An effect assessment of microplastics and nanoplastics interacting with androstenedione on mosquitofish (Gambusia affinis).

An increasing number of microplastics have been detected in aquatic environments, causing various damage to organisms. The size of microplastics affects the toxicity once they enter the organisms. Meanwhile, there is an increasing variety of Endocrine-disrupting chemicals (EDCs) present in aquatic environments. Androstenedione (AED) is a typical EDC. In this study, we used polystyrene microspheres of 80 nm (NPs) and 8 µm (MPs) as materials to simulate environmental contaminants in the aquatic environment with AED. We used female mosquitofish (Gambusia affinis) as the research object to investigate the effects of microplastics on fish in waters containing AED. We compared different sizes of particles accumulation in some tissues of fish and variation of enzyme activities (SOD, LDH, CAT), and the content of MDA in the gut. MPs, NPs, and AED combined exposure test investigated mRNA profiles of immune-related genes (IL-1ß, IL-6, IL-8, IL-10) and hormone receptor genes (ARα, ARß, ERα, ERß) in the liver of fish. Our results indicated that MPs emerged in various tissues (gill, gut, and liver) of mosquitofish. Besides, NPs and MPs caused enteric abnormal enzyme activity after 48 h of exposure, which was particularly pronounced in the MPs-AED group. MPs induced significant upregulation of inflammatory factors and gonadal factor genes after 96 h of exposure, which was more pronounced when co-exposed with AED. In conclusion, NPs and MPs caused mechanisms of immune damage and inflammatory response. MPs were found to be more likely to cause adverse reactions than NPs, and these responses were enhanced by the combined effects of AED. This study demonstrated that AED can exacerbate the negative effects of MPs and NPs on mosquitofish. It provided an important basis for the effective assessment of MPs and NPs on bioaccumulation and biochemical status of mosquitofish. Additionally, it serves as a foundation to investigate the interactive effects of microplastics and EDCs in living organisms.

Androstenedione (a Natural Steroid and a Drug Supplement): A Comprehensive Review of Its Consumption, Metabolism, Health Effects, and Toxicity with Sex Differences.

Androstenedione is a steroidal hormone produced in male and female gonads, as well as in the adrenal glands, and it is known for its key role in the production of estrogen and testosterone. Androstenedione is also sold as an oral supplement, that is being utilized to increase testosterone levels. Simply known as "andro" by athletes, it is commonly touted as a natural alternative to anabolic steroids. By boosting testosterone levels, it is thought to be an enhancer for athletic performance, build body muscles, reduce fats, increase energy, maintain healthy RBCs, and increase sexual performance. Nevertheless, several of these effects are not yet scientifically proven. Though commonly used as a supplement for body building, it is listed among performance-enhancing drugs (PEDs) which is banned by the World Anti-Doping Agency, as well as the International Olympic Committee. This review focuses on the action mechanism behind androstenedione's health effects, and further side effects including clinical features, populations at risk, pharmacokinetics, metabolism, and toxicokinetics. A review of androstenedione regulation in drug doping is also presented.

Histological and transcriptional effects of androstenedione in adult zebrafish (Danio rerio).

As a natural androgen, androstenedione (AED) may pose potential risks to aquatic organisms due to its ubiquitousness in aquatic environments. Here we assessed the adverse effects of AED on histology of gonads, as well as mRNA expression levels of 34 genes concerned with hypothalamic-pituitary-gonadal (HPG) axis, germ-cell differentiation and sex differentiation in zebrafish (Danio rerio). Adult zebrafish were exposed to solvent control and three measured concentrations of 0.2, 2.3 and 23.7 µg/L AED for 60 days. The results showed that AED did not induce any obvious histological effects in the ovaries and testes. Of the investigated genes, transcriptional expression levels of amh and cyp11c1 genes in the ovaries of females were significantly increased by AED at 2.3 or 23.7 µg/L. However, different exposure concentrations of AED significantly inhibited mRNA expression of gnrh3, atf4b1 and cyp19a1b in the brain of males. In the testes of males, AED at 2.3 µg/L led to a significant induction of sox9b gene, but it at 23.7 µg/L down-regulated nr5a1b gene. These observed transcriptional changes indicated that AED could pose potential androgenic effects in zebrafish.

Acute exposure to 4-OH-A, not PCB1254, alters brain aromatase activity but does not adversely affect growth in zebrafish.

Acute developmental exposure to pharmaceuticals or environmental contaminants can have deleterious, long lasting effects. Many of these compounds are endocrine disruptors (EDCs) that target estrogen signaling, with effects on reproductive and non-reproductive tissues. We recently reported that zebrafish larvae transiently exposed to the pharmaceutical EDC 4-OH-A display visual deficits as adults. Here, we examine whether these long-term effects are due to compound-induced morphological and/or cellular changes. Zebrafish aged 24 h, 48 h, 72 h, or 7 days post-fertilization (larvae) or 3-4mos (adults) were exposed to either 4-OH-A or PCB1254 for 24 h. After that time, notochord length, eye diameter, inter-eye distance, and heart rate were measured from larvae; and aromatase (estrogen synthase) activity was measured in homogenates of adult brain tissue. In general, indices of larval growth and development were not altered by 24 h exposure to either compound. 4-OH-A potently inhibited aromatase activity, while PCB1254 did not, with inhibition continuing even after removal from treatment. These results support differential function of EDCs and indicate that developmental exposure to 4-OH-A causes sustained inhibition of aromatase, which could be associated with altered adult behaviors.

Transcriptional effects of androstenedione and 17α-hydroxyprogesterone in zebrafish embryos.

Steroid hormones in the aquatic environment may pose a risk to fish health. Here we evaluated effects of two different class steroids that frequently occur in the aquatic environment, the androgen androstenedione (A4) and the progestin 17α-hydroxyprogesterone (17-OHP4). Zebrafish embryos were exposed to four concentrations of A4 and the positive control testosterone and to 17-OHP4, and transcriptional changes were determined at 96 h post fertilization (hpf) and 120 hpf. Transcriptional changes of 18 selected genes were assessed upon exposure to measured concentrations of 0.004, 0.046, 0.62 and 6.56 µg/L A4. Significant induction of the genes encoding sulfotransferase (sult2st3) and aromatase (cyp19b) occurred in 120 hpf embryos at 6.56 µg/L A4 and 1 µg/L testosterone. Additionally, cyp2k7 was significantly induced in two of three independent experiments. 17-OHP4 did not induce physiological effects (muscle contraction, heart rate, hatching success, swimming activity) at concentrations between 0.01 and 10 µg/L. Of the analyzed 15 genes, slight transcriptional alterations occurred for the genes encoding progesterone receptor, aromatases (cyp19a) and (cyp19b) and cyp2k7 at 10 µg/L. Our study highlights sult2st3, cyp19b and cyp2k7 as potential markers of androgen exposure in fish and indicates that 17-OHP4 is not likely to pose a risk for fish at environmental concentrations.

Masculinization and reproductive effects in western mosquitofish (Gambusia affinis) after long-term exposure to androstenedione.

Androstenedione (AED) is a naturally occurring steroid hormone. It is metabolized to potent androgens, which may induce androgenic effects in fish. However, little is known whether and how the androgens interfere with the fish gonadal development and reproduction. This study aimed at demonstrating the effects of long-term AED exposure on reproduction and development in mosquitofish (Gambusia affinis). The growth, development and several morphological endpoints, including the segment number and length of anal fin, histological changes of gonads and liver, were evaluated in mosquitofish during development from fertilized embryo to adulthood (180 days) after exposure of AED at environmentally relevant concentrations. We found that the growth (length, body weight and condition factor) of fish was negatively correlated with AED concentration in females, but not in males. The significant elongation of the ray and increment of segment numbers in the anal fin, were detected in all mosquitofish after exposure. Moreover, AED exposure (0.4gµ/L) caused damages in gonads and reduced the number of pregnant females. These findings indicate that AED has adverse effects on the growth and development of the western mosquitofish after long-term exposure (180d). Long-term exposure (180d) to AED, including environmentally relevant concentration (0.4µg/L and 4µg/L), induced masculinization in female mosquitofish under the experimental conditions.

From the Cover: Genomic Effects of Androstenedione and Sex-Specific Liver Cancer Susceptibility in Mice.

Current strategies for predicting carcinogenic mode of action for nongenotoxic chemicals are based on identification of early key events in toxicity pathways. The goal of this study was to evaluate short-term key event indicators resulting from exposure to androstenedione (A4), an androgen receptor agonist and known liver carcinogen in mice. Liver cancer is more prevalent in men compared with women, but androgen-related pathways underlying this sex difference have not been clearly identified. Short-term hepatic effects of A4 were compared with reference agonists of the estrogen receptor (ethinyl estradiol, EE) and glucocorticoid receptor (prednisone, PRED). Male B6C3F1 mice were exposed for 7 or 28 days to A4, EE, or PRED. EE increased and PRED suppressed hepatocyte proliferation, while A4 had no detectable effects. In a microarray analysis, EE and PRED altered >3000 and >670 genes, respectively, in a dose-dependent manner, whereas A4 did not significantly alter any genes. Gene expression was subsequently examined in archival liver samples from male and female B6C3F1 mice exposed to A4 for 90 days. A4 altered more genes in females than males and did not alter expression of genes linked to activation of the mitogenic xenobiotic receptors AhR, CAR, and PPARα in either sex. A gene expression biomarker was used to show that in female mice, the high dose of A4 activated the growth hormone-regulated transcription factor STAT5b, which controls sexually dimorphic gene expression in the liver. These findings suggest that A4 induces subtle age-related effects on STAT5b signaling that may contribute to the higher risk of liver cancer in males compared with females.

Effects of androstenedione exposure on fathead minnow (Pimephales promelas) reproduction and embryonic development.

High concentrations (300 ng/L) of androstenedione (A4) were identified in snowmelt runoff from fields fertilized with manure from livestock feeding operations in Wisconsin, USA. In fishes, A4 is an active androgen and substrate for biosynthesis of functional androgens (e.g., testosterone and 11-ketotestosterone) and estrogens (e.g., estradiol-17ß). Thus, A4 has the potential to be a powerful endocrine disruptor. This hypothesis was tested by exposing reproductively mature fathead minnows to 0.0 ng/L, 4.5 ng/L, 74 ng/L, and 700 ng/L A4 for 26 d in a flow-through system. Various reproductive endpoints were measured including fecundity, fertilization success, secondary sexual characteristics, gonadosomatic index (GSI), and hepatic vitellogenin messenger RNA (mRNA) expression. In addition, fertilized embryos from the reproduction assay were used in an embryonic development assay to assess A4 effects on development and hatchability. In males, A4 significantly increased Vtg mRNA expression (estrogenic effect), significantly reduced GSI, and had no effect on tubercle expression (p = 0.067). In females, A4 induced tubercle development (androgenic effect) with no effects on GSI. Fecundity was not significantly impacted. Exposure to A4 had no effect on fertilization, embryonic development, or hatchability. These data indicate that exogenous A4, at environmentally relevant concentrations, can significantly modulate the reproductive physiology of the fathead minnows in a sex-specific manner and that A4 should be monitored as an endocrine disruptor.

A novel androstenedione derivative induces ROS-mediated autophagy and attenuates drug resistance in osteosarcoma by inhibiting macrophage migration inhibitory factor (MIF).

Osteosarcoma is a common primary bone tumor in children and adolescents. The drug resistance of osteosarcoma leads to high lethality. Macrophage migration inhibitory factor (MIF) is an inflammation-related cytokine implicated in the chemoresistance of breast cancer. In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. The inhibition of MIF by DSTD promoted autophagy by inducing Bcl-2 downregulation and the translocation of HMGB1. N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced ROS-mediated autophagy to rescue cell death. However, in the presence of chemotherapy drugs, DSTD enhanced the chemosensitivity by decreasing the HMGB1 level. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.

Heterocyclic ring extension of androstenedione: synthesis and cytotoxicity of fused pyran, pyrimidine and thiazole derivatives.

The reaction of androstenedione with either malononitrile or ethyl cyanoacetate and aromatic aldehydes 2a-c gave the pyran derivatives 4a-f, respectively. On the other hand, the reaction of androstenedione with thiourea and the aromatic aldehydes 2a-c gave the pyrimidine derivatives 6a-c, respectively. Compound 6b reacted with 2-bromo-1-arylethanone derivatives 7a-d to give the indeno[2,1-e]thiazole derivatives 8a-d. Some of the produced compounds were used for further heterocyclization reactions. The cytotoxicity of the newly obtained products was evaluated against some cancer cell lines and a normal cell line.

Test concentration setting for fish in vivo endocrine screening assays.

Fish in vivo screening methods to detect endocrine active substances, specifically interacting with the hypothalamic-pituitary-gonadal axis, have been developed by both the Organization for Economic Co-operation and Development (OECD) and United States Environmental Protection Agency (US-EPA). In application of these methods, i.e. regulatory testing, this paper provides a proposal on the setting of test concentrations using all available acute and chronic data and also discusses the importance of avoiding the confounding effects of systemic toxicity on endocrine endpoints. This guidance is aimed at reducing the number of false positives and subsequently the number of inappropriate definitive vertebrate studies potentially triggered by effects consequent to systemic, rather than endocrine, toxicity. At the same time it provides a pragmatic approach that maximizes the probability of detecting an effect, if it exists, thus limiting the potential for false negative outcomes.

Androstenedione induces abnormalities in morphology and function of developing oocytes, which impairs oocyte meiotic competence.

OBJECTIVE: To obtain insight into the effects of androstenedione on ovarian folliculogenesis and oogenesis. DESIGN: Experimental study. SETTING: St. Marianna University School of Medicine. ANIMAL(S): Prepubertal (14-day-old) BDF1 female mice. INTERVENTION(S): Early secondary follicles were isolated from the ovaries and were cultured individually in vitro with or without androstenedione (10(-11) to 10(-5) M) for 12 days. Thereafter, the follicles were treated with hCG and epidermal growth factor (EGF). MAIN OUTCOME MEASURE(S): Diameters and morphology of follicles and oocytes; E(2) and P secretion; and chromatin configuration and expression of growth differentiation factor 9 (GDF9) in oocytes were examined. RESULT(S): Early secondary follicles developed to the preovulatory stage. Androstenedione treatments increased the follicle diameters, reduced survival rates of follicles, and promoted the formation of follicles with abnormal morphology, including misshapen oocyte. The secretion of E(2) and P was significantly higher in androstenedione-exposed follicles. Androstenedione prevented the alteration in chromatin configuration and reduced oocyte GDF9 expression. When follicles cultured with androstenedione were treated with hCG and EGF, the first polar body exclusion, chromosome alignment on metaphase plate, and spindle assembly were inhibited in the oocytes. CONCLUSION(S): These results demonstrate that excess androgen induces abnormalities in the morphology and function of developing oocytes, which impairs oocyte meiotic competence.

Toxicity and carcinogenicity of androstenedione in F344/N rats and B6C3F1 mice.

Androstenedione was marketed as a dietary supplement to increase muscle mass during training. Due to concern over long-term use, the NTP evaluated the subchronic and chronic toxicity and carcinogenicity of androstenedione in male and female F344/N rats and B6C3F1 mice. In subchronic studies, dose limiting effects were not observed. A chronic (2-year) exposure by gavage at 10, 20, or 50 mg/kg in rats and male mice, and 2, 10, or 50 mg/kg in female mice (50 mg/kg, maximum feasible dose) was conducted. Increased incidences of lung alveolar/bronchiolar adenoma and carcinoma occurred in the 20 mg/kg male rats and increases in mononuclear cell leukemia occurred in the 20 and 50 mg/kg female rats, which may have been related to androstenedione administration. In male and female mice, androstenedione was carcinogenic based upon a significant increase in hepatocellular tumors. A marginal increase in pancreatic islet cell adenomas in male (50 mg/kg) and female (2, 10, 50 mg/kg) mice was considered to be related to androstenedione administration. Interestingly, incidences of male rat Leydig cell adenomas and female rat mammary gland fibroadenomas decreased. In conclusion, androstenedione was determined to be carcinogenic in male and female mice, and may have been carcinogenic in rats.

Synthesis and biological evaluation of 16E-arylidenosteroids as cytotoxic and anti-aromatase agents.

Taking into consideration the structural requirements for cytotoxicity and aromatase inhibition, several new 16E-arylidenosteroidal derivatives have been prepared and evaluated for their cytotoxic and aromatase inhibitory activity. The new steroidal analogues 3, 5-8 and 11 exhibited significant cytotoxic effects when screened against three cancer cell lines, MCF-7 (breast), NCl-H460 (lung) and SF-268 central nervous system (CNS) at 100 µM and sensible cytotoxic effects subsequently in sixty cancer cell lines derived from nine cancers types (leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate and breast cancers). The imidazolyl substituted steroidal derivatives 5 and 7 exhibited strong inhibition of the aromatase enzyme with 16-[4-{3-(imidazol-1-yl)propoxy}-3-methoxybenzylidene]-5-androstene-3ß,17ß-diol (7) displaying 13 times more potency in comparison to aminoglutethimide.

Toxicology and carcinogenesis studies of androstenedione (CAS No. 63-05-8) in F344/N rats and B6C3F1 mice (gavage studies).

UNLABELLED: Androstenedione is an androgen steroid that is normally synthesized within men and women and may be metabolized to a more potent androgen or estrogen hormone. It was nominated to the National Toxicology Program for study due to concern for adverse health effects associated with its chronic use as a dietary supplement by athletes (prior to the banning of its over the counter sales). In order to evaluate its subchronic and chronic toxicity, male and female F344/N rats and B6C3F1 mice were administered androstenedione (98% pure) by gavage for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, rat bone marrow cells, and mouse peripheral blood erythrocytes. 2-WEEK STUDY IN RATS: groups of five male and five female rats were administered 0, 1, 5, 10, 20, or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for 12 days. All rats survived to the end of the study, and the mean body weights of dosed groups were similar to those of the vehicle control groups. The development of cytoplasmic vacuoles within centrilobular hepatocytes in male rats was the only treatment-related effect observed. 2-WEEK STUDY IN MICE: groups of five male and five female mice were administered 0, 1, 5, 10, 20, or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for 12 days. One vehicle control female, one 20 mg/kg female, and one 50 mg/kg female died early due to gavage accidents. There were no significant chemical-related histopathological or mean body weight changes. 3-MONTH STUDY IN RATS: groups of 10 male and 10 female core study rats were administered 0, 1, 5, 10, 20, or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for 14 weeks; additional groups of 10 male and 10 female clinical pathology study rats received the same doses for 23 days. All rats survived to the end of the study. The mean body weights of the 20 mg/kg female group was significantly greater than those of the vehicle control group and there was significant increased weight gain in the 1, 20, and 50 mg/kg female groups. Female thymus weights were significantly increased in the 20 and 50 mg/kg groups, which may be related to the increase in mean body weight. The numbers of sperm per mg cauda epididymis in the 10, 20, and 50 mg/kg male groups and the total number of sperm per cauda epididymis in 50 mg/kg males were significantly less than those of the vehicle controls. No treatment-related histological lesions were observed in males or females. 3-MONTH STUDY IN MICE: groups of 10 male and 10 female mice were administered 0, 1, 5, 10, 20, or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for 14 weeks. Except for one 10 mg/kg female that died early due to a dosing accident, all mice survived to the end of the study. The mean body weights of dosed groups were similar to those of the vehicle control groups. The number of spermatids per mg testis and the total number of spermatids per testis in 20 mg/kg males were significantly greater than those of the vehicle controls. Sperm motility in 50 mg/kg males was significantly lower than that in the vehicle controls. The incidences of x-zone atrophy of the adrenal cortex, an androgen-sensitive endpoint, were significantly increased in females administered 5 mg/kg or greater. There were also significant decreases in the incidences of x-zone cytoplasmic vacuolization in 20 and 50 mg/kg females. The incidences of bone marrow hyperplasia were significantly increased in 5 and 50 mg/kg males. 2-YEAR STUDY IN RATS: groups of 50 male and 50 female rats were administered 0, 10, 20, or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for at least 104 weeks. Survival of 10 mg/kg males was significantly greater than that of the vehicle controls. The mean body weights of 20 and 50 mg/kg females were greater than those of the vehicle controls after weeks 17 and 9, respectively. The incidences of mononuclear cell leukemia were significantly increased in 20 and 50 mg/kg females and significantly decreased in 20 and 50 mg/kg males. Incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in 20 mg/kg males. The incidence of testicular interstitial cell adenoma (including bilateral) was significantly decreased in 50 mg/kg males. In females, the incidences of mammary gland fibroadenoma were significantly decreased in the 20 and 50 mg/kg groups, the incidences of mammary gland hyperplasia were significantly decreased in all dosed groups, and the incidences of mammary gland cyst were significantly decreased in the 10 and 50 mg/kg groups. In the liver of males, the incidences of basophilic focus in all dosed groups, the incidence of clear cell focus in the 20 mg/kg group, and the incidence of eosinophilic focus in the 50 mg/kg group were significantly increased. The incidences of pancreatic islet hyperplasia and atrophy of the exocrine pancreas were significantly increased in 50 mg/kg females. 2-YEAR STUDY IN MICE: groups of 50 male and 50 female mice were administered 0, 2 (females only), 10, 20 (males only), or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for at least 104 weeks. Survival of dosed groups was similar to that of the vehicle control groups. Mean body weights of 10 and 50 mg/kg females were generally less than those of the vehicle controls after weeks 81 and 17, respectively. The incidences of hepatocellular adenoma in males and females were significantly increased in the 50 mg/kg groups. In females, the incidences of hepatocellular carcinoma were significantly increased in all dosed groups. Incidences of hepatocellular adenoma or carcinoma (combined) in males and females were significantly increased in the 50 mg/kg groups. Incidences of hepatoblastoma were marginally increased in dosed males. Incidences of multiple hepatocellular adenomas and carcinomas were significantly increased in 10 and 50 mg/kg males, and there was an increased incidence of multiple hepatocellular adenomas in 50 mg/kg females. The incidence of eosinophilic focus was significantly increased in 50 mg/kg males, and the incidences of mixed cell focus and cytoplasmic vacuolization were significantly increased in 50 mg/kg females. There was a marginally increased incidence of pancreatic islet adenoma in 50 mg/kg males and in 10 and 50 mg/kg females, with an earlier day of first incidence in males. The incidences of clitoral gland hyperplasia and clitoral gland duct dilatation were significantly increased in 10 and 50 mg/kg females. The incidence of glomerular metaplasia of the kidney was significantly increased in 50 mg/kg females, and the incidences of cytoplasmic alteration of the submandibular salivary gland were significantly increased in all dosed female groups. The increased incidences of cytoplasmic alteration of the submandibular salivary gland and glomerular metaplasia of the kidney in female mice indicated a masculinizing effect from androstenedione treatment. In 50 mg/kg females, the incidence of malignant lymphoma was significantly decreased. GENETIC TOXICOLOGY: androstenedione was not mutagenic in either of two independent bacterial mutation assays conducted with and without exogenous metabolic activation. No significant increases in the frequencies of micronucleated polychromatic erythrocytes, indicators of chromosomal damage, were observed in bone marrow of male rats administered androstenedione by gavage once daily for 3 consecutive days. Results of a peripheral blood erythrocyte micronucleus test in mice, in which androstenedione was administered by gavage for 3 months, were negative in males but judged to be equivocal in females due to a small increase (twofold over background) in micronucleated normochromatic erythrocytes observed at the highest dose administered (50 mg/kg). CONCLUSIONS: under the conditions of these 2-year gavage studies, there was equivocal evidence of carcinogenic activity of androstenedione in male F344/N rats based on increased incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined). There was equivocal evidence of carcinogenic activity of androstenedione in female F344/N rats based on increased incidences of mononuclear cell leukemia. There was clear evidence of carcinogenic activity of androstenedione in male B6C3F1 mice based on increased incidences of multiple hepatocellular adenoma and hepatocellular carcinoma and increased incidence of hepatoblastoma. There was clear evidence of carcinogenic activity of androstenedione in female B6C3F1 mice based on increased incidences of hepatocellular adenoma and hepatocellular carcinoma. Increased incidences of pancreatic islet adenoma in male and female mice were also considered chemical related. Androstenedione administration caused increased incidences in nonneoplastic lesions of the liver in male and female rats and mice; pancreatic islets and exocrine pancreas of female rats; and clitoral gland, kidney, and submandibular salivary gland of female mice. Decreases in the incidences of testicular interstitial cell adenoma in male rats, mammary gland fibroadenoma, cysts, and hyperplasia in female rats, and malignant lymphoma in female mice were considered related to androstenedione administration. Synonyms: Andro; androst-4-ene-3,17-dione; 4-androstene-3,17-dione; delta-4-androstene-3,17-dione; delta-4-androstenedione; 3,17-dioxoandrost-4-ene; 17-ketotestosterone; SKF 2170 Trade names: Androtex, Fecundin.

Effects on guppy brain aromatase activity following short-term steroid and 4-nonylphenol exposures.

Brain estrogen production, performed by the enzyme aromatase, can be disrupted/affected in teleost fish exposed to endocrine disruptors found in polluted aquatic environments. The guppy (Poecilia reticulata) was previously studied and confirmed to suffer negative effects on reproductive behaviors following inhibition of the brain aromatase reaction. Here adult guppies (Poecilia reticulata) of both genders were subjected to known endocrine disruptors: the androgen androstenedione (A), the synthetic estrogen 17alpha-ethinylestradiol (EE(2)), and the estrogenic surfactant 4-nonylphenol (NP), at high (50 microg/L) and at environmentally relevant concentrations (10 ng/L EE(2), 5 microg/L NP, and 0.7 microg/L A) for 2 weeks followed by measurements of brain aromatase activity (bAA). In the adult males, bAA was stimulated by A and EE(2) at 50 microg/L. Female activity was also stimulated by the higher estrogenic treatment. At environmentally relevant concentrations only the EE(2) treatment affected bAA, and only in males. The alkylphenolic substance NP produced no effect in either of the experiments, not on males nor females. The results indicate that short-term steroid treatments have stimulatory effects on guppy brain aromatase even at concentrations that can be found in the environment. We thus suggest bAA of adult guppies to be a suitable bioindicator of endocrine disruptors.

The developmental effects of a municipal wastewater effluent on the northern leopard frog, Rana pipiens.

Wastewater effluents are complex mixtures containing a variety of anthropogenic compounds, many of which are known endocrine disruptors. In order to characterize the developmental and behavioral effects of such a complex mixture, northern leopard frogs, Rana pipiens, were exposed to a range of concentrations (0%, 10%, 50%, 100%) of municipal wastewater effluent from the egg stage through metamorphosis. The estrogenic activity of the effluent was quantified by the calculation of an estradiol (E2) equivalency (EEQ) factor and was determined to be equivalent to 1.724+/-2.103ng/L E2. Individuals from the 50% and 100% wastewater treatments took significantly longer to reach metamorphosis than individuals in the 0% and 10% treatments. An increased incidence of male testicular oocytes was observed in the 50% and 100% treatments when compared to the control treatment. Morphological changes in the thyroid glands of 100% wastewater-treated individuals were also noted. No effects of wastewater exposure on growth, sex ratio, swim speed, startle response, or female gonadal development were observed. These results suggest that municipal wastewater effluent can alter the timing of the metamorphic process and impact male sexual development in R. pipiens.

Micronucleus induction in V79 cells by the anabolic doping steroids desoxymethyltestosterone (madol) and 19-norandrostenedione.

The abuse of anabolic steroids for doping raises concerns. Many of these compounds have never been examined for their toxicological properties. Aside from hormonal (androgenic) activity, anabolic steroids may also exert genotoxic effects. In the present study, we determined the potencies of the "designer steroid" madol (MAD) and the anabolic prohormone 19-norandrostenedione (NA) to induce micronuclei in V79 cells in vitro. CREST analysis was used to differentiate between aneugenic and clastogenic mechanisms of micronucleus induction. Cytotoxicity of the steroids and their influence on the cell cycle were assessed in parallel. In addition, the ability of MAD and NA to increase production of reactive oxygen species and to induce apoptosis were studied. Both agents caused a concentration-dependent increase in the rates of micronuclei in V79 cells, exceeding a doubling of the background micronucleus rates of untreated controls, which was evident at 27microM and 29microM for MAD and NA, respectively. The steroid-induced micronuclei were predominantly kinetochor (CREST)-negative, pointing to a clastogenic mode of action. As cytotoxicity of both compounds is weak (IC(20) value of 300microM for NA and IC(10) of 100microM for MAD), cytotoxicity was unlikely to contribute to their genotoxicity. The observed genotoxicity of both compounds was due neither to apoptosis induction nor to production of reactive oxygen species. However, the ability of both steroids to induce micronuclei appears related to their lipophilicity. Therefore, a "non-specific" chromosomal genotoxicity of MAD and NA, based on hydrophobic interactions, appears likely. This could well result in biologically relevant increases in chromosomal damage as soon as critical concentrations of the agents are reached in vivo. Regarding the current misuse of the steroids for doping, the uncontrolled administration of very high doses must be considered. Therefore it cannot be ruled out that MAD and NA present genotoxic hazards under current misuse conditions by athletes in sports or in body building.

Acute and chronic effects of testosterone and 4-hydroxyandrostenedione to the crustacean Daphnia magna.

Steroid compounds have been globally detected in surface waters. The ecological impacts of these biologically active chemicals are largely unknown. Toxicity of testosterone and 4-hydroxyandrostenedione was assessed for the freshwater cladoceran Daphnia magna. Acute toxicity tests showed that 6.20 mg L(-1) of testosterone, the highest concentration tested, did not have effect on the daphnids, whereas 4-hydroxyandrostenedione had an EC(50) of 4.26 mg L(-1). Chronic toxicity tests were carried out using survival, body length, fecundity, and fertility as endpoints. Long-term testosterone exposure reduced D. magna fecundity and fertility at concentrations ranging from 0.31 to 2.48 mg L(-1). The significant decrease in fecundity was associated with an increase in aborted eggs. Long-term 4-hydroxyandrostenedione exposure at 0.84 mg L(-1) increased the mortality of the neonates. The chronic toxicity effects were observed at concentrations higher than the measured environmental concentrations of these compounds. Nevertheless, the reproductive impairment of the daphnids is likely to occur at environmental levels as an ultimate response to long-term exposure.

The prohormone 19-norandrostenedione displays selective androgen receptor modulator (SARM) like properties after subcutaneous administration.

One of the most frequently misused steroid precursors (prohormones) is 19-norandrostenedione (4-estrene-3,17-dione, NOR), which is, after oral administration, readily metabolised to nortestosterone, also known as nandrolone (durabolin). In this study we have characterised molecular mechanisms of its action determined its tissue specific androgenic and anabolic potency after subcutaneous (s.c.) administration and investigated potential adverse effects. Receptor binding tests demonstrate that NOR binds with high selectivity to the AR. The potency of NOR to transactivate androgen receptor (AR) dependent reporter gene expression was 10 times lower as compared to dihydrotestosterone (DHT). In vivo experiments in orchiectomised rats demonstrated that s.c. treatment with NOR resulted only in a stimulation of the weight of the levator ani muscle; the prostate and seminal vesicle weights remained completely unaffected. Like testosterone, administration of NOR resulted in a stimulation of AR and myostatin mRNA expression in the gastrocnemius muscle. NOR does not affect prostate proliferation, the liver weight and the expression of the tyrosine aminotransferase gene (TAT) in the liver. Summarizing these data it is obvious that NOR, if administrated s.c. and in contrast to its metabolite nandrolone, highly selectively stimulates the growth of the skeletal muscle but has only weak androgenic properties. This observation may have relevance with respect to therapeutic aspects but also doping prevention.