Evaluation of the Primary Humoral Immune Response Following Exposure of Male Rats to 17β-Estradiol or Flutamide for 15 Days

TOXICOLOGICAL SCIENCES ARTICLE NO.

46, 75– 82 (1998)

TX982554

Evaluation of the Primary Humoral Immune Response Following Exposure of Male Rats to 17b-Estradiol or Flutamide for 15 Days Gregory S. Ladics, Charlene Smith, Susan C. Nicastro, Scott E. Loveless, Jon C. Cook, and John C. O’Connor DuPont Haskell Laboratory for Toxicology and Industrial Medicine, P.O. Box 50, Newark, Delaware 19714 Received April 23, 1998; accepted July 29, 1998

mg/kg/day FLUT based on the increased relative thymus weights that were judged to be compound-related. In the Tier I male battery, responses to FLUT included decreased absolute epididymis and relative accessory sex gland unit weights and hormonal alterations (increased serum T, DHT, E2, and LH, and decreased follicle stimulating hormone levels). The LOAEL for the reproductive indices was 0.25 mg/kg/day FLUT based on the hormonal alterations seen at this level; no NOAEL was established. Based on these data, the reproductive and not the immune system appears to be the primary target organ of toxicity in young adult male rats treated with either 17b-E2 or FLUT. © 1998 Society of Toxicology. Key Words: humoral immune function; endocrine; 17b-estradiol; flutamide; rat.

Evaluation of the Primary Humoral Immune Response Following Exposure of Male Rats to 17b-Estradiol or Flutamide for 15 Days. Ladics, G. S., Smith, C., Nicastro, S. C., Loveless, S. E., Cook, J. C., and O’Connor, J. C. (1998). Toxicol. Sci. 46, 75– 82. There is a concern that certain industrial chemicals found in the environment may mimic or antagonize endogenous hormones and adversely affect the endocrine as well as the immune system. The objective of this study was to determine if exposure of Crl:CD (SD)BR male rats to 17b-estradiol (17b-E2), an estrogen receptor agonist, or flutamide (FLUT), an androgen receptor antagonist, would significantly alter the primary IgM humoral immune response to sheep red blood cells (SRBC). This study was conducted in the context of a male in vivo Tier I battery designed to identify endocrine-active compounds (EACs). The Tier I male battery consists of organ weights coupled with a comprehensive hormonal assessment. Rats were dosed by the intraperitoneal route for 15 days with vehicle or 0.001, 0.0025, 0.0075, or 0.050 mg/kg/day 17b-E2 or 0.25, 1, 5, or 20 mg/kg/day FLUT. Six days prior to termination, selected rats were injected intravenously with SRBC for assessment of humoral immune function. Spleen cell number and spleen and thymus weights were obtained. Serum was analyzed for anti-SRBC IgM antibody by using an enzyme-linked immunosorbent assay. At 0.050 mg/kg/day 17b-E2, mean final body and absolute thymus weights were significantly decreased to 84 and 65% of control, respectively. 17b-E2 did not significantly alter spleen weight, spleen cell number, or the primary IgM humoral immune response to SRBC. The no-observed-adverse-effect level (NOAEL) for immune system alteration was 0.050 mg/kg/day 17b-E2 since the decrease in absolute thymus weight was judged to be secondary to the decrements in body weight. In the Tier I male battery, responses to 17b-E2 included decreased absolute testis and epididymis weights, decreased relative accessory sex gland unit weights, hormonal alterations (decreased serum testosterone (T), dihydrotestosterone (DHT), and luteinizing hormone (LH), and increased serum prolactin and E2 levels). The lowestobserved-adverse-effect level (LOAEL) for the reproductive indices was 0.001 mg/kg/day 17b-E2 based on the hormonal alterations seen at this level; no NOAEL was established. Exposure to FLUT did not significantly alter mean final body, spleen, or absolute thymus weights, spleen cell number, or the primary IgM humoral immune response to SRBC. A significant increase (118% of control) in relative thymus weight was observed at 20 mg/kg/ day FLUT. The NOAEL for immune system alteration was 5

There is an increasing concern that certain persistent, biocumulative agricultural products and industrial chemicals found in the environment may mimic or antagonize endogenous hormones in wildlife (reviewed in Colborn et al., 1993; Ankley et al., 1997) and humans (Birnbaum, 1994; Kavlock et al., 1996). A number of adverse health effects have been observed in laboratory animals exposed to endocrine-active compounds (EACs). Such EACs include diethylstilbestrol (DES) (McLachlan, 1993), polychlorinated biphenyls (PCBs) (Juarez et al., 1994), dioxins (Rier et al., 1993), and organochlorine compounds such as 1,1,1-trichloro-2,2bis(p-chlorophenyl)ethane (DDT) and its metabolites (Thomas, 1975; Brown and Lamartiniere, 1995), chlordecone (Gellert and Wilson, 1979), and methoxychlor (Gray et al., 1989; Cooper et al., 1986). Some investigators have hypothesized that reported increases in certain cancers (mammary gland, prostate, testicular) and developmental abnormalities (male urogenital defects), as well as alleged decreases in sperm counts in humans, may be attributed, in part, to exposure to EACs in the environment (Birnbaum, 1994). Except for a few examples, such as human exposure to DES (Herbst et al., 1971; Gill et al., 1979) or wildlife exposure to DDT (Bitman and Cecil, 1970), a causal relationship between specific EACs and adverse health effects on humans, fish, and wildlife has not been well established (Safe, 1995; Crisp et al., 1997). In general, there has been inadequate exposure assessment of EACs for many of the effects that have been reported in both wildlife and humans. Despite the lack of a well-established link between EACs 75

1096-6080/98 $25.00 Copyright © 1998 by the Society of Toxicology. All rights of reproduction in any form reserved.

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and adverse health effects, EACs have received a great deal of attention within the scientific community, the government, and the popular press. In order to address the proposed hypothesis of EAC exposure and adverse health effects, the United States Environmental Protection Agency (EPA) sponsored a workshop in April 1995 entitled, “Research Needs for the Risk Assessment of Health and Environment Effects of Endocrine Disruptors” (Kavlock et al., 1996). The objective of the workshop was to identify research gaps for EACs and to prioritize future research activities. Although the highest priorities for research on EACs were determined to be reproductive toxicology and carcinogenesis, Kavlock and co-workers (1996) indicated that “the incidence of effects on the nervous and immune systems may be underestimated at this point because of the incomplete characterization of the biologic effects of endocrine disruptors.” In February 1997, the Risk Assessment Forum of the EPA prepared a document entitled, “Special Report on Environmental Endocrine Disruption: An Effects Assessment and Analysis” to evaluate the strengths and weaknesses of currently available data on EACs (Crisp et al., 1997). The principal health effects reported following exposure to EACs included carcinogenesis, reproductive toxicity, neurotoxicity, as well as immunotoxicity. Increasingly, investigators have demonstrated a highly complex bidirectional interrelationship between the immune and neuroendocrine systems (reviewed in Fuchs and Sanders, 1994; Besedovsky and Del Rey, 1996; Chryssikopoulos, 1997; Tomaszewska and Przekop, 1997). Historically, most interactions between the immune and neuroendocrine systems have been associated with the suppressive effects of glucocorticoid hormones on the immune system (Claman, 1972; Sobhon and Jivasattham, 1974; Fauci and Dale, 1974; Hrushesky, 1994). Recent data indicate, however, that products of the immune system (i.e., cytokines) affect neuroendocrine functions (Koenig, 1991; Roy, 1994; Rivest and Laflamme, 1995). Immune cells have also been found to produce various peptide and protein hormones such as growth hormone (GH), luteinizing hormone (LH), prolactin (PRL), adrenocorticotropin (ACTH), and thyrotropin-stimulating hormone (TSH) (Blalock, 1989; Gaillard, 1995). Additionally, various hormone receptors have been found on the cells of the immune system and a number of hormones have been reported to enhance (e.g., GH, TSH, and PRL), attenuate (e.g., gonadal steroids and endogenous opioids), or suppress (e.g., glucocorticoids and ACTH) responses of the immune system (Gaillard, 1995; Weigent and Blalock, 1987). Since it is beyond the scope of this article to review the relationship(s) between the immune and endocrine systems, the reader is referred to the several review articles listed above which have addressed this topic. The objective of this study was to determine if exposure of Crl:CD (SD)BR male rats to 17b-estradiol (17b-E2), an estrogen receptor agonist, or flutamide (FLUT), an androgen receptor antagonist, would significantly alter a number of immune system parameters. 17b-E2 and FLUT were used as bench-

mark compounds to help predict potentially adverse responses to the immune system following exposure to less-well-characterized environmental chemicals with estrogen-like or antiandrogen-like activity, respectively. Spleen and thymus weights, spleen cell number, and the primary IgM humoral immune response to sheep red blood cells (SRBC) were evaluated. This study was conducted using a satellite group of rats from an ongoing validation of a Tier I male in vivo battery designed to identify EACs (Cook et al., 1997). Data on the effects of 17b-E2 on the reproductive/endocrine system have been published previously (O’Connor et al., 1998a). The effects of FLUT on the reproductive/endocrine system are reported in the accompanying paper (O’Connor et al., 1998b). The Tier I male battery consists of organ weights coupled with microscopic evaluations and a comprehensive hormonal assessment (Cook et al., 1997). It is designed to identify compounds that have the potential to act as agonists or antagonists to the estrogen, androgen, progesterone, or dopamine receptors, steroid biosynthesis inhibitors (aromatase, 5a-reductase, and testosterone (T) biosynthesis), or compounds that alter thyroid function (Cook et al., 1997; O’Connor et al., 1998a,b). The effects of 17b-E2 and FLUT on the immune system were compared to those seen in the reproductive/endocrine system to determine which is the primary target organ of toxicity in young adult male rats. MATERIALS AND METHODS Test materials. Materials were obtained from the following manufacturers: 17b-E2 (98 –100% purity) and FLUT (100% purity), Sigma Chemical Company (St. Louis, MO); Certified, Irradiated Rodent Diet 5002 PMI Feeds, Inc. (St. Louis, MO); methylcellulose, Fisher Scientific (Springfield, NJ); sterile SRBC in Alsevers solution was obtained from Rockland (Gilbertsville, PA). Hanks’ balanced salt solution (HBSS) and 1.0 M Hepes were purchased from GIBCO Laboratories (Grand Island, NY). Test species. Male Crl:CD BR rats were acquired from Charles River Laboratories, Inc. (Raleigh, NC). Rats were approximately 63 days old upon receipt. Upon arrival, rats were housed in stainless steel, wire-mesh cages suspended above cage boards and were fed irradiated PMI Feeds, Inc., Certified Rodent Diet 5002 and provided with tap water (United Water Delaware) ad libitum. Animal rooms were maintained on a 12-h light/dark cycle (fluorescent light), a temperature of 23 6 2°C, and a relatively humidity of 50 6 10%. After a quarantine period of approximately 1 week, rats that displayed adequate weight gain and freedom from clinical signs were divided by computerized, stratified randomization into four (FLUT treatment groups) or five (17b-E2 treatment groups) groups of eight rats so that there were no statistically significant differences among group body weight means. Rats were clinically normal and free of antibody titers to pathogenic murine viruses and mycoplasma and free of pathogenic endo- and ectoparasites and bacteria. Study design. All rats were weighed daily and cageside examinations were done to detect moribund or dead rats. At each weighing, rats were individually handled and examined for abnormal behavior or appearance. 17b-E2 and FLUT were prepared in 0.25% methylcellulose vehicle and administered by intraperitoneal (ip) injection at approximately 0900 h daily. All dosing solutions were made within 3 days of study start and were prepared weekly for the duration of the study. Dosing solutions were stored at 4°C. Rats were dosed for 15 days with vehicle, or 0.001, 0.0025, 0.0075, or 0.050 mg/kg/day 17b-E2, or 0.25, 1, 5, or 20 mg/kg/day FLUT. The dose volume was 2.0 ml/kg body weight. Six days prior to study termination, rats designated for assessment of the primary humoral immune response (8/dose group) were injected into a tail

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TABLE 1 Immune System Assessment: Final Body and Organ Weights and Total Spleen Cell Number

Compound 17b-Estradiol

Flutamide

Dosage (mg/kg/day)

Final body (grams)

Final body (% of control)

Spleen (grams)

Spleen (% body weight)

Thymus (grams)

Thymus (% body weight)

Total spleen cell number (3108)

0 0.001 0.0025 0.0075 0.050 0 1 5 20

417 6 15a 389 6 11 389 6 8 391 6 7 349 6 11* 409 6 9 377 6 8 376 6 11 393 6 13

100 93 93 94 84 100 92 92 96

1.1 6 0.09 1.1 6 0.09 1.1 6 0.06 1.0 6 0.04 0.97 6 0.09 0.97 6 0.07 1.0 6 0.04 1.0 6 0.08 0.92 6 0.05

0.24 6 0.01 0.27 6 0.02 0.28 6 0.01 0.27 6 0.01 0.28 6 0.02 0.24 6 0.01 0.28 6 0.01 0.27 6 0.02 0.23 6 0.01

0.55 6 0.04 0.49 6 0.03 0.54 6 0.04 0.54 6 0.02 0.36 6 0.04* 0.44 6 0.04 0.40 6 0.04 0.43 6 0.05 0.52 6 0.04

0.13 6 0.01 0.13 6 0.01 0.13 6 0.01 0.14 6 0.00 0.10 6 0.01 0.11 6 0.01 0.10 6 0.01 0.11 6 0.01 0.13 6 0.01*

5.2 6 0.55 3.8 6 0.55 5.0 6 0.73 4.7 6 0.69 4.3 6 0.63 1.9 6 0.39 2.3 6 0.37 3.3 6 0.68* 2.7 6 0.57

Means 6 standard error. * Significantly different (P # 0.05) from control by Jonckheere’s test for trend.

a

vein with 0.5 ml of 4 3 108 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). Animals were euthanized by carbon dioxide anesthesia and exsanguination on the afternoon of test day 115. Immune system assessment. On the morning of test day 115, rats were injected with 17b-E2 or FLUT approximately 2 h prior to termination. Between 1300 and 1500 h, rats were euthanized, the spleen and thymus were removed and weighed, and relative (to body weight) organ weights calculated. A single-cell suspension was prepared from half of the spleen in HBSS containing 1.0 M Hepes (GIBCO) by first cutting the spleen halves into several pieces and then placing the pieces in a Stomacher Lab Blender (Seward Medical Ltd., London, UK). The splenic cell suspension was placed into a conical tube and inverted, and debris allowed to settle for 5 min while the tube was kept on ice. 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. Serum samples were analyzed for SRBC-specific IgM antibody using an ELISA as described by Temple and co-workers (1993). The SRBC-specific serum IgM antibody titers were reported as log2 to normalize the data. Statistical analyses. Mean final body and organ weights and SRBCspecific IgM levels were analyzed using Jonckheere’s trend test in a step-down manner to determine which dose groups were significantly different from the control groups (Marcus et al., 1976; Hochberg and Tamhene, 1987). If a significant dose–response trend was detected, data from the top dose group were excluded and the test repeated until no significant trend was detected. Significance was judged at P # 0.05.

Comparison of Body and Lymphoid Organ Weights, Spleen Cell Number, and the Primary Humoral Immune Response to SRBC of Control and FLUT-Treated Animals Exposure to FLUT did not significantly alter mean final body, absolute or relative spleen, or absolute thymus weights. A statistically significant increase (118% of control) in relative thymus weight was observed at 20 mg/kg/ day FLUT. Additionally, 5 mg/kg/day FLUT increased spleen cell number to 173% of control (Table 1). The primary humoral immune response to SRBC was not significantly altered by FLUT (Fig. 2).

RESULTS

Comparison of Body and Lymphoid Organ Weights, Spleen Cell Number, and the Primary Humoral Immune Response to SRBC of Control and 17b-E2-Treated Animals Exposure to 0.050 mg/kg/day 17b-E2 decreased mean final body and absolute thymus weights to 84 and 65% of control, respectively. Absolute and relative spleen weights, spleen cell number, and relative thymus weights were not significantly altered by 17b-E2 (Table 1). The primary humoral immune response to SRBC was not significantly altered by 17b-E2 (Fig. 1).

FIG. 1. Assessment of the primary IgM antibody response to SRBC following exposure to 17b-E2. Male rats (8/group) were exposed to a daily intraperitoneal dose of 0, 0.001, 0.0025, 0.0075, or 0.050 mg/kg 17b-E2 for 15 days. Animals received SRBC by intravenous injection 6 days prior to termination for assessment of the humoral immune response. At termination, serum was obtained and analyzed for IgM antibody specific for SRBC by an ELISA as described under Materials and Methods. Results are reported as the log2 of the SRBC-specific serum IgM titers.

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FIG. 2. Assessment of the primary IgM antibody response to SRBC following exposure to FLUT. Male rats (8/group) were exposed to a daily intraperitoneal dose of 0, 1, 5, or 20 mg/kg FLUT for 15 days. Animals received SRBC by intravenous injection 6 days prior to sacrifice for assessment of the humoral immune response. At sacrifice, serum was obtained and analyzed for IgM antibody specific for SRBC by an ELISA as described under Materials and Methods. Results are reported as the log2 of the SRBC-specific serum IgM titers.

DISCUSSION

Although there have been a number of studies evaluating the effects of EACs on the immune system, few have examined the effects of EACs on the immune, endocrine, and reproductive systems within the context of the same study. Furthermore, many of the studies examining EACs and the immune system have involved relatively short-term exposures to high (i.e., supraphysiological or pharmacological) dosages of compound. For instance, Seaman and Gindhart (1979) reported that sustained high serum levels of 17b-E2 in mice (93 normal) decreased natural killer cell activity, while Kita and co-workers (1989) found that high serum 17b-E2 (103 normal) decreased the host resistance of mice. Treatment with supraphysiological doses of sex steroid hormones has been reported to produce immunotoxicity (Grossman, 1984; Ahmed et al., 1985). Exposure of mice to pharmacological levels of DES and 17b-E2 produced thymic atrophy and bone marrow hypocellularity (Barnes et al., 1983; Luster et al., 1984a,b). A number of potential EACs that are environmental pollutants such as DDT, chlordecone, PCB mixtures, and 2,3,7,8-tetrachlordibenzo-pdioxin (TCDD) have also been reported to produce immunotoxicity (Kirkvliet, 1984; Smialowicz et al., 1985; Thomas and Faith, 1985; Banerjee et al., 1986). Similar to our data, Chapin and co-workers (1997) found that the primary alteration of perinatal/juvenile exposure to the estrogenic pesticide methoxychlor was to the reproductive and not the immune system. The objective of this study was to evaluate the effects of low-dose 17b-E2 or FLUT exposure on a number of immune system parameters in the context of our Tier I male in vivo

battery (Cook et al., 1997; O’Connor et al., 1998a,b). By doing so, the immune, endocrine, and reproductive systems have been examined within the context of the same study design. The reproductive/endocrine effects of 17b-E2 (O’Connor et al., 1998a) and FLUT (O’Connor et al., 1998b) are reported in detail elsewhere, but the no-observed-adverse-effect levels (NOAEL) have been summarized (Table 2) to facilitate comparisons with immune effects described in this report. Results of the current study indicate that low-dose exposure to 17b-E2 did not adversely affect the immune system. At 0.050 mg/kg, 17b-E2 significantly decreased absolute thymus weights. Estrogen-induced thymic atrophy has been well documented in the literature (Greenman et al., 1977; Kalland et al., 1978; Boorman et al., 1980). The decrease in thymus weight observed in our study, however, was accompanied by a significant decrease in body weight. Although the decreased thymus weight may be due, in part, to a direct effect of 17b-E2 on the thymus, the significant alteration in body weight observed at 0.050 mg/kg 17b-E2 suggests a nonspecific toxic effect. In contrast to our findings, Meyers and Petersen (1985) reported that ip administration of 0.075 or 0.750 mg/kg/day estradiol 3 days prior to antigen administration increased IgM antibody titers during a primary antibody response. The differences observed between our results and those of Meyers and Petersen (1985) may be due to the use of higher dosages of estradiol, different rat strains, and/or differences in the route, dose, and timing of antigen administration. High-dose 17b-E2 has been reported to increase antibody responses in rodents (reviewed in Forsberg, 1984; Schuurs and Verheul, 1990). Biegel and co-workers (1998) fed ad libitum rats a diet that contained 0.05–50 ppm of 17b-E2 for 90 days. Splenic cell counts, spleen weight, and absolute numbers of splenic lymphocyte populations were decreased by 17b-E2 in both males and females. The 17b-E2-induced decreases in spleen weight and cell numbers were attributed to an adjustment in regulated body weight (Biegel et al., 1998). Both synthetic and natural estrogens decrease the growth and body weight of rodents, resulting in decreases in food intake and an adjustment in regulated body weight (Coling and Herberg, 1982; Finkelstein, 1986; Hart, 1990). It is important to note that significant effects on body weights were observed in our studies at relatively low doses of 17b-E2 [0.050 mg/kg/day for 15 days ip (O’Connor et al., 1998a) or 2.5 ppm in diet (mean daily intake of 0.139 and 0.173 mg/kg/day for males and females, respectively) for 90 days (Biegel et al., 1998)]. Our data suggest that immunotoxicological studies in rodents with estrogenic compounds need to be carefully interpreted in the context of significant body weight changes in order to distinguish between indirect immunotoxicity due to overt toxicity and direct impairment of the immune system. Exposure to 20 mg/kg/day FLUT significantly increased relative thymus weight to 118% of control. Absolute thymus weight was also increased to 118% of control, albeit nonsignificantly. The occurrence of androgen receptors in the thymus

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TABLE 2 Summary of the Tier I Male Battery: The Effect of 17b-Estradiol and Flutamide Treatment on Reproductive Indices 17b-Estradiola

Flutamideb

Endpoint

Response

NOAELc (mg/kg/day)

Response

NOAELc (mg/kg/day)

Final body weight Organ weights Liver weight (% body weight) Testes weight (grams) Accessory sex gland unit (% body weight) Epididymides (grams) Serum hormones Testosterone (ng/ml) Estradiol (pg/ml) Dihydrotestosterone (pg/ml) Prolactin (ng/ml) Follicle-stimulating hormone (ng/ml) Luteinizing hormone (ng/ml)

2 (88)d

0.0075

2 (96)

—

— 2 2 2

(99) (79) (32) (66)

— 0.0075 0.0025 0.0075

1 2 2 2

(107) (106) (47) (78)

5.0 — 1.0 5.0

2 1 2 1 — 2

(3) (337) (30) (999) (88) (70)

0.001e 0.0075 0.001e 0.001e — 0.001e

1 1 1 2 1 1

(464) (163) (343) (122) (203) (255)

0.25e 5.0 0.25e — 1.0 1.0

a

O’Connor et al. (1998a). O’Connor et al. (1998b). c No-observed-adverse-effect level. d Compound increased (1), decreased (2), or did not affect (—) the end point response. The percentage of control is in parentheses and represents the greatest change observed for the end point. e These values represent lowest-observed-adverse-effect levels. b

has been demonstrated (McCruden and Stimson, 1981; Kumar et al., 1995). Furthermore, exposure to various androgenic hormones has been reported to produce thymolysis (Grossman, 1984; Kumar et al., 1995), while administration of FLUT significantly blocked the thymolytic effect of androgens (Kumar et al., 1995). Perhaps by inhibiting androgen-induced thymolysis, FLUT allows for the stimulation of thymic growth. Interestingly, hypertrophy of the thymus has been observed following gonadectomy (Sobhon and Jirasattham, 1974; Comsa et al., 1982; Grossman, 1984). Exposure to 5 mg/kg/ day FLUT significantly increased spleen cell number. The effect of FLUT on spleen cell number, however, was not observed in the context of a dose–response. Additionally, spleen weight was not significantly altered. Therefore, the biological relevance of this effect is not clear. When the NOAELs for the reproductive/endocrine effects (Table 2) (O’Connor et al., 1998a,b) are compared with the immune responses, the data suggest that the reproductive system was the primary target of 17b-E2 and FLUT rather than the immune system. For 17b-E2, the NOAEL for immune system alteration was 0.050 mg/kg since no compound-related findings were observed. In contrast, no NOAEL was established for the reproductive indices with 17b-E2 (Table 2). The lowest-observed-adverse-effect level (LOAEL) was 0.001 mg/ kg/day based on the statistically significant alterations in serum hormone levels (T, DHT, LH, and PRL). For FLUT, the NOAEL for immune function alteration was 5 mg/kg based on the increased relative thymus weight. In contrast, no NOAEL was established for the reproductive indices with FLUT (Table

2). The LOAEL was 0.25 mg/kg/day based on the statistically significant alterations in serum hormone levels (T and DHT). Although our data suggest that the reproductive and not the immune system is the primary target organ of toxicity in young adult male and female rats following low-dose exposure to EACs up to 90 days (i.e., 17b-E2), it is premature to conclude that the immune system is not a primary target organ of toxicity. There are no published studies that examine the potential immunotoxicity of low-dose chronic exposure to EACs. The need for these data is particularly important due to the highly lipophilic, persistant nature of many manmade EACs found in the environment and their subsequent ability to bioaccumulate. Studies are needed to assess the effects of lowdose chronic exposures to EACs on the various components of the immune system: humoral, cell-mediated, and innate immunity. In addition, the developing immune system of the fetus or progeny may be substantially more susceptible to exposure to environmental immunotoxicants than adults (Blair, 1981; reviewed by Holladay and Luster, 1994, 1996); however, limited data are available. DES produced immunotoxicity in adult mice at mg/kg doses (Luster et al., 1980; Holsapple et al., 1983), while pre- or neonatal exposure of mice to mg/kg doses of DES resulted in immunotoxicity (Kalland and Forsberg, 1978; Kalland et al., 1979). In addition, DES-induced immunotoxicity in adults is reversible (Holsapple et al., 1983), while DES exposure during prenatal or early postnatal development resulted in long-term, often permanent, changes in immune responses (Luster et al., 1979; Kalland et al., 1979; Kalland,

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1980; Ways et al., 1980). Similarly, prenatal exposure of mice to chlordane resulted in persistant immunotoxicity (SpykerCranmer et al., 1982; Barnett et al., 1985), while exposure of adults to chlordane did not produce significant alterations in immunocompetence (Johnson et al., 1986). Maternal exposure to mg/kg doses of TCDD or DES during gestation resulted in altered thymocyte differentiation in fetal mice (Holladay et al., 1991, 1993). Prenatal T exposure of NZB/NZW mice altered splenic T-cell subsets, mitogen-induced proliferation, and the subsequent expression of an autoimmune disease (Walker et al., 1996). In summary, these and previous data (Biegel et al., 1998) suggest that the reproductive and not the immune system is the primary target organ of toxicity in young adult male and female rats. However, additional immunotoxicological studies involving chronic and in utero/neonatal exposure to low-dose EACs need to be conducted. Furthermore, the immune, endocrine, and reproductive systems should be examined in the context of the same study design to determine which is the primary target organ of toxicity for other EACs. ACKNOWLEDGMENTS The authors thank Vivian Thompson, Bryan Crossley, Stephen Novak, Christine Glatt, Suzanne Craven, and Denise Janney for their technical support.

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