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.     

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.     

First detection of CYP1A1 and CYP2B induction in Mediterranean cetacean skin biopsies and cultured fibroblasts by Western blot analysis

The need to develop powerful tools to detect exposure and effects of POPs and emerging contaminants in Mediterranean cetaceans led us to develop a suite of sensitive non-lethal biomarkers in integument biopsies of free-ranging animals. In order to propose induction of CYP1A1 and CYP2B, detected by Western blot analysis, as biomarkers of exposure to OCs, PAHs and PBDEs, a three-phase experimental protocol (in vitro experiments, calibration experiments and field applications) was followed using fibroblast cell cultures and biopsies of Mediterranean Stenella coeruleoalba and Tursiops truncatus. This methodology was confirmed to be sensitive and stable in comparison to previous methods used to detect CYP1A1 in biopsies, enabling analysis of several inducible proteins in non-lethal samples and analysis of material from stranded animals.     

Interaction of CYP1A1 inducers in liver of rainbow trout and eelpout

The hepatic CYP1A1 (ethoxyresorufin-O-deethylase (EROD) and protein level) in rainbow trout and eelpout was induced by isosafrole, β-naphthoflavone, 3,3′,4,4′-tetrachlorobiphenyl and mixtures of two of the compounds. A potentiation effect of the CYP1A1 response was observed when isosafrole was given together with β-naphthoflavone, but not when isosafrole was given with 3,3′,4,4′-tetrachlorobiphenyl.     

Effect of exposure to benzo[a]pyrene on SODs, CYP1A1/1A2- and CYP2E1 immunopositive proteins in the blood clam Scapharca inaequivalvis

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.     

Estradiol and estriol suppress CYP1A expression in rainbow trout primary hepatocytes

Hepatic levels of the pollutant inducible enzyme, CYP1A, are strongly suppressed in spawning female fish, a phenomenon attributed to high plasma levels of the female sex steroid hormone, estradiol. To evaluate the contribution of estrogen metabolites to estradiol-mediated CYP1A regulation, we treated primary hepatocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss) with vehicle, 17beta-estradiol, or the estrogen metabolite, estriol, alone and in combination with each other and with the potent CYP1A inducer, benzo[a]pyrene (B[a]P). We found dose-dependent suppression of B[a]P-induced CYP1A activity by both steroids relative to controls. At 10(-7) M doses, estradiol and estriol suppressed B[a]P-induced CYP1A activity by 3- and 2-fold, respectively. Although not statistically significant, mean basal CYP1A activity levels were 15- and 13-fold lower in estradiol and estriol treated hepatocytes, respectively, relative to vehicle treated controls. Combining doses of estradiol and estriol failed to produce synergistic suppression of either basal or B[a]P-induced CYP1A activity relative to treatment with either steroid alone. The observed suppression is well below the often strong suppression observed in spawning female fish. We conclude that factors in addition to estradiol and estriol are likely involved in producing sexual dimorphism in CYP1A expression observed in spawning fish.     

体外方法研究海洋药物911和971对大鼠CYP2E1的影响

研究海洋药物911,971对大鼠CYP2E1是否有抑制或诱导作用,以及这种作用是否具有性别差异。取大鼠,雌雄各半,随机分为6个组：Ⅰ)雄性对照组、Ⅱ)雄性911组、Ⅲ)雄性971组;Ⅳ)雌性对照组、Ⅴ)雌性911组、Ⅵ)雌性971组,均采用钙离子沉淀法制备肝微粒体。在各组肝微粒体中同时给予一定剂量的探针药物及目标药物,进行孵育。于不同时间点取样,测定该探针药物的剩余浓度并计算其体外半衰期。结果表明：药物911对CYP2E1活性无影响,971对雌性大鼠CYP2E1活性无影响,但对雄性大鼠的CYP2E1有显著诱导作用。由此可得出：药物对CYP2E1的影响存有明显的性别差异,971在同各种与CYP2E1代谢有关的药物合用时,应充分考虑其在不同性别间的差异,以避免可能出现的毒性反应或不良反应。     

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.     

甘草和连翘对牙鲆CYP3A 活性影响的研究

研究甘草(Glycyrrhiza uralensis)和连翘(Forsythia suspensa)对牙鲆(Paralichthys olivaceus)肝细胞色素P4503A(CYP3A)活性的影响。将牙鲆随机分为3组,即对照组、甘草组和连翘组,对照组每日口灌0.9%的生理盐水,试验组每日1次口灌甘草30 mg/kg和连翘100 mg/kg,连续口灌6 d,第7天时,采用直接测定法和间接测定法(探针药物)进行CYP3A酶活性的测定。结果显示:在肝微粒体水平上,甘草和连翘均可明显提高红霉素-N-脱甲基酶活性,分别提高了1.79倍、4.87倍。与对照组相比,甘草组和连翘组氨苯砜的半衰期分别降低了4.51%(P0.05)和36.8%(P0.01);药时曲线下面积分别减小了8.28%(P0.05)和32.3%(P0.01);总清除率分别增加了5.63%(P0.05)和99.3%(P0.01)。说明连翘对牙鲆CYP3A的活性具有极显著诱导作用,甘草诱导作用不显著。提示其他药物与连翘合用时,其有效性和安全性可能会受到影响。     

Quantitation of CYP1A expression in two flatfish species showing different prevalences of contaminant-induced hepatic disease

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.     

Estrogenic and CYP1A response of mummichogs and sunshine bass to sewage effluent

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.     

Teratogenesis in Fundulus heteroclitus embryos exposed to a creosote-contaminated sediment extract and CYP1A inhibitors

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.     

Hepatic CYP1A levels and EROD activity in English sole: biomonitoring of marine contaminants in Vancouver Harbour

To assess chemical contaminant stress in the marine environment, ethoxyresorufin-O-deethylase (EROD) activity and cytochrome P450 1A (CYP1A) expression were measured in 88 English Sole (Pleuronectes vetulus) collected during May and June 1999 from four sites in Vancouver Harbour and at an expected reference site outside the harbour. Hepatic microsomes were prepared from the fish and analyzed for total CYP content, EROD activity, and CYP1A protein levels. Hepatic EROD activity and CYP1A protein levels were elevated in fish from two sites in the inner harbour. A comparison with sediment chemistry data showed that fish with increased EROD activity and CYP1A levels came from sites containing relatively high levels of polycyclic aromatic hydrocarbons and polychlorinated biphenyls. Unexpectedly high levels of EROD activity and CYP1A protein were also found in fish from a reference site near Gibsons, in Howe Sound. The elevated EROD activity and CYP1A expression in fish from this site cannot be explained by the chemical analysis data collected.     

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.     

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 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.