Induce Vitellogenin Production Research Articles

Analysis of vitellogenin by histochemical method as an indicator of estrogenic effect in male Danio rerio exposed to metals.

Several chemicals present in the aquatic environment have the ability to alter the endocrine system of aquatic organisms, including the metals cadmium (Cd) and zinc (Zn). In males, a tool to analyze this effect is the vitellogenin (VTG) detection, a protein with defined function in the yolk production and subsequent aid to embryo-larval development. This protein is produced just by females and can be detected by simple and cheap methods such as histochemical method. Thus, this study aimed to analyze the capacity of Cd and Zn to induce VTG production in Danio rerio males and determine whether the histochemical labeling method is efficient to study estrogenic effects in this species. For this, D. rerio adult males were exposed chronically (21days) to 0.25 and 1μg/L of Cd and 120 and 180μg/L of Zn, values allowed by the Brazilian and American legislation to aquatic life protection. After which, the organisms were submitted to a histological process as preparation to liver VTG marking by histochemical method and subsequent qualitative analysis of this protein in zebrafish's liver. After analyzes, it was possible observed that Cd and Zn are capable of inducing VTG production in D. rerio malesandthat the histochemistry method is efficient for detection of estrogenic effects in this species.

English

Aquatic organisms in the coastal regions are often exposed to many anthropogenic effluents. Kuwait Bay receives considerable quantities of sewage and industrial effluents, and thus, naturally, many endocrine disrupting chemicals (EDCs) find their way into the coastal waters. These EDCs could influence the physiology of the aquatic organisms inhabiting the coastal region. Response of two important local fish of aquaculture importance, bluefin bream (Sparidentax hasta) and yellowfin bream (Acanthopagrus latus) was evaluated by injecting a standard vitellogenin inducer, 17 β-estradiol (17 BEST).   The inducer was used at 2, 4 and 6 µg/fish (injected intraperitoneally) to induce vitellogenin production in juvenile fish (150-200 g).  There was a dose-dependent Vtg induction response as evidenced by a qualitative immune-dot assay. All levels significantly enhanced Vtg gene expression, as early as one-hour post injection. The expression was noticed to be the highest in liver tissue at 6 h post injection. The bluefin bream was used for amplification of the Vtg gene yielding 282 bp product which was sequenced. This indicated a 97% sequence similarity with the Vtg gene sequence of European seabream, S. aurata. Key words: Induction, Vtg, EDCs, Kuwait, fish, effluent. &nbsp

Modeling the loading and fate of estrogens in wastewater treatment plants

AbstractEndocrine-disrupting compounds may produce infertility, nervous system disorders, and improper functioning of the immune system in humans and wildlife. Estrogens are classified as the most potent and common endocrine-disrupting compounds and the major point source for estrogen is municipal wastewater. Monitoring of estrogen is challenging, expensive, and intermittent; and therefore, the focus of this work is modeling estrone, 17β-estradiol, and 17α-ethynylestradiol concentrations from wastewater treatment plants in Calgary and Edmonton, Alberta, and Brandon, Manitoba. Demographic groups, excretion rates, population estimates, average daily flows, calculated estrogen transformation, calibration, calculated influent-to-effluent reduction percentages, and a treatment unit removal matrix are used to determine loading estimations of estrogen. Predicted average concentrations for EE2 and E2 in all the study sites exceed the threshold concentrations that could induce vitellogenin production by order of 1...

Does anti-androgen, flutamide cancel out the in vivo effects of the androgen, dihydrotestosterone on sexual development in juvenile Murray rainbowfish (Melanotaenia fluviatilis)?

The aim of the present study was to investigate if the effects of the androgen, dihydrotestosterone (DHT) on the sexual development in juvenile Murray rainbowfish (Melanotaenia fluviatilis) are canceled out by the anti-androgen, flutamide. Fish (60 days post hatch) were exposed to 250ng/L of DHT, 25μg/L of flutamide (Flu-low), 250μg/L of flutamide (Flu-high), DHT+Flu low and DHT+Flu high. After 35 days of exposure, lengths and weights of the fish were measured and the condition factor (CF) calculated; vitellogenin (VTG) concentrations were measured in tail tissue; sex steroid hormones (17β-estradiol [E2] and 11-keto testosterone [11-KT]) were measured in the head tissue and abdominal regions were used in histological investigation of the gonads. Treatment with DHT reduced the body-length of both male and female fish, an effect which was canceled out by low and high concentrations of flutamide. However, flutamide (low or high) could not nullify the DHT-induced reduction in the CF in either sex. The E2 levels were reduced only in female fish after exposure to DHT but returned to normal after treatment with Flu-high. DHT increased the levels of 11-KT and decreased the E2/11-KT ratio in both sexes. Flu-high, but not Flu-low, could nullify these effects. Both DHT and flutamide (low or high) induced VTG production and this effect persisted when both chemicals were co-administered. Treatment with DHT did not affect gonadal cell development in the testes. However, the female fish treated with DHT contained ovaries in early-vitellogenic stage in comparison to the pre-vitellogenic ovaries in control fish. Co-treatment with flutamide (low or high) resulted in oocyte atresia. The results from the present study suggest that treatment with Flu-high could cancel out DHT-induced effects only on the hormonal profile and body-length in both male and female fish. Juvenile fish co-treated with DHT and flutamide (low or high) had high VTG levels and low CF. In addition, the ovaries in female fish were atretic. These data represent potential adverse effects on the ability of the fish to reproduce successfully.

Biological responses of marine flatfish exposed to municipal wastewater effluent

There is increasing concern over the presence of pharmaceutical compounds, personal care products, and other chemicals collectively known as contaminants of emerging concern (CECs) in municipal effluents, yet knowledge of potential environmental impacts related to these compounds is still limited. The present study used laboratory exposures to examine estrogenic, androgenic, and thyroid-related endocrine responses in marine hornyhead turbot (Pleuronichthys verticalis) exposed to CECs from municipal effluents with 2 degrees of treatment. Fish were exposed for 14 d to environmentally realistic concentrations of effluent (0.5%) and to a higher concentration (5%) to investigate dose responses. Plasma concentrations of estradiol (E2), vitellogenin (VTG), 11-keto testosterone, and thyroxine were measured to assess endocrine responses. Contaminants of emerging concern were analyzed to characterize the effluents. Diverse types of effluent CECs were detected. Statistically significant responses were not observed in fish exposed to environmentally realistic concentrations of effluent. Elevated plasma E2 concentrations were observed in males exposed to ammonia concentrations similar to those found in effluents. However, exposure to ammonia did not induce VTG production in male fish. The results of the present study highlight the importance of conducting research with sentinel organisms in laboratory studies to understand the environmental significance of the presence of CECs in aquatic systems.

Aluminum Inhibits Vitellogenin Production via Toxic Effects on Hepatocytes in the Rockfish Sebastes schlegelii

Effects of aluminum (Al) on plasma vitellogenin (VTG), alkaline-labile phosphorus (ALPP), calcium (Ca), glutamate pyruvate transaminase (GPT), the hepatosomatic index (HSI), and hepatic Al concentration were examined in estradiol-17β (E 2 )-administered immature rockfish Sebastes schlegeli. Fish were injected intraperitoneally with E 2 (5 mg/kg body weight [BW]) and/or Al (0, 0.1, 1, 5, and 10 mg/kg BW) and plasma and liver samples were extracted 7 days later. After sodium dodecyl sulfate polyacrylamide gel electrophoresis, the relative amount of VTG was determined by integrated optical density. VTG accounted for 23.6% of the total proteins in the control group, but this value decreased with increasing Al administration. Al reduced the concentrations of ALPP and Ca in a concentration-dependent manner and significant reduction occurred at Al concentrations greater than 5 mg/kg. The concentration of GPT increased in a concentration-dependent manner in all Al-administered rockfish. The concentrations of Al in the liver also increased, and HSI was decreased, in a concentration-dependent manner. These results suggest that Al inhibits E 2 -induced VTG production by being toxic to hepatocytes in marine fish.

Poultry litter-induced endocrine disruption in fathead minnow, sheepshead minnow, and mummichog laboratory exposures

Animal feeding operations in the United States produce more than 500 million tons of manure annually. Disposal of poultry waste via application as fertilizer results in substantial runoff of poultry litter-associated contaminants (PLAC). Of particular concern are sex steroids, 17β-estradiol, estrone and testosterone, responsible for sex differentiation and development of reproductive structures. In a series of laboratory assays, mature male and mixed-sex larval/juvenile fish were continuously exposed to environmentally relevant PLAC solutions. Effects on gonads were assessed histologically, and vitellogenin (VTG) induction was measured as a gauge of estrogenicity. Twenty-one-day exposures to laboratory-generated PLAC solutions routinely induced VTG in mature male Pimephales promelas. Vitellogenesis in Fundulus heteroclitus only occurred at the highest tested PLAC concentration, and Cyprinodon variegatus were unresponsive at any tested concentration. All species produced considerable VTG in response to a 17β-estradiol-positive control. A pronounced feminization was seen in P. promelas when exposed to PLAC as larvae but not when exposed as juveniles. Runoff from a poultry litter-amended field cropped under standard agronomic practices induced significant VTG in male P. promelas. Results indicate that environmentally relevant PLAC concentrations exhibit endocrine activity sufficient to induce VTG production in male fish and possibly affect sex ratios in resident fish populations.

Induction of vitellogenin production in male tilapia ( Oreochromis mossambicus) by commercial fish diets

Mozambique tilapia, ( Oreochromis mossambicus), are a euryhaline teleost and an important biological model species. Captive male tilapia frequently have high levels of the estrogen-induced yolk precursor protein vitellogenin (Vg), a common indicator of exposure to estrogenic compounds. Sex steroids are found in commercial fish diets, but relatively few studies have examined the relationship between commercial diets and Vg production. In a fasting experiment to ascertain a dietary role in male Vg production, plasma Vg was reduced to negligible levels after 2 weeks of fasting, while no change in estrogen receptor (ER) expression was seen. When male tilapia were fed a squid-based diet that replaced the commercial trout diet, plasma Vg was reduced to undetectable levels over 40 days, concomitant with significant reductions in hepatic expression of Vgs A, B, and C, and ERβ, compared with control fish fed commercial trout diet. Female tilapia fed the squid-based for 20 days had no change in these parameters. When male tilapia were fed a defined, soy-based diet, plasma Vg reduced to 20% of levels in fish given either commercial trout diet or a defined, fishmeal-based diet. Overall, results from these studies suggest that estrogens in a commercial trout diet induce vitellogenin production by increasing expression of Vg, but not ER genes in male tilapia.

Detection of estrogenic activity in municipal wastewater effluent using primary cell cultures from three-spined stickleback and chemical analysis

Environmental estrogens are substances that imitate the effects of endogenous estrogens. Effluents from municipal wastewater treatment plants are known to contain substances with estrogenic activity including steroidal estrogens and xenoestrogens. In the current study, a combination of biological and chemical analysis was applied to determine the estrogenic activity in municipal wastewater effluents in Finland. The male three-spined stickleback ( Gasterosteus aculeatus) hepatocyte assay with vitellogenin induction as an endpoint was used for the detection of estrogenic activity in solid phase extracts of wastewater effluents, and 17β-estradiol (E2) as a positive control. The wastewater extracts and E2 were found to induce vitellogenin production. The extracts were also subjected to chromatographic fractionation and the collected fractions were assayed. The only active fraction was the one in which E2, estrone and ethynylestradiol were eluted. Its activity corresponded to the activity of the original wastewater extract. The LC–MS/MS analyses of the wastewater extracts showed that the concentration of estrone was about 65 ng L −1, the concentration of E2 was less than 1 ng L −1, while estriol and 17α-ethynylestradiol could not be detected. These findings showed that the activity of the wastewater extracts and the chromatographic fraction was much higher than the activity which could have been expected on the base of the chemical analysis. This strongly indicates that other compounds, possibly acting by additivity or synergism, are playing a major role in the induced vitellogenin production by the hepatocytes.

Functional characterization of estrogen receptor subtypes, ERα and ERβ, mediating vitellogenin production in the liver of rainbow trout

The estrogen-dependent process of vitellogenesis is a key function on oviparous fish reproduction and it has been widely used as an indicator of xenoestrogen exposure. The two estrogen receptor (ER) subtypes, ERα and ERβ, are often co-expressed in the liver of fish. The relative contribution of each ER subtype to modulate vitellogenin production by hepatocytes was studied using selected compounds known to preferentially interact with specific ER subtypes: propyl-pyrazole-triol (PPT) an ERα selective agonist, methyl-piperidino-pyrazole (MPP) an ERα selective antagonist, and diarylpropionitrile (DPN) an ERβ selective agonist. First, the relative binding affinity of the test compounds to estradiol for rainbow trout hepatic nuclear ER was determined using a competitive ligand binding assay. All the test ligands achieved complete displacement of specific [ 3H]-estradiol binding from the nuclear ER extract. This indicates that the test ligands have the potential to modify the ER function in the rainbow trout liver. Secondly, the ability of the test compounds to induce or inhibit vitellogenin production by primary cultures of rainbow trout hepatocytes was studied. Estradiol and DPN were the only compounds that induced a dose-dependent increase on vitellogenin synthesis. The lack of vitellogenin induction by PPT indicates that ERα could not have a role on this reproductive process whereas the ability of DPN to induce vitellogenin production supports the participation of ERβ. In addition, this hypothesis is reinforced by the results obtained from MPP plus estradiol. On one hand, the absence of suppressive activity of MPP in the estradiol-induced vitellogenin production does not support the participation of ERα. On the other hand, once blocked ERα with MPP, the only manifestation of agonist activity of estradiol would be achieved via ERβ. In conclusion, the present results indicate that vitellogenin production is mainly mediated through ERβ, implying, furthermore that compounds which only exhibit ERα selectivity are not detected by vitellogenin bioassay.

Ethoxyresorufin-O-deethylase (EROD) and vitellogenin (VTG) in flounder ( Platichthys flesus): System interaction, crosstalk and implications for monitoring

The extent to which biological systems interact in fish from multi-contaminant areas needs to be understood for full interpretation of monitoring data. This study investigates the interaction between two biomarkers, ethoxyresorufin-O-deethylase (EROD) activity and plasma vitellogenin (VTG) in the European flounder ( Platichthys flesus). Flounder were exposed to several waterborne EROD inducers and estrogenic chemicals on their own and in binary combinations. Each experimental exposure was for 10 days. The estrogenic chemicals suppressed PAH-mediated EROD induction. Ethynylestradiol (EE2) and nonylphenol (NP) had threshold concentrations of EROD inhibition similar to those at which they induced VTG production. Estradiol (E2), however, showed an ability to suppress EROD at a concentration much lower than that at which VTG was induced. This established that, although EE2 is a more potent VTG inducer than E2, it is less potent in its ability to inhibit EROD activity. The PAH, dibenz[ a, h]anthracene (DbA), showed no effect on the VTG induction caused by EE2 and E2. A small effect was noted with NP at threshold concentrations for VTG induction. Archived data on flounder hepatic EROD activity collected during estuarine monitoring were reassessed in light of the project findings. It is hypothesised that published EROD monitoring data may be an underestimation of effects if it is assumed that estrogen-mediated MFO suppression is occurring in wild populations. A greater understanding of system interaction and other factors, including genetics, that influence biomarker response to contaminants would be required to interpret biomarker monitoring data.

Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances

Concern over possible adverse effects of endocrine-disrupting compounds on fish has caused the development of appropriate testing methods. In vitro screening assays may provide initial information on endocrine activities of a test compound and thereby may direct and optimize subsequent testing. Induction of vitellogenin (VTG) is used as a biomarker of exposure of fish to estrogen-active substances. Since VTG induction can be measured not only in vivo but also in fish hepatocytes in vitro, the use of VTG induction response in isolated fish liver cells has been suggested as in vitro screen for identifying estrogenic-active substances. The main advantages of the hepatocyte VTG assay are considered its ability to detect effects of estrogenic metabolites, since hepatocytes in vitro remain metabolically competent, and its ability to detect both estrogenic and anti-estrogenic effects. In this article, we critically review the current knowledge on the VTG response of cultured fish hepatocytes to (anti)estrogenic substances. In particular, we discuss the sensitivity, specificity, and variability of the VTG hepatocyte assay. In addition, we review the available data on culture factors influencing basal and induced VTG production, the response to natural and synthetic estrogens as well as to xenoestrogens, the detection of indirect estrogens, and the sources of assay variability. The VTG induction in cultured fish hepatocytes is clearly influenced by culture conditions (medium composition, temperature, etc.) and culture system (hepatocyte monolayers, aggregates, liver slices, etc.). The currently available database on estrogen-mediated VTG induction in cultured teleost hepatocytes is too small to support conclusive statements on whether there exist systematic differences of the VTG response between in vitro culture systems, VTG analytical methods or fish species. The VTG hepatocyte assay detects sensitively natural and synthetic estrogens, whereas the response to xenoestrogens appears to be more variable. The detection of weak estrogens can be critical due to the overshadow with cytotoxic concentrations. Moreover, the VTG hepatocyte assay is able to detect antiestrogens as well as indirect estrogens, i.e substances which require metabolic activation to induce an estrogenic response. Nevertheless, more chemicals need to be analysed to corroborate this statement. It will be necessary to establish standardized protocols to minimize assay variability, and to develop a set of pass-fail criteria as well as cut-offs for designating positive and negative responses.

Seasonal Variations in Plasma Concentrations of Sex Steroid Hormones and Vitellogenin in Wild Male Japanese Dace (Tribolodon hakonensis) Collected from Different Sites of the Jinzu River Basin

In order to clarify the seasonal variations of plasma sex steroid hormones and vitellogenin (VTG) concentrations in the wild male Japanese dace, Tribolodon hakonensis, we measured plasma levels of testosterone (T), 11-ketotestosterone (11-KT), estradiol-17 beta (E2) and VTG, as well as spermatogenetic stages and gonadosomatic index (GSI). Wild Japanese dace were collected from different sites of the Jinzu River basin (including the Takahara River and the Itachi River). The fish from Toyama Bay were also measured the spermatogenetic stages, GSI and VTG levels. The seasonal variations of the hormone levels were discussed in the relationship with various environmental factors. In landlocked fish of the Takahara River, the plasma concentrations of T and E2 reached the highest levels in May and June. In the fish collected from the Itachi River, plasma concentrations of T, 11-KT and E2 reached the highest levels during breeding season of April and May. Sexual maturation, evaluating from GSI and the spermatogenetic stages, proceeded earlier in the fish population at Toyama Bay, and afterward it was followed in the fish population at the Takahara River, in associated with a rise of environmental water temperature at fish captured sites. In the male dace, low but detectable levels of plasma E2 were measured and there were significantly positive correlations between E2 level and the levels of GSI, VTG or T. These results suggest that E2 might be a necessary sex steroid hormone related to gonad maturation, and that circulating E2 may induce VTG production in the wild male Japanese dace.

Quantitative measurement of fathead minnow vitellogenin by liquid chromatography combined with tandem mass spectrometry using a signature peptide of vitellogenin.

Vitellogenin (VTG) has been proposed as a sensitive biomarker of exposure to environmental estrogenic contaminants that induce VTG production in oviparous species. Enzyme-linked immunosorbent assay (ELISA) methods are currently widely used to measure the VTG levels. In this paper, a new liquid chromatography combined with tandem mass spectrometry (LC/ESI-MS/MS) method for the quantitative analysis of VTG in the plasma of fathead minnows exposed to 17alpha-ethinylestradiol (EE2) has been developed. This method includes, first, the selection of the signature peptide, which involves sodium dodecyl sulfate-polyarylamide gel electrophoresis separation, in-gel digestion, LC/ESI-MS/MS analysis with an ion trap mass spectrometer, and sequence determination with the TurboSEQUEST MS/MS database application; second, optimization of the selected signature peptide in multireaction monitor (MRM) mode with a triple quadrupole mass spectrometer; and third, trypsin digestion of plasma and VTG quantitation via MRM-mode LC/ESI-MS/MS. A series of plasma samples from fathead minnows following exposure to EE2 was assayed. A good correlation was found when EE2-induced plasma samples from fathead minnows were analyzed with ELISA and the described new method. Although used here with fathead minnow, the new LC/ESI-MS/MS method could be easily applied to the analysis of VTG expressed in any other fish species. Quantitation of VTG by this method was found to be highly specific and linear. The absence of potential artifactual measurements of VTG at low exposure levels could also be critical in future studies that evaluate weakly estrogenic compounds in aquatic species.

11-ketotestosterone potentiates estrogen-induced vitellogenin production in liver of Japanese eel (Anguilla japonica)

Isolated hepatocytes from male Japanese eel at various stages of human chorionic gonadotropin (HCG)-induced spermatogenesis were cultured in the presence of estradiol-17β (E2). Hepatic vitellogenin (VTG) production in response to E2 increased during testicular development. This indicates that VTG production is influenced by gonadal maturity not only in females but also in males, where serum levels of 11-ketotestosterone (11KT) increase during testicular development. Recently, it has been confirmed that significant levels of 11KT are detected in maturing female eel. Therefore, in a next experiment, effect of 11KT on E2-induced VTG production was examined in vitro. As a result, in both male and female hepatocytes, 11KT enhanced E2-induced VTG production, although 11KT alone failed to induce VTG production.

Induction of Plasma Vitellogenin Synthesis by the Commercial Fish Diets in Male Goldfish(Carassius auratus) and Dietary Phytoestrogens.

In this study, we analyzed contents of phytoestrogens (genistein, daidzein, equol, and coumestrol) in two commercial fish diets [a diet for trout (TD) and a diet for ornamental carp (CD)] using Liquid Chromatography-Mass Spectroscopy/Mass Spectroscopy (LC-MS/MS), and these contents were compared with that of a casein-based formulated fish diet (FD) which does not contain soya bean or fish meal. We also analyzed phytoestrogen contents in commercial infant casein- and soya bean-based diets. The contents of phytoestorogens were generally high in CD, TD, and soya milk, and low or non-detectable in FD and casein-based milks. Among these samples, CD showed the highest phytoestrogen contents: genistein, 390800 ng/g; daidzein, 416800 ng/g; coumestrol, 1325 ng/g; equol, 6.4 ng/g. We also determined the estrogenic activity of the fish diets using male goldfish by measuring plasma vitellogenin (VTG) levels as a biomarker of estrogen exposure. When male goldfish were fed one of these diets for 31 days, plasma VTG was detected in CD-fed fish (78.01 ± 48.18 μg/ml) and TD-fed fish (3.51 ± 3.83 μg/ml), whereas plasma VTG was not detected in FD-fed fish (less than 0.040 μg/ml). These results indicate that the commercial fish diets examined contain a large amount of phytoestrogens and showed estrogenic activity that were strong enough to induce VTG production in male goldfish. It is necessary to eliminate estrogenic substances other than test chemicals in the screening test system for estrogenic endocrine-disrupting chemicals (EDCs). Since the formulated diet developed in the present study contain less phytoestrogens than the commercial fish diets and has low estrogenic activity, it is suggested that VTG production using male goldfish in combination with the low estrogen fish diet is a good in vivo system for evaluation of estrogenic effects of EDCs.

Effects of chloro-s-triazine herbicides and metabolites on aromatase activity in various human cell lines and on vitellogenin production in male carp hepatocytes.

We investigated a potential mechanism for the estrogenic properties of three chloro-s-triazine herbicides and six metabolites in vitro in several cell systems. We determined effects on human aromatase (CYP19), the enzyme that converts androgens to estrogens, in H295R (adrenocortical carcinoma), JEG-3 (placental choriocarcinoma), and MCF-7 (breast cancer) cells; we determined effects on estrogen receptor-mediated induction of vitellogenin in primary hepatocyte cultures of adult male carp (Cyprinus carpio). In addition to atrazine, simazine, and propazine, two metabolites--atrazine-desethyl and atrazine-desisopropyl--induced aromatase activity in H295R cells concentration-dependently (0.3-30 microM) and with potencies similar to those of the parent triazines. After a 24-hr exposure to 30 microM of the triazines, an apparent maximum induction of about 2- to 2.5-fold was achieved. The induction responses were confirmed by similar increases in CYP19 mRNA levels, determined by reverse-transcriptase polymerase chain reaction. In JEG-3 cells, where basal aromatase expression is about 15-fold greater than in H295R cells, the induction responses were similar but less pronounced; aromatase expression in MCF-7 cells was neither detectable nor inducible under our culture conditions. The fully dealkylated metabolite atrazine-desethyl-desisopropyl and the three hydroxylated metabolites (2-OH-atrazine-desethyl, -desisopropyl, and -desethyl-desisopropyl) did not induce aromatase activity. None of the triazine herbicides nor their metabolites induced vitellogenin production in male carp hepatocytes; nor did they antagonize the induction of vitellogenin by 100 nM (EC(50) 17beta-estradiol. These findings together with other reports indicate that the estrogenic effects associated with the triazine herbicides in vivo are not estrogen receptor-mediated, but may be explained partly by their ability to induce aromatase in vitro.

Induction of vitellogenin gene transcription in vitro by juvenile hormone in Blattella germanica

In the cockroach Blattella germanica, the synthesis of vitellogenin is juvenile hormone III (JH III)-dependent. We have studied the effect of JH III upon vitellogenin gene expression in periovaric fat bodies incubated in vitro. Periovaric fat bodies were obtained from cardioallatectomized females. The response to JH III was measured in terms of vitellogenin and vitellogenin mRNA after 7 h of incubation. A hormonal concentration as low as 1 nM was enough to induce vitellogenin production and its release to the medium, whereas the concentration of 10 nM produced the maximal effects. Although the response of the vitellogenin gene to JH III is fast and efficient, it seems that the action is mediated by protein factors, given that cycloheximide treatment impairs the hormonal effect. The presence in the medium of brain extract (0.5 equivalents), corpora cardiaca (one pair) or hypertrehalosemic hormone (10 −7 or 10 −8 M), partially inhibited the response to JH III.

Effects of Chloro-s-Triazine Herbicides and Metabolites on Aromatase Activity in Various Human Cell Lines and on Vitellogenin Production in Male Carp Hepatocytes

We investigated a potential mechanism for the estrogenic properties of three chloro-s-triazine herbicides and six metabolites in vitro in several cell systems. We determined effects on human aromatase (CYP19), the enzyme that converts androgens to estrogens, in H295R (adrenocortical carcinoma), JEG-3 (placental choriocarcinoma), and MCF-7 (breast cancer) cells; we determined effects on estrogen receptor-mediated induction of vitellogenin in primary hepatocyte cultures of adult male carp (Cyprinus carpio). In addition to atrazine, simazine, and propazine, two metabolites--atrazine-desethyl and atrazine-desisopropyl--induced aromatase activity in H295R cells concentration-dependently (0.3-30 microM) and with potencies similar to those of the parent triazines. After a 24-hr exposure to 30 microM of the triazines, an apparent maximum induction of about 2- to 2.5-fold was achieved. The induction responses were confirmed by similar increases in CYP19 mRNA levels, determined by reverse-transcriptase polymerase chain reaction. In JEG-3 cells, where basal aromatase expression is about 15-fold greater than in H295R cells, the induction responses were similar but less pronounced; aromatase expression in MCF-7 cells was neither detectable nor inducible under our culture conditions. The fully dealkylated metabolite atrazine-desethyl-desisopropyl and the three hydroxylated metabolites (2-OH-atrazine-desethyl, -desisopropyl, and -desethyl-desisopropyl) did not induce aromatase activity. None of the triazine herbicides nor their metabolites induced vitellogenin production in male carp hepatocytes; nor did they antagonize the induction of vitellogenin by 100 nM (EC(50) 17beta-estradiol. These findings together with other reports indicate that the estrogenic effects associated with the triazine herbicides in vivo are not estrogen receptor-mediated, but may be explained partly by their ability to induce aromatase in vitro.

A proposed multigeneration protocol for Japanese medaka ( Oryzias latipes) to evaluate effects of endocrine disruptors

Definitive data on reproductive impairment of chronically exposed populations may be required to assess the appropriateness of the existing test methods for hazard identification and prioritization of endocrine modulators. Multigeneration toxicity testing protocols for wildlife receptors are lacking. To help address this gap we describe a multigeneration fish assay using the freshwater fish, Japanese medaka ( Oryzias latipes). This test species has been used for the evaluation of carcinogenic, teratogenic and reproductive effects and is sensitive to estrogen exposure producing ovo-testis, altered biochemical parameters and phenotypic characteristics. Due to the short life cycle, a multigeneration test with medaka can be conducted in 1 year. Endpoints evaluated include: survival, growth, sex ratio, fecundity, embryonic lesion occurrence, embryonic stage development, gonadal and hepatic somatic indices, histopathology and biochemical parameters. As new endpoints are developed they can be incorporated into the protocol. Results of a positive control (17β-estradiol) study are presented to give an indication of the baseline associated with various test endpoints and to highlight the importance of nutrition in the experimental design. 17β-Estradiol treatment induced vitellogenin production in male and female medaka, feminized males, and disrupted egg production. The proposed protocol provides researchers with an effective multigeneration fish test that can be used to examine potential effects of stressors at the population, individual, cellular and subcellular level.