A NOVEL REPORTER GENE- BASED BIOASSAY SYSTEM FOR DIOXINS - PowerPoint PPT Presentation

A NOVEL REPORTER GENE- BASED BIOASSAY SYSTEM FOR DIOXINS DETERMINATION IN SOLID WASTE Bin Zhao 06/28/2019 State Key Laboratory of Environmental Chemistry and Ecotoxicology Research Center for Eco-Environmental Sciences Chinese Academy of Sciences

Health Effects of Dioxins • Immunotoxicity Cl • Hepatotoxicity Cl O • Wasting Syndrome O Cl Cl • Dermal Toxicity Dioxins & Dioxin-like • Teratogenicity Coumounds • Lethality • Endocrine Disruption • Tumor Promotion UNEP • Porphyria • Induction of Gene Expression

Dioxin Emission Reduction EUROPE USA and JAPAN CHINA Emission standard Emission standard Emission standard Waste incineration MSIWs GB 18485-2001 1994 MSIWs 59 FR 48198 2000/76/EC 2000 HWIs 65 FR 47937 HIWs GB 18484-2001 Foodstuffs Landfill of IWs 2006/1881/EC EPA 2005 GB 16889-2008 Industrial emissions The Inventory 2010/75/EU Steelmaking Plant of Sources of Dioxin GB 28664-2012 EU Dioxin Emission Goverment of Japan Waste water Inventory Stage I 2000. Dioxin GB 3544-2008 GB 18918-2002 Emission standard EU Dioxin Emission In Various fields Inventory Stage II

Detection Methods for Dioxins HRGC-HRMS Expensive Strict condition Concentration of congeners Low throughput TEF----TEQ BIOASSAY • Simple operation EROD • Efficiency ELISA • High throughput Reporter gene

AhR-based Reporter Gene Bioassay Toxicological mechanism of dioxins: Dioxins AhR signaling pathway AhR AhR Reporter AhR ARNT Gene AhR ARNT Luciferase DRE CYP1A1 gene LUCIFERASE + Luciferin Luminis cence CALUX (Chemically Activated LUciferase gene eXpression )

Bioassay Development and Application European Union 2005 Belgium NVRIP dioxins detection in feed and soil 2009 Revision EC No152/2009 DR-CALUX in feed US EPA SW846- 2011 EC No 252/2012 DR-CALUX in food 4435,TOX 21 2018 China Draft comments (HJ) in solid waste Japan JIS K 0463-2009 Environmental samples 2013 Chile SAG CALUX in birds AU Queensland government air pollution assessment

China: development of bioassay Frequency of DRE Sequence around DRE Key ： AHR-dependent transcription efficiency

Construction of Novel plasmid 40 DMSO MDL: 0.1 pM 35 35 RLU ( ×10 4 ) 1nM TCDD 30 30 25 25 RLU (X 20 Control) 20 15 15 10 10 5 5 0 0 10 -10 10 -11 10 -12 10 -13 10 -14 10 -8 10 -9 Reference TCDD (M) Our Plasmid pCL-CR2 ： ultra-sensitivity China Patent 201410099507.7

Novel cell-based biosensor 6000 MDL: 0.1 pM DMSO 50 1nM TCDD RLU ( ×10 4 ) 40 4000 RLU 30 20 2000 10 0 0 Reference Our Sensor DMSO 10 -14 10 -13 10 -12 TCDD （ M ） CBG2.8D 0.1 pM: 0.003 pg/assay China Patent: 201410100654.1

CBG2.8D Bioassay System 0.1pM Stability of standard curve Minimum response to TCDD standard curve of TCDD  Stability  Repeatability  reproducibility S standard O operating P procedure

Application of CBG2.8D Bioassay System Fly ash Samples extraction and cleaning Fly ash of Municipal Solid Waste Fly ash of Hazardous Waste n=6 n=45 Unpublished data Correlation between bioassay and HRGC-HRMS in Fly ash samples

China National Standard 2015 年度国家环境保护标准计划项 目 Listed as China National Monitoring Method for Solid Waste Management

Application of CBG2.8D Bioassay System Detection of dioxins in Beijing atmospheric particulates samples n=45 Zhang et al. Environ. Sci. Technol. 2018

Application of CBG2.8D Bioassay System C B G 2 .8 D C e ll B io a s s a y (p g B E Q /g fa t) Detection of Dioxins in Food Samples 12 10 8 f(x) = 1,1x + 0,55 R² = 0,91 Milk 6 4 2 0 0 1 2 3 4 5 6 7 8 HRGC-HRMS analysis (pg-TEQ/g fat) n= 13 9,6 CBG 2.8D Cell Bioassay 8 (pg BEQ/g ww) 6,4 4,8 3,2 Seafood 1,6 0 f(x) = 0 0,1 0,2 0,3 0,4 0,5 0,6 R² = 0 HRGC-HRMS analysis (pg-TEQ/g ww) New standard curve n= 18

Application of CBG2.8D Bioassay System Toxicity Effect Assessment Pollut ants Zhang et al. Environ. Sci. Technol. 2018 Ginsenoside Activity TAA Natural Screening Screening Product Tryptophan metabolites Health Risk Metabo Assessment Kyn lites Indole compounds Liu et al. Nat. Commun. 2017

Dioxin Research in China Dioxin Emissions and Human Exposure in China: A Brief History of Policy and Research EHP 2011 119, A112-113 “Because of the historical focus of basic research on environmental pollutant levels rather than on environmental health. It is important to further encourage and enhance basic environmental health research in China in order to provide a scientific basis for environmental policies to protect the environment, ecological systems, and human health.” COLLABORATIONS !!!

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