鱼肝EROD酶活力诱导作为二的水生态毒理学指标

利用离体ＥＲＯＤ法对严家湖各氧化塘鱼肝中二恶英的毒性效应进行了定量测定,同时与ＨＲＧＣ／ＨＲＭＳ－ＭＩＤ化学分析结果和野外活体暴露鱼肝中ＥＲＯＤ活力诱导结果进行了比较,研究发现,各氧化塘中鱼肝的离体、活体ＥＲＯＤ测定结果与化学分析结果之间有着极好的相关性。这不仅提示了鱼肝细胞色素Ｐ４５０系统以ＥＲＯＤ酶活力诱导指示可作为二恶英的水生态毒理学指标的可靠性和性。同时也表明了活体ＥＲＯＤ和离体ＥＲＯＤ生     

利用离体大白鼠肝癌细胞的EROD诱导指示二恶英的复合毒性效应

离体条件下,以大白鼠肝癌细胞株H4IIE的7－乙氧基－3－异吩恶唑酮－脱乙基酶（EROD）活力诱导作为毒性指标,测定了2,3,7,8－TCDD单独存在以及与一定浓度的2,3,7,8,－TCDF,OCDD,PCB126和PCB77分别共存下的EROD活力,并用TEF和独立作用模型（independence)两种方法对实验结果进行了评估。利用TEF评估的结果表明实验的TEQ值和理论计算的TEQ值十分接近,复合毒性表现为加合作用（additivity),这一结果与用独立作用模型评估的结果完全一致。研究结果不仅证实了TEF评估方法的有效性和利用模型方法评估二恶英类化合物复合毒性的可行性,同时还表明离体条件下,大白鼠肝癌细胞株H4IIE的EROD酶活力诱导适用于化合物的复合毒性的研究。     

褐菖鲉肝CYP 1A作为生物标志物监测厦门海域石油污染状况

以褐菖鲉为实验鱼类,以鱼肝微粒体CYP1A 生物标志物(EROD活性和CYP1A蛋白表达量)为指标,在厦门海域开展了两次野外监测实验,研究EROD活性和CYP1A蛋白表达量的变化,以及它们与海水和沉积物中石油类和重金属含量之间的相关性。结果表明,在现场属于一类海水的石油类浓度(0.0121-0.0242 mg/L)条件下,石油类就能够显著诱导褐菖鲉肝EROD活性和CYP1A蛋白表达量,鱼肝EROD活性和CYP1A蛋白表达量与海水中石油类含量均呈现极显著正相关,CYP1A蛋白表达量比EROD活性较为敏感和稳定。此外,在监测实验中,尚未发现这两种生物标志物受所监测海区的海水和沉积物重金属含量的影响。因此,利用褐菖鲉肝微粒体EROD活性和CYP1A蛋白表达量作为生物标志物监测海洋石油类及其PAHs污染是可行的,在海洋环境石油类污染监测及其生化效应评价中具有重要的应用价值。而且,把这两种生物标志物结合起来加以研究并推广应用将更有意义。     

用EROD研究苯并芘和六氯苯的联合毒性作用

用7-乙氧基异叻唑酮-脱乙基酶(EROD)检测的方法,研究了苯并芘和六氯苯对日本青鳉肝脏EROD酶的比活力的影响。结果表明,苯并芘和六氯苯对EROD酶的比活力均有激活作用,在实验浓度范围内,EROD酶的比活力与两者浓度之间存在剂量-效应关系。苯并芘和六氯苯表现为一定的协同作用。实验同时发现日本青鳉在六氯苯和苯并芘中暴露后,EROD酶的比活力开始有一个短暂的降低,然后持续升高。对六氯苯和苯并芘暴露的最佳时间进行了探讨。     

蔗糖脂肪酸脂对离体和活体大豆蔗糖酶活力和构象的影响

研究了蔗糖脂肪酸脂(SFE)对离体和活体大豆蔗糖酶活力和构象的影响。当SFE的浓度大于1.0mmol/L后,开始激活离体蔗糖酶的活力。在大豆的开花期和结荚期,SFE可以增加蔗糖酶的活力。荧光实验结果表明,在小于2mmol/L时,SFE不改变蔗糖酶的荧光最大发射峰峰位和峰高。施用SFE后,大豆叶片中果糖、葡萄糖和蔗糖酶的含量,分别是对照的170%±10%,190%±10%,和260%±20%;而蔗糖的含量几乎不变。对蔗糖酶 的SDS-凝胶电泳图进行扫描分析的结果表明,经SFE处理后的蔗糖酶含量比对照高两倍左右。这些结果说明SFE可以显著增加活体蔗糖酶的活力,但活力的增加既不是因为蔗糖酶构象的改变,也不是蔗糖(作为底物)诱导所致,而是SFE增加了大豆叶片中蔗糖酶的含量引起的。     

从无芒稗花粉活力评价其与转基因抗除草剂水稻基因漂移的可能性

为给转基因抗除草剂水稻和稗草间的基因漂移研究提供必要的信息,探寻了无芒稗(Echinochloacrusgalli var.mitis)花粉活力及其测定的最佳方法,并利用该方法测定了无芒稗开花盛期后离体和活体条件下不同时间的花粉活力。结果表明无芒稗开花盛期取样离体条件下花粉活力下降较快,3 h后花粉活力只有5.41％,活体条件下花粉活力下降较慢,3 h后仍有17.31％的花粉有活力。这说明无芒稗花粉在水稻开花时仍有部分保持活力,因而存在潜在的基因漂移可能性。同时用最佳培养基法测定了不同条件处理下(紫外灯照射,反复冻融,黑暗放置)无芒稗花粉活力,结果表明无芒稗花粉在不利环境条件下活力难以保持。     

两种光敏化合物对斜纹夜蛾超氧物歧化酶影响的比较研究(英文)

室内测试了斜纹夜蛾Ｓｐｏｄｏｐｔｅｒａ ｌｉｔｕｒａ Ｆ． ４龄幼虫超氧物歧化酶的活性,并就α－三噻吩（α－ｔｅｒｔｈｉｅｎｙｌ）和化合物５,即１－苯基－４－（３,４－亚甲基二氧）苯基－丁二炔等两种光敏化合物对其产生的影响进行了比较研究。结果表明,近紫外光照（３００－４００ｎｍ）基本不影响对照幼虫超氧物歧化酶活体（ｉｎｖｉｖｏ）活力,但对其离休（ｉｎ ｖｉｔｒｏ）活力有抑制作用。经光敏化合物处理后,在紫外光照下,超氧物歧化酶活体活力基本不受化合物５的影响,但能被α－三噻吩抑制。离体情况下,两种化合物均促进其活力,α－三噻吩尤甚。表明无论在离体还是活体情况下,幼虫对α－三噻吩均比化合物５具有更高的光敏性。对两种光敏化合物可能的作用机理进行了探讨。     

50例兔在体和离体器官组织射频介电性能的测量与分析

根据同轴传输线反射原理,建立起计算机控制的生物组织介电测量系统;在６０—３０００ＭＨｚ频率范围对５０例兔器官组织进行了有效的在体和离体介电测量。测量结果的统计分析表明,不同兔器官组织之间介电参数的最大差异可达３０·４％;在体和离休测量得出的介电常数之间未见显著性差异,但电导率有频率依赖性的显著差异,因此将人体的离体介电参数延用到实际活体场合时,必须慎重。     

节节麦远缘杂种胚的组织培养(简报)

利用活体-离体胚培养和胚愈伤组织诱导、再生植株技术有效地克服了节节麦远缘杂种胚的败育,高效产生了节节麦与硬粒小麦-簇毛麦双倍体、六倍体小黑麦、小麦、大麦间的杂种植株,从而为节节麦种质利用提供了技术方法。     

植物胚胎学实验方法（一）：花粉生活力的测定

采集的花粉和经贮存的花粉,在使用之前必须作生活力的测定以鉴定其质量。测定花粉生活力的方法主要有三种。即离体萌发、活体萌发和用氧化还原染料的非萌发分析测定法。这里介绍三种经常用的和在短时间可以得到结果的方法:花粉离体萌发法,用氧化还原染料染色法和荧光素二醋酸脂的荧光方法。     

Noncompetitive mixed-type inhibition of rainbow trout CYP1A catalytic activity by clotrimazole

Over the past two decades a number of antifungal imidazole derivatives have been approved for use in agricultural. The purpose of this study was to characterize the interaction of a model antifungal imidazole compound with a cytochrome P450 isozyme in a species of fish. Clotrimazole inhibited rainbow trout (Oncorhyncus mykiss) hepatic CYP1A-catalyzed ethoxyresorufin O-deethylase (EROD) activity in vivo and in vitro. Although clotrimazole inhibited EROD activity in vivo, it did not effect CYP1A mRNA levels. Addition of clotrimazole to microsomes produced a type II binding spectrum and clotrimazole was determined to be a noncompetitive mixed-type inhibitor of EROD activity with an IC₅₀ of 190 nM. Since antifungal imidazole compounds may be co-applied with other pesticides, inhibition of cytochrome P450 activity by antifungal imidazole compounds may lead to unexpected toxicological interactions.     

Baculovirus-insect cell production of bioactive porcine FSH

The in vitro and in vivo bioactivity of recombinant porcine FSH (rpFSH) produced from insect cells through use of a baculovirus expression system were studied and compared with those of natural FSH preparations. Determination of in vitro bioactivity, using the rat Sertoli cell aromatase bioassay, indicated that rpFSH is as active as purified pituitary FSH. Determination of in vivo bioactivity, using the mouse uterine weight bioassay, indicated that rpFSH is as active as purified pituitary FSH. Using the mouse Leydig cell testosterone bioassay, it was demonstrated that the intrinsic LH bioactivity of rpFSH is negligible. The increases in ovarian and uterine weight, and the stimulation in follicular growth in immature hypophysectomized rats induced by rpFSH supplemented with hCG were comparable to those induced by natural FSH preparations. Furthermore, rpFSH alone in hypophysectomized mice stimulated preantral follicular growth to preovulatory stages, and the subsequent injection of hCG caused ovulation. These results demonstrate that in vitro and in vivo biological characteristics of rpFSH produced from baculovirus-insect cells are indistinguishable from those of FSH isolated from natural sources.     

Determination of cytochrome P450 1A activities in mammalian liver microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) in liver microsomes from human, monkey, rat and mouse by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than previous methods using a spectrofluorimeter and fluorescence plate reader. The detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol/assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver microsomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in liver microsomes from all animal species exhibited a monophasic kinetic pattern, whereas the pattern of EROD activities in rat and mouse liver microsomes was biphasic. In addition, the method could determine the non-inducible and 3-methylcholanthrene-inducible activities of EROD and MROD in rat and mouse liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with cytochrome CYP1A isoforms in mammals.     

Disparate results between proliferation rates of surgically excised prostate tumors and an in vitro bioassay using sera from a positive randomized controlled trial

Abstract

In vitro bioassay has been used extensively to test the effects of culturing cancer cells in sera from humans participating in dietary interventions, i.e, studies of modified intake of nutrients for the purpose of reducing cancer risk or progression. It has been hypothesized that cell proliferation rates determined by the in vitro bioassay indicate whether modification of dietary intake could decrease cancer cell growth in vivo. It has been suggested, however, that the in vitro bioassay may not correlate with tumor cell proliferation rates in prostate cancer. We investigated the concordance of cell proliferation rates from surgically excised prostate tumor tissue with the in vitro bioassay using sera from matched patients. We used samples from an earlier randomized clinical trial that showed that supplementation with flaxseed significantly inhibited prostate cancer cell proliferation rates in vivo as indicated by Ki67 staining in tumor specimens. Proliferation rates of LNCaP, DU145 and PC3 cell lines cultured in 10% human sera from participants in the flaxseed trial were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Spearman's Rho correlation coefficients (ρ) indicated no association between Ki67 staining in prostate tumors and the in vitro bioassay for the three cell lines. These disparate findings suggest that the in vitro bioassay may not provide an accurate assessment of the environment in vivo.     

Sulfur-free parathyroid hormone analogues containing D-amino acids: biological properties in vitro and in vivo

Three sulfur-free analogues of bovine parathyroid hormone (bPTH) containing D-amino acids were synthesized by the solid-phase method and their biological properties compared in an in vitro bioassay (rat renal adenylate cyclase assay), a receptor assay for parathyroid hormone (PTH) (canine renal membranes), and an in vivo bioassay (chick hypercalcemia assay). The analogue [Nle8,Nle18,D-Tyr34]-bPTH-(1-34)-amide, which was found to be more than 4 times as potent in vitro as unsubstituted PTH, is the most potent analogue of PTH yet synthesized. The enhanced potency was largely attributable to increased affinity for the PTH receptor. In vivo, however, this analogue was only one-third as potent as bPTH-(1-34). Cumulative evidence suggests that the nearly 15-fold decline in the relative potency when the compound was assayed in vivo is due to the substitution of norleucine for methionine. The other analogues, [D-Val2,Nle8,D-Tyr34]bPTH-(1-34)-amide and [D-Val2,Nle8,Nle18,D=Tyr34]bPTH-(2-34)-amide, were only weakly active in vitro and in vivo, indicating that substitution with D-amino acids at the NH2 terminus of PTH causes markedly diminished receptor affinity. In fact, the placement of a D-amino acid at the NH2 terminus is more deleterious to biological activity than is omission of amino acids at positions 1 and 2.     

Implications for genetic toxicology of the chromosomal breakage syndromes

The activation of oncogenes and our knowledge of the chromosome breakage syndromes show that both intragenic mutations and chromosomal aberrations are important in carcinogenesis. Each suggests that an agent could produce genetic changes in a tissue without producing cancer there, if the types of genetic change do not match: chromosomal aberrations may be irrelevant in the mammary epithelium but be very significant in the bone marrow, and vice versa. This has vital implications for genetic toxicology: (1) both gene mutations and chromosomal aberrations should be measured, and (2) carcinogens may be mutagenic in tissues in which they are not carcinogenic. One might therefore expect in vivo assays for mutagenicity to correlate rather well with cancer bioassays; unfortunately, the bioassays themselves seem faulty. If cancer bioassays are valid, they would be reproducible. If bioassays are reproducible, they would be internally consistent. The information supplied by Tennant et al. (1987) for their validation of in vitro assays gives data from both sexes in rats and mice for 70 chemicals. When the data are analyzed site-by-site, positive results were not replicated in the other sex or in the other species much of the time: in half the cases the other sex does not give the same result; in two-thirds of the cases the other species does not give the same result. There are 3 potential explanations for these differing results: (1) genuine sex-specific carcinogens are common, (2) genuine species-specific carcinogens are common, or (3) the bioassay does not replicate well, i.e., is erratic. The third possibility best explains the data. The apparent inability of short-term in vitro tests to discriminate well between carcinogens and non-carcinogens may be more a reflection of the cancer bioassays that were used to determine which chemicals were carcinogenic than any defect in the assays. In this situation in vivo assays can scarcely be expected to do better even if they are better.     

A sialylation-sensitive cell-based in vitro bioassay for erythropoietin; incorporation of the galactose-binding Erythrina crista-galli lectin.

The in vivo biological activity of erythropoietin (Epo) is dependent on its being adequately sialylated. Current in vitro bioassays for Epo do not correlate with the in vivo bioassays as the former do not take into account the role the liver plays in clearing desialylated glycoproteins from the circulation. Here we describe a sialylation-sensitive cell-based Epo bioassay. In the first instance, Epo activity in vitro was measured using proliferation of AS-E2 cells, and in vivo using the polycythaemic mouse bioassay. Activity in vivo was progressively abolished by controlled desialylation, whereas activity in vitro was essentially unaffected. Incorporation of an incubation step with a solid-phase galactose-binding lectin (Erythrina crista-galli), effectively mimicking passage through the liver in vivo, renders the in vitro bioassay sensitive to desialylation, such that Epo desialylated almost to completion had <10% of the activity of untreated Epo. These studies offer proof of principle, that rational manipulation of in vitro bioassays can allow prediction of activity in vivo without the use of live animals.     

Evaluation of chromosomal damage by flow cytometry in turbot (Scophthalmus maximus L.) exposed to fuel oil

Flatfishes, turbots (Scophthalmus maximus), were injected intraperitoneally with two doses of fuel oil number 2. Biliary metabolites were evaluated by fixed fluorescence to verify the efficiency of intoxication. Ethoxyresorufin-O-deethylase (EROD) activity was compared with chromosomal damage measured by flow cytometry. The analysis of biliary metabolites showed a good dose–response relation and constitutes a clear reference for the subsequent measurements. Comparing flow cytometry and EROD results, a shorter delay of response for EROD activity was obtained, but chromosomal damage was significant only after 1 week. The persistence of the EROD response was shorter, while the genotoxic signal still persisted after 1 month. The measurement of chromosomal damage allowed a good differentiation between the two tested doses. In the case of EROD activity, the results were less clear. The results suggest that within a few weeks after exposure to fuel oil number 2, the measurements of chromosomal damage by flow cytometry can be used to detect a dose-dependant genotoxic response in fish.