Endocrine active agents: implications of adverse and non-adverse changes.
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The US Environmental Protection Agency (EPA) is currently in the process of developing screening and testing methodologies for the assessment of agents that may possess endocrine-like activity--the so-called endocrine disruptors. Moreover, the EPA has signaled its intention of placing information arising from such studies on the worldwide web. This has created significant interest in how such information may be used in risk assessment and by policymakers and the public in the potential regulation or deselection of specific chemical agents. The construction of lists of endocrine disruptors, although fulfilling the requirements of some parties, is really of little use when the nature of the response, the dose level employed, and the lifestage of the test species used are not given. Thus, we have already seen positive in vitro information available on the interaction with a receptor being used as a key indicator when the results of large, high quality in vivo studies showing no adverse changes have been ignored. Clearly a number of in vitro systems are available to ascertain chemical interaction with specific (mainly steroid) hormone receptors including a number of reporter gene assays. These assays only provide indicators of potential problems and should not be, in isolation, indicators of toxicity. Likewise, short-term in vivo screens such as the uterotrophic and Hershberger studies are frequently conducted in castrated animals and thus indicate the potential for a pharmacological response in vivo rather than an adverse effect. A number of new end points have been added to standard rodent testing protocols in the belief of providing more sensitivity to detect endocrine related changes. These include the measurement of anogenital distance (AGD), developmental landmarks [vaginal opening (VO), preputial separation (PPS)], and in some studies the counting of nipples and areolae on males. AGD, VO, and PPS are all affected by the size of the pup in which they are measured and should always be compared using bodyweight as a covariate. The historical control database for such changes is gradually growing, albeit that if pups are not individually identified it becomes problematic to associate any change with a specific malformation or to assess whether a delay or advance in, for example, developmental landmarks is biologically significant. Agents that significantly reduce AGD in males (it is an androgen-dependent variable) frequently have other more adverse changes associated with this end point (eg, reproductive tract malformations), but a 2 to 3% change in AGD although measurable is unlikely to be biologically of importance and in isolation would not necessarily be considered adverse. Retention of thoracic nipples in male rat pups is also an indicator of impaired androgen status. Recent studies have also shown that this retention for some endocrine active chemicals is permanent. Thus, the presence of a permanent structural change that is rarely found in adult control animals could be considered a malformation and therefore a developmental adverse effect on which risk assessment decisions could be made. The advent of multigeneration reproduction studies as the definitive studies for the assessment of the dose-response relationships and risk assessment for endocrine disruptors has shown that current testing protocols may be inadequate to reliably detect the adverse effects of concern as only 1 adult/sex/litter is examined. A number of the effects on reproductive development although, due to an in utero exposure, will not be manifest until after puberty or at adulthood. The use of only a limited number of animals to examine such changes, particularly for weaker acting materials indicates that some agents may have been examined in well-conducted, modern protocols but have insufficient power to detect low incidence phenomena (eg, a 5% incidence of malformations). (+info)
Effects of perinatal exposure to flutamide on sex hormones and androgen-dependent organs in F1 male rats.
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Flutamide, which has antiandrogenic properties, was administered to pregnant rats, and effects on male offspring were examined. Crj: CD (SD) IGS (SPF) females were administered flutamide (0.15, 0.6, 2.5, 10.0, 100 mg/kg, p.o.) from gestation Day 14 to post parturition Day 3. The number of pups, body weights, clinical features, anogenital distance (AGD), nipple retention, testicular descent, and urogenital malformation in F1 males were examined. Hormone measurement, necropsy and histopathological examination were carried out at post-neonatal Day 4 (PND 4) and PND 60. Sperm analysis was also carried out at PND 60. Decrease in body weight was seen in the 100 mg/kg group and the AGD was decreased at 2.5 mg/kg and above. Retention of nipples, hypospadia, vaginal pouches, penis malformation, unilateral ectopic testis, and decrease of organ weights (prostate, seminal vesicles, levator ani muscle plus bulbocavernosus muscle, testis) were observed at 10 mg/kg and above. Testicular testosterone (T) was increased significantly with 100 mg/kg at PND 4 and tendencies for increase were observed in serum T, LH and FSH at 10 mg/kg and more at the same time point. In contrast, elevated levels of LH and FSH were seen with 100 mg/kg at PND 60. Histopathological examination revealed defects or hypoplastic changes of genital organs (> or = 10 mg/kg), squamous metaplasia (10 mg/kg) or mucification (100 mg/kg) of the urethral diverticulum epithelium and inflammation of genital organs (100 mg/kg). Though only undescended testes lacked spermatogenesis at 10 mg/kg, atrophic change of seminiferous tubules and azoospermia were observed in the 100 mg/kg group, despite testicular descent. Perinatal administration of flutamide affected F1 male rats at 2.5 mg/kg and above. In addition to urogenital malformation, 100 mg/kg flutamide caused high LH and FSH levels at PND 60. This study indicates that the most sensitive parameter is AGD, whereby reduction was observed at 2.5 mg/kg. A clear no-effect level (NOEL: 0.6 mg/kg) was obtained in this perinatal study of an antiandrogenic chemical. (+info)
Evaluation of the 20-day pubertal female assay in Sprague-Dawley rats treated with DES, tamoxifen, testosterone, and flutamide.
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The Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) has recommended the rodent pubertal female assay as a Tier I test to detect potential endocrine disrupters (EDs). This assay is designed to screen estrogenic activity in immature rats exposed to chemicals during sexual maturation. The aim of this study was to evaluate whether this assay can detect the EDs with effects brought about through various mechanisms. Immature Sprague-Dawley female rats (21 days of age) were dosed daily for 20 days by oral gavage (DES, tamoxifen, and flutamide) or sc injection (testosterone). The mean age at vaginal opening (VO) was 32.3 +/- 0.5 days in control rats. Although VO was unaffected by DES at doses of 0.2 and 1.0 microg/kg, a high dose of DES (5.0 microg/kg) significantly advanced the age at VO to 24 days. Both tamoxifen (50 and 200 microg/kg) and flutamide (25 mg/kg) also significantly accelerated VO to 27.8 +/- 0.5, 25.1 +/- 0.1, and 26.1 +/- 0.1, respectively. However, testosterone dose-dependently delayed VO (exposure to 1.0 mg/kg extended VO to 37.3 +/- 0.8 days, and VO did not occur in 2 of 10 animals by the time of necropsy at 41 days of age). Estrous cyclicity was monitored in rats from VO to necropsy. Irregular cycles were observed in the groups treated with DES (5.0 microg/kg), tamoxifen (200 microg/kg), testosterone (1.0 mg/kg), and flutamide (25 mg/kg). High dose of DES showed a persistent estrus state throughout the entire observation period. In addition, the number of days in diestrus was increased by tamoxifen (200 microg/kg) and flutamide (25 mg/kg) treatments. Significant decreases in ovarian weight were observed in 5.0 microg/kg DES (64% of control), 25 mg/kg flutamide (76% of control), and 200 microg/kg tamoxifen (47% of control). Testosterone also significantly decreased the ovarian weights in all treatment groups. Uterine weights were also decreased significantly at high doses of tamoxifen (200 microg/kg, 39% of control) or testosterone (1.0 mg/kg, 47% of control). In hormone analysis, tamoxifen significantly increased serum E(2) levels at 50 microg/kg. The mean serum levels of TSH were significantly increased in tamoxifen (10 and 50 microg/kg), testosterone (0.2 mg/kg), and flutamide (1.0 and 25 mg/kg) treatment groups compared with the control. However, serum T(4) levels were significantly reduced by testosterone. Furthermore, serum T(3) levels were significantly increased in DES, tamoxifen (10 and 50 microg/kg), testosterone (1.0 mg/kg), and flutamide (1.0 and 5 mg/kg). Our data demonstrate that the rodent pubertal female assay is useful for identifying potential EDs having not only estrogenic/antiestrogenic but also androgenic/antiandrogenic activities. However, further validation study is necessary to identify chemicals that operate through other action mechanisms, including steroid biosynthesis inhibitors and thyroid inhibitors. Moreover, additional data on other compounds with weak endocrine disrupting activity will be required to further characterize the sensitivity of the female pubertal assay. (+info)
Low serum testosterone level predicts worse response to endocrine therapy in Japanese patients with metastatic prostate cancer.
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Patients with prostate cancer generally respond to androgen withdrawal therapy, but progression to androgen-independence is frequently observed later. To examine whether pretreatment serum androgen status could predict disease progression in metastatic prostate cancer, pretreatment serum testosterone, histological grade, extent of bony metastasis, serum prostate-specific antigen (PSA) response to hormone therapy, and prognosis of the 40 patients with untreated metastatic prostate cancer who received endocrine therapy were evaluated. Although there were no differences in age, pretreatment PSA level, extent of bony disease and histological grade between patients with normal testosterone and those with low testosterone, PSA response after endocrine therapy was better in normal testosterone group. There was a significantly longer interval to disease progression in patients with normal testosterone than in those with low testosterone. The patients with metastatic prostate cancer with low serum testosterone were in the high risk group of worse response to endocrine therapy. Additional therapy might be considered in those patients. (+info)
Expression of aquaporin 9 in the adult rat epididymal epithelium is modulated by androgens.
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Reabsorption of fluid and solutes across the epithelium lining the male excurrent duct is important for adequate sperm maturation, concentration, and storage. Water channels contribute to water movement across epithelia in many tissues. Aquaporin 9 (AQP9) is abundantly expressed in the apical membrane of principal cells that line the epididymis, and in reabsorptive and secretory epithelial cells of the male reproductive tract. In this study we show that the nonsteroidal antiandrogen flutamide, given to adult rats at a dose of 50 mg x kg(-1) x day(-1) for 2 wk via osmotic minipumps significantly decreased the amount of AQP9 in the epididymis. This down-regulation was observed by immunofluorescence of cryostat tissue sections and by Western blotting of epididymal brush border membrane preparations. In addition, castrated adult rats showed lower levels of epididymal AQP9 compared with adult controls, whereas systemic testosterone treatment of castrated adult rats induced a recovery of the expression of AQP9 to control levels. These data indicate that the expression of AQP9, a likely candidate for apical transepithelial fluid and solute transport in several regions of the male reproductive tract, is modulated by androgens in the adult rat epididymis. (+info)
Cytotoxicity of flutamide and 2-hydroxyflutamide and their effects on CYP1A2 mRNA in primary rat hepatocytes.
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AIM: To compare the cytotoxicity of flutamide and its active metabolite 2-hydroxyflutamide and their effects on cytochrome P-450 1A2 mRNA in primary rat hepatocytes. METHODS: After the isolation of hepatocytes a nd t he primary incubation for 4 h, flutamide and 2-hydroxyflutamide were added respectively to the medium at the concentration of 10, 20, and 50 mg/L and incubated for 8 h. Cytotoxicity of hepatocytes was assessed by Trypan blue exclusion, lactate dehydrogenase (LDH) leakage, percentage of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) release, and reduced glutathione (GSH). The effect of flutamide and 2-flutamide on the CYP1A2 mRNA level was further analyzed by Northern blot. RESULTS: After incubation for 8 h, cell viability was observed by Trypan blue exclusion. The increase of ALT and AST activity and the decrease of glutathione content were also noted at 10, 20, and 50 mg/L of flutamide and 50 mg/L of 2-hydroxyflutamide as compared with normal rat hepatocytes. Induction of CYP1A 2 mRNA were 2-, 5-, and 7.5-fold at 10, 20, and 50 mg/L of flutamide and 3.5-fold at 50 mg/L of 2-hydroxyflutamide. CONCLUSION: Cytotoxicity of flutamide and its effect on CYP1A2 mRNA were stronger than those of its active metabolite 2-hydroxyflutamide in primary rat hepatocytes. (+info)
Androgens protect against apolipoprotein E4-induced cognitive deficits.
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Compared with apolipoprotein (apo) E2 and E3, apoE4 increases the risk of Alzheimer's disease (AD), but it remains unknown how apoE4 affects neuronal function. ApoE4 interacts with female gender, further increasing the risk of AD and decreasing treatment response. Female mice are also more susceptible to apoE4-induced impairments of spatial learning and memory than male mice. To assess the role of sex steroids in this process, we studied mice deficient in mouse apoE (Apoe(-/-)) and expressing human apoE4 or apoE3 in the brain at comparable levels. Even brief periods of androgen treatment improved the memory deficits of female apoE4 mice. Female apoE3 mice had no memory deficits and did not benefit from the treatment. ApoE4 male mice, which performed normally in a water-maze test at baseline, developed prominent deficits in spatial learning and memory after blockade of androgen receptors (ARs), whereas apoE3 male mice did not. Untreated apoE4 mice had significantly lower cytosolic AR levels in the neocortex than wild-type, Apoe(-/-), and apoE3 mice. Improved memory in androgen-treated female apoE4 mice was associated with increased cytosolic AR levels. Our findings suggest that apoE4 contributes to cognitive decline by reducing AR levels in the brain, and that stimulating AR-dependent pathways can reverse apoE4-induced cognitive deficits. (+info)
Effect of local heating of rat testes after suppression of spermatogenesis by pretreatment with a GnRH agonist and an anti-androgen.
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The effects of local heating of rat testes, in which spermatogenesis had been suppressed with injections of a GnRH agonist and an anti-androgen, were examined. Although the detrimental effects of heating were not as marked as those found in the testes of non-injected rats, the testes in which spermatogenesis was suppressed also showed a significant reduction in mass, the number of spermatozoa, tubular diameter and the percentage of normal tubular cross-sections at day 35 after heating. The results indicate that heating has an effect on cells in the testis other than those shown to be most susceptible to heat, namely pachytene spermatocytes and early spermatids, which were absent or markedly reduced in number when spermatogenesis was suppressed. The long-term effects of heating on the above parameters, as reported in a previous study, were also confirmed. However, in testes in which spermatogenesis was suppressed at the time of heating, there appeared to be no or a reduced long-term impairment of spermatogenesis, as determined by testis mass, the percentage of qualitatively normal tubules and epididymal sperm counts. (+info)