Chemical Industry Institute of Toxicology, 6 Davis Drive, Research Triangle Park, North Carolina 277092137
Received August 16, 1999; accepted November 19, 1999
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ABSTRACT |
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Key Words: methyl tert-butyl ether (MTBE); Leydig cell tumors; hormonal feedback; hypothalamus-pituitary-testicular axis (HPT).
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INTRODUCTION |
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MTBE, a nongenotoxic chemical carcinogen (International Program on Chemical Safety, 1998), may alter endocrine feedback mechanisms, which under chronic exposure conditions may result in Leydig cell tumorigenesis. Some nonDNA-reactive compounds have been shown to perturb the hormones that regulate the HPT axis, resulting in increased serum luteinizing hormone (LH) levels and induction of LCT in rats (Clegg et al., 1997
). A sustained increase in LH, the primary trophic hormone regulating testosterone synthesis in Leydig cells, is thought to be mitogenic for Leydig cells (Christensen and Peacock, 1980
); therefore, investigation of LH levels is warranted in this study.
MTBE has also been shown to cause adverse effects in endocrine tissues in female mice. A decreased incidence of uterine endometrial cystic hyperplasia was demonstrated in CD-1 mice exposed with 8000 ppm MTBE for 18 months (Bird et al., 1997). MTBE also caused a significant decrease in uterine weight in B6C3F1 mice exposed to 7814 ppm MTBE for 3 or 21 days and a significant increase in estrogen metabolism of mice gavaged with 1800 mg MTBE/kg/day for 3 days (Moser et al., 1996
). Following chronic exposure to 8000 ppm MTBE, female B6C3F1 mice exhibited decreased uterine, ovary, and pituitary weights, and increases in estrous cycle length (Moser et al., 1998
). These responses may provide a basis for assessing the ability of MTBE to alter the endocrine system in rats.
As the mode of induction for MTBE-induced LCT in rats is unknown, risk assessment estimations for MTBE by the United States Environmental Protection Agency assume that the increased incidence of tumors is of concern for humans. This concern will persist unless a species-specific mode of action for MTBE-induced LCT or other endocrine effects not operating in humans at anticipated levels of exposure can be elucidated. A potential mechanism for MTBE-induced LCT by an endocrine modulator needs to be fully characterized, as a significant number of humans are exposed to MTBE either via air or through contamination of drinking water supplies (National Science and Toxicology Council, 1997).
The objective of this study was to determine whether MTBE functions as an endocrine-active compound in male rats, particularly in relation to the potential of MTBE to alter the functions of the HPT axis. Persistent alterations in the endocrine system due to MTBE administration could contribute to the formation of LCT observed in chronic studies (Belpoggi et al., 1995). The current study design was constructed to evaluate several, but not all, modes of action that would affect the hormonal control of Leydig cell growth. This study was based on a previous 15-day, intact adult male assay that investigated the following modes of action: estrogen, androgen, progesterone, or dopamine receptor agonism or antagonism; steroid biosynthesis inhibition (testosterone biosynthesis, 5
-reductase, and aromatase); and alteration of thyroid function (Cook et al., 1997b
; O'Connor et al., 1998b
). These studies using nongenotoxic chemicals known to cause LCT demonstrated that a 15-day exposure would result in alterations in hormones critical to regulation of the HPT axis. Because a previous study demonstrated a decrease in plasma testosterone in male Sprague-Dawley rats treated orally with 800 mg MTBE/kg/day for 28 days (Day et al., 1998
), a 28-day MTBE treatment was used along with a 15-day treatment to compare possible length of exposure differences. These data presented in this manuscript indicate that MTBE alters endocrine-sensitive parameters in adult male rats. However, these responses were mild and primarily observed at high doses that exceed the concentrations causing LCT in male rats and far exceed those encountered in human exposures.
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MATERIALS AND METHODS |
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Animals.
Seven-week-old male Sprague-Dawley rats were obtained from Charles River Laboratories (Raleigh, NC) and allowed to acclimate for 14 days prior to use. Rats were randomized by weight (n = 15/dose group) and housed individually in stainless steel and glass 1-m3 Hazelton H1000 inhalation chambers (Lab Products, Inc., Maywood, NJ). Because MTBE has a high vapor pressure and is readily exhaled, one H1000 chamber was used for each oral dose level to prevent any possible cross- contamination of exposure groups that could occur from expired MTBE. Total chamber airflow through each H1000 chamber was maintained at approximately 225 l/min (1215 air changes/h), with the chamber temperature and relative humidity controlled to 23 ± 1°C and 3070%, respectively. All environmental parameters were recorded every 30 min for a 24-h period by an Infinity Building Automation System (Andover Controls Corporation, Andover, MA). Rats received pelleted food (NIH-07, Zeigler Bros., Gardners, PA) and deionized, filter-purified water ad libitum. A 12-h lightdark cycle was maintained. These studies were performed under National Research Council guidelines (1996) and were approved by the Institutional Animal Care and Use Committee of the Chemical Industry Institute of Toxicology (CIIT).
Study design.
Three experiments were conducted. In Experiment A, rats (231383 g) were dosed with 250, 500, 1000, or 1500 mg MTBE/kg/day or vehicle (corn oil) by gavage for 28 consecutive days using a dosing volume of 3 ml/kg body weight. In Experiment B, rats were treated with 1500 mg MTBE/kg/day or vehicle for 15 consecutive days. This study was included to compare hormonal changes at 15 and 28 days. Experiment A and B were initiated on the same day. If similar responses were observed in Experiment A and B, then a dose-response study would be conducted at 15 days and further investigations would use the shorter treatment period in their design. Based on the results from the initial 15- and 28-day studies, an additional dose-response study (Experiment C) was conducted in which rats (258324 g) were dosed with 250, 500, or 1000 mg MTBE/kg/day or vehicle for 15 days, as described above. The 1500-mg MTBE/kg/day dose group was omitted due to adverse clinical effects of MTBE observed in the 15- and 28-day studies.
The MTBE doses were selected based on previous oral toxicity studies, including a 28-day study in Fischer 344 rats (IIT Research Institute, 1992), a 14- and 90-day study in Sprague-Dawley rats (Robinson et al., 1990
), and a chronic study in Sprague-Dawley rats (Belpoggi et al., 1995
). The dosing schedule was based on a previous study in Sprague-Dawley rats in which a decrease in plasma testosterone after administration of 800 mg MTBE/kg for 28 days was observed (Day et al., 1998
). Body weights were collected every 34 days throughout the study and on the final day of dosing. Animals were sacrificed by decapitation between 9:00 A.M. and 12:00 P.M. to limit the effects of interanimal circadian rhythm variations.
Additional male rats (234348 g) were injected subcutaneously twice daily for 10 days with either 10 mg flutamide or vehicle (1,2-propanediol) (Kassim et al., 1997; van der Schoot, 1992
). The total injection volume was 0.25 ml (n = 10 rats/dose group). Because flutamide treatment increases testosterone and LH secretion, both serum and testicular interstitial fluid (TIF) measurements from these animals were used to verify that changes in hormone concentrations could be measured.
Radioimmunoassays.
Rats were sacrificed by decapitation 1 h following the last dose, trunk blood was collected, and serum was prepared by centrifugation (600 x g, 30 min, 10°C). The left testis was removed, and four small incisions were made around the caudal pole of the tunica. TIF was collected under slow centrifugation for 1 h (100 x g) at 10°C (Rehnberg, 1993). Serum and TIF were frozen at 80°C until analysis.
Testosterone, 17ß-estradiol, dihydrotestosterone (DHT), triiodothyronine (T3), and thyroxine (T4) radioimmunoassay kits were purchased from Diagnostics Systems Laboratories, Inc. (Webster, TX). Rat luteinizing hormone (LH), rat follicle-stimulating hormone (FSH), rat thyroid-stimulating hormone (TSH), and rat prolactin radioimmunoassay kits were purchased from Amersham Life Science (Buckinghamshire, England). All these hormones were measured in the serum, and only testosterone was measured in the TIF (Experiments A and B). In flutamide-treated animals, testosterone was measured in the serum and TIF, whereas LH was measured only in the serum. In Experiment C, serum testosterone, LH, prolactin, and TIF testosterone were measured. Assays were performed according to the manufacturer's directions.
Histologic evaluation.
Liver, epididymides, testes, kidneys, dorsal lateral prostate, ventral prostate, pituitary gland, adrenal glands, and seminal vesicles (seminal vesicles and coagulating glands plus fluids) were removed and weighed. The right testis was fixed with Bouin's fixative for 6 h, nicked, and returned to Bouin's fixative for 18 h. The right epididymidis was fixed with Bouin's solution for 24 h. The liver, right kidney, and both adrenal glands were fixed in 10% neutral-buffered formalin for 72 h. All tissues were washed and maintained in 50% ethanol for 48 h following fixation and then stored in 70% ethanol for 1 week until they were embedded in paraffin.
Hematoxylin-eosin (H&E)-stained sections of liver, right testis, right kidney, and adrenal glands were examined for lesions using a grading scale from 0 to 4. Scores were assigned as follows: 0, absence of lesions; 1, minimal lesions; 2, mild lesions; 3, moderate lesions; and 4, marked to severe lesions.
Statistical analysis.
Body weights were evaluated using a repeated-measures analysis. Organ weights, serum, and TIF hormone levels were analyzed by a one-way analysis of variance (ANOVA). Dunnett's test was used to determine significant differences between treatment and control. Trend analyses were performed using Jonckheere's test for trend. The significance level for all analyses was 0.05.
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RESULTS |
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Body and Organ Weights
A statistically significant reduction in the rate of body weight gain (8893% of control) was observed in the 1500 mg MTBE/kg/day rats starting on treatment day 15 and persisting through 28 days (Fig. 1). No statistical differences in body weight were observed in the other groups, although the group averages for MTBE-treated rats were less than the control group. No statistically significant differences in body weights were observed in rats treated for 15 days (data not shown). Rats treated with 1500 mg MTBE/kg/day exhibited temporary labored breathing after dosing and appeared dehydrated throughout the study, as indicated by a lack of skin elasticity; however, no deaths were attributed to MTBE toxicity. Accidental deaths, which were primarily confined to the 1500 mg MTBE/kg/day dose group and occurred immediately following dosing, were attributed to the difficulty administering MTBE, a noxious irritating chemical. Autopsies confirmed red mottled lungs and punctured esophagi in all cases of death, which included six rats in the 28-day study (Experiment A) and four rats in the 15-day study (Experiment B). Rats found dead or moribund were excluded during data analysis.
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DISCUSSION |
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In the current study, MTBE caused body and organ weight changes in rats treated with 1500 mg MTBE/kg/day. Depression in body weight gain and increases in kidney weights in the 15-day study were consistent with a previous 2-week oral gavage study in Sprague-Dawley rats (Robinson et al., 1990). Kidney histopathology demonstrated protein droplet nephropathy in MTBE-treated rats, which is consistent with the increase in kidney weight and the previously reported study (Robinson et al. 1990
). In an MTBE inhalation study,
2u-globulin accumulation was identified in protein droplets in the kidney of Fischer 344 rats (Prescott-Mathews et al., 1997
). The only other histologic abnormality observed was centrilobular hypertrophy in the liver. This hypertrophic response is consistent with the increased liver weight, which might indicate induction of cytochrome P450s, UDP-glucuronyltransferase, or peroxisomes (Cook et al., 1997b
). Centrilobular hypertrophy was previously observed in a chronic 2-year MTBE inhalation study using Fischer 344 rats and was attributed to adaptive responses associated with the increased metabolic capacity of the liver and not toxicity (Bird et al., 1997
). Testes, adrenal glands, and pituitary gland weight increases were confined to animals treated with 1500 mg MTBE/kg/day. Slight increases in testis weight at 714 and 1071 mg MTBE/kg/day were observed in the 14- but not 90-day gavage study, and no testicular histopathology was reported (Robinson et al., 1990
). The authors attributed these increases in testis weight to spontaneous pathologic changes that did not result directly from MTBE administration. Testis weight changes reported in this study occurred only at 1500 mg MTBE/kg/day. No changes in testis weight or histopathology were observed at 1000 mg MTBE/kg/day, the dose shown to cause LCT in chronically treated male Sprague-Dawley rats (Belpoggi et al., 1995
). The apparent signs of clinical toxicity such as dehydration and depressed activity of the rats may have confounded the responses such as organ weights and histopathology observed in animals treated with 1500 mg MTBE/kg/day.
Prolactin levels measured in serum decreased in rats treated with 1500 mg MTBE/kg/day for 15 but not 28 days. Even though the role of prolactin in the maintenance of Leydig cell function is still debated, investigators accept that prolactin exerts a stimulatory effect on testicular LH, human chorionic gonadotropin, and prolactin receptors in many species (Saez and Lejeune, 1996). Because the negative feedback control of prolactin secretion is governed by the interaction of prolactin with the prolactin receptor, the observed decrease in prolactin in this study would likely lessen the response of circulating LH on the testis. Typically, alterations in prolactin levels are associated with several factors, including exercise, stress, and circadian rhythms, and result in the increased release of prolactin from the anterior pituitary (Reichlin, 1998
). It is unclear from these studies if and how the decrease in prolactin levels following MTBE administration contributes to the disruption of the HPT axis regulation.
In the 15-day treated animals, testosterone was significantly decreased in the serum and TIF with 1500 mg MTBE/kg/day. Serum and TIF hormonal changes measured at one point in time are extremely variable due to the pulsatile secretion of hormones, namely LH and testosterone (Sharpe, 1988). Secretion of LH from the anterior pituitary triggers the production of testosterone in Leydig cells, resulting in normal testosterone levels in the rat from 0.5 to 15.0 ng/ml in the serum and 802500 ng/ml in the TIF (Sharpe, 1988
). This common variability often complicates the interpretation of hormonal alterations; however, the decrease in testosterone levels was statistically significant and consistent in that there was both a decrease in serum and TIF in the same dose group. Measurement of serum testosterone levels accounts for the balance between testosterone release from the testis and testosterone metabolism and clearance from the liver and kidney. Although MTBE administration may affect testosterone metabolism and clearance, the most plausible explanation for the decrease in serum testosterone is that there is a decrease in its release from the testis. The decrease in testosterone levels in the TIF appears to be a local response in the testis and not a direct affect on the HPT axis. The potential for a local response in the testis needs to be investigated in vitro using isolated Leydig cell. In addition, the hormonal changes observed in this study were evaluated at one time point following administration. The influence of dynamic changes may need to be evaluated in further investigations to assess the potential mode of action for MTBE-induced LCT.
The mechanism of formation for LCT has been studied with a number of nongenotoxic carcinogens. In most cases, the mode of action has been attributed to alterations in the HPT axis, which results in elevated LH (Clegg et al., 1997; Cook et al., 1997a
). In this study, MTBE did not cause an increase in LH, but a statistically significant dose-related decrease was apparent in animals treated for 28 days. In addition, no significant change in LH was observed in MTBE-treated intact or orchiectomized rats dosed for 28 or 5 days, respectively (Allgaier and de Peyster, 1999
; Day et al., 1998
). The observed decrease in LH may be associated with t-butyl alcohol, a metabolite of MTBE. t-Butyl alcohol administration to male Sprague-Dawley rats resulted in a dose-related decrease in plasma LH (Chapin et al., 1980
). However, long-term consequences of this suppression are unlikely, as LCT were not observed in a 2-year TBA carcinogenicity study in rats and mice (Cirvello et al., 1995
).
The investigation of other endocrine-active compounds in intact male Sprague-Dawley rats, such as ICI-182,780 (estrogen receptor antagonist), flutamide (androgen receptor antagonist), ketoconazol (testosterone biosynthesis inhibitor), finasteride (5-reductase inhibitor), and anastrozol (aromatase inhibitor), demonstrated that distinct end-point fingerprints for each mechanism of action for perturbations of the HPT axis could be determined (O'Connor et al., 1998a
). These fingerprints, which incorporate endocrine-sensitive tissue weight changes and alterations in hormone concentration, have been outlined by O'Connor et al. (1998a). The results from the current study do not clearly identify a mode or modes for MTBE-induced endocrine activity, indicating that weak endocrine-active compounds may be difficult to identify with this approach. The results from this study and others do not support the hypothesis that MTBE causes significant alterations in the HPT axis, nor do they indicate a potential mode of action for the increase in LCT with chronic exposure.
In studying the intact male rat, most of the significant changes in body and organ weight and hormone measurements were confined to animals receiving 1500 mg MTBE/kg/day. As clear signs of clinical toxicity were apparent in these animals throughout the study, caution must be used when interpreting these high-dose responses. In addition, the dose at which these effects were observed (1500 mg MTBE/kg/day) exceeds the dose used in the bioassay (1000 mg MTBE/kg/day) where LCT were apparent at 2 years (Belpoggi et al., 1995). Exclusion of the high-dose responses demonstrates that MTBE appears to elicit a weak, if any, effect on the endocrine system of male rats with a notable change only in T3 and not T4 serum concentrations. T3 changes may be attributed to increased liver cell numbers, consistent with increased liver weight, which may enhance the hepatic metabolism of thyroid hormones. A variety of chemicals and drugs have been shown to induce hepatic metabolizing enzymes, which results in decreased circulating thyroid hormones (Biegel et al., 1995
; Johnson et al., 1993
). Future studies need to investigate the ability of MTBE to induce the hepatic metabolism and excretion of thyroid hormones.
Our results indicated that oral administration of MTBE for 15 or 28 days at the high doses resulted in mild changes in hormone levels and endocrine-sensitive tissues in the rat; however, increased LH levels, which commonly but not always cause LCT, were not observed in male rats dosed with MTBE. These mild changes do indicate that the endocrine system is perturbed following MTBE-treatment; however, it is unclear whether these perturbations during chronic treatment would result in LCT formation. Data gathered here will be useful in developing a longer-term administration study to assess the relationship between dynamic hormonal changes and Leydig cell hyperplasia and tumor formation.
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ACKNOWLEDGMENTS |
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NOTES |
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This project was supported in part by grant number 1 F32 ES0586501 from the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH.
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