星豹蛛四个羧酸酯酶基因克隆及溴氰菊酯胁迫下的表达模式

【目的】探究星豹蛛Pardosa astrigera羧酸酯酶基因PaCarE1-4是否与其代谢溴氰菊酯有关。【方法】采用RT-PCR技术克隆星豹蛛4个羧酸酯酶基因PaCarE1-4 cDNA序列,通过生物信息学软件分析其序列特性。采用RT-qPCR技术测定这4个羧酸酯酶基因在星豹蛛雌雄成蛛不同组织(头胸部、腹部和足)以及在不同浓度(LC₁₀=5.151 mg/L; LC₃₀=8.619 mg/L; LC₅₀=12.311 mg/L)溴氰菊酯胁迫12 h和LC₃₀浓度溴氰菊酯胁迫2, 4, 8, 12, 24和48 h雄成蛛中的相对表达水平。【结果】克隆获得星豹蛛羧酸酯酶基因PaCarE1-4(GenBank登录号分别为MZ643212, MZ643214, MZ643215和 MZ643216)的全长cDNA序列,开放阅读框(ORF)分别长1 653, 1 803, 1 827和1 818 bp,分别编码550, 600, 608和605个氨基酸。组织表达谱结果表明,PaCarE1和PaCarE2在星豹蛛雌雄成蛛腹部中的表达量最高,且在雄成蛛腹部中的表达量高于雌成蛛中的;PaCarE3和PaCarE4在雌雄成蛛头胸部中的表达量最高,且PaCarE3在雌成蛛头胸部中的表达量高于雄成蛛中的,PaCarE4在雄成蛛头胸部中的表达量高于雌成蛛中的。LC₃₀浓度溴氰菊酯胁迫12 h诱导了星豹蛛雄成蛛中PaCarE1的表达,LC₁₀和LC₃₀浓度溴氰菊酯胁迫12 h诱导了PaCarE2的表达。LC₃₀浓度溴氰菊酯胁迫不同时间后,与对照组(丙酮处理组)相比,星豹蛛雄成蛛中PaCarE4的表达量与对照组均无显著差异,而PaCarE1的表达量在处理后2, 8和12 h, PaCarE2的表达量在处理后12 h,以及PaCarE3的表达量在处理后24 h显著上调。【结论】羧酸酯酶基因PaCarE1, PaCarE2和PaCarE3可以被溴氰菊酯诱导表达,表明其可能参与星豹蛛对溴氰菊酯的代谢过程。本研究结果有助于了解星豹蛛对外源物质的代谢机理,为这一捕食性天敌的保护提供了新思路。     

应用微核试验和单细胞凝胶电泳技术来检测农药对青蛙蝌蚪及成体的遗传毒性

应用青蛙红细胞微核试验和单细胞凝胶电泳试验研究了两种新型杀虫剂 -吡虫啉和抑食肼对青蛙蝌蚪和成体的遗传毒性 ,结果表明 :当吡虫啉为 2mg/L时 ,蝌蚪红细胞微核率与对照组相比 ,无显著性差异 (p >0 .0 5) ;浓度升高到 8mg/L时 ,微核率与对照组相比 ,有显著性差异 (p <0 .0 5) ;当浓度为 3 2mg/L时 ,微核率与对照组相比 ,有极显著性差异 (p <0 .0 1) ;并有明显的剂量 -效应关系 (r =0 .9843 )。而抑食肼在浓度为 2 .5mg/L和 10mg/L时 ,微核率与对照组相比 ,无显著性差异 (p >0 .0 5) ;当浓度增至 40mg/L时 ,微核与对照组相比 ,有极显著性差异 (p <0 .0 1) ;吡虫啉与抑食肼各浓度组对青蛙红细胞的DNA损伤与阴性对照组相比 ,都有极显著性差异 (p <0 .0 1) ,且具有明显的剂量 -效应关系 (r =0 .960 ,r=0 .990 )。     

防治扶桑绵粉蚧化学农药的筛选及其防治效果

为寻求防治扶桑绵粉蚧Phenacoccus solenopsis Tinsley有效化学药剂,在室内测定了9种化学农药对该虫的致死效果,在此基础上,选出4种农药进行了田间防治试验.结果表明,室内条件下三氯杀螨醇、阿维菌素、阿维-氯氰、联苯菊酯4种药剂对成虫的校正死亡率在23.7%～66.7%之间,明显低于对1龄若虫的作用效齐果;高效氯氟氰菊酯、吡虫啉、啶虫脒和毒死蜱4种化学药剂,在田问施药后第5天的防治效果可达到91%以上,其中啶虫脒和毒死蜱的防治效果分别达到95.6%和98.2%.在防治应用中,建议推荐高效氯氟氰菊酯、吡虫啉、啶虫脒、马拉硫磷和毒死蜱5种药剂,同时应注意针对不同的虫态有必要适当调整用药浓度(就除害处理而言)和施药次数,以达到理想的防治效果.     

喷施农药对银耳生长的影响及膳食暴露风险评估

为制订银耳(Tremella fuciformis)栽培中农药合理使用的建议,以我国银耳主栽菌株Tr01为对象,研究8组常用农药对其生长的影响,采用食品安全指数法评估长期膳食银耳导致的农药残留慢性暴露健康风险。结果表明,除咪鲜胺乳油和哒螨灵、啶虫脒微乳剂的部分处理外,其余6组农药对银耳子实体生长并无显著影响。银耳中的农药残留水平与农药种类及喷施模式密切相关,当农药残留水平低于GB 2763-2019标准中蔬菜的农药最大残留限量时,成人和儿童长期膳食银耳的慢性健康风险商(cHQ)分别为0.001～0.174和0.002～0.191,风险水平可接受。结合我国农药使用现状,建议银耳栽培中应禁用乙酰甲胺磷、毒死蜱、克百威,减少阿维菌素、咪鲜胺、异丙威的使用频率,在合适的安全间隔期下可以使用联苯菊酯、啶虫脒、吡虫啉、哒螨灵。     

甲硝唑诱导蚕豆根尖细胞微核的研究

本实验研究了甲硝唑诱导蚕豆根尖细胞微核的效应。实验表明：(1)浓度为0.1、6、12、40和500 mg/L甲硝唑均能使蚕豆根尖细胞微核率显著增加,且蚕豆根尖细胞微核率和甲硝唑浓度之间存在明显的剂量-效应关系;(2)甲硝唑能引起DNA损伤,具有分子诱变剂的性能。     

啶虫脒污染下土壤微生物多样性

避开传统的分离培养过程,采用现代分子生物学方法探讨了杀虫剂啶虫脒污染条件下旱地土壤微生物种群多样性.通过对不同培养时间、不同浓度啶虫脒污染下旱地土壤微生物进行DGGE基因多样性的分子指纹图谱分析,发现随着培养时间不同,各处理之间的土壤微生物基因多样性出现了一定的差异.但在整个试验过程中,正常田间使用浓度（0.5mg kg^-1干土）的啶虫脒对土壤微生物群落的影响不明显,DGGE图谱条带与对照没有明显差异,土壤微生物基因多样性没有明显下降,这说明在旱地中使用正常田间浓度的啶虫脒不会对微生物群落造成较大的影响,高浓度啶虫脒对土壤微生物群落基因多样性有一定的影响,但是影响时间不长.在培养第五周时,浓度为5 mg kg^-1干土的土样出现了特异性条带,为对照所没有,其他处理浓度染色暗淡.经序列比对分析,与来自土壤的Uncultured bacterium具有100﹪的相似率,可能为不可培养或未培养过的细菌种.     

蚯触圈源啶虫脒降解菌D35的分离鉴定及其降解条件优化

【背景】啶虫脒等新烟碱类杀虫剂的残留易对非靶标生物造成伤害,投加高效降解细菌进行生物强化,可促进其快速降解。【目的】从蚯触圈中分离筛选啶虫脒降解菌并优化其降解条件,提高降解效率。【方法】制备蚯触圈基质富集筛选降解菌;通过生理生化特征和16S rRNA基因序列分析对其进行鉴定;利用单因素筛选、Plackett-Burman试验、最陡爬坡试验及Box-Behnken design试验优化菌株降解条件。【结果】分离得到1株啶虫脒降解菌D35,可在72 h内降解55.46%初始浓度为50 mg/L的啶虫脒,将其鉴定为一株假单胞菌(Pseudomonas sp.)。优化得到菌株降解啶虫脒的最佳环境条件为：胰蛋白胨10.19 g/L、温度为30℃、接种量为5.24%,pH 7.0、初始农药浓度50 mg/L,在此条件下72 h内菌株降解率为80.21%,较未优化前提高了24.75%。【结论】本研究对分离筛选新烟碱类杀虫剂降解菌的方法进行了探索,获得的菌株D35可高效降解啶虫脒,为快速消除环境中啶虫脒污染提供了新的微生物资源。     

啶虫脒防治葡萄斑叶蝉的残留安全性分析

为了解啶虫脒在葡萄上使用的安全性,采用高效液相色谱的方法,研究了啶虫脒于葡萄不同物候期防治斑叶蝉时,在果实与叶片上的残留动态。结果表明:3%啶虫脒微乳剂在葡萄开花期至硬核期叶片上的半衰期为3.55～3.93d,在葡萄着色期至成熟期果实上的半衰期为4.14～5.60d,虽然在果实中降解稍慢,但最终残留量相差较小。按推荐剂量22.5g·hm-2(a.i.)和加倍剂量45g·hm-2(a.i.)各施3%啶虫脒微乳剂3次,间隔期7d,末次施药后7d葡萄果实中的残留量均小于0.2mg·kg-1,14d残留量均小于0.1mg·kg-1,本方法的最低检出量为0.5ng,最低检出浓度为0.01mg·kg-1。参照美国、韩国与日本的最大残留限量(MRL),药后7d葡萄果实是安全的。建议用3%啶虫脒微乳剂在葡萄开花期防治斑叶蝉的第1代若虫、成虫,硬核期至着色期防治第2代若虫、成虫,最多使用3次,用量为22.5g·hm-2(a.i.),安全间隔期为7d。     

荧光物示踪法测定重复施药胁迫下食虫沟瘤蛛的摄食量动态

定量评价农药对天敌的影响是IPM研究的重要内容之一,由于对天敌的捕食量缺乏有效的定量测量方法,农药对天敌摄食功能影响仍处于定性水平的研究,误差基数较大。本文采用一种定量的测定方法——荧光物示踪法,以稀土元素铕作为荧光示踪物质,定量地测定了食虫沟瘤蛛在重复接受噻嗪酮喷施后,其存活个体在药后13天内摄食量的变化趋势,且把药后食虫沟瘤蛛的死亡率加以考虑,计算药后整个试验食虫沟瘤蛛群体的摄食量变化。结果表明：任何浓度任何一次施药后2天左右食虫沟瘤蛛存活个体的捕食量均急剧下降;药物浓度较低时,第1次施药对食虫沟瘤蛛的影响最大;药物浓度较高时,食虫沟瘤蛛存活个体药后摄食量恢复周期较长。药后一段时间内整个食虫沟瘤蛛种群的摄食率相当低下,高浓度农药处理的食虫沟瘤蛛组3次施药后13天内的平均种群摄食率只有正常状况下的1/4左右（24.44%）,推荐浓度及低于推荐浓度的食虫沟瘤蛛种群摄食率也仅为对照组的1/3左右（38.69%,36.52%）。因此在对水稻虫害防治时,应当尽可能地避免使用化学农药,充分发挥天敌对害虫数量的调控作用;当害虫数量超过经济阈值而必须施药时,也应当掌握农药用药剂量和次数的尺度,以利于食虫沟瘤蛛等稻田天敌的生理恢复和群落重建。     

高效氯氰菊酯和啶虫脒对螟黄赤眼蜂繁殖的亚致死效应

螟黄赤眼蜂 Trichogramma chilonis是害虫生物防治中一种重要的卵寄生蜂,其在防治害虫的同时,也会受到田间杀虫剂的影响。本研究选择高效氯氰菊酯和啶虫脒亚致死剂量,以两性生命表法计算种群参数,揭示了这两种药剂亚致死剂量对该种群生长、繁殖的影响。试验测定了高效氯氰菊酯和啶虫脒对螟黄赤眼蜂的亚致死剂量LC20值分别为0.119和1.091 mg/L。研究结果显示,经亚致死浓度LC20的啶虫脒处理后,螟黄赤眼蜂的寄生卵量显著低于对照（P<0.05）,寿命(1.17 d)显著缩短,种群参数（内禀增长率 rm、周限增长率λ、净生殖力R0和世代平均历期T）均低于对照,其中净生殖力R0（27.573）显著低于对照（P<0.05）。经亚致死剂量LC20的高效氯氰菊酯处理后,螟黄赤眼蜂的单雌产卵量显著高于对照(P<0.05)。试验结果表明高效氯氰菊酯的亚致死剂量对螟黄赤眼蜂的增殖有一定的刺激作用,而亚致死剂量的啶虫脒则会影响螟黄赤眼蜂的寄生能力,在螟黄赤眼蜂盛发期,田间施用啶虫脒时,应注意其残留量对螟黄赤眼蜂的影响。     

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg)

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.     

Investigations on DNA damage and frequency of micronuclei in occupational exposure to electromagnetic fields (EMFs) emitted from video display terminals (VDTs)

The potential effect of electromagnetic fields (EMFs) emitted from video display terminals (VDTs) to elicit biological response is a major concern for the public. The software professionals are subjected to cumulative EMFs in their occupational environments. This study was undertaken to evaluate DNA damage and incidences of micronuclei in such professionals. To the best of our knowledge, the present study is the first attempt to carry out cytogenetic investigations on assessing bioeffects in personal computer users. The study subjects (n = 138) included software professionals using VDTs for more than 2 years with age, gender, socioeconomic status matched controls (n = 151). DNA damage and frequency of micronuclei were evaluated using alkaline comet assay and cytochalasin blocked micronucleus assay respectively. Overall DNA damage and incidence of micronuclei showed no significant differences between the exposed and control subjects. With exposure characteristics, such as total duration (years) and frequency of use (minutes/day) sub-groups were assessed for such parameters. Although cumulative frequency of use showed no significant changes in the DNA integrity of the classified sub-groups, the long-term users (> 10 years) showed higher induction of DNA damage and increased frequency of micronuclei and micro nucleated cells.     

Assessment of DNA damages in lymphocytes of agricultural workers exposed to pesticides by comet assay in a cross-sectional study

Purpose: To assess the predictive power of the comet assay in the context of occupational exposure to pesticides.

Materials and methods: The recruited subjects completed a structured questionnaire and gave a blood sample. Exposure to pesticides was measured by means of an algorithm based on Dosemeci’s work (Agricultural Health Study). Approximately 50 images were analyzed for each sample via fluorescence microscopy. The extent of DNA damage was estimated by tail moment (TM) and is the product of tail DNA (%) and tail Length.

Results: Crude significant risks (odds ratios, ORs) for values higher than the 75th percentile of TM were observed among the exposed subjects (score?>?1). The frequency of some confounding factors (sex, age and smoking) was significantly higher among the exposed workers. A significant dose–effect relationship was observed between TM and exposure score. Significant high-risk estimates (ORs), adjusted by the studied confounding factors, among exposure to pesticides and TM, % tail DNA and tail length were confirmed using unconditional logistic regression models.

Conclusions: The adjusted associations (ORs) between the comet parameters and exposure to pesticides were significant. The sensitivity of the comet test was low (41%), the specificity (89%) and the predictive positive value (0.77) were found acceptable.     

Genotoxicity of cadmium chloride in human lymphocytes evaluated by the comet assay and cytogenetic tests

Peripheral blood lymphocytes were tested in vitro for genotoxic effects of cadmium chloride. Whole blood samples of four healthy, non-smoking subjects were preincubated with CdCl2 in concentrations of 10(-4), 10(-3), and 5 . 10(-3) mol/L for three hours before the cells were assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay) or cultivated for chromosomal aberrations (CA), sister chromatid exchanges (SCE), and the micronucleus (MN) test. The comet assay showed notable interindividual differences. The results of the cytogenetic tests showed an increase in the frequency of CA, MN, and SCE with CdCl2 in the treated cultures, yet none was able to show a correlation between concentrations of cadmium chloride and the frequency of damages. The MN slides were stained with Giemsa and with DNA fluorochrome 4', 6'-diamidino-2-phenylindole (DAPI). The frequency of MN in slides stained with DAPI was significantly higher than in those stained with Giemsa, which might be due to an underestimation of small micronuclei in Giemsa-stained slides.     

Low sensitivity of the comet assay to detect acetaldehyde-induced genotoxicity

Acetaldehyde (AA) is known to induce DNA-protein cross-links (DPX) and other genotoxic and mutagenic effects in cultured mammalian cells. Compared to formaldehyde (FA), AA is a very weak inducer of DPX and increased DPX levels are only measured at high, cytotoxic concentrations by different methods. Besides DPX, AA also induces DNA-DNA cross-links. Because the comet assay is increasingly used for the detection of cross-linking agents, we characterized the effects of AA in the comet assay in relation to cytotoxicity and other genetic endpoints such as the induction of sister chromatid exchange (SCE) and micronuclei (MN). The standard alkaline comet assay did not indicate induction of DNA strand-breaks by AA in a range of concentrations from 0.2 to 20 mM. AA at a concentration of 20 mM was clearly cytotoxic and reduced cell growth and population doubling to less than 50% of the control. Using the comet assay modification with proteinase K, slightly enhanced DNA migration was measured in comparison to treatment with AA only. No significant induction of cross-links by AA (measured as reduction of gamma ray-induced DNA migration) was determined by the comet assay. A small and reproducible but statistically not significant effect was measured for the AA concentration 20 mM. A clear and concentration-related increase in the frequency of sister chromatid exchange (SCE) and micronuclei (MN) was already measured at lower concentrations (0.2 and 0.5 mM, respectively). These results suggest that the comet assay has a low sensitivity for the detection of AA-induced DNA lesions leading to the induction of SCE and MN.     

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg)

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.     

Genotoxic effects of styrene-7,8-oxide in human white blood cells: comet assay in relation to the induction of sister-chromatid exchanges and micronuclei

Styrene is used in the production of plastics, resins and rubber. The highest human exposures to styrene take place by inhalation during the production of fiberglass reinforced plastics. Styrene is metabolized mainly in the liver to styrene-7,8-oxide (SO), its principal in vivo mutagenic metabolite. In this study, human peripheral white blood cells were exposed to several SO concentrations (10-200 microM) in order to evaluate its genotoxic properties by means of comet assay, sister-chromatid exchanges (SCE) and cytokinesis-blocked micronucleus (MN) test, in addition to determine its clastogenic or aneugenic properties by combining MN with fluorescence in situ hybridization (FISH) procedures. Our results show that SO induces DNA damage, SCE and MN in human leukocytes in vitro at concentrations above 50 microM, and that there is a strong relationship between DNA damage, as measured by the comet assay, and cytogenetic damage induced by SO at the doses employed. SO shows preferentially a clastogenic activity and produces a cytostatic effect at high doses, reflected by the significant decrease of the calculated proliferation indices. A good dose-effect relationship is obtained in the three tests performed at the concentration range assayed.     

Examination of various biomarkers measuring genotoxic endpoints from Barcelona airport personnel

Three different biomarkers: sister-chromatid exchanges (SCE), micronuclei (MN), and the Comet assay, were used to evaluate different kinds of genetic damage in peripheral blood lymphocytes from 34 male workers at Barcelona airport, exposed to low levels of hydrocarbons and jet fuel derivatives. The control group consisted of 11 unexposed men. We also investigated the ras p21 protein levels in plasma, in order to evaluate whether the ras gene could serve as a suitable potential marker of carcinogenic pollution in occupationally exposed cohorts. SCE and MN analyses failed to detect any statistically significant increase in the airport workers when compared with the controls, and in fact, the frequency of binucleated cells with MN in the exposed group was significantly lower than that obtained in the control. However, slight but significant differences in the mean comet length and genetic damage index were observed between the exposed and control groups when using the Comet assay. There were no statistically significant differences between both groups in p21 plasma levels. Smoking was shown to affect significantly both SCE and high frequency cells (HFC) in the exposed group.