Use of the three-spined stickleback (Gasterosteus aculeatus) as a sensitive in vivo test for detection of environmental antiandrogens

We have previously shown that exposure to exogenous androgens causes female sticklebacks (Gasterosteus aculeatus) to produce the glue protein, spiggin, in their kidneys. This protein can be quantified by an enzyme-linked immunosorbent assay developed and validated at the Centre for Environment, Fisheries and Aquaculture Science. Here we report the development of an in vivo test for the detection of environmental antiandrogens. The system involves the simultaneous exposure of female sticklebacks to 17alpha-methyltestosterone (a model androgen) at 500 ng/L and suspected environmental antiandrogens over a period of 21 days. The spiggin content of the kidneys is then measured, and any antiandrogenic activity is evaluated by comparing the spiggin levels of female fish exposed to antiandrogens to those of female fish exposed solely to the model androgen. The assay detects the antiandrogenic activity of flutamide, vinclozolin (both used at 250 microg/L), linuron (at 150 microg/L), and fenitrothion (at 15 and 150 microg/L). These results provide the first evidence of in vivo antiandrogenic activity of both linuron and fenitrothion in teleosts. Although there are other suggested fish species that could be used for this purpose, the stickleback is the only widely available species in which it is now possible to study both estrogenic and antiandrogenic end points in the same individual. Furthermore, the species is endemic and ubiquitous in Europe, and it possesses many ecological traits that make it better suited than other potential species for field research into endocrine disruption.     

Parasite frequency and liver anomalies in three-spined stickleback,Gasterosteus aculeatus (L.), after long-term exposure to pulp mill effluents in marine mesocosms

Model ecosystems, simulating a Baltic Sea littoral habitat, were used for evaluating the effects of different effluents from kraft pulp mill industries on fish. Populations of newly hatched three-spined stickleback (Gasterosteus aculeatus) were continuously exposed to four different types of pulp mill effluents in a flowthrough sea water system. After 51/2 months of exposure, the sticklebacks were collected for a skin parasite count and histological examination of the gills and liver. The frequencies of two types of skin-dwelling ciliates of the generaTrichodina andApiosoma were higher in the exposed fish populations, particularly in those exposed to the high effluent doses (diluted 400 times). Anomalies in the liver cell structure were observed as necrotic cells, nuclear pyknosis, vacuolation, and fat accumulation. The two effluents from the factories producing unbleached softwood and bleached hardwood without oxygen pre-bleaching affected the sticklebacks most, both in respect to ciliate abundance and to liver anomalies.     

Transfer of nodularin to three-spined stickleback (Gasterosteus aculeatus L.), herring (Clupea harengus L.), and salmon (Salmo salar L.) in the northern Baltic Sea

Nodularin (NODLN) is a hepatotoxin produced by the cyanobacterium Nodularia spumigena, which occurs regularly in the Baltic Sea. The primary aim of this study was to study the transfer of NODLN to three-spined stickleback (Gasterosteus aculeatus L.), herring (Clupea harengus membras L.), and salmon (Salmo salar L.), which were caught from the northern Baltic Sea between August 2002 and August 2003. Liquid chromatography mass spectrometry (LC-MS) was used for NODLN analysis. NODLN was found in both herring (0–90 μg kg⁻¹ dw) and three-spined sticklebacks samples (2.8–700 μg kg⁻¹ dw). The recovery for the spiked stickleback samples in vitro was 28%. Only 1 salmon of a total of 10 contained a small amount of NODLN (10 μg kg⁻¹ dw). However, the high concentrations in individual stickleback suggest that possible transfer to higher trophic levels deserves more research.     

Disruption of male reproductive behavior in threespine stickleback Gasterosteus aculeatus exposed to 17 beta-estradiol

In past years we have witnessed decreased reproductive capacity in many wildlife species due to exposure to chemicals with endocrine-disrupting properties. In this laboratory experiment male threespine sticklebacks Gasterosteus aculeatus exposed to 17 beta-estradiol (2.0 micrograms/g) dispersed in peanut oil, on days 1, 7, 14, and 21, showed impaired paternal care compared to control fish that were exposed to peanut oil only. There were no differences between the two groups in number of males that built nests or in courtship displays. However, exposed males started nest building significantly later than control males. This study suggests that some but not all essential traits of male reproductive behavior may be altered as a result of exposure to 17 beta-estradiol. To reveal harmful effects of chemicals with suggested reproductive-disrupting properties it is thus important to take a wide variety of variables related to reproductive behavior into consideration.     

Comparative efficacy of the threespine stickleback (Gasterosteus aculeatus) and the mosquitofish (Gambusia affinis) for mosquito control.

The effectiveness of the threespine stickleback as a mosquito control agent was compared to that of the mosquitofish in 28-m2 earthen ponds during 26-wk experiments where the 2 fish were stocked alone and together. Relative to ponds without fish, the stickleback was not effective for controlling larval mosquito populations; however, sticklebacks reduced the abundance of Culex pupae. Mosquitofish provided significant levels of control whether stocked alone or concurrently with the stickleback. As compared to mosquitofish alone, mosquito control was not significantly enhanced when both fish were stocked together. Mortality of adult sticklebacks was related to a gradient of increasing water temperature across the ponds rather than the direct effects of other abiotic factors such as low dissolved oxygen concentrations or biotic interactions with the mosquitofish. The stickleback exhibited a lower thermal tolerance and slower population recruitment as compared to the mosquitofish populations,which reproduced successfully in water > 33 degrees C and grew rapidly. Stickleback biomass either declined or increased slightly (approximately 50% of initial stocking weight). Mosquitofish biomass increased 33- to 38-fold at rates averaging between 0.079 and 0.095 g wet weight/g/day and total wet weight per pond at 6 wk after stocking did not differ significantly between the 2 mosquitofish treatments.     

The uptake and loss of dissolved cadmium by the stickleback, Gasterosteus aculeatus L

The whole-body uptake of cadmium by the three-spined stickleback (Gasterosteus aculeatus) has been measured after exposure of fish to 2.5 and 5.0 mg Cd2+/liter hard water and equal amounts of 2.0 mg Cd2+ plus 2 mg Zn2+/liter and 4.0 mg Cd2+ plus 4.0 mg Zn2+/liter hard water, respectively. Fish absorbed and retained cadmium while the uptake and accumulation of zinc was depressed. The loss of absorbed cadmium was quicker in fish previously exposed to cadmium plus zinc solutions used together. This phenomenon is discussed in relation to the ability of fish to excrete absorbed cadmium.     

Histological changes in the kidneys and gills of the stickleback, Gasterosteus aculeatus L, exposed to dissolved cadmium in hard water

Damage to the kidneys and gills of the three spined stickleback Gasterosteus aculeatus have been investigated, using light microscopy after exposure of fish to 2 to 6 mg Cd2+ liter-1 hard water (299 mg liter-1 as CaCO3). Cytological breakdown of kidneys and gill tissue appears to be the cause of death, since oxygen uptake and cadmium excretion are impaired, leading to tissue hypoxia and fish poisoning.     

Detection of environmental androgens: a novel method based on enzyme-linked immunosorbent assay of spiggin,the stickleback (Gasterosteus aculeatus) glue protein

We report the development and validation of a novel in vivo biomarker test for waterborne androgens. During breeding, male sticklebacks (Gasterosteus aculeatus) manufacture a glue protein, spiggin, in their kidneys that they use to build their nests. Spiggin production is under the control of androgens. Until now, however, it has only been possible to quantify its production by measurement of the height of kidney epithelial cells. In the present study, we report the development of an enzyme-linked immunosorbent assay (ELISA) for spiggin and demonstrate its application to the measurement of spiggin in the kidneys of female sticklebacks that have been exposed to androgens in water. Results from the ELISA procedure revealed a strong correlation with measurement of kidney epithelial cell height (r2 = 0.93). However, the ELISA was much quicker and had a considerably higher response range (100,000-fold vs fourfold). Clear, graded responses in spiggin production were obtained by exposing intact females to increasing concentrations of 17a-methyltestosterone and 5alpha-dihydrotestosterone over three-week test periods. The lowest effective concentrations for these two steroids were 100 ng/L and 3 micorg/L, respectively. Female sticklebacks that were exposed to pulp mill effluent also produced spiggin in their kidneys. Possession of an androgen-regulated protein by the female stickleback makes it a unique bioassay organism for detecting androgenic contamination in the aquatic environment.     

Accumulation of metals in the tissues of three spined stickelback (Gasterosteus aculeatus) from natural fresh waters

Three spined sticklebacks (Gasterosteus aculeatus) were sampled from five different watercourses in Flanders (Belgium). Concentrations of cadmium, lead, copper, and zinc were measured in water, sediment, food, and fish tissues. Considered as food was the taxonomic groups of macroinvertebrates predominately present in the stomach of the sticklebacks at the different sites. Analyzed tissues were gill, liver, and muscle. To determine the relative importance of the different uptake routes, multiple linear regression models were constructed. Accumulated metal levels in the tissues were related to metal levels in sediment, water, and food. Generally, the amount of variation in accumulated metal levels that could be explained by these models was limited, with coefficients of determination not exceeding 0.57. Cadmium levels in the three tissues were solely related to levels in invertebrates. The same was true for copper in liver. In the other cases the tissue levels could be related only to sediment and/or water levels. Depending on the metal, levels were highest in gill or in liver. In all cases lowest metal levels were measured in muscle.     

Content of notification: Exposure to the pesticide chlordimeform

Using occupational data for over 3,400 primary liver cancer cases diagnosed between 1980 and 1984 reported to the Shanghai Cancer Registry, and employment information from the 1982 census for the Shanghai population, standardized incidence ratios were computed to generate leads to occupational risks of liver cancer. Among men, a statistically significant excess number of cases was observed for chemical processors, textile workers, wood workers, blacksmiths and machine-tool operators, and material handlers and dock workers. Increased incidence of liver cancer also was observed among female transport equipment operators. These findings indicate that a number of similar occupations are associated with increased risk of primary liver cancer in western countries and China. Although causal inferences cannot be drawn from these data, our study adds to the limited evidence of the potential role of occupational exposures in liver carcinogenesis. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Exposure of pesticide applicators and support personnel toO-ethylO-(4-nitrophenyl) phenylphosphonothioate (EPN)

Applicator personnel were monitored during aerial and ground applications of EPN to cotton in Mississippi and Arizona. Respiratory exposure based on an 8-hr workday averaged 11g for pilots, 15g for loaders, and 39g for ground applicators. Respiratory exposure of flagmen, monitored during a complete application cycle, averaged 317g/8 hr. Mean 8-hr dermal exposures were 2.1 mg for pilots, 6.3 mg for loaders, 117.7 mg for flagmen, and 7.5 mg for ground applicators. The levels of exposure were affected by route of exposure, body region, method of application, job function, attitude and experience, duration of exposure, weather conditions, and accidents. Recommendations are made to reduce the exposure of pilots, loaders, and ground applicators by modification of procedures and use of personal protective equipment. Because flagmen had the greatest exposure, a recommendation is made to substitute their function with fixed landmarks; if this is not possible, flagmen must wear the proper protective equipment.     

Exposure to nanoparticle-rich diesel exhaust affects hippocampal functions in mice

Epidemiological studies have indicated associations between day-to-day particulate air pollution and increased risks of various adverse health outcomes. Although an association between exposure to diesel exhaust particles (DEPs) and the development of pulmonary inflammation has been reported, there are limited reports on the neurotoxic effects of DEPs, particularly those of nanoparticle-rich diesel exhaust (NRDE). In this minireview, we highlighted the effects of NRDE which was generated in the National Institute for Environmental Studies, on hippocampus-dependent spatial learning ability and the expression of memory-function-related genes, neurotrophins, and proinflammatory cytokines in a mouse model.     

Exposure to the herbicide acetochlor alters thyroid hormone-dependent gene expression and metamorphosis in Xenopus Laevis

A growing number of substances released into the environment disrupt normal endocrine mechanisms in a wide range of vertebrates. Little is known about the effects and identities of endocrine-disrupting chemicals (EDCs) that target thyroid hormone (TH) action, particularly at the cellular level. Frog tadpole metamorphosis depends completely on TH, which has led to the suggestion of a metamorphosis-based assay for screening potential EDCs. A major mechanism of TH action is the alteration of gene expression via hormone-bound nuclear receptors. To assess the gene expression profiles in the frog model, we designed a novel multispecies frog cDNA microarray. Recently, the preemergent herbicide acetochlor was shown to accelerate 3,5,3 -triiodothyronine (T3)-induced forelimb emergence and increase mRNA expression of thyroid hormone ss receptors in ranid tadpoles. Here we show that T3-induced metamorphosis of Xenopus laevis, a species commonly used in the laboratory, is accelerated upon acute exposure to an environmentally relevant level of acetochlor. The morphologic changes observed are preceded by alterations in gene expression profiles detected in the tadpole tail, and the nature of these profiles suggest a novel mechanism of action for acetochlor.     

Exposure to fluorotrichloromethane (R-11)

Summary Three volunteers were exposed to fluorotrichloromethane (R-11) under experimental conditions. Solvent levels in ambient and alveolar air, in blood and urine were measured. The mean concentration of R-11 in ambient air was 657 ml/m³. The average values of pulmonary retention and solvent levels in alveolar air and blood were 18.2%; 537 ml/m³ and 2.8 mg/l. Interindividual variations of these parameters are negligible. R-11 concentrations in urine—in contrast to blood or alveolar air—depend on the dose taken up. After termination of exposure, R-11 concentrations in alveolar air and in blood are excreted with biological half-lives of seven and eleven minutes respectively during the first phase of elimination and with 1.8 and 1.0 h respectively during the second phase of elimination. Though ambient monitoring should, in most cases, be sufficient for the prevention of occupational diseases, the R-11 concentration in alveolar air seems to be the best parameter if biological monitoring seems to be necessary.     

Cyproterone acetate affects protamine gene expression in the testis of adult male rat

The temporal effects of oral administration of cyproterone acetate (CPA), a progestational androgen receptor blocker, were studied on the fertility of adult male rat sires, at a dose of 20 mg kg-1 day-1 after 15 days of gavage. The treatment reduced the fertility and weights of accessory sex glands, without altering the serum levels of luteinizing hormone, follicle-stimulating hormone (FSH) and testosterone (T). Sperm counts were significantly reduced after treatment. Several changes were evident in caput epididymal sperm chromatin in treated rats. The in vitro decondensation rates of sperm chromatin and total fluorescent acridine orange (AO) dye uptake were enhanced. The fluorescent AO dye uptake by the double- and single-stranded sperm chromatin increased. The uptake of thiol-specific monobromobimane fluorescent dye by sperm chromatin was significantly reduced. Sperm of treated rats exhibited hypoprotamination. Protamine levels in the testis were significantly reduced after treatment. Androgen-binding protein (ABP) expression was significantly reduced in testis after treatment. A slight but significant increase was observed in cyclic AMP immunoexpression in testis after treatment. The expression and levels of transition proteins 1 (TP1) and 2 (TP2) as well as cyclic AMP response element modulator protein-tau were maintained at control levels in the testis of treated rats. The present study reports that androgen receptor occupation by CPA preferentially reduces the levels of spermatidal protamine in testis and spermatozoa involved in nuclear chromatin condensation. It is inferred that ABP could be mediating the effects of T in modulating the sequential expression of TPs and protamines during nuclear chromatin condensation. It is likely that indirect effects of T involve its aromatization in spermatids.     

Conjugated linoleic acid affects lipid composition, metabolism, and gene expression in gilthead sea bream (Sparus aurata L)

To maximize growth, farmed fish are fed high-fat diets, which can lead to high tissue lipid concentrations that have an impact on quality. The intake of conjugated linoleic acid (CLA) reduces body fat in mammals and this study was undertaken to determine the effects of dietary CLA on growth, composition, and postprandial metabolic variables in sea bream. Fish were fed 3 diets containing 48 g/100 g protein and 24 g/100 g fat, including fish oil supplemented with 0 (control), 2, or 4% CLA for 12 wk. Feed intake, specific growth rate, total body fat, and circulating somatolactin concentration were lower in fish fed CLA than in controls. Feed efficiency was greater in fish fed 2% CLA than in controls. Liver triglyceride concentrations were higher in fish fed 4% CLA and muscle triglyceride concentrations were lower in fish fed both CLA diets than in controls. Hepatic fatty acyl desaturase and elongase mRNA levels in fish fed CLA were lower than in controls. Metabolic differences between controls and CLA-fed fish were observed at 6 h but not at 24 h after the last meal, including lower postprandial circulating triglyceride concentrations, higher hepatic acyl-CoA-oxidase, and lower L-3-hydroxyacyl-CoA dehydrogenase activities in CLA-fed fish than in controls. Dietary CLA did not affect enzymes involved in lipogenesis including hepatic fatty acid synthase and malic enzyme, but it decreased glucose 6-phosphate dehydrogenase activity at 24 h, but not at 6 h after feeding. The data suggest that CLA intake in sea bream has little effect on hepatic lipogenesis, channels dietary lipid from adipose tissue to the liver, and switches hepatic mitochondrial to peroxisomal beta-oxidation.     

Retinal degeneration in rats exposed to an organophosphate pesticide (fenthion)

Pigmented (Long-Evans) and albino (Wistar) rats were chronically exposed to an organophosphate pesticide (fenthion). Fenthion (50 mg/kg) was administered subcutaneously twice a week for 1 year; the total dosage for each animal ranged from 1.6 to 1.8 g. Concurrent with the fenthion administration, the amplitude of the scotopic electroetinogram (ERG) gradually declined, disappearing by the 12th month in all treated pigmented rats. For the albino experimental rats, however, the ERG amplitude disappeared as early as the 6th month in 7 out of 15 treated animals. Funduscopically, degeneration of the retina was observed in all rats when ERG responses had disappeared. Histopathological studies confirmed degeneration of the sensory retina and marked abnormalities in the pigment epithelium cells. Treated pigmented rats also had reduced a rhodopsin concentration in the retina by the 3rd month even though the photoreceptors were structurally normal. Interestingly, the plasma vitamin A levels remained normal and liver stores of vitamin A actually increased during the course of the study. Levels of butylcholinesterase in plasma and liver, on the other hand, were extremely reduced after 3 months of fenthion treatment. In general, the biochemical and functional (ERG) changes appeared before any structural damage could be detected in the retina.     

Trivalent chromium alters gene expression in the mummichog (Fundulus heteroclitus)

Mummichogs (Fundulus heteroclitus) were used as a model fish species to study the effects of trivalent chromium exposure. To ascertain chromium's effects, we examined altered gene expression by differential display between fish exposed in the laboratory and fish collected from a chromium-impacted estuarine site. Twenty differentially expressed genes were found from either laboratory-exposed fish or in fish collected from the field site. Database sequence searches indicated that several of these genes are highly homologous to known sequences, including a fatty acid-binding protein (FABP), cytochrome P4502N2 (CYP2N2), and a precursor to the translation initiation factor eIF2B. Verification of the differentially expressed genes by real-time polymerase chain reaction (PCR) revealed that the fatty acid-binding protein was repressed to a 3.6-times greater extent (3.6-fold) in the field-site animals as compared to a reference site, eIF2B was repressed 2-fold, and an expressed sequence tag (EST) termed A31 was induced 2.6-fold. In the laboratory-exposed animals, A31 was also induced between 2- and 4-fold. However, in contrast to the field-site fish, FABP was upregulated in the chromium-exposed animals. We hope to be able to use A31 as a biomarker for ascertaining the impacts of chromium exposure on fish.     

Exposure of small mammals,in particular the wood mouse Apodemus sylvaticus,to pesticide seed treatments

Field exposure of small mammals to fungicide-treated wheat seed was investigated over three weeks following drilling on fields near York, United Kingdom. Seed consumption by small mammals trapped on and immediately adjacent to the drilled fields was quantified by measuring the amount of seed in the stomach. In addition, exposure to one seed-treatment, fluquinconazole, was quantified by measuring residues of the fungicide in the stomach, liver, and intestine. The wood mouse, Apodemus sylvaticus, was the dominant species caught on the fields and the only species found to have consumed measurable quantities of seed. Voles, Microtus agrestis and Clethrionomys glareolus, were caught in small numbers, almost exclusively in the field hedge, and showed no evidence of having consumed seed. Stomach-contents analysis revealed that more than 80% of animals trapped in the hedge adjacent to the field had consumed no wheat seed, whereas 98% had consumed less than 10% (by stomach volume). Ninety percent of animals trapped on the field had consumed seed, although 90% of these animals had less than 20% seed in the stomach. Residues of the fungicide in the stomach, intestine, and liver were lower than would be expected for the amount of seed consumed, possibly because of dehusking of the seeds by mice. The relevance of these findings when assessing exposure (and risk) posed by seed treatments to wild mammals is discussed.