Effect of tributyltin on the development of ovary in female cuvier (Sebastiscus marmoratus)

Organotin compounds, such as tributyltin (TBT) used as an antifouling biocide, can induce masculinization in female mollusks. However, few studies addressing the effect of TBT in fish have been reported. This study was conducted to investigate effects of TBT at environmental levels (1, 10, 100ng/L) on the development of ovary in female cuvier. TBT exposure elevated testosterone levels, increased the ratio of testosterone to 17beta-estradiol and decreased 17beta-estradiol levels in ovaries after 50 days compared to the control. Three stages of follicles (primary growth stage, yolk vesicle stage, vitellogenic stage) were observed in the ovaries of cuvier at the control and 1ng/L TBT group. The ovaries at the 10ng/L TBT group were characterized by the lack of vitellogenic stage follicles and instead had higher proportions of primary growth stage follicles. 100ng/L TBT resulted in follicles that were entirely at the earliest (primary growth stage) stages of development. There was a significant increase in apoptotic ovarian follicular cells judged by TUNEL-positive cell at the 10ng/L TBT group. The TUNEL-positive follicles were observed at the 100ng/L TBT group. The result in the present study showed that TBT at environmentally realistic concentrations can inhibit the ovarian development in fish. Besides the changes of sex hormone induced by TBT, apoptosis appears to be one mechanism affecting ovarian development.     

Tributyltin impairs the coronary vasodilation induced by 17β-estradiol in isolated rat heart

Triorganotins, such as tributyltin (TBT), are environmental contaminants that are commonly used as antifouling agents for boats. However, TBT is also known to alter mammalian reproductive functions. Although the female sex hormones are primarily involved in the regulation of reproductive functions, 17β-estradiol also protects against cardiovascular diseases, in that this hormone reduces the incidence of coronary artery disease via coronary vasodilation. The aim of this study was to examine the influence of 100 ng/kg TBT administered daily by oral gavage for 15 d on coronary functions in female Wistar rats. Findings were correlated with changes in sex steroids concentrations. Tributyltin significantly increased the baseline coronary perfusion pressure and impaired vasodilation induced by 17β-estradiol. In addition, TBT markedly decreased serum 17β-estradiol levels accompanied by a significant rise in serum progesterone levels. Tributyltin elevated collagen deposition in the heart interstitium and number of mast cells proximate to the cardiac vessels. There was a positive correlation between the increase in coronary perfusion pressure and incidence of cardiac hypertrophy. In addition, TBT induced endothelium denudation (scanning electron microscopy) and accumulation of platelets. Moreover, TBT impaired coronary vascular reactivity to estradiol (at least in part), resulting in endothelial denudation, enhanced collagen deposition and elevated number of mast cells. Taken together, the present results demonstrate that TBT exposure may be a potential risk factor for cardiovascular disorders in rats.     

Influence of triphenyltin exposure on the hypothalamus-pituitary-gonad axis in male Sebastiscus marmoratus

Both triphenyltin (TPT) and tributyltin (TBT) have been used as ingredients of antifouling biocides. However, far fewer studies addressing the reproductive toxicity of TPT on fishes are available than for TBT. The present study was conducted to investigate the effects of TPT at environmentally relevant concentrations on testicular development in male rockfish Sebastiscus marmoratus and to gain insight into its mechanism of action. After exposure for 48 days, the gonadosomatic index had decreased, and there was a reduced number of mature sperm and an abundance of the late stages of spermatocysts in the testes. Although the testosterone levels in the testes were elevated and the 17β-estradiol levels were decreased, spermatogenesis was suppressed. The activity of γ-glutamyl transpeptidase (which is used as a Sertoli cell marker) was decreased after TPT exposure, and serious interstitial fibrosis was observed in the interlobular septa of the testes exposed to TPT. The increased expression of cGnRH-II (chicken-II type gonadotropin-releasing hormone) and sGnRH (salmon-type GnRH), and the decreased expression of LHβ (luteinizing hormone) in the fish brains were detected. The expression of FSHβ (follicle-stimulating hormone) was decreased at day 21, while was increased slightly at day 48. The changes of cGnRH-II, sGnRH, FSHβ and LHβ mRNA levels might have mainly resulted from the alteration of the sex steroids via feedback mechanisms. The decrease of the FSHβ mRNA might have been one of the reasons causing the dysfunction of Sertoli cells, which play a critical role during spermatogenesis. The results suggested that TPT could perturb the function of hypothalamus-pituitary-gonad axis, and inhibiting the spermatogenesis.     

Involvement of sex hormonal regulation of K+ channels in electrophysiological and contractile functions of muscle tissues

Sex hormones, such as testosterone, progesterone, and 17β-estradiol, control various physiological functions. This review focuses on the sex hormonal regulation of K⁺ channels and the effects of such regulation on electrophysiological and contractile functions of muscles. In the cardiac tissue, testosterone and progesterone shorten action potential, and estrogen lengthens QT interval, a marker of increased risk of ventricular tachyarrhythmias. We have shown that testosterone and progesterone in physiological concentration activate KCNQ1 channels via membrane-delimited sex hormone receptor/eNOS pathways to shorten the action potential duration. Mitochondrial K⁺ channels are also involved in the protection of cardiac muscle. Testosterone and 17β-estradiol directly activate mitochondrial inner membrane K⁺ channels (Ca²⁺ activated K⁺ channel (K_Ca channel) and ATP-sensitive K⁺ channel (K_ATP channel)) that are involved in ischemic preconditioning and cardiac protection. During pregnancy, uterine blood flow increases to support fetal growth and development. It has been reported that 17β-estradiol directly activates large-conductance Ca²⁺-activated K⁺ channel (BK_Ca channel) attenuating arterial contraction. Furthermore, 17β-estradiol increases expression of BK_Ca channel β1 subunit which enhances BK_Ca channel activity by DNA demethylation. These findings are useful for understanding the mechanisms of sex or generation-dependent differences in the physiological and pathological functions of muscles, and the mechanisms of drug actions.     

Sex differences in dizocilpine (MK-801) neurotoxicity in rats

The sex differences in the clinical signs and the distribution of astrocytic glial fibrillary acidic protein (GFAP) induced by an N-methyl-d-aspartate antagonist, dizocilpine (MK-801), were examined. A single intraperitoneal injection of MK-801 (5 mg/kg body weight) caused a prolonged recumbency (35-40 h), leading to a severe loss of body weight in female rats, in contrast to a light effect in males, independent of age. Early salivation or lacrimation was also severe in females and delayed bloody lacrimation was observed in females only. The pretreatment with 17β-estradiol (0.1 or 1.0 mg/kg body weight) made early signs worse in both sexes, but a remarkable mortality (20-40%) was observed in females only. The treatment with MK-801 greatly enhanced GFAP expression in retrospenial cortex of both sexes with a higher enhancement in females. The MK-801-induced expression of GFAP was further increased by the pretreatment with 17β-estradiol (1 mg/kg body weight) in females. Overall, the expression of GFAP in the retrospenial cortex of rats treated with MK-801 appeared to be higher in females than males, somewhat in parallel with more severe clinical signs in females. The results indicate the higher sensitivity of female rats to MK-801 neurotixicity, and the possible involvement of 17β-estradiol in the sex differences of the sensitivity.     

Effects of benzo(a)pyrene exposure on killifish (Fundulus heteroclitus) aromatase activities and mRNA

Cytochrome P450 aromatase (CYP19) plays an important role in steroid homoeostasis by converting androgens to estrogens. To evaluate the effects of benzo(a)pyrene (BaP), a model carcinogenic PAH and AhR ligand, on aromatase mRNA expression and enzyme activity, adult Fundulus were exposed to water-borne BaP (1 and 10 microg/L) for 15 days, and embryos were exposed to 10 microg/L for 10 days. Effects of BaP were examined by tissue, gender, and season in adults. Constitutively, the sexes did not have significantly different CYP19A2 mRNA levels, however females had higher brain aromatase activity. Female control killifish had more than 700-fold more CYP19A1 mRNA in their gonads compared to males. Within brain tissue of both sexes, there was 100-fold more CYP19A2 mRNA compared to CYP19A1. In ovary, CYP19A1 predominated by approximately 30-fold over the CYP19A2, but in testis there was relatively more CYP19A2. In embryos there was approximately 5-fold higher CYP19A2 expression. Due to high inter-individual variability, a significant effect of BaP treatment by gender, season or age was not observed for either aromatase mRNA. However, ovarian aromatase activity was significantly decreased by 10 microg/L BaP, while female brain activity was increased following winter exposure. These findings suggest that the aromatase enzyme is a potential target for disruption of fish developmental and reproductive physiology by BaP.     

Effects of ECF-Kraft pulp mill effluent treated with fungi (Rhizopus oryzae) on reproductive steroids and liver CYP1A of exposed goldfish (Carassius auratus)

The toxicity of bleached Kraft pulp mill effluents (BKME) is usually attributed to chemical compounds which are produced and released throughout various stages of pulp and paper production. The main objective of the present work was to detect sub-lethal responses of goldfish (Carassius auratus) to secondary treated BKME which was treated with Rhizopus oryzae. A total of 96 carps (C. auratus; 11 ± 3 g) were exposed to different concentrations of the post-treated effluent (0, 1, 10, 25, 50, and 100%), in 28 days semi-static tests. Several biomarkers were then evaluated to assess the toxicological effects: induction of CYP1A (metabolic processes of organic compounds in liver), change in steroid profiles (11-Ketotestosterone, 17β-estradiol), histopathology of liver and gonads and somatic indices (GSI, HSI) for endocrine disruption and other physiological disturbances. The most significant results show an induction of CYP1A in both sexes and a decrease of 17β-estradiol concentrations in females. Histopathological changes such as liver tissue degeneration were observed in fish exposed to 50 and 100% of the BKME. Although the BKME was biologically treated there are some chemical compounds in the effluent that are capable to affect fish physiology, however, a clear evidence for endocrine disruption was not found.     

Molecular and cellular effects of chemicals disrupting steroidogenesis during early ovarian development of brown trout (Salmo trutta fario)

A range of chemicals found in the aquatic environment have the potential to influence endocrine function and affect sexual development by mimicking or antagonizing the effects of hormones, or by altering the synthesis and metabolism of hormones. The aim of this study was to evaluate whether the effects of chemicals interfering with sex hormone synthesis may affect the regulation of early ovarian development via the modulation of sex steroid and insulin‐like growth factor (IGF) systems. To this end, ex vivo ovary cultures of juvenile brown trout (Salmo trutta fario) were exposed for 2 days to either 1,4,6‐androstatriene‐3,17‐dione (ATD, a specific aromatase inhibitor), prochloraz (an imidazole fungicide), or tributyltin (TBT, a persistent organic pollutant). Further, juvenile female brown trout were exposed in vivo for 2 days to prochloraz or TBT. The ex vivo and in vivo ovarian gene expression of the aromatase (CYP19), responsible for estrogen production, and of IGF1 and 2 were compared. Moreover, 17β‐estradiol (E2) and testosterone (T) production from ex vivo ovary cultures was assessed. Ex vivo exposure to ATD inhibited ovarian E2 synthesis, while T levels accumulated. However, ATD did not affect ex vivo expression of cyp19, igf1, or igf2. Ex vivo exposure to prochloraz inhibited ovarian E2 production, but did not affect T levels. Further prochloraz up‐regulated igf1 expression in both ex vivo and in vivo exposures. TBT exposure did not modify ex vivo synthesis of either E2 or T. However, in vivo exposure to TBT down‐regulated igf2 expression. The results indicate that ovarian inhibition of E2 production in juvenile brown trout might not directly affect cyp19 and igf gene expression. Thus, we suggest that the test chemicals may interfere with both sex steroid and IGF systems in an independent manner, and based on published literature, potentially lead to endocrine dysfunction and altered sexual development. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 199–206, 2014.     

Effect of testosterone replacement or duration of castration on baroreflex bradycardia in conscious rats

Background

In this study, we tested the hypothesis that 17β-estradiol contributes to testosterone-mediated restoration of baroreflex-mediated bradycardia in short-term (3 weeks) castrated rats. Further, a reported increase in serum testosterone after long-term (6 weeks) castration constituted a basis for testing the hypothesis that a spontaneous increase in serum testosterone or androstenedione in this model causes a commensurate increase in baroreflex-mediated bradycardia.

Results

Testosterone (1 week) replacement enhanced baroreflex-mediated bradycardia in short-term castrated rats without changing 17β-estradiol level. A spontaneous recovery of baroreflex-mediated bradycardia occurred following long-term castration, although circulating testosterone and androstenedione remained suppressed.

Conclusion

The data suggest: 1) 17β-Estradiol does not contribute to testosterone restoration of the baroreflex-mediated bradycardia in short-term castrated rats. 2) The long-term modulation of baroreflex-mediated bradycardia occurs independent of androgens, or the baroreflex mechanism may become adapted to low levels of circulating androgens.     

Sex reversal of the amphibian, Xenopus tropicalis, following larval exposure to an aromatase inhibitor

Aromatase is a steroidogenic enzyme that catalyzes the conversion of androgens to estrogens in vertebrates. Modulation of this enzyme's activity by xenobiotic exposure has been shown to adversely affect gonad differentiation in a number of diverse species. We hypothesized that exposure to the aromatase inhibitor, fadrozole, during the larval development of the tropical clawed frog, Xenopus tropicalis, would result in masculinization of the developing female gonad. Tadpoles were exposed to fadrozole at nominal concentrations from 1 to 64 microg/L in a flow-through system from < 24 h post-fertilization (Nieuwkoop Faber (NF) stage 15-20) to metamorphosis (NF stage 66). At metamorphosis, morphologically examined gonads indicated complete masculinization of all tadpoles at concentrations of 16 microg/L and above and a significant bias in sex ratio towards males at concentrations of 1 microg/L and above. No effects on time to metamorphosis, body mass, or body length were observed. A random subsample of frogs was raised to reproductive maturity (39 weeks post-fertilization) in control water. All frogs exposed as tadpoles to 16 microg/L fadrozole or greater possessed testes at sexual maturity. Intersexed gonads characterized by the presence of both testicular and ovarian tissue were observed in 12% of frogs in the 4 microg/L treatment. No differences in estradiol, testosterone, or vitellogenin plasma concentrations were observed in exposed males or females compared to controls. Females in the 4 microg/L treatment possessed a significantly greater percentage of pre-vitellogenic oocytes than controls and were significantly smaller in body mass. No differences in sperm counts were observed in exposed males compared to controls. Results from this study demonstrate that larval exposure to an aromatase inhibitor can result in the complete masculinization of female gonads. These masculinized females are phenotypically indistinguishable from normal males at adulthood. Lower levels of aromatase inhibition resulted in intersexed gonads and possible female reproductive impairment at adulthood. These results indicate that exposure of amphibians to xenobiotics capable of inhibiting aromatase would result in adverse reproductive consequences.     

Ovotoxicity and PPAR-mediated aromatase downregulation in female Sprague-Dawley rats following combined oral exposure to benzo[a]pyrene and di-(2-ethylhexyl) phthalate

The aim of the present study was to determine the ovotoxicity of female Sprague-Dawley (SD) rats exposed to benzo[a]pyrene (B[a]P) and di-(2-ethylhexyl) phthalate (DEHP), either alone or in combination; the molecular mechanism and the combined effects were also evaluated. Female rats were given intragastric administration of control (corn oil), B[a]P (5 and 10mg/kg), DEHP (300 and 600 mg/kg) and B[a]P+DEHP (at 5mg/kg and 300 mg/kg respectively, or at 10mg/kg and 600 mg/kg respectively) on alternate days for 60 days. Relative ovary weight, estrous cycle, 17β-estradiol blood level, ovarian follicle populations, granulosa cell apoptosis, and gene and protein expression of P450Arom and PPAR were investigated. Our study demonstrated that the combination of B[a]P and DEHP exerts ovotoxicity in female rats and suppression of sex hormone secretion and homeostasis, which is associated with prolonged estrous cycles, decreases in ovarian follicle populations and granulosa cell apoptosis involving a PPAR-mediated signaling pathway of action of the two chemicals. In addition, based on qualitative assessment of the combined toxicity, no interaction effects were observed following combined B[a]P and DEHP administration.     

Polysubstrate monooxygenase activity and sex hormones in pre- and postspawning rainbow trout (Salmo gairdneri)

The hepatic microsomal cytochrome P-450 content and polysubstrate monooxygenase (PSMO) activities as well as plasma levels of sex hormones in gonadally mature rainbow trout (Salmo gairdneri) were studied.The plasma level of testosterone in prespawning female fish exceeded that of male fish. In both sexes, the testosterone level was at its highest in March and decreased considerably before the spawning time. The 17 β-estradiol content in female fish showed similar changes.The hepatic cytochrome P-450 content was at its lowest in prespawning fish in March, and significantly lower in females than in males. The sex difference in the cytochrome P-450 content disappeared after spawning. The absorption maximum of the reduced cytochrome-CO-complex varied from 449–450 nm without any correlation to the sex of the fish or the cytochrome P-450 content.In PSMO activities (benzo[a]pyrene, 7-ethoxycoumarin, aminopyrine and ethylmorphine as substrates), a significant sex difference could be observed during the prespawning period. In all cases the activity was lower in females than in males. The 7-ethoxy-resorufin deethylase activity did not, however, show any sex difference. Benzo[a]pyrene hydroxylase and 7-ethoxyresorufin deethylase activities in both sexes increased gradually during the pre- and postspawning times, being at their highest level in June. However, aminopyrine demethylase activity increased considerably during the prespawning period, and then decreased during and after spawning.The results indicate that changes in monooxygenase activities are dependent on the substrate used and the state of spawning, refering to the presence of multiple forms of cytochrome P-450 in the rainbow trout liver. Prior to spawning the rainbow trout is probably most sensitive to direct acting chemicals and most resistant to activation requiring compounds due to the much lowered levels of cytochrome P-450 and several monooxygenase activities.     

Brain cytochrome P450 aromatase activity in roach (Rutilus rutilus): seasonal variations and impact of environmental contaminants

P450 aromatase catalyses the conversion of C19 androgens to C18 estrogens which is thought to be essential for the regulation of the reproductive function. In this study, brain aromatase activity (AA) was measured monthly over a reproductive cycle in wild roach (Rutilus rutilus) sampled in a reference site in Normandy. AA peaked during the breeding season, reaching 35 fmol mg(-1)min(-1) in both male and female fish, and was low during the rest of the year except for a significant rise in October. AA was correlated with ovary maturation (measured either as gonado-somatic index or by histological analysis of the gonads) and plasma sex-steroid levels (11-ketotestosterone in males and 17-β-estradiol in females). Measurements of AA in polluted sites showed that activity was significantly upregulated in sites with fish showing high levels of plasma vitellogenin and large proportion of intersexuality (20-50%) thus suggesting the occurrence of estrogenic compounds and their involvement in AA modulation.     

Comparative embryotoxicity of pulp mill extracts in rainbow trout (Oncorhynchus mykiss), American flagfish (Jordanella floridae) and Japanese medaka (Oryzias latipes)

This study evaluated the effects of Chilean pulp mill effluent extracts (untreated, primary and secondary treated pulp mill effluents), along with steroid standards (testosterone and 17β-estradiol) and a wood extractive standard (beta-sitosterol) on developing post-fertilized fish embryos. Our study included a cold freshwater species, rainbow trout (Oncorhynchus mykiss), and two warm freshwater species American flagfish (Jordanella floridae) and Japanese medaka (Oryzias latipes). Embryotoxicity results included delay in time to hatch and decreased hatchability but no significant egg and larvae mortality was observed in the pulp mill extract exposed embryos. By contrast, significant early hatching and increased hatchability were observed in beta-sitosterol exposed embryos, along with high mortality of testosterone exposed embryos across species. Teratogenic responses were observed in medaka embryos in all treatments. Abnormalities were detected starting at development stages 19-20 (2-4 somite stages) and included optical deformities (micro-opthalmia, 1 or 2 eyes) and lack of development of brains and hearts. Additionally, phenotypic sex identification of surviving offspring found female-biased sex-ratios in all treatments except testosterone across species. Overall, our study indicated that Chilean pulp and paper mill extractives caused embryotoxicity (post-fertilized embryos) across species and irrespective of the effluent treatment. The effects were mainly associated with delayed time to hatch, decreased hatchability, and species-specific teratogenesis.     

Tributyltin impairs the reproductive cycle in female rats

Triorganotins are environmental contaminants, commonly used in antifouling agents for boats, that bioaccumulate and thus are found in mammals and humans due to ingestion of contaminated seafood diets. The importance of triorganotins as environmental endocrine disruptors and consequent reproductive toxicity in different animal models is well known; however, the adverse effects on reproductive cycle are less well understood. The potential reproductive toxicity of tributyltin (TBT) on regular reproductive cycling of female rats was examined. Wistar female rats (12 wk old, weighing approximately 230 g) were divided into two groups: control (vehicle, ethanol 0.4%) and tributyltin (100 ng/kg/d, 7 d/wk, for 16 d by gavage). Tributyltin significantly decreased the cycle regularity (%), duration of the reproductive cycle, the proestrus and diestrus phases, and number of epithelial cell in proestrus phase. TBT also increased the duration of metestrus and the number of cornified cells in this phase. Ovary weight and serum 17β-estradiol levels decreased markedly, accompanied by a significant increase in progesterone levels. Histological analysis showed apoptotic cells in corpus luteum and granulosa cells layer, with cystic follicles after TBT exposure. Tributyltin also elevated number of atretic follicles and corpoa lutea. The micronucleus (MN) test, using Chinese hamster ovary cells, demonstrated a concentration-dependent mutagenic effect of TBT, and at 2.0?×?10(-2)ng/ml most of the cells were nonviable. The toxic potential of TBT over the reproductive cycle may be attributed to changes found in the ovarian weight, unbalanced levels of sexual female hormones, and number of ovarian follicles and corpora lutea.     

Reduction of spermatogenesis in mice after tributyltin administration

Organotin compounds, such as tributyltin (TBT) used as an antifouling biocide, can induce masculinization in female mollusks. However, few studies addressing the effect of TBT on spermatogenesis in mammalian have been reported. This study was conducted to investigate the effects of TBT at low doses (0.5, 5, and 50mug/kg, respectively) on spermatogenesis in mice as exposed from puberty and gave insight into the mechanism. After exposure for 30 days, the gonadosomatic index (GSI) was significantly decreased. The testosterone levels in the testes were not altered and the 17beta-estradiol levels were significantly decreased in a dose-dependent manner, spermatogenesis of the testis was significantly inhibited. Estrogen receptor (ER-alpha and ER-beta) levels in testes of the mice exposed to TBT were decreased in a dose-dependent manner. The results suggest that ER play an important role in TBT-mediated inhibition of spermatogenesis.     

Dicamba affects sex steroid hormone level and mRNA expression of related genes in adult rare minnow (Gobiocypris rarus) at environmentally relevant concentrations

Dicamba is a benzoic acid herbicide that has been detected in surface and ground water. The herbicide has been shown to have cytogeneic and DNA damaging effects and to cause organ damage in mammals; however, little is known about the endocrine disrupting effects of dicamba in fish. In this study, histological changes, plasma vitellogenin (VTG) and sex hormone levels, and mRNA expression of sex steroid hormone‐related genes were determined in adult rare minnow exposed to environmentally relevant concentrations of dicamba (0, 0.05, 0.5, 5, and 50 μg/L) for 40 days. The results showed inhibition of spermatogenesis in male testes and ovarian degeneration in females. Plasma 17β‐estradiol (E2) levels were significantly increased in both genders, and plasma VTG levels were significantly increased in males (p < 0.05). These results indicate that sex hormone homeostasis and normal reproduction of fish could be affected by dicamba. Moreover, the mRNA levels of vtg were significantly upregulated in the livers and gonads of both male and female rare minnows (p < 0.05). The downregulation of cytochrome P450c19a (cyp19a) and steroidogenic acute regulatory protein (star) mRNA levels, and the upregulation of cytochrome P450c17 (cyp17) mRNA levels were observed in the livers and ovaries (p < 0.05). The results of the mRNA analysis suggest that changes in steroid hormone‐related gene expression could serve as a regulatory mechanism to maintain sex hormone homeostasis. Overall, dicamba exposure could result in histological lesions, plasma VTG increases, changes in sex hormone levels, and alterations of hormone‐related gene expression. Therefore, dicamba should be considered to be a potential endocrine disruptor. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 693–703, 2015.