Lead and Aroclor 1254 disrupt reproductive neuroendocrine function in Atlantic croaker

We have previously shown that lead (lead chloride) and a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) can alter hypothalamic serotonin (5-hydroxytryptamine, 5-HT) content, and pituitary gonadotropin II (GTH II) release in vitro, in the Atlantic croaker (Micropogonias undulatus). In the present study we investigated whether impairment of the hypothalamic 5-HT pathway involves alterations in tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, or monoamine oxidase (MAO), the catabolic enzyme. Aroclor 1254 (1 mg/kg body wt. for 30 days) significantly inhibited hypothalamic TPH activity without altering MAO activity, and caused a significant decline in 5-HT content. On the other hand, lead exposure (15 mg/kg body wt. for 30 days) only induced a slight decrease in hypothalamic 5-HT content and TPH activity, and a minor increase in MAO activity. However, both Aroclor 1254 and lead significantly inhibited the GTH II response to stimulation by a luteinizing hormone-releasing hormone analog (LHRHa) in vivo and caused reduced gonadal growth. These results demonstrate that impairment of hypothalamic serotonin metabolism by Aroclor 1254 involves inhibition of 5-HT synthesis, whereas lead does not exert a profound influence on 5-HT metabolism. The decline in 5-HT availability due to reduced 5-HT synthesis in the PCB-exposed fish may result in disruption of the stimulatory 5-HT-GnRH pathway controlling GTH II secretion leading to impairment of gonadal growth.     

Disruption of neuroendocrine function in atlantic croaker exposed to Aroclor 1254

To investigate the effects of a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) on reproductive neuroendocrine function, male Atlantic croaker (Micropogonias undulatus) were exposed to the PCB mixture in the diet (0.1 mg/100. g body wt/day) for 30 days during gonadal recrudescence. Concentrations of the monoamines (5-hydroxytryptamine (5-HT), dopamine (DA) and norepinephrine (NE)) and their major metabolites (5-hydroxyindolacetic acid, dihydroxyphenylacetic acid and 3-methoxytyramine) were measured in the preoptic-anterior hypothalamus (POAH) and medial and posterior hypothalamus (MPH) using HPLC with electrochemical detection. There was a significant decline in 5-HT and DA concentrations and an increase in their metabolite to parent amine ratios in both the POAH and MPH of Aroclor 1254-exposed fish. In addition, Aroclor 1254 exposure resulted in the loss of the in vitro gonadotropin (GtH) response to stimulation by a luteinizing hormone-releasing hormone analog (LHRHa). The reduced availability of 5-HT in the nerve terminals may be responsible for the loss of pituitary GtH response to stimulation by LHRHa. These results support the hypothesis that Aroclor 1254-induced alterations in pituitary gonadotropin release may be mediated, at least partially, by altered hypothalamic serotonergic activity.     

Reproductive endocrine function in female atlantic croaker exposed to pollutants

To examine whether xenobiotics impair teleost reproduction by altering reproductive endocrine function, steroid hormone secretion and ovarian growth were investigated in female Atlantic croaker (Micropogonias undulatus) after chronic oral administration with sublethal doses of three classes of reproductive toxins (lead, benzo[a]pyrene and Aroclor 1254). All the sublethal treatments significantly impaired ovarian growth as assessed by the gonadosomatic index. Reduced ovarian growth was accompanied by a significant decline in circulating 17β-estradiol levels in fish exposed to lead and benzo[a]pyrene. All three toxicants significantly decreased plasma testosterone levels. However, the steroidogenic capacity of ovarian tissue in vitro was not altered by xenobiotic exposure. The data suggest that the decreased ovarian growth in croaker after pollutant exposure may be a consequence of the decline in plasma 17β-estradiol levels. Further, this decrease in circulating levels of gonadal steroids does not appear to be caused by a direct effect of the chemicals on ovarian steroid synthesis.     

Transport and accumulation of organochlorines in the ovaries of Atlantic croaker (Micropogonias undulatus)

The role of vitellogenin in the transport of organochlorines was investigated in Atlantic croaker (Micropogonias undulatus) by exposing them through the diet to o,p′-DDT at a concentration of 10.8 μg/100 g fish/day for 14 days or Aroclor 1254 (PCB) at a concentration of 0.5 mg/100 g fish/day for 30 days during gonadal recrudescence. Tissue samples were taken from the fish at various times after initial exposure, and o,p′-DDT and PCBs were extracted in acetonitrile and analyzed by gas chromatography. Analysis of the ovarian tissue collected 2 and 3 weeks from the start of exposure revealed that the o,p′-DDT concentration increases as the gonadosomatic index (GSI) increases (r² = 0.63), with accumulation ranging from less than 1% to as much as 8% of the total dosage. Interestingly, o,p′-DDT did not accumulate in the testes during the same exposure period. Accumulation of PCBs was found to be 40 times higher in the ovaries than in the testes. Gel filtration of plasma from exposed females showed that o,p′-DDT elutes in the low density lipoprotein and vitellogenin fractions. Control plasma incubated with o,p′-DDT at 4 °C for 16 h followed by chromatography on Sepharose 6B gave similar results with an o,p′-DDT concentration of 0.6μg/mg protein in the vitellogenin fraction. Furthermore, both o,p′-DDT and PCBs were found to bind to purified croaker vitellogenin. These results suggest that lipoproteins, including vitellogenin, are involved in the transport and accumulation of organochlorines in the ovaries of exposed fish.     

Resistance may be an important mechanism by which marine microbes respond to environmental toxicants

The hypothesis, that at least certain marine microbial organisms respond to toxic stress by the development of resistance, was tested using the hypotric marine ciliate Euplotes vannus Muller as the test organism. Resistance to polychlorinated biphenyls (PCB, Aroclor 1254) was developed in E. vannus by exposing the animals to progressively higher PCB concentrations during a period of several months. The resistance to PCB persisted for at least 80 days (greater than 40 generations) after final exposure. This suggests either that genetic selection or persistent (lasting over many cell division cycles) phenotypic trait modification, possibly in the form of Dauermodification, had occurred. If resistance were widespread among marine microbial organisms in polluted environments it would be an important consideration in evaluating the long-term biological impact of both natural and man-induced chemical stress.     

Factors affecting the elimination of PCBs in the marine copepod Acartia tonsa

The effects of feeding, egg laying, and fecal pellet production on the elimination of polychlorinated biphenyls (PCBs) from the marine copepod Acartia tonsa were studied in a series of experiments. Copepods were exposed to ¹⁴C-labelled Aroclor 1254 and allowed to depurate in clean seawater. Copepods fed during depuration eliminated PCBs more rapidly than unfed copepods whether or not the original PCB exposure medium had contained food. Both eggs and fecal pellets contained PCBs during depuration, with the weight specific concentration of PCB in the eggs (up to 407 ppm, dry weight) exceeding four times that in the females that produced them. Female copepods eliminated PCBs twice as rapidly as males, indicating that egg production is an important route for PCB elimination.     

Effects of Aroclor 1254 on reproductive organ weight, sperm quality and testicular mitochondria oxidative stress of Boleophthalmus pectinirostris

Boleophthalmus pectinirostris is an amphibious economic fish and wildly distributed in the southeast coast of China. In this study, Aroclor 1254 was intraperitoneally injected into B. pectinirostris with 1, 2 and 4 μg/(g·d) for 28 d to assay the reproductive organ weight, the sperm quality (sperm concentration and motility), and the testicular mitochondrial testicular mitochondria oxidative stress. The results show that the sperm number and motility in seminal vesicles, the absolute weight of testes and seminal vesicles of B. pectinirostris treated with 2 and 4 μg/(g·d) Aroclor 1254 decreased significantly as compared to the controls (p <0.05), while those treated with 1 μg/(g·d) Aroclor 1254 had no significant effects on these indictors. For the relative weight of reproductive organs, significant reduction (p <0.05) was only observed in the seminal vesicles of B. pectinirostris treated with 4 μg/(g·d). SOD activities and GSH levels in all the Aroclor 1254 treatments were significantly lower than those of the controls (p <0.05). The activities of CAT, GPx, GR and the levels of Vit C also decreased significantly in comparison with the controls (p <0.05) at the higher dose of 2 and 4 μg/(g·d) Aroclor 1254 treatments. In addition, both H₂O₂ level and MDA content in testicular mitochondria of B. pectinirostris had a close correlation with Aroclor 1254 dosage, and were significantly higher than the controls (p <0.05). Those indicate that Aroclor 1254 can induce the oxidative stress of testicular mitochondria, and impair the reproductive function of male B. pectinirostris.     

Induction of DNA strand breakage and apoptosis in the eel Anguilla anguilla

The ability of benzo[a]pyrene, Aroclor 1254, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone to induce DNA strand breaks (SB) and apoptosis in erythrocytes of the European eel (Anguilla anguilla) was investigated following by in vivo exposure. DNA damage was evaluated by the Comet assay, while the diffusion assay was used to investigate the induction of apoptosis 7 days after a single intraperitoneal administration. 2-3-7-8-Tetrachlorodibenzo-p-dioxin induced the highest genotoxic effect, followed by benzo[a]pyrene, while the other two substances had limited effects. A significant induction of apoptosis was observed at the highest doses after exposure to benzo[a]pyrene, when DNA damage was also elevated. The occurrence of apoptotic cells after exposure to Aroclor, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone was quite variable and did not show clear dose-related responses. The role of oxidative stress in mediating DNA damage was also discussed.     

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.     

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 μg 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 μg 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 tissues compared to the same tissues in the undosed control.     

Regulation of hepatic glutathione S-transferase expression in flounder

The major glutathione S-transferase isoform of flounder liver, an antigenically related structural homologue of plaice GST-A, also displays mRNA homology. A 901bp cRNA probe for plaice GST-A cross-hybridised to a 1100bp flounder mRNA on northern blot analysis. The plaice antibody and cRNA probes were used to study effects of inducer treatment on GST-A expression in flounder liver. Six days after PAH treatment (3-methylcholanthrene) total hepatic GST activity was halved, levels of GST-A were 80% and GST-A mRNA levels were 25% of controls. A commercial PCB mixture (Aroclor 1254^TM) had little effect on total GST or GST-A levels despite halving GST-A mRNA levels. An epoxide, trans-stilbene oxide induced total GST activity 1·4 fold and GST-A protein levels 1·8-fold and its mRNA levels 3-fold. This reduced expression of the major flounder hepatic GST by agents which induce cytochrome P4501A1 may modulate cytoxicity of environmental pollutants in this species.     

An in vitro procedure for evaluation of early stage oxidative stress in an established fish cell line applied to investigation of PHAH and pesticide toxicity

Oxidative stress by increased production of reactive oxygen species such as superoxide has been implicated in the toxicity of PCB's and non-target toxicity of many pesticides. We report the development of a microplate-based method for determination of early stage oxidative stress using an established cell line (EPC) from a skin tumour of carp Cyprinus carpio L. and 2',7'-dichlorodihydrofluorescein diacetate (H(2)-DCFDA) as a fluorescent probe for detection of reactive oxygen species (ROS) formation. Sublethal concentrations of the herbicide Paraquat, an established redox cycling agent and a crude PCB mixture, Arochlor 1254 elicited a linear increase in ROS formation over 2 h exposure which was some 45- and 10-fold higher, respectively, than attributable to basal respiration, confirming the suitability and response of the test system. Whilst in vivo studies in mammals have implicated early stage oxidative stress in the toxicity of pesticides, we did not observe an increase in ROS production after exposure of EPC cells to sublethal concentrations of Carbaryl, 2,4-DDT, Lindane or Malathion implying that this is not the causative mechanism of acute toxicity in this fish cell line. The apparent involvement of oxidative stress in their mammalian toxicity may therefore be an indirect effect or dependent upon compound metabolism.     

Differential gene expression in the brain of Sebastiscus marmoratus in response to exposure to polychlorinated biphenyls (PCBs)

Effects of exposure to polychlorinated biphenyls (PCBs) on Sebastiscus marmoratus were investigated using a suppression subtractive hybridization method. A total of 108 gene sequences were identified as having the potential for being differentially expressed, and 45 could be identified with homologous database sequences. Functions with which they were associated included long-term potentiation and neurotransmitter release, neuroendocrine, mitosis and cell proliferation, energy-related metabolism, general metabolism, signal protein, hemopoiesis system, immune system, and structure. The expression of 17 of these genes was analyzed in the brain using real time fluorescent quantitative PCR. The present study provided a basis for studying the response of fish to PCB exposure and allowed the characterization of new potential neurotoxicol biomarkers of PCB contamination in seawater.     

Differential gene expression in the brain of Sebastiscus marmoratus in response to exposure to polychlorinated biphenyls (PCBs)

Effects of exposure to polychlorinated biphenyls (PCBs) on Sebastiscus marmoratus were investigated using a suppression subtractive hybridization method. A total of 108 gene sequences were identified as having the potential for being differentially expressed, and 45 could be identified with homologous database sequences. Functions with which they were associated included long-term potentiation and neurotransmitter release, neuroendocrine, mitosis and cell proliferation, energy-related metabolism, general metabolism, signal protein, hemopoiesis system, immune system, and structure. The expression of 17 of these genes was analyzed in the brain using real time fluorescent quantitative PCR. The present study provided a basis for studying the response of fish to PCB exposure and allowed the characterization of new potential neurotoxicol biomarkers of PCB contamination in seawater.     

大黄鱼性早熟问题的研究Ⅱ.不同药物对性早熟的影响

本文研究了促性腺激素释放激素类似物和紫萁两种药物对大黄鱼性早熟的影响。结果表明,在雌性,低和高剂量类似物组与低剂量紫萁组大黄鱼早熟率比对照组分别下降66％和33％,而高剂量紫萁组与对照组间没有差异;在雌必性,低剂量类似物组和低剂量紫萁组早熟率比对照组下降37％,高剂量紫萁组与对照组之间则没有差异。这些结果可为药物防止大黄鱼性早熟提供一条有效的途径。     

Development of two novel CYP-antibodies and their use in a PCB exposure experiment with Mytilus edulis

In an attempt to learn more about the cytochrome P450 (CYP) system of mussels, we used protein databases and alignment software to extract highly conserved CYP sequences. From these alignments synthetic peptides were produced and used for rabbit immunisation, which yielded polyclonal antibodies against the CYP families 2 and 4. The antibodies were evaluated with Western Blot and ELISA assays, using digestive gland microsomal samples from the mussel Mytilus edulis. Western Blots revealed immunoreactions for both antibodies. The anti-CYP2 sequence rendered one major immunopositive protein of approximately 49 kDa size, and weak signals for proteins of approximately 41 and 56 kDa size. The anti-CYP4 sequence rendered two major bands of approximately 56 and 59 kDa size, and also a weak immunoreaction with a protein of approximately 43 kDa size. ELISA rendered only weak signals even with a 1:50 dilution of IgG-purified serum. A 10-day exposure to Aroclor 1254 did not appear to affect any of the immunopositive proteins, while total PCBs in soft bodies increased from 14-40 ng/g DW in controls to 373-638 ng/g DW in exposed mussels.     

多氯联苯(PCB1254)对栉孔扇贝消化盲囊和鳃丝抗氧化系统及脂质过氧化水平的影响

本实验研究了浓度梯度为0.5,1.0,和10.0μg/L的多氯联苯化舍物(PCB1254)处理30d栉孔扇贝消化盲囊和鳃丝SOD,CAT和GPx活性和脂质过氧化水平的变化.研究结果表明:低浓度组(0.5μg/L和1.0μg/L)的抗氧化酶活力能够被PCB1254显著的诱导,但是在高浓度组,抗氧化酶快速被诱导升高然后随着...     

Application of real time PCR determination to assess interanimal variabilities in CYP1A induction in the European flounder (Platichthys flesus)

In both laboratory experiments and field investigations with fish a large interanimal variability in CYP1A expression has been observed which may be attributed to variations in environmental inducer exposure and/or inducer response. We are carrying out laboratory investigations to assess the contribution of a potential genetic component in inducer response of flounder (Platichthy sflesus) CYP1A to PCB exposure and in this paper we report development of a sensitive quantitative RT-PCR procedure (real time PCR) where accumulation of the intercalated dye SYBR Green I is followed during cycling. Preliminary experiments using this procedure with artificially reared Arochlor 1254-treated flounders showed large interanimal differences in response for a single family group indicating that variability does have a genetic basis.     

Mechanism of Gonadal Precocity in Cultured Large Yellow Croaker,Pseudosciaena Crocea: A Study of Microstructure and Submicrostructure of Leydig Cell and Sertoli Cell in Testis

Microstructure and submicrostructure of Leydig cell and Sertoli cell in the testis of gonadal precocity and immaturation in cultured large yellow croaker, Pseudosciaena crocea, are studied using histology and electron microscopic technique. The results indicate that the fine structure of the two kinds of cells in different development stages presents an obvious difference. The smooth endoplasmic reticular and tubular mitochondria of Leydig cell and Sertoli cell are well developed in the testis of gonadal precocity, but poorly developed in the testis of immaturation. We suggest that the reason for gonadal precocity in the large yellow croaker may be related to the earlier development and maturation of Leydig cell and Sertoli cell.