Biotransformation enzyme activities and PCDD/PCDF levels in pike caught in a Swedish Lake

Recently, analysis of organochlorines in sediment and in pike from Lake Vänern, Sweden, showed a north-south gradient of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). In the present study, good correlations were found between muscle 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents and liver ethoxyresorufin-O-deethylase (EROD) activities (and P4501A levels) in pike caught at three sampling sites along the north-south gradient in the lake. This comparison between tissue levels of PCDD/PCDF and EROD activity (and P4501A) may indicate that TCDD or structural analogs could be responsible for the observed induction of the pike P450 system. It must, however, be taken into account that the analysed contaminants often do not appear alone but are, rather, accompanied by a range of other substances which may be inducers or antagonists.     

Biochemical and physiological disturbances in fish inhabiting coastal waters polluted with bleached kraft mill effluents

An extensive trial to examine physiological and biochemical changes in perch (Perca fluviatilis) inhabiting coastal waters polluted by bleached kraft mill effluents has been carried out. The results from this investigation show profound effects of bleached kraft mill effluents on several fundamental biochemical and physiological functions on four different sampling occasions. Typical symptoms in perch from the polluted areas were reduced gonad growth, enlarged liver, and very strong induction of certain cytochrome P-450-dependent activities. Elevated content of ascorbic acid in liver tissue and strongly affected carbohydrate metabolism show that the effluent causes metabolic disorders. Marked effects on the white blood cell pattern indicate a suppressed immune defence. Alterations in the red blood cell status and the ion balance suggest an impaired gill function. This extensive symptom picture is strengthened by the relatively good agreement with the effects previously observed in fish exposed to bleached kraft mill effluents (BKME) in the laboratory.^1–3     

The DNA de-methylating agent 5-azacytidine does not restore CYP1A induction in PCB resistant Newark Bay killifish (Fundulus heteroclitus)

Newark Bay (NB) killifish (Fundulus heteroclitus) have been chronically exposed to environmental contaminants that activate the aryl hydrocarbon receptor (AHR) and are tolerant to toxic effects and CYP1A induction provoked by AHR ligands. Resistance to CYP1A induction could be due to an epigenetic mechanism such as DNA methylation. We measured in-ovo CYP1A catalytic activity (ethoxyresorufin-O-deethylase, EROD) in NB and reference site killifish embryos aqueously exposed to various concentrations of the de-methylating agent 5-azacytidine, 5-AC (5, 50 and 500 μ(micro)M) with or without 0.2 μ(micro)g/l of the CYP1A inducer 3,3^′,4,4^′,5 pentachlorobiphenyl (IUPAC PCB126). Neither PCB126 alone, nor PCB126 plus 5-AC, induced EROD above levels in vehicle treated Newark Bay fish. In reference site fish, the same PCB126 dose provoked a 7.4-fold EROD induction relative to controls. We conclude that Newark Bay killifish are resistant to CYP1A induction by co-planar PCBs during early embryological development and our data suggests that DNA methylation does not play a critical role in resistance to CYP1A induction in this model.     

Vitellogenin-associated maternal transfer of exogenous and endogenous ligands in the estuarine fish, Fundulus heteroclitus

Sequestration of large quantities of vitellogenin (VTG) is critical for proper oocyte development in most oviparous vertebrates. While previous studies have shown a general correlation between oocyte growth and the accumulation of various exogenous and endogenous ligands, few studies have attempted to elucidate the role VTG plays in this maternal transfer. In the present study, we have demonstrated that oocytic accumulation of [³H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (³H-TCDD) and [¹⁴C]-benzo(a)pyrene (¹⁴C-BaP) by gravid Fundulus heteroclitus is directly correlated with oocyte maturational status. A positive correlation was observed between oocyte maturational state (size) and both total quantity (total pmoles) as well as concentration (pmoles/g tissue) of TCDD and BaP. Further, the bi-phasic accumulation of both TCDD and BaP mirrors that previously observed for protein sequestration by cultured Fundulus oocytes. Additionally, both TCDD and BaP were associated with VTG in vivo. HPLC analysis of serum extracts has shown that VTG associates with both parent BaP and BaP metabolites. Studies with ¹²⁵I-T₄ (thyroxine) also suggest that this critical hormone also associates with VTG in vivo. Ongoing studies are examining the VTG-associated transport and oocytic sequestration of ¹²⁵I-T₄ in gravid Fundulus.     

Regulation of hepatic metallothionein in estradiol-treated rainbow trout

Metallothionein has previously been shown to be regulated during the period of exogenous vitellogenesis (Olsson et al. (1987); Fish Physiol, Biochem., 3(1), 39–47). Following estradiol injection of rainbow trout it has been shown that hepatic metallothionein remains at basal levels until the vitellogenin mRNA levels begin to decline and Zn is released from high-molecular-weight proteins (Olsson et al. (1989); Biochem.J., 257, 555–559), In the present study the effects of estradiol treatment on the metal-inducibility of metallothionein have been investigated. Estradiol-treated fish that were injected with low doses of cadmium or zinc did not respond by induction of metallothionein.     

Application of a sensitive chemiluminescent technique for comparison of cytochrome P4501A induction in hepatic and intestinal tissues of fish exposed to bleached kraft mill effluent

In conjunction with an environmental assessment of biologically-treated bleached kraft mill effluent (BKME) in a Western Canadian river, data indicated that lipophilic compounds were transferred to the mountain whitefish Prosopium williamsoni through ingestion of filter-feeding benthic caddisflies. P4501A induction was correlated with lipophilic body burdens, not with indices of recent BKME exposure. P4501A contents in hepatic and intestinal tissues of BKME-exposed whitefish were compared during a follow-up collection. Ethoxyresorufin O-deethylase (EROD) activity was not detectable in intestines; P-450 spectral analysis indicated denaturation had occurred. Use of enhanced chemiluminescence (ECL) immunoblotting achieved at least 10-fold greater sensitivity over colorimetric methods and indicated that some intestines did contain P4501A protein. No correlation of intestinal P4501A with hepatic EROD activity or P4501A content was found. Application of the ECL technique significantly improves the analytical detection limits of P4501A immunoblotting. Analysis of historical samples from this site will further probe water-borne versus dietary routes of uptake of BKME-related P4501A inducers in this species.     

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.     

Unbleached pulp mill effluents affect cytochrome P450 monooxygenase enzyme activities

The polysubstrate monooxygenase system has been shown to be highly responsive to chemical pollution. The present study summarizes the enzyme based biomonitoring of the waste waters released by a pulp mill producing unbleached pulp and paperboard. Cytochrome P4501A enzyme activities of feral and caged fish, as well as cultures of fish hepatocytes, were tested. The 7-ethoxyresorufin-O-deethylase (EROD) activity was clearly induced in fish hepatocytes exposed to biotreated unbleached pulp mill effluent fractions in vitro. The effluent increased EROD activities also in feral perch, compared with controls. Caging experiments showed similar effects to those seen in feral fish: however, the maximal induction coefficients observed were higher. Unbleached effluents contain compounds that are able to affect the P4501A activities in fish.     

The cytochrome P450 1A1 response in fish: application of immunodetection in environmental monitoring and toxicological testing

We have developed polyclonal and, recently, monoclonal antibodies against the aromatic hydrocarbon-inducible P450 1A1 form purified from Atlantic cod (Gadus morhua). A simple, indirect enzyme-linked immunosorbent assay (ELISA) based on these antibodies has been developed in our laboratory and tested in numerous experiments with both field-collected and laboratory-exposed fish of different species. The exposure situations studied to date include complex mixtures such as polychlorinated biphenyls (PCBs), dioxins and water-soluble oil fractions, as well as defined compounds such as CB congeners, TCDD, and pesticides. Factors affecting the induction response, including sexual maturation and dietary factors, have also been investigated. The ELISA technique generally shows good correlation with contaminant exposure and catalytic measurements, but has also given new and important information in certain cases where catalytic measurements failed to reveal effects.     

Mechanisms of imposex induction in the mud snail, Ilyanassa obsoleta: TBT as a neurotoxin and aromatase inhibitor

The occurrence of imposex, imposition of male sex characteristics on female snails, has been extensively documented throughout the world. Tributyltin (TBT) and other organotins have been causally linked to imposex induction at levels as low as 2 ng/l. There are several proposed mechanisms of action. First, TBT has been shown to be neurotoxic and to accumulate in snail ganglia. Peptide hormones control sexual differentiation in gastropods, and one hypothesis is that TBT acts as a neurotoxin to abnormally release the peptide hormone Penis Morphogenic Factor (PMF). However, PMF has not been characterized to date. The neuropeptide APGWamide significantly induces imposex in the mud snail, Ilyanassa obsoleta, at 10⁻¹⁶ moles sub-cutaneous (SQ) injection over 2 weeks, and could be the PMF in this species. A second hypothesis is that TBT inhibits aromatase activity leading to increased testosterone levels and decreased estradiol. In vitro studies with snail digestive gland microsomes showed that TBT-dosed snails not exhibiting imposex had a 52% reduction in aromatase activity. Although the role of vertebrate sex steroids is not known in gastropods, it is possible that the combination of changes in peptide and steroid hormones may lead to imposex induction at extremely low doses of TBT.     

Monitoring of the chemical pollution of the river Rhône through measurement of DNA damage and cytochrome P4501a induction in chub (Leuciscus cephalus)

The in vivo response of freshwater fish exposed to pollutants was assessed using two biomarkers, 7-ethoxyresorufin-O-deethylase (EROD) induction and DNA single strand breaks. Chub (Leuciscus cephalus) were caught in spring and in fall at various locations in the river Rhône watershed. EROD activity was measured in the liver while DNA damage was evaluated in chub erythrocytes using the recently developed Comet assay. Chemical contamination was evaluated both in fish muscle (PCBs) and in sediment (PCBs, PAHs, heavy metals) collected at each sampling station. Sex of individuals was shown to influence the level of EROD activity but not the level of DNA damage. The EROD activity as well as the DNA damage were found to be higher in the mostly contaminated stations compared to the reference one. This study shows that multibiomarker-based approach provides complementary informations about early effects in feral fish exposed to complex chemical pollution and highlights the interest of the Comet assay in genotoxicity assessment.     

Maternal transfer of bioactive polychlorinated aromatic hydrocarbons in spawning chinook salmon (Oncorhynchus tschawytscha)

The biological potency (relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin. TCDD) of planar polychlorinated aromatic hydrocarbons (PCHs) in extracts of eggs and flesh from spawning female chinook salmon (Oncorhynchus tschawytscha) from Lake Michigan was determined by measuring the induction of 7-ethoxyresorufin O-deethylase activity in H-4-II-E rat hepatoma cells. TCDD-equivalents in flesh and egg samples ranged from 0 to 115·8 pg/g, and were approximately 5-fold greater in eggs than in flesh. These results suggest that the maternal transfer of PCHs may play a role in determining the reproductive success of Lake Michigan chinook salmon.     

Regulation of Tilapia metallothionein gene expression by heavy metal ions

Tilapia is a common fish species inhabiting inland waters and estuarine regions in Hong Kong and Southeast Asia, and useful for bio-monitoring of metal pollution. Metallothionein (MT) gene expression in fish tissues has been useful to sub-lethal risk assessment as biomarker of exposure to metal ions in fishes inhabiting metal contaminated area. To investigate metal inductions of Tilapia MT gene expression in vivo, Tilapias were injected with different concentrations of heavy metals and tissues were then removed for quantitative PCR assay using mimic PCR methods. All of the metal ions tested (Cu²⁺, Cd²⁺, Hg²⁺, Ni²⁺, Pb²⁺ and Zn²⁺) were able to induce hepatic MT mRNA levels. Renal MT mRNA levels of Cd²⁺ and Zn²⁺ treated fish was not induced with significant fold induction, however MT mRNA levels in gills were sensitive to the administrations of these metal ions. These data indicated that Tilapia MT mRNA levels in gills and liver are sensitive biomarker of exposure to various metal ions.     

Acute sublethal toxicity of MV Rena contaminants (heavy fuel oil,oil dispersant and cryolite) to finfish and rock lobster

ABSTRACT

The wreck of the MV Rena in October 2011 discharged large quantities of potentially toxic materials in the coastal marine environment of the Bay of Plenty, New Zealand. We evaluated sublethal effects of the water soluble fractions of physically and chemically dispersed heavy fuel oil (HFO) and the water soluble fraction of cryolite on the haematology of subadult or adult snapper (Chrysophrys auratus), spotted wrasse (Notolabrus celidotus) and red rock lobster (Jasus edwardsii). Small but transient effects occurred, the most significant of which were changes in circulating immune cell populations in fish and lobster that recovered by the conclusion of a 96-h exposure period. No post-exposure effects were observed during a 10-day recovery in toxicant-free seawater. Corexit 9500 oil dispersant increased polycyclic aromatic hydrocarbon (PAH) uptake from HFO in fish and lobster by around two-fold and four-fold, respectively, and caused a larger change in immune cell abundance in snapper than exposure to HFO alone. While depuration of PAHs was more rapid in fish than lobster, significantly elevated PAH levels were still evident after 10 days of depuration.     

Characterization and molecular analysis of hepatic microsomal UDP-glucuronosyltransferases in the marine fish, Pleuronectes platessa

Here we report the results of the first molecular enzymological study of fish UDP-glucuronosyltransferase (UDPGT). UDPGT activities of plaice liver microsomes were greatest for planar phenols and there was low but measurable conjugation of bilirubin, testosterone and androsterone. A highly purified preparation was isolated which possessed activities towards 1-naphthol, bilirubin and testosterone, containing several molecular weight species of M_r 52–56 kDa. On immunoblot analysis these proteins cross-reacted with a polyspecific anti-rat UDPGT antibody, suggesting that a number of plaice UDPGT isoforms with epitopes in common with the corresponding rat enzymes had been purified.     

The effects of the ‘Braer’ oil spill, Shetland Isles, Scotland, on P4501A in fanned Atlantic salmon (Salmo salar) and the common dab (Limanda limanda)

This paper describes the response of two fish species, the Atlantic salmon (Salmo salar) and the common dab (Limanda limanda) to the oil spilled from the Braer tanker which grounded on the southern tip of Shetland, Scotland, on 5 January 1993. Both the immediate sublethal effects and the long-term response to the oil which accumulated in sediments around the Shetland Isles, Scotland, are examined. The primary response measured was the induction of detoxification enzymes and the relationship of the levels or activities of these enzymes to the concentration and distribution of aromatic hydrocarbons measured in sediments, water and fish. The results show that, immediately following the spill, there was a marked induction of Cytochrome P4501A enzymes in salmon, indicative of exposure to bioavailable aromatics. In dab there was evidence of induction at some sites in January 1993 immediately following the spill but, subsequently, no induction has been observed in fish caught in the vicinity of Shetland despite the very high concentrations of oil measured in sediments at some locations. This would indicate that the polyaromatic hydrocarbons in these sediments are unlikely to be directly bioavailable to fish. The effect of the oil spill on hepatic pathology in dab was investigated and shows that pathology predictive of neoplasia, such as basophilic foci and vacuolation, were observed in fish from the most contaminated sites in 1994 but not in 1993. The incidence of this pathology appeared to correlate with the degree of contamination at the sites, but additional studies are required to establish whether this was a consequence of the initial impact and exposure from waterborne oil at the time of the spill or whether it was due to the continued exposure to oil from the sediments.     

Induction of cytochrome P4501A and biliary PAH metabolites in European eel Anguilla anguilla: Seasonal, dose- and time-response variability in field and laboratory conditions

Induction of cytochrome P4501A in fish is a well-known indicator of exposure to polycyclic aromatic hydrocarbons (PAHs) and determination of PAH metabolites in bile by fixed wavelength fluorescence (FF) or synchronous fluorescence spectroscopy (SFS), has become an useful method in monitoring programs. In this work the relationship between cytochrome P4501A (EROD activity) and levels of biliary PAH metabolites was measured in the European eel Anguilla anguilla, in both field and laboratory conditions: organisms were sampled on a seasonal basis from the Orbetello lagoon (Tuscany) to characterize the natural variability of these biological parameters, while in laboratory eels were intraperitoneally injected with benzo[a]pyrene to investigate temporal and dose-dependent induction patterns. Results showed that induction of cytochrome P450 and accumulation of PAHs metabolites in bile are not necessarily correlated either in field, or in laboratory investigations; different seasonal changes were measured in natural conditions and slight variations in dose and time response patterns were also obtained in laboratory exposures.     

Feeding habits of Solea senegalensis in earthen ponds in Sado estuary

The senegale sole, Solea senegalensis Kaup, 1858, is a commercially valuable fish and it is beginning to be cultured in a semi-intensive way in fish farms in southern European countries. The present study was initiated to investigate the diet of S. senegalensis under semi-extensive conditions. The feeding habits of sole were studied in two earthen ponds of a fish farm in the Sado estuary. In one earthen pond, artificial fish ration was given. S. senegalensis feeds on few prey items, its diet is mainly composed of insect larvae (Chironomus salinarus) and polychaeta (Hediste diversicolor). The diet composition of this species suggests feeding specialization, by consuming mainly annelids and insect larvae and by avoiding other items, extremely abundant in the environment, such as gastropods. In the water reservoir where ration was given, some fish consumed simultaneously benthic organism together with ration. However, benthic organisms seem to be the most important component of S. senegalensis diet.     

牙鲆gnrh基因的表达分析

本文通过qPCR方法,研究了我国重要海水鱼类——牙鲆（Paralichthys olivaceus）雌、雄成体组织以及性腺分化期的脑、性腺中gnrh1、gnrh2和gnrh3的表达水平。结果显示,gnrh1分布于所有检测的组织：在脑、垂体、肾脏和肝脏中高表达,在性腺中表达相对较低;gnrh2则在雌、雄性的肝脏以及雄性的肠、肾脏、眼睛和头肾中;而gnrh3只在雄性的肾脏、眼睛和肌肉中检测到微弱表达。人工诱导的牙鲆雌核发育鱼苗分别用17β-雌二醇（E₂）和17α-甲基睾酮（MT）处理,使其分化为雌性或雄性个体表型。在性腺分化期的脑中,gnrh1的表达呈现上升趋势,在全长（total length,TL）4 cm鱼苗中,E₂组的表达显著高于MT组（P<0.05）;gnrh2在2 cm TL时E₂组显著高于MT组（P<0.05）,随后在E₂和对照组的表达水平均出现下降趋势;gnrh3在2 cm TL时E₂和对照组的表达均显著高于MT组（P<0.05）,自6 cm TL时MT和E₂组的表达开始下降。在性腺中,三种gnrh在性腺分化前2 cm TL表达量都相对较高,随后呈现下降趋势。综上,推测牙鲆gnrh1可能参与了性腺分化的启动、分化过程及性腺发育,gnrh2主要参与了性腺分化的启动,gnrh3主要参与了性腺分化的启动和分化过程。     

外源激素对养殖牙鲆血浆睾酮和雌二醇含量的影响研究

雄性牙鲆单独注射促黄体激素释放激素类似物(LHRH-A)(每千克质量的注射量,下同)25μg/kg或人绒毛膜促性腺激素(hCG)1 000 IU/kg,血浆睾酮(T)含量在注射后6 h大幅度降低,12h时达到最低值,以后逐渐回升;LHRH-A与hCG联合注射[LHRH-A(12.5μg/kg)+hCG(500 IU/kg)],注射后6 h血浆T含量剧烈下降,24 h时达到最低点,以后缓慢回升;对照组雄鱼血浆T含量随着采样进行而呈现降低趋势.雌鱼单独LHRH-A后,血浆雌二醇(E₂)水平逐渐下降,24 h时达到最低值,30 h后略有回升;单独注射hCG后,血浆E₂含量缓慢下降,30 h达到最低值;LHRH-A与hCG联合注射,注射后6 h血浆E2含量逐渐下降,24 h时达到最低点,30 h时缓慢回升.对照组雌鱼血浆E₂水平也随着采样的进行而逐渐降低.结果表明:单独或联合注射LHRH-A和hCG降低了雌、雄牙鲆血浆E₂和T含量,与对照组结果相同.