Reporter genes produce a protein product in transfected cells that can be easily measured in intact or lysed cells and they have been extensively used in numerous basic and applied research applications.Over the past ...Reporter genes produce a protein product in transfected cells that can be easily measured in intact or lysed cells and they have been extensively used in numerous basic and applied research applications.Over the past 10 years,reporter gene assays have been widely accepted and used for analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like compounds in various types of matrices,such as biological,environmental,food and feed samples,given that high-resolution instrumental analysis techniques are impractical for large-scale screening analysis.The most sensitive cell-based reporter gene bioassay systems developed are the mechanism-based CALUX(Chemically Activated Luciferase Expression) and CAFLUX(Chemically Activated Fluorescent Expression) bioassays,which utilize recombinant cell lines containing stably transfected dioxin(AhR) responsive firefly luciferase or enhanced green fluorescent protein(EGFP) reporter genes,respectively.While the current CALUX and CAFLUX bioassays are very sensitive,increasing their lower limit of sensitivity,magnitude of response and dynamic range for chemical detection would significantly increase their utility,particularly for those samples that contain low levels of dioxin-like HAHs(i.e.,serum) .In this study,we report that the addition of modulators of cell signaling pathways or modification of cell culture conditions results in significant improvement in the magnitude and overall responsiveness of the existing CALUX and CAFLUX cell bioassays.展开更多
Objective To improve the chemically-activated luciferase expression (CALUX)bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms ofDLCs. Method A recombinant vector was construc...Objective To improve the chemically-activated luciferase expression (CALUX)bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms ofDLCs. Method A recombinant vector was constructed and used to transfect humanhepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor(AhR)-meditated luciferase gene expression. The reliability of luciferase induction in thiscell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection timewas examined and a comparison was made by using the commonly used ethoxyresoufin-O-deethylase (EROD) activity induction assay. Result The results suggested that theluciferase activity in recombinant cells was peaked at about 4 h and then decreased to astable activity by 14 h after TCDD treatment. The detection limit of this cell line was0.11pmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%.Conclusion The luciferase induction is 30-fold more sensitive than EROD induction, thedetection time is 68 h shorter and the detection procedure is also simpler.展开更多
[Objectives]To determine the content of organic pollutants by CALUX bioassay.[Methods]According to JJF 1059.1-2012 Technical Specification for Evaluation and Expression of Uncertainty in Measurement,the determination ...[Objectives]To determine the content of organic pollutants by CALUX bioassay.[Methods]According to JJF 1059.1-2012 Technical Specification for Evaluation and Expression of Uncertainty in Measurement,the determination results of organic pollutants were analyzed,various sources of uncertainty that may be introduced in the detection process were analyzed,and the mathematical model of uncertainty was established.Type A and B evaluation methods were used to calculate the components of uncertainty and extended uncertainty.[Results]In the range of 95%confidence interval,the determination result of organic pollutants was 6.59 pg/g fat,and the extended uncertainty was 0.0764 pg/g fat,so the determination results of organic pollutants could be expressed as(6.59±0.0764)pg/g fat(k=2).[Conclusions]This study can provide a basis for more accurate determination of organic pollutants.展开更多
This review compiles information on PCDD/F-and PCB-contaminated eggs from 20 years of global egg monitoring around emission sources in four continents conducted by the International Pollutants Elimination Network(IPEN...This review compiles information on PCDD/F-and PCB-contaminated eggs from 20 years of global egg monitoring around emission sources in four continents conducted by the International Pollutants Elimination Network(IPEN)and Arnika as well as a compilation of data from scientific literature.IPEN monitored 127 pooled egg samples including samples from 113 chicken flocks at potential PCDD/F-and PCB-contaminated sites around priority sources listed in the Stockholm Convention(e.g.waste incinerators,metal industries,cement plants,and open burning).99(88%)of pooled egg samples were above the EU maximum limits for PCDD/Fs(2.5 pg PCDD/F-TEQ/g fat)or the sum of PCDD/Fs and dioxinlike PCBs(5 pg PCDD/F-PCB-TEQ/g fat).Children consuming such eggs exceed the tolerable weekly intake(TWI).This demonstrates that close to 90%of these areas were not safe for the production of free-range eggs.Sixteen out of the 113 egg samples(14%)were contaminated above 50 pg TEQ/g fat and exceeded the EU maximum limit more than 10 times.From the 26 pooled egg samples around incinerators 24(92%)exceeded the limit with a mean of 43.1 pg TEQ/g fat(2.6e234 pg TEQ/g).All 21 egg samples around metal industries(4.4e112.6 pg TEQ/g fat)were above limits with mean concentration of 26.0 pg TEQ/g fat.Also all 7 egg samples measured at e-waste recycling sites were above limits(mean 308 pg TEQ/g fat).In 58(51%)pooled egg samples the PCB-TEQ was above 5 pg TEQ/g fat exceeding the EU maximum limit with dioxin-like PCBs alone.This highlights the role of commercial PCBs for global contamination with dioxin-like compounds.It was discovered that around metal industries,shredder plants,open burning sites of e-waste and dump sites,a high share of contamination was caused by dl-PCBs.This clearly shows severe PCB release from the end-of-life management of PCB-containing equipment in developing countries.Also highly contaminated eggs were found at many sites where plastic was incinerated.The highest contaminated egg sample ever measured came from an e-waste site in Ghana and had 856 pg TEQ/g fat plus 300 pg TEQ from brominated dioxins(PBDD/Fs).Other extreme PCDD/F contaminations of eggs were found at a chlor-alkali site(514 pg TEQ/g fat),Agent Orange contaminated areas in Vietnam(490,249 and 246 pg TEQ/g fat)and e-waste sites(568 and 520 pg TEQ/g fat).Where DR CALUX®bioassay revealed higher TEQ compared to measured PCDD/F-PCB-TEQ in IPEN studies,polybrominated PBDD/F were also measured and detected up to 300 pg TEQ/g fat at e-waste sites.One positive outcome from the IPEN studies is that all 10 pooled supermarket eggs in developing countries were below regulatory limit.Policy recommendations are made including:a systematic assessment of areas around PCDD/Fs and PCBs sources;measures for reduction of exposures of populations;urgent control of emission sources including PCB equipment,the open burning of plastic,and the use of plastic as fuel in boilers/incinerators in developing countries without air pollution control.Furthermore,soil limits need to be re-assessed and lowered for free-range poultry.展开更多
基金support from the National Institutes of Environmental Health Sciences Superfund Basic Research Grant(ES004699(MSD))the California Agricultural Experiment Station,the Startup Fund of 100 Talents Program of Chinese Academy of Sciences+1 种基金the National Natural Science Foundation of China (20921063)the National Basic Research Program(2010CB933500)
文摘Reporter genes produce a protein product in transfected cells that can be easily measured in intact or lysed cells and they have been extensively used in numerous basic and applied research applications.Over the past 10 years,reporter gene assays have been widely accepted and used for analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like compounds in various types of matrices,such as biological,environmental,food and feed samples,given that high-resolution instrumental analysis techniques are impractical for large-scale screening analysis.The most sensitive cell-based reporter gene bioassay systems developed are the mechanism-based CALUX(Chemically Activated Luciferase Expression) and CAFLUX(Chemically Activated Fluorescent Expression) bioassays,which utilize recombinant cell lines containing stably transfected dioxin(AhR) responsive firefly luciferase or enhanced green fluorescent protein(EGFP) reporter genes,respectively.While the current CALUX and CAFLUX bioassays are very sensitive,increasing their lower limit of sensitivity,magnitude of response and dynamic range for chemical detection would significantly increase their utility,particularly for those samples that contain low levels of dioxin-like HAHs(i.e.,serum) .In this study,we report that the addition of modulators of cell signaling pathways or modification of cell culture conditions results in significant improvement in the magnitude and overall responsiveness of the existing CALUX and CAFLUX cell bioassays.
文摘Objective To improve the chemically-activated luciferase expression (CALUX)bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms ofDLCs. Method A recombinant vector was constructed and used to transfect humanhepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor(AhR)-meditated luciferase gene expression. The reliability of luciferase induction in thiscell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection timewas examined and a comparison was made by using the commonly used ethoxyresoufin-O-deethylase (EROD) activity induction assay. Result The results suggested that theluciferase activity in recombinant cells was peaked at about 4 h and then decreased to astable activity by 14 h after TCDD treatment. The detection limit of this cell line was0.11pmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%.Conclusion The luciferase induction is 30-fold more sensitive than EROD induction, thedetection time is 68 h shorter and the detection procedure is also simpler.
文摘应用化学诱导荧光素酶表达基因法(chemical activated luciferase gene expression,CALUX)调研了小清河(济南段)主要支流的多种内分泌干扰物综合激素效应,明确了4类内分泌干扰物的含量水平及分布特征。结果表明,调研河流的二噁英类、雌激素类和肾上腺皮质激素类的年平均毒性当量浓度范围为0.020~0.040 ng 2,3,7,8-TCDD TEQ·L^(-1)、0.007~0.050 ng E2-eq·L^(-1)和1.8~9.6 ng Dex-eq·L^(-1)。二噁英类和雌激素类的含量水平相对较低,雄激素类内分泌干扰物的污染风险较低,肾上腺皮质激素类是其内分泌干扰物污染的最大贡献者。二噁英类检出水平无明显的季节性和区域性差别,雌激素类季节性变化明显,春、冬两季检出率高,肾上腺皮质激素类分布的季节性分布不明显,但区域性分布规律明显,农业区支流的检出频次和含量水平明显低于居民区和商业区支流。
基金Supported by Special Project of "Grassland Talents" in Inner Mongolia
文摘[Objectives]To determine the content of organic pollutants by CALUX bioassay.[Methods]According to JJF 1059.1-2012 Technical Specification for Evaluation and Expression of Uncertainty in Measurement,the determination results of organic pollutants were analyzed,various sources of uncertainty that may be introduced in the detection process were analyzed,and the mathematical model of uncertainty was established.Type A and B evaluation methods were used to calculate the components of uncertainty and extended uncertainty.[Results]In the range of 95%confidence interval,the determination result of organic pollutants was 6.59 pg/g fat,and the extended uncertainty was 0.0764 pg/g fat,so the determination results of organic pollutants could be expressed as(6.59±0.0764)pg/g fat(k=2).[Conclusions]This study can provide a basis for more accurate determination of organic pollutants.
文摘This review compiles information on PCDD/F-and PCB-contaminated eggs from 20 years of global egg monitoring around emission sources in four continents conducted by the International Pollutants Elimination Network(IPEN)and Arnika as well as a compilation of data from scientific literature.IPEN monitored 127 pooled egg samples including samples from 113 chicken flocks at potential PCDD/F-and PCB-contaminated sites around priority sources listed in the Stockholm Convention(e.g.waste incinerators,metal industries,cement plants,and open burning).99(88%)of pooled egg samples were above the EU maximum limits for PCDD/Fs(2.5 pg PCDD/F-TEQ/g fat)or the sum of PCDD/Fs and dioxinlike PCBs(5 pg PCDD/F-PCB-TEQ/g fat).Children consuming such eggs exceed the tolerable weekly intake(TWI).This demonstrates that close to 90%of these areas were not safe for the production of free-range eggs.Sixteen out of the 113 egg samples(14%)were contaminated above 50 pg TEQ/g fat and exceeded the EU maximum limit more than 10 times.From the 26 pooled egg samples around incinerators 24(92%)exceeded the limit with a mean of 43.1 pg TEQ/g fat(2.6e234 pg TEQ/g).All 21 egg samples around metal industries(4.4e112.6 pg TEQ/g fat)were above limits with mean concentration of 26.0 pg TEQ/g fat.Also all 7 egg samples measured at e-waste recycling sites were above limits(mean 308 pg TEQ/g fat).In 58(51%)pooled egg samples the PCB-TEQ was above 5 pg TEQ/g fat exceeding the EU maximum limit with dioxin-like PCBs alone.This highlights the role of commercial PCBs for global contamination with dioxin-like compounds.It was discovered that around metal industries,shredder plants,open burning sites of e-waste and dump sites,a high share of contamination was caused by dl-PCBs.This clearly shows severe PCB release from the end-of-life management of PCB-containing equipment in developing countries.Also highly contaminated eggs were found at many sites where plastic was incinerated.The highest contaminated egg sample ever measured came from an e-waste site in Ghana and had 856 pg TEQ/g fat plus 300 pg TEQ from brominated dioxins(PBDD/Fs).Other extreme PCDD/F contaminations of eggs were found at a chlor-alkali site(514 pg TEQ/g fat),Agent Orange contaminated areas in Vietnam(490,249 and 246 pg TEQ/g fat)and e-waste sites(568 and 520 pg TEQ/g fat).Where DR CALUX®bioassay revealed higher TEQ compared to measured PCDD/F-PCB-TEQ in IPEN studies,polybrominated PBDD/F were also measured and detected up to 300 pg TEQ/g fat at e-waste sites.One positive outcome from the IPEN studies is that all 10 pooled supermarket eggs in developing countries were below regulatory limit.Policy recommendations are made including:a systematic assessment of areas around PCDD/Fs and PCBs sources;measures for reduction of exposures of populations;urgent control of emission sources including PCB equipment,the open burning of plastic,and the use of plastic as fuel in boilers/incinerators in developing countries without air pollution control.Furthermore,soil limits need to be re-assessed and lowered for free-range poultry.
