2种烷基多环芳烃对仿刺参CYP450和p53基因表达的影响研究

海洋环境中多环芳烃类(PAHs)主要来源于海洋溢油事故以及沿海石油化工企业的废水排放,国内外大量研究发现海洋中的多环芳烃对海洋生物造成了潜在的生态风险。为了揭示不同浓度多环芳烃类污染物对海参的生态毒理效应,将仿刺参(Apostichopus japonicus)分别暴露于不同浓度的2种烷基多环芳烃3-甲基菲(5、10、100μg·L~(-1))和2-甲基蒽(5、10、50μg·L~(-1))中,检测暴露3 d、7 d和14 d后,3-甲基菲和2-甲基蒽胁迫下仿刺参CYP450和p53基因的相对表达量。结果表明,3-甲基菲和2-甲基蒽胁迫下,仿刺参CYP450和p53基因的表达均对毒物产生了不同程度的响应。与对照组相比,3-甲基菲各处理组对仿刺参CYP450和p53基因的表达均产生显著的抑制作用(P0.05); 2-甲基蒽各处理组对仿刺参CYP450和p53基因的表达影响作用不同,暴露7 d后,2-甲基蒽各处理组对仿刺参CYP450基因的表达表现出抑制作用,对p53基因的表达表现出诱导作用。相同浓度与时间胁迫下,2-甲基蒽对仿刺参CYP450和p53基因表达的影响比3-甲基菲的影响大。上述研究结果表明,3-甲基菲和2-甲基蒽均可不同程度影响仿刺参CYP450和p53基因的表达,且与3-甲基菲相比,2-甲基蒽对仿刺参CYP450和p53基因表达的影响较明显。上述结果为多环芳烃类污染物对仿刺参的生物毒性评价提供了基础数据。     

The profile of antibiotics resistance and integrons of extended-spectrum beta-lactamase producing thermotolerant coliforms isolated from the Yangtze River basin in Chongqing

The spreading of extended-spectrum β-lactamases (ESBL)-producing thermotolerant coliforms (TC) in the water environment is a threat to human health but little is known about ESBL-producing TCs in the Yangtze River. We received 319 ESBL-producing stains obtained from the Chongqing basin and we investigated antibiotic susceptibility, bla gene types and the presence of integrons and gene cassettes. 16.8% of TC isolates were ESBL-producing bacteria and bla_TEM+CTx-M was the predominant ESBL type. 65.2% of isolates contained class 1 integrons, but only 3 carried intI 2. Gene cassettes were amplified and sequenced. aadA, drfA, cmlA, sat1, aar3 and two ORF cassettes were found. In conclusion, Yangtze River is heavily polluted by ESBL-producing TC bacteria and the combined bla gene type could enhance antibiotic resistance. Class 1 integrons were widespread in ESBL-producing isolates and play an important role in multi-drug resistance. Characterization of gene cassettes could reveal the dissemination of antibiotic resistance genes.     

Characterization of the first step involved in enzymatic pathway for microcystin-RR biodegraded by Sphingopyxis sp. USTB-05

Enzymes encoded by genes biodegrading microcystins (MCs) can help reveal the function of genes and biodegradation pathway of MCs. Here the first and important gene (USTB-05-A, 1,008 bp) involved in biodegradation of microcystin-RR (MC-RR) was cloned from Sphingopyxis sp. USTB-05 and firstly expressed in Escherichia coli BL21 (DE3) with an expression vector of pGEX4T-1 successfully. The nucleotide sequences of cloned USTB-05-A possessed 92.5% homology to that of mlA reported in Sphingomonas sp. strain ACM-3962. The deduced amino acid sequences containing the cleavage sites of 26th (alanine) and 27th (leucine) showed 83% identical to that of MlrA. The cell-free extract (CE) of recombinant E. coli BL21 (DE3) containing USTB-05-A had high activity for biodegrading MC-RR. Initial MC-RR of 40 mg L⁻¹ was completely biodegraded under total protein of 350 mg L⁻¹ within 0.25 h. A product derived from MC-RR appeared distinctly with the decrease of MC-RR peak on the profile of HPLC. The product (m/z 1056.5) had molecular weight of 18 higher than that of MC-RR (m/z 1038.7). The findings provided the positive evidences that biodegradation of MC-RR began with the breakage of cyclic MC-RR and then it was converted to linear MC-RR as the first product catalyzed by first enzyme of Sphingopyxis sp. USTB-05.     

Determination of dioxin concentrations in fish and seafood samples using a highly sensitive reporter cell line, DR-EcoScreen cells

There is a strong need for the development of relatively rapid and low-cost bioassays for the determination of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), and dioxin-like polychlorinated biphenyls (dl-PCBs) in environmental and food samples. In this study, we applied a reporter gene assay using DR-EcoScreen cells (DR-cell assay), which is highly sensitive to dioxins, to the determination of PCDD/Fs and dl-PCBs in fish and seafood samples. The PCDD/Fs and dl-PCBs were extracted from homogenated samples (10 g) of 30 fish and shellfish, purified by clean-up procedure using a multilayered silica gel column and an alumina column, and applied to DR-cell assay. Interestingly, the bioanalytical equivalent (BEQ) values obtained from the DR-cell assay [<0.1 ∼ 5.4 pg BEQ g⁻¹ wet weight (ww)] were closely correlated with the toxicity equivalent (TEQ) values from conventional high-resolution gas chromatography/high-resolution mass spectrometry (HRGC-HRMS) analysis (r² = 0.912), and the slope of regression line was 0.913. Therefore, we multiplied the BEQ values from the DR-cell assay by a conversion coefficient (1.095, the reciprocal of 0.913) to approximate the TEQ values from the HRGC-HRMS analysis. Furthermore, we used this DR-cell assay to perform a prescreening test of PCDD/Fs and dl-PCBs in 16 fish and seafood samples purchased from a supermarket, revealing that a sample from the fatty flesh of a bluefin tuna exceeded 8 pg TEQ g⁻¹ ww (the European Union-tolerance limit). Taken together, these results suggest that the DR-cell assay might be applicable as a rapid and low-cost prescreening method to determine dioxin levels in fish and seafood samples.     

Tissue bioaccumulation patterns, xenobiotic biotransformation and steroid hormone levels in Atlantic salmon (Salmo salar) fed a diet containing perfluoroactane sulfonic or perfluorooctane carboxylic acids

In the present study, groups of juvenile Atlantic salmon (Salmo salar) were fed gelatine capsules containing fish-food spiked with PFOA or PFOS (0.2 mg kg⁻¹ fish) and solvent (methanol). The capsules were given at days 0, 3 and 6. Blood, liver and whole kidney samples were collected prior to exposure (no solvent control), and at days 2, 5, 8 and 14 after exposure (Note: that day 14 after exposure is equal to 7 d recovery period). We report on the differences in the tissue bioaccumulation patterns of PFOS and PFOA, in addition to tissue and compound differences in modulation pattern of biotransformation enzyme genes. We observed that the level of PFOS and PFOA increased in the blood, liver and kidney during the exposure period. Different PFOS and PFOA bioaccumulation patterns were observed in the kidney and liver during exposure- and after the recovery periods. Particularly, after the recovery period, PFOA levels in the kidney and liver tissues were almost at the control level. On the contrary, PFOS maintained an increase with tissue-specific differences, showing a higher bioaccumulation potential (also in the blood), compared with PFOA. While PFOS and PFOA produced an apparent time-dependent increase in kidney CYP3A, CYP1A1 and GST expression, similar effects were only temporary in the liver, significantly increasing at sampling day 2. PFOA and PFOS exposure resulted in significant decreases in plasma estrone, testosterone and cortisol levels at sampling day 2, and their effects differed with 17α-methyltestostrerone showing significant decrease by PFOA (also for cholesterol) and increase by PFOS. PFOA significantly increased estrone and testosterone, and no effects were observed for cortisol, 17α-methyltestosterone and cholesterol at sampling day 5. Overall, the changes in plasma steroid hormone levels parallel changes in CYP3A mRNA levels. Given that there are no known studies that have demonstrated such tissue differences in bioaccumulation patterns with associated differences in toxicological responses in any fish species or lower vertebrate, the present findings provide some potential insights and basis for a better understanding of the possible mechanisms of PFCs toxicity that need to be studied in more detail.     

微生物降解拟除虫菊酯类农药残留的研究进展

随着拟除虫菊酯类农药使用量不断增加,产生的农药残留问题对生态环境和人类健康造成了危害.对降解拟除虫菊酯类农药的微生物种类、降解酶和降解机制及降解酶基因克隆和构建工程菌等方面进行综述,旨在为研究和开发微生物降解拟除虫菊酯类农药残留提供参考.     

城市生活污水中志贺氏菌ipaH毒力基因的定量PCR检测

基于ipaH毒力基因的实时荧光定量PCR检测,建立适合城市污水中志贺氏菌的定量检测方法。使用从临床分离出的志贺氏菌构建重组质粒作为实时荧光定量PCR的标准品。在ipaH基因模板量2.58×100～2.58×106copy范围内具有良好的线性关系,每100 mL水样中含有2.58×101copy以上的ipaH基因即可被检出。从西安市生活污水分离得到2株野生型志贺氏菌,分析ipaH基因数量和菌体数量的关系,从而确定ipaH基因定量检测志贺氏菌的可行性。该方法灵敏、快速、特异性好,适用于城市生活污水中志贺氏菌的检测。     

Effects of estrone on the early life stages and expression of vitellogenin and estrogen receptor genes of Japanese medaka (Oryzias latipes)

The fertilized eggs of Japanese medaka (Oryzias latipes) were exposed to estrone (E1) at 5–5000 ng L⁻¹ for 15 d, and the hatched fry were exposed continuously to the same concentrations for the additional 15 d. Adverse effects on hatchability, time to hatching, and gross abnormalities occurred at 50 ng L⁻¹ or above. Then the fry were divided into a continual exposure group, and a water recovery group. When the fry were exposed to E1 for another 60 d, there was a decrease in the hepatosomatic index (HSI) of males and the influence disappeared in the water recovery group. The gonadosonatic index (GSI) of females at 500 ng L⁻¹ decreased significantly in another 60 d exposure. While the fry were maintained in dechlorinated tap water for 60 d, a significant decrease in female GSI was observed at 50 ng L⁻¹ or above. An increased GSI was found in males in both continual exposure and water recovery groups at all E1 treatments. Quantitative RT-PCR showed that vitellogenin-I (Vtg-I) gene expressions in the female liver were significantly down-regulated at 50 ng L⁻¹ in the continual exposure group, and at 500 ng L⁻¹ in the water recovery group, while male Vtg-I genes were significantly up-regulated for all E1 treatments. In addition, all E1 treatments caused sex reversal of males. These results suggest that E1 at 5 ng L⁻¹ or above have unrecoverable impacts on the gonadal growth and development of medaka, even if only early life stages were exposed to E1.     

phlb基因在普通小麦与簇毛麦杂种F1中的作用

通过CS、CSph1b与Haynaldiavillosa杂交,幼胚拯救,获杂种F1,其结实率分别为6．67％（12／18）、625％（25／400）对杂种F1植株花粉母细胞减数分裂中期Ⅰ染色体行为观察发现CS×H.villosa杂种F1平均每PMC仅有1．61条染色体形成二价体和三价体,平均构型为2n＝28＝26．391＋0．79Ⅱ＋0．007Ⅲ,平均染色体交叉数为0．84;而CSph1b×H．villosa杂种F1每PMC中有14．43条染色体发生联会,形成二价体和多价体,平均约型为2n＝28＝13．551Ⅰ＋5．95Ⅱ＋0．55Ⅲ＋0．22Ⅳ,为9．72,其中56％以上的PMC具1～4个多价体（三、四价体）表明Ph1b基因强烈诱导了普通小麦与H.villosa间的部分同源染色体配对杂种F1育性极差,自交均不结实.CS×H.villosa杂种F1与CS回交结实率为6．67％（4／60）,但CSph1b×H．villosa杂种F1与CS、CSph1b回交极难,结实率仅为0．45％（6／1320）．CSph1b×H．villosa杂种已与普通小麦回交成功,为利用ph1b实现簇毛麦优良基因直接对小麦遗转移奠定了基础．