Biotransformation enzyme activities in the olfactory organ of rainbow trout (Oncorhynchus mykiss). Immunocytochemical localization of cytochrome P4501A1 and its induction by β-naphthoflavone

Olfaction is a crucial function in most fish species, but little is known about biotransformation enzymes in the olfactory organ. This study demonstrates that biotransformation enzymes usually found in the rainbow trout liver, are present in the olfactory organ as well. While microsomal cytochrome P450 reductase, p-nitrophenol hydroxylase and cytosolic glutathioneS-transferase presented similar levels in both the olfactory organ and the liver, microsomal 7-ethoxyresorufinO-deethylase (EROD), 7-ethoxycoumarinO-deethylase, and 7-pentoxyresorufinO-deethylase were much lower in the olfactory organ (77-, 35-, 200-times respectively). Furthermore, microsomes from the olfactory organ were able to perform testosterone hydroxylation only in the 16α-position while testosterone was hydroxylated in the 16β-position by liver microsomes. Using polyclonal antibodies raised against perch cytochrome P4501A1, the immunoreactive protein was shown to be strongly expressed in various cellular types forming the nonsensory epithelium. Some immunostaining was also reported in the nonsensory cellular elements constituting the sensory epithelium, while olfactory receptor cells failed to show cytochrome P4501A1-immunoreactivity. Finally, the exposure of rainbow trout to waterborne β-naphthoflavone (0.1 μg ml⁻¹) for 2 or 4 days resulted in a higher induction of EROD activity in the olfactory organ compared to the liver. The presence of biotransformation enzymes in the olfactory organ of rainbow trout addresses the question of their involvement in the detoxication/toxication of pollutants as well as in the olfactory function.     

嘉庚蛸精子发生的超微结构

在研究GCL细胞中EROD催化底物反应时间的基础上分别以三种数学模型拟合，分析比较了诱导剂TCDD、PCB、PB、BaP、3-MC、BNF对GCL细胞EROD酶活的剂量．效应曲线，结果表明Log-Normal模型能较好的拟合药酶诱导前升后降的钟形效应曲线，所得方程参数值p100和EC100可用于药物代谢酶的诱导研究；Gentox模型拟合度相对较低且其EC50值显著高于半效应浓度；Logistic模型只能拟合曲线诱导上升部分而且嬲50和P100与Log-Normal模型相比显著偏高。进一步研究结果显示诱导剂TCDD对GCL细胞作用时间越长，EC100越大，P100越小，但在作用48h后即达到较大诱导效应，延长时间并无显著加强作用。     

Catalytic activity and immunochemical quantification of hepatic cytochrome P-450 in β-naphthoflavone and isosafrol treated rainbow trout (Oncorhynchus mykiss)

In addition to catalytical assays, immunochemical techniques have recently been employed to measure induction of the cytochrome P-450 (P450) monooxygenase system in fish with polyaromatic hydrocarbons (PAH). In the present study, polyclonal antibodies were raised against rainbow trout P450IA1. Levels of rainbow trout P450IA1 determined using protein blotting- and ELISA procedures were compared with levels of 7-ethoxyresorufin-O-deethylase (7-EROD) activity in liver microsomes from rainbow trout. These comparisons showed that values of P450A1 were positively correlated (r=0.99 and r=0.97) with 7-EROD activities. In addition, the effects of isosafrol (ISF) or -naphthoflavone (NF) treatments on P450 levels in rainbow trout liver were investigated using immunochemical and catalytical methods. ISF treatment induced 7-EROD activity as well as 7-methoxycoumarin-O-demethylase-, 7-ethoxycoumarin-O-deethylase-, 7-propoxy-coumarin-O-depropylase and 7-butoxycoumarin-O-debutylase activities, although to a lesser extent, compared with the NF treatment. In contrast, immunochemical quantification of rainbow trout P450IA1 protein revealed a slightly different pattern. ISF appeared to be a weak inducer of P450IA1 in rainbow trout compared with NF. In addition, the degree of inhibition of 7-alkoxycoumarin-O-dealkylase activities in ISF microsomes differed from that measured in control- and NF microsomes. The discrepancies between catalytic and immunochemical estimates of rainbow trout P450IA1 in ISF treated fish in addition to differencs between specific inhibitory pattern by specific polyclonal antibodies raised against rainbow trout P450IA1, indicate that important differences exists between the responses induced by NF- and ISF treatments in the rainbow trout liver.Part of this work was presented at the 6th International Conference on Biochemistry and Biophysics of Cytochrome P-450, Vienna, Austria, July 3–8, 1988.     

The cytochrome P450 system of atlantic salmon (Salmo salar): I. Basal properties and induction of P450 1A1 in liver of immature and mature fish

The major components of the cytochrome P450 (P450) system in liver microsomes of Atlantic salmon were studied using spectrophotometric, catalytic and immunochemical techniques. In juvenile fish sampled during the winter season, high basal activities of 7-ethoxyresorufin O-deethylase (EROD) were found. The K_m for 7-ethoxyresorufin was 0.4 µM, and V_max 1.23 nmol/min/mg protein in juvenile fish. In mature fish sampled from the same group of fish in December, EROD activity was barely detectable (20–30 pmol/min/mg protein). Treatment with the P450 1A1 inducer -naphthoflavone (BNF) resulted in almost 2-fold induction of total P450, and 30–40-fold induction of EROD activity in immature fish. A similar fold increase was seen in mature fish. The differences in EROD activity between untreated and BNF-treated fish, was accompanied by similar differences in a P450 1A1 cross-reacting protein (M_r=58,000 D) in immunochemical studies using rabbit anti-cod P450 1A1 IgG. However, judging from these studies, the levels of P450 1A1-protein in mature salmon far exceeded those accounted for by the measured EROD activity in comparison to immature fish (both before and after BNF-treatment), indicating inhibiting effects of sex steroids on the measured activity. This effect was not seen on 7-ethoxycoumarin O-deethylase activity. A long-term storage experiment indicated that Atlantic salmon liver microsomes can be stored for 2 years at –80°C in 20% glycerol without losing more than 20–40% of its catalytic activity.Parts of this work were presented at the 5th International Symposium on Responses of Marine Organisms to Pollutants, April 1989 in Plymouth, United Kingdom (Larsen and Goksøyr 1989).     

Immunochemical relationships of cytochrome P4503A-like proteins in teleost fish

Multiple P450 proteins have been purified from several teleost species, including rainbow trout (Oncorhynchus mykiss), scup (Stenotomus chrysops) and Atlantic cod (Gadus morhua). Identity, relationships and/or functions have been established in these fish species for the cytochrome P4501 As. Information about the structure, function, regulation and relationships of other piscine cytochrome P450 (CYP) proteins is sparse. In the present study we have focused on constitutively expressed CYP forms, P450con and LMC5 isolated from rainbow trout, P450A from scup, and P450b from Atlantic cod, and we consider evidence for the relationship of these proteins to mammalian members of the CYP3A subfamily. Reciprocal western blot analysis shows that P450con and LMC5, isolated from rainbow trout in two different laboratories, are closely related and ostensibly identical proteins. These trout proteins show specific reciprocal cross-reactivity with scup P450A, and polyclonal antibodies (PAb) to the trout and scup proteins both recognize cod P450b, indicating that rainbow trout P450con/LMC5, scup P450A and cod P450b are immunochemically-related proteins. In analyses of liver microsomes of trout, scup and cod, PAb to trout P450con/LMC5 and scup P450A recognize only bands that are identical in migration to the CYP proteins purified from these species, and which were used as immunogens. These CYP proteins purified from fish are each immunochemically-related to mammalian CYP3A proteins, showing recognition by PAb to human CYP3A4 and to rat CYP3A1. PAb to the mammalian CYP3As also recognize the same bands in liver microsomes from these fish species as seen by PAb to the fish proteins. These results strongly suggest that these fish proteins are members of theCYP3 gene family and probably theCYP3A subfamily. Although sequence analysis is required before their designation in the CYP3A subfamily can be confirmed and specified, we refer to these as CYP3A-like. Immunoblot analyses of hepatic microsomes from other fish species with PAb to scup P450A and trout P450con show that multiple CYP3A-like proteins are expressed in liver of several species, including killifish (Fundulus heteroclitus) and winter flounder (Pleuronectes americanus). Important questions still remain to be addressed concerning CYP3A structure, multiplicity, physiological function, regulation and metabolism of endogenous as well as exogenous substrates in fish.Part of this study was presented at the 10th International Symposium on Microsomes and Drug Oxidations. Toronto, Canada, July 18–21, 1994.     

CYP1A1 immunolocalization in the olfactory organ of rainbow trout and its possible induction by β-naphthoflavone: analysis in adults and embryos around hatching

Cytochrome P4501A1 (CYP1A1) isoform, which is known as being of major toxicological significance, has been well-studied in the mammalian olfactory mucosa. Only few studies have dealt with this biotransformation system in the fish olfactory organ which is particularly vulnerable to waterborne xenobiotics since sensory neurons are in direct contact with the aquatic environment. The present immunocytochemical study describes the cellular and subcellular distributions of CYP1A1 in the olfactory organ of rainbow trout in both adults and embryos around hatching. The enzyme inducibility in response to a 4-day exposure to waterborne -naphthoflavone (0.1 mg l^–1), a model inducer of CYP1A1, was also examined. In untreated adult fish, CYP1A1 was almost exclusively expressed in the nonsensory epithelium which covers the edges and the tip of the lamellae. Both goblet and ciliated nonsensory cells appeared immunoreactive. In -naphthoflavone-treated fish, in addition to a strong labeling in the nonsensory epithelium, ciliated nonsensory cells in the olfactory epithelium appeared well-labeled. Four days before hatching, only a few cells were weakly stained in the placodal epithelium of some embryos. By 7 days post-hatching, the enzyme expression was increased in the olfactory pit and it was restricted to ciliated nonsensory cells. No evident CYP1A1 induction was detected in either embryos or alevins. Results suggest the presence of a two-line CYP1A1 biotransformation system in the adult fish olfactory organ: a basal level of enzyme expression insured by the nonsensory epithelium and an additional line in which the sensory epithelium is activated in response to CYP1A1 inducers. This system might take place during development in parallel with the onset of the nonsensory epithelium.     

三倍体雌性虹鳟性腺发育阶段细胞色素相关基因的表达

Cyp19a1b、Cyp11a1、Cyp11b2、Cyp17a1、Hsd3b1等细胞色素相关基因能够调节硬骨鱼类性类固醇的合成,对性腺发育和性别决定产生影响。本研究以全雌三倍体虹鳟(Oncorhynchus mykiss)为研究对象,正常雌性二倍体虹鳟为对照,选取31~68 dpf(days post fertilization)时间段的虹鳟仔鱼脑组织,采用q RT-PCR和酶联免疫的方法研究以上几种基因的表达状况和脑芳香化酶的活性变化,以期探明导致三倍体雌性虹鳟性腺发育异常的关键原因。q RTPCR结果显示,二倍体中Cyp19a1b在30~50 dpf时表达量上调并且维持在较稳定水平,但50~56 dpf时表达量逐渐下调,之后56~68 dpf表达量持续上调;三倍体中Cyp19a1b表达量在30~35 dpf开始上调,35~47 dpf逐渐下调,47~55 dpf开始第二次上调,之后维持在较稳定水平直至68 dpf,但三倍体Cyp19a1b的表达量显著(P0.05)低于同期二倍体的。二倍体Cyp11a1表达量在34 dpf出现峰值,三倍体Cyp11a1在38 dpf时出现峰值。二倍体Hsd3b1表达量在33~42 dpf时维持在较高水平,在38 dpf时出现高峰;三倍体Hsd3b1表达量在47~59 dpf时较高,在49 dpf出现高峰。二倍体中Cyp11b2在37 dpf出现峰值,之后开始下调;三倍体在40 dpf出现峰值,之后逐渐下调,但三倍体Cyp11b2表达量显著低于同期二倍体。二倍体Cyp17a1的表达量在35~46 dpf时逐渐上升,在45 dpf时达到高峰之后直至69 dpf逐渐下降,并且维持在较为平稳的水平上;但是在相同的实验条件下未检测到同一时期三倍体Cyp17a1的表达量。酶联免疫结果显示,在40 dpf时二者的脑芳香化酶活性到达高峰,但在40~60 dpf时期,二倍体虹鳟脑芳香化酶活性显著(P0.05)高于三倍体虹鳟,尤其在45~50 dpf时,该酶活性分别较三倍体的高1.15倍和1.12倍。以上结果表明三倍体虹鳟早期性腺发育迟缓的原因之一是Cyp19a1b、Cyp11a1、Cyp11b2、Cyp17a1、Hsd3b1等基因的表达晚于二倍体,且表达量低于二倍体,造成雌二醇不能正常合成,最终导致性腺发育迟缓。     

NADH-dependent cytochrome b5 reductase and NADPH methemoglobin reductase activity in the erythrocytes of Oncorhynchus mykiss

Methemoglobin is oxidized hemoglobin that cannot bind to or dissociate from oxygen. In fish, it is most commonly caused by exposure to excess nitrites and can lead to abnormal swimming, buoyancy, or death. The methemoglobin concentration in mammals is determined by the balance of oxidizing agents versus reducing enzymes in erythrocytes. The objective of our studies was to characterize the enzymes that reduce methemoglobin in fish erythrocytes. Whole blood was collected from healthy rainbow trout. Methemoglobin was induced in vitro by NaNO₂ exposure. Methemoglobin reduction in controls was compared to reduction in samples with added NADH, NADPH, or NADPH and methylene blue. Rainbow trout whole blood was also fractionated into cytosol, microsomal, and mitochondria/plasma membranes/nuclei fractions. The fractions were compared for NADH-dependent cytochrome b5 reductase (CB5R) activity and for nitrite induction of methemoglobin. The CB5R activity in rainbow trout erythrocytes was compared to the CB5R activity in equine, feline, and canine erythrocytes. Rainbow trout erythrocytes had significant NADPH methemoglobin reductase activity in the presence of methylene blue (P < 0.001). The CB5R activity was greatest (P < 0.001) in the plasma membrane/mitochondria/nuclei fraction. The CB5R activity in rainbow trout erythrocytes was not significantly different than canine or equine activity but was significantly lower than feline CB5R activity (P < 0.0001). Methemoglobin in rainbow trout erythrocytes can be reduced by CB5R or NADPH-dependent methemoglobin reductase. Unlike mammalian anuclear erythrocytes, which are dependent on soluble CB5R, the nucleated RBCs of rainbow trout use membrane-bound CB5R to reduce methemoglobin.     

Immunochemical cross-reactivity ofβ-naphthoflavone-inducible cytochrome P450 (P450IA) in liver microsomes from different fish species and rat

Antibodies prepared against the major -naphthoflavone (BNF)-inducible cytochrome P450 (P450) forms from three species of fish (rainbow trout, Atlantic cod, and scup) well separated in teleost phylogeny, were used to investigate the immunochemical relatedness of liver microsomal P450 in different species of BNF-treated fish and rat. Rabbit polyclonal IgG against all three P450s and mouse monoclonal antibodies prepared against scup P450E were employed in this study. Liver microsomes were prepared from BNF-treated specimens of hagfish, herring, rainbow trout, cod, scup, perch, plaice and rat. With Western blotting it was shown that the various antibodies cross-reacted with a protein band in liver microsomes in the P450-region of each of the BNF-treated fish species. The apparent molecular weight of the cross-reacting proteins showed differences within the range 54,000–59,000 daltons. The effects of the different antibodies on the microsomal BNF-inducible 7-ethoxyresorufin O-deethylase (EROD) activity gave inhibition patterns that reflected to a certain extent the phylogenetic relationship of the species investigated. In rat microsomes a protein band of relative molecular mass similar to rat P450c (Mr=54,000) was recognized by all antibodies. In addition, a second band of lower molecular mass was strongly recognized by anti-cod P450c antibodies, and faintly stained with anti-rainbow trout P450LM4b IgG and anti-scup P450E MAb 1-12-3. This band could correspond to rat P450d, the isosafrole-inducible rat isoenzyme. Considering the long separate evolutionary history of some of these fishes (50–200 million years), the results demonstrate that certain antigenic epitopes in the BNF-inducible P450 isoenzymes have been strongly conserved during the evolution of fish species. These conserved epitopes seem however not to be directly involved in the measured EROD activities. Furthermore, the results suggest that the BNF-inducible P450s in fish contain regions with structural similarity to the homologous counterpart that has evolved through gene duplication into a P450 family in mammals containing at least two gene products (the P450IA gene family).     

基于ITS1基因分析亚东鲑(♂)、虹鳟(♀)及其F_1代遗传关系

虹鳟、棕鳟均为优质水产养殖品种,亚东鲑为棕鳟的同物异名,是19世纪西藏由欧洲引进。为探索杂种优势,对亚东鲑和虹鳟进行杂交试验,对其子一代幼鱼与虹鳟、棕鳟进行了体长和体质量测定。采用ITS1基因分析其遗传关系,并与巴基斯坦引进的棕鳟对比研究。结果发现F_1代体长、体质量增长快于虹鳟、棕鳟,其遗传多样性高于亚东鲑和虹鳟,略低于棕鳟,表明F1代具有一定杂种优势。此外,棕鳟较高的遗传多样性也反映出从巴基斯坦引进的发眼卵遗传背景较好,具有进一步养殖选育的潜力。