Effects of 2,3,7,8-TCDD and benzo[a]pyrene on modulating vitellogenin expression in primary culture of crucian carp (Carassius auratus) hepatocytes

Vitellogenin (Vtg) is the precursor of yolk protein. Its expression and secretion are estrogen-regulated and are crucial for oocyte maturation. An in vitro xenoestrogen screening model was established by measuring Vtg induction in cultured primary hepatocytes from crucian carp. Vtg production was detected by biotin-avidin sandwich ELISA method while Vtg and cytochrome P4501A1 (CYP1A1) mRNA induction were measured by semi- quantitative PCR-primer dropping technique. Vtg and Vtg mRNA were dose-dependently induced by diethylstilbestrol (DES, 0.2-200 ng/mL) in hepatocytes of crucian carp. Co-treatment of the DES-induced hepatocytes with either 2,3,7,8-TCDD (TCDD, 0.1-4 pg/mL) or benzo[a]pyrene (B[a]P, 5-1000 ng/mL) resulted in a reduction of Vtg production and an increment of CYP1A1 mRNA expression both in a dose dependent manner, indicating the anti-estrogenic effects of the compounds. However, at lower tested concentrations, TCDD (0.1, 0.2 pg/mL), B[a]P (5 ng/mL) seemed to have a potentiating effect on Vtg expression and secretion, although by their own these compounds had no observable estrogenic effect on Vtg induction. Tamoxifen (a selective estrogen receptor modulators, 1 nmol/L-1 mumol/L), and P-naphtho-flavone (beta-NF, an aryl hydrocarbon receptor inducing compounds, 2.5-1000 ng/mL) also were employed to study the possible interactions in DES-induced Vtg expression. In co-treatment of the DES-induced hepatocytes with beta-NF or tamoxifen, the decrease in Vtg production did parallel induction of CYP1A1 for beta-NF, but tamoxifen inhibited Vtg induction did not parallel induced CYP1A1 expression in all test concentrations. On the contrary, it was found that in co-treatment of the TCDD-induced hepatocytes with DES, TCDD induced CYP1A1 mRNA production was inhibited by DES also. These results implicated a possible cross talk between estrogen receptor- and aryl hydrocarbon receptor-mediated pathways in the hepatocytes.

Nucleic Acid-Induced Aggregation and Pyrene Excimer Formation

Nucleic acid was found to induce the aggregation of the positively charged pyrene probe (compound 1); as a result, strong pyrene excimer emission was observed. The intensity of the excimer emission was dependent on the concentration of the pyrene probe and the oligonucleotide length, sequence, and concentration. These results suggest a new strategy for label-free nucleic acid-based biosensing applications.

Response of integrated biomarkers of fish (Lateolabrax japonicus) exposed to benzo[a]pyrene and sodium dodecylbenzene sulfonate

Fish Lateolabrax japonicus were exposed to 0.1 and 1 mg/L of anion surfactant sodium dodecylbenzene sulfonate (SDBS) and to 2 and 20 mu g/L of benzo[a]pyrene (B[a]P) for 6, 12, and 18 days, with control and solvent control groups. Liver antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), and glutathione S-transferase (GST), were determined; brain acetyleholinesterase (AChE) and liver inducible nitric oxide synthase (iNOS) activities were also measured. The results indicated that (1) L. japonicus avoided oxidative damage through antioxidant systems; (2) SOD, GPx, and GSH were induced, and GST was inhibited and then induced by B[a]P exposure; and (3) CAT, GPx, and AChE were induced while NOS was inhibited, and GST was induced and then inhibited by SDBS stress in experimental period. (c) 2005 Elsevier Inc. All rights reserved.

Comparative studies of AOT and NaDEHP by fluorescence technique and z-potential (ζ) measurements

The aggregation behaviors of two surfactants with the same hydrophobic tail, sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and sodium bis(2-ethylhexyl)phosphate (NaDEHP), have been investigated by the fluorescence technique and z-potential (ζ) measurements. Five fine peaks of the pyrene molecule fluorescence spectroscopy appear in the surfactant solution, and the micropolarity at which pyrene locates is monitored from the intensity ratio of the first (I1) and the third peak (I3). A wide peak around 475 nm, the emission spectra of the excimer of pyrene molecules, is observed in the NaDEHP solution, while this is not found for the AOT system. The value of I1/I3 decreases in a more limited concentration range for the AOT system than for NaDEHP, indicating that small aggregates can be more easily formed by NaDEHP molecules. The z-potential results for the aggregates formed by the two surfactants show that the interaction between AOT and PVP is stronger than that between NaDEHP and PVP.

Determination of PAH, PCB, and OCP in water from the Three Gorges Reservoir accumulated by semipermeable membrane devices (SPMD)

Bioavailable water concentrations of polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB) and organochlorine pesticides (OCP) were measured in the water column from Three Gorges Reservoir (TGR) collected in May 2008 using semipermeable membrane devices (SPMDs). The sampling sites spanned the whole reservoir from the upstream Chongqing to the great dam covering more than 600 km long distance with water flow velocities ranging from <0.05 to 1.5 m s(-1). This is the first experience of SPMD application in the biggest reservoir in the world. The results of water sampling rates based on performance reference compounds (PRC) were tested to be significantly correlated with water flow velocities in the big river. Results of back-calculated aqueous concentrations based on PRC showed obvious regional variations of PAH, PCB and OCP levels in the reservoir. Total PAH ranged from 13.8 to 97.2 ng L-1, with the higher concentrations occurring in the region of upstream and near the dam. Phenanthrene, fluoranthene, pyrene and chrysene were the predominant PAH compounds in TGR water. Total PCB ranged from 0.08 to 0.51 ng L-1, with the highest one occurring in the region near the dam. PCB 28, 52, 101, 138, 153, 180, 118 were the most abundant PCB congeners in the water. The total OCP ranged from 2.33 to 3.60 ng L-1 and the levels showed homogenous distribution in the whole reservoir. HCH, DDT and HCB, PeCB were the major compounds of OCP fingerprints. Based on water quality criteria, the TGR water could be designated as being polluted by HCB and PAH. Data on PAH, PCB and OCP concentrations found in this survey can be used as reference levels for future POP monitoring programmes in TGR. (C) 2009 Elsevier Ltd. All rights reserved.

Primary cultured cells as sensitive in vitro model for assessment of toxicants-comparison to hepatocytes and gill epithelia

In an effort to develop cultured cell models for toxicity screening and environmental biomonitoring, we compared primary cultured gill epithelia and hepatocytes from freshwater tilapia (Oreochromis niloticus) to assess their sensitivity to AhR agonist toxicants. Epithelia were cultured on permeable supports (terephthalate membranes, "filters") and bathed on the apical with waterborne toxicants (pseudo in vivo asymmetrical culture conditions). Hepatocytes were cultured in multi-well plates and exposed to toxicants in culture medium. Cytochrome P4501A (measured as 7-Ethoxyresorufin-O-deethylase, EROD) was selected as a biomarker. For cultured gill epithelia, the integrity of the epithelia remained unchanged on exposure to model toxicants, such as 1,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene B[a]P, polychlorinated biphenyl (PCB) mixture (Aroclor 1254), and polybrominated diphenyl ether (PBDE) mixture (DE71). A good concentration-dependent response of EROD activity was clearly observed in both cultured gill epithelia and hepatocytes. The time-course response of EROD was measured as early as 3 h, and was maximal after 6 h of exposure to TCDD, B [alp and Aroclor 1254. The estimated 6 h EC50 for TCDD, B [a]P, and Aroclor 1254 was 1.2x10(-9), 5.7x10(-8) and 6.6x10(-6) M. For the cultured hepatocytes, time-course study showed that a significant induction of EROD took place at 18 h, and the maximal induction of EROD was observed at 24 h after exposure. The estimated 24 It EC50 for TCDD, B[a]P, and Aroclor 1254 was 1.4x10(-9), 8.1x10(-8) and 7.3x10(-6) M. There was no induction or inhibition of EROD in DE71 exposure to both gill epithelia and hepatocytes. The results show that cultured gill epithelia more rapidly induce EROD and are slightly more sensitive than cultured hepatocytes, and could be used as a rapid and sensitive tool for screening chemicals and monitoring environmental AhR agonist toxicants. (c) 2006 Elsevier B.V. All rights reserved.

Cultured gill epithelial cells from tilapia (Oreochromis niloticus): a new in vitro assay for toxicants

A culture gill epithelium from seawater-adapted tilapia (Oreochromis niloticus) was developed for testing PAHs and dioxin-like contaminants in seawater. The epithelia consists two to three layers of epithelial cells incorporating both pavement cells and mitochondria-rich cells (MRCs). Polarity and a stable transepithelial resistance (TER) were maintained. and closely resembled those in fish gills in vivo. The tightness (integrity) of the epithelia remained unchanged upon exposure to benzo[a]pyrene (B[a]P). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4',5-pentachlorobiphenyl (PCB#126), while a concentration-dependent response of EROD activity in the epithelia was induced within 18-24 h when the apical side was exposed to these toxicants. The 24 h EC50 of EROD activity was 2.77 x 10(-7) M for PCB#126, 1.85 x 10(-7) M for B[a]P and 7.38 x 10(-10) M for TCDD. showing: that the preparation was not only sensitive to PAHs and dioxin-like compounds, but also able to produce inductive potency of AhR agonists that generally agreed with those derived from other established in vitro and in vivo systems. The results suggest, that the cultured gill epithelia from seawater-adapted tilapia may serve as a simple. rapid and cost-effective tool for assessing exposure and potential effects of toxicants in marine waters. (C) 2004 Elsevier B.V. All rights reserved.

Dioxin-like components in human breast milk collected from Hong Kong and Guangzhou

The H4IIE rat hepatoma cell line was employed as a cell model to screen 7-ethoxyresorufin O-deethylase (EROD)-TCDD equivalents (EROD-TEQ) of human breast milk samples collected from Hong Kong and Guangzhou, China. The screening methods employed a 96-well plate spectrofluorometer-EROD assay. For cell-line validation, our results demonstrated a dose-dependent increase in the Ah receptor-mediated response (i.e., CYP1A1 mRNA and EROD) of the cells upon exposure to a number of known Ah receptor agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzothiophene, benzo[a]pyrene, and beta-naphthaflavone. TCDD induced CYP1A1 mRNA and EROD was in a close positive correlation (r = 0.98). For the screening of dioxin-like compounds, breast milk samples collected during lactation weeks 3-5 were used. One hundred (from Hong Kong) and 48 (from Guangzhou) breast milk samples were assayed, of which 65% and 68% of the samples, respectively, showed detectable dioxin-like activities using the H4IIE cell EROD screening method. For sixty-five samples from Hong Kong the mean EROD-TEQ values ranged from 58.1 to 96.5 pg/g of milk fat for those aged 21-36 years while 32 samples from Guangzhou had mean values of 98.8-202.1 pg/g of milk fat. In comparisons of the EROD-TEQ values for different age groups from both cities, there were no significant differences (P < 0.05). However, the mean and median EROD-TEQ values of the Guangzhou population were in general higher than those of the Hong Kong population. The results of the present study indicate that it is feasible to use the H4IIE cell-line as a model for screening dioxin-like compounds in human breast milk. In addition, the method is rapid and cost-effective, particularly for a routine and high-throughput sample screening analysis, compared to the costly and time-intensive chemical analytical techniques. (C) 2003 Elsevier Inc. All rights reserved.

Relationship between tissue concentrations of polycyclic aromatic hydrocarbons and DNA adducts in green-lipped mussels (Perna viridis)

Green-lipped mussels (Perna viridis) were collected from a site in Hong Kong which is relatively free from polycyclic aromatic hydrocarbon (PAH) contamination, and maintained in situ at this and three other sites with different degrees of PAH contamination. The transplanted mussels were retrieved after a 30-day field exposure. DNA adducts in the gill tissues were quantified, and tissue concentrations of benzo[a]pyrene as well as total PAHs (with potential carcinogenicity) determined for individual mussels. Results indicate that (1) tissue concentration of PAHs and adduct levels in mussels collected from a single site can be highly variable; and (2) adduct levels were related to tissue concentrations of benzo[a]pyrene as well as total PAHs of individual animals.

多环芳烃胁迫下农田土壤微生物特征及高效芘降解菌筛选

本文通过传统和现代微生物生态学方法，从土壤微生物数量、土壤底物诱导呼吸强度和微生物群落多样性角度，评价了长期污灌所致PAHs污染对土壤微生物特征的影响。结果发现沈抚灌区农田PAHs总量在表层为612.3-6362.81μg•kg-1干土，在亚表层为319.5-4318.51μg•kg-1干土。土壤微生物主要类群、功能群数量、微生物生物量碳和代谢商与土壤PAHs污染程度无明显相关性，土壤底物诱导呼吸强度和所试土壤酶与PAHs含量呈显著正相关，微生物商与PAHs含量呈显著负相关，可以作为土壤PAHs污染评价的敏感生化指标之一。污染稻田土壤细菌群落中优势菌群为β-和γ-变形细菌亚纲的成员，中度PAHs土壤的分支杆菌多样性指数较重度和轻度的略高，PAHs污染使一种或几种分支杆菌得到富集。长期污水灌溉造成土壤固氮细菌种群多样性降低，清水灌溉一段时间后，固氮细菌种群结构得到不同程度的恢复，但是，即使清灌不能使其种群结构得到完全恢复。 通过富集得到一株高效降解芘的细菌N12，经鉴定确认为分支杆菌。经10天培养菌株可将100mg•l-1芘降解97.84%。还可降解菲、苊、芴，不能降解萘、蒽和苯并[a]芘。污染土壤修复实验表明，单一接种菌剂对芘的降解率为57.42%，含N12的混合接种菌剂对芘的降解率为61.11%。

苯并（a）芘的微生物降解及其酶蛋白应答研究

本文以苯并(a)芘为目标污染物，探讨了母体化合物BaP及其次生代谢产物的连续降解的方法、降解过程和微生物的酶蛋白应答，并运用种子（小麦、白菜和萝卜）根伸长生长实验，考查了BaP的不同降解时期次生代谢产物造成的复合污染整体效应，旨在探讨BaP及其次生代谢产物的降解影响因素，减少其环境累积，为BaP污染环境的全面修复提供实验依据和理论基础。 在实验条件下，运用HPLC鉴定出真菌FZSY-1降解BaP的同时生成了三个次生代谢产物BP1,6-quinone, BP7,8-diol 和 3-OHBP；同时鉴定出真菌FZSY-2降解BaP的同时生成了两个代谢产物BP1,6-quinone 和 3-OHBP。 驯化微生物与氧化剂（KMnO4）的耦合降解系统对BaP及其代谢产物的连续降解效果好于单纯微生物降解。三个次生代谢产物中，BP1,6-quinone在环境中最易累积。同时提出了微生物与氧化剂协同的作用可以有效促进环境中持久有机污染物（尤其是高浓度，小面积污染）的连续降解。对于FZSY-1与氧化剂（KMnO4）耦合降解BaP，在TW80存在下，与对照（未加TW80）相比，在降解的前期（3天取样），BaP及其代谢产物的降解相对滞后；而在降解的后期（12天取样），BaP及其代谢产物的降解高于对照。 在不同BaP浓度下，检测了四种酶，C120、C230、CAT和PPO。三株细菌的CAT酶活与BaP的浓度无关；三株细菌的C230酶活都比较高；三株细菌的PPO酶活均较低。加入共代谢底物(琥珀酸钠)后，与对照(未加入共代谢底物)相比，C120、C230酶活明显提高。 以BaP以及FZSY-1（BZSY-2）降解BaP不同时期的复合降解产物（BaP，M6，M12，CK）为目标污染物，它们对小麦种子根伸长的抑制作用顺序为：M6﹥BaP﹥M12﹥CK。BaP，M6，M12，CK对白菜和萝卜种子根伸长的抑制作用顺序和小麦相同；同一目标污染物（M6）对这几种供试种子（小麦、白菜和萝卜）根伸长的抑制作用顺序为小麦﹥白菜﹥萝卜；三种植物种子根伸长抑制作用均表现为：真菌的M6﹥细菌的M6，真菌的M12﹥细菌的M12。

辽河干流底质微生物群落结构及芘降解菌研究

辽宁省是以石油化工、煤炭化工和钢铁工业为主的重工业基地。辽河流域近年来经济发展迅速，城市化水平不断提高，但由于产业结构的不合理和污染治理水平的相对滞后，致使辽河流域水体污染严重。对辽河流域水体污染状况、污染物化学与生物学的相互作用、微生物群落结构与功能的关系开展调查研究，对开展污染水体的生物生态修复具有重要的指导意义。 本论文选取辽河流域干流8个水文监测站点的不同时期（丰水期和平水期）底质为研究对象，调查了有机污染物（总油TPHs）和有毒污染物（多环芳烃PAHs）污染程度以及主要来源；采用变性梯度凝胶电泳（DGGE）和磷脂脂肪酸（PLFA）两种分析方法，对其微生物群落结构及多样性进行了分析；并以13C标记的菲和芘为代谢底物，以PLFA为生物标记物，采用气相色谱-稳定同位素比率质谱（GC-c-IRMS）分析技术，鉴定了底质样品中参与菲和芘代谢的主要微生物类群；并利用不同的吸附性载体进行了芘降解菌的富集和筛选。 研究结果表明： 1）平水期总石油烃污染比丰水期严重，TPH 含量分别在276.1～560.6mg/kg（平水期）和157.9～462.2mg/kg（丰水期）之间，辽河入海口TPH污染最重；PAHs含量分别在124.1～270.4ug/kg（平水期）和93.5～209.1ug/kg（丰水期）之间；主要来源于石油类污染物和化石燃料的热解，汽车尾气的污染等。 2) 采用DGGE和PLFA两种方法分析微生物群落结构得到基本一致的结果。微生物多样性与总石油烃含量、总多环芳烃含量无显著相关性，多样性指数是多种污染物和环境因子综合影响的结果。 3) 稳定同位素代谢示踪实验表明，底质中存在菲和芘的降解菌群；参与菲和芘降解的微生物均以G-细菌为主，真菌次之；G+细菌和放线菌也参与代谢；参与菲和芘代谢的菌群有一定的相似性。 4) 利用不同吸附性载体从污染底质样品中筛选到6株降解菌。

细菌对芘的降解特性及其降解性质粒研究

本研究通过富集培养，从沈抚灌区石油污染土壤中分离到两株以芘为惟一碳源和能源生长的细菌，分别命名为ZHL-4和B61。经过形态观察、生理生化实验与16S rDNA序列分析鉴定，ZHL-4和B61分别为苍白杆菌属（Ochrobactrum）和假单胞菌属（Pseudomonas）。 ZHL-4和B61均对芘具有较强的降解能力，在不添加任何其它碳源的情况下，分别在7d内将10mg•L-1芘降解了71.8%和67.4%，各加入500mg•L-1的葡萄糖和酵母膏作为共代谢底物后，7d内对芘的降解率分别提高到86.8%和89.9%。 经电泳检测，菌株ZHL-4和B61均存在内生质粒。质粒消除实验表明，消除质粒后的菌株ZHL-4和B61不能利用芘进行生长；将质粒转化入大肠杆菌中后，转化子获得了在芘固体培养基上生长的能力，初步证明两株细菌的内生质粒是与芘代谢有关的降解性质粒，其降解芘的基因位于质粒上，这与其它高分子量PAHs降解菌的降解基因位于染色体上不同。 通过设计引物、PCR扩增，菌株ZHL-4和B61并不具有已报道的芘降解基因nidA，这表明ZHL-4和B61具有可能不同于nidA基因的新的芘降解基因，有待于进一步研究。