Effects of cucl2 on UV‐mutagenesis and on DNA damage in a restriction fragment of E. Coli gpt∗

CuCl₂ does not cause Trp⁺ reversion in E. coli WP2. However, when the bacteria are exposed to CuCl₂ and UV‐irradiated, a greater than 3‐fold enhancement of mutagenesis (compared to UV alone) is seen at 30 μMCuCl₂, and significant enhancement is seen even at 3 μM. The mechanism for this comutagenic effect was studied using a restriction fragment of the E. coli gpt gene. Whereas UV or CuCl₂ alone caused few strand breaks, UV + CuCl₂ induced breaks at every site. This reaction was blocked by KI, a free radical scavenger. While UV alone induced alkali‐labile sites, UV+ CuCl₂ induced many more such sites and altered the sequence specificity. We suggest that at least some of the comutagenic effect might be due to hydroxyl radical formed via a Fenton reaction.     

Modified pretreatment method for total microbial DNA extraction from contaminated river sediment

Extraction of high-quality microbial DNA from contaminated environmental samples is an essential step in microbial ecological study. Based on previously published methods for soil and sediment samples, a modified pretreatment method was developed for extracting microbial DNA from heavily contaminated river sediment samples via selection of optimal pretreatment parameters (i.e., reagent solution, reaction duration, and temperature). The pretreatment procedure involves washing the river sediment sample for three times with a solution containing 0.1 mol·L^-1 ethylene diamine tetraacetic acid (EDTA), 0.1 mol·L^-1 Tris (pH 8.0), 1.5 mol·L^-1 NaCl, 0.1 mol·L^-1 NaH₂PO₄, and Na₂HPO₄ at 65°C with 180 r·min^-1 for 15 min to remove humic materials and heavy metals prior to the employment of standard DNA extraction procedures. We compared the results of standard procedure DNA extraction following pretreatment, without pretreatment, and with using a commercial PowerSoil^TM DNA Isolation Kit. The results indicated that the pretreatment significantly improved the DNA quality based on DNA yield, DNA fragment length, and determination of prokaryotic diversity. Prokaryotic diversity exhibited in the DNA with the pretreatment was also considerably higher than that extracted with the PowerSoil^TM DNA Isolation Kit only. The pretreatment method worked well even with a small amount of sediment sample (0.25 g or even lower). The method provides a novel, simple, cost-effective tool for DNA extraction for microbial community analysis in environmental monitoring and remediation processes.     

Evaluation of genotoxicity of combined soil pollution by cadmium and phenanthrene on earthworm

The DNA-damaging effects of the combined pollution of heavy metal and organic contamination on earthworms were evaluated by single cell gel electrophoresis （SCGE） assay. Earthworms Eisenia andrei were exposed to single or combined test compounds in different doses of cadmium （Cd） 5, 10, 50 mg/kg and phenanthrene （Phe） 0.5, 2.5, 12.5 mg/kg with a treatment of 14 d. In SCGE assay, isolated coelomcytes and electrophoresis were employed to determine DNA damage degree after a 14-d treatment by test compounds. The results showed that there was a significant statistical difference between earthworms treated with Cd combined Phe with them treated alone with Cd or Phe. The Olive tail moment （OTM） of SCGE assay using earthworm coelomcytes appears to be a sensitive biomarker for evaluating exposure to genotoxic compounds. These tests also revealed that the interaction between Cd and Phe to DNA damaging effects was negative, and was strongly dependent on the concentration of pollution. This study corresponds to exploratory phase in order to reveal interaction effects of heavy metal and organic contamination on earthworms and then to set up more in-depth analysis to increase progressively the understanding of the genotoxicity mechanisms involved.     

应用物种DNA条形码识别太湖流域部分底栖无脊椎动物种类

将物种DNA条形码实际应用于太湖流域底栖无脊椎动物分类,并与形态学分类结果比对,结果表明,DNA条形码技术可应用于本流域底栖无脊椎动物分类,但现阶段无法替代形态学鉴定,主要原因是太湖流域底栖无脊椎动物的绝大多数物种COⅠ基因特征序列是未知的或是BLAST所拥有的分类程度不够,提出在今后的研究中应探索建立太湖流域底栖无脊椎动物COⅠ基因数据库。     

Analysis of micronucleated erythrocytes in heron nestlings from reference and impacted sites in the Ebro basin (N.E. Spain)

The frequency of micronuclei (MN) in peripheral erythrocytes was tested for 59 heron nestlings (Ardea purpurea, Egretta garzetta and Bubulcus ibis) sampled at two areas (polluted and reference) on the River Ebro (NE Spain) and at its Delta during Spring 2006. Flow-cytometry analysis revealed higher (three- to six-fold) MN counts in samples from the most polluted site relative to samples from the reference area. Samples from the Delta showed intermediate values. Age, morphometric parameters (weight, tarsus size and bill-head length) and maturation status showed no significant differences among the different populations for each species; nor were they correlated with MN levels. The data suggest that elevated levels of MN in chicks in impacted areas reflected the chemical pollution of their nesting sites. The use of nestlings for this assay appears to be a convenient, non-destructive method to assess the impact of pollution in natural bird populations.     

DNA碱解旋速率法及其在DNA损伤研究中的应用

对DNA碱解旋速率法的细胞DNA经碱解旋后通过羟基磷灰石(HA)柱分离单、双链DNA时的洗脱次数、温度以及DNA回收效果进行了研究。结果表明,用0.125mol/L和0.25mol/L磷酸钾缓冲液各3ml,先后各洗脱一次,即可满足细胞DNA链断裂研究的要求。洗脱双链DNA时的温度为60℃即可。~3H-DNA与细胞其它组分一起通过HA柱时,~3H-DNA的回收率达94％左右。应用这一技术对电离辐射引起的中国田鼠肺细胞和小鼠脾细胞DNA单链断裂损伤与照射剂量的关系进行了探讨。结果表明,DNA单链断裂程度与照射剂量呈正相关。     

六价铬致小鼠DNA损伤及肝肾氧化应激的实验研究

为研究六价铬(Cr(Ⅵ))摄入后对机体造成的损伤,将重铬酸钾(K2Cr2O7)以25、50、100 mg·kg-1对小鼠进行灌胃,染毒时间为1 d、3 d和5 d.检测外周血淋巴细胞DNA损伤及肝肾组织活性氧自由基(ROS)水平、脂质过氧化产物丙二醛(MDA)含量,以及超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性.实验结果表明,经Cr(Ⅵ)染毒1 d、3 d和5 d后小鼠外周血淋巴细胞DNA损伤及肝ROS水平与对照组相比均有明显升高,肝脏抗氧化酶活性也有显著改变,但MDA含量差异不显著,而肾组织中各指标均未见有明显改变.由此认为,经口摄入Cr(Ⅵ)后能导致DNA损伤并且对肝脏造成氧化应激.     

Modulation of the DNA repair system and ATR-p53 mediated apoptosis is relevant for tributyltininduced genotoxic effects in human hepatoma G2 cells

The toxic effects of tributyltin (TBT) have been extensively documented in several types of cells, but the molecular mechanisms related to the genotoxic effects of TBT have still not been fully elucidated. Our study showed that exposure of human hepatoma G2 cells to 1-4 μmol/L TBT for 3 hr caused severe DNA damage in a concentration-dependent manner. Moreover, the expression levels of key DNA damage sensor genes such as the replication factor C, proliferating cell nuclear antigen and poly (ADP-ribose) polymerase-1 were inhabited in a concentration-dependent manner. We further demonstrated that TBT induced cell apoptosis via the p53-mediated pathway, which was most likely activated by the ataxia telangiectasia mutated and rad-3 related (ATR) protein kinase. The results also showed that cytochrome c, caspase-3, caspase-8, caspase-9, and the B-cell lymphoma 2 were involved in this process. Taken together, we demonstrated for the first time that the inhibition of the DNA repair system might be more responsible for TBT-induced genotoxic effects in cells. Then the generated DNA damage induced by TBT initiated ATR-p53-mediated apoptosis.     

活性污泥中PHA合成优势菌的筛选及产物结构分析

聚羟基脂肪酸酯(PHA)是一种具有广阔应用前景的生物可降解塑料,活性污泥中存在大量PHA合成菌,通过好氧/微好氧/沉淀的方式对污泥进行驯化,使PHA占污泥MLVSS的比例提高到21.19%。用BOX-PCR、ERIC-PCR和REP-PCR指纹图谱技术,对驯化前后污泥样品的菌群组成进行比较分析,结合尼罗蓝荧光平板法和菌落原位杂交技术,筛选得到活性污泥中PHA合成的优势菌株CAMP11。分类鉴定表明CAMP11属于芽孢杆菌属,FT-IR与NMR碳谱分析表明其合成的产物为羟基丁酸与羟基戊酸的共聚物(PHBV)。将菌株CAMP11回注入污泥驯化液,可使PHA占污泥MLVSS的比例进一步提高到32.08%。     

热处理重组质粒DNA的复性及其在水环境中的降解特性

为了考察热处理后的重组DNA进入水环境后可能存在的环境风险,以pET-28b质粒为材料,以质粒相对转化效率为指标,考察了热变性重组质粒DNA的复性可能性,并在此基础上构建了人工模拟水环境系统,研究了热变性质粒DNA在水环境中的降解速率和影响因素.结果表明,热变性pET-28b质粒经过30min后,其转移活性可以得到恢复,在4～37℃之间,温度越高越有利于热变性质粒的复性.热处理过程中未被降解的质粒DNA进入水环境后,在pH值为7、8时降解速率相对较慢,在pH值为5、6、9的条件下,降解较快,但在任何pH下,1.0h后仍存在未降解的质粒DNA.水环境中的NaCl对热处理质粒DNA有一定的保护作用,而且这种作用是随着NaCl质量分数的提高而增强.进入水环境中的热变性DNA有足够的时间复性,从而可能发生基因转移.因此,这些热处理的质粒DNA进入环境后理论上存在一定的生态风险.