Inhibition of metamorphosis in tadpoles of Xenopus laevis exposed to polybrominated diphenyl ethers (PBDEs).

Tadpoles of the African clawed frog, Xenopus laevis were exposed, beginning at stage 50, to a commercial pentabromodiphenyl ether mixture (DE-71) through the diet. Subsequent experiments were conducted using a single intraperitoneal injection at stage 58 with limited quantities of two purified brominated diphenyl ether (BDE) congeners, BDE47 and BDE99 and DE-71 to determine the relative potency of these BDE congeners within the commercial mixture. Significant inhibition of tail resorption, delayed metamorphosis and impacts on skin pigmentation were observed in Xenopus exposed to DE-71 in the diet at nominal doses of 1000 and 5000 microgg(-1) of food. The estimated time required for 50% of the tadpoles to complete metamorphosis was significantly lengthened in Xenopus exposed to a dietary concentration of 1 microg DE-71 per gram of food. Analysis of PBDEs (sum of 32 congeners) in Xenopus from the treatment with 5000 microgg(-1) of DE-71 indicated that the frogs accumulated an average of 1030 microgg(-1) (wet weight) of PBDEs. In the intraperitoneal injection trials, similar inhibitory responses were observed in Xenopus injected with DE-71 at a nominal dose of 60 microg per tadpole, or injected with BDE47 at a nominal dose of 100 microg per tadpole. No responses were observed in Xenopus injected with BDE99 at doses up to 100 microg per tadpole. Complete inhibition of metamorphosis was observed only in the highest DE-71 dietary treatment. The results of this study are consistent with a mechanism of action of PBDEs involving competitive inhibition of binding of thyroid hormones to transporter proteins, although the mechanism cannot be definitively determined from this study. The observed effects may have occurred through other mechanisms, including sublethal toxicity. The doses used in this study are greater than the levels of PBDEs to which anurans are exposed in the environment, so further studies are required to determine whether exposure to PBDEs at environmentally relevant concentrations can affect frog metamorphosis.

Effects of the isoflavones genistein and equol on the gonadal development of Japanese medaka Oryzias latipes.

The estrogenic isoflavone compound genistein recently has been found in the effluents of sewage treatment plants and pulp mills, and the related compound equol has been detected in the runoff from agricultural fields treated with hog manure. Waterborne exposures of Japanese medaka (Oryzias latipes) to equol from soon after hatch to approximately 100 days posthatch induced gonadal intersex (i.e., testis-ova) in males at incidences of 10 and 87% in equol treatments of 0.4 and 0.8 micro g/L, respectively. Exposure to the highest test concentration of genistein, 1,000 micro g/L, also caused a low incidence (i.e., 12%) of gonadal intersex in male medaka. The ovaries of female medaka from both equol and genistein treatments showed delayed oocyte maturation, atretic oocytes, an enlarged ovarian lumen, proliferation of somatic stromal tissue, and primordial germ cells; responses were concentration dependent. Alterations to externally visible secondary sex characteristics occurred in medaka exposed to both equol and genistein. In treatments with 1,000 micro g/L genistein, 72% of male medaka (as identified by the gonadal phenotype) showed feminized secondary sex characteristics. Gonadal intersex and alterations to secondary sex characteristics have been noted in several fish populations around the world. This laboratory study indicates that isoflavone compounds should be considered candidate estrogenic compounds that may be involved in the alteration of sexual development in feral fish populations.

Endocrine disruption and altered gonadal development in white perch (Morone americana) from the lower Great Lakes region.

High prevalences of gonadal intersex have been observed in wild fish populations in areas affected by domestic and industrial effluents. For this study, fish were collected in 1998 from the Cootes Paradise region of Hamilton Harbour in western Lake Ontario, Canada, to determine whether gonadal abnormalities, including intersex, were present in young of the year (YOY) fish. No gonadal abnormalities were observed in goldfish (Carassius auratus), common carp (Cyprinus carpio), gizzard shad (Dorosoma cepedianum), brown bullhead (Ictalurus ameiurus), pumpkinseed (Lepomis gibbosus), and bluegill (Lepomis macrochirus). However, intersex gonads were observed in 8 of 16 male white perch (Morone americana) examined in this survey. Subsequently, in 1999 and 2000 white perch estimated to be YOY to approximately 2 years of age were collected from Cootes Paradise and from two other sites in the lower Great Lakes region. Gonadal intersex was observed in male white perch collected from the Bay of Quinte (22-44%) and Lake St. Clair (45%), although the prevalence and the extent of the intersex condition were lower relative to the 83% prevalence in white perch collected in Cootes Paradise. Intersex was not observed in hatchery-reared white perch or in white perch collected from an uncontaminated reference site (i.e., Deal Lake) in the United States. An analysis of plasma collected in the spring of 2002 from male adult white perch in Cootes Paradise revealed high concentrations of vitellogenin, ranging from 49 to 1,711 microg/mL. These observations indicate that male white perch are exposed to estrogenic endocrine-disrupting substances that may be responsible for the induction of gonadal intersex.

Effects of the antiandrogens, vinclozolin and cyproterone acetate on gonadal development in the Japanese medaka (Oryzias latipes).

This study was focused on determining the effects of exposure to antiandrogens on the gonadal development of Japanese medaka (Oryzias latipes). Test compounds included the fungicide, vinclozolin and the clinical antiandrogen, cyproterone acetate. Newly hatched medaka were exposed to aqueous solutions of vinclozolin (2500 microg/l) and the vinclozolin fungicide formulation, Ronilan (1000 and 5000 microg/l) and cyproterone acetate (1 and 10 microg/l), for 3 months. Histological evaluation of the gonadal tissues of exposed fish indicated that the 5000 microg/l concentration of the vinclozolin formulation (Ronilan) induced a low incidence of intersex (i.e. testis-ova) and the 2500 microg/l concentration of vinclozolin-affected spermatogenesis in males. Also, the vinclozolin treatments induced moderate ovarian atresia. Cyproterone acetate also induced a low incidence of testis-ova, but in contrast to the vinclozolin treatment the amount of ovarian tissue in the testis-ova was equal to or greater than the amount of testicular tissue. In the cyproterone acetate treatments, both oogenesis and spermatogenesis were moderately inhibited at all test concentrations. The results of this study indicate that antiandrogens have the potential to alter testicular development and gametogenesis in fish. However, research is needed to determine the mechanisms by which antiandrogens affect fish.

Alterations to gonadal development and reproductive success in Japanese medaka (Oryzias latipes) exposed to 17alpha-ethinylestradiol.

The Japanese medaka (Oryzias latipes) was used as an in vivo model to evaluate the effects of exposure to the synthetic estrogen 17alpha-ethinylestradiol (EE2) on reproductive behavior and reproductive success in fish. Exposures to EE2 began within 2 to 5 d posthatch and continued until medaka were sexually mature, between four and six months of age. Among male and female medaka exposed to EE2 at nominal concentrations of 0.2 and 2 ng/L, mating behavior and reproductive success were normal in reproductive trials. However, reproductive behavior (i.e., copulations) was suppressed in the treatment with 10 ng/L EE2. Among 19 males exposed to 10 ng/L EE2 and placed with unexposed females in reproductive trials, 16 males did not copulate and reproductive success was very low. None of the females exposed to 10 ng/L of EE2 participated in reproductive behavior with unexposed males. The reproductive trials in combination with a histological survey indicated that male fish with gonadal intersex (i.e., testis-ova) were still capable of reproductive behavior and could fertilize eggs. Even though females exposed to 10 ng/L EE2 had poor reproductive success, their ovaries showed normal development and oogenesis. These data are relevant to observations of intersex in feral fish populations. Although intersex in male fish may be an indicator of exposure to estrogenic compounds, it appears that the presence of oocytes in testicular tissue may not directly impact the reproductive capability of the male fish. However, it is clear that concentrations of endocrine disrupting chemicals (EDCs) that cause gonadal intersex are sufficient to reduce reproductive performance.

Feminization of female leukophore-free strain of Japanese medaka (Oryzias latipes) exposed to 17beta-estradiol.

The recently developed female leukophore-free (FLFII) strain of Japanese medaka (Oryzias latipes) carries DNA markers for the identification of genotypic sex. Information regarding genotypic sex is useful for tests in which endocrine-disrupting compounds may masculinize or feminize fish. In the present study, methods were developed to automate DNA extraction and profiling for rapid determination of genotypic sex. Adequate amounts of DNA were isolated by robotic extraction procedures from the caudal fin. New primers were developed to include an 18-base pair segment that is in the X chromosome of female medaka but is absent in the Y chromosome of male medaka. Automated profiling methods with 96-well plates permitted analysis of the genomic sex of medaka at rates of up to 500 fish/d. We investigated the sensitivity of the FLFII strain to the feminizing effects of the potent estrogen 17beta-estradiol (E2), and we compared this sensitivity to that of a wild strain that has been used widely in the study of endocrine-disrupting compounds. All FLFII medaka exposed to 1 microg/L of E2 (n = 50) had the female gonadal phenotype (i.e., ovaries), and all but one wild-strain medaka exposed to 1 microg/L of E2 (i.e., 47 of 48 fish) had the female gonadal phenotype, indicating that the FLFII and wild strains have approximately equal sensitivities to the feminizing effects of E2. Analysis of the genotype of FLFII medaka confirmed that 100% of fish with the male genotype had been feminized to the female gonadal phenotype. The FLFII strain is an excellent teleost model for detecting feminization or masculinization of fish, and automated methods can be used for rapid analysis of the genotypic sex of FLFII medaka.

Simultaneous measurement of uptake and elimination of cadmium by caddisfly (Trichoptera: Hydropsychidae) larvae using stable isotope tracers.

The use of stable isotopes coupled with inductively coupled plasma-mass spectrometry provides a unique opportunity to investigate pathways of metals in aquatic invertebrates. In this study, the simultaneous uptake and elimination of cadmium (Cd) in stream-dwelling caddisfly (Trichoptera: Hydropsychidae) larvae was measured in laboratory experiments with stable isotopes of Cd. In one experiment, animals were exposed to low levels (0.5 microg/L) of 114Cd in filtered river water and uptake was measured for 9 d, followed by 9 d during which the larvae were exposed to either 114Cd, 113Cd, or filtered river water. In a second experiment, the initial exposure concentration of 114Cd varied from 0.1 to 1.6 microg/L for 5 d, followed by 5 d during which the larvae were exposed to the same respective levels of 113Cd. The purpose of the two experiments was to test whether or not elimination of 114Cd from Hydropsyche larvae was the same in the presence or absence of Cd in the water and also whether or not elimination rates were dependent on exposure concentration. The results indicated that elimination of Cd by Hydropsyche larvae (elimination rate constant approximately 0.21-0.24/d) in the presence of Cd in the water was the same as the depuration when no metal was present. Furthermore, the elimination rate was independent of exposure concentrations, ranging between 0.1 and 1.6 microg/L. Thus, the stable isotope tracer technique enabled the simultaneous measurement of uptake and elimination of Cd in hydropsychid larvae and may have the potential to facilitate the labeling of various compartments (e.g., water, sediment, and food) in the aquatic environment.

Increased kidney, liver, and testicular cell death after chronic exposure to 17alpha-ethinylestradiol in medaka (Oryzias latipes).

Sublethal effects observed in fish exposed to environmental estrogens may be mediated via stimulation of cell death. To investigate whether cell death is induced in fish after chronic exposure to estrogenic chemicals, Japanese medaka (Oryzias latipes) were exposed from hatch until sexual maturity to 10 ng/L 17alpha-ethinylestradiol (EE2) or acetone solvent (control). Cell death was evaluated in blinded histological sections of whole medaka using terminal dideoxynucleotidyl-mediated dUTP nick end-labeling (TUNEL), which labels nuclei of cells containing apoptotic or necrotic (fragmented) DNA. The major impact of EE2 exposure in both male and female medaka was to significantly increase the number of TUNEL-positive hepatocytes and kidney tubule cells compared to control. Cell morphology was consistent with apoptosis in the liver and cloudy swelling or necrosis in the tubule cells. The number of TUNEL-positive interstitial (hematopoietic) and glomerular cells was significantly greater in the kidneys of EE2-exposed male, but not female, medaka. The EE2 exposure also significantly increased the number of TUNEL-positive testicular cells in medaka compared to corresponding controls, namely Leydig cells, Sertoli cells, spermatocytes, and spermatids. In medaka with gonadal intersex, areas of fibrosis and areas containing female gonadal cells were relatively unstained with TUNEL. No effect of EE2 exposure on the number of TUNEL-positive ovarian somatic cells or on the rate of female ovarian follicle atresia was found. These results suggest that chronic exposure to EE2 in medaka is hepatotoxic and nephrotoxic in both sexes, whereas gonadal toxicity is specific to males.

Decreased apoptosis in the forebrain of adult male medaka (Oryzias latipes) after aqueous exposure to ethinylestradiol.

Endocrine disrupting compounds (EDCs), especially those that are estrogenic, are an issue of growing concern because they may ultimately adversely affect wildlife survival. 17-beta-Estradiol and its synthetic counterpart, 17-alpha-ethinylestradiol, two common EDCs, are associated with intersex conditions and impaired male reproductive behavior in fish. Male and female Japanese medaka (Oryzias latipes) were exposed to 10 ng/l ethinylestradiol for 6 months. Using terminal dideoxynucleotidyl-mediated dUTP nick end-labeling (TUNEL) to quantitate cell death, we found that ethinylestradiol-exposed males had significantly fewer apoptotic cells in the forebrain compared to untreated males and exposed females. Our results show that the effects of ethinylestradiol exposure are highly variable among individuals of the same species and even within tissues of the same individual. Thus, when examining the effects of EDCs on natural populations, data from a variety of tissues should be examined and the interpretation of any effects should include consideration of tissue-specific processes.