饥饿对金鱼卵巢型和脑型芳香化酶基因表达的影响研究

采用RT-PCR技术,研究了金鱼性腺型芳香化酶基因(CYP19A)和脑型芳香化酶基因(CYP19B)的表达对饥饿胁迫的响应机制.研究结果表明:正常投喂的雌性金鱼中CYP19A基因mRNA在卵巢、肝脏、肾脏、肌肉、鳃、头肾、脾脏、脑、肠和心脏10个组织均有表达,表达量呈现递减趋势;饥饿胁迫后,CYP19A基因mRNA在脑和卵巢中表达的变化规律基本一致,即饥饿5d时表达量显著降低(P<0.05),随后表达水平维持稳定.正常投喂的雌性金鱼CYP19B基因mRNA在脑、头肾、性腺、脾、肌肉和心脏6个组织中表达呈现递减趋势;CYP19B基因mRNA在脑和卵巢中的表达量,在饥饿5d时表达量显著降低(P<0.05),随后表达水平维持稳定.这些结果说明,雌性金鱼在饥饿条件下,性激素合成关键酶基因在脑和卵巢中的表达量显著降低,可能影响卵巢发育。     

Catalytic function of avian cytochrome P450 1A4 and 1A5 (CYP1A4 and CYP1A5) enzymes heterologously expressed using in vitro yeast system

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.     

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.     

Cloning a new cytochrome P450 isoform (CYP356A1) from oyster Crassostrea gigas

We have cloned the full-length cDNA of the first member of a new cytochrome P450 (CYP) family from the Pacific oyster Crassostrea gigas. This new CYP gene was obtained based on an initial 331bp fragment previously identified among the list of the differentially expressed genes in oysters exposed to untreated domestic sewage. The full-length CYP has an open reading frame of 1500bp and based on its deduced amino acid sequence was classified as a member of a new subfamily, CYP356A1. A phylogenetic analysis showed that CYP356A1 is closely related to members of the CYP17 and CYP1 subfamilies. Semi-quantitative RT-PCR was performed to analyze the CYP356A1 expression in different tissues of the oyster (digestive gland, gill, mantle and adductor muscle). Results showed slightly higher CYP356A1 expression in digestive gland and mantle, than the other tissues, indicating a possible role of the CYP356A1 in xenobiotic biotransformation and/or steroid metabolism.     

Comparison of cytochrome P450 in three butterflyfish species: Ecological implications of elevated CYP2B and CYP3A in Chaetodon capistratus

The relationship between cytochrome P450 and feeding on terpenoid-rich gorgonian corals was investigated in a species of tropical butterflyfish and compared with two other sympatric congeners that do not feed on gorgonians. Fish were collected from non-polluted waters in Belize and the levels of two cytochrome P450 isozymes (CYP2B and CYP3A) were immunoquantitated in addition to quantification of total P450. Chaetodon capistratus regularly feeds on gorgonian corals and has higher levels of total hepatic microsomal cytochrome P450 than C. ocellatus or C. striatus. The content of hepatic P450 (0.588–0.794 nmol mg⁻¹) in C. capistratus is among the highest ever reported in teleosts from non-polluted waters and is significantly greater than detected in C. ocellatus or C. striatus. Chaetodon capistratus also had a larger hepatic index (g liver per g fish) and more microsomal protein (mg protein per g liver), factors that translate into 3.3- to 8-fold more total P450 per g fish. Sexual differences in total P450 were observed between male and female C. capistratus, but not among the other species. The contents of proteins detected by immunoassay with polyclonal anti-scup P450B (CYP2B) and anti-human P4503A (CYP3A) were 2- to 10-fold and 2- to 20-fold greater, respectively, in C. capistratus than in the congeneric species. CYP2 and CYP3 gene families in mammals are thought to have evolved partially in response to dietary allelochemicals. These results suggest that these P450 isozymes may also be important in marine teleosts that feed on terpenoid-rich prey.     

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.     

雌牙鲆CYP19A基因启动子“CpG”岛区的单核苷酸多态性及与繁殖性能的关联分析

芳香化酶基因(CYP19)编码的芳香化酶是雄激素转化为雌激素的关键酶,在鱼类性腺发育及繁殖内分泌过程中起着重要的作用。本研究采用聚合酶链式反应-单链构象多态(PCR-SSCP)和放射性免疫方法(RAI)对61尾雌牙鲆进行了CYP19A基因启动子CpG岛区多态性检测,并将其与繁殖性能进行了关联分析。CpG岛区存在3种类型的基因型(pattern1、pattern2、pattern3),经测序发现CpG岛区有以下突变:T-1927插入或缺失、C-1899插入或缺失、T-1772C、C-1766T、TGTCAAC-1752~-1744插入或缺失(11个SNPs连锁突变)。方差分析结果表明:此位点对雌二醇(E2)水平的效应达到显著(P<0.05)水平,经多重比较分析,具有pattern1基因型的个体的雌激素(E2)水平显著低于pattern2和pattern3基因型个体的雌激素水平(P<0.05),同时pattern1基因型的个体的mRNA表达量显著低于pattern2和pattern3基因型个体的mRNA表达量(P<0.05)。以上结果表明,CYP19A基因调控区甲基化位点突变可能导致芳香化酶的表达量,进而影响牙鲆繁殖内分泌过程;同时也阐明了这些与牙鲆繁殖性状相关的SNPs可以用于生产实践中进行标记辅助选择具有优良繁殖性能的牙鲆。     

CYP1A and phase II responses of Scophthalmus maximus upon long-term repeated exposure to B(a)P and B(a)P-PCB77 mixtures

In a controlled laboratory experiment, fish in two treatment groups were exposed to B(a)P or B(a)P+PCB77 repeatedly over 1 year. Fish were held in a flow-through system and sampled on day 0, week 3, and on months 3, 6, 9, and 12. Each sampling (except week 3) was followed by exposure in water under static conditions. Exposure was terminated by restoring the flow-through within a maximum period of 24 h. Hepatic CYP1A remained significantly induced in juveniles even 3 months after the first exposure. The induction response in liver was more persistent in comparison to the gill response in juveniles. Sex influenced CYP1A expression in both organs of control fish and in both exposure groups. Suppression of CYP1A occurred in females even under repeated exposure. Liver GST activity showed sex differences, control females having higher activities than control males on the same sampling day. GST activity showed increases and decreases under both exposures possibly indicating the overriding influence of some other factors. Increased levels of fluorescent aromatic compounds (FACs) were detected in bile only in females exposed to B(a)P and PCB77 for 9 months. Liver UDPGT activity, length, weight, and condition showed no differences related to exposure or gender. The study was supported by Norwegian Research Council and the Ministry of Oil and Energy in Norway.     

Effects of three fungicides alone and in combination on glutathione S-transferase activity (GST) and cytochrome P-450 (CYP 1A1) in the liver and gill of brown trout (Salmo trutta)

In order to evaluate the gill glutathione S-transferase (GST) activity as a biomarker of effect of fungicide exposure in juvenile brown trout (Salmo trutta), the fungicides propiconazole [(R,S)-1-[2-(2,4-diclophenyl)-4-propyl-1,3-dioolan-2-ylmetyl]-1H-1,2,4-triazole] and fenpropimorph [(+/-)-cis-4-[3-(4-tert-butylphenyl)-2-metyl propyl]-2,6 dimetylmorfolinc] were administrated in the water separately and together in a static system (80 microg/l for each pesticide) for 5 days. The combined fungicides gave a significant decrease in gill GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB), whilst hepatic GST-activity was not significantly changed. Furthermore, continuous exposure to 540 ug/l thiabendazole[2-(thiazol-4'-yl)benzimidazole] in a flow-through system for 4 days significantly increased the gill glutathione S-transferase (GST) activity towards CDNB, whilst hepatic GST and cytochrome P450 (CYP 1A) activities were not increased by the treatment.     

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.     

CYP1A- and CYP3A-immunopositive protein levels in digestive gland of the mussel Mytilus galloprovincialis from the Mediterranean Sea

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.     

Effects of three fungicides alone and in combination on glutathione S-transferase activity (GST) and cytochrome P-450 (CYP 1A1) in the liver and gill of brown trout (Salmo trutta)

In order to evaluate the gill glutathione S-transferase (GST) activity as a biomarker of effect of fungicide exposure in juvenile brown trout (Salmo trutta), the fungicides propiconazole {(R,S)-1-[2-(2,4-diclophenyl)-4-propyl-1,3-dioolan-2-ylmetyl]-1H-1,2,4-triazole} and fenpropimorph {(±)-cis-4-[3-(4-tert-butylphenyl)-2-metyl propyl]-2,6 dimetylmorfolinc} were administrated in the water separately and together in a static system (80 μg/l for each pesticide) for 5 days. The combined fungicides gave a significant decrease in gill GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB), whilst hepatic GST-activity was not significantly changed. Furthermore, continuous exposure to 540 ug/l thiabendazole{ 2-(thiazol-4′-yl)benzimidazole} in a flow-through system for 4 days significantly increased the gill glutathione S-transferase (GST) activity towards CDNB, whilst hepatic GST and cytochrome P450 (CYP 1A) activities were not increased by the treatment.     

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 micro(micro)M) with or without 0.2 micro(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.