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1.
The main objective of this study was to apply a set of sensitive non-lethal biomarkers in skin biopsies of fin whales (Balaenoptera physalus) to evaluate the toxicological status of this mysticete in the Pelagos Sanctuary (Mediterranean Sea) and in the Gulf of California (Sea of Cortez-Mexico). We developed a “multi-trial diagnostic tool” (based on field and in vitro studies), combining molecular biomarkers (western blot of CYP1A1, CYP2B) and gene expression (qRT-PCR of HSP70, ERα, AHR, E2F-1) with the analysis of OCs, PAHs and PBDEs. The study revealed a higher level of toxicological stress in the Mediterranean fin whales.  相似文献   

2.
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.  相似文献   

3.
Abstract. Endocrine Disrupting Chemicals (EDCs) have the potential to alter hormone pathways that regulate reproductive processes in wildlife and fisheries. In this research the unexplored hypothesis that Mediterranean top predator species (such as large pelagic fish and marine mammals) are potentially at risk due to EDCs is investigated. In the Mediterranean environment, top predators accumulate high concentrations of polyhalogenated aromatic hydrocarbons (PHAHs) and toxic metals, incurring high toxicological risk. Here we illustrate the need to develop sensitive biomarkers for evaluation of toxicological risk in top marine predators (Xiphias gladius, Thunnus thynnus thynnus) and non-lethal techniques, such as non-destructive biomarkers, for the hazard assessment of threatened species exposed to EDCs, such as marine mammals ( Stenella coeruleoalba, Tursiops truncatus, Delphinus delphis and Balaenoptera physalus ).  相似文献   

4.
CYP1A-immunopositive protein can be elevated in response to planar PAHs and PCBs in Mytilus sp. digestive gland whilst CYP3A-immunopositive protein has been associated with testosterone 6beta-hydroxylation in fish. Levels of CYP1A- and CYP3A-immunopositive protein were determined in Mytilus galloprovincialis digestive gland microsomes collected from 12 sites in the Mediterranean Sea during May and September 2001. CYP1A-immunopositive protein was significantly highest at contaminated sites whilst CYP3A-immunopositive protein was significantly lowest. A weak negative correlation (r2 = 0.21) was seen between CYP1A- and CYP3A-immunopositive protein. Little evidence of differences at the different sampling times was observed. These results confirm previous work indicating elevation of CYP1A-immunopositive protein in Mytilus sp. digestive gland at contaminated sites. Further study is required to characterise CYP3A-like expression in Mytilus and to elucidate the consequences of possible CYP3A-like down-regulation at contaminated sites.  相似文献   

5.
Various studies have revealed high concentrations of contaminants such as organochlorines (OCs) and heavy metals in Mediterranean cetaceans. A geographical trend of contamination (PCBs and DDTs) has been found for striped dolphin (Stenella coeruleoalba). In this study we used a non-lethal approach (skin biopsy) to investigate bioaccumulation of OCs, including polychlorobiphenyls (PCBs), DDTs, polychlorodibenzo-p-dioxins (PCDDs), polychlorodibenzofurans (PCDFs), trace elements (Hg, Cd, Pb) and CYP1A activity (BPMO) in nine striped dolphins sampled in the Aeolian area (Sicily - Italy) in summer 2002. The arithmetic mean value of BPMO activity in this group was 43.46 AUF/g tissue/h. This value is approximately 3 times and 5 times lower, respectively, than the value found in the Ionian and in the Ligurian groups. Skin biopsies of striped dolphins emerged as a suitable material for assessing the toxicological status of the various Mediterranean groups.  相似文献   

6.
Biomarkers of organochlorine exposure, such as the induction of cytochrome P450 1A (CYP1A), can be used to assess the impact of environmental contaminants on the health of free-ranging marine mammal populations. The objective of the present study was to measure CYP1A in skin and liver biopsies obtained from live harbour seals (Phoca vitulina). Twelve harbour seal pups, aged three to five weeks, were captured from the Fraser River estuary, British Columbia, Canada, and temporarily held in captivity. Skin ( approximately 60 mg) and liver ( approximately 40 mg) biopsies, obtained while seals were under general anaesthesia, yielded sufficient tissue for the measurement of CYP1A by immunoblot analysis and ethoxyresorufin O-deethylase activity. A short-term exposure experiment, in which harbour seals (n=3) were treated orally with beta-naphthoflavone (BNF), resulted in increased hepatic and cutaneous CYP1A protein levels, consistent with observations in other mammals. This study is the first to measure CYP1A in skin and liver biopsies from live harbour seals and to report in vivo BNF-associated CYP1A induction in a marine mammal. The results demonstrate that microsamples collected using minimally-invasive techniques can provide toxicologically-relevant information form marine mammals.  相似文献   

7.
Various studies have revealed high concentrations of contaminants such as organochlorines (OCs) and heavy metals in Mediterranean cetaceans. A geographical trend of contamination (PCBs and DDTs) has been found for striped dolphin (Stenella coeruleoalba). In this study we used a non-lethal approach (skin biopsy) to investigate bioaccumulation of OCs, including polychlorobiphenyls (PCBs), DDTs, polychlorodibenzo-p-dioxins (PCDDs), polychlorodibenzofurans (PCDFs), trace elements (Hg, Cd, Pb) and CYP1A activity (BPMO) in nine striped dolphins sampled in the Aeolian area (Sicily – Italy) in summer 2002. The arithmetic mean value of BPMO activity in this group was 43.46 AUF/g tissue/h. This value is approximately 3 times and 5 times lower, respectively, than the value found in the Ionian and in the Ligurian groups. Skin biopsies of striped dolphins emerged as a suitable material for assessing the toxicological status of the various Mediterranean groups.  相似文献   

8.
CYP1A-immunopositive protein can be elevated in response to planar PAHs and PCBs in Mytilus sp. digestive gland whilst CYP3A-immunopositive protein has been associated with testosterone 6β-hydroxylation in fish. Levels of CYP1A- and CYP3A-immunopositive protein were determined in Mytilus galloprovincialis digestive gland microsomes collected from 12 sites in the Mediterranean Sea during May and September 2001. CYP1A-immunopositive protein was significantly highest at contaminated sites whilst CYP3A-immunopositive protein was significantly lowest. A weak negative correlation (r2=0.21) was seen between CYP1A- and CYP3A-immunopositive protein. Little evidence of differences at the different sampling times was observed. These results confirm previous work indicating elevation of CYP1A-immunopositive protein in Mytilus sp. digestive gland at contaminated sites. Further study is required to characterise CYP3A-like expression in Mytilus and to elucidate the consequences of possible CYP3A-like down-regulation at contaminated sites.  相似文献   

9.
The contaminant-induced cytochrome P450 (CYP) members in minke whale (Balaenoptera acutorostrata) can be potential biomarkers of the contaminant exposure and toxic effects. In this study, we constructed a cDNA library from the liver of minke whale from the North Pacific, and further screened a total of 6930 clones randomly selected in the library for the isolation of cDNA clones encoding novel members of CYP superfamily. The screening revealed the isolation of six novel CYP cDNA clones that are classified into CYP1A, CYP2C, CYP2E, CYP3A, CYP4, and CYP4A subfamilies. The BLAST homology search using the partial cDNA fragments of four CYP subfamilies (CYP1A, CYP2C, CYP2E and CYP4A) demonstrated that the minke whale CYPs were most closely related to pig CYPs (81-91%). Identification of multiple CYP genes in marine mammal species such as minke whale will provide new insights into the metabolic or toxicological functions of individual CYP members.  相似文献   

10.
English sole (Pleuronectes vetulus) and starry flounder (Platichthys stellatus) are two sympatric flatfish species which show markedly different responses to chemical contaminant exposure. Whereas English sole develop hepatic neoplasms, the prevalences of which are strongly linked to exposure to polycyclic aromatic hydrocarbons (PAHs), evidence of neoplasia is virtually nonexistent in starry flounder, even those residing in areas with very high levels of PAH in the sediments. Because PAHs are activated to genotoxic forms by the action of cytochrome P450 1A (CYP1A) in teleosts, we are examining the hypothesis that variation in the hepatic expression of this major inducible cytochrome P450 isozyme may contribute to the differential contaminant response. These two species were captured in the Duwamish Waterway, a contaminated area of Puget Sound, Washington. Catalytic activity of CYP1A [determined as aryl hydrocarbon hydroxylase (AHH) activity] was measured in the liver to quantitatively assess the relative induction of CYP1A in these two species, and AHH activity was significantly higher in English sole than in starry flounder (p = 0.015). Cellular expression of CYP1A was determined by immunohistochemical localization of tissues, using an avidin-biotin peroxidase complex method, with polyclonal rabbit anti-cod CYP1A. The results showed a markedly reduced expression of CYP1A in hepatocytes of starry flounder, which is consistent with the apparent resistance of this species to the development of hepatocellular neoplasia. This reduced expression of CYP1A in hepatocytes of contaminant-exposed fish was previously seen in oyster toadfish from the Elizabeth River, Virginia, and this species is also apparently resistant to hepatocellular neoplasia.  相似文献   

11.
Normal operation of oil well platforms results in the discharge of produced formation water (PFW). The expression of CYP1A, CYP2M1- and 2K1-like proteins was examined for use as possible biomarkers of PFW exposure. A pilot study on the Northwest Shelf of Australia had indicated that PFW contamination possibly contributes to induction of CYP1A-like proteins in Gold-Spotted Trevally (Carangoides fulvoguttatus). The pilot study samples were re-examined for CYP1A, and, in addition, CYP2K1/2M1-like proteins. In a subsequent caged fish study in the same location a second species, Stripey seaperch (Lutjanus carponotatus), caught at a clean site, were distributed to three caging sites in a PFW gradient from the Harriet A production platform: A (near-field), B (far-field) and C (a non-impacted reference site). Fish were sampled at time (T) T = 0, T = 3 and T = 10 days. Significant increases of CYP1A, one CYP2K1- and two CYP2M1-like proteins were noted at Site A at T = 10d. For another CYP2K1-like protein, a significant increase was observed at Site A only at T = 3d. These results support a previous study indicating that CYP1A protein is sensitive to PFW exposure. Importantly, statistically significant environmental induction of both CYP2M1- and CYP2K1-like proteins in tropical fish due to PFW exposure had not previously been described and induction of enzymes in the CYP2 family suggest new biomarkers for PFW. In addition, the novel response of one CYP2K-like protein requires further verification, but offers promise for improved monitoring of sub-lethal responses in marine organisms.  相似文献   

12.
Mummichogs, Fundulus heteroclitus, an estuarine fish with a relatively small home range found along the eastern coast of the United States are well-suited to monitoring contaminant effects, including those of polycyclic aromatic hydrocarbons (PAHs). One of the common PAHs in estuaries is pyrene. We report here on efforts to develop multiple biomarkers of pyrene exposure in this species. Adult male mummichogs were exposed in the laboratory to the weak aryl hydrocarbon receptor (AhR) agonist pyrene at 0, 30, or 50 microg/L in 7-day static renewal exposures. The RNA was extracted from livers and alterations in mRNA expression were assessed by subtractive hybridization and differential display in order to produce multiple biomarkers of pyrene exposure. Genes demonstrating differential expression were confirmed by quantitative-PCR (Q-PCR) and include cytochrome P-450 1A (CYP1A), a putative hepatocyte growth factor activator, a X-ray inducible retrotransposon, and several expressed sequenced tags (ESTs). Some of these genes represent new biomarkers of pyrene exposure and potential biomarkers of PAH exposure. Therefore, similar changes were investigated at a Superfund site in Charleston, SC. Mummichogs from a creosote contaminated site and from a reference site (North Inlet National Estuarine Research Reserve near Georgetown, SC) were trapped, RNA extracted from the livers, and Q-PCR performed. Many of the genes differentially expressed following pyrene exposure were not altered at the creosote contaminated site in comparison to the reference site. However, CYP1A and an EST were induced. CYP1A induction at Diesel Creek indicates that this population of fish does not demonstrate refractory CYP1A phenotypes observed at several sites with high levels of AhR agonists. Ultimately, we anticipate that the use of multiple biomarkers of PAH exposure will provide useful information on the potential effects of toxicants.  相似文献   

13.
The goal of these experiments was to explore the relationship between cytochrome P4501A (CYP1A) induction and the teratogenicity of sediments from the Atlantic Wood Industries Superfund site (Elizabeth River, VA) in Fundulus heteroclitus embryos. In these experiments we used embryos spawned from reference site adults to assess CYP1A activity and teratogenicity induced by aqueous Elizabeth River sediment extracts (ERSE). Embryo exposures to ERSE induced CYP1A activity and caused deformities, including pericardial edema, heart elongation and tail shortening. Co-exposures with various CYP1A inhibitors significantly decreased CYP1A activity and increased the teratogenicity of the sediment extract. Potential mechanisms for this increased toxicity are discussed herein.  相似文献   

14.
The present study clarifies the enzymatic properties of two avian cytochrome P4501A (CYP1A) paralogs, CYP1A4 and 1A5, using a yeast-based vector system. Recombinant CYP1A4 and 1A5 proteins from common cormorant (Phalacrocorax carbo) were expressed in yeast cells, and showed typical reduced CO-difference spectra with a peak at 446 nm. Kinetic analysis of O-dealkylase of methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin catalyzed by the CYP1A enzymes revealed that Vmax value for ethoxyresorufin-O-deethylase (EROD) activity was much higher than that for the other three O-dealkylase activities for both isozymes. Interestingly, remarkable substrate specificity of the CYP1As was observed for O-dealkylation of benzyloxyresorufin and methoxyresorufin; CYP1A4 was highly specific for catalyzing benzyloxyresorufin-O-debenzylase activity, whereas CYP1A5 was more efficient in catalyzing methoxyresorufin-O-demethylase activity. The present study also measured CYP1A-dependent EROD activity in the presence of 2,3,7,8-tetrachlorodibenzofuran (TCDF) to evaluate the ability of this dioxin-like congener to inhibit the EROD activity. One hundred nanomolar TCDF noncompetitively inhibited CYP1A5-dependent EROD activity, although no inhibitory effect was detected for CYP1A4-dependent EROD activity. These results indicate that the avian CYP1A paralogs have different affinities for substrate and inhibitor, thus suggesting their distinct physiological and toxicological roles.  相似文献   

15.
The gulf killifish, Fundulus grandis, was used to determine the influence of biological rhythms on three biomarker responses. We first developed monoclonal antibodies against the model's immunoglobulins and vitellogenin in order to measure antibody responses and vitellogenesis, respectively. We then treated adults with 10, 1, 1, and 10 ppm of Aroclor 1254, tribuyltin, 3-methylcholanthrene, and nonyl-phenol, respectively, in mixtures over a 16-week period. The study followed Vibrio anguillarum-specific antibody responses, hepatic CYP1A, and plasma vitellogenin levels in the morning and again in the evening at 2-week intervals. The contaminated diet suppressed secondary antibody responses, but only in the morning. The contaminated diet also altered CYP1A, but not vitellogenesis. In addition, fish in the control group exhibited daily and seasonal differences in specific antibody levels and CYP1A induction. Moreover, circulating vitellogenin levels in control males sampled in the morning increased throughout the exposure, but remained below those of females. This study underscores the need to consider normal physiological rhythms when employing biomarkers in toxicology.  相似文献   

16.
Recent studies demonstrating feminization of effluent-exposed wild-caught male fish in the UK have prompted much research regarding the estrogenic activity of effluent from municipal sewage treatment plants (MSTPs). To investigate the estrogenicity and cytochrome P450 1A (CYP1A) induction potency of MSTP effluent, two species of fish, adult male mummichogs, Fundulus heteroclitus, and juvenile sunshine bass, Morone saxatilis x Morone chrysops, were exposed to un-chlorinated effluent (75% effluent, 25% seawater) from a large MSTP in Yonkers, NY, USA. After a 21-day static-daily (75%) renewal exposure, significant elevations over controls were observed in levels of vitellogenin (VtG) in plasma (1730%) and liver (131%) in effluent-exposed sunshine bass. In contrast, hepatic VtG was not elevated in mummichogs; plasma VtG was not measured in this species. Effluent exposure elevated hepatic CYP1A protein (140-145%) and ethoxyresorufin-O-deethylase (EROD) activity (408-598%) in both species. These findings suggest ontogenetic and/or species differences in response to estrogenic compounds in MSTP effluent. Furthermore, the elevation of CYP1A in response to sewage effluent exposure indicates the presence of additional compounds that may alter xenobiotic and/or steroid biotransformation in fish.  相似文献   

17.
The effects of water-borne exposure to benzo[a]pyrene (36 h; celite-bound 0.44 mg L(-1) B[a]P) on cytochrome P450 (CYP) and superoxide dismutases (SODs) were examined in digestive gland of the blood clam, Scapharca inaequivalvis. B[a]P accumulation and elimination were rapid, with maximum whole-body concentrations of 1.78 ng g(-1) wet wt after 12 h of treatment, followed by a progressive decline to 0.89 ng g(-1) at 36 h. The presence of B[a]P resulted in an increase in total CYP of digestive gland microsomes from 54+/-14 to 108+/-21 pmol/mg protein (mean+/-SD; p<0.05, 24 h). Increases were also seen in microsomal CYP1A1/1A2-immunopositive protein (50.5 kDa app. mol. wt; p<0.05), but not CYP2E1-immunopositive protein (49 kDa app. mol. wt.), indicating a specific response of the former isoform. Exposure to B[a]P produced a steady increase in Mn-SOD digestive gland activity (p<0.01; p<0.05) but no significant change in Cu/Zn-SOD activity. The respective proteins, measured by western blotting, were not significant induced after B[a]P exposure. Cu/Zn-SOD and Mn-SOD activities were correlated with total CYP levels (r=0.96 and 0.63, respectively), indicating a role for CYP in reactive oxygen species (ROS) production during exposure. Both 'NADPH-independent' and NADPH-dependent metabolism of B[a]P by digestive gland microsomes was seen, producing mainly 1,6-, 3,6- and 6,12-diones, with some phenols and 7,8-dihydrodiol; putative protein adducts were also formed. Redox cycling of the diones may also have contributed to ROS production, leading to the increased SOD activities.  相似文献   

18.
The goal of these experiments was to explore the relationship between cytochrome P4501A (CYP1A) induction and the teratogenicity of sediments from the Atlantic Wood Industries Superfund site (Elizabeth River, VA) in Fundulus heteroclitus embryos. In these experiments we used embryos spawned from reference site adults to assess CYP1A activity and teratogenicity induced by aqueous Elizabeth River sediment extracts (ERSE). Embryo exposures to ERSE induced CYP1A activity and caused deformities, including pericardial edema, heart elongation and tail shortening. Co-exposures with various CYP1A inhibitors significantly decreased CYP1A activity and increased the teratogenicity of the sediment extract. Potential mechanisms for this increased toxicity are discussed herein.  相似文献   

19.
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.  相似文献   

20.
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.  相似文献   

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