Induction of zona radiata and vitellogenin genes in estradiol and nonylphenol exposed male sheepshead minnows (Cyprinodon variegatus)

Several genes normally induced by estradiol (E(2)) in female fish, those for vitellogenins (VTGs) and zona radiata proteins (ZRPs), are also inducible in males exposed to estrogenic chemicals. Male sheepshead minnows (SHM) were exposed to both E(2) and para-nonylphenol (NP), at several doses and times to determine a dose-response. Quantitative real time PCR was used to measure mRNA for VTG1, VTG2, ZRP2 and ZRP3. Both E(2) and NP elicited a dose-response increase in all of the mRNAs tested. Exposure to both chemicals resulted in VTG2 expression at about a 10-fold lower level than VTG1, and ZRP2 expression at a lower level than ZRP3.     

Induction of zona radiata and vitellogenin genes in estradiol and nonylphenol exposed male sheepshead minnows (Cyprinodon variegatus)

Several genes normally induced by estradiol (E₂) in female fish, those for vitellogenins (VTGs) and zona radiata proteins (ZRPs), are also inducible in males exposed to estrogenic chemicals. Male sheepshead minnows (SHM) were exposed to both E₂ and para-nonylphenol (NP), at several doses and times to determine a dose-response. Quantitative real time PCR was used to measure mRNA for VTG1, VTG2, ZRP2 and ZRP3. Both E₂ and NP elicited a dose-response increase in all of the mRNAs tested. Exposure to both chemicals resulted in VTG2 expression at about a 10-fold lower level than VTG1, and ZRP2 expression at a lower level than ZRP3.     

Cellular responses and disease expression in oysters (Crassostrea virginica) exposed to suspended field contaminated sediments.

Exposure of oysters to water soluble fractions derived from field-contaminated sediments (FCS) containing predominantly lower molecular weight organic aromatic compounds, has been previously demonstrated to enhance pre-existing infections caused by the protozoan parasite, Perkinsus marinus (Dermo), and the prevalence of experimentally induced infections. To further explore the role of pollution on the onset and progression of disease, effects of suspended FCS from an estuarine creek in Virginia, USA, dominated by higher molecular weight polycyclic aromatic hydrocarbons (PAHs) on cellular responses and Dermo disease expression in oysters (Crassostrea virginica) were examined. Sediments were collected from a PAH polluted estuarine creek in Virginia, USA. To test effects on cellular response, oysters from Maine were exposed daily to 0, 1.0, 1.5, or 2.0 g suspended FCS (corresponding to 0, 70.2, 105, or 140 microg PAHs, respectively) for 5, 10, 20, and 40 days. Hemocyte activities and plasma lipid, protein and lactate dehydrogenase (LDH) levels were then measured. Exposure stimulated neutral red uptake, MTT reduction, and 3H-leucine incorporation in oyster hemocytes at various exposure times, but did not affect the plasma protein, lipid and LDH levels. To test effects on Dermo expression, oysters from a Dermo enzootic area, with an initial estimated infection prevalence of 39%, were exposed daily to 0, 1.0, 1.5, or 2.0 g suspended FCS (corresponding to 0, 75.0, 113, or 150 microg PAHs, respectively) for 30 days. Exposure enhanced disease expression in oysters. However, no significant change was noted in any measured cellular or humoral parameters.     

Survival of juvenile spot (Leiostomus xanthurus) exposed to bromochlorinated and chlorinated sewage in estuarine waters

The acute toxicity of chlorinated and bromochlorinated treated sewage waste was determined with spot, Leiostomus xanthurus. The halogenated sewage waste was generated in a pilot plant located at the James River Sewage Treatment Plant, Newport News, Va. The 96 h and 144 h LC50's were 0·23 mg/l (measured residual) for chlorine and 0·25 mg/l (measured residual) for bromine chloride. These concentrations are considerably higher than those which would be produced by optimal halogenation rates for disinfection or maximum superhalogenation rates permitted by pilot plant equipment and normal effluent dilution rates.     

Estrogenic responses of larval sunshine bass (Morone saxatilis x M. Chrysops) exposed to New York City sewage effluent

To determine the estrogenicity of effluents from sewage treatment plants (STPs) to larval fish, 2-day-old sunshine bass were exposed to effluents from three STPs serving New York City (NYC), varying in size and treatment level. Estrogenic response was evaluated by measuring vitellogenin (VTG) and estrogen receptor (ER) expression in cytosolic fractions of whole body homogenates. Concentrations of the presumptive endocrine disruptors in the effluents were also measured. VTG and ER levels in sewage-exposed fish were 3-5 times that observed in controls. Combined concentrations of estradiol and estrone ranged from 5 to 13 ng/l and nonylphenol-ethoxylate metabolites (NPEOs: 4-nonylphenol, and 1-, 2-, and 3-nonylphenol-ethoxylates) ranged from 180 to 470 microg/l in chlorinated effluent. Results indicate that both ER and VTG can be used as biomarkers for endocrine disruption in larval fish, and that 4-day exposure to sewage effluent is sufficient to elicit significant expression of these markers in sunshine bass larvae. The extremely higher concentrations of NPEOs found in effluent relative to hormones (approximately 40,000-fold) indicates that surfactant metabolites may be contributing significantly to the estrogenic effects observed.     

Determining the sensitive developmental stages of intersex induction in medaka (Oryzias latipes) exposed to 17 beta-estradiol or testosterone.

Certain environmentally persistent compounds can adversely affect reproduction by acting as steroid hormone agonists or antagonists. The goal of the present study was to determine the developmental stage most susceptible to exogenous hormone (estradiol and testosterone) exposure using a small teleost model. In the first (pilot study) of two experiments, medaka (Oryzias latipes), at varying developmental stages, were bath-exposed to 5 micrograms/l 17 beta-estradiol for 24 h. At 5 months of age, fecundity, fertility and embryo and larval viability (reproductive success) were investigated in control and exposed groups. Fish at 1, 1.5, 2 and 5.5 months of age were also sampled, processed and examined histologically for gonadal alteration. No significant differences in mortality, gonadal morphology, body weight, sex-ratio or time to maturity were seen between control and exposed fish. At 5 months, however, when exposure groups were compared to controls, significant differences were seen in reproductive success and viability of offspring. A second experiment exposed embryo stage 10, and 1-, 7- and 21-day-old larvae for 6 days to 15 micrograms/l 17 beta-estradiol or 100 micrograms/l testosterone. No significant differences were seen at 5 months in mortality, body weight, or time to sexual maturity. However, sex-ratios were significantly biased toward female in the stage 10, 1- and 7-day post-hatch estradiol exposure groups. No significant changes in sex-ratio were associated with testosterone exposure at any developmental stage. Further, intersex gonads were observed in fish from all groups exposed to 15 micrograms/l estradiol. Only those fish exposed as newly hatched fry or at 1 week post-hatch displayed intersex gonads following 100 micrograms/l testosterone exposure. Data from these experiments show that newly hatched fry are that life stage most sensitive to hormone exposure and the most appropriate to use in determining effects of known endocrine-disrupting compounds.     

PCB uptake and accumulation by oysters (Crassostrea virginica) exposed via a contaminated algal diet.

Reproductively active oysters were fed daily with 0.2 g algal paste containing 0, 0.1, and 1.0 microgram polychlorinated biphenyls (PCBs) (1:1:1 mixture of Aroclor 1242, 1254 and 1260) for either 15 or 30 days, and accumulation of PCBs in different organ tissues and eggs assessed. The effects of PCB exposure on lipid content, lipid class and fatty acid composition were also evaluated. PCBs were accumulated by the oysters and transferred to the eggs. PCB accumulation in oysters was dose, time and tissue dependent. Mean PCB contents were 3150, 1970, and 250 ng/g dry wt., respectively, in the visceral mass, gills + mantle and muscle of oysters fed algal paste containing 1.0 microgram PCBs for 30 days. The PCBs in the eggs from the same oysters reached 671 ng PCBs/g dry wt. Feeding oysters with PCB-sorbed algal paste for 30 days significantly increased phospholipid and free fatty acid contents in gills + mantle tissue compared to the same tissues in the undosed control.     

Neophrynichthys heterospilos,a new species of fathead sculpin (Scorpaeniformes: Psych rol utidae) from New Zealand

A second species of Neophrynichthys is described from 20 specimens collected from the Campbell Plateau, south of South Island, New Zealand. It differs from its sole congener, N. latus, in having 25–26 pectoral rays (versus 22–24), cirri on head more than twice as large, half as few (c. 50 versus >100), and generally absent from the mid‐region behind the eyes (versus being relatively dense), and in spotted colour pattern covering much less than 50% of the dorsal surface of the head (versus >50%). This new species is the sixth psychrolutid known from New Zealand. The genus Neophrynichthys is formally diagnosed by two synapomorphic characters unique among psychrolutids: having light spots on a dark ground colour and having an open fourth and fifth infraorbital, fifth and sixth cranial arch, medial extrascapular, and posterior tabular of the pterotic.     

Kinetics of Cd accumulation and elimination in tissues of juvenile rockfish (Sebastes schlegeli) exposed to dietary Cd

Cadmium (Cd) accumulation and elimination were investigated in the tissues (gill, intestine, kidney, lever and muscle) of juvenile rockfish, Sebastes schlegeli, after sub-chronic dietary Cd exposure (0, 0.5, 5, 25 and 125 mg/kg). No mortality occurred during the sub-chronic exposure to dietary Cd. Specific growth rates of the rockfish estimated by weight and length were significantly different from those of the control, and a significant inverse relationship was observed between weight gain and the exposure concentration of dietary Cd at 25, 125 mg/kg. Cd accumulation in the tissues increased with exposure periods and concentrations for the 60 days of dietary Cd exposure. Cd accumulation in the intestine of rockfish was higher than other tissues, and the order of Cd accumulation in tissues were intestine>kidney approximately liver>gill>muscle. Accumulation factors showed an increase with the exposure period and an inverse relationship between the accumulation factor and the exposure concentrations in the gill, intestine, liver and muscle, but not in kidney. Cd elimination in tissues of rockfish decreased during the 30 days of depuration except kidney and muscle. Intestine showed the fastest elimination rates of Cd at all concentrations compared with other tissues.     

Reduced survivorship of Himasthla (Trematoda, Digenea)-infected cockles (Cerastoderma edule) exposed to oxygen depletion

Bivalve populations from inshore waters often accommodate a diverse trematode fauna that may have a variety of effects on host specimens. In particular, larval trematodes that grow or reproduce within their host are known to be severe pathogens, whereas trematodes utilising bivalves only for encystment are thought to be relatively benign. Yet this may depend on the environmental conditions, and it can be expected that such trematodes in concert with other stress agents can be detrimental to host organisms. To examine the impact of such larval trematodes on hosts subjected to stress, we studied the digenetic trematode Himasthla elongata and one of its second intermediate hosts, the bivalve Cerastoderma edule. Experimentally infected cockles and non-infected cockles were exposed to oxygen depletion, whereupon we measured their burrowing ability and survivorship. After 30 h of hypoxia, the survival of infected cockles was significantly reduced compared to non-infected cockles, whereas no effect of parasites on cockles under normoxic conditions was found. In addition, parasites tended to reduce the burrowing ability of cockles exposed to hypoxia but the effect was not clear. The effect of parasites and possible ecological consequences are discussed and it is suggested that the combined effects of parasites and oxygen deficiency may explain some hitherto unexplained cases of mass mortalities in bivalve populations.     

Gonadal alterations in male eelpout (Zoarces viviparus) exposed to ethinylestradiol and trenbolone separately or in combination

To evaluate the interaction between 17β-trenbolone (TB) and 17α-ethinylestradiol (EE2) in relevant environmental concentrations, male eelpout Zoarces viviparus were exposed in a flow-through seawater-system for 21 days to 5 ng l⁻¹ EE2, 5 ng l⁻¹ or 20 ng l⁻¹ TB or to combinations of both compounds. The effects on hepatosomatic index (HSI), gonadosomatic index (GSI) and gonadal histology were studied. No significant effects on HSI were observed in any treatment; in contrast, decreased GSI was observed in males exposed to EE2 alone or in combination with TB compared to controls (p < 0.05). The histology revealed that the males were in the beginning of spermatogenesis. Males from the control group and some from the TB groups showed tubules with cysts containing spermatogonia, spermatocytes and spermatids; however, some testes of males exposed to TB showed slight to moderate interstitial fibrosis. Nevertheless, the most severely affected were males exposed to EE2 showing marked interstitial fibrosis, necrosis of germinal cells and reduced number of spermatocytes and spermatogonia in the cyst. Likewise, increased tubule number and proportionally decreased tubule diameter were observed in the testis of all EE2 exposed groups (p < 0.05). Finally, a similar tubule number was observed in males exposed to EE2 + 20 ng l⁻¹ TB compared to control (p > 0.05). This study shows that EE2 dramatically disrupts the spermatogenesis and low doses of 17β-trenbolone are unable to effectively counteract the morphological effects of EE2.     

Esterases as biomarkers in Nereis (Hediste) diversicolor exposed to temephos and Bacillus thuringiensis var. israelensis used for mosquito control in coastal wetlands of Morbihan (Brittany,France)

Since 1998, a biomonitoring programme has been implemented to assess the potential impact of chemical mosquito control on macroinvertebrates of the coastal wetlands of Morbihan (Brittany, France). Acetylcholinesterase and carboxylesterases were used as biomarkers to assess the effects of Abate 500e (a.i. temephos) and Vectobac 12 AS (a.i. endotoxins of Bacillus thuringiensis var. israelensis, Bti) in Nereis (Hediste) diversicolor. Esterase inhibition revealed a marked impact of temephos, suggesting preferential contamination of the worms through the food. In Bti-exposed N. diversicolor, random variations of esterase activities were observed, that could not be attributed to the larvicide. However, esterases only reflected indirect physiological effects of Bti, and further investigations are needed to identify biomarkers more specific of Bti endotoxins.     

Biomarker responses in Macoma nasuta (Bivalvia) exposed to sediments from northern San Francisco Bay

Our study investigates biomarker responses and survival of Macoma nasuta exposed to sediments collected from six locations in northern San Francisco Bay. Biomarkers analyzed were stress proteins (hsp70) in gill, mantle and digestive gland, lysosomal membrane damage and histopathologic lesions. Sediments and clam tissues were analyzed for a comprehensive suite of heavy metals and trace organic pollutants. Sediment grain size and organic carbon content were determined. Clams accumulated metals, polyaromatic hydrocarbons (PAHs) and organochlorine pesticides (aldrin and p,p(')-DDT and its metabolites p,p(')-DDD and p,p(')-DDE). Pearson and Spearman correlation analysis revealed that mortality, hsp70 in gill and histopathologic lesion scores in gonads, and lysosomal membrane damage were significantly correlated with tissue concentrations of DDT and/or its metabolites. Tissue concentrations of metals, in particular nickel, chromium, and copper, were associated with macrophage aggregates in digestive gland and germ cell necrosis. Cadmium was linked to mortality and lysosomal membrane damage.     

Altered glutathione status in atlantic croaker (Micropogonias undulatus) tissues exposed to lead

Metals which induce the synthesis of metallothioneins, such as cadmium and mercury, also elevate glutathione (GSH) concentrations in fish tissues.^1,2 To determine whether metals which do not stimulate metallothionein synthesis increase GSH levels, the GSH status of Atlantic croaker tissues was examined after oral administration of lead. Lead significantly increased GSH levels in the intestine and liver, but not in kidney or brain tissues. Lead was not bound to GSH and the increase in GSH did not appear to be linked to lead accumulation in these tissues. Investigations of hepatic GSH synthesis and turnover in vitro using a radiolabelled amino acid precursor, C¹⁴ glycine, showed that the elevation of GSH was due to an increase in GSH synthesis rather than decreased utilization of GSH. These studies suggest that the interactions between GSH and lead are indirect and involve a stimulatory effect of the metal on the activity of the enzymes for GSH synthesis.     

0#柴油水溶性成分对僧帽牡蛎DNA损伤的初步研究

在实验条件下,将僧帽牡蛎(Ostrea cucullata)分别置于含有低(0．5mg/L)、中(2mg／L)和高(5mg／L)3种浓度0^#柴油水溶性成分的海水中,利用碱解旋法测定消化腺和鳃INA损伤的程度。结果表明,INA损伤的程度随污染时间的延长而增加,并存在一定的剂量-效应关系;解除污染后,DNA损伤恢复到对照水平。DNA损伤可作为监测海洋石油污染的生物标志物。     

Perturbation of gene expression and steroidogenesis with in vitro exposure of fathead minnow ovaries to ketoconazole

Ketoconazole is a fungicidal drug that inhibits function of cytochrome P450s in the synthesis of steroids. To examine if inhibition of P450 function affects gene expression in a dynamic manner, we conducted in vitro exposures of ovary tissue from fathead minnows (Pimephales promelas) to 0.5 microM ketoconazole to investigate effects on steroid production and gene expression over time. Expression of four key steroidogenesis genes was examined at 1, 6, and 12h of exposure. 11 beta- and 20 beta-hydroxysteroid dehydrogenases were down regulated at 1h and Cytochrome P450 17 was down-regulated at 12h, consistent with the absence of steroid production. In contrast, cytochrome P450 19A was up-regulated at 6h, indicating feedback regulation. Microarray analysis of 12h exposures indicated enrichment of biological processes involved in neurotransmitter secretion, lymphocyte cell activation, sodium ion transport, and embryonic development. These data suggest that, with the exception of cytochrome P450 19A, these steroid metabolic genes are regulated in a feed forward manner and that the effects of ketoconazole may be broader than anticipated based on the mechanism of action alone.     

苯并(a)芘影响黑鲷肝脏EROD活性变化的动力学研究

研究了黑鲷(Sparusmacro cephalus)在实验生态条件下暴露于不同浓度苯并(a)芘(benzo(a)pyrene,BaP)后,肝脏乙氧基异吩噁唑酮脱乙基酶(EROD)活性在不同暴露时相的动力学变化情况.EROD活性采用动力学方法测定.实验结果表明,暴露于较低质量浓度BaP(0.5,1.0μg/L)黑鲷肝脏的EROD活性在2 d后极显著高于对照组(P<0.01),2.0μg/L组则在2 h后极显著高于对照组,并且随暴露时间的延长,诱导程度表现出不同的下降趋势,而暴露于高质量浓度组(5.0 μg/L)的肝脏EROD活性极显著性诱导出现在12h.总体上较高质量浓度组(2.0,5.0μg/L)EROD活性出现极显著性诱导的时间比低质量浓度组(0.5,1.0μg/L)早,诱导程度也比低质量浓度组的高.在净化实验中各暴露组肝脏EROD活性均下降,与空白对照组比较均没有显著性差异,说明试验浓度没有超出严重损伤肝脏自身恢复系统的范围,在此浓度范围内污染的鱼通过净化可清除BaP污染.