In vivo and in vitro effects of flutamide and diethylstilbestrol on fetal testicular steroidogenesis in the rat

Prenatal testosterone surge is considered crucial for physiological masculinization of male progeny. Disorders in sex steroid hormone balance during the fetal development may interfere with male reproductive health later in life. In this study, we have investigated in utero and in vitro effects of flutamide (FLU) and diethylstilbestrol (DES) on fetal rat testicular steroidogenesis. In utero exposure to FLU 25mg/kg or DES 0.02mg/kg had no obvious effects on ED 19.5 rat testicular testosterone and progesterone production, StAR protein or AR protein expression. However, when ED 19.5 rat testis were cultured for 180min in the presence of 0.1, 1, 10 and 100mg/l of FLU or DES, the highest doses of both compounds were capable of disturbing steroidogenesis. To study the rate of the changes seen in testicular steroidogenesis after 180min, time-series experiments, in which intact testes were cultured with FLU 100mg/l or DES 100mg/l for 30, 60 or 120min, were performed. In vitro time-series experiments revealed that changes in steroidogenesis occur very fast. Experiments with FLU brought further evidence to the hypothesis that ARs have negative autocrine role in developing Leydig cells.     

Effects of neonatal exposure to 4-tert-octylphenol, diethylstilbestrol, and flutamide on steroidogenesis in infantile rat testis.

Exposure of neonatal testis, populated by fetal-type Leydig cells, to endocrine-active compounds may have far-reaching consequences. Our aim was to resolve the sensitivity of testosterone synthesis of infant rat (Sprague-Dawley) testis to diethylstilbestrol (DES; 0.1-1.0 mg/kg), 4-tert-octylphenol (OP; 10-100 mg/kg), and Flutamide (FLU; 2.0-25 mg/kg) given by daily sc injections from birth to postnatal day 4. Testes and serum were collected on day 14 when body and testis weight, testicular histology, circulating testosterone, LH and FSH levels, and steroidogenic acute regulatory protein (StAR) and 3beta-hydroxy-steroid-dehydrogenase (3beta-HSD) protein levels were determined. DES at each dose and FLU at 25 mg/kg dose reduced testis weight and the diameter of seminiferous cords. FLU caused some Leydig cell hyperplasia. Plasma testosterone was reduced in all DES animals, LH elevated in DES 0.5 mg/kg and FLU 25 mg/kg animals, and FSH reduced in the DES 1.0 mg/kg group. Basal testicular ex vivo progesterone and human chorionic gonadotropin (hCG)-stimulated testosterone production were decreased in DES animals. Despite a decrease in hCG-induced cyclic adenosine-3',5'-monophosphate (cAMP) production, intratesticular testosterone was increased in the FLU 10 and 25 mg/kg groups. OP 100 mg/kg elevated hCG-induced progesterone production only. No changes were seen in 3beta-HSD protein levels in any treatment group. StAR levels were reduced in DES animals. The results indicate the sensitivity of postnatal fetal-type Leydig cells to endocrine-active compounds. Suppression of StAR expression level was an early sign of the DES-induced steroidogenic lesion. FLU-induced changes suggest the importance of androgen receptor-mediated regulation of testosterone synthesis in the postnatal rat testis. Octylphenol appeared less effective in bringing about acute steroidogenic changes.     

In vitro effects of diethylstilbestrol, genistein, 4-tert-butylphenol, and 4-tert-octylphenol on steroidogenic activity of isolated immature rat ovarian follicles

Isolated rat ovarian follicles grow and produce steroid hormones in vitro and so provide a good model for studying the effects of hormonally active compounds on follicular steroidogenesis. We have evaluated the effects of diethylstilbestrol (DES), genistein (GEN) and two alkylphenols, 4-tert-butylphenol (BP) and 4-tert-octylphenol (OP) on the growth, survival, and steroid hormone and cAMP production by isolated 14-day-old rat (Sprague-Dawley) ovarian follicles. During a 5-day culture, FSH was obligatory for follicle growth and increased estradiol and testosterone secretion in a dose-dependent manner. DES (10(-6) M) caused the strongest decline in estradiol and testosterone levels but did not have detectable effects on either cAMP production or aromatase enzyme activity. GEN caused a prominent decrease in cAMP and testosterone levels without significant changes in secreted estradiol. The latter, apparently, was due to a dose-dependent stimulation of aromatase enzyme activity in the presence of genistein. Both BP and OP decreased estradiol and testosterone secretion in a dose-dependent manner while no effect on aromatase activity was observed. OP, unlike BP, decreased forskolin-induced cAMP levels. Xenoestrogens at the used concentrations did not interfere with the growth and survival of the follicles. The results indicate that isolated ovarian follicles representing intact morphological and functional units offer a sensitive model system for elucidating the female-specific reproductive effects of environmental chemicals.     

Long-lasting effects of lindane on mouse spermatogenesis induced by in utero exposure

Long-lasting effects on mouse spermatogenesis induced by prenatal exposure to the insecticide lindane have been investigated by conventional reproductive endpoints complemented by the flow cytometric (FCM) DNA content analysis of testis cells and by the Sperm Chromatin Structure Assay (SCSA). Two lindane dose levels, 15 and 25 mg/kg bw, and diethylstilboestrol (DES, 10 microg/kg bw) as positive control, were administered daily by gavage to pregnant CD1 mice on gestation days (GD) 9-16. Reproductive endpoints were evaluated on F1 male mice on postnatal day (PND) 60; additionally, animals treated with lindane 25 mg/kg per day and DES were examined on PND 100 to evaluate the possible reversibility of the effects. On PND 60, lindane and DES caused a reduction in the sperm head count and concentration, with recovery in older lindane 25 mg/kg per day animals (PND 100). By contrast, the DES group exhibited a greater reduction in the sperm head count on PND 100 than on PND 60. Changes in biochemical parameters in the testes, lactate dehydrogenase-C(4) (LDH-C(4)), and sorbitol dehydrogenase (SDH) activities, were also observed in adult treated F1 mice. Furthermore on PND 60, the FCM analysis revealed changes in the pattern of testicular germ cell distribution, especially in the haploid subcompartment, in the lindane 25 mg/kg per day group. A dose-dependent increase in chromatin abnormalities of the epididymal sperm was also shown by SCSA. These changes recovered on PND 100. Preliminary qualitative examination did not reveal any significant difference in the structure of testicular tissue; however, there were suggestions of a moderate increase in number and size of Leydig cells in both DES- and lindane-treated animals. The partial reversibility of these effects and the lack of structural modification of the testicular tissue as evidenced by histopathologic assessment suggest a functional impairment of sperm production and maturation, possibly associated with changes induced by lindane on factors affecting intratesticular steroidogenesis.     

Acetaldehyde acutely impairs canine testicular testosterone secretion

Utilizing a method for perfusion of the in vivo isolated canine testis we have examined basal and human chorionic gonadotropin (hCG)-stimulated testosterone production of testes acutely exposed to ethanol and acetaldehyde. Ethanol infused at concentrations of 0.2 g/dl to 0.6 g/dl did not alter basal or hCG-stimulated testicular testosterone production of one testis when compared to the saline-infused control testis of the same animal. However, acetaldehyde infused at a concentration of 0.2 mg/dl, a level similar to that found in humans drinking moderate amounts of ethanol, significantly impaired hCG-stimulated testicular testosterone production. It is concluded that acetaldehyde acutely impairs hCG-stimulated testicular testosterone secretion by a direct effect on the testis, but ethanol does not.     

The alkylphenols 4‐nonylphenol, 4‐tert‐octylphenol and 4‐tert‐butylphenol aggravate atopic dermatitis‐like skin lesions in NC/Nga mice

Phthalate esters in plastics act as adjuvants for immunoglobulin production, which aggravates allergic disease. However, the effects of alkylphenols (used as plasticizers and surfactants) on atopic dermatitis have not been studied in detail. Therefore, the goal of the present study was to investigate the effects of the alkylphenols 4‐nonylphenol (NP), 4‐tert‐octylphenol (OP) and 4‐tert‐butylphenol (BP) in a murine model of atopic dermatitis. NC/Nga mice were intraperitoneally administered NP, OP or BP and were subcutaneously injected with mite allergen in one ear to induce atopic dermatitis‐like skin lesions (ADSLs). The condition of the skin was observed, and the levels of immunoglobulin in serum and inflammatory cytokines in lesions were determined. NP exacerbated mite allergen‐induced ADSLs according to dose. OP and BP also significantly exacerbated skin lesions but not as a function of dose. Alkylphenols tended to increase the levels of IgE and antigen‐specific IgG₁ in serum. Further, the treatment of the alkylphenols increased the expression in lesions of inflammatory cytokines, interleukin‐4 and monocyte chemotactic protein‐3. Thymic stromal lymphopoietin levels increased according to ADSL severity. In contrast, the levels of the T‐helper 1 cytokines (interleukin‐18 and interferon‐gamma) decreased. NP, OP or BP may enhance T‐helper 2‐type immune responses in NC/Nga mice, which aggravates mite allergen‐induced ADSLs. Therefore, the uptake of very low levels of alkylphenols may contribute to the increase in the incidence of atopic dermatitis. Copyright © 2013 John Wiley & Sons, Ltd.     

Demonstration of a functional blood-testis barrier to acetaldehyde. Evidence for lack of acetaldehyde effect on ethanol-induced depression of testosterone in vivo

In vitro studies have shown that acetaldehyde is a more potent inhibitor of testicular steroidogenesis than ethanol. The present study examined the in vivo role of acetaldehyde in ethanol-induced reduction of testosterone by (1) determining the levels of acetaldehyde to which the testes were exposed subsequent to acute ethanol administration to mice; and (2) examining the effect of ethanol on testosterone in animals subsequent to drug pretreatment which decreased or increased ethanol-derived acetaldehyde. Ethanol-induced (3 g/kg) depression of testosterone was dependent upon gonadotropin stimulation. The increase in hCG-induced testosterone was suppressed (P less than 0.01) in ethanol- as compared to saline-treated animals [39.8 +/- 2.6 (S.E.M.) vs 28.1 +/- 2.3 ng/ml]. Pargyline (100 mg/kg) or cyanamide (8.4 mg/kg) increased (P less than 0.05) plasma and testicular acetaldehyde, while having no effect on the testosterone response to ethanol. Similarly, 4-methylpyrazole (25 mg/kg) reduced blood and testicular acetaldehyde to nondetectable levels, while having no effect on testosterone. Testicular acetaldehyde was lower (P less than 0.001) than plasma levels (14 +/- 2 vs 2.0 +/- 0.2 microM). This functional blood-testis barrier to acetaldehyde could be explained by testicular aldehyde dehydrogenases in the mitochondria (Km for acetaldehyde = 1.5 microM) and in the cytosol (Km = 123 microM) whose maximal activities totaled to more than 25-fold greater than that of testicular alcohol dehydrogenase (ADH). ADH was concentrated in the Leydig cells, while aldehyde dehydrogenase was evenly distributed in the testis. Ethanol prevented further hCG-induced rises in testosterone rather than inhibiting testosterone production to below pre-ethanol values. The above data argue against a significant role of acetaldehyde in the in vivo response of testosterone to ethanol. Ethanol appears to impair gonadotropin-testicular receptor interaction in vivo.     

Infantile 4-tert-octylphenol exposure transiently inhibits rat ovarian steroidogenesis and steroidogenic acute regulatory protein (StAR) expression

Phenolic compounds, such as 4-tert-octylphenol (OP), have been shown to interfere with rat ovarian steroidogenesis. However, little is known about steroidogenic effects of infantile OP exposure on immature ovary. The aim of the present study was to investigate the effects of infantile OP exposure on plasma FSH, LH, estradiol, and progesterone levels in 14-day-old female rats. The effect on ovarian steroidogenic acute regulatory protein (StAR) and FSH receptor (FSHr) expression was analyzed by Western blotting. Ex vivo analysis was carried out for follicular estradiol, progesterone, testosterone, and cAMP production. Sprague-Dawley rats were given OP (0, 10, 50, or 100 mg/kg) subcutaneously on postnatal days 6, 8, 10, and 12. On postnatal day 14, plasma FSH was decreased and progesterone increased significantly at a dose of 100 mg OP/kg. In addition, the highest OP dose advanced the time of vaginal opening in puberty. OP had no effect on infantile LH and estradiol levels or ovarian FSHr content. Ovarian StAR protein content and ex vivo hormone and cAMP production were decreased at all OP doses compared to controls. However, hormone levels recovered independent on FSH and even increased above the control level during a prolonged culture. On postnatal day 35, no statistically significant differences were seen between control and OP-exposed animals in plasma FSH, LH, estradiol, and progesterone levels, or in ovarian StAR protein content. The results indicate that the effect of OP on the infantile ovary is reversible, while more permanent effects in the hypothalamus and pituitary, as described earlier, are involved in the reduction of circulating FSH levels and premature vaginal opening.     

Lack of effect on rat testicular organogenesis after in utero exposure to 3-monochloropropane-1,2-diol (3-MCPD)

3-Monochloropropane-1,2-diol (3-MCPD) is a food-born contaminant known to display toxic effects on male reproduction, producing infertility in rats and humans. Using the rat as a model, we investigated whether or not testicular organogenesis, which, in the rat species, occurs during the second half of gestation, was at particular risk regarding 3-MCPD toxicity. Pregnant rats were given daily doses of 5, 10 or 25 mg/kg BW of 3-MCPD from days 11.5-18.5 postcoitum (dpc). On 19.5 dpc, testes were removed from fetuses for histological examination and testosterone analysis. Eight genes were selected among the differentiation markers of testicular cell lineages, and their expression was studied by RT-PCR. The levels of 3-MCPD and its main metabolite, beta-chlorolactic acid, were assayed in fetal tissues and dam plasma. Our results show a statistically significant decrease in the mean body weight gain of pregnant rats treated with 10 and 25 mg/kg BW of 3-MCPD. Fetal testes exposed to 3-MCPD exhibited normal histology and produced testosterone at levels that were similar to controls. In addition, 3-MCPD did not alter gene expression in the fetal testes. This lack of effect occurred under conditions where 3-MCPD and beta-chlorolactic acid were found to readily cross the placental barrier and diffuse throughout the fetal tissues. Our findings indicate that 3-MCPD has minimal effect on rat testicular organogenesis.     

Adult exposure to diethylstilbestrol induces spermatogenic cell apoptosis in vivo through increased oxidative stress in male hamster

Effects of diethylstilbestrol (DES), an endocrine disrupting chemical, on abnormal spermatogenesis were studied in adult hamster using daily subcutaneous injection of 0.01, 0.1 and 1.0mg/kg body weight for 1 week. Testicular weight and seminiferous tubular area gradually decreased as dosage increased to 1.0mg/kg DES. Germ cells were rarefied and showed irregular distribution in seminiferous tubules. Apoptosis was pronounced among spermatocytes and spermatids at the 1.0mg/kg dose level. Antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and total antioxide capacity (T-AOC) markedly decreased and malondialdehyde (MDA) concentration significantly increased in the testes. These results suggest that DES (1.0mg/kg) induces testicular oxidative stress and spermatogenic apoptosis in adult male hamsters to extend findings shown for prenatal and/or neonatal exposure.     

Rosiglitazone, an agonist of peroxisome proliferator-activated receptor-gamma, prevents contralateral testicular ischaemia-reperfusion injury in prepubertal rats

1. Rosiglitazone plays a positive role in the reparation of ischaemia-reperfusion (I/R) injury in different tissues. Thus, we examined its biochemical and histological effects on the contralateral testes to determine whether exogenous rosiglitazone affords any protection against testicular damage. 2. Forty-eight prepubertal male Wistar-Albino rats were divided into six groups. Testicular torsion was created by rotating the right testis 720 degrees in a clockwise direction for 5 h in all groups except group I, which was the sham-control group. In group II, bilateral orchiectomy was performed following the torsion period. After detorsion both testes were removed in the fifth hour in group III and on the seventh day in group IV. In group V, one-shot rosiglitazone (4 mg/kg) was administered 40 min before detorsion and both testes were removed following the torsion period. In group VI, rosiglitazone was administered (4 mg/kg) 40 min before detorsion and for 7 days, and then both testes were harvested. The tissue levels of malondialdehyde (MDA) were measured and mean testicular biopsy score (MTBS) and mean seminiferous tubule diameter (MSTD) were examined. Immunoexpression of endothelial nitric oxide synthase (eNOS) in testes tissues was investigated by immunohistochemical studies. 3. In the contralateral testis, the MTBS and MSTD values of group VI were significantly higher than those in group IV. Immunohistochemically, mild eNOS immunostaining was present in the germ cells of the contralateral testes in group IV after I/R. In group VI, intense eNOS immunoreactivity was seen in the contralateral testes. 4. Rosiglitazone reduces contralateral testicular damage formed after unilateral testicular torsion and alleviates the oxidative events.     

Inhibition of intratesticular testosterone synthesis by inorganic lead

The alterations in testicular testosterone synthesis produced by exposure to inorganic lead were investigated in BALB/c+ mice. Lead concentration in blood and testes and the levels of testosterone and delta 4-androgen biosynthesis pathway precursors (4-androstenedione, 17-hydroxyprogesterone, and progesterone) were measured in animals which were exposed to lead acetate in the drinking water (366 mg/l, 0.97 +/- 0.12 mg lead/animal/day) during 6 months. The results showed a significant reduction of the intratesticular testosterone levels after 30 days of exposure and of the androstenedione levels after 150 days. Intratesticular progesterone and hydroxyprogesterone levels showed no changes during the assay.     

Cimetidine-induced Leydig cell apoptosis and reduced EG-VEGF (PK-1) immunoexpression in rats: Evidence for the testicular vasculature atrophy

The antiulcer drug cimetidine has shown to cause changes in the testicular microvasculature of adult rats. Since Leydig cells (LCs) produce the pro-angiogenic factor, EG-VEGF (endocrine gland-derived vascular endothelial growth factor), also known as prokineticin 1 (PK-1), this study examined the effect that cimetidine might have on LCs in testes with damaged vasculature. Rats received intraperitoneal injections of 100 mg/kg of cimetidine (cimetidine group) or saline vehicle (control group) for 50 days. Serum testosterone levels were measured by chemiluminescence immunoassay and testicular sections were subjected to TUNEL and immunohistochemical reactions for caspase-3, 17β-HSD6, CD163 (ED2 macrophage), PK-1 and androgen receptor (AR). LCs in the cimetidine group showed TUNEL and caspase-3 positive labeling and apoptotic ultrastructural features. Moreover, the presence of 17β-HSD6-positive inclusions inside macrophages and the reduced number of LCs, AR immunoreactivity and serum testosterone levels correlated with a decrease in either the number of PK-1-immunostained LCs or PK-1 immunoreactivity. Although it is not clear which cell type is the primary target of cimetidine in the testicular interstitial compartment, these findings support a direct link between cimetidine-induced testicular vascular atrophy and LCs damage.     

Mechanisms underlying the anti-androgenic effects of diethylhexyl phthalate in fetal rat testis

Diethylhexyl phthalate (DEHP) is widely used as a plasticizer in consumer products and is known to disturb the development of the male reproductive system in rats. The mechanisms by which DEHP exerts these effects are not yet fully elucidated, though some of the effects are related to reduced fetal testosterone production. The present study investigated the effects of four different doses of DEHP on fetal testicular histopathology, testosterone production and expression of proteins and genes involved in steroid synthesis in fetal testes. Pregnant Wistar rats were gavaged from GD 7 to 21 with vehicle, 10, 30, 100 or 300 mg/kg bw/day of DEHP. In male fetuses examined at GD 21, testicular testosterone production ex vivo and testicular testosterone levels were reduced significantly at the highest dose. Histopathological effects on gonocytes were observed at 100 and 300 mg/kg bw/day, whereas Leydig cell effects were mainly seen at 300 mg/kg bw/day. Quantitative RT-PCR revealed reduced testicular mRNA expression of the steroidogenesis related factors SR-B1, StAR, PBR and P450scc. Additionally, we observed reduced mRNA expression of the nuclear receptor SF-1, which regulates certain steps in steroid synthesis, and reduced expression of the cryptorchidism-associated Insl-3. Immunohistochemistry showed clear reductions of StAR, PBR, P450scc and PPARgamma protein levels in fetal Leydig cells, indicating that DEHP affects regulation of certain steps in cholesterol transport and steroid synthesis. The suppression of testosterone levels observed in phthalate-exposed fetal rats was likely caused by the low expression of these receptors and enzymes involved in steroidogenesis. It is conceivable that the observed effects of DEHP on the expression of nuclear receptors SF-1 and PPARgamma are involved in the downregulation of steroidogenic factors and testosterone levels and thereby underlie the disturbed development of the male reproductive system.     

Effects of alkylphenols on bone metabolism in vivo and in vitro

Alkylphenols are endocrine disruptors that show estrogen-like effects in various wildlife species. However, little information is available about the action of these chemicals on bone metabolism. We investigated the effects of alkylphenols, such as nonylphenol (NP) and octylphenol (OP), on the formation of bone using several culture systems for osteoclasts and osteoblasts, as well as in vivo experiments. NP and OP dose-dependently inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) in cocultures of mouse spleen cells or mouse bone marrow cells with ST2 cells. However, beta-estradiol at 10(-9)M to 10(-6)M did not affect this process. In contrast, neither compound affected the proliferation and differentiation of rat calvarial osteoblast-like cells (ROB cells). When NP or OP (0.1mg/kg body weight) was administered subcutaneously to pregnant mice at 10 days, 12 days and 14 days post-coitus, fetuses at 17.5 days post-coitus showed stimulation of sternebrae bone calcification. Our findings suggest that alkylphenols have critical effects on the formation of bone by non-estrogenic effects.     

Serum and testicular testosterone and androgen binding protein profiles following subchronic treatment with carbendazim

While the general toxicity of the benzimidazole pesticides for mammals is low, one of these compounds, carbendazim (MBC), causes degeneration of testicular tissue and decreases spermatogenic activity at doses well below the LD50 value. A study conducted by S. D. Carter, R. A. Hess, and J. W. Laskey (1987, Biol. Reprod. 37, 709-717) showed that treatment with 400 mg/kg/day MBC resulted in severe seminiferous tubular atrophy and infertility. Since spermatogenesis is an androgen-dependent process, we characterized the effects of MBC (0-400 mg/kg/day) on the endocrine function of the rat testes. Following subchronic (85 day) exposure, serum hormones (TSH, LH, FSH, and Prl) were measured as were androgen binding protein (ABP) and testosterone in testicular fluids (interstitial fluid and seminiferous tubule fluid). In addition, the functional capacity of the Leydig cell to secrete testosterone was assessed in vitro following an hCG challenge. Subchronic treatment with MBC at doses of 50-100 mg/kg/day had no effect on pituitary or testicular hormone concentrations: 200 mg/kg/day elevated the testosterone concentration in the seminiferous tubule fluid and the ABP concentration in both the interstitial fluid and the seminiferous tubule fluid without affecting serum testosterone or ABP concentrations. The 400 mg/kg/day dose resulted in increased concentration of both testosterone and ABP in the interstitial fluid and seminiferous tubule fluid and elevated serum ABP, with no change in serum testosterone. This endocrine profile is consistent with the testicular atrophy and "Sertoli cell-only" syndrome seen in these animals as reported by Gray et al. (1987, Toxicologist 7, 717). We conclude that seminiferous tubule fluid testosterone may be a result of two factors: (1) increased interstitial fluid testosterone concentrations and (2) decreased testosterone outflow from the testis to the general circulation. Also, increased ABP in the interstitial fluid may reflect a change in the relative secretion of ABP into the interstitial fluid and the seminiferous tubules.     

Anti-estrogen prevents xenoestrogen-induced testicular pathology of eelpout (Zoarces viviparus)

Estrogenic alkylphenols have been shown to affect the reproductive system of male fish causing induction of vitellogenin synthesis and altered testis structure. However, it is still unknown whether the histopathological effects on the testes is mediated by the estrogen receptor or if it represent general toxicopathological effects. In the present study, the effects of different concentrations of the estrogenic chemical 4-tert-octylphenol on vitellogenin (Vtg) synthesis and testicular structure were investigated in the eelpout Zoarces viviparus during spermatogenesis. Adult male eelpout were exposed to 4-tOP (nominal concentrations: 10, 50 or 100 microg l(-1)) or 17beta-estradiol (E2; 0.5 microg l(-1)) in a continuous flow-through system for 3 weeks. A group of fish were exposed to 4-tOP (50 microg l(-1)) concomitantly with the anti-estrogen ZM 189,154 (20 microg g(-1) week(-1), i.p.). The Vtg concentration in plasma was measured by enzyme-linked immunosorbent assay. The testicular structure was examined by light microscopy and gamma-glutamyl transpeptidase (gamma-GTP) activity was measured in the testes. The testicular localization of gamma-GTP was analysed by enzyme histochemistry. A marked increase in the plasma Vtg concentration was observed after exposure to the actual concentration of 35 microg l(-1) 4-tOP (nominal concentration, 50 microg l(-1)), 63 microg l(-1) 4-tOP (nominal concentration, 100 microg l(-1)) or E2. Co-treatment with ZM 189,154 totally abolished the 4-tOP-dependent induction of Vtg synthesis. Exposure to 4-tOP or E2 caused a marked reduction in the testis mass and severely affected the testicular development and structure including the Sertoli cells (based on histology and gamma-GTP activity), resulting in impairment of spermatogenesis and degeneration of lobular structures. Other cellular abnormalities such as accumulations of yellowish-brown pigmented cells and increased interstitial fibrosis in the testes was also observed in the exposed fish. In the groups exposed to the nominal concentrations of 50 or 100 microg l(-1) all fish had severely affected testes, while both normal, moderately and severely affected testes were found in the group exposed to the nominal concentration of 10 microg l(-1). Co-treatment with ZM 189,154 abolished part of these 4-tOP-induced effects on the testicular growth and histological structure. The study demonstrates that an anti-estrogen can abolish effects on the testis caused by estrogenic chemicals, providing evidence that some of the effects are mediated by the estrogen receptor.     

Evaluation of acute hepatotoxic effects exerted by environmental estrogens nonylphenol and 4-octylphenol in immature male rats

Nonylphenol (NP) and 4-Octylphenol (4OP) have shown estrogenic properties both in vivo and in vitro. Researchers have known for years that estrogens induce a wide number of hepatotoxic actions in rodents. In order to study the acute hepatic effects exerted by NP and 4OP on rat liver the following endpoints were evaluated: relative liver weight (RLW), morphology, cell cycle and ploidy status, monooxygenase enzymes content and levels of both, cytosolic estrogen receptor (cER) and microsomal binding sites for estrogens (mEBS). Immature male Sprague-Dawley rats were injected intraperitoneally (i.p.) with 60 mg/kg of NP or 4OP for 1, 5 or 10 days. Despite the fact that RLW of the animals was not modified but any treatment, the histopathological study revealed the presence of an increase in the percentage of both, mitotic activity and Ki-67-labeling index (LI) in the livers from animals treated with alkylphenols in absence of any degenerative lesion. Furthermore, all the livers from alkylphenols-treated groups showed the presence of abnormal mitosis and c-mitosis. Although the levels of both, cER and cytochrome P450 (Cyt. P450) were not affected by any treatment, concentration of the mEBS was decreased after 10 days of treatment with alkylphenols. These findings taken together suggest that the exposition to alkylphenols induce cell proliferation and spindle disturbances and that these compounds are capable of modulating the expression of putative membrane receptors for estrogens.     

Epigallocatechin-3-gallate counters cisplatin toxicity of rat testes

Context: Epigallocatechin-3-gallate (EG), the main active flavonoid in green tea, has well-known anti-inflammatory, antioxidant, and anti-apoptotic activities.

Objective: The EG protection against testicular injury induced by cisplatin was studied in Sprague–Dawley rats.

Materials and methods: Cisplatin (10?mg/kg, i.p) was given as a single injection to rats. EG was given at 40 and 80?mg/kg/day, i.p., for 5 days, starting the same day of cisplatin insult. Serum testosterone, and testicular malondialdehyde, total antioxidant status, nitric oxide, interleukin-6, interleukin-1β, cytochrome C, Bax/Bcl-2 ratio, and caspase-3 were measured. In addition, testicular histopathological examination and immunohistochemical expression of testicular tumour necrosis factor-α were evaluated.

Results: Cisplatin, compared to the control, significantly decreased serum testosterone (6.48?±?0.7 vs. 50.8?±?4.91?ng/10?mL), and testicular tissue antioxidant status (17.3?±?1.21 vs. 64.12?±?5.4?μmol/g), and significantly increased interleukin-6 (85.81?±?6.11 vs. 38.2?±?2.79?pg/100?mg), interleukin-1β (98.09?±?8.31 vs. 32.52?±?2.08?pg/100?mg), malondialdehyde (74.5?±?5.88 vs. 23.8?±?1.91?nmol/g), nitric oxide (104.98?±?8.5 vs. 52.68?±?5.12?nmol/100?mg), cytochrome C (5.97?±?0.33 vs. 1.6?±?0.99?ng/mg protein), Bax/Bcl-2 ratio (4.01?±?0.38 vs. 0.71?±?0.0), and caspase-3 (3.2?±?0.21 vs. 0.98?±?0.08?O.D. 405?nm) in rat testes. EG (40 and 80?mg/kg, respectively) caused significant increases of serum testosterone (33.9?±?2.89 and 47.88?±?4.4?ng/10?mL), and testicular antioxidant status (47.1?±?3.92 and 58.22?±?3.58?μmol/g), and significant decreases of interleukin-6 (57.39?±?4.2 and 48.18?±?3.98?pg/100?mg), interleukin-1β (65.12?±?5.88 and 41.96?±?3.51?pg/100?mg), malondialdehyde (42.3?±?3.9 and 28.67?±?2.49?nmol/g), nitric oxide (70.6?±?6.79 and 61.31?±?5.18?nmol/100?mg), cytochrome C (3.4?±?0.27 and 2.21?±?0.18?ng/mg protein), Bax/Bcl-2 ratio (1.49?±?0.14 and 1.1?±?0.09), and caspase-3 (2.1?±?0.17 and 1.48?±?0.13?O.D. 405?nm) in testes of cisplatin-treated rats. Additionally, both doses of EG significantly ameliorated the histopathological injury and reduced tumour necrosis factor-α expression in rat testes.

Conclusion: EG can afford testicular protection in cisplatin-challenged rats by its antioxidant, antinitrative, anti-inflammatory and antiapoptotic effects.     

Effects of maternal 4-tert-octylphenol exposure on the reproductive tract of male rats at adulthood

Increases in human male reproductive disorders (testicular cancer, cryptorchidism, hypospadias, and low sperm counts) might stem from increased exposure of the developing male to environmental estrogens. In the present study, we investigated the effects of octylphenol (OP), an estrogenic compound, exposure on the male reproductive system during the fetal period in which the development of reproductive organs and sexual differentiation occurs. Male rats were treated with OP in utero at doses of control (vehicle), 100 or 250 mg/kg/day. After birth, male rats were allowed to grow until adulthood, and then testes, epididymides, and prostate glands were investigated histopathologically. Sperm counts and percentage of abnormal sperm were determined. Seminiferous and epididymal round tubules were evaluated for tubule diameter, lumen diameter, and height of tubule epithelium. Treatment with OP caused abnormalities in the histology of the testis and epididymis and induced atrophy of prostate glands tubules. Although there were no differences in sperm counts among treatment groups, abnormal sperm percentages in the high dose group increased significantly. The results of this study demonstrate that maternally injected OP causes adverse effects on male reproductive tract at adulthood, especially on sperm structure.