Exposure to sublethal concentrations of tributyltin reduced survival, growth, and 20-hydroxyecdysone levels in a marine mysid.

Tributyltin (TBT) is as an antifouling organotin compound used in boat paints. Although organotin-based antifouling agents have been banned on a global scale, the mode of action of TBT has been studied in numerous aquatic species because of its toxicity, persistence, bioaccumulation potential, and endocrine-disrupting characteristics. In this study, we conducted 96-h acute toxicity tests wherein we exposed juvenile and adult marine mysids to waterborne TBT. Over 4 weeks of exposure, mortality was dose-dependently increased in juveniles and adult mysids. To test sublethal effects of TBT on juvenile development, newborn juvenile mysids were exposed to 1, 5, or 10 ng L-1 TBT for 4 weeks. Subsequently, we measured morphological growth parameters and quantified the hormone ecdysterone (20-hydroxyecdysone: 20E), which controls molting in mysids. The lengths of the whole body, antennal scale, exopod, endopod, and telson were significantly smaller in the 5 and/or 10 ng L-1 TBT-exposed juvenile mysids than in control and DMSO-exposed groups. Levels of 20E were significantly lower at 5 and 10 ng L-1 TBT exposures. Additionally, the number of newly hatched juveniles was significantly lower from females previously exposed to 10 ng L-1 TBT. Our results indicate sublethal concentrations of TBT have inhibitory effects on the survival, growth, and production of juveniles. The lower 20E levels could be strongly associated with TBT-triggered inhibition.

Release of chitobiase as an indicator of potential molting disruption in juvenile Daphnia magna exposed to the ecdysone receptor agonist 20-hydroxyecdysone.

During arthropod molting, the old exoskeleton is degraded and recycled by the molting fluid. Chitobiase, a major chitinolytic enzyme in the molting fluid, has been widely used as a biomarker to indicate endocrine disruption of molting in arthropods under environmental stress. Although release of chitobiase was extensively studied in organisms exposed to molting-inhibiting chemicals, enzymic association with molting and response of the molting hormone receptor, ecdysone receptor (EcR), is not well understood. The present study was therefore conducted to identify potential linkages between release of chitobiase, molting frequency, and EcR activation in a freshwater crustacean Daphnia magna after short-term (96 hr) exposure to endogenous molting hormone 20-hydroxyecdysone (20E). A suite of bioassays was used for this purpose, including the chitobiase activity, molting frequency, viability, and in vitro EcR activation. Effect concentrations were compared between different assays analyzed. Results showed that exposure to 20E reduced chitobiase release and molting frequency in a concentration-dependent manner. Exposure to as low as 250 nM 20E significantly decreased release of chitobiase after 72 hr exposure, whereas adverse effects on molting frequency and incomplete molting-associated mortality required higher 20E exposure concentrations. The EcR reporter assay further demonstrated that as low as 100 nM 20E may activate EcR in vitro. Data suggest that release of chitobiase may be employed as a sensitive indicator of potential molting disruption in crustaceans after exposure to EcR agonists such as 20E.

The Sublethal Effects of ß-Ecdysterone, a Highly Active Compound from Achyranthes bidentata Blume, on Grape Phylloxera, Daktulosphaira vitifoliae Fitch.

Grape phylloxera, Daktulosphaira vitifoliae (Fitch) (Hemiptera, Phylloxeridae), is a very destructive insect pest of grapevines. Intercropping of Achyranthes bidentata Blume (f. Amaranthaceae) and Vitis spp. grapevines can be useful to control this pest. In the present study, the toxicity of 22 compounds, known to be present in A. bidentata, to grape phylloxera was evaluated. All treatments were toxic towards grape phylloxera but the degree of toxicity differed between treatments. Among the 22 tested compounds, several of which proved toxic towards grape phylloxera. However ß-ecdysterone had higher toxic effects against grape phylloxera, with LC50 values of 175.73 mg a.i. liter-1. In addition, we assessed the sublethal effects of LC10, LC20 and LC40 of ß-ecdysterone on grape phylloxera. The fourth instar and adult developmental periods and total life span were significantly prolonged by LC40 of ß-ecdysterone. Fecundity decreased when grape phylloxera were exposed to LC20 and LC40 of ß-ecdysterone. In addition, LC40 of ß-ecdysterone decreased the intrinsic rate of increase (rm) and the finite rate of increase (λ) and prolonged the population doubling time (DT). The net reproductive rate (R0) was significantly reduced by both the LC20 and LC40 ß-ecdysterone treatments. Our results demonstrated that ß-ecdysterone had higher toxic effects and significant sublethal effects on grape phylloxera, and showed potential control of grape phylloxera.

Sida tuberculata (Malvaceae): a study based on development of extractive system and in silico and in vitro properties.

Sida tuberculata (Malvaceae) is a medicinal plant traditionally used in Brazil as an antimicrobial and anti-inflammatory agent. Here, we aimed to investigate the different extractive techniques on phytochemical parameters, as well as to evaluate the toxicity and antioxidant capacity of S. tuberculata extracts using in silico and in vitro models. Therefore, in order to determine the dry residue content and the main compound 20-hydroxyecdysone (20E) concentration, extracts from leaves and roots were prepared testing ethanol and water in different proportions. Extracts were then assessed by Artemia salina lethality test, and toxicity prediction of 20E was estimated. Antioxidant activity was performed by DPPH and ABTS radical scavenger assays, ferric reducing power assay, nitrogen derivative scavenger, deoxyribose degradation, and TBARS assays. HPLC evaluation detected 20E as main compound in leaves and roots. Percolation method showed the highest concentrations of 20E (0.134 and 0.096 mg/mL of extract for leaves and roots, respectively). All crude extracts presented low toxic potential on A. salina (LD50 >1000 µg/mL). The computational evaluation of 20E showed a low toxicity prediction. For in vitro antioxidant tests, hydroethanolic extracts of leaves were most effective compared to roots. In addition, hydroethanolic extracts presented a higher IC50 antioxidant than aqueous extracts. TBARS formation was prevented by leaves hydroethanolic extract from 0.015 and 0.03 mg/mL and for roots from 0.03 and 0.3 mg/mL on egg yolk and rat tissue, respectively (P<0.05). These findings suggest that S. tuberculata extracts are a considerable source of ecdysteroids and possesses a significant antioxidant property with low toxic potential.

The phytoecdysteroid ß-ecdysone is genotoxic in Rodent Bone Marrow Micronuclei and Allium cepa L. Assays.

ETHNAOPHARMACOLOGIAL RELEVANCE: In South America, the ß-ecdysone ecdysteroid has been found in species of the genus Pfaffia Mart. Due to the similar morphology of its roots to the Panax ginseng C. A. Mey. (Korean ginseng), some species of this genus has been known as Brazilian ginseng and have been used as tonic and aphrodisiac, as well as for the treatment of diabetes and rheumatism. AIM OF THE STUDY: Here we report a cytogenotoxic evaluation of ß-ecdysone (a natural ecdysteroid found in plants) in Rodent Bone Marrow Micronuclei and Allium cepa Assays. MATERIALS AND METHODS: Three ß-ecdysone (pure) concentrations (based in human therapeutic dosage) were used in the Micronucleus Assay. The animals were treated during two consecutive days. Micronucleated cells were counted in 2000 polychromatic erythrocytes per animal. For A. cepa L. Assay, one ß-ecdysone concentration was analyzed. The onions bulbs were exposed for 24h. RESULTS: The Micronucleus Assay showed genotoxic effects for all treatments, expressed by an increase of micronucleated cells. In A. cepa L. Assay, cell abnormalities associated to the malfunction/non-formation of mitotic spindle (aneugenic effect) and chromosomal bridges (clastogenic effect) were observed. CONCLUSIONS: The results indicate a cytogenotoxic activity of ß-ecdysone. Therefore, the popular use of Pfaffia and others species containing ß-ecdysone should be considered with caution.

Sida tuberculata (Malvaceae): a study based on development of extractive system and in silico and in vitro properties

Sida tuberculata (Malvaceae) is a medicinal plant traditionally used in Brazil as an antimicrobial and anti-inflammatory agent. Here, we aimed to investigate the different extractive techniques on phytochemical parameters, as well as to evaluate the toxicity and antioxidant capacity of S. tuberculata extracts using in silico and in vitro models. Therefore, in order to determine the dry residue content and the main compound 20-hydroxyecdysone (20E) concentration, extracts from leaves and roots were prepared testing ethanol and water in different proportions. Extracts were then assessed by Artemia salina lethality test, and toxicity prediction of 20E was estimated. Antioxidant activity was performed by DPPH and ABTS radical scavenger assays, ferric reducing power assay, nitrogen derivative scavenger, deoxyribose degradation, and TBARS assays. HPLC evaluation detected 20E as main compound in leaves and roots. Percolation method showed the highest concentrations of 20E (0.134 and 0.096 mg/mL of extract for leaves and roots, respectively). All crude extracts presented low toxic potential on A. salina (LD50 >1000 µg/mL). The computational evaluation of 20E showed a low toxicity prediction. For in vitro antioxidant tests, hydroethanolic extracts of leaves were most effective compared to roots. In addition, hydroethanolic extracts presented a higher IC50 antioxidant than aqueous extracts. TBARS formation was prevented by leaves hydroethanolic extract from 0.015 and 0.03 mg/mL and for roots from 0.03 and 0.3 mg/mL on egg yolk and rat tissue, respectively (P<0.05). These findings suggest that S. tuberculata extracts are a considerable source of ecdysteroids and possesses a significant antioxidant property with low toxic potential.

[20-Hydroxyecdysone--plant adaptogen: an anabolic effect, possible use in sports nutrition].

In the review the presentation about plant adaptogens--biologically active compounds is given. Its administration can help to achieve non-specific state of high resistance. The hypothetical mechanism of action: adaptogens are prostressors, reducing excessive increase of stress mediators in the following stress exposure. The features of adaptogenic effect of phytoecdysteroids, polyhydroxylated sterols, which are analogs of hormones of molting and metamorphosis of arthropodas, and are structurally similar to glucocorticoids on the example of the most widely studied phytoecdysteroid--20-hydroxyecdysone--are described. The results of studies of anabolic action of 20-hydroxyecdysone in experiments on laboratory animals and the possible explanation (existing in the modern scientific literature) of the mechanism of this phenomenon are discussed. Scientific publication testifying on the application of phytoecdysteroids to remove chronic fatigue syndrome, reducing nerve and muscle fatigue, improve memory and attention processes are presented. The prospects of using the 20-hydroxyecdysone in the composition of food supplements and specialized products for athletes are discussed.

Vitellogenin-like proteins in the freshwater amphipod Gammarus fossarum (Koch, 1835): functional characterization throughout reproductive process, potential for use as an indicator of oocyte quality and endocrine disruption biomarker in males.

This work focused on the validation of biological specificity of the quantitative LC-MS/MS assay by checking the natural variability of Vg levels during the reproductive cycle in Gammarus fossarum (i.e., including oogenesis and embryogenesis). Laboratory tests were performed for 21 days under controlled conditions to assess Vg changes in male and female gammarids after exposure to chemical stress. Females were exposed to two crustacean hormones, 20-hydroxyecdysone (0.01, 1 and 100 µg L⁻¹) and methyl-farnesoate (0.01, 1 and 100 µg L⁻¹). No effect was recorded for 20-hydroxyecdysone, whereas in females exposed to methyl-farnesoate a deleterious impact on Vg production was observed. Males were exposed to crustacean hormones 20-hydroxyecdysone (0.01, 1 and 100 µg L⁻¹) and methyl-farnesoate (0.01, 1 and 100 µg L⁻¹), the insecticide methoxyfenozide (0.001, 0.1 and 10 µg L⁻¹), the fungicide propiconazole (0.001, 0.1, 10 and 1000 µg L⁻¹), and the pharmaceutical products benzophenone, carbamazepine, cyproterone, and R-propranolol (0.001, 0.1, 10 and 1000 µg L⁻¹). Induction of Vg synthesis was recorded in males exposed to cyproterone, methoxyfenozide, methyl-farnesoate, and propiconazole. Finally, we validated the function of the ILIPGVGK peptide used to track vitellogenin in G. fossarum across reproductive processes (vitellogenesis and embryogenesis), and results confirmed the energy reserve role of Vg during embryo development. We show that oocyte surface measurement is directly related to Vg levels in the oocyte, constituting a reliable indicator of egg quality in G. fossarum. Consequently, it could be used as a reliable tool for biomonitoring programs. We recorded induction of Vg in male G. fossarum; however, the possible use of this tool as a specific biomarker of exposure to endocrine disruption should be confirmed in further studies.

Tributyltin synergizes with 20-hydroxyecdysone to produce endocrine toxicity.

One of the great challenges facing modern toxicology is in predicting the hazard associated with chemical mixtures. The development of effective means of predicting the toxicity of chemical mixtures requires an understanding of how chemicals interact to produce nonadditive outcomes (e.g., synergy). We hypothesized that tributyltin would elicit toxicity in daphnids (Daphnia magna) by exaggerating physiological responses to 20-hydroxyecdysone signaling via synergistic activation of the retinoid X receptor (RXR):ecdysteroid receptor (EcR) complex. Using reporter gene assays, we demonstrated that RXR, alone, is activated by a variety of ligands including tributyltin, whereas RXR:EcR heterodimers were not activated by tributyltin. However, tributyltin, in combination with the daphnid EcR ligand 20-hydroxyecdysone, caused concentration-dependent, synergistic activation of the RXR:EcR reporter. Electrophoretic mobility shift assays revealed that tributyltin did not enhance the activity of 20-hydroxyecdysone by increasing binding of the receptor complex to a DR-4 DNA-binding site. Exposure of daphnids to elevated concentrations of 20-hydroxyecdysone caused premature and incomplete ecdysis resulting in death. Tributyltin exaggerated this effect of exogenous 20-hydroxyecdysone. Further, exposure of daphnids to tributyltin enhanced the inductive effects of 20-hydroxyecdysone on expression of the 20-hydroxyecdysone-inducible gene HR3. Continuous, prolonged exposure of maternal daphnids to concentrations of tributyltin resulted in mortality concurrent with molting. Taken together, these results demonstrate that xenobiotics, such as tributyltin, can interact with RXR to influence gene expression regulated by the heterodimeric partner to RXR. The result of such interactions can be toxicity due to inappropriate or exaggerated hormonal signaling. The application of the in vitro/in vivo approach used in this study is discussed in relation to modeling of nonadditive interactions among constituents of chemical mixtures.

Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna).

The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.

A comparative study examining the cytotoxicity of inducible gene expression system ligands in different cell types.

Inducible gene expression systems are being used in many in vitro and in vivo applications for target discovery, target validation and as components in exploratory therapeutic agents. Ideally, the ligands, which activate the systems, are benign so that the effects can be strictly attributed to the induced protein. As a first step to defining the potential effects of these inducers, we tested three of them, doxycycline, muristerone A and mifepristone (for tet-, ecdysone- and progesterone antagonist-inducible systems respectively), for toxicity across a panel of normal cells and cancer cell lines. In contrast to both muristerone A and mifepristone that showed no significant toxicity on any of the tested cells, we observed that doxycycline induced cell death in selected cancer and primary cell lines. The different susceptibility of cell lines to the ligands commonly used in these inducible systems suggests that it is important to consider the effects of the inducers prior to their use in experimental in vitro cell culture systems.

Daphnia magna responses to a vertebrate estrogen receptor agonist and an antagonist: a multigenerational study.

Whereas ecological risk assessments rely on standardized aquatic toxicity tests to assess ecological hazards, these techniques have limited utility for endocrine-active compounds, including select pharmaceuticals. Due to structural similarity between of vertebrate estrogens and ecdysone, previous studies suggest that endocrine-active pharmaceuticals may interfere with invertebrate endocrine systems, while other investigations do not support these suggestions. We assessed effects of the pharmaceuticals 17alpha-ethinylestradiol and faslodex, model therapeutics designed to interact with vertebrate estrogen receptors, on endocrine biomarkers and transgenerational life-history parameters of a model invertebrate, Daphnia magna. Identical studies were performed with 20-hydroxyecdysone and testosterone, which served as positive controls for ecdysteroid receptor agonism and antagonism, respectively. Results from this study at biochemical, individual and population levels suggest that a mammalian estrogen receptor agonist and antagonist did not act through the ecdysone receptor in D. magna. Acute-to-chronic ratios based on various chronic responses ranged from 2.59 to 5.18 for 17alpha-ethinylestradiol and 1.29-12.9 for faslodex. Toxicity exerted by these therapeutics on D. magna likely resulted from non-endocrine-mediated responses. Mechanism-specific biomarkers, multigenerational designs and population growth models may be useful to assess organismal and population level responses to low-level exposures, which may serve to reduce uncertainty in future hazard assessments of invertebrate responses to endocrine-active pharmaceuticals in the environment.

Shell protein characteristics and vitellogenin-like proteins in brine shrimp Artemia franciscana exposed to municipal effluent and 20-hydroxyecdysone.

We developed biomarkers to monitor the endocrine-disrupting potential of contaminants and municipal effluents in aquatic arthropods. Artemia fransciscana shrimp were cultured and exposed to increasing concentrations of 20-hydroxyecdysone (20HE) and solid phase (C-8) municipal effluent extract (MEE) for 48 h at 20 degrees C. The levels of vitellogenin (Vtg)-like proteins, alkali-labile phosphates in total proteins and acetylcholinesterase (AChE) activity were determined in soft tissues. The levels of acid-soluble, alkali-soluble (sclerotin), neutral-soluble (arthropodin) proteins and chitin were determined to characterize the maturation state of shells. Both 20HE and the municipal effluent extract readily increased the total activity of acetylcholinesterase, alkali-labile phosphates in proteins and vitellogenin-like proteins in brine shrimp. In shells, 20HE and the effluent extract increased the proportion of chitin in shells and acid-soluble proteins but were not statistically significant for the latter. The proportion of sclerotin was increased by 20HE but was not changed by the effluent extract. The proportion of arthropodin was decreased by both 20HE and the effluent extract. Correlation analysis revealed that, as expected, the proportion of acid-soluble protein for biomineralisation and chitin levels were positively correlated and the proportion of arthropodin and sclerotin were negatively correlated in shells exposed to 20HE. Principal component analysis revealed that the proportion of chitin, arthropodin, sclerotin and acid-soluble proteins explained 63% of the responses. Shell protein and carbohydrate contents, controlled by the molting hormone 20HE, could be used as efficient markers for ecdysial properties of various contaminants. The municipal effluent appears to contain ecdysial-like chemicals that are capable of producing changes in shell protein composition that are similar to 20HE.

Comparative toxicity and ecdysone receptor affinity of non-steroidal ecdysone agonists and 20-hydroxyecdysone in Chironomus tentans.

Ecdysone agonists belonging to the bisacylhydrazine class of compounds are a new generation of insecticidal compounds that cause premature lethal molts in susceptible intoxicated insects. While two of the bisacylhydrazines (coded as RH-5992 and RH-2485) are predominantly toxic to lepidopteran pests, RH-5849, which has not been commercialized, has a broader spectrum of toxicity. We have carried out toxicity bioassays with last (4th) instar Chironomus tentans L. larvae, radioligand binding assays using bacterial fusion proteins of C. tentans ecdysone receptor and ultraspiracle (CtEcR, CtUSP), and C. tentans imaginal disc development assays to compare the relative potencies of the three bisacylhydrazine compounds as well as of 20-hydroxyecdysone (20E). In all three assays, the potency of the three bisacylhydrazines was in the order RH-2485>RH-5992>RH-5849. While in toxicity assays 20E was ineffective, most likely due to rapid metabolism, it was more potent than RH-5849 but less so than RH-5992 and RH-2485 in imaginal disc assays. In summary, we compared the potencies of the ecdysone agonists for C. tentans at three levels: whole organism, imaginal discs and the receptor level, and our results indicate that the increased toxicity of the non-steroidal ecdysone agonists for C. tentans has a high correlation to the affinity of these compounds for CtEcR/CtUSP bacterially expressed proteins. Our results, though, do not exclude reasons of metabolic stability of the compounds in C. tentans, which we have not investigated in this report.