Mechanisms of extrahepatic tumor induction by peroxisome proliferators in male CD rats.

Wyeth-14,643 (WY) and ammonium perfluorooctanoate (C8) belong to a diverse class of compounds which have been shown to produce hepatic peroxisome proliferation in rodents. From previous work, WY, but not C8, has been shown to produce hepatocellular carcinoma in rats, while C8 has been shown to produce Leydig cell adenomas. In addition, based on a review of bioassay data a relationship appears to exist between peroxisome-proliferating compounds and Leydig cell adenoma and pancreatic acinar cell hyperplasia/adenocarcinoma formation. To further investigate the relationship between peroxisome-proliferating compounds and hepatic, Leydig cell, and pancreatic acinar cell tumorigenesis, a 2-year feeding study in male CD rats was initiated to test the hypothesis that peroxisome proliferating compounds induce a tumor triad (liver, Leydig cell, pancreatic acinar cell), and to examine the potential mechanism for the Leydig cell tumors. The study was conducted using 50 ppm WY and 300 ppm C8. The concentration of WY in the diet was decreased to 25 ppm on test day 301 due to increased mortality. In addition to the ad libitum control, a second control was pair-fed to the C8 group. Interim sacrifices were performed at 1- or 3-month intervals. Peroxisome proliferation measured by beta-oxidation activity and cell proliferation were measured in the liver and testis at all time points and in the pancreas beginning at the 9-month time point (cell proliferation only). Serum hormone concentrations (estradiol, testosterone, LH, FSH, and prolactin) were also measured at each time point. Increased relative liver weights and hepatic beta-oxidation activity were observed in both the WY- and C8-treated rats at all time points. In contrast, hepatic cell proliferation was significantly increased only in the WY-treated group. Neither WY nor C8 significantly altered the rate of Leydig cell beta-oxidation or Leydig cell proliferation when compared to the control groups. Moreover, the basal rate of beta-oxidation in Leydig cells was approximately 20 times less than the rate of hepatic beta-oxidation. There were no biologically meaningful differences in serum testosterone, FSH, prolactin, or LH concentrations in the WY- and C8-treated rats when compared to their respective controls. There were, however, significant increases in serum estradiol concentrations in the WY- and C8-treated rats at 1, 3, 6, 9, 15, 18, and 21 months. At 12 months, only the C8-treated rats had elevated serum estradiol concentrations when compared to the pair-fed control. Histopathological evaluation revealed compound-related increases in liver, Leydig cell, and pancreatic acinar cell tumors in both WY- and C8-treated rats. The data support the hypothesis that the peroxisome-proliferating compounds induce the previously described tumor triad. In addition, both C8 and WY produced a sustained increase in serum estradiol concentrations that correlated with the potency of the 2 compounds to induce Leydig cell tumors (i.e., WY caused a more consistent sustained increase in serum estradiol throughout the entire study, and more specifically at the end of the study, than did C8). This study suggests that estradiol may play a role in enhancement of Leydig cell tumors in the rat, and that peroxisome proliferators may induce tumors via a non-LH type mechanism.

Effects of 5-bromo-2'deoxyuridine (BrdU) implants on hepatic cytochrome P-450 content and beta-oxidation activity in rats and mice.

Standard regulatory toxicity tests are frequently supplemented with additional compound specific analysis. Analysis of hepatic cytochrome P-450 content, hepatic beta-oxidation activity (biochemical analysis), and cell proliferation rates are examples of these analyses that are included when past experience or similarity to other compounds, suggest that a presently tested compound may have an effect. Until now, separate subsets of animals have been designated for cell proliferation analysis and biochemical analysis, because it was unknown if implantation of 5-bromo-2'deoxyuridine (BrdU) filled osmotic pumps (BrdU implants) would effect the rate of hepatic-beta or hepatic cytochrome P-450 content. The purpose of the current study was to determine if BrdU implants had an effect on hepatic cytochrome P-450 content, beta-oxidation activity, or the measurement of these enzymes in rats and mice. The BrdU was administered through subcutaneous osmotic pump implants. The rate of hepatic peroxisomal beta-oxidation was not altered in male or female rats or mice with the BrdU implants when compared to those of the control groups. The total hepatic cytochrome P-450 content was also not altered in male or female rats or mice with the BrdU implants when compared to those of the control groups. BrdU implants do not appear to have an effect on the rate of hepatic peroxisomal beta-oxidation or the total hepatic cytochrome P-450 content in male or female rats and mice. It can be concluded that in future studies, rats or mice which are designated for cell proliferation analysis using BrdU implants are also suitable for use in evaluating chemically induced effects on hepatic peroxisomal beta-oxidation activity and/or total hepatic cytochrome P-450 content.

Screening methods for thyroid hormone disruptors.

The U.S. Congress has passed legislation requiring the EPA to implement screening tests for identifying endocrine-disrupting chemicals. A series of workshops was sponsored by the EPA, the Chemical Manufacturers Association, and the World Wildlife Fund; one workshop focused on screens for chemicals that alter thyroid hormone function and homeostasis. Participants at this meeting identified and examined methods to detect alterations in thyroid hormone synthesis, transport, and catabolism. In addition, some methods to detect chemicals that bind to the thyroid hormone receptors acting as either agonists or antagonists were also identified. Screening methods used in mammals as well as other vertebrate classes were examined. There was a general consensus that all known chemicals which interfere with thyroid hormone function and homeostasis act by either inhibiting synthesis, altering serum transport proteins, or by increasing catabolism of thyroid hormones. There are no direct data to support the assertion that certain environmental chemicals bind and activate the thyroid hormone receptors; further research is indicated. In light of this, screening methods should reflect known mechanisms of action. Most methods examined, albeit useful for mechanistic studies, were thought to be too specific and therefore would not be applicable for broad-based screening. Determination of serum thyroid hormone concentrations following chemical exposure in rodents was thought to be a reasonable initial screen. Concurrent histologic evaluation of the thyroid would strengthen this screen. Similar methods in teleosts may be useful as screens, but would require indicators of tissue production of thyroid hormones. The use of tadpole metamorphosis as a screen may also be useful; however, this method requires validation and standardization prior to use as a broad-based screen.

90-day feeding and one-generation reproduction study in Crl:CD BR rats with 17 beta-estradiol.

Over the past several years, there has been increasing concern that chemicals and pesticides found in the environment may mimic endogenous estrogens, potentially producing adverse effects in wildlife and human populations. Because estrogenicity is one of the primary concerns, a 90-day/one-generation reproduction study with 17 beta-estradiol was designed to set dose levels for future multigenerational reproduction and combined chronic toxicity/oncogenicity studies. The purpose of these studies is to evaluate the significance of a range of responses as well as to provide benchmark data for a risk assessment for chemicals with estrogen-like activities. This 90-day/one-generation reproduction study was conducted in male and female Crl:CD BR rats using dietary concentrations of 0, 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol. Endpoints were chosen in order to evaluate both subchronic and reproductive toxicity. In addition, several mechanistic/biochemical endpoints were evaluated for their usefulness in follow-up studies. In the P1 generation, dietary administration of 2.5, 10, and 50 ppm 17 beta-estradiol produced dose-dependent decreases in body weight, body weight gain, food consumption, and food efficiency. At 10 and 50 ppm 17 beta-estradiol, minimal to mild nonregenerative anemia, lymphopenia, decreased serum cholesterol (50 ppm only), and altered splenic lymphocyte subtypes were also observed in the P1 generation. Additionally, at these concentrations, there were changes in the weights of several organs. Evidence of ovarian malfunction, characterized by reduced numbers of corpora lutea and large antral follicles, was observed at 2.5 ppm 17 beta-estradiol and above. Other pathologic changes in males and females fed 10 and 50 ppm 17 beta-estradiol included centrilobular hepatocellular hypertrophy; diffuse hyperplasia of the pituitary gland; feminization of the male mammary glands; mammary gland hyperplasia in females; increased number of cystic follicles in the ovary; hypertrophy of the endometrium and endometrial glands in the uterus; degeneration of seminiferous epithelium; and atrophy of the testes and the accessory sex glands. In the reproduction portion of this study, rats fed 10 or 50 ppm 17 beta-estradiol did not produce litters. While there was no evidence that the 50 ppm treated rats mated, 33.3% of the rats fed 10 ppm mated but did not produce litters. No effects on mating and fertility indices were observed in rats fed 0.05 and 2.5 ppm 17 beta-estradiol. Pup weights at birth were statistically decreased relative to control in the groups fed 0.05 and 2.5 ppm 17 beta-estradiol. Weights of the rats in the 0.05 ppm group recovered by postnatal day 4 and remained similar to control throughout the remainder of the study. The mean gestation length of the 0.05 ppm group was slightly, albeit not statistically significantly, shorter (0.5 days) than that of the control group, which may have contributed to the decrease in birth weight of the 0.05 ppm group. In contrast, the weights of the F1 generation rats fed 2.5 ppm 17 beta-estradiol remained decreased relative to the control group throughout the study. Parental administration of 17 beta-estradiol did not alter anogenital distance in male or female pups. The onset of sexual maturation, as measured by day of preputial separation in males and day of vaginal opening in females, was delayed in male rats fed 2.5 ppm (by 8.2 days) and was hastened in female rats fed 0.05 and 2.5 ppm (by 1.6 and 8.8 days, respectively). The age at vaginal opening ranged from 26 to 37, 26 to 35, and 21 to 25 days for rats fed 0, 0.05, and 2.5 ppm 17 beta-estradiol, respectively. Hence, the range of age at vaginal opening was similar between the control and 0.05 ppm group. The organ weight and pathologic alterations observed in the adult F1 generation rats were similar to those observed in the P1 generation rats. (ABSTRACT TRUNCATED)

Effects of 17 beta-estradiol on serum hormone concentrations and estrous cycle in female Crl:CD BR rats: effects on parental and first generation rats.

The recently passed Food Quality Protection Act of 1996 requires the U.S. EPA to implement screening strategies for endocrine active compounds (EACs) within the next 2 years. Interpreting results from screening tests is complicated by the absence of traditional dietary rodent bioassay data with model estrogenic compounds such as 17 beta-estradiol. Thus, a 90-day/one-generation reproduction study with 17 beta-estradiol was designed to: (1) provide such baseline data; (2) set dose levels for multigeneration reproduction and combined chronic toxicity/oncogenicity studies; and (3) evaluate various mechanistic/biochemical endpoints for inclusion in these follow-up studies. The current article describes the effects of dietary administration of 0, 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol on the serum hormone concentrations and estrous cyclicity of female Crl:CD BR rats and evaluates a sampling strategy for measuring serum hormone levels in cycling female rats. Serum hormones were measured at three time points during a 90-day dietary exposure (1 week, 28 days, and 90 days) and in the F1 generation rats on postnatal day 98. Over the course of the 90-day feeding study for the P1 generation and from postnatal days 21 to 98 for the F1 generation, the estrous cycle was monitored daily in 10 rats/group. In P1 generation rats, dietary administration of 2.5, 10, and 50 ppm 17 beta-estradiol produced a dose-dependent increase in serum estradiol (E2) concentrations at all time points. In contrast, administration of 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol produced a dose-dependent decrease in serum progesterone (P4) concentrations on test day 90, which correlated with an absence of corpora lutea and ovarian atrophy. At 10 and 50 ppm 17 beta-estradiol, serum luteinizing hormone (LH) concentrations were consistently decreased at all time points and were decreased at 2.5 ppm on test day 90. Serum prolactin (PRL) concentrations were increased at 50 ppm 17 beta-estradiol on test day 90. Serum follicle stimulating hormone (FSH) concentrations were either similar to the control levels or minimally changed at all time points. No F1 generation rats were produced at 10 or 50 ppm 17 beta-estradiol. In F1 generation rats, serum E2 concentrations were increased and P4 concentrations were decreased at a dietary concentration of 2.5 ppm 17 beta-estradiol, while serum concentrations of LH, FSH, and PRL were similar to the control. Dietary administration of 17 beta-estradiol at concentrations of 2.5 (both generations) and 10 and 50 ppm (P1 generation only) produced marked effects on the estrous cycle: decreased number of cycles, increased mean cycle length, and decreased number of normally cycling rats. The estrous cyclicity of rats fed 2.5 ppm 17 beta-estradiol appeared more severely affected in rats of the F1 generation than in rats of the P1 generation. Whether this increase in severity is related to an in utero exposure and/or greater mean daily intake of 17 beta-estradiol in the F1 generation rats in the postnatal period is unclear. Another goal of this study was to evaluate whether a single time point sampling strategy using cycling female rats could be used to detect compound-related changes in serum hormone concentrations. In evaluating a sampling strategy for measuring serum hormone levels, it appears that detection of compound-related alterations in serum hormone concentrations can be best detected by sampling during diestrus. Since the stage of the cycle dramatically influences hormone concentrations, large sample sizes (n = 50) are needed if serum hormone measurements are not matched with the stage of the cycle. The data indicate that this strategy of measuring serum hormone concentrations has utility in detecting compound-related effects within the confines of a traditional guideline study (subchronic, chronic, or multigenerational reproduction study).

Effects of dietary 17 beta-estradiol exposure on serum hormone concentrations and testicular parameters in male Crl:CD BR rats.

A 90-day/one-generation reproduction study was conducted in male and female Crl:CD BR rats using dietary levels of 0, 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol. The goals of this study were to set dose levels and evaluate several mechanistic endpoints for inclusion in multigeneration reproduction and combined chronic toxicity/oncogenicity studies with 17 beta-estradiol. In this report we discuss the effects of dietary 17 beta-estradiol exposure on serum hormonal levels and sperm parameters from P1 and F1 male rats. Sperm parameters were also evaluated in recovery P1 and F1 male rats that were fed control diets for 105 and 103 days, respectively, following 97 and 86-94 days of estradiol exposure, respectively. Measurement of Sertoli cell number from F1 male rats was performed to test the hypothesis that in utero exposure to estrogens will decrease Sertoli cell number and sperm production. Other findings from this 90-day/one-generation reproduction study are summarized elsewhere. 17 beta-Estradiol produced a dose-dependent decrease in body weight in P1 male rats at > or = 2.5 ppm and in the F1 male rats at 2.5 ppm. This decrease in body weight was due to a combination or reduced food consumption and food efficiency. In the recovery P1 males, body weight increased in the affected groups, albiet not to control levels, due to food consumption returning to control levels accompanied by an increase in food efficiency. However, in F1 males there was no corresponding rebound in body weight. In the P1 rats, exposure to 17 beta-estradiol decreased testis and epididymis weights in the 10 and 50 ppm groups, while no effects were seen in the P1 2.5 ppm group. In contrast, epididymis weights in the F1 and F1 recovery 2.5 ppm groups were statistically decreased; however, there were no histopathological effects observed. The decreases in testis weights in the P1 generation correlated with histopathologic evidence of interstitial cell atrophy and seminiferous tubule degeneration and reduced sperm production. Correlative changes in the epididymides of P1 rats were characterized by oligospermia or aspermia, the presence of germ cell debris in the lumen of tubules, and atrophy of epididymal tubules. 17 beta-Estradiol decreased testicular spermatid numbers, epididymal sperm numbers, and sperm motility in the P1 males in the 10 and 50 ppm groups, but not in the 2.5 ppm group. Following a 105-day recovery period in the P1 males, all sperm parameters and reproductive organ weights returned to control values except for the epididymal sperm count. Overall, the decline in testicular spermatid and epididymal sperm numbers in the P1 rats correlated with the reduced organ weights and the observed histopathological changes and appeared primarily related to the decrease in serum testosterone levels. In the F1 rats, no significant decreases were noted in the testicular spermatid number but a slight decrease in epididymal sperm number was seen in the 2.5 ppm group, which showed no evidence of recovery. Using morphometric analysis, no change was seen in the number of Sertoli cell nuclei per testis in F1 males. The pattern of hormonal responses seen in this study was characteristic of an estrogen receptor agonist such as 17 beta-estradiol: increased serum prolactin and decreased testosterone, luteinizing hormone, and follicle stimulating hormone levels. The data demonstrate that in utero and postnatal dietary administration of 17 beta-estradiol at levels which increased serum estradiol levels to approximately 400% of control and decreased testosterone levels to 33% of control did not reduce the number of Sertoli cell nuclei per testis.

Sensitivity of a Tier I screening battery compared to an in utero exposure for detecting the estrogen receptor agonist 17 beta-estradiol.

A Tier I screening battery for detecting endocrine active compounds (EACs) has been evaluated for its ability to identify 17 beta-estradiol, a pure estrogen receptor agonist. In addition, the responses obtained with the Tier I battery were compared to the responses obtained from F1 generation rats from a 90-day/one-generation reproduction study with 17 beta-estradiol in order to characterize the sensitivity of the Tier I battery against the sensitivity of an in utero exposure for detecting EACs. The Tier I battery incorporates two short-term in vivo tests (5-day ovariectomized female battery; 15-day intact male battery) and an in vitro yeast transactivation system (YTS) for identifying compounds that alter endocrine homeostasis. The Tier I female battery consists of traditional uterotrophic endpoints coupled with biochemical and hormonal endpoints. It is designed to identify compounds that are estrogenic/antiestrogenic or modulate dopamine levels. The Tier I male battery consists of organ weights coupled with microscopic evaluations and a comprehensive hormonal assessment. It is designed to identify compounds that have the potential to act as agonists or antagonists to the estrogen, androgen, progesterone, or dopamine receptor; steroid biosynthesis inhibitors (aromatase, 5 alpha-reductase, and testosterone biosynthesis); or compounds that alter thyroid function. The YTS is designed to identify compounds that bind to steroid hormone receptors (estrogen, androgen, and progesterone) and activate gene transcription. The profile generated for 17 beta-estradiol was characteristic of the responses expected with a pure estrogen receptor agonist. In the female battery, responses to 17 beta-estradiol included increases in uterine fluid imbibition, uterine weight, estrus conversion, uterine stromal cell proliferation, uterine epithelial cell height, uterine progesterone receptor content, serum prolactin and estradiol levels, and decreases in uterine estrogen receptor content and follicle stimulating hormone and luteinizing hormone levels. In the male battery, responses to 17 beta-estradiol included decreases in absolute testis and epididymides weights, decreases in relative weights for androgen-dependent tissues (prostate, seminal vesicles, and accessory sex gland unit), hormonal alterations (decreased serum testosterone, dihydrotestosterone, and LH and increased serum prolactin levels), and microscopic alterations of the testis and epididymides. In the YTS for the estrogen receptor, 17 beta-estradiol had an EC50 value of 7.2 x 10(-9) M, while DHT and progesterone had little cross-activation. The androgen and progesterone receptor systems were less selective in that 17 beta-estradiol activated these systems within 3 orders of magnitude of the primary ligand. In the 90-day/one-generation reproduction study, responses to dietary administration of 17 beta-estradiol included alterations in organ weights, developmental landmarks, and hormonal levels. Comparison of the responses obtained with our Tier I battery and an in utero exposure demonstrates that the Tier I screening battery is as sensitive as an in utero exposure for detecting 17 beta-estradiol-induced alterations in hormonal homeostasis.

Induction of coagulation effects by Wyeth-14,643 in Crl:CD BR rats.

A two-year mechanistic bioassay in male Crl:CD BR rats was initiated with 50 ppm Wyeth-14,643 (WY) to investigate the relationship between peroxisome proliferating compounds and Leydig cell adenoma formation. After 154 days, the survival rate in the WY group decreased below control levels. After 300 days, the dose was lowered to 25 ppm for the remainder of the study. Gross examination of WY-treated rats either found dead or euthanized in extremis revealed hemorrhages at several sites. To investigate this observation, blood was then collected on test day 281 from 10 randomly selected control and WY-treated rats and a clinical pathological examination was performed. The WY-treated rats had significantly decreased red blood cell count, hemoglobin, and hematocrit, and elevated platelet counts. In the WY-treated rats, prothrombin times in undiluted plasma were similar to the controls, but were markedly prolonged in 2 of 10 rats when the plasma samples were diluted to 25%. Subsequently, blood was collected prior to sacrificing WY-treated rats which were exhibiting clinical signs of anemia. These rats had prolonged prothrombin times, activated partial thromboplastin time, and thrombin clot time when compared to laboratory historical control data (116.7 vs 13.3, 116.4 vs 13.7, and 42.4 vs 25.7 seconds, respectively). In a subsequent, ongoing study, Vitamin K was added to control and WY-treated diets (100 ppm). No survival differences between control and WY-treated rats occurred through 260 days in this second study. These new data suggest that deaths in the WY-treated group in our initial study were due to a vitamin K deficiency. The role of increased serum estradiol, its effects on blood coagulation, and enhanced hepatic cell proliferation in the vitamin K-dependent coagulation processes warrant further investigation.

Effect of the peroxisome proliferator, ammonium perfluorooctanoate (C8), on hepatic aromatase activity in adult male Crl:CD BR (CD) rats.

The incidence of Leydig cell adenomas increases in CD rats fed for 2 years with the hepatic peroxisome proliferator, ammonium perfluorooctanoate (C8). Treatment with C8 increased the serum concentration of estradiol in 2-week gavage studies, and feeding studies at various time points up to 2 years, and was also accompanied by increases in liver weight and hepatic beta-oxidation activity. Since peroxisome proliferators induce both hepatic beta-oxidation and specific cytochrome P450 enzymes, C8 may also induce aromatase (cytochrome P450-19A1), the cytochrome P450 monooxygenase which converts androgens to estrogens. This hypothesis was investigated in the present study. Adult male CD rats were dosed daily by gavage for 14 days with 0, 0.2, 2, 20, or 40 mg C8/kg body wt. An additional group, the pair-fed control, was fed at a rate matched to the daily consumption by the 40 mg C8/kg group. Treatment with C8 produced a dose dependent decrease in body weight, and increases in absolute and relative liver weights, and in the protein yield of hepatic microsomes. These C8-induced changes were associated with a 2-fold increase in the serum concentration of estradiol and up to a 16-fold increase in total hepatic aromatase activity. A significant linear correlation was established between serum estradiol and total hepatic aromatase activity. The absolute weights and the aromatase activity of the testes were not affected by C8. Hepatic peroxisomal beta-oxidation activity and the microsomal concentration of total cytochrome P450 were also increased by C8. A comparison of estimated EC50 values suggested that these parameters may be less sensitive to induction by C8 than hepatic aromatase activity. Co-incubation of control liver microsomes with C8 in the aromatase assay for 2 hr dose dependently reduced the apparent aromatase activity. This inhibition of aromatase in vitro but increase in vivo was further investigated using cultured rat hepatocytes. Decreases in aromatase activity were found after up to 42 hr of treatment with C8, but the enzyme activity was increased almost 2-fold after 66 hr. The results of this study suggest that the increased serum concentration of estradiol produced by C8 in rats is at least partly due to a direct effect on the liver to increase synthesis of estradiol through induction of aromatase cytochrome P450 in the endoplasmic reticulum.

Subchronic toxicity study in rats with 1-methyl-3-propylimidazole-2-thione (PTI): effects on the thyroid.

A 90-day gavage study was performed to evaluate the subchronic toxicity of 1-methyl-3-propylimidazole-2-thione (PTI) when administered to Crl:CD BR rats. PTI is a chemical catalyst and is structurally similar to the thioureas, which are known to adversely affect the thyroid. Therefore, this study was designed to investigate the effects of PTI on the thyroid. Male and female rats were dosed with 0, 5, 10, 25, or 75 mgPTI/kg/day for 13 weeks. Clinical pathology examinations and pathology examination were performed and the following were measured periodically: serum T3, T4, and TSH, hepatic UDP-glucuronyltransferase activity, and cell proliferation of the thyroid and liver. Under the conditions of this study, the overall no-observed-adverse-effect level (NOAEL) for the subchronic effects of PTI in male and female rats was 10 mg PTI/kg/day. The NOAEL was based on the effects on the thyroid gland in male and female rats dosed with 25 and 75 mg PTI/kg/day, as well as the hepatic centrilobular fatty change, increased severity of chronic progressive nephropathy, fatty change in the adrenal medulla, and the substantial reduction in body weight and body weight gain. The primary target organs were the thyroid and liver. Alterations in thyroid hormones (T3, T4, and TSH) occurred predominantly at 25 and 75 mg/kg/day. Toxicologically significant alterations in T3, T4, and TSH levels, cell proliferation, and UDP-glucuronyltransferase activity occurred in rats dosed with 25 and 75 mg/kg/day, which correlated with organ weight and histopathological effects. Additionally, the effect of PTI on thyroid peroxidase activity, a key step in thyroid hormone synthesis, was evaluated in vitro using microswine thyroid microsomes. PTI was shown to inhibit thyroid peroxidase, with an IC50 of 0.02 M. These data suggest that PTI enhances the excretion of T4 via induction of glucuronyltransferase and inhibits thyroid hormone synthesis via a direct affect on thyroid peroxidase. Both of these effects contribute to the disruption of the hypothalamic-pituitary-thyroid axis and result in sustained elevation of TSH and the corresponding thyroid hypertrophy and hyperplasia.

Effects of ammonium perfluorooctanoate on Leydig cell function: in vitro, in vivo, and ex vivo studies.

Ammonium perfluorooctanoate (C8) produced a dose-dependent increase in Leydig cell adenomas in Crl:CD BR (CD) rats fed 0, 30, or 300 ppm for 2 years. Administration of C8 to adult male CD rats, by gavage for 14 days, produced decreased serum and testicular interstitial fluid testosterone levels and increased serum estradiol levels. The C8-mediated decrease in the serum testosterone levels appeared to be due to a lesion at the level of the testis. These endocrine changes may play a role in the C8 induction of Leydig cell tumors. In the present work, C8 was examined for its ability to (1) directly affect Leydig cells in vitro using isolated Leydig cells from untreated rats and ex vivo using Leydig cells isolated from C8-treated rats, (2) affect testicular interstitial fluid hormone levels, and (3) induce aromatase activity. These studies were conducted to investigate the hypothesis that C8 produces an increase in estradiol by inducing cytochrome P450 XIX (aromatase), which converts testosterone to estradiol, and that the elevated estradiol levels ultimately produce Leydig cell hyperplasia and adenoma formation by acting as a mitogen or enhancing growth factor secretion. In the in vivo and ex vivo studies, adult male CD rats were gavaged with either 0 or 25 mg/kg/day C8 for 14 days. In addition to the ad libitum control, a second control group was pair-fed to the 25 mg/kg/day C8 group. In the in vitro and ex vivo studies, Leydig cells were isolated from testes of adult males by collagenase digestion followed by enrichment over Percoll gradients. A dose-dependent decrease in testosterone levels was observed in hCG-stimulated Leydig cells treated in vitro with C8 for 5 hr (IC50 approximately 200 microM). In contrast, ex vivo studies demonstrated an increase in testosterone production in hCG-stimulated Leydig cells from C8-treated rats when compared to Leydig cells isolated from either the ad libitum or pair-fed control rats. The in vitro data demonstrate that C8 directly inhibits testosterone release from Leydig cells, while the ex vivo data demonstrate that this inhibition is reversible.(ABSTRACT TRUNCATED AT 400 WORDS)

Investigation of a mechanism for Leydig cell tumorigenesis by linuron in rats.

In a previously conducted 2-year study, a concentration-dependent increase in Leydig cell adenomas was observed in Crl:CD BR(CD) rats fed diets containing the herbicide linuron. Linuron has been shown to be negative in a battery of six tests for genotoxicity; therefore, a nongenotoxic mechanism of tumorgenesis was investigated. Linuron is structurally related to the nonsteroidal antiandrogen, flutamide. Flutamide has also been shown to produce Leydig cell tumors within 1 year, presumably due to sustained hypersecretion of luteinizing hormone (LH) which occurs following disruption of the hypothalamic-pituitary-testicular (HPT) axis. To investigate whether linuron possesses antiandrogenic activity, sexually immature and mature CD rats were administered either 200 mg/kg linuron or 10 mg/kg flutamide (positive control) for 2 weeks. Accessory sex organs were weighed and serum hormone levels were measured to assess androgen status and alterations in the HPT axis. Serum from a multigeneration reproduction study with linuron was also analyzed for serum hormone levels. In addition, competitive receptor binding studies were conducted to evaluate the ability of linuron to bind to the androgen receptor. Linuron decreased accessory sex organ weights in sexually immature and mature linuron-treated rats. Increased serum estradiol and LH levels were observed in sexually mature linuron-treated rats. Serum estradiol and LH levels were also elevated in P1 and F1 male rats from the multigeneration reproduction study. These accessory sex organ and hormonal changes are consistent with those seen with the antiandrogen flutamide, the only exception being serum testosterone, which was elevated following exposure to flutamide but not to linuron. The inability of linuron to increase testosterone levels may reflect the lower potency of linuron as an antiandrogen compared with that of flutamide, which is a potent antiandrogen. Additionally, linuron competed with [3H]testosterone for binding to the androgen receptor. The IC50 data for competition to the androgen receptor suggest that linuron is approximately 3.5 times less potent than flutamide. These data are consistent with the effects seen with flutamide and demonstrate that linuron is a less potent antiandrogen than flutamide. Collectively, these data support the hypothesis that linuron produces Leydig cell tumors via an antiandrogenic mechanism where sustained hypersecretion of LH appears to be responsible for the development of Leydig cell hyperplasia and adenomas.

Comparison of the effects of Wyeth-14,643 in Crl:CD BR and Fisher-344 rats.

Wyeth-14,643 (WY) belongs to a diverse class of compounds which have been shown to produce hepatic peroxisome proliferation and hepatocellular carcinoma in rodents. Based on a review of bioassay data, a relationship appears to exist between peroxisome proliferating compounds and Leydig cell adenoma formation. Most rat bioassays with peroxisome proliferators have been conducted in the Fisher-344 (F344) rat, which has a high spontaneous incidence of Leydig cell adenomas. Thus, it was necessary to use an alternative animal model to investigate this relationship further. Therefore the Crl:CD BR (CD) rat, which has a low spontaneous Leydig cell adenoma incidence, was chosen for a 2-year feeding study with WY. Before initiating this 2-year feeding study, it was necessary to investigate whether any strain differences existed between CD and F344 rats with respect to WY-induced peroxisome and cell proliferation. In this study, male CD and F344 rats were fed diets containing 0, 50, or 1000 ppm WY for 21 days. Peroxisome proliferation in the liver and testis was determined biochemically by measuring beta-oxidation activity and was confirmed ultrastructurally. Serum hormone levels and cell proliferation rates in the liver and testis were also measured. In addition, basal beta-oxidation activity and cell proliferation rates were compared between the CD and F344 rats. A significant decrease in final body weight was observed in the 1000 ppm WY groups for both CD and F344 rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of insulin-like growth factor-I responses in MCF-7 cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds.

Insulin-like growth factor-I (IGF-I) stimulated the growth and [3H]thymidine uptake in MCF-7 human breast cancer cells grown in serum- and growth factor-inactivated serum-containing media. Cotreatment of the cells with IGF-I plus 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a significant decrease in mitogen-induced cell proliferation and [3H]thymidine uptake. Similar effects were observed for cells treated with 2,3,7,8-TCDD and IGF-I plus 17 beta-estradiol. The relative antimitogenic activities of 2,3,7,8-TCDD and related compounds followed the order 2,3,7,8-TCDD greater than 2,3,7,8-tetrachlorodibenzofuran (TCDF) greater than 1,2,7,8-TCDF greater than 1,3,7,8-TCDD which was similar to their aryl hydrocarbon (Ah) receptor binding affinities. The results showed that 2,3,7,8-TCDD did not alter the IGF-I receptor mRNA levels or the KD values for binding of [125I]IGF-I to the IGF-I receptor in MCF-7 cells. However, 2,3,7,8-TCDD significantly decreased the number of IGF-I-induced IGF-I receptor binding sites and this may play a role in the growth-inhibitory properties of 2,3,7,8-TCDD and related compounds and in the 'cross-talk' between the two endocrine-response pathways.

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) as an antiestrogen in human and rodent cancer cell lines: evidence for the role of the Ah receptor.

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) is a relatively nontoxic analog of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Treatment of aryl hydrocarbon (Ah)-responsive MCF-7 human breast cancer cells with 100 nM MCDF resulted in the inhibition of 17 beta-estradiol-induced proliferation and the secretion of the 34-, 52-, and 160-kDa proteins. After treatment of the cells with 17 beta-[3H]estradiol, 100 nM of MCDF caused a decrease in the accumulation of the radiolabeled nuclear estrogen receptor (ER) complex in these cells. In parallel experiments, the antiestrogenic effects of MCDF were also determined in Ah-responsive wild-type Hepa 1c1c7 cells and Ah-nonresponsive class 1 and class 2 mutant cells. Treatment of the wild-type cells with 17 beta-[3H]estradiol and 100 nM MCDF caused a decrease in the accumulation of radiolabeled nuclear ER complex in these cells whereas no significant effects were observed in the mutant cells as determined by velocity sedimentation analysis. Comparable results were obtained using ER antibodies to measure the decrease in immunoreactive nuclear ER. In addition, both actinomycin D and cycloheximide inhibited the MCDF-mediated decrease of nuclear ER levels in the Hepa 1c1c7 wild-type cells. Although 100 nM MCDF did not induce cytochrome P-450-dependent monooxygenases in the MCF-7 or Hepa 1c1c7 cell lines, incubation of nuclear extracts from the MCF-7 cells treated with 100 nM MCDF with a synthetic consensus dioxin responsive element (an oligonucleotide duplex of 26 bases) gave a retarded band in a gel-retardation assay. The data suggest that the antiestrogenic effects of MCDF does not require the induction of the CYP1A1 gene expression but may involve the induction of other genes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds as antioestrogens: characterization and mechanism of action.

In the female Sprague-Dawley rat uterus 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds exhibited a broad spectrum of antioestrogenic responses. For example 2,3,7,8-TCDD inhibited the 17 beta-oestradiol-induced uterine wet weight increase, peroxidase activity, oestrogen and progesterone receptor levels, epidermal growth factor (EGF) receptor binding, and EGF receptor and c-fos protooncogene mRNA levels. The aryl hydrocarbon (Ah) receptor was identified in the rat uterus and the antioestrogenic activities of TCDD and related compounds were structure-dependent. In parallel studies, the effects of TCDD as an antioestrogen in MCF-7 human breast cancer cells was also investigated. TCDD inhibited the 17 beta-oestradiol-induced proliferation of these cells and the secretion of the 34-, 52- and 160-kDa proteins. Treatment of MCF-7 cells with 1 nM [3H]-17 beta-oestradiol resulted in a rapid accumulation of nuclear oestrogen receptor (ER) complexes. Pretreatment of the cells with TCDD caused a rapid decrease in nuclear ER binding activity and immunoreactive protein; moreover, the structure-dependent potencies of TCDD and related compounds as antioestrogens were similar to their Ah receptor binding affinities. TCDD also caused a decrease in nuclear ER levels in wild-type Ah-responsive Hepa 1c1c7 cells but was inactive in Ah non-responsive mutant Hepa 1c1c7 cells. Moreover, in the wild-type cells, both actinomycin D and cycloheximide blocked the effects of TCDD. 6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) has previously been characterized as a TCDD antagonist in rodents and in transformed rodent cell lines. However, like TCDD, MCDF also exhibited a broad spectrum of antioestrogenic activities in both the female Sprague-Dawley rat uterus and MCF-7 cells. MCDF is relatively non-toxic compared to TCDD and is being investigated as a compound which may be clinically useful for the treatment of mammary cancer.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell growth and the secretion of the estrogen-induced 34-, 52- and 160-kDa proteins in human breast cancer cells.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the growth of estrogen-responsive MCF-7 human breast cancer cells in the presence of 17 beta-estradiol was determined. After treatment with 17 beta-estradiol (1 nM), TCDD (10 nM) and 17 beta-estradiol (1 nM) plus TCDD (10 nM) the cells were monitored daily for cell growth and DNA content for 7 days. The results showed that TCDD inhibited cell proliferation and DNA content of untreated cells and inhibited the 17 beta-estradiol-stimulated cell proliferation and increase in cellular DNA content. In contrast, TCDD did not effect the growth of estrogen non-responsive MDA-MB-231 human breast cancer cells. TCDD (0.1-10 nM) also caused a concentration-dependent decrease in the 17 beta-estradiol-induced proliferation in MCF-7 cells. The effects of TCDD on the 17 beta-estradiol-induced secretion of the 52-kDa protein (i.e. procathepsin D), the 34-kDa (cathepsin D) and 160-kDa proteins were also determined in the MCF-7 and MDA-MB-231 human breast cancer cell lines. The levels of the proteins were determined by autoradiographic analysis of the incorporation of [35S]methionine into the secreted proteins which were separated by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of MCF-7 cells with 17 beta-estradiol (1 nM), TCDD (10 and 100 nM) and 17 beta-estradiol (1 nM) plus TCDD (10 nM) resulted in levels of the 52-kDa protein which were 497, 63.6, 98.1 and 66.3%, respectively, of the corresponding levels observed in control (untreated) cells. Using the same concentrations, the levels of the 34-kDa protein secreted into the media were 372, 42.3, 64.0 and 43.8% of control values, respectively, and the corresponding levels of the 160-kDa protein were 381, 52.9, 71.2 and 76.6% of the control values, respectively. In contrast, treatment of MDA-MB-231 cells with 17 beta-estradiol (1 nM), TCDD (10 and 100 nM) and 17 beta-estradiol (1 nM) plus TCDD (10 nM) resulted in a 31-39% reduction in the secretion of the 52-kDa protein however these effects were not statistically different from the control values. In addition, the treatments did not cause any significant effects on the secretion of the 34- and 160-kDa proteins by MDA-MB-231 cells. These results clearly confirm and extend the range of antiestrogenic effects caused by TCDD in estrogen-responsive MCF-7 cells and indicate that the MDA-MB-231 cells are not responsive to the antiestrogenic effects of TCDD.

2,2',4,4',5,5'-hexachlorobiphenyl as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL/6J mice.

At doses as high as 750 to 1000 mumol/kg, 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP) did not cause fetal cleft palate, suppress the splenic plaque-forming cell response to sheep red blood cells, or induce hepatic microsomal ethoxyresorufin O-deethylase (EROD) in C57BL/6J mice. Despite the lack of activity of HCBP in eliciting any of these aryl hydrocarbon (Ah) receptor-mediated responses, competitive binding studies indicated that HCBP competitively displaced 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) from the murine hepatic cytosolic receptor. Cotreatment of C57BL/6J mice with TCDD (3.7 nmol/kg) and HCBP or 4,4'-diiodo-2,2',5,5'-tetrachlorobiphenyl (I2-TCBP) (400 or 1000 mumol/kg) showed that both compounds partially antagonized TCDD-mediated cleft palate and immunotoxicity (i.e., suppression of the splenic plaque-forming cell response to sheep red blood cells), and HCBP antagonized TCDD-mediated hepatic microsomal EROD induction. Thus, HCBP and I2-TCBP, like the commercial polychlorinated biphenyl mixture Aroclor 1254, were partial antagonists of TCDD action in C57BL/6J mice; however, it was also apparent from the results that Aroclor 1254 was the more effective antagonist at lower doses. Using [3H]TCDD, it was also shown that some of the effects of HCBP on TCDD-mediated cleft palate may be due to the decreased levels of TCDD found in the fetal palates after cotreatment with TCDD and HCBP. 4,4'-[125I2]diiodo-2,2',5,5'-tetrachlorobiphenyl ([125I2]TCBP) of high specific activity (3350 Ci/mmol) was synthesized and used to investigate the direct binding of this compound to the murine hepatic Ah receptor or other cytosolic proteins. No direct specific binding was observed between 125I2-TCBP and any cytosolic proteins using a sucrose density gradient assay procedure. These results contrasted with previous studies with Aroclor 1254 that suggested that this mixture acted as a competitive Ah receptor antagonist.

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL/6 mice.

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and TCDD plus MCDF were administered to C57BL/6 mice and their effects on several aryl hydrocarbon (Ah) receptor-mediated responses including hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction, immunotoxicity and teratogenicity were determined. MCDF did not induce hepatic microsomal AHH and EROD at doses up to 500 mumol/kg, however, co-administration of MCDF (50 mumol/kg) with a dose of TCDD which elicited a submaximal induction response (i.e. ED80-100, 15 nmol/kg) resulted in some small but significant inhibition of the induction of hepatic microsomal AHH and EROD (14 and 17%, respectively) compared to that observed with TCDD alone. Co-administration of TCDD and other doses of MCDF (10, 100, 200 or 500 mumol/kg) did not effect the induction response. These results were in contrast to the effectiveness of MCDF as an antagonist of the induction of AHH and EROD by TCDD in the rat (up to 50% inhibition of monooxygenase induction). Administration of MCDF (4, 20 and 40 mumol/kg) to C57BL/6 mice caused some inhibition of the splenic plaque-forming cell response to sheep erythrocytes only at the highest dose (26% decrease); the interaction of MCDF (4, 20 and 40 mumol/kg) and an immunotoxic dose of TCDD (3.7 nmol/kg) resulted in significant protection from the immunotoxic effects of TCDD at the 2 higher dose levels of MCDF. Similarly, MCDF (400 mumol/kg) did not cause cleft palate in mice but at this dose level MCDF afforded some protection from TCDD (20 micrograms/kg)-mediated cleft palate in mice. However, studies utilizing [3H]TCDD suggested that the protective effects may be due to modulation of TCDD reaching the palate in the co-treated animals (MCDF plus TCDD). Although both MCDF and Aroclor 1254 were both weak Ah receptor agonists in C57BL/6 mice, the former compound was much less effective as a TCDD antagonist. The observed species-specific effects for these 2 TCDD antagonists may be related species-dependent differences in receptor structure and receptor-ligand (i.e. agonist or antagonist) interactions.