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1.
Elskus AA 《Marine environmental research》2004,58(2-5):463-467
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. 相似文献
2.
H. Jonsson K. Viken Sandnes D. Schiedek R. Schneider B. E. Grsvik A. Goksyr 《Marine environmental research》2004,58(2-5):655
In an attempt to learn more about the cytochrome P450 (CYP) system of mussels, we used protein databases and alignment software to extract highly conserved CYP sequences. From these alignments synthetic peptides were produced and used for rabbit immunisation, which yielded polyclonal antibodies against the CYP families 2 and 4. The antibodies were evaluated with Western Blot and ELISA assays, using digestive gland microsomal samples from the mussel Mytilus edulis. Western Blots revealed immunoreactions for both antibodies. The anti-CYP2 sequence rendered one major immunopositive protein of ≈49 kDa size, and weak signals for proteins of ≈41 and 56 kDa size. The anti-CYP4 sequence rendered two major bands of ≈56 and 59 kDa size, and also a weak immunoreaction with a protein of ≈43 kDa size. ELISA rendered only weak signals even with a 1:50 dilution of IgG-purified serum. A 10-day exposure to Aroclor 1254 did not appear to affect any of the immunopositive proteins, while total PCBs in soft bodies increased from 14–40 ng/g DW in controls to 373–638 ng/g DW in exposed mussels. 相似文献
3.
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 μ(micro)M) with or without 0.2 μ(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. 相似文献
4.
为了了解苯并[a]芘(BaP)对鱼类细胞色素P4501A1(CYP1A1)表达的影响,以褐菖鲉(Sebasticus marmoratus)为实验材料,采用体内实验,研究其在经过不同浓度(0.1、1、10、20、50mg/kg鱼体重量)的BaP诱导后,鱼体肝脏研究CYP1A1基因表达的情况,筛选出后续时间-效应实验中BaP注射的最佳浓度,研究BaP诱导6h、12h、1d、3d、7d后(质量浓度为20mg/kg鱼体重量)鱼体肝脏CYP1A1酶活性、基因表达和蛋白表达的情况。结果表明:剂量-效应实验中,20mg/kg鱼体重量为最佳浓度,此浓度下,基因表达在各组中变化最显著。时间-效应实验中,较空白对照组而言,染毒6h、12h和1d后,EROD酶活性显著增加。3d后开始下降,与对照组相比变化不大,7d后酶活性又发生上调。半定量RT-PCR结果表明,各染毒组与对照组相比,CYP1A1基因表达量都发生了上调,呈现先上升后下降的趋势。其中,6h和12h组相对表达量极显著增加,1d后开始下降且与3d和7d组相比变化不明显。Western blot结果表明,蛋白表达量在染毒12h后表现出显著的诱导效应,随着时间的延长略有回落,但与对照组相比仍有显著性差异。研究表明:BaP对褐菖鲉CYP1A1具有较强的诱导作用。一定质量浓度的BaP注射于褐菖鲉不同的时间后,能诱导褐菖鲉活体EROD酶活性、CYP1A1基因m RNA表达及蛋白表达,并随着时间的延长呈现先诱导后抑制的趋势。这说明BaP作为诱导剂对CYP1A1酶活性和蛋白表达的作用机制可能与调控CYP1A1的转录水平有关。 相似文献
5.
Rapid amplification of cDNA ends(RACE) and real-time polymerase chain reaction(RT-PCR) were carried out to analyze the CYP4 gene expression in polychaete Marphysa sanguinea exposed to benzo[a]pyrene(BaP) in this study. The full length of MsCYP4 cDNA was 2 470 bp, and it encoded 512 amino acids. The deduced amino acid sequence showed 47% identity with CYP4 F from frog Xenopus tropicalis and shared high homology with other known CYP4 sequences. To analyse the role of CYP4 in protecting M. sanguinea from BaP exposure, three BaP groups were established: 0.5, 5 and 50 μg/L. Polychaetes were sampled after 3, 7 and 12 d. At 0.5 μg/L, the effect of BaP on MsCYP4 gene expression increased with time prolonged. MsCYP4 gene expression curve showed Ushaped trend with time in 5 and 50 μg/L BaP groups. Therefore, MsCYP4 gene may play an important role in maintaining the balance of cellular metabolism and protecting M. sanguinea from BaP toxicity. 相似文献
6.
Application of the luciferase cell culture bioassay for the detection of refined petroleum products 总被引：1，自引：0，他引：1
A luciferase cell culture-based bioassay, developed to detect 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like activity of halo-genated and polycyclic aromatic hydrocarbons, was optimized to detect refined petroleum products and to determine their relative inducing potency. Quality control standards from 32 refined products (gasolines and diesels, jet fuels, lubricating oils, fuel oils and weathered products) and three commercial products were evaluated. Induction equivalents (I-EQs) were determined by direct comparison of the EC50 and EC20 values (based on the median and 20% TCDD maximal response, respectively) from dose-response curves for each product to those obtained with TCDD. Most petroleum products were active in the luciferase bioassay, with those products composed of fractions produced later in the distillation process (i.e. fuel oils) inducing higher levels. Additionally, weathering of products reduced their induction potency. Based on the high I-EQ estimates of many products, biological effects associated with exposure may have been previously underestimated using other diagnostic methods. 相似文献
7.
二噁英类化合物(dioxin-like compounds,DLCs)包括多环芳烃(polycyclic aromatic hydrocar-bons,PAHs)中的多氯代二苯对二噁英(polychlori-nated dibenzo-p-dioxins,PCDDs)、多氯代二苯对呋喃(polychlorinated dibenzofurans,PCDFs)及卤代芳香化合物(halogenated aromatic comp 相似文献
8.
黄斑篮子鱼去毒相关基因的克隆与肝脏组成型表达分析 总被引：1，自引：0，他引：1
从基因水平探讨海洋鱼类对海洋藻毒素的去毒分子机理。采用RT-PCR法成功克隆了黄斑篮子鱼Siganus oramin肝脏I时相代谢酶细胞色素P450 1A(CYP1A)、II时相代谢酶alpha型谷胱甘肽S-转移酶(GSTA)和rho型谷胱甘肽S-转移酶(GSTR)、热休克蛋白70 (HSP70)、alpha 1型钠钾ATP酶(ATP1A1)及β-肌动蛋白(beta-actin, ACT)基因cDNA核心序列，序列分别长879 bp、582 bp、588 bp、660 bp、749 bp和554 bp。序列同源性分析发现，属鲈形目的黄斑篮子鱼CYP1A、GSTA和GSTR与同属鲈形目的牙鲆Paralichthys olivaceus、欧洲鲽Pleuronectes platessa、真鲷Pagrus major、鲤形目的斑马鱼Brachydanio rerio 相应氨基酸序列同源性较高，CYP1A和GSTA与非洲爪蟾(两栖类)、鸡(鸟类)、小鼠、大鼠和人(哺乳类)相应氨基酸序列同源性低，这可能与鱼类I、II时相去毒酶基因承担水环境毒素去毒代谢的特殊功能有关；而HSP70、ATP1A和β-肌动蛋白在鱼类、两栖类、鸟类、哺乳类中均有较高的同源性，这可能与这些基因在机体中承担的最基本的生命功能相关。应用半定量RT-PCR的方法，以β-肌动蛋白作为外参照，在指数期增长范围内分别得到了CYP1A、GSTA、GSTR、HSP70和ATP1A1 mRNA与β-肌动蛋白mRNA (%)的比值，确定黄斑篮子鱼肝脏去毒相关基因的组成型表达水平。其中，黄斑篮子鱼肝脏CYP1A、GSTA和GSTR基因组成型表达相对较高，HSP70和ATP1A1基因组成型表达相对较低，这可能与不同基因在黄斑篮子鱼海洋藻毒素去毒分子机理中承担的作用相关，为海洋藻毒素在海洋鱼类中的积聚及代谢去毒分子机制的研究提供了相关数据。 相似文献
9.
利用简并引物扩增及RACE全长克隆技术, 克隆得到绿鳍马面鲀(Navodon septentrionalis)CYP19a基因cDNA全长序列。通过多序列比对, 发现具有芳香化酶特定保守序列, 包括一个I-螺旋区, 一个Ozol肽区, 一个亚铁血红素结合区域以及一个芳香化酶特异性结合区域。通过RT-PCR技术检测了其在绿鳍马面鲀成鱼各组织表达的情况, 发现其CYP19a基因只在卵巢中有表达; 同时也分析了其在不同卵巢发育期的表达情况, 发现CYP19a在卵黄发生后期表达量达到最高值, 卵巢退化吸收期表达量达到最低值。 相似文献
10.
三疣梭子蟹(Portunus trituberculatus)CYP2基因的cDNA克隆及表达分析 总被引：1，自引：0，他引：1
通过RT-PCR及Smart?TM Race技术,首次克隆了三疣梭子蟹(Portunus trituberculatus)CYP2基因cDNA全长序列。该基因cDNA全长1662bp,编码一个由492个氨基酸组成的多肽,预测理论等电点为6.348,分子量大小为56.68kD。氨基酸序列中含有CYP基因家族所特有的K螺旋保守序列(ExxR)和血红素结合区(FxxGxxxCxG)。经氨基酸序列比对及系统进化树分析发现,与岸蟹(Carcinus maenas)的同源性最高,达到75%。实时荧光定量PCR结果表明,CYP2基因在肝胰腺、鳃、肌肉、血淋巴、心脏和眼柄中均有分布,在肝胰腺中表达量最高。肌肉注射磺胺嘧啶后,三疣梭子蟹高、中、低三剂量组CYP2基因表达较对照组都有上调,并具有时间差异性,低剂量组表达量逐渐降低,趋于对照组,中剂量组和高剂量组表达量先升高后降低,6h后同一时间点,均是高剂量组表达最高,低剂量组最低。表明磺胺嘧啶可诱导三疣梭子蟹CYP2基因,CYP2基因可能参与三疣梭子蟹的药物代谢反应。 相似文献