三种环境激素对四种海洋微藻的急性毒性效应研究

近年来,海洋环境激素污染日益严重,为了考察环境激素对海洋微藻的生物毒性效应,进而评估其对海洋生态系统的影响,该实验研究了三氯卡班、邻苯二甲酸二丁酯、三丁基氯化锡三种环境激素对海洋小球藻(Chlorella sp.)、眼点拟微绿球藻(Nannochloropsis ocutala)、球形棕囊藻(Phaeocystis globosa)和东海原甲藻(Prorocentrum donghaiense )4种海洋微藻的急性毒性效应。结果表明,三种环境激素均可显著抑制该4种微藻的生长。三氯卡班对4种微藻的96 h-EC₅₀分别为108.19 μg·L^-1、63.21 μg·L^-1、60.73 μg·L^-1和57.58 μg·L^-1;邻苯二甲酸二丁酯对4种微藻的96 h-EC₅₀分别为1.42 mg·L^-1、1.02 mg·L^-1、1.47 mg·L^-1 和1.21 mg·L^-1;三丁基氯化锡对4种微藻的96 h-EC₅₀分别为3.5 μg·L^-1、4.36 μg·L^-1、0.6 μg·L^-1和0.6 μg·L^-1。三种环境激素对四种海洋微藻的毒性强弱顺序为三丁基氯化锡>三氯卡班>邻苯二甲酸二丁酯。     

九种海洋微藻总脂含量及脂肪酸组成分析

从广东沿海分离出9 种海洋微藻(包括6 种绿藻, 2 种金藻, 1 种硅藻), 在低氮并持续通入二氧化碳(1% CO2)条件下测试其总脂含量, 对其中总脂含量超过其干重45%的海洋微藻进行脂肪酸组成分析并优化反应条件。结果表明,9 种海洋微藻的总脂含量存在着明显差异, 介于8.9%-55.77%之间。其中, 海洋小球藻Chlorella sp.、眼点拟微绿球藻Nannochloropsis oculata、绿色巴夫藻Pavlova viridis 和微拟球藻Nannochloropsis sp.的总脂含量超过干重的45%, 而且, 这4 种富油海洋微藻的C16 和C18 脂肪酸含量丰富, C16:0、C16:1(n-7)、C18:1(n-9)含量较高, C14-C18 长链脂肪酸的含量超过总脂肪酸含量的80%。4 种高脂微藻在30 ℃高温培养条件下比生长速率均超过1.0 d−1。研究结果可为高温富油海洋微藻的工业化培养提供参考依据。     

微藻叶绿素荧光值与传统生长指标的关联性研究

利用TD-700型叶绿素荧光仪测定了6种不同海洋和淡水微藻,即锥状斯氏藻(Scrippsiella trochoidea)、球形棕囊藻(Phaeocystis globosa)、三角褐指藻(Phaeodactylum tricornutum)、眼点拟微绿球藻(Nannochloropsis oculata)、四列藻(Tetraselmis tetrethele)和雨生红球藻(Haematococcus pluvialis)的叶绿素荧光值,同时采用经典方法同步测定其细胞密度和叶绿素a含量。统计分析结果显示,微藻细胞密度、叶绿素a含量、叶绿素荧光值三者之间具有显著或极显著的正相关关系。因此,利用叶绿素荧光仪测定微藻生物量的方法是切实可行的,具有快捷方便、灵敏度高的优点。     

两阶段氮源补料策略促进微拟球藻EPA高效积累

为促进微拟球藻EPA高效积累,本研究探索了光照强度和氮源种类对微拟球藻生理生化及EPA相对含量的影响;根据光照和氮源实验结果设计两阶段氮源补料策略,并优化了氮源补料时间。结果表明:光强从1 000 lux增加至9 000 lux,微拟球藻比生长速率从0.25 d~(-1)增加至0.54 d-1,油脂含量从11.4%增加至20.2%,而EPA相对含量从25.5%降低至13.1%;培养10 d后乙酸铵组生物量和油脂含量最高,分别为0.41 g·L~(-1)和14.3%,硝酸钠组EPA相对含量最高,为27.1%;两阶段硝酸钠加乙酸铵培养模式,最佳乙酸铵补料时间为第11 d,培养20 d最终生物量及EPA相对含量分别为0.68 g·L~(-1)和24.3%。采用两阶段氮源补料策略能促进微拟球藻高效积累EPA。     

HPLC-ELSD快速测定微藻中性脂

溶剂法提取微藻油脂不同于植物种子油脂,它是全细胞的提取物,成分非常复杂,存在与甘油三酯(TAG)在色谱保留性质上相近的低极性物质,干扰TAG的测定。建立了基于二醇基柱及蒸发光检测器、正己烷-异丙醇为流动相的快速测定微藻中性脂的方法。对该方法进行评价,结果显示,测定的TAG、游离脂肪酸(FFA)、甘油二酯(DAG)、甘油一酯(MAG)线性相关系数均大于0.99,方法重复性好。湛江等鞭金藻及微拟球藻样品中TAG加标回收率为96.2%~113.1%,相对标准偏差(RSD)为0.46%~4.8%。将本方法测定湛江等鞭金藻及微拟球藻中TAG的含量并与传统的固相萃取(SPE)及常用的TLC/GC测定TAG的方法进行比较,相比上述两种方法,该方法前处理简单、灵敏度高,可快速准确测定微藻中TAG的含量。     

缺氮和高光对集球藻光合生理与油脂积累的影响

微藻是生产生物柴油理想的生物资源,对产油微藻的生理生化特性的研究具有重要意义。本文以一种耐碱性产油微藻集球藻为研究对象,将其置于两种光强水平(100和600μmol photons·m~(-2)·s~(-1))下培养,探讨缺氮对集球藻光合生理及油脂积累的影响。结果表明:缺氮培养下,集球藻光系统II的最大光化学效率F_v/F_m下降,电子从QA-到QB的电子传递受阻,光合作用受到抑制,生长表现缓慢,蛋白质含量降低,而油脂含量增加;在高光的耦合作用下,其光合作用进一步受到抑制,蛋白质含量下降,但油脂含量与低光缺氮条件下相比,没有显著变化。研究表明,缺氮使集球藻碳同化进入油脂合成代谢,增加其油脂含量,而光强对油脂积累的诱导效应不明显。     

响应面法对微拟球藻产微藻蛋白的发酵条件优化

以稳定期微藻蛋白浓度为评价指标,利用响应面设计对微拟球藻(Nannochloropsis gaditana)的分批发酵条件进行优化。在单因素试验的基础上,选取温度、p H、搅拌速度及通气量为影响因子,采用四因素三水平的Box-Benhnken中心组合法设计试验。结果表明:微拟球藻的最佳发酵条件为温度30℃、p H 6.9、搅拌速度340 r/min以及通气量0.65 vvm,在此优化条件下得到微藻蛋白浓度为6.18 g/L,与模型预测值基本相符,较优化前提高了9.18%。     

2,4-表油菜素内酯和赤霉素对微拟球藻产油率及脂肪酸组成的影响

分别研究了2,4-表油菜素内酯(EBR)、赤霉素(GA_3)对微拟球藻大洋种生长、产油率及脂肪酸组成的影响。结果表明,5、15 mg/L EBR能显著促进微拟球藻生长和提高其产油率,产油率较对照组分别增加了7.85%和13.97%,但对单位质量藻细胞总脂含量影响不大。50 mg/L EBR则显著抑制该藻生长。5、15和50 mg/L GA_3均显著促进微拟球藻生物量积累。50 mg/L GA_3处理组生物量最高(0.337 g/L),但总脂积累受到一定抑制。总体来看,15 mg/L GA_3对微拟球藻产油率提升效果最显著,达8.184 mg/(L·d)。添加EBR(5、15 mg/L)和GA_3(5、15、50 mg/L)浓度后,饱和及单不饱和脂肪酸(16∶0、16∶1、18∶1)占比下降,多不饱和脂肪酸(18∶2、20∶4和20∶5)占比显著提高。     

富油能源微藻的筛选及产油性能评价

为了筛选具有产油潜力的能源微藻,以实验室保藏的20株淡水和海洋微藻(绿藻门18株,真眼点藻纲1株,硅藻纲1株)为研究对象,利用光径为3 cm柱状光生物反应器通气分批培养,通过测定微藻培养物的生物量和总脂含量等指标,从中筛选生长速度快、生物量和总脂含量高的微藻。结果表明:20株微藻的生物量和总脂含量分别在1.81~7.88g/L和16.0%~55.9% dw(% Dry weight)之间,筛选得到具有产油潜力的微藻9株,分别是栅藻(Scenedesmus sp.)(6.34g/L,55.9% dw)、麻织绿球藻(Chlorococcum tatrense)(5.93g/L,46.9% dw)、眼点拟微绿球藻(Nannochloropsis oculata)(7.88g/L,35.0% dw)、油面绿球藻(Chlorococcum oleofaciens)(5.58g/L,45.9% dw)、多形拟绿球藻(Pseudochlorococcum polymorphum)(6.10g/L,40.0% dw)、八月衣藻(Chlamydomonas augustae)(5.78g/L,40.5% dw)、椭圆小球藻(Chlorella ellipsoidea)(5.56g/L,40.7%dw)、椭圆绿球藻(Chlorococcum ellipsoideum)(5.41g/L,38.0% dw)、雪绿球藻(Chlorococcum nivale)(5.55g/L,36.3% dw),其中最具产业化潜力的微藻为栅藻(Scenedesmus sp.),其总脂收获量和单位体积总脂产率分别为3.5 g/L和218.7mg/L·d。     

富碳培养对海洋富油微藻油脂积累特性的影响

实验针对三株海洋富油微藻:球等鞭金藻（Isochrysis galbana CCMM5001）、一种等鞭金藻（Isochrysis sp. CCMM5002）和一种微拟球藻（Nannochloropsis sp. CCMM7001）,研究了它们在通入0.03%（空气）、5%、10%三个浓度CO2培养条件下的生长特性,同时考察了其总油脂及中性脂的累积情况。结果显示,富碳培养有利于这三株海洋微藻的生长,但最适生长的CO2浓度不同。球等鞭金藻（Isochrysis galbana CCMM5001）和等鞭金藻（Isochrysis sp. CCMM5002）在通入10% CO2时具有最大产率,分别达到（182.287.07） mg/（Ld）和（164.227.10） mg/（Ld）,而微拟球藻在通入5%时具有最大产率,达到（122.251.17） mg/（Ld）,随着CO2浓度的增加,三株海洋微藻的总脂含量和中性脂含量有明显提高。在通入10% CO2条件下,球等鞭金藻（Isochrysis galbana CCMM5001）、等鞭金藻（Isochrysis sp. CCMM5002）和微拟球藻（Nannochloropsis sp. CCMM7001）的总脂含量分别达到（45.154.03）%、（47.151.20）%和（41.201.69）%;从中性脂的累积规律来看,三株藻均在平台期的累积达到最大,脂肪酸分析结果表明三株藻种适合制备生物柴油的C14-C18系脂肪酸相对含量在不同CO2条件下基本保持不变,维持在90%左右。实验结果显示,研究的藻株作为富油高固碳优良藻株,具备用于海洋生物质能耦合CO2减排开发的潜力。       

Benzo(a)pyrene diolepoxide adducts to albumin in workers exposed to polycyclic aromatic hydrocarbons: association with specific CYP1A1, GSTM1, GSTP1 and EHPX genotypes

We investigated whether the presence of (+)-anti-benzo(a)pyrene diolepoxide adducts to serum albumin (BPDE-SA) among workers exposed to benzo(a)pyrene (BaP) and unexposed reference controls was influenced by genetic polymorphisms of cytochrome P4501A1 (CYP1A1), microsomal epoxide hydrolase (EHPX), glutathione S-transferases M1 (GSTM1) and P1 (GSTP1), all involved in BaP metabolism. Exposed workers had significantly higher levels of adducts (0.124 ± 0.02 fmol BPTmg^-1 SA, mean ± SE) and a higher proportion of detectable adducts (40.3%) than controls (0.051 ± 0.01 fmol BPT mg^-1 SA; 16.1%) (p = 0:014 and p = 0:012). Smoking increased adduct levels only in occupationally exposed workers with the GSTM1 deletion (GSTM1 null) (p = 0:034).

Smokers from the exposed group had higher adduct levels when they were CYP1A1 *1/*1 wild-type rather than heterozygous and homozygous for the variant alleles (CYP1A1 *1/*2 plus *2/*2) (p = 0:01). The dependence of BPDE-SA adduct levels and frequency on the CYP1A1 *1/*1 genotype was most pronounced in GSTM1-deficient smokers. Exposed workers with GSTM1 null/GSTP1 variant alleles had fewer detectable adducts than those with the GSTM1 null/GSTP1*A wild-type allele, supporting for the first time the recent in vitro finding that GSTP1 variants may be more effective in the detoxification of BPDE than the wild-type allele. Logistic regression analysis indicated that occupational exposure, wild-type CYP1A1*1/*1 allele and the combination of GSTM1 null genotype+EHPX genotypes associated with predicted low enzyme activity were significant predictors of BPDE-SA adducts. Though our findings should be viewed with caution because of the relatively limited size of the population analysed, the interaction between these polymorphic enzymes and BPDE-SA adducts seems to be specific for high exposure and might have an impact on the quantitative risk estimates for exposure to polycyclic aromatic hydrocarbons.     

Influence of GSTM1, GSTT1, GSTP1, and EPHX gene polymorphisms on DNA adduct level and HPRT mutant frequency in coke-oven workers

To evaluate the influence of individual susceptibility factors on the level of polyaromatic (PAH) hydrocarbon DNA adducts and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutants in peripheral lymphocytes, 70 coke-oven workers exposed to PAH were genotyped for four metabolic enzyme polymorphisms of potential importance in PAH metabolism. The examined genetic polymorphisms concerned glutathione S-transferases M1 (GSTM1; gene deletion; 96 workers), T1 (GSTT1; gene deletion), P1 (GSTP1; Ile→Val substitution at codon 104 or Ile→Val at codon 104 and Val→Ala at codon 113), and microsomal epoxide hydrolase (EPHX; Tyr→His substitution at codon 113 and His→Arg at codon 139). The workers were classified in a high- and low-exposure group on the basis of urinary concentration of 1-pyrenol. The GSTM1 null genotype increased the number of DNA adducts in smoking coke-oven workers with high PAH exposure. DNA adducts were affected by PAH-exposure in non-smokers and in GSTM1 null smokers and by smoking in GSTM1 null individuals. In a multiple linear regression analysis, the interaction of the GSTM1 genotype was statistically significant (p=0.04) with smoking (yes/no) and of borderline significance (p=0.06) with PAH-exposure (high/low). As smoking also increased urinary 1-pyrenol, the genotype modification seemed to concern DNA adducts due to smoking rather than occupational exposure. GSTT1 positive individuals showed an elevated level of DNA adducts in comparison with GSTT1 null subjects (p=0.04), and EPHX genotypes associated with slow hydroxylation reaction yielded a higher (p=0.05) HPRT mutant frequency than fast EPHX genotypes; these findings were, however, based on small numbers of subjects and need to be clarified in further studies. In conclusion, our findings indicate that homozygous deletion of GSTM1 results in an increased sensitivity to genotoxic PAHs in tobacco smoke, which is seen as an increase in aromatic DNA adducts in blood mononuclear cells.     

Analysis of genetic variants of the poly(ADP-ribose) polymerase-1 gene in breast cancer in French patients

Previous studies have provided evidence for an association between exposure to high levels of air pollution and increased DNA damage in human sperm. In these studies DNA damage was measured using the sperm chromatin structure assay (SCSA) wherein the percentage of sperm with abnormal chromatin/fragmented DNA is determined and expressed as % DNA fragmentation index (%DFI). Here we extend these observations to address the following hypothesis: men who are homozygous null for glutathione-S-transferase M1 (GSTM1−) are less able to detoxify reactive metabolites of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) found in air pollution. Consequently they are more susceptible to the effects of air pollution on sperm chromatin. Using a longitudinal study design in which men provided semen samples during periods of both low (baseline) and episodically high air pollution, this study revealed a statistically significant association between GSTM1 null genotype and increased SCSA-defined %DFI (beta = 0.309; 95% CI: 0.129, 0.489). Furthermore, GSTM1 null men also showed higher %DFI in response to exposure to intermittent air pollution (beta = 0.487; 95% CI: 0.243, 0.731). This study thus provides novel evidence for a gene–environment interaction between GSTM1 and air pollution (presumably c-PAHs). The significance of the findings in this study with respect to fertility status is unknown. However, it is biologically plausible that increases in %DFI induced by such exposures could impact the risk of male sub/infertility, especially in men who naturally exhibit high levels of %DFI.     

Genetic polymorphisms in GSTM1, GSTT1, GSTP1, GSTM3 and the susceptibility to gallbladder cancer in North India

The glutathione S-transferase (GSTs) are polymorphic supergene family of detoxification enzymes that are involved in the metabolism of numerous potential carcinogens. Several allelic variants of polymorphic GSTs show impaired enzyme activity and are suspected to increase the susceptibility to various cancers. To find out the association of GST variants with risk of gallbladder cancer, the distribution of polymorphisms in the GST family of genes (GSTT1, GSTM1, GSTP1, and GSTM3) were studied in 106 cancer patients and 201 healthy controls. Genotypes were analysed by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP). The frequencies of GSTM1 null and GSTM3*BB genotypes did not differ between patients and controls. The overall frequency of GSTT1 null was lower in cases as compared with controls (p=0.003, Odds ratio (OR) = 0.2, 95% confidence interval (CI), 0.1-0.6). After sex stratification, the GSTT1 null frequency was reduced only in female patients (p=0.008, OR = 0.2, 95% CI = 0.1-0.6). However, the GSTP1, ile/val genotype and the val allele were significantly higher in cases than controls (p=0.013, OR = 1.9, 95% CI = 1.1-3.1; p=0.027, OR = 1.5, 95% CI = 1.0-2.1), respectively. To study gene-gene interactions, a combined risk of gallbladder cancer due to ile/val or val/val were calculated in combination with null alleles of GSTM1 and GSTT1 or the *B allele of GSTM3, but there was no enhancement of risk. Gallstones were present in 57.5% of patients with gallbladder cancer, but there were no significant differences between allelic/genotype frequencies of the studied GST genes polymorphisms between patients with or without gallstones. To best of our knowledge, this is the first paper showing ile/val genotypes and val allele of GSTP1 to be associated with higher risk of gallbladder cancer.     

Interaction between smoking, GSTM1 deletion and colorectal cancer: results from the GSEC study

Cigarette smoking has inconsistently been associated with an increased risk of colorectal cancer. One of the enzymes responsible for the detoxification of the carcinogenic compounds present in tobacco smoke is glutathione S-transferase-μ (GST-μ). The gene that codes for this enzyme is GSTM1. In this study, we evaluated the associations and interaction between GSTM1 deletion, smoking behaviour and the development of colorectal cancer. We performed a pooled analysis within the International Collaborative Study on Genetic Susceptibility to Environmental Carcinogens (GSEC). We selected six studies on colorectal cancer, including 1130 cases and 2519 controls, and restricted our analyses to Caucasians because the number of patients from other races was too limited. In addition we performed a meta-analysis including the studies from the GSEC database and other studies identified on MEDLINE on the same subject. The prevalence of the GSTM1 null genotype was within the range reported in other studies: 51.8% of the cases had the GSTM1 null genotype versus 56.6% of the controls. No significant association between the GSTM1 null genotype and colorectal cancer was found (odds ratio 0.92, 95% confidence interval 0.73-1.14). Our results suggest a possible positive association between lack of the GST-μ enzyme and colorectal cancer for non-smoking women (odds ratio 1.47, 95% confidence interval 0.80-2.70). There was no interaction between the effects of smoking and GSTM1 genotype on colorectal cancer risk in men and women (χ²=0.007, p=0.97). Our findings do not support an association between the GSTM1 null genotype and colorectal cancer. In addition, we did not find any modification of the smoking-induced colorectal cancer risk by GSTM1 genotype     

共生菌促进斜生栅藻生长和油脂合成

从斜生栅藻藻际分离共生菌群并进行16S rDNA序列鉴定,构建藻菌共培养体系,并检测斜生栅藻生长、生理生化及产油特性。共分离出7个菌群/株,包括微球菌(菌株1-1、1-2和1-3)、假单胞菌(菌株2-1和2-2)、微小杆菌(菌株-3)和葡萄球菌(菌株-4)。微球菌(菌株1-2)和假单胞菌(菌株2-1)为优势促生菌株,能显著促进微藻生长及色素和油脂积累。斜生栅藻与微球菌(菌株1-2)1∶10共培养体系培养8 d后,栅藻生物量高达4.27 g·L^-1,比对照增加了46.0%;叶绿素a、叶绿素b和类胡萝卜素分别比对照提高12.1%、16.7%和25.0%;含油量为25.7%,比对照增加了14.0%,单不饱和油酸含量(16.4%)也显著高于对照。另一个优异共培养体系是斜生栅藻与假单胞菌(菌株2-1)1∶5共培养体系,在培养8 d后,微藻生物量、叶绿素a、叶绿素b和类胡萝卜素分别比对照提高47.9%、16.0%、17.5%和19.9%;总油脂(27.1%)和单不饱和油酸含量(18.2%)分别比对照增加了20.4%和64.0%。表明藻际共生菌假单胞菌和微球菌可分别与斜生栅藻互作,能够显著促进斜生栅藻生物量和优质油脂的富集,可应用于栅藻商业生产。     

过表达GDP-D-甘露糖焦磷酸化酶(GMP)基因提高灵芝多糖的生产

灵芝多糖是灵芝的主要生物活性成分之一,具有多种药理活性,但灵芝多糖的低产量限制了其广泛应用.相关研究表明,灵芝中过表达多糖生物合成相关基因能够提高多糖产量,但过表达GDP-D-甘露糖焦磷酸化酶(GMP)基因提高灵芝多糖产量未有报道.本研究克隆获得了灵芝gmp基因,并成功构建出了过表达gmp基因的工程菌株(GMP菌株),...     

互补交配型配对诱导蛹虫草有性子实体形成

前期我们获得了优良性状蛹虫草Cordyceps militaris野生菌株W141436,其子实体虫草素含量高达3.72 mg/kg干重,子实体多糖高达6.7 g/100 g干重,但在大规模应用中发现它发生退化。针对蛹虫草人工栽培退化问题,我们提出蛹虫草子实体形成是有性生殖过程,其两种交配型发生交配是蛹虫草子实体形成的必要条件。本文基于交配型基因分子标记研究了优良性状蛹虫草野生菌株W141436的退化机制。具体地,采用单孢子分离的方法对蛹虫草野生分离株W141436的子囊孢子进行分离,得到了72个单孢菌株。进一步采用PCR方法对单孢菌株及亲本菌株进行了交配型基因类型鉴定。在72个单孢菌株中,28株为Mat1-1类型交配基因型,31株为Mat1-2类型交配基因型,13株含有与亲本相同的交配型基因。根据鉴定结果,对2株Mat1-1型(SP28、SP33)和2株Mat1-2型(SP15、SP32)菌株进行了栽培实验。结果表明,形成子实体的栽培用亲本菌株同时含有Mat1-1和Mat1-2两种交配类型基因,即只有含不同交配型基因的菌株具有发育为子实体的能力,而含同种交配型基因的菌株则不能发育为子实体。本研究为防止蛹虫草在大规模种植中退化提供了理论依据。     

Polymorphisms in GSTT1 and p53 and urinary transitional cell carcinoma in south-western Taiwan: A preliminary study

Little is known about the relevance of genetic polymorphisms to arsenic-related bladder cancer. A preliminary case-control study was conducted to explore the association between genetic polymorphisms of GSTT1, p53 codon 72 and bladder cancer in southern Taiwan, a former high arsenic exposure area. Fifty-nine urinary transitional cell carcinoma (TCC) patients from a referral centre in south-western Taiwan and 81 community controls matched on residence were recruited from 1996 to 1999. A questionnaire was administered to obtain arsenic exposure and general health information. Genotypes of p53 codon 72 and GSTT1 were analysed by polymerase chain reaction-restriction fragment length polymerase. The combined variant genotypes (heterozygous or homozygous variant) of p53 codon 72 and GSTT1 null were observed in 29% of cases and in 44% of controls, respectively. In this preliminary study, bladder cancer risk was slightly elevated for subjects carrying the variant genotype of p53 codon 72 or in subjects carrying the GSTT1 null genotype. Variants in p53 codon 72 increased the risk of bladder cancer among smokers. However, the results were not statistically significant and larger confirmatory studies are needed to clarify the role of candidate gene polymorphisms and bladder cancer risk in arsenic exposed populations.