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.     

Hepatic hemosiderosis in buffalo fish (Ictiobus spp.)

Hepatocellular hemosiderosis was observed in several species of fish associated with mixed chemical pollution in a Mississippi River Basin ecosystem. Three species of buffalo fish—smallmouth buffalo (Ictiobus bubalus), bigmouth buffalo (Ictiobus cyprinellus), and black buffalo (Ictiobus niger)—were collected from a contaminated site, Devil's Swamp, and a control site, Tunica Swamp, both near Baton Rouge, Louisiana. Liver, kidney and spleen were examined histologically. Hepatocellular iron overload was observed in smallmouth buffalo and bigmouth buffalo from Devil's Swamp. Smallmouth buffalo from Tunica Swamp had no stainable iron in the hepatocytes. Bigmouth buffalo from Tunica Swamp had trace amounts of stainable iron in the hepatocytes. Black buffalo from both Devil's Swamp and Tunica Swamp had no stainable iron in the hepatocytes. Iron could accumulate in sediment where it enters the food chain through benthic organisms. Analyses of water and sediment from both study sites revealed similarly high concentrations of iron. The pollutants in the Devil's Swamp may have affected the iron absorption and metabolism in smallmouth buffalo and bigmouth buffalo. The variation in the presence of stainable iron in the liver of three species of buffalo fish may indicate significant interspecies variation in the iron absorption and metabolism under conditions of mixed pollution.     

Small-scale variations in mussel (Mytilus spp.) dynamics and local production

A mussel bed was sampled monthly at four intertidal levels (mid: 2.15; mid-low: 1.65; low: 1.2 and sublittoral fringe: 0.7 m from chart datum) from July 1979 to July 1980 at Pointe-Mitis in the St. Lawrence estuary. A strong spring reduction of abundance (both in density and biomass) suggested that the mussel bed was being degraded. Community perturbation was attributed to ice scour. Partial reestablishment of the mussel bed (all age classes) was observed during late spring and early summer and occurred mainly at the mid-low intertidal level. Changes in the size structure of the mussel bed with level suggest that the annual windstorm regime may be an important factor in the dynamics of the bed. Mean body mass decreased at the three lower shore levels but increased at the highest shore level. Overall, net secondary production (assessed by the increment-summation method) was negative due to the decrease in mean body mass. Annual production rates (kJ m⁻² y⁻¹) from the mid intertidal level to the sublittoral fringe were 1130, − 4072, − 4013 and − 3258, respectively, while P/B ratios (y⁻¹) were 0.17, − 0.69, − 0.50 and − 0.45. The calculated production and the productivity (potential production) are compared and used to provide insight into the condition of the mussel bed.     

Chromatophoromas and Related Hyperplastic lesions in Pacific rockfish (Sebastes spp.)

Pacific rockfish were sampled from Cordell Bank, off central California, from 1985 to 1990. Hyperplastic and neoplastic lesions, involving dermal chromatophores, were observed in five species; yellowtail rockfish (Sebastes flavidus), bocaccio (S. paucispinis), olive rockfish (S. serranoides), widow rockfish (S. entomelas), and chilipepper rockfish (S. goodei). Yearly prevalences were highest, but static, in S. pancipinis, while prevalences in S. flavidus have risen steadily. The majority of lesions were black, but white, red, orange, yellow, and mixed color variants were also seen. Lesions were found in skin, fins, lips, gingiva, longue, urogenital papilla, and in the conjunctira and cornea of the eye. Flat lesions were consistent with either melanophore (black), erythrophore (red), or xanthophore (yellow) hyperplasia. Neoplastic lesions included melanophoromas, amelanotic melanophoromas, xanthophoromas, erythrophoromas, and mixed chromatophoromas. Electron microscopy did not reveal the presence of viruses and the etiology of the lesions has not been determined.     

Natural modulation of hepatic metallothionein and cytochrome P4501A in flounder, Platichthys flesus L.

The use of biomarkers such as cytochrome P4501A (CYP1A) or metallothionein (MT) for pollution monitoring is based on the assumption that an increased activity or concentration is primarily caused by exposure to contaminants. Previous studies have shown that the response of biomarkers may be affected by factors such as season, temperature, gender, nutritional status or size. The objective of the present study was to identify natural factors that affect the hepatic activity of CYP1A and hepatic concentration of MT in flounder (Platichthys flesus L.). Thirty flounder were sampled at each of two sites in the Hvaler archipelago, southern Norway, monthly through one year. A set of variables were recorded for each sampling and individual including water temperature, salinity, external and internal lesions, intestinal content, sex, size and organ weights. Hepatic CYP1A activity (EROD), MT and metal concentrations were determined for each individual flounder. The influence of environmental and endogenous factors on the response of these two biomarkers was assessed in multiple regression models. For both biomarkers, 50–60% of the total variability could be explained from factors directly related to season, gender and maturation. Season contributed significantly in the model for EROD, as did external lesions. Size, condition and diet did not contribute greatly when the above factors were included. The results confirm previous findings that season, gender and maturation must be taken into account in biomarker monitoring, but also indicate that other factors such as external lesions should be considered.     

The use of polyclonal antibodies raised against rat and trout cytochrome P450 CYP1A1 orthologues to monitor environmental induction in the channel catfish (Ictalurus punctatus)

Induction of hepatic cytochrome P450-dependent microsomal mono-oxygenase by xenobiotics is a well-established phenomenon in teleost fish. As in laboratory mammals, fish possess multiple forms of cytochrome P450 that display overlapping substrate specificity. One such isoform, CYP1A1, which has been cloned and sequenced from rainbow trout, has been shown to be orthologous to rat CYP1A1 and, as in mammals, is inducible up to several hundred-fold by planar aromatic hydrocarbons, PCBs and dioxins. It has been suggested that induction of CYP1A1 orthologues might provide a sensitive biomonitor for environmental pollution by mixtures of such compounds. In the current study, polyclonal antibodies directed against CYP1A1 purified from rat and trout liver were used to monitor induction of the CYP1A1 orthologue in hepatic microsomes from the fresh water species, the channel catfish (Ictalurus punctatus). Catfish from a local fish farm were induced in the laboratory by three daily injections of 50 mg/kg of the PCB mixture Aroclor 1254 and compared with fish taken from a site in central Arkansas—the Bayou Meto, known to be polluted with dioxin. Hepatic microsomal activities towards ethoxyresorufin (EROD) and pentoxyresorufin (PROD) were measured and Western blot analysis carried out with the two antibodies. EROD was elevated in both the Aroclor-treated fish and in the Bayou Meto fish compared with untreated fish farm controls; smaller but significant increases were observed in PROD. Spearman's rank correlations of 0·74 and 0·89 were observed between EROD and immunoquantified cross-reactivity towards the rat CYP1A1 and trout CYP1A1 antibodies.     

Quantification of cytochrome P4501A1 and catalytic activities in liver microsomes of isosafrol- and β-Naphthoflavone-treated rainbow trout (Oncorhynchus mykiss)

The effects of isosafrol (ISF) or β-naphthoflavone (βNF) treatments on cytochrome P450 (P450) levels in rainbow trout liver were investigated using immunochemical and catalytic techniques. The discrepancies in catalytic activities and ELISA quantification of rainbow trout P4501A1 protein levels between ISF- and βNF-treated fish indicate that important differences exist between the responses induced by βNF and ISF treatments in the rainbow trout liver.     

Distribution of cytochrome P4501A (CYP1A) in the tissues of Baltic ringed and grey seals

Information about the expression of CYP1A in wildlife species is essential for understanding the impact of organochlorine exposure on the health status of an exposed population. Therefore, we aimed at characterising a putative CYP1A enzyme expression in both hepatic and extrahepatic tissues of ringed and grey seals from the Baltic Sea and from less polluted waters. The cellular localisation of CYP1A was identified using a monoclonal antibody against scup P4501A1 (MAb 1-12-3). Immunohistochemical staining showed the highest level of CYP1A expression in liver hepatocytes, and the second highest level in the endothelial cells of capillaries and larger blood vessels in the liver and other organs. The most frequent and strongest staining was found in Baltic ringed seals. Although CYP1A-positive staining was observed in only a few tissues in the other seal populations, it was more intense in Baltic grey seals than in Canadian grey seals. The CYP1A enzyme activity, expressed as ethoxyresorufin O-deethylation (EROD), followed a similar tissue distribution and geographical pattern as the immunohistochemistry with clearly elevated EROD activities in most tissues of both Baltic seal populations. Immunochemical characterisation by immunoblotting confirmed the presence and elevation pattern of a putative CYP1A protein in ringed and grey seals, supporting our findings using other methods. The evenly distributed elevation of CYP1A expression among most of the tissues examined indicates that Baltic seals are exposed to CYP1A inducing agents affecting the whole body. This may result in an increased or decreased toxic potential of foreign substances, which may ultimately determine the biological effects of the contaminants.     

Seasonal abundance, distribution and recruitment of mud crabs (Scylla spp.) in replanted mangroves

The abundance and distribution of mud crabs were studied in a replanted mangrove forest in Buswang, Aklan, Philippines. Two fishing gears, lift nets and bamboo traps, were used to monitor relative abundance of Scylla spp. populations from March 2002 to December 2003 inside the mangrove forest. A third gear, a stakenet set across a creek, was used to monitor crabs migrating out of the mangroves during the ebb tide. Scylla olivacea formed 99.3% and 70.3% of the catch in the mangrove and the stakenet, respectively. The percentage of Scylla tranquebarica increased from <1% in the mangrove catches to 29% in the stakenet. Scylla serrata was present at very low levels in both catches. The lack of modal progression in the size–frequency plots and the year-round catch rate of gravid females suggested that recruitment was constant throughout the year. Even though relative abundance decreased over the study period indicating that the stock is being over-exploited, mud crab production is more than equivalent to that of most natural mangroves.     

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.     

Accumulation and effects of benzo(a)pyrene on cytochrome P450 1A in waterborne exposed and intraperitoneal injected juvenile turbot (Scophthalmus maximus)

Juvenile turbot (Scophthalmus maximus) were exposed to benzo(a)pyrene (BaP) for 14 d using a glass bead generator flow-through system. Exposure was followed by a recovery period of 16 d. The highest BaP concentration in the edible portion of the fish, 16.5 ± 4.3 μg BaP/kg, was observed on the first day. Then concentrations dropped following first-order kinetics. BaP was below detection level at the end of the experiment. A statistically significant increase in bile fluorescence was observed from day 9 until the end of the experiment, suggesting the elimination of BaP metabolites by this route. No significant differences between control and exposed fish in EROD activity and CYP1A concentration, measured by immunodetection method, were observed. Intraperitoneal injection of 2.5 mg BaP/kg in juvenile turbot induced EROD activity. Under waterborne experimental conditions, bile fluorescence was observed to be a more sensitive biomarker of BaP exposure than EROD activity and CYP1A measurement.     

Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.     

Natural expansion and experimental manipulation of seagrass (Zostera spp.) abundance and the response of infaunal invertebrates

A study of an expanding seagrass bed on the south-west coast of British Columbia, Canada involved documentation and explanation of the pattern of expansion of the vegetation as well as documentation and experimental investigation of the accompanying changes in the distribution of infaunal invertebrates. Expansion followed a major environmental change, improved water clarity initiated in 1969 when a causeway blocked access to the site for silty Fraser River water. The original eelgrass (Zostera marina) bed expanded landward over 30 m year⁻¹, probably causing more and more water to be retained in the bed at low tide and thus improving its own habitat. More rapid expansion occurred from 1979–1983 after Z.japonica colonized at the landward edge of the eelgrass bed and in a separate intertidal area. Expansion ceased around 1983 coincident with, but probably independent of, further construction.Burrowing shrimp (Callianassa californiensis) decreased in abundance between 1977 and 1984 in areas where the two seagrasses colonized. Short-term experiment over one summer and fall showed that removal of all shoots allowed adult shrimp and tube worms to colonize the sediment while addition of shoots of Z. japonica by transplanting caused temporary decreases in shrimp abundance. After a few weeks an established shrimp population destroyed the transplants. Among reasonable alternate hypotheses for the decline in Callianassa, the effect of sediment texture can be eliminated, but either direct negative effects of seagrass, i.e. inhibition of the burrowing of adult shrimp or of settlement of juveniles, or indirect effects, i.e. the harboring of more predators of shrimp in seagrass beds, deserve further study.     

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.     

重组别藻蓝蛋白(rAPC)的纯化与鉴定

报道了重组大肠杆菌表达的别藻蓝蛋白的下游生产工艺研究,并对产物进行了分析鉴定。工程菌株P1先在NBSBioFlo3000型5L自动发酵罐进行高密度发酵,融合蛋白获得了高效表达,且主要在细胞内以可溶形式存在。纯化前先将获得的菌体超声破碎,然后经硫酸铵沉淀、疏水层析和离子交换层析三步纯化,接着对纯化的重组别藻蓝蛋白(rAPC)进行SDS-PAGE、PAGE、Westernblot等电点分析。证明已获得纯度为96.4%的rAPC,其等电点为6.0,并且具有与天然APC相似的抗原性。     

单体牡蛎诱导变态的研究

为了得到壳型规则、大小均一的单体牡蛎,作者以葡萄牙牡蛎(Crassostrea angulata)和长牡蛎(Crassostrea gigas)眼点幼虫为材料,从肾上腺素(EPI)浓度梯度、处理时间梯度和眼点幼虫密度梯度3个因素诱导产生不固着变态的单体牡蛎。结果表明,葡萄牙牡蛎用EPI处理24 h的最适浓度为5×10–4 mol/L,不固着变态率为72.8%,在该最适浓度下,最佳处理时间为12 h,不固着变态率为82.7%。长牡蛎用EPI处理6 h的最适浓度为5×10–5 mol/L,不固着变态率为53.2%,在该最适浓度下,最佳处理时间为8h,不固着变态率为56.8%,眼点幼虫密度在80个/m L以下EPI处理效果没有显著性差异。对长牡蛎幼虫进行后续生长测定,结果显示EPI处理组幼虫的壳长、壳高和存活率要高于对照组,表明EPI可能促进牡蛎幼虫变态长出次生壳并提高其生存能力。