L08_Dioxin-like compounds and TEF concept TH 2024.pdf

6/6/2024 L08: Dioxin-like compounds and TEF concept Timo Hamers 1 Set-up of this lecture  Dioxins and DL compounds Sources/Use and characteristics Exposure Effects Mode of action...

6/6/2024 L08: Dioxin-like compounds and TEF concept Timo Hamers 1 Set-up of this lecture  Dioxins and DL compounds Sources/Use and characteristics Exposure Effects Mode of action Toxic equivalency factor (TEF) concept 2 2 1  6/6/2024 Sources of PCDDs and PCDFs Cly Clx Side products from O  Combustion processes  Organochlorine production O  Leaded fuel PolyChlorinated DibenzoDioxins (PCDDs)  Metal industry Cly Clx O Never been produced on purpose Emissions: PolyChlorinated DibenzoFurans (PCDFs)  1989: 960 g TEQ per year  1999: 50 g TEQ per year 29 g TEQ newly formed 21 g TEQ from pentachlorophenol applications 3 3 PolyChlorinated Biphenyls (PCBs) Cly Clx General structure Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl PCB-19 PCB-52 PCB-122 Cl Cl Cl Cl Cl Cl Cl Cl Cl PCB-28 PCB-153 4 4 2  6/6/2024 Sources of PCBs Produced on purpose 1930-1980 Very stable compounds – Resistant against electrical, thermal, chemical or biological breakdown Used in closed applications, e.g. Cl Cl – insulating fluids in transformers and capacitors – thermal conductors in heat transfer systems Used in open applications, e.g. Cl Cl – plasticizers – flame retardants – solvents Cl Biomagnification in the food chain (top predators) Toxic properties Ban on production Still an environmental problem – “leak” from existing applications and waste – Stockholm convention: “persistent organic pollutants” (POPs) – use in existing equipment completely eliminated by 2025 – unintentional production should be prevented 5 5 Substitution pattern of PCBs M’ O’ O M O = ortho M = meta P = para P’ P  In principle: 209 different congeners possible Not all are stable compounds Not all congeners are dioxinlike  PCBs are always produced as technical mixtures 6 6 3  6/6/2024 Structure PCBs M’ O’ O M O = ortho Cl P’ P M = meta P = para Cl Cl Cl non-ortho PCB Cl Cl Cl Cl Cl ClCl Cl Cl mono-ortho PCB Cl Cl Cl Cl Cl ClCl Cl Cl Cl Cl Cl Cl di-ortho PCB 7 7 What are dioxin-like compounds? Term “dioxinlike compounds” refers to a group of halogenated compounds: Cly Clx O  dioxins (PCDDs) O Cly Clx O  furans (PCDFs) Cly Clx  dioxinlike PCBs 8 8 4  6/6/2024 Examples of dioxins, furans, and DL-PCBs Cl Cl Cl Cl Cl Dioxin Dibenzofuran PCB-126 Cl Cl Cl Cl Cl PCB-118 9 9 Properties of dioxinlike compounds  Very persistent against breakdown  Bioaccumulative (hydrophobic)  Same mode of action  Same clinical symptoms  Toxicity is additive: can be summed together 10 10 5  6/6/2024 Most PCBs are non-dioxinlike (NDL-PCBs) Cl Cl Cl PCB-28  Reprotoxic Cl Cl PCB-52  Developmental toxic Cl Cl Cl Cl Cl PCB-101  Thyroid hormone disrupting  Immunotoxic Cl Cl Cl Cl PCB-118  Neurotoxic Cl Cl Cl Cl Cl Cl Cl Cl PCB-138  Probably carcinogenic Cl Cl Cl Cl Cl PCB-153 Cl Cl Cl Cl Cl Cl Cl PCB-180 Cl Cl 11 11 Stockholm Convention 2001 Persistent Organic Pollutants (POPs) are organic chemical substances, that is, they are carbon-based. They possess a particular combination of physical and chemical properties such that, once released into the environment, they:  remain intact for exceptionally long periods of time (many years);  become widely distributed throughout the environment as a result of natural processes involving soil, water and, most notably, air;  accumulate in the fatty tissue of living organisms including humans, and are found at higher concentrations at higher levels in the food chain; and  are toxic to both humans and wildlife. http://www.youtube.com/watch?v=keOLiXOoFDU 12 12 6  6/6/2024 Exposure to dioxinlike compounds  Natural origin  Intentional poisoning  Disaster  Illegal activities (Belgian chicken crisis in WG04)  Background levels 14 14 Natural origin  Use of marl clay in potato selection  Fires or volcanic activity when the marl clay layers were formed, millions of years ago Case study for working class bioassays Wednesday 12 June; 13:30-15:15 15 15 7  6/6/2024 Intentional poisoning of Viktor Yushchenko Viktor Yushchenko (Joesjtsjenko), president Oekraïne 16 16 Agent orange (Vietnam war)  Chlorophenoxy herbicides 2,4-D; 2,4,5-T; MCPA Contaminated with PCDDs 18 18 8  6/6/2024 Background levels in the foodchain 21 21 Background exposure in the Arctic: Grasshopper effect of POPs  Persistent organic pollutants (POPs) as PCBs, DDT, etc move from hotter regions to colder regions  Alternating processes of volatilisation and condensation (grasshopping) 22 22 9  6/6/2024 Example of foodchain accumulation by PCBs PCB-concentration unit Water 0.000002 mg/l Sediment 0.005-0.16 mg/kg dw Fytoplankton 8 mg/kg lipid Zooplankton 10 mg/kg lipid Evertebrates 5-11 mg/kg lipid Marine fish 0.1-37 mg/kg lipid Marine birds 110 mg/kg lipid Marine mammals 160 mg/kg lipid Source: RSU, 1980 23 23 Food is main source of human TEQ exposure 24 24 10  6/6/2024 Background levels in humans  Lipophilic Cl Cl  Accumulating in fat tissue Cl Cl  Excretion Cl Cl Slow through blood → urine Quick through breast milk 25 25 Effects of dioxins in different species ATSDR, 1998 26 26 11  6/6/2024 Effects of dioxins in different species ATSDR, 1998 27 27 Mechanism of action of dioxin-like compounds (I) Ah Receptor (AhR) theory: induction of gene expression cell nucleus UGT-gene Ah-receptor CYP450-gene UGT-mRNA Dioxinlike CYP450-mRNA compounds CYP450 UGT OH O-Gluc OH OH O-Gluc OH O-Gluc OH 29 29 12  6/6/2024 Mechanism of action of dioxin-like compounds (II) Ah Receptor (AhR) theory: effects on gene expression and phosphorylation Induction of gene expression Effects on Induction of phosphorylation gene of kinases expression 30 30 Cumulative risk of dioxinlike compounds  Large differences in AhR activating potency between congeners  Exposure is always to complex mixtures  What is the cumulative risk of mixtures? Toxic Equivalency Factor (TEF) concept: an instrument to express the relative potency of individual dioxin congeners compared to the most potent dioxin 2,3,7,8-TCDD 31 31 13  6/6/2024 How to determine a TEF value? Response (RLU) 2,3,7,8-TCDD (pM) concentration in the well Formula: TEFx = ED502,3,7,8-TCDD/ ED50x 32 32 WHO-list of TEF-values 33 33 14  6/6/2024 GC-HRMS analysis dioxins 34 34 Calculating the total dioxin-like activity of a mixture  TCDD EQuivalent (TEQ) concentration  Indirect: Calculate TEQ concentration based on results from GC-HRMS-analysis: TEQ(total) = Σn{(TEF)(1,n) x [congener](1,n)} GC-HRMS method Compound 1: concentration 1 x TEF1 = TEQ1 Compound 2: concentration 2 x TEF2 = TEQ2 Compound 3: concentration 3 x TEF3 = TEQ3 Compound n: concentration n x TEFn = TEQn + Total dioxin toxicity of mixture: SumTEQ 35 35 15  6/6/2024 TCDD EQuivalent Concentration (TEQ) pg/g lipid TEF: Potency relative to TCDD (dimensionless number) TEQ: Potency of a compound or mixture expressed as a TCDD concentration with equivalent (similar) potency (not dimensionless!) 36 36 TEF concept = Concentration Addition  Concentration addition ∑ 𝑇𝑈 = + +  TEF concept ∑ 𝑇𝐸𝑄 = 𝑇𝐸𝐹 ∗ 𝐶 + 𝑇𝐸𝐹 ∗ 𝐶 + 𝑇𝐸𝐹 ∗ 𝐶 ∑ 𝑇𝐸𝑄 = ∗𝐶 + ∗𝐶 + ∗𝐶 ∑ = + + = ∑ 𝑇𝑈  So: ∑ 𝑇𝐸𝑄 = ∑ 𝑇𝑈 ∗ 𝐸𝐶50 37 37 16  6/6/2024 Requirements for compound inclusion in TEF concept  Very persistent against breakdown  Bioaccumulative (hydrophobic)  Toxicity: Same mechanism of action Same clinical symptoms Toxicity is additive: can be summed together 38 38 TEQ calculation GC-HRMS method Compound 1: concentration 1 x TEF1 = TEQ1 Compound 2: concentration 2 x TEF2 = TEQ2 Compound 3: concentration 3 x TEF3 = TEQ3 Compound n: concentration n x TEFn = TEQn + Total dioxin toxicity of mixture: SumTEQ Bioassay method Direct measurement of TEQ value of sample  39 39 17  6/6/2024 Reporter gene assay (slide from L10) reporter-gene Receptor responsive gene reporter gene mRNA Receptor Corresponding ligand mRNA Corresponding Reporter protein protein Easy read-out, e.g. reporter protein Is fluorescent Converts substrate into colored product Converts substrate into luminescent product 40 40 Mechanism of action of dioxin-like compounds (I) Many xenobiotics induce their own biotransformation cell nucleus UGT-gene Ah-receptor CYP450-gene UGT-mRNA Dioxinlike CYP450-mRNA compounds CYP450 UGT OH O-Gluc OH OH O-Gluc OH O-Gluc OH 41 41 18  6/6/2024 Principle of DR-CALUX bioassay Cl Cl Cl Cl luciferase-gene Cl Cl Ah-receptor CYP1A-gene luciferase-mRNA Dioxinlike CYP1A-mRNA compounds CYP1A luciferase Luciferin 42 42 Dose-response curve: backward use 110 100 Eg. Response to complex mixture 90 80 70 Respons 60 50 40 30 20 10 0 0.001 0.01 Equivalent concentration 0.1 1 10 Concentration 43 43 19  6/6/2024 TEQ assessment  Indirect via HR-GC/MS analysis plus TEF concept Is the relative  Direct via DR-CALUX measurement potency (REP) of individual GC-HRMS method congeners in the Compound 1: concentration 1 x TEF1 = TEQ1 DR-LUC bioassay Compound 2: concentration 2 x TEF2 = TEQ2 comparable to Compound 3: concentration 3 x TEF3 = TEQ3 the TEF value? Compound n: concentration n x TEFn = TEQn + Total dioxin toxicity of mixture: SumTEQ Bioassay method Direct measurement of TEQ value of sample  44 44 Determine Relative Potency (REP) in DR-CALUX Response (RLU) 2,3,7,8-TCDD (pM) concentration in the well Formula: REPx = EC502,3,7,8-TCDD/ EC50x 45 45 20  6/6/2024 WHO-TEF-values vs REP-values DR CALUX assay PCDDs and PCDFs PCBs Structure WHO-TEF CALUX- CALUX-REP IUPAC No. Structure WHO-TEF CALUX-REP Furans non-ortho PCBs 2,3,7,8-TCDF 0.1 0.32 81 3,4,5,3'-TCD 0.0001 0.0001 1,2,3,7,8-PeCDF 0.05 0.21 77 3,4,3',4'-TCB 0.0005 0.0013 2,3,4,7,8-PeCDF 0.5 0.5 126 3,4,5,3',4'-PeCB 0.1 0.067 1,2,3,4,7,8-HxCDF 0.1 0.13 169 3,4,5,3',4',5'-HxCB 0.01 0.0034 1,2,3,6,7,8-HxCDF 0.1 0.039 2,3,4,6,7,8-HxCDF 0.1 0.18 mono-ortho PCBs 1,2,3,7,8,9-HxCDF 0.1 0.11 118 2,4,5,3',4'-PeCB 0.0001 0.0000073 1,2,3,4,6,7,8-HpCDF 0.01 0.032 114 2,3,4,5,4'-PeCB 0.0005 0.000048 1,2,3,4,7,8,9-HpCDF 0.01 0.041 105 2,3,4,3',4'-PeCB 0.0001 0.000012 OCDF 0.0001 0.0001 167 2,4,5,3',4',5'-HxCB 0.00001 0.00001 156 2,3,4,5,3',4'-HxCB 0.0005 0.00021 Dioxins 157 2,3,4,3',4',5'-HxCB 0.0005 0.00008 2,3,7,8-TCDD 1 1 189 2,3,4,5,3',4',5'-HpCB 0.0001 0.0001 1,2,3,7,8-PeCDD 1 0.54 1,2,3,4,7,8-HxCDD 0.1 0.3 1,2,3,6,7,8-HxCDD 0.1 0.14 1,2,3,7,8,9-HxCDD 0.1 0.066 1,2,3,4,6,7,8-HpCDD 0.01 0.05 OCDD 0.0001 0.0001 Ahlborg et al., 199 4; Hosoe et al., 2002 46 46 Working group VR-CALUX  DR-CALUX bioassay Dioxin Responsive Chemically Activated LUciferase eXpression VR: virtual reality (computer practicum) Goes along with this lecture  Goal Learn about PCBs and dioxins and their toxicity Understand the principle of the DR-CALUX assay Know the set-up and execution of a bioassay experiment Understand how to use bioassay results for risk assessment  How does it work? (see CANVAS instruction) http://www.bio.vu.nl/~vr-calux/design/ Be prepared until paragraph 3.2.3 Finalization in working group: Wednesday June 12th: 13:30-15:15 47 47 21  6/6/2024 Goals: after this lecture, you…  Know what dioxins and dioxin-like (DL) compounds are  Know their sources and properties (including POPs), routes of exposure and adverse effects  Know differences in species sensitivity and congener potencies  Understand the mode of action via Ah-receptor Induced gene expression Changes in phosphorylation status of transcription factors  Can apply the TEF/TEQ concept Calculate TEQ based on individual congener’s level  Understand how a reporter gene assay can be used to determine TEQ values  Can prepare for the VR-CALUX working group 48 48 22