) spermatids in Stage IX to XIII tubules was present in the testes of 6/10 rats dosed with 100 mg/kg/day.

PB did not affect any of the reproductive organ weights at the dose levels tested. Microscopically, slight spermatid retention was present in the testes of 4/15 rats administered 100 mg/kg/day PB. This change was characterized by retention of late-step spermatids in stage X–XII tubules. Retained spermatids were present at both the luminal and basilar areas of the seminiferous epithelium. No microscopic changes were observed in the epididymides.

PTU did not affect any of the reproductive organ weights, and there were no microscopic alterations of the testis or epididymides at the dose levels tested.

Reproductive hormone concentrations (Table 3Go).
FAD decreased serum T (48, 30, 18, and 16% of control at 1, 5, 10, and 25 mg/kg/day, respectively) and DHT (41, 23, 15, and 13% of control at 1, 5, 10, and 25 mg/kg/day, respectively) concentrations in a dose-dependent manner, with statistically significant decreases at all dosages. Serum FSH concentrations were significantly increased at 25 mg/kg/day (121% of control). Serum E2, LH, and PRL concentrations were not affected by FAD treatment at the dose levels tested.


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TABLE 3 Intact Male Assay: Reproductive Hormone Concentrations
 
KETO decreased serum T (73, 54, 31, and 12% of control at dosages of 10, 25, 75, and 100 mg/kg/day, respectively) and DHT (75, 67, 37, and 27% of control at 10, 25, 75, and 100 mg/kg/day, respectively) concentrations in a dose-dependent manner, with statistically significant decreases at all dosages. Serum E2 concentrations were significantly increased at 100 mg/kg/day (130% of control). Serum FSH (122, 136, and 136% of control, respectively) and LH (134, 148, and 141% of control, respectively) concentrations were significantly increased at concentrations of 25, 75, and 100 mg/kg/day. Serum PRL concentrations were not affected by FLUT treatment at the dose levels tested.

PB caused a statistically significant decrease in serum PRL (69 and 52% of control, respectively) and LH (81 and 87% of control, respectively) concentrations at 50 and 100 mg/kg/day. Serum DHT concentrations were significantly decreased at all dosages (67, 79, 69, and 60% of control at dosages of 5, 25, 50, and 100 mg/kg/day, respectively). Serum T, E2, and FSH concentrations were not affected by PB treatment at the dose levels tested.

PTU increased serum FSH concentrations in a dose-dependent manner, with statistically significant increases at 1, 10, and 20 mg/kg/day (120, 135, and 147% of control, respectively). Serum T, DHT, E2, LH, and FSH concentrations were not affected by PTU treatment at the dose levels tested.

Thyroid weight, thyroid hormone concentrations, and thyroid histopathology (Table 4Go).
FAD administration caused a statistically significant decrease in serum T4 concentrations at all dose levels (73, 73, 68, and 66% of control at 1, 5, 10, and 25 mg/kg/day, respectively). Thyroid weight and serum TSH and T3 concentrations were not affected by FAD treatment at the dose levels tested. There were no compound-related microscopic changes in the thyroid gland.


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TABLE 4 Intact Male Assay: Thyroid Parameters
 
KETO administration caused a statistically significant decrease in serum T3 concentrations at 75 and 100 mg/kg/day (78 and 82% of control, respectively). Serum T4 concentrations were decreased in a dose-dependent manner and were significantly decreased at 25, 75, and 100 mg/kg/day (74, 55, and 45% of control, respectively). Serum TSH concentrations were not affected by KETO treatment at the dose levels tested. Some equivocal evidence of hypertrophy of thyroid follicular epithelium was present in a few rats administered 100 mg/kg/day KETO; however, thyroid weight was not affected. In addition, the microscopic changes were not convincingly outside the spectrum of thyroid gland histology observed in control male rats.

PB administration significantly increased relative thyroid weights at all dose levels (125, 125, 150, and 125% of control at 5, 25, 50, and 100 mg/kg/day, respectively). Serum T3 concentrations were decreased in a dose-dependent manner and were statistically significant at all dosages (88, 77, 75, and 53% of control at 5, 25, 50, and 100 mg/kg/day, respectively). Serum T4 concentrations were decreased in a dose-dependent manner and were significantly decreased at 25, 75, and 100 mg/kg/day (80, 73, and 47% of control, respectively). Serum TSH concentrations increased in a dose-dependent manner and were significantly increased at 25, 50, and 100 mg/kg/day (141, 143, and 145% of control, respectively). In the thyroid gland, slight colloid depletion (pale staining of colloid) was present in 3/15 in the 100 mg/kg/day groups. Equivocal evidence of hypertrophy of follicular epithelium was also present in affected thyroids.

PTU administration significantly increased relative thyroid weights at all dose levels (140, 240, 360, and 280% of control at dosages of 0.1, 1, 10, and 20 mg/kg/day, respectively). Serum T3 concentrations were significantly decreased at 1, 10, and 20 mg/kg/day (35, 26, and 29% of control, respectively). Similarly, serum T4 concentrations were decreased at dosages of 1, 10, and 20 mg/kg/day (2, 0, and 0% of control, respectively). Serum TSH concentrations were increased at dosages of 1, 10, and 20 mg/kg/day (351, 377, and 345% of control, respectively). Microscopic changes consistent with excess TSH stimulation of the thyroid gland were present in the thyroid gland of rats at all concentrations of PTU. At 1 mg/kg/day and above, thyroid changes were similar and consisted of diffuse hypertrophy and hyperplasia of follicular epithelial cells. Affected thyroid follicles were lined by columnar epithelial cells and had reduced lumenal diameter and pale-staining or absent colloid. Hypertrophy was associated with diffuse hyperplasia characterized primarily by crowding of nuclei along follicular basement membranes. Focal stratification of nuclei and papillary proliferations were less common. Consistent with the hyperplastic response was a slight increase in mitotic figures. At 0.1 mg/kg/day, changes in the thyroid were primarily limited to diffuse pale staining of colloid with equivocal hypertrophy of follicular epithelium in some animals.

Immune system assessment (Table 5Go and Fig. 1Go).
Exposure of rats to KETO or PB did not significantly alter mean final body weights. Relative spleen weights were increased (128% of control) in rats treated with 100 mg/kg/day KETO. Both absolute and relative spleen weights were significantly increased in rats treated with 100 mg/kg/day PB (125% and 129% of control, respectively). Spleen cell number was also increased (117% of control), albeit nonsignificantly, in the 100 mg/kg/day PB group (Table 5Go). The primary humoral immune response to SRBC was not significantly altered by KETO or PB (Fig. 1Go). As expected, the known immunosuppressant agent, CY, decreased the humoral immune response to SRBC (22–38% of control; data not shown).


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TABLE 5 Immune System Assessment: Final Body and Organ Weights and Total Spleen Cell Number
 


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FIG. 1. Assessment of the primary IgM antibody response to SRBC following exposure to KETO or PB. Male rats (10/group) were exposed to a daily oral gavage dose of either 0, 10, 25, 75, or 100 mg/kg/day KETO or 0, 5, 25, 50, or 100 mg/kg/day PB for 15 days. Animals received SRBC by iv injection 6 days prior to termination for assessment of the humoral immune response. At termination, sera were obtained and analyzed for IgM antibody specific for SRBC by an ELISA as described in Materials and Methods. Results are reported as the log2 of the SRBC-specific serum IgM titers.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this report, we examined four EACs with distinct endocrine activities in order to evaluate the sensitivity and specificity of the 15-day intact male assay for detecting EACs. The EACs included the steroid biosynthesis inhibitors FAD and KETO, and the thyroid modulators PB and PTU. For each compound, the "fingerprint" of the responses was compared to the expected pattern of responses based on the known mechanism of action. Responses for KETO, PB, and PTU were also compared to the responses that were obtained in previous studies where the compounds were administered via ip injection (O’Connor et al., 1998aGo, 1999bGo). In addition, an evaluation of immune system endpoints was conducted on a subset of animals dosed with either KETO or PB. Finally, the data from the current report were compared to data from the pubertal male and female assays in order to facilitate comparisons of the three screening assays. The readers are also referred to the accompanying report (O’Connor et al., 2002Go), which presents data from 6 additional EACs (antiandrogens) that were evaluated via oral compound administration in the 15-day intact male assay.

Detection of Steroid Biosynthesis Inhibitors
KETO (a testosterone biosynthesis inhibitor) has been previously examined in the intact male assay by the ip route of administration (O’Connor et al., 1998aGo). In the intact male assay, responses to KETO included decreased weights for all androgen-dependent tissues, hormonal alterations, and microscopic alterations of the testis (spermatid retention). KETO inhibits T biosynthesis by binding to the heme iron of the three cytochrome P450 isozymes of the testosterone biosynthetic pathway (Feldman, 1986Go; Morita et al., 1990Go). Therefore, Leydig cells are unable to produce sufficient quantities of T. As a result, LH and FSH release from the pituitary are increased to stimulate T production. Secondary to the decrease in T, serum levels of DHT (the major metabolite of T), and E2 (the aromatization product of T) are also decreased. As a result of the decreased androgen levels, the weights for the androgen-dependent tissues are also decreased. Therefore, the data for the current experiment are characteristic for a testosterone biosynthesis inhibitor, namely decreased weights for the androgen-dependent tissues and hormonal alteration (decreased T, DHT, and E2; increased LH and FSH).

The overall fingerprint of the effects were consistent with previous data for KETO when administered via ip injection (O’Connor et al., 1998aGo). Overall the sensitivity of the assay was very similar regardless of the route of compound administration, although there were some minor differences. By the ip route of administration, the maximal effects on organs’ weights were slightly more pronounced than those observed by the oral route. However, the greater effects observed by the ip route of administration were accompanied by more severe body weight effects. The serum hormone data was less consistent than the organ weight data. KETO administration by the ip route of administration resulted in more pronounced effects on the gonadotropins. For example, at 75 mg/kg/day KETO serum LH concentrations were increased to 191 and 148% of control with compound administration via ip or oral gavage, respectively. However, the effects on the steroid hormones were more pronounced when KETO was administered via oral gavage. At 75 mg/kg/day via ip injection, serum T and DHT concentrations were decreased to 41 and 55% of control, respectively. At 75 mg/kg/day via oral gavage, serum T and DHT concentrations were decreased to 31 and 37% of control, respectively. The reason for the differential responses for the serum hormones is unclear; however, when examining all the data, the sensitivity of the two routes of administration are essentially equivalent.

The pattern of the responses observed with FAD (an aromatase inhibitor) were similar to those observed for KETO: decreased ASG unit and seminal vesicle weights and hormonal alterations (decreased serum T and DHT; increased serum FSH). Interestingly, serum E2 concentrations were not decreased by FAD treatment. The reason for the lack of an effect on serum E2 concentrations is unknown, and was unexpected since aromatase inhibition is the primary pharmacologic activity of FAD (Nunez et al., 1996Go). The mechanism(s) for the decrease in ASG and seminal vesicle weights is also unclear, but is consistent with the data generated for another aromatase inhibitor, anastrozole (Dukes et al., 1996Go; O’Connor et al., 1998aGo). Furthermore, the decrease in ASG unit and seminal vesicle weights is a pattern that has been replicated in our laboratory using two proprietary compounds with aromatase inhibition activity (data not shown); therefore, it seems likely that these effects are true compound-related effects that are characteristic of aromatase inhibitors. Overall, the data were consistent with a compound with steroid biosynthesis inhibition activity, although with an aromatase inhibitor, we would also have expected decreased serum E2 concentrations.

Detection of Thyroid Modulators
Chemicals that alter thyroid function can act by altering synthesis, release, transport, or metabolism of thyroid hormones, or by acting as thyroid hormone receptor agonists or antagonists (reviewed in Capen, 1997Go). In the current report, PB, a hepatic enzyme inducer that enhances the clearance of thyroid hormones (reviewed in Capen, 1996Go, 1997Go), and PTU, a thyroid hormone synthesis inhibitor (reviewed in Capen, 1996Go, 1997Go) and 5‘-deiodinase inhibitor (Kohrle, 1990Go), were used to characterize the ability of intact male assay to detect weak and potent thyroid toxicants, respectively. Both compounds have been previously evaluated in the 15-day intact male assay via ip administration (O’Connor et al., 1999bGo). The current report provides a basis for comparing differences in sensitivity between the ip and oral routes of compound administration for the 15-day intact male assay. Overall, the endpoint profiles obtained for PB and PTU were characteristic for their mechanism(s) of action. Both PB and PTU displayed the typical profile of thyroid effects for compounds that induce follicular cell hypertrophy/hyperplasia in rodents, namely increased thyroid weight, and hormonal alterations (decreased T3 and T4; increased TSH). As expected, PB administration increased relative liver and thyroid weight, and caused changes in thyroid hormones (increased TSH; decreased serum T3 and T4). Definitive microscopic changes were limited to effects on colloid and mild hypertrophy at the highest dosage. The increase in TSH is a secondary response following PB administration due to the clearance of serum T3 and T4 via increased biliary excretion as a result of enhanced UDP-GT activity (McClain et al., 1989Go). Similar to PB, the effects of PTU on thyroid parameters included increases in relative thyroid weight, as well as changes in thyroid hormones (increased TSH; decreased serum T3 and T4). Microscopic changes of the thyroid gland were more severe than those observed with PB, demonstrating the higher thyrotrophic potency of PTU as compared to PB (reviewed in Capen, 1996Go, 1997Go). The potency difference between PB and PTU are also underscored by the magnitude of the changes in thyroid weight and hormonal alterations, where PTU causes greater changes in all parameters than PB. The differences in potency of PB and PTU are reflective of their mechanism(s) of action, and help to illustrate the ability of the intact male assay to detect thyroid toxicants that can act via multiple pathways, and have marked potency differences. PTU, the more potent of the two thyroid modulators, acts directly at the thyroid gland to inhibit synthesis of thyroid hormone. In contrast, PB alters thyroid hormone homeostasis via increased hepatic clearance of circulating thyroid hormones. Overall, the endpoint profiles obtained for PB and PTU were similar to the effects that were observed when the compounds were administered via ip injection, and the severity of the effects were similar by both routes of administration (i.e., the sensitivity of the intact male assay was equivalent for the two routes of administration; O’Connor et al., 1999bGo).

The initial expectation was that FAD and KETO would not alter thyroid function, since neither of them have been shown to induce follicular cell hypertrophy/hyperplasia in long-term rodent studies (Physician’s Desk Reference, 1999). However, similar to previous studies with a variety of EACs looking at thyroid parameters in the 15-day intact male assay (O’Connor et al., 1998aGo,bGo, 1999aGo,O’Connor et al., bGo, 2000aGo,O’Connor et al., bGo), thyroid hormone levels were affected by both FAD and KETO. It is hypothesized that the decreases in T3 and T4 observed with KETO may be due to liver enzyme induction based on the increases in relative liver weights. These data are similar to the effects observed when KETO was administered via ip injection (O’Connor et al., 1998aGo). In contrast to ip injection, when administered via oral gavage, serum TSH levels were not significantly increased by KETO, although there is a trend towards increased serum TSH concentrations. This is most likely due to the lower dosages that were achieved and/or decreased absorption of KETO in the oral gavage study. The mechanism(s) for the changes in T4 concentrations after FAD administration are unclear. Based on these data for thyroid parameters, and as previously discussed by the authors (O’Connor et al., 1999bGo), data interpretation based solely on changes in thyroid hormone levels is not sufficient for identifying true thyroid modulators. For example, of the 28 compounds that we have evaluated in the intact male assay, 27 of the compounds produced some statistically significant change in at least one of the thyroid hormones. Hence, many compounds can transiently alter thyroid hormone homeostasis in rodents without resulting in long-term (i.e., adverse) thyroid effects. Therefore, one must be particularly cognizant of the fact that thyroid hormone concentrations are very easily perturbed by a wide variety of chemicals, and a large proportion of these will not be thyroid toxicants in long-term studies. A more comprehensive approach such as the one taken by the 15-day intact male assay, including thyroid weight, thyroid hormone analyses, and microscopic evaluation of the thyroid gland appears prudent when screening for compounds that target the thyroid gland. Thyroid modulators would be identified based on a weight-of-evidence approach using all the thyroid endpoints included in the 15-day intact male assay.

Comparison of the Intact Male Assay and Other Screening Assays
The results from the current report were also used to help facilitate a comparison of the assays that differ between the three proposed EDSTAC screening batteries (Table 1Go). Of the four EACs examined in the current report (Table 6Go), all four have been evaluated in the pubertal male assay (Table 7Go), and FAD, KETO, and PTU have been evaluated in the pubertal female assay (Table 8Go). The pubertal assays are apical tests that rely primarily on evaluation of age at pubertal onset and organs weights for identifying EACs (EDSTAC, 1998Go). Limited hormonal assessment and histopathological evaluation are also included to enhance the sensitivity of the assays. The primary markers for pubertal onset in the male and female assays are age at preputial separation (PPS; androgen-dependent; Stoker et al., 2000Go) and vaginal opening (VO; estrogen-dependent; Goldman et al., 2000Go), respectively.


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TABLE 6 Summary of Results from the Intact Male Assay
 

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TABLE 7 Summary of Results from Pubertal Male Assays Conducted in Other Laboratories
 

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TABLE 8 Summary of Results from Pubertal Female Assays Conducted in Other Laboratories
 
Of the two steroid biosynthesis inhibitors evaluated in the current report, KETO was identified as an EAC in the pubertal male assay, and both FAD and KETO were identified as EACs in the pubertal female assay. FAD administration did not alter any of the endpoints in the pubertal male assay, a finding that may not be surprising given the role of estrogens in male sexual development. However, the inability of the pubertal male assay to detect FAD may be a reflection of the dose that was administered (6 mg/kg/day) when compared to the dose that was employed in the intact male assay (25 mg/kg/day; Marty et al., 2001bGo). The female pubertal assay identified FAD based on decreased uterine weight and delay in the age at VO (Marty et al., 1999Go).

Consistent with the data for the intact male assay, KETO was detected in the pubertal male assay by decreased epididymis and ASG weights (Marty et al., 2001bGo). Surprisingly, KETO did not alter the age at PPS or serum T/DHT levels in the pubertal male assay at 25 mg/kg/day, levels that resulted in hormonal alterations in the intact male assay. The hormonal data are consistent with other reports that illustrate that mature animals are more sensitive than immature animals to alterations in serum hormone levels (Cook et al., 1993Go; Viguier-Martinez et al., 1983aGo,bGo). KETO was also detected in the pubertal female assay by decreased uterine weight and a delay in the age at VO (Marty et al., 1999Go), most likely the result of the broad-spectrum inhibitory effects of KETO on cytochrome P450 enzymes of the steroid biosynthetic pathway (Feldman, 1986Go).

Similar to the data for the intact male assay, both PB and PTU were detected in the pubertal assays. For the PTU experiments, the severe body weight effects that were observed in both pubertal assays (dose of 240 mg/kg/day) confounded interpretation of the results, and true endocrine effects could not be differentiated from effects due to general toxicity (Marty et al., 1999Go, 2001aGo). This was particularly problematic since only one (or two for PB) dose group was performed for each compound. Dietary restriction experiments should also be performed for both pubertal assays in order to help differentiate between effects due to general toxicity and true endocrine-mediated effects. However, in both pubertal assays, PTU increased relative thyroid weight and caused microscopic alterations of the thyroid gland consistent with a true thyroid toxicant. In the pubertal male assay, PTU also produced the typical hormonal pattern for a thyroid modulator (i.e., increased TSH and decreased T4). Most of the responses to PB in the pubertal male assay were similar to that observed in the intact male assay both in positive responses and the magnitude of the effects. For example, in both assays the increases in thyroid weight and incidence/severity of thyroid microscopic alterations were similar. However, the alterations in serum hormones were more pronounced in the intact male assay than in the pubertal male assay. Interestingly, the broad effects on reproductive hormones that were observed in the intact male assay were supported by the data from the pubertal male assay, where testis weight was decreased (Marty et al., 2001aGo). Consistent with other hormonal data from the screening assays, the PB-induced effects on serum hormones (T and DHT) were not observed in the pubertal male assay as they were in the intact male assay.

Assessment of Immune Function
KETO did not significantly alter the primary humoral immune response to SRBC. Studies with humans have also reported that KETO at therapeutic concentrations did not alter serum immunoglobulin levels, total haemolytic complement, or serum C3 or C4 levels (Van Rensburg et al., 1983Go) but reduced in vitro lymphocyte proliferation to several mitogens (Marchall et al., 1981Go). Kim and coworkers, however, indicated that oral exposure of mice to 160 mg/kg/day KETO for 14 days significantly suppressed the primary humoral immune response to SRBC (Kim et al., 1999Go). In contrast to our findings, Kim and coworkers (1999) also reported that KETO significantly decreased body weight parameters and spleen and liver weights compared to control. In our hands, KETO did not alter body weights but significantly increased both spleen and liver weights. The differences observed between our results and those of Kim and coworkers (1999) may be due to the use of a higher dose of KETO and/or different species.

Exposure to PB by gavage for 14 days did not significantly alter the primary humoral immune response to SRBC. These results are consistent with those of Deyo and coworkers. (1994) in which the exposure of mice for four days to 80 mg/kg/day PB by ip injection did not alter the primary antibody response to SRBC (Deyo et al., 1994Go). Similarly, the SRBC antibody response of mice treated ip for three days with PB was not significantly changed (Matulka et al., 1996Go). PB has also been reported not to alter lymphocyte responses to phytohaemagglutinin stimulation (Spiers et al., 1987Go). Exposure to 100 mg/kg/day KETO or PB significantly increased spleen weights. The mechanism by which KETO increased spleen weights is not known. However, PB is a known inducer of cytochrome P450 (Parkinson, 1996Go) and cytochrome P450 has been detected in the spleens of rodents (Ciaccio and DeVera, 1975Go; Griffin, 1986Go). The subchronic exposure regimen utilized in this study may have induced splenic cytochrome P450 activity with the resulant increase in spleen weight.

Conclusions
In the current study, the 15-day intact male assay definitively identified all four of the EACs examined, namely, the steroid biosynthesis inhibitors FAD and KETO and the thyroid modulators PB and PTU. The data for KETO, PB, and PTU complement the data that has been previously reported by the authors, where each compound was evaluated in the 15-day intact male assay using the ip route of compound administration (O’Connor et al., 1998aGo, 1999bGo). Overall, the sensitivity (i.e., the dose required to elicit similar magnitude responses) of the assay for detecting these three EACs was similar for both routes of administration (i.e., ip injection vs. oral gavage). The ability of the intact male assay and the pubertal male and female assays to detect the four EACs evaluated in the current report appear to be very similar. The one advantage the intact male assay has over the pubertal assays is the addition of the comprehensive hormonal assessment, which not only provides a very sensitive battery of endpoints but also allows differentiation of mode-of-action. In the intact male assay, serum hormone measurements are typically the most sensitive endpoint. Clearly, more EACs must be evaluated in all of the potential screening assays to more fully understand the strengths and limitations of each. Furthermore, these and previous data (Biegel et al., 1998Go; Ladics et al., 1998Go) with EACs suggest that the reproductive and endocrine systems and not the immune system are the primary target organs of toxicity in young adult rats. However, it is too early to conclude that the immune system is not a primary target. Additional immunotoxicological studies involving chronic and in utero/neonatal exposure to low-dose EACs need to be conducted.

The data from the current report, in addition to the > 25 compounds that have already been examined using the 15-day intact male assay, support this assay as a viable alternative to the screening model recommended by EDSTAC (EDSTAC, 1998Go; O’Connor et al., in pressGo). The authors envision the 15-day intact male assay as one component of a Tier I screening battery for identifying EACs (O’Connor et al., 2002Go). One clear benefit to a screening battery utilizing the 15-day intact male assay over the apical approach recommended by EDSTAC is that it is a mode-of-action screening assay, with the ability not only to identify potential EACs, but to determine a specific mode of action. By identifying the potential mode of action, this Tier I battery focuses the direction of future testing on critical endpoints to be included in Tier II studies that will be used to define dose-response curves and no observed adverse effect levels (NOAELs)/no observed effect levels (NOELs) for the compound. As we gain more understanding of the effects EACs have on the environment and humans, mode-of-action data will become a critical component in decisions to reduce the associated risks. Additional data will need to be developed to further define the strengths and limitations of the 15-day intact male assay versus the other potential screening models proposed by EDSTAC (EDSTAC, 1998Go).


    ACKNOWLEDGMENTS
 
We acknowledge the invaluable advice of Dr. A. Michael Kaplan. We thank Vivian Thompson, Bryan Crossley, Suzanne Craven, Charlene Smith, Michele Egan, and Denise Janney for their technical support.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (302) 366-5003. E-mail: john.c.oconnor{at}usa.dupont.com. Back


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