Detection of endocrine disrupters: evaluation of a Fish Sexual Development Test (FSDT)

Managed by the Organisation for Economic Co-operation and Development (OECD), a comprehensive work is carried out in numerous laboratories to develop test guidelines for the detection of endocrine disrupting chemicals in humans, and various animal species. Development of tests to detect chemicals with endocrine disrupting properties in fish is a part of that work. A Fish Sexual Development Test (FSDT) (an extension of the existing OECD TG 210, fish early life stage toxicity test), proposed as an international test guideline for the detection of endocrine disrupting chemicals, was evaluated by water exposure of juvenile zebrafish to the three natural estrogens: estrone, 17beta-estradiol, and estriol and the synthetic androgen trenbolone (trenbolone acetate). As endpoints, vitellogenin induction and histological changes including changes in sex ratios were investigated. The sex ratio was significantly altered towards females from 49 ng/l estrone, 54 ng/l 17beta-estradiol and 22 microg/l estriol, respectively. An all male population was observed from exposure to 9.7 ng/l trenbolone and above. Significant vitellogenin induction in whole body homogenate was measured after exposure to 14 ng/l estrone, 54 ng/l 17beta-estradiol and 0.6 mug/l estriol, respectively. Significant vitellogenin reduction was measured after exposure to 193 ng/l trenbolone or higher. The present results provide strong evidence that the FSDT is a sensitive test toward estrogenic and especially androgenic exposure and the validation of the FSDT as an OECD test guideline should continue.     

Elimination kinetic of 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate ester metabolites in calves' urine

Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C(18) SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CCalpha) for steroids were below 0.1 microg L(-1), except for 19-norandrosterone (CCalpha=0.7 microg L(-1)) and estrone (CCalpha=0.3 microg L(-1)). Kinetics of elimination of the administered 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate were established by monitoring 17beta-estradiol, 17alpha-estradiol, estrone and 17beta-nandrolone, 17alpha-nandrolone, 19-noretiocholanolone, 19-norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17alpha-estradiol and 17alpha-nandrolone (>20 and 2 mg L(-1), respectively after 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate administration) whereas 17beta-estradiol, estrone, 17beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the microg L(-1) level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17beta form.     

Elevation of Striatal Dopamine Receptors by Estrogen: Dose and Time Studies

Administration to male rats of a single dose of 17 beta-estradiol valerate (8-500 micrograms/rat) or implantation of a pellet containing 17 beta-estradiol (0.5-50 mg/rat) increased serum 17 beta-estradiol levels in a dose-dependent relationship when measured on the sixth day after administration. At the same time, after these doses, the serum rat prolactin (rPRL) levels were doubled and the striatal 3,4-dihydroxyphenylethylamine (DA, dopamine) receptor densities were increased 20%. A single dose of 17 beta-estradiol valerate of 4 micrograms/rat or less did not alter serum 17 beta-estradiol or rPRL levels or the striatal DA receptor density. After the single injection of 17 beta-estradiol valerate (125 micrograms/rat) the serum 17 beta-estradiol levels peaked at 1 day, the serum rPRL levels peaked at 2 days, and the striatal DA receptor density elevation peaked from 4 to 8 days. Implantation of a pellet containing 17 beta-estradiol (25 mg/rat) produced a constant elevation of serum 17 beta-estradiol levels from 1 to 10 days. Whereas the serum rPRL levels were continuously elevated about two-fold, the densities of the striatal DA receptors were increased significantly by 20-25% only from 4 to 8 days after pellet implantation. These results indicate that striatal DA receptor density rises and returns to control levels during the constant elevation of serum 17 beta-estradiol and rPRL levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma levels of dehydroepiandrosterone and 17 beta-estradiol after intramuscular administration of Gynodian-Depot in 3 women

Gynodian-Depot is well suited for treatment of the characteristic symptoms accompanying the menopause in women. The plasma levels of prasterone and 17 beta-estradiol after intramuscular injection of Gynodian-Depot in women were studied using a radioimmunological method. The maximum active ingredient concentrations were found in the plasma of the subjects only a few days after administration. The depot action lasted on an average 14 days (17 beta-estradiol) and 18 days (prasterone) after administration, respectively. Prasterone enanthate is completely hydrolysed into the free steroid and the fatty acid.     

On the role of 17 alpha-estradiol and 17 beta-estradiol in the proliferation of MCF7 and T47D-A11 human breast tumor cells

A comparative study of the proliferative effect of 17 beta-estradiol and 17 alpha-estradiol on human estrogen-sensitive cell lines was performed. When using charcoal-dextran stripped human female sera-supplemented media the administration of the hormones, 17 alpha-estradiol at 3 X 10(-10)M, and 17 beta-estradiol at 3 X 10(-11)M, resulted in a ten-fold increase in cell yield when compared with non-estrogen supplemented controls after cells were grown for periods between 10 to 14 days. No significant metabolization of 17 alpha-estradiol into 17 beta-estradiol occurred as measured by the E2 levels in the supernatants of the cell culture flasks. Increased concentrations of 17 beta-estradiol and 17 alpha-estradiol added to the media bathing C7MCF7-173 cells resulted in a triggering of a partially successful shut-off effect; this phenomenon was not observed with T47D-All cells. These results are compatible with predictions stemming from the indirect and direct negative working hypothesis for the regulation of cell proliferation.     

Androgen receptors in skeletal muscle cytosol from sheep treated with trenbolone acetate

Female sheep were implanted with the anabolic agent trenbolone acetate and the effects of the treatment on the skeletal muscle androgen receptor studied. In two separate trials the binding capacity/mg skeletal muscle protein was reduced. In trial 1 the animals were implanted (80 mg trenbolone acetate) at the start and after 5 weeks of the trial and samples taken one week later while in the second trial samples were taken 15 days after a single implant (80 mg trenbolone acetate). The ability of a variety of steroids and steroid-like compounds to displace [3H] testosterone and [3H] methyltrienolone was also studied. The androgen receptor was also partially purified using heparin-sepharose chromatography The involvement of the anddrogen receptor in the mode of action of trenbolone is discussed.     

Comparative morphological studies of lamb and calf Sertoli cells treated with anabolic agents.

The morphological alterations caused by anabolic steroids (oestradiol and trenbolone acetate) on the Sertoli cells of testicles in animals for human consumption (lambs and calves) were studied. The morphopathological study of the treated lambs revealed delayed development of the seminiferous tubules, which was marked by signs of immaturity and even degeneration of Sertoli cells. The main morphopathological alterations affecting the Sertoli cells in calves occurred as hyperfunction symptoms marked by increased nuclei and smooth endoplasmic reticulum.     

Behaviour of free-ranging beef cows and calves

The aim of this project was to describe the behaviour of free-ranging cows and calves after birth and during growth to the age of 6 months. Ten bull and 10 heifer calves were followed from birth until first suckling. Calves were observed to record their position in the field once a day until 10 days of age. Focal observations of 5 bull and 5 heifer calves were made from 27 to 167 days of age.

Of the cows studied, 2 separated completely from the herd at calving. The calving sites were randomly distributed in the area available. After birth all cows licked their calves. The amount of licking between 0 and 30 min was significantly greater than that between 30 and 60 min after birth. The duration of the first suckling was significantly longer for heifer calves than for bull calves. Eleven of 17 calves changed area during the first day after birth. The duration of cows licking calves did not change significantly from 27 to 167 days of age, and was not correlated to duration of licking immediately after birth. Suckling frequency per hour, suckling time per hour and duration of each suckling did not change significantly from 27 to 167 days of age. Bull calves from 27 to 167 days of age had a significantly higher frequency than heifer calves of sniffings towards adult cows other than the mother and a significantly higher frequency of mountings of adult cows. Cows and calves spent more time together when the calf was a female than when it was a male, and more time when the weaning weight was low than when it was high.     

Regulation of leptin gene expression and secretion by steroid hormones.

Previous work has shown that 17 beta-estradiol is the primary ovarian signal regulating body weight and adiposity, although its mechanisms of action remain unclear. We hypothesized that 17 beta-estradiol could enhance leptin levels as a mechanism of its anorectic effects. Administration of 5 microg 17 beta-estradiol subcutaneously (s.c.) for 2 days significantly elevated leptin mRNA levels in adipose tissue as compared to vehicle controls (P < 0.003). A time-course administration of estrogen showed increased mRNA levels in adipose tissue between 6 and 12 h after estrogen injection as compared to vehicle controls (P < 0.03). Corresponding to the increased leptin mRNA levels at 6 and 12 h, elevated plasma leptin levels were observed at 12 h after estrogen administration as compared to controls (P < 0.05). Administration of progesterone (1 mg/rat) after estradiol injection did not enhance the elevated leptin mRNA levels in adipose tissue. Serum leptin levels from cycling rats did not differ significantly between metestrous and proestrous animals. In conclusion, the present studies demonstrate that 17 beta-estradiol can regulate leptin gene expression and secretion in the female rat, thus providing a better understanding of the possible anorectic effect of estrogens.     

Changes in the mutagenic and estrogenic activities of 17beta-estradiol after treatment with nitrite

We determined the changes in the mutagenic and estrogenic activities of 17beta-estradiol after a nitrite treatment. Nitrite-treated 17beta-estradiol showed mutagenic activities toward Salmonella typhimurium strains TA 100 and TA 98. We confirmed that nitrite-treated 17beta-estradiol generated radicals from the results of an analysis of electron spin resonance. By applying an instrumental analysis, we identified 2-nitro-17beta-estradiol to have been formed in the reaction mixture. 2-Nitro-17beta-estradiol did not exhibit mutagenic activities toward Salmonella typhimurium strains, suggesting that other mutagens might have been formed in the reaction mixture. The clastogenic properties of nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol were analyzed by a micronucleus test with male ICR mice. Nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol induced a significantly higher frequency of micronucleated reticulocytes in mice. The estrogenic activity of 2-nitro-17beta-estradiol was found to be lower than that of 17beta-estradiol. These data suggest that a daily oral intake of 17beta-estradiol and nitrite might induce the formation of mutagenic compounds in our body.     

IGF-I mRNA levels in bovine satellite cell cultures: effects of fusion and anabolic steroid treatment

Androgenic and estrogenic steroids enhance muscle growth in a number of species; however, the mechanism by which anabolic steroids enhance muscle growth is not known. Castrated male cattle (steers) provide a particularly good model system in which to study the effects of anabolic steroids on muscle growth because they respond dramatically to treatment with both estrogens and androgens. The goal of this study was to determine if treatment of bovine satellite cell (BSC) cultures with 17beta-estradiol (E(2)) or trenbolone (a synthetic androgen) directly affects proliferation rate or level of mRNA for estrogen receptor (ER)-alpha, androgen receptor, and growth factors that have been shown to affect muscle growth (insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-3, and myostatin). BSC cultures were established from the semimembranosus muscles of steers and then treated for 48 h with various concentrations of E(2) or trenbolone ranging from 0.001 to 10 nM. IGF-I mRNA levels in proliferating BSC cultures were significantly increased at 0.01 (1.9-times control values, P < 0.02) and at 0.1, 1, and 10 nM E(2) (2.9-, 3.5-, and 3.5-times control values, respectively, P < 0.0001). Additionally both 1 and 10 nM trenbolone increased IGF-I mRNA levels to 1.7-times control values (P < 0.02). ER-alpha mRNA was detectable in BSC cultures, and levels were increased (2.3-times control levels, P < 0.001) in cultures treated with 0.001 nM E(2) but not in cultures treated with higher concentrations of E(2). Androgen receptor mRNA levels also were increased (1.5-times control levels, P < 0.02) in cultures treated with 0.001 nM trenbolone but not by treatment with higher concentrations of trenbolone. Levels of IGFBP-3 were increased (1.4-times control values, P < 0.02) by treatment with 0.001 nM E(2) but not by treatment with high concentrations of E(2). Myostatin mRNA levels were not affected by any concentration of either of the steroids. Although, levels of IGF-I mRNA were 10-times greater (P < 0.02) in fused BSC cultures than in proliferating cultures, treatment of fused cultures for 48 h with 10 nM E(2) increased IGF-I mRNA levels (2.5-times control levels, P < 0.02). Both E(2) and trenbolone increased (3)H-thymidine incorporation rate (1.5-times control levels, P < 0.001) in BSC cultures in media containing serum from which IGFBP-3 had been removed by anti-IGFBP-3 affinity chromatography. In summary, treatment of BSC cultures with either E(2) or trenbolone increased IGF-I mRNA level and proliferation rate, thus, establishing that these steroids have direct anabolic effects on cells present in the BSC culture.     

Effect of anabolics on bovine granulosa-luteal cell primary cultures

Granulosa cell tumours are observed with increased frequency among calves slaughtered in Northern Italy. The use of illegal anabolics in breeding was taken into account as a cause of this pathology. An in vitro approach was used to detect the possible alterations of cell proliferation induced by anabolics on primary cultures of bovine granulosa-luteal cells. Cultures were treated with different concentrations of substances illegally used in cattle (17beta-estradiol, clenbuterol and boldione). Cytotoxicity was determined by means of MTT test, to exclude toxic effects induced by anabolics and to determine the highest concentration to be tested. Morphological changes were evaluated by means of routine cytology, while PCNA expression was quantified in order to estimate cell proliferation. Cytotoxic effects were revealed at the highest concentrations. The only stimulating effect on cell proliferation was detected in boldione treated cultures: after 48 h treated cells, compared to controls, showed a doubled expression of PCNA. In clenbuterol and 17beta-estradiol treated cells PCNA expression was similar to controls or even decreased. As the data suggest an alteration in cell proliferation, boldione could have a role in the early stage of pathogenesis of granulosa cell tumour in cattle.     

The action of trenbolone acetate, a synthetic anabolic steroid, on ovarian function in the guinea pig

The action of trenbolone acetate, a synthetic anabolic steroid, on ovarian function was investigated in the guinea pig. Certain comparisons were made with testosterone, the naturally occurring androgen, administered as the phenylpropionate ester. Two milligrams trenbolone acetate per kg given subcutaneously on alternate days for 20 days blocked oestrous cyclicity and ovulation in 9 of 10 animals. A similar effect was shown by 2.2 mg of testosterone phenylpropionate. Treatment of trenbolone acetate-treated animals with exogenous gonadotrophins suggested that the production of follicle-stimulating hormone had been suppressed. Signs of abnormality were seen in the livers of animals receiving 2 mg trenbolone acetate and 2.2 mg testosterone phenylpropionate.     

Effects of natural and synthetic estrogens and progestins on glycogen deposition in female mice

Glycogen deposition and glucose tolerance were examined in female mice after 24 days of oral treatment with natural (17 beta-estradiol and progesterone) and synthetic (ethinyl estradiol and norethisterone acetate) sex steroids, administered individually and in estrogen-progestin combination. Doses were 5 micrograms/kg/day for estrogens and 1 mg/kg/day for progestins. Compared with diestrus control mice, each treatment increased glycogen deposition in liver, uterus, heart and biceps femoris muscle. 17 beta-Estradiol produced the greatest increments. Progesterone produced considerably smaller increments and antagonized the glycogenic effects of 17 beta-estradiol. Ethinyl estradiol and norethisterone acetate generally induced similar changes in glycogen deposition. Treatments containing 17 beta-estradiol improved glucose tolerance. Although glucose tolerance was not significantly altered by the other sex steroid treatments, the changes in glycogen deposition indicate important effects on tissue carbohydrate metabolism.     

Additional effects of postpuberal estrogen injections on the vaginal epithelium in neonatally estrogenized mice

In the ovariectomized mice given 10 injections of 100 micrograms 17 beta-estradiol at intervals of 2 weeks from 60 days of age, the vaginal epithelium was atrophic when killed more than 2 months after the last injection. If mice given 3 daily injections of 20 micrograms 17 beta-estradiol from the day of birth were similarly treated with estradiol after postpuberal ovariectomy, the vaginal epithelium was stratified and hyperplastic at autopsy performed more than 2 months later. These changes in the epithelium persisted for at least 30 days after transplantation of the vaginae to normal ovariectomized hosts. Neonatal treatments only did not produce such persistent vaginal changes. In view of these results, additional effects of neonatal and postpuberal injections of estrogen on the vaginal epithelium are evident. However, effects of such neonatal and postpuberal injections of estrogen might be transient on the uterine epithelium, since abnormal proliferation was not observed in it.     

Effects of anabolics on the thermostimulated current spectrum in calf muscles

The effects of growth substances (zeranol, trenbolone) on calf muscle (pectoralis transversus) have been studied with a recent physical method (thermostimulated current spectroscopy). This method appears promising for detecting meat from calves treated with such hormonal substances. The authors have hypothesized that the observed differences are related to protein modification, the nature of which is yet unknown. A complementary work is now in progress.     

Effects of estrogen on venous function in rats with chronic heart failure

The effect of 17beta-estradiol on venous function was investigated in ovariectomized rats with heart failure. Rats (50-60 days old) were ovariectomized and implanted with 60-day-release pellets that contain 17beta-estradiol (1.5 mg) or vehicle. The left coronary artery was ligated 7 days later. Another group of ovariectomized rats was given vehicle pellets and then a sham operation was performed. The rats were studied while under pentobarbital anesthesia at 7 wk after ligation. Ligated rats, relative to sham groups, had lower mean arterial pressure (MAP, -34 mmHg) and cardiac output (CO, -38%); higher arterial resistance (R(A), +12%) and venous resistance (R(V), +116%); mean circulatory filling pressure (MCFP, +40%) and left ventricular end-diastolic pressure (LVEDP, +11 mmHg); and similar cardiovascular responses to norepinephrine (NE). Treatment of ligated rats with 17beta-estradiol increased CO (+16%); reduced R(A) (-16%), R(V) (-35%), MCFP (-23%), and LVEDP (-3 mmHg); and augmented MAP, R(V,) and MCFP responses to NE. Therefore, 17beta-estradiol reduced MCFP, and this reduced preload (LVEDP). 17beta-Estradiol decreased R(V), which, along with decreased R(A) (afterload), led to an increase in CO. 17beta-Estradiol likely augmented vasoconstriction to NE through an improvement on the cardiovascular status.     

Control of oestrus in primiparous sows

The weaning to service records were analysed for 69 primiparous sows in the University College herd. More large white sows had returned to service within 7 days of weaning than crossbreds (O<0.05). However, there was no significant difference in the mean interval from weaning to heat in Large White or crossbred animals (10.5 vs 13.3 days). Sows with a weaning to service interval of less than 12 days had an average litter size of 10.3 piglets compared with 10.9 piglets when the interval was greater than 12 days. In attempts to gain control over the interval from weaning to oestrus 63 primiparous sows were allocated to either control or 20 mg allyl trenbolone per head per day for either 3 or 7 days starting on the day of weaning. Following a 7-day allyl trenbolone regime, no sow showed signs of oestrus prior to day 6 post-withdrawal, but 82% of sows were in oestrus 6–8 days post treatment, compared with 17% of controls. Results indicate that oestrus can be synchronized in about 80% of treated animals in the period of 3–8 days post-treatment. Pregnancy rates and average litter sizes were 82.9, 88.2, 88.9 and 10.5, 10.7 and 11.0 for the three treatments, respectively.     

Ostertagia ostertagi: establishment of patent infections in calves by intravenous inoculation

In two experiments calves raised free of parasite infection were given parenteral injections of Ostertagia ostertagi infective larvae to determine if patent infections might result. Patency was achieved by intravenous injection of larvae. Ten calves of different age (3–8 months) and sex were given intravenous, subcutaneous, and intraperitoneal injections of infective larvae. These calves were not necropsied; patency was based on fecal egg counts. Six calves injected intravenously all achieved patency (17–21 days after inoculation). Two calves each were inoculated subcutaneously and intraperitoneally; patency was observed in none. Calves given primary intravenous inoculation responded with higher levels of ova production to subsequent oral challenge inoculation.In a second experiment, six 2–3-month-old calves were injected intravenously with 186,000 infective larvae. Calves were killed at 2, 7, 12, 20 and 30 days after inoculation and had a tissue reaction in the lungs to migrating larvae characterized by focal granulomas, interstitial thickening of alveolar walls, and some hemorrhage. Infective larvae were recovered from the lungs at 2, 7 and 12 days, fourth stage larvae from the abomasum at 7 and 12 days, and adults only from the abomasum at 20 and 30 days. It was considered that larvae reached the abomasum by way of the trachea and by then being swallowed. Clinical signs of disease were not observed.     

Testicular toxicity and mutagenicity of steroidal and non-steroidal estrogens in the male mouse.

The mutagenicity and toxicity of diethylstilbestrol (DES), 17 beta-estradiol and zeranol on the male mouse germ cells were investigated with meiotic micronucleus assays in vivo and in vitro, sperm-head abnormality test and morphometry. Further, the developmental effects of DES on testicular morphology were explored. Micronucleus induction was observed at 10(-7) M concentration of DES and 17 beta-estradiol in vitro, but other treatments yielded negative results. The micronucleus assay in vivo revealed a small number of micronuclei in early haploid spermatids 17 days after a single subcutaneous injection of DES 50 mg/kg, whereas estradiol and zeranol gave negative results. The sperm-head abnormality rates were significantly elevated 5 weeks after treatments with high doses of DES, 17 beta-estradiol and zeranol, and testicular morphometry revealed transient changes in the volume densities of testicular tissue components. Prenatal and neonatal estrogen administration resulted in permanent alterations in seminiferous epithelium and dilatation of the rete testis, but did not affect micronucleus or sperm-head abnormality rates. The mutagenicity and toxicity of hormones in the mouse testis paralleled the hormonal activity of these compounds. Early estrogenization was the most sensitive toxicity test, followed by in vitro meiotic micronucleus induction, whereas the sperm-head abnormality assay and morphological analysis did not reveal subtle changes.