Effects of Sublethal Concentrations of Insecticides on the Fecundity of Sogatella furcifera (Hemiptera: Delphacidae) via the Regulation of Vitellogenin and Its Receptor.

White-backed planthopper (Sogatella furcifera, Hemiptera: Delphacidae) is an important migratory pest of rice. It causes severe economic losses by reducing crop production. Vg and VgR are important proteins that help in the successful reproduction of insects and have been studied in many insects. To understand the molecular mechanisms underlying the effects of insecticides on white-backed planthopper reproduction, we studied the expression profiles of SfVg, SfVg-like, and SfVgR in white-backed planthopper exposed to insecticides. SfVg and SfVgR silencing inhibited the ovarian development, number of eggs laid by, and hatching rate of white-backed planthopper. Thiamethoxam LC10 significantly inhibited SfVg-like and SfVgR expression. In contrast, triazophos LC25 significantly promoted SfVg, SfVg-like, and SfVgR expression and increased vitellogenin content in white-backed planthopper. These results demonstrate that insecticides can regulate the reproduction of white-backed planthopper by altering the expression of SfVg and SfVgR, thereby affecting the population density of white-backed planthopper. These findings build a foundation for improving our understanding of the molecular mechanisms underlying the effects of insecticides on the reproduction and resurgence of pests.

In vivo effects of PCB-126 and genistein on vitellogenin expression in zebrafish.

In this study, the vitellogenin (Vtg) modulation by genistein and polychlorinated biphenyl-126 (PCB-126) exposure in zebrafishes has been investigated. Both PCB-126 and genistein have been identified as aquatic pollutants and can further increase estrogenicity of waterways. Vtg is egg yolk precursor protein release by the hepatocytes during vitellogenesis. This process occurs normally in the hepatocytes in response to the activation with the estrogens such as 17-ß-estradiol. Our immunohistochemical findings showed a Vtg expression that increases at 12 h and at 72 h in the liver of treated fishes with both PCB-126 and genistein, individually and in combination. Furthermore, for the first time, also hepatic stellate cells (HSC) in the liver parenchyma were strongly positive for vitellogenin.

Occurrence and Biological Effects of Endocrine Disrupting Chemicals in the Yellow River (Zhengzhou Section).

Estrogenic activities of river water from four representative cross-sections of the Yellow River (Zhengzhou section) and their effects on reproduction and development of fish were assessed. MVLN assay showed estradiol equivalents of river water from Yiluohe, Xinmanghe, Qinhe and Huayuankou cross-sections were 1.09 ± 0.11, 0.72 ± 0.01, 1.19 ± 0.19 and 0.80 ± 0.04 ng/L, respectively. Significant vitellogenin (VTG) inductions were observed in adult male Japanese madaka (Oryzias latipes) after 30 days of exposure to river water from Yiluohe and Qinhe cross-sections (p < 0.05). Hepatic-somatic index was significantly elevated in fish exposed to water from Qinhe cross-section (p < 0.05). A significant delay in time to hatching was observed in embryos treated by water from Xinmanghe cross-section (p < 0.05). Significant lower survivals were observed in fish treated by water from Yiluohe and Xinmanghe cross-sections after a full life cycle exposure (p < 0.05). Exposure of water from Yiluohe and Qinhe cross-sections induced significantly elevated VTG levels in the first sexually mature male fish (p < 0.05). Both the in vitro and in vivo bioassay demonstrate endocrine disrupting chemicals exist in the Yellow River (Zhengzhou section) and fish in Yiluohe and Qinhe cross-sections can be at a risk of reproductive and developmental impairment.

Effects of chronic exposures to the herbicides atrazine and glyphosate to larvae of the threespine stickleback (Gasterosteus aculeatus).

Atrazine (ATR) and glyphosate (GLY) are among the most widely used herbicides in Canada, yet there is relatively little information concerning their toxicity to early life stages of marine fish. The threespine stickleback (Gasterosteus aculeatus) reproduces in coastal habitats which receive runoff of pesticides during the summer, the peak season of herbicide use. Sticklebacks have biomarkers for effects of both estrogenic and androgenic contaminants. Stickleback adults from a clean reference site were allowed to reproduce in the laboratory and the fertilized eggs were incubated until hatching. Larval sticklebacks (<24h old) were exposed for 42 d to four concentrations (0.1, 1, 10 and 100 µg/l) of either ATR or GLY, a seawater control, a carrier (acetone) control and positive controls for estrogenic (0.05 µg/l ethinylestradiol, EE2) and androgenic (3 µg/l dihydrotestosterone, DHT) effects. The survivors were measured (length, wet weight) then conserved for biochemical (vitellogenin, VTG, and the male nest-protein spiggin, SPG) and histological (phenotypic sex determination) analyses. There were no significant effects of ATR and GLY exposures on larval survival or growth. Exposure to 3 µg DHT/l resulted in a significant effect on growth (body lengths) but did not induce SPG, possibly because of DHT degradation after the 24h solution renewal. VTG was induced after the EE2 exposure, yet neither ATR nor GLY induced production of VTG and SPG. The proportion of mixed sex individuals was higher in the positive controls compared to the negative controls. A single mixed sex individual was found in the group exposed to the lowest dose of atrazine and none in glyphosate expositions. We conclude that these herbicides do not show estrogenic or androgenic effects to early life stages of sticklebacks at environmentally realistic concentrations.

Exposure to monocrotophos pesticide during sexual development causes the feminization/demasculinization of the reproductive traits and a reduction in the reproductive success of male guppies (Poecilia reticulata).

Monocrotophos is a highly toxic organophosphorus pesticide that has been confirmed to be an endocrine-disrupting chemical. To evaluate the influence of this pollutant on the reproductive system of male fish, we studied the sex steroid levels, reproductive traits, sex ratio, and reproductive success in male guppies (Poecilia reticulata) exposed to 40% monocrotophos pesticide at the nominal concentrations of 0.01, 0.10, and 1.00 mg/L for 90 days from birth to adulthood in a semi-static exposure system. Radioimmunoassay and western blot analyses demonstrated that the long-term exposure to monocrotophos pesticide during the sexual development of male guppies caused a significant increase in 17ß-estradiol levels and consequently induced vitellogenin synthesis, suggesting the feminization of the males. Monocrotophos pesticide also caused a significant decrease in testosterone levels, which consequently inhibited testis growth and reduced the sperm count and the area and intensity of their sexually attractive orange spots, which collectively indicated the significant demasculinization of the male sexual characteristics. Furthermore, these changes in the sexual characteristics at the cellular and organ levels translated into ecologically important effects on the reproductive success at the individual level, as measured by a decrease in offspring production and survival rate. The present study provides the first evidence that monocrotophos pesticide can cause severe reproductive abnormalities in fish due to its endocrine-disrupting action.

Effects of the UV filter benzophenone-3 (oxybenzone) at low concentrations in zebrafish (Danio rerio).

Organic UV filters including benzophenone-3 (BP-3) are widely used to protect humans and materials from damage by UV irradiation. Despite the environmental occurrence of BP-3 in the aquatic environment, little is known about its effects and modes of action. In the present study we assess molecular and physiological effects of BP-3 in adult male zebrafish (Danio rerio) and in eleuthero-embryos by a targeted gene expression approach focusing on the sex hormone system. Fish and embryos are exposed for 14 days and 120 hours post fertilization, respectively, to 2.4-312 µg/L and 8.2-438 µg/L BP-3. Chemical analysis of water and fish demonstrates that BP-3 is partly transformed to benzophenone-1 (BP-1) and both compounds are accumulated in adult fish. Biotransformation to BP-1 is absent in eleuthero-embryos. BP-3 exposure leads to similar alterations of gene expression in both adult fish and eleuthero-embryos. In the brain of adult males esr1, ar and cyp19b are down-regulated at 84 µg/L BP-3. There is no induction of vitellogenin expression by BP-3, both at the transcriptional and protein level. An overall down-regulation of the hsd3b, hsd17b3, hsd11b2 and cyp11b2 transcripts is observed in the testes, suggesting an antiandrogenic activity. No histological changes were observed in the testes after BP-3 treatment. The study leads to the conclusion that low concentrations of BP-3 exhibit similar multiple hormonal activities at the transcription level in two different life stages of zebrafish. Forthcoming studies should show whether this translates to additional physiological effects.

Effects of triclosan and triclocarban, two ubiquitous environmental contaminants, on anatomy, physiology, and behavior of the fathead minnow (Pimephales promelas).

Triclosan (TCS) and triclocarban (TCC) are two common antimicrobial agents found in many personal care products and subsequently are detected ubiquitously in wastewater effluent and receiving waters. Both compounds are of recent regulatory interest due to their omnipresence in the environment, including in humans. Although TCS and TCC have been suggested to be endocrine active, little information exists about their effects on organismal end points in development (growth, escape performance), anatomy (morphological indices, histology), physiology (vitellogenin), and behavior of exposed aquatic organisms. In this study, newly hatched fathead minnows were exposed for 12 days, and mature male and female fathead minnows (Pimephales promelas) were exposed for 21 days to environmentally realistic concentrations (nanograms per liter) of these two compounds singularly and in mixtures. At the end of the exposure, larvae were assessed for growth and predator-avoidance performance, and a subset of mature fish was assessed for plasma vitellogenin induction, expression of secondary sexual characteristics, relative size of liver and gonads, and histopathological changes to both organs. The remaining exposed mature fish were placed in breeding pairs of one male and one female minnow from the same treatment to assess their ability to defend a nest site and reproduce. Exposure to either antimicrobial compound, alone or as a mixture, caused no changes to larval fish, gonad size, or vitellogenin concentrations in mature fathead minnows. In contrast, decreased aggression was seen in adult male fathead minnows exposed to TCC (1.6 µg/l) or a mixture (560 ng/l TCS + 179 ng/l TCC and 1.6 µg/l TCS + 450 ng/l TCC). Decreased aggression would likely decrease their ability to defend and hold a nest site needed for spawning and reproduction. Substantial variability was found in the severity of observed effects within treatments, suggesting that environmentally realistic concentrations of these compounds may only affect particularly sensitive individuals.

Exposure to 17α-ethynylestradiol causes dose and temporally dependent changes in intersex, females and vitellogenin production in the Sydney rock oyster.

Although mounting evidence suggests exposure to estrogenic contaminants increases vitellogenin production in molluscs, demonstration of dose-response relationships and knowledge of the temporal nature of the vitellogenin response with continual exposure is currently lacking for biomarker utility. To address this knowledge gap, adult Sydney rock oysters, Saccostrea glomerata, were exposed to a range of environmentally relevant concentrations of 17α-ethynylestradiol (EE2) (0, 6.25, 12.5, 25 or 50 ng/l) in seawater under laboratory conditions. Vitellogenin induction and gonadal development was assessed following 4, 21 and 49 days exposure to EE2. Vitellogenin was found to increase in a dose dependent manner with EE2 exposure for females (4 and 49 days) and males (4 and 21 days). Histological examination of gonads revealed a number of individuals exhibited intersex (ovotestis) in 50 ng/l EE2 (after 21 days) and in 6.25 and 12.5 ng/l EE2 (after 49 days). Furthermore, a significant shift towards females was observed following 49 days exposure at 50 ng/l EE2 suggesting estrogenic exposure is capable of facilitating a progression for protandric males from male-intersex-female gametal status. Increases in female vitellogenin (4 days) were predictive of later increases in female developmental stages at 21 days and increases in oocyte area following 49 days. Male vitellogenin (4 days) was predictive of decreased male percentages and lower male developmental stages at 49 days. Vitellogenin in S. glomerata is a predictive biomarker of estrogenic exposure and effect if sampled soon after exposure and at the commencement of a gonadal development cycle.

Assessing the combination effects of environmental estrogens in fish.

The method on combined effects of environmental estrogens and mixture environmental risk assessment was discussed. Batch tests were conducted to assess the in vivo potency of mixtures of estrogens using plasma vitellogenin concentrations in male crucian carp (Carassius carassius) as the endpoint. A nonlinear regression was determined on the concentration response relationship for the single chemical of 17ß-estradiol (E(2)), 17α-ethynylestradiol (EE(2)), 4-tert-octylphenol (OP), bisphenol A (BPA), and that of the mixed compounds at equipotent concentrations (E(2)-EE(2), E(2)-EE(2)-OP-BPA), the mixture was tested using a fixed-ratio design. On the basis of statistical selection criteria, the best-fit model is chosen individually for each set of data. Furthermore, the bootstrap methodology is applied for constructing confidence intervals for the estimated effect concentrations. The combined effects of the mixture can be predicted using biomathematical models based on the concentration and potency of the individual mixture components. The finding of non-monotonic dose-response relationship and the combined effects can be accurately predicted in whole range of exposure concentration by the reference models, whereas the outcome of simple effect summation with a great deal of indetermination. Results suggested that there can be a risk of mixture effects. The potential impact of components on mixture would depend predominantly on its concentration, the mixture ratio, and its relative potency. Existing environmental risk assessment procedures are limited in their ability to evaluate the combined effects of chemical mixtures, therefore further improvement is needed.

Endocrine disruption in wild populations of chub (Leuciscus cephalus) in contaminated French streams.

The aim of this study was to assess endocrine disruptive effects in wild population of fish in five French rivers selected to represent different pollution contexts at two seasons (summer and fall). For that purpose, a panel of biometrical parameters (length, weight, and gonado-somatic index: GSI) and biochemical (ethoxyresorufin-O-deethylase: EROD, vitellogenin: VTG, and brain aromatase) and histological biomarkers (gonads histology) were used in chub (Leuciscus cephalus), a common cyprinid fish species. In fish from the reference site, EROD activity and VTG levels were low at the two seasons. Brain aromatase activities (AAs) were similar to other species and increased with increasing GSI and gonad maturation. Among the four contaminated sites, the Jalle d'Eysines River was the most impacted site. At this site, fish were exposed to estrogenic substances as demonstrated by the VTG induction in males and the arrest of development of the gonads that led to lower brain AA compared to fish from the reference site. In fish from other contaminated sites, EROD activity was induced as compared to fish from the reference site and some males had elevated concentrations of VTG. Moreover, the presence of aromatase-inhibiting compounds was demonstrated in the sediments of three contaminated sites, even if the precise nature of contaminants is not known. This study provides new data concerning endocrine disruption in wild fish populations inhabiting French rivers and demonstrates that measurements of in vivo and in vitro aromatase could be used as biomarkers of endocrine disruption in field studies.

Short-term exposure to sublethal tebuconazole induces physiological impairment in male zebrafish (Danio rerio).

The aim of the present study was to assess the physiological response of male zebrafish Danio rerio to the fungicide tebuconazole and recovery in fungicide-free water. Acute toxicity tests were carried out and the median lethal concentration (LC(50)) from 24 to 96 h was calculated. The fish were exposed to a sublethal fungicide concentration of 230 microg/L for 7 or 14 days and allowed to recover for 7 or 14 more days, respectively. Whole-body levels of vitellogenins, triglycerides, cholesterol, glucose, lactate and proteins as well as the activities gamma-glutamil transpeptidase (gamma-GT), alanin aminotransferase (AlAT), alkaline phosphatase (AP) and lactate dehydrogenase (LDH) were assayed; corpulence factor (k) was also calculated. Fish exhibited significant increase of vitellogenins (Vtg), which continued to increase after 14 days of recovery. Levels of glucose, lactate, cholesterol and triglycerides increased after 7 and 14 days of exposure. Finally, cholesterol and glucose recovered after 14 days of recovery whereas triglycerides and lactate continued to be elevated. Proteins and k remained unaltered the entire experiments. AAT, AlAT and AP enhanced during exposure and did not recover at the end (except AlAT). A longer recovery period should be necessary to re-establish fish physiology. These results alert about the multiple disruptive physiological actions that tebuconazole may have on fish.

Effects of 17alpha-ethynylestradiol on early-life development, sex differentiation and vitellogenin induction in mummichog (Fundulus heteroclitus).

Fertilized mummichog eggs retrieved from 17alpha-ethynylestradiol (EE(2)) exposed adult fish were raised in concentrations of EE(2) ranging from 0 to 100 ng/L (100 ng/L EE(2) estimated to have actual average exposure concentrations of 30% of nominal; 0.1-10 ng/L were below detect throughout 24-h exposure period) for 61 weeks post-hatch. Eggs exposed at 100 ng/L hatched sooner, the larvae were longer, and survival of juvenile fish from hatch to study termination was greater than all other treatments, though fewer hatched at this treatment. Sex ratios were skewed (>80% female phenotype) at 100 ng/L EE(2), and some gonadal male fish displayed female secondary sex characteristics. Condition factor, gonadosomatic index (GSI), and liver somatic index (LSI) were found to decrease in both sexes between 52 and 61 weeks post-hatch. Female fish had increased hepatic vitellogenin (VTG) at 52 weeks post-hatch. When exposed to 1, 10 and 100 ng/L EE(2), female fish had a higher proportion of vitellogenic follicles in the ovarian tissue. Males exposed at 100 ng/L may have had disruption at some endpoints (GSI, VTG) that is masked due to reduced sample size compared to other treatments. Fish exposed to concentrations of EE(2) at or below 10 ng/L showed inconsistent effects on development and reproductive potential. This study indicates the potential for population-level effects at the high range of environmental EE(2) at concentrations equivalent to those at which consistent effects in fecundity in the adult mummichog reproductive test have been measured. This work demonstrates that chronic EE(2) exposure causes developmental effects at concentrations similar to those which cause effects in the shorter-term adult mummichog reproductive test. Effects are at higher concentrations than have been noted for freshwater model species. Whether this is because of species sensitivity or due to differences between freshwater and saltwater availability of EE(2) or its uptake requires further study.

Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor.

BACKGROUND: Several chemicals in the environment have the potential to inhibit aromatase, an enzyme critical to estrogen synthesis. OBJECTIVES: The objective of this study was to provide a detailed characterization of molecular and biochemical responses of female fathead minnows to a model aromatase inhibitor, fadrozole (FAD). METHODS: Fish were exposed via water to 0, 3, or 30 microg FAD/L for 8 days and then held in clean water for 8 days, with samples collected at four time points during each 8-day period. We quantified ex vivo steroid production, plasma steroids, and plasma vitellogenin (Vtg) concentrations and analyzed relative transcript abundance of 10 key regulatory genes in ovaries and 3 in pituitary tissue by real-time polymerase chain reaction. RESULTS: Ex vivo 17beta-estradiol (E2) production and plasma E2 and Vtg concentrations were significantly reduced after a single day of exposure to 3 microg or 30 microg FAD/L. However, plasma E2 concentrations recovered by the eighth day of exposure in the 3-microg/L group and within 1 day of cessation of exposure in the 30-microg/L group, indicating concentration- and time-dependent physiologic compensation and recovery. Concentration-dependent increases in transcripts coding for aromatase (A isoform), cytochrome P450 side-chain cleavage, steroidogenic acute regulatory protein, and follicle-stimulating hormone receptor all coincided with increased E2 production and recovery of plasma E2 concentrations. CONCLUSIONS: Results of this research highlight the need to consider compensation/adaptation and recovery when developing and interpreting short-term bioassays or biomarkers or when trying to predict the effects of chemical exposures based on mode of action.

Effects of adrenergic agents on the expression of zebrafish (Danio rerio) vitellogenin Ao1.

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins, traditionally considered to be estrogen-responsive precursors of the major egg yolk proteins, expressed and synthesized mainly in hepatic tissue. The inducibility of VTGs has made them one of the most frequently used in vivo and in vitro biomarkers of exposure to estrogen-active substances. A significant level of zebrafish vtgAo1, a major estrogen responsive form, has been unexpectedly found in heart tissue in our present studies. Our studies on zebrafish cardiomyopathy, caused by adrenergic agonist treatment, suggest a similar protective function of the cardiac expressed vtgAo1. We hypothesize that its function is to unload surplus intracellular lipids in cardiomyocytes for "reverse triglyceride transportation" similar to that found in lipid transport proteins in mammals. Our results also demonstrated that zebrafish vtgAo1 mRNA expression in heart can be suppressed by both alpha-adrenergic agonist, phenylephrine (PE) and beta-adrenergic agonist, isoproterenol (ISO). Furthermore, the strong stimulation of zebrafish vtgAo1 expression in plasma induced by the beta-adrenergic antagonist, MOXIsylyl, was detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). Such stimulation cannot be suppressed by taMOXIfen, an antagonist to estrogen receptors. Thus, our present data indicate that the production of teleost VTG in vivo can be regulated not only by estrogenic agents, but by adrenergic signals as well.

Effects of atrazine on hepatic metabolism and endocrine homeostasis in rainbow trout (Oncorhynchus mykiss).

The herbicide atrazine (ATZ) is one of the most widely used pesticides in the world and is now under scrutiny for its alleged capacity to disrupt the endocrine system. Exhibiting negligible interaction with the estrogen receptor (ER), ATZ's mode of action remains to be elucidated. ATZ may act as an inducer of the enzyme aromatase, which converts androgens to estrogens, although other mechanisms should also be taken into consideration such as impairment of hepatic metabolism. Therefore we administered juvenile rainbow trout (Oncorhynchus mykiss) a dose of either 2 or 200 microg ATZ/kg, or of carrier control phosphate buffered saline (PBS) and we measured plasma concentrations of testosterone (T), 17beta-estradiol (E2) and vitellogenin (Vtg) 6 days after exposure. Simultaneously we analyzed hepatic gene expression of cytochrome P450 (CYP) 1A and pi-class glutathione S-transferase (GST-P), and catalase (CAT) activity. Although sex steroid levels showed no significant alterations, we found a dose-dependent increase in Vtg and a concomitant decrease in CYP1A. There was no effect of ATZ on GST-P mRNA levels but GST-P was positively correlated with CYP1A. Also, CYP1A was negatively correlated with liver CAT and E2, and varied with T concentrations in a hormetic manner. The results showed that ATZ can alter hepatic metabolism, induce estrogenic effects and oxidative stress in vivo, and that these effects are linked.

Estrogenic activity of ternary UV filter mixtures in fish (Pimephales promelas) - an analysis with nonlinear isobolograms.

Numerous estrogenic compounds are present in aquatic environments, but currently it is not well understood how compounds that differ in maxima and slope of their individual dose-response curves contribute to the overall mixture effect. In order to better understand such interactions we investigated 3 commonly used UV filters, for their estrogenic mixture activity and analysed their joint effects by using the concentration addition (CA) concept. Thereby, we extended the method of isoboles for analysis of 3 compounds that differ in maxima and slopes of their dose-response curves. 3-Benzylidene camphor (3BC), benzophenone-1 (BP1) and benzophenone-2 (BP2) are estrogenic in fish and act as pure- or partial estrogen receptor alpha agonists. First we exposed juvenile fathead minnows for 14 days to six concentrations of each UV filter alone to determine vitellogenin (VTG) induction curves, calculate equi-effective mixture concentrations and predict mixture effects. For 3BC, BP1 and BP2 significant VTG-induction occurred at 420, 2668, and 4715 microg/L, respectively. BP2 displayed a full dose-response curve, whereas 3BC and BP1 showed submaximal activity of 70 and 78%, respectively. Second, we exposed fish to 6 equi-effective mixtures (EC-NOEC, EC1, EC5, EC10, EC20, EC30) of these UV filters. Significant VTG-induction occurred at EC5 and higher. Submaximal activity of 67% as compared to the control EE2 (100 ng/L) was reached. The curves for the observed and predicted mixture activity agreed for mixture levels (EC10 to EC30), however, at EC-NOEC, EC1 and EC5, lower activity was observed than predicted by CA. Detailed isobolographic analysis indicate additivity at EC10 to EC30, and antagonism at low levels (EC-NOEC to EC5). Our data show for the first time, that for compounds with differences in maxima and slope, considerably more mixture combinations are additive than previously thought. This should be taken into account for hazard and risk assessment of UV filters and xenoestrogens.

Effects of 17alpha-ethynylestradiol on hormonal responses and xenobiotic biotransformation system of Atlantic salmon (Salmo salar).

Pharmaceuticals are ubiquitous pollutants in the aquatic environment where their potential effects on non-target species like fish has only recently become subject of systematic investigations. In the present study, experiments were undertaken to examine the effects of a synthetic pharmaceutical endocrine disruptor, ethynylestradiol (EE2), given in water at 5 or 50 ng/L and sampled at days 0 (control), 3 and 7 after exposure, on hepatic phase I and II biotransformation and hormonal pathways of juvenile salmon using quantitative (real-time) polymerase chain reaction (qPCR), Vtg ELISA and 7-ethoxyresorufin O-deethylase (EROD) catalytic activity. Our data show that EE2 produced time- and concentration-specific modulation of estrogen receptor isoforms (ERalpha, ERbeta) and androgen receptor-beta (ARbeta). EE2 produced a concentration-specific induction of vitellogenin (Vtg) and zona radiata protein (Zr-protein) at day 3 after exposure. At day 7, Vtg and Zr-protein mRNA (and plasma Vtg protein) expression were significantly decreased in the group given 5 ng EE2/L, compared to dimethyl sulfoxide (DMSO) control group. In the xenobiotic biotransformation pathway, EE2 produced a significant increase of aryl hydrocarbon receptor-alpha (AhRalpha) at day 3 in the group given 5 ng EE2/L and AhRbeta was decreased at the same concentration at day 7. While CYP3A was not significantly affected by EE2 exposure, the CYP1A1, AhR nuclear translocator (Arnt) and AhR repressor (AhRR) mRNA showed an apparent EE2 concentration and time-dependent decrease. The expression of uridine diphosphoglucuronosyl transferase (UGT) and glutathione S-transferase class pi-like (GSTpi-like) mRNA were decreased after exposure to 50ng EE2/L at both day 3 and 7 after exposure. The effect of EE2 on the CYP1A1 gene expressions paralleled effect on EROD and AhRR mRNA, suggesting a direct role of EE2 in controlling cellular detoxification machinery. Interestingly, the carrier vehicle, DMSO produced significant time-dependent induction of estrogenic (ERalpha, Vtg and Zr-protein) responses, compared with blank (i.e. without DMSO) controls at day 7 post-exposure. The effect of DMSO totally underscored the observed EE2 effect at day 7 after exposure. In general, these findings support previous reports on the endocrine effects of EE2, in addition to effects on hepatic biotransformation system. In view of the data presented here and our recent studies, the use of DMSO as carrier vehicle in endocrine toxicological experimental studies should be re-evaluated.

Effects of the UV filter benzophenone-2 on reproduction in fish.

The UV filter benzophenone-2 (BP-2) is largely used in personal care products such as cosmetics and in numerous other materials for UV protection. Like other UV filters, BP-2 has been found to be estrogenic in vitro and in vivo, but potential effects on reproduction of fish are unknown. In this study, we evaluate whether BP-2 affects important reproductive parameters such as fecundity, gametogenesis and secondary sex characteristics. After a pre-exposure period of 19 days, reproductively mature fathead minnows (Pimephales promelas) were exposed to 0.002, 0.1, 1.2, 5.0 and 9.7 mg/L BP-2 for 15 days. BP-2 was accumulated in fish up to 3.1 microg/g body weight. In males, a dose-dependent vitellogenin induction and decrease in the number of nuptial tubercles occurred. Moreover, significant dose-related effects on gonads of male and female fish were observed. At concentrations of 1.2 mg/L and higher, spermatocyte and oocyte development was significantly inhibited in male and female fish, respectively. Testes of exposed males had much fewer spermatocytes and ovaries of exposed females had much fewer mature and more atretic follicles. Reproduction was negatively affected in a dose-dependent manner with a decrease in egg production at 5.0 mg/L and a complete cessation of spawning activity at 9.7 mg/L BP-2. Our findings show significant estrogenic effects of the common UV filter BP-2 on vitellogenin induction, secondary sex characteristics, gonadal development, and reproduction in fish.

Effects of 17alpha-ethynylestradiol on EROD activity, spiggin and vitellogenin in three-spined stickleback (Gasterosteus aculeatus).

The three-spined stickleback (Gasterosteus aculeatus) has quantifiable biomarkers of exposure to estrogens (vitellogenin), androgens (spiggin) and aryl hydrocarbon receptor (AhR) agonists (EROD activity) and is therefore a promising test species for biomonitoring of reprotoxic chemicals in aquatic environments. In this study we evaluated the effects of 17alpha-ethynylestradiol (EE(2)) on EROD activity, induction of vitellogenin and spiggin, hepatosomatic index (HSI), ovarian somatic index (OSI) and nephrosomatic index (NSI). Adult male and female three-spined sticklebacks were exposed to concentrations of 0-170 ng EE(2)/l (measured concentrations) in a flow-through system for 21 days. Exposure to 170 ng EE(2)/l resulted in a significant 8- and 9-fold induction of gill EROD activity in males and females, respectively. In livers, EROD activity expressed in relation to microsomal protein content was suppressed due to a significant increase in microsomal protein content. Hepatic EROD activity per se expressed as picomol/min was not affected by exposure to EE(2). The lowest observed effect concentration for induction of vitellogenin in males was 53.7 ng EE(2)/l. In females, vitellogenin levels were significantly higher in those exposed to 170 ng EE(2)/l compared to controls. Spiggin production was significantly inhibited and NSI lower in males exposed to 170 ng EE(2)/l. In both females and males LSI was significantly higher in fish exposed to 170 ng EE(2)/l than in controls. In females exposed to 170 ng EE(2)/l, OSI was significantly lower and NSI higher than controls. The observed results from this study show that a synthetic estrogen can affect the well-known biomarker of exposure for dioxin-like compounds, EROD activity, and further that this response can differ between tissues. These findings are important for interpretation of biomonitoring data.

Effects of alkylphenols on the reproductive system of Atlantic cod (Gadus morhua).

Produced water, a by-product of offshore oil production, contains significant amounts of alkylphenols (APs). Many studies have shown that APs cause endocrine disruption in marine organisms, but relatively little is currently known about their long-term effects on the biology of pelagic fish. Here, we describe in detail the effects of APs on the reproductive potential of first-time spawning Atlantic cod (Gadus morhua). Cod were fed with feed paste containing four APs (4-tert-butylphenol, 4-n-pentylphenol, 4-n-hexylphenol and 4-n-heptylphenol), at a range of concentrations, for either 1 or 5 weeks. AP-exposed fish were compared to unexposed fish and to fish fed paste containing natural estrogen (17beta-estradiol). Our results showed that in female fish AP exposure impaired oocyte development, reduced estrogen levels, and delayed the estimated time of spawning by 17-28 days. Similarly, in male fish, we observed that AP-exposure reduced 11-keto-testosterone concentrations, and caused a small induction of VTG levels. We also observed impaired testicular development, with an increase in the amount of spermatogonia and a reduction in the amount of spermatozoa present. Taken together these results suggest that APs released into the sea via produced water may have a negative influence on the overall reproductive fitness of cod populations.