柴油分散液对马粪海胆细胞色素P450 活性的影响

试验研究了不同质量浓度下(5、20、50 mg/L)三组柴油分散液,对马粪海胆的肠体、性腺、体液三个部位的抗氧化还原系统细胞色素P450活性变化的影响。结果表明,随着污染暴露时间的增加,三个部位的细胞色素P450活性均呈现出先被诱导、后被抑制的规律,并且油浓度越高,出现诱导和抑制效应的时间越早,活性变化的幅度也越大。海胆P450活性变化在一定程度上能够反映油污染的强度及其对海洋生物的毒性效应,可作为海洋石油烃污染监测的毒理学指标。     

Partial purification and properties of cytochrome P450 from digestive gland microsomes of the common mussel, Mytilus edulis L.

Cytochrome P450 from the digestive gland of M. edulis was partially purified by sodium cholate solubilization, 4–15% polyethylene glycol fractionation, and octyl-Sepharose affinity, DEAE-Sephacel ion-exchange and hydroxylapatite chromatography (yields of up to 7–10%). Three peaks were resolved by DEAE-Sephacel chromatography (termed peaks 1–3). P450 specific content was increased from 26 to 800 pmol per mg protein, and the ratio of P450 content to NADPH-cytochrome c (P450) reductase activity reduced by a factor of 250. Oxidised spectrum λmax of P450 was 410.5 ± 1.5 nm. Type II difference spectra were seen with both type II (clotrimazole, metyrapone) and type I (α-naphthoflavone, 7-ethoxycoumarin) compounds. Western blotting with polyclonal anti-P4501A from perch (Perca fluviatilis) gave a single band of approximately 54 kDa molecular weight. A reconstituted system containing peak 2 or 3, rat liver P450 reductase, lipid and NADPH metabolised benzo[a]pyrene to diones, diols, phenols and putative protein adducts. Peak 2 plus cumene hydroperoxide was indicated to produce diones and protein adducts only. Peak 2 alone was indicated to produce diones and phenols. The major free metabolites in all cases were diones (53–100%). The results indicate the existence of a P4501A-like enzyme in M. edulis, possibly with unusual properties as indicated by the difference spectra, metabolism in absence of NADPH and added P450 reductase, and predominance of diones.     

Microsomal cytochrome P-450 function in fish evaluated with polyclonal and monoclonal antibodies to cytochrome P-450E from scup (Stenotomus chrysops)

Monooxygenase reactions catalyzed by cytochromes P-450 are paramount in the oxidative metabolism of many xenobiotics, determining both the persistence and effects of numerous types of compounds. Immunological probes are proving useful in evaluating the functions of P-450 isozymes in microsomal preparations from many species. The regulation of specific isozymes by endogenous and exogenous factors can also be evaluated with such probes. Here we describe studies on the activities apparently catalyzed by induced P-450 in fish, evaluated with both polyclonal and monoclonal antibodies to cytochrome P-450E, the apparent major β-naphthoflavone(BNF) or methylcholanthrene(MC)-inducible isozyme purified from scup (S. chrysops) liver.     

Catalytic properties of cytochrome P-450 in hepatopancreas of the spiny lobster, Panulirus argus

These studies were designed to determine the metabolic capability of the microsomal cytochrome(s) P-450 in spiny lobster hepatopancreas, and to determine how chemicals which selectively modify mammalian monooxygenase activity catalyzed by different cytochrome P-450 isozymes affect the spiny lobster cytochrome P-450. We used a washing procedure to concentrate the hepatopancreas microsomal cytochrome P-450 and remove the inhibitors of monooxygenase activity which are normally present in microsomes. The resulting reparation (MI fraction) was used to determine monooxygenase activity towards benzo[a]pyrene, benz-phetamine, 7-ethoxyresorufin in the presence of either cumene hydroperoxide or NADPH and vertebrate liver cytochrome P-450 reductase. Benzphetamine was the best substrate for the lobster cytochrome P-450, whereas 7-ethoxyresorufin was metabolized very slowly. Studies with chemical modifiers showed that the responses of the lobster cytochrome(s) P-450 were not similar to those of any of the well-characterized cytochrome P-450 isozymes purified from mammalian liver.     

Indications of P450 monooxygenase activities in beluga (Delphinapterus leucas) and narwhal (Monodon monoceros) from patterns of PCB, PCDD and PCDF accumulation

Selective accumulation of PCDDs, PCDFs and PCB congeners was investigated in beluga (Delphinapterus leucas), narwhal (Monodon monoceros) and several species at the same trophic level in two areas of Canada. There was a much higher relative abundance of meta-para-unsubstituted PCBs in the cetaceans compared with other species. This suggests that activity of cytochrome P450 monooxygenase enzymes causing CYP2B-type metabolism was relatively low in beluga and narwhal. Lower relative levels of 2,3,7,8-TCDD occurred in beluga from both areas, and selective reduction of toxic non-ortho PCBs occurred in the highly PCB-contaminated St Lawrence beluga. These compounds are potent inducers of cytochrome P450 CYP 1A-type enzymes, suggesting that such an enzyme with the capability of metabolizing TCDD-like substrates is present in beluga and narwhal. SIMCA principal components analysis of PCB congener patterns showed that the two cetaceans were in a statistically significant different class than the other species. Variables (congeners) with the greatest separation power between classes were mainly those defined by the selective metabolism rules.     

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.     

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.     

Cytochrome P450 differences in normal and imposex-affected female whelk Buccinum undatum from the open North Sea

Normal and imposex-affected female Buccinum undatum were sampled from the open North Sea at three locations, one with low, and two with high shipping densities. Cytochrome P450 components and P450 aromatase activity were determined in the microsomal fractions isolated from pooled digestive gland/gonads. Cytochrome P450 aromatase activity was significantly higher (P < 0.05) in normal females collected in the low shipping density area (1,325 +/- 295 fmol/h/mg protein) than levels from imposex animals from a high shipping density area (620 +/- 287 fmol/h/mg protein). A negative correlation was found between aromatase activity and organotin body burden (r = -0.99). Levels of CYP450, cytochrome b5 and NADPH cytochrome c reductase activity did not show differences among groups. This is the first field evidence of depressed aromatase activity in imposex affected females, although additional research under laboratory controlled conditions is required to fully understand the mechanisms underlying the development of imposex in this species.     

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.     

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.     

三斑石斑鱼(Epinephelus trimaculatus)两种芳香化酶cDNA的克隆及其表达的组织特异性

通过设计引物,从三斑石斑鱼的卵巢中克隆鉴定出性腺芳香化酶(EtP450arom a)和脑芳香化酶(EtP450arom b)的cDNA序列,EtP450arom a开放阅读框为1557bp,编码519个氨基酸残基;EtP450arom b开放阅读框为1530bp,共编码510个氨基酸残基;性腺芳香化酶(EtP450arom a)和脑芳香化酶(EtP450arom b)的氨基酸序列的同源性为63.3%,并且包含了芳香化酶经典的功能保守结构。构建了芳香化酶的系统进化树。采用半定量RT-PCR方法研究了性腺芳香化酶(EtP450arom a)和脑芳香化酶(EtP450arom b)在雌性成鱼各组织中的mRNA的表达情况。组织表达结果显示,性腺芳香化酶EtP450arom a只在性腺中特异性表达,而脑芳香化酶EtP450arom b主要在脑区和垂体中表达,表明三斑石斑鱼两种芳香化酶基因的组织特异性及功能的差异。     

Metabolic activation of polycyclic aromatic hydrocarbons by fish liver cytochrome P-450

The incidence of hepatoma, epidermal and other forms of cancerous growths in fish is well documented (Halver, 1967; Matsushima & Sugimura, 1976; Dawe et al., 1964). In fish, as in mammals, cancer may be a result of metabolically activated carcinogens. In mammals the primary enzyme system involved in the activation of environmental carcinogens is the cytochrome P-450 linked mixed-function oxidase (MFO). The hepatic microsomes of the species offish studied contain variable levels of cytochrome P-450. Liver microsomes of the trout Salmo trutta lacustris are surprisingly active in metabolising benzo-[a]pyrene (BP) and other compounds preferentially metabolised by polycyclic aromatic hydrocarbon (PAH)-specific cytochrome P-450. This finding may be significant, because it is apparent that the detrimental effects of PAHs require metabolic activation.We have studied the production of reactive intermediates of BP by following their binding to DNA and by measuring the specific nucleotide-BP metabolite complexes by chromatography. Untreated S. trutta liver microsomes catalyse the production of reactive intermediates of BP which bind to nucleotides of DNA at a rate that is 3–4 times higher than that catalysed by control rat liver microsomes. The most prominent DNA-BP metabolite adducts produced by trout liver microsomes are the nucleoside adduct of BP-7, 8-diol-9,10-epoxide and 9-OH-BP-4,5-oxide and other phenol oxides. In contrast to the trout, another fish species, roach (Rutilus rutilus), has extremely low activity. Although the in vitro binding of BP to DNA is not strictly correlated to in vivo binding or biological effects, our results show that reactive intermediates are formed by trout liver and thus the prerequisite for chemical carcinogenesis or mutagenesis is ful filled. This is further supported by the fact that in Ames's test, trout liver preparations produce mutagenic products from promutagens.     

Immunohistochemical localization of cytochrome P-450E in liver, gill and heart of scup (Stenotomus chrysops) and rainbow trout (Salmo gairdneri)

Monoclonal antibody directed against a major β-naphthoflavone (BNF)-induced form of teleost cytochrome P-450, P-450E (equivalent to P-450c in rat) was used to immunolocalize this enzyme in liver, gill and heart of scup and trout. Liver sections from both species showed P-450E in the cytoplasm of hepatocytes. No regional differences were observed which might indicate zonation of cytochrome P-450E within subpopulations of hepatocytes. Scup exocrine pancreatic cells were only weakly positive. In the gill of both fish, cytochrome P-450E was restricted to the endothelium (pillar cells) of secondary lamellae, where fluorescence appeared as a chain in longitudinal sections through lamellae and as star-shaped clusters in en face views. Sections of ventricular wall in both species revealed P-450E was restricted to endothelium at margins of muscle bands limiting heart ventricular lumen. Localization in the specific cells of these and other organs may be fundamentally important in understanding the role of cytochrome P-450E.     

Isolation of CYP2L2 and two other cytochrome P450 sequences from a spiny lobster, Panulirus argus, hepatopancreas cDNA library

Cytochrome P450 monooxygenases constitute an enzyme superfamily, with at least 74 known families. Members of CYP families 1–4 are important in the phase 1 metabolism of lipophilic xenobiotics, such as those found in contaminated marine environments. Previous studies (James et al. Arch. Biochem. Biophys. (1996) 329, 31–38) showed that a major form of P450 in spiny lobster, Panulirus argus, hepatopancreas was CYP2L1, a new sub-family, and that there was evidence for other P450 forms in hepatopancreas. We now report the sequence of a second member of this subfamily, named CYP2L2, present in P. Argus hepatopancreas. The deduced amino acid sequences of CYP2L1 and CYP2L2 share 54.7% sequence identity and an additional 13.6% of the sequences show conservative substitutions. Analysis of the sequences of CYP2L1, CYP2L2 and other representative CYP2 family members (from rat and mouse sub-families 2A, 2B, 2D and 2E) showed that the crustacean sequences clustered together. In addition to CYP2L2, cDNA clones of 66 to 117 base pairs from the 5′ coding region of two more P450 isoforms were isolated from the spiny lobster cDNA library. The deduced amino acid sequence of one of these additional cDNA clones was identical to the first 22 amino acids of the N-terminal sequence of a P450 protein previously isolated from hepatopancreas microsomes. These studies confirm earlier biochemical evidence that the hepatopancreas contains multiple forms of cytochrome P450.     

大黄鱼(Pseudosciaena crocea)细胞色素P450 CYP4F7基因的克隆及表达分析

CYP4F7是细胞色素P450超基因家族中CYP4F亚家族中的同工酶之一,而细胞色素P450在通过电子传递参与生物体代谢和转化内源和外源化合物方面起着重要的作用。以本实验室构建的大黄鱼消减杂交cDNA文库中623bp的CYP4F7片段设计引物,以大黄鱼的总RNA为模板,利用RACE-PCR的方法,克隆鉴定出了大黄鱼细胞色素P450 CYP4F7基因。结果表明:基因全长1934bp,5'端非编码区59bp,3'端非编码区264bp,开放阅读框1611bp,编码536个氨基酸。序列分析表明大黄鱼的CYP4F7氨基酸序列与舌齿鲈的相似性为91%。利用RT-PCR方法研究CYP4F7在大黄鱼各组织中的表达特征,结果表明CYP4F7在肝脏、脾脏中表达量最高,在心脏、肾脏、头肾、鳃丝中表达量次之。另外,CYP4F7在注射了细菌脂多糖的大黄鱼各组织中的表达量均低于正常的大黄鱼,认为细菌脂多糖可能是CYP4F7表达的抑制剂,说明CYP4F7可能与鱼类免疫反应有一定相关性。     

In vitro mechanistic differences in benzo[a]pyrene–DNA adduct formation using fish liver and mussel digestive gland microsomal activating systems

Turbot (Scophthalmus maximus) and mussel (Mytilus edulis) microsomes were incubated with DNA to examine if microsomal in vitro metabolism of BaP could result in DNA adducts detected by ³²P-postlabelling. Turbot DNA was incubated with benzo[a]pyrene (BaP), NADPH and microsomal activating systems prepared from either livers of unexposed turbot, turbot exposed to BaP or β-naphthoflavone (ß-NF) or digestive glands from mussels. The β-NF activating system generated the highest levels of DNA adducts detected in this study (451.7 adducts per 10⁸ nucleotides) and were distributed in three discrete adduct TLC spots, one of which (97% of the total adducts) co-migrated with the ³²P-postlabelled BaP 7,8-diol, 9,10-epoxide-N²-guanine adduct. Fewer adducts (P <0.05) were generated by BaP-induced microsomes (9.4–30.6 adducts per 10⁸ nucleotides) but levels were higher (P <0.05) than those generated from untreated fish (3.5 adducts per 10⁸ nucleotides). Co-incubation with 500 μM α-naphthoflavone (α-NF) resulted in 97–99% inhibition in adduct formation implicating cytochrome P450-dependent (CYP) bioactivation however there was some evidence for carry over of BaP in the liver microsomal preparations from BaP injected fish. In contrast to the fish activating systems, no DNA adducts were observed when mussel microsomes were incubated with BaP, DNA and NADPH.     

Effects of petroleum hydrocarbons on the hepatic cytochrome P450 1A1 system in rainbow trout

Effects on the hepatic cytochrome P450 1A1 system were investigated in juvenile rainbow trout i.p. injected with three different aromatic containing fractions: kerosene, light gas oil or heavy gas oil, originated from distilled North Sea crude oil. Kerosene treatment resulted in no effect on the P450 1A1 system, light gas oil injection caused a weak induction of EROD activities and heavy gas oil treatment resulted in a prominent induction of EROD activities as well as accumulation of CYP1A1 mRNA and P450 1A1 protein levels. The effects of heavy gas oil were compared with effects of β-napthoflavone (β-NF) on the P450 1A1 system. It was obvious that important discrepancies seemed to exist between EROD activities and corresponding CYP1A1 mRNA and P450 1A1 levels in rainbow trout treated with either heavy gas oil or β-NF i.e. heavy gas oil treatment resulted in higher specific EROD activities (EROD/P450 1A1) compared to β-NF. GC-MS analyses revealed that liver and bile from heavy gas oil treated rainbow trout in addition to naphthalene also contained polycyclic aromatic hydrocarbons such as phenanthrenes, anthracene, pyrenes, fluoranthene benz(a)anthracene and chrysene, while none of these compounds were detected in control trout.     

Nomenclature for hydrocarbon-inducible cytochrome P450 in fish

A growing number of studies concerning organic chemical pollutant induction of cytochrome P450 mixed function oxidases in fish stimulate a need for common terminology. Similar names for proteins might be based on similarities in function, in regulation, and in their structure. Under the current guidelines for P450 proteins, classification is based on measured or inferred amino acid sequence. At present, a single teleost hydrocarbon-inducible P450 (rainbow trout) can conclusively be termed 1A1: a second (scup P450E) can be so termed on the basis of partial sequence. The lack of primary sequence information makes it difficult to assign a specific identity to hydrocarbon inducible P450s in other fish. Nevertheless, the sum of information on catalytic activities, introduction and immunological cross-reactivities provides a weight of evidence sufficient to assume (but not prove) gene family (I) and subfamily (A) identities for hydrocarbon-inducible proteins in many species. Reference to these proteins as P4501 or IA (or CYPI or IA) in fish species where sequence data are lacking is suggested as more appropriate than reference to them as P450IAI (or CYPIAI). Additional primary sequence data are required to further define orthologous relationships among P450IA genes in fish, and to determine whether IAI is a correct designation for a hydrocarbon-inducible P450 in many species.     

7-ethylresorufin O-deethylase activity and level of DNA-adducts in trout treated with benzo(a)pyrene

In fish, as well as in mammals, it is well known that the cytochrome P450-dependent oxidative metabolism of xenobiotics can generate DNA-reactive species. Moreover, this metabolism is known to be inducible by several compounds of environmental significance, such as polychlorobiphenyls, polycyclic aromatic hydrocarbons (PAHs) and dioxins. Consequently, we studied the relationship between the degree of induction of the cytochrome P4501A, expressed as that of 7-ethylresorufin O-deethylase (EROD) activity, and the level of DNA-adducts, using the post-labelling assay, in the liver of rainbow trout exposed to benzo(a)pyrene (a representative PAH). The results showed a significant 2- to 4-fold increase in EROD activity 2, 4 and 8 days after treatment, paralleled by an increase in DNA-adduct levels. This work further emphasizes the involvement of cytochrome P4501A in the metabolism of benzo(a)pyrene into genotoxic metabolites in rainbow trout.