L07 Mixture Toxicity PDF (2024)

6/6/2024 L07 Mixture toxicity Timo Hamers 1 Set-up of this lecture Effect summation Independent action Concentration addition Isobologram method Parallel dose-response curves and...

6/6/2024 L07 Mixture toxicity Timo Hamers 1 Set-up of this lecture Effect summation Independent action Concentration addition Isobologram method Parallel dose-response curves and relative potencies 2 2 1  6/6/2024 Exercise  Imagine two compounds A and B  [A]=cA causes 30% effect; [B]=cB causes 10% effect What is the combined effect of cA and cB? Effect summation? 30% + 10% = 40% Appears to 37%... 3 3 Exercise  Imagine two compounds A and B  [A]=cA causes 40% effect; [B]=cB causes 30% effect What is the combined effect of cA and cB? Effect summation? 30% + 40% = 70% Appears to 58%... 4 4 2  6/6/2024 Exercise  Imagine two compounds A and B  [A]=cA causes 40% effect; [B]=cB causes 70% effect What is the combined effect of cA and cB? Effect summation? 40% + 70% = 110%??? Appears to 82%... 5 5 Conclusion effect summation  Effect summation is an overestimation, even exceeding 100% effect level!  Why is it an overestimation?  Because you only live once!  Despite what others want you to believe...  6 6 3  6/6/2024 Independent Action  cA causes 40% effect; cB causes 70% effect If only exposed to A: 60% chance of not being affected Additional exposure to B: only 70% of the remaining 60% can be affected So only 30% of remaining 60% has chance of being non- affected pnon-affected = 0.6 x 0.3 = (1-0.4) x (1-0.7) paffected = 1 - pnon-affected = 1 - 0.6 x 0.3 = 1 - (1-0.4) x (1-0.7) = 0.82 NB: multiply chances (0-1), not percentages (0-100)! 7 7 Independent Action, alternative  cA causes 40% effect; cB causes 70% effect Test population pA=0.4 pB=0.7 1-pA=0.6 (1-pA) x (1-pB) =0.6x0.3=0.18  pnon-affected = (1-pA) x (1-pB) = (1-0.4) x (1-0.7) = 0.6x0.3=0.18  paffected = 1- (1-pA) x (1-pB) = 1 – 0.6x0.3 = 1 – 0.18 = 0.82 NB: calculate with chances (0-1), not percentages (0-100)! 8 8 4  6/6/2024 Independent Action  cA causes 40% effect; cB causes 70% effect Test population pA=0.4 pAB= pB=0.7 0.4x0.7= 0.28  paffected = pA + pB - pAB = 0.4 + 0.7 – 0.4x0.7 = 1.1-0.28=0.82 NB: calculate with chances (0-1), not percentages (0-100)! 9 9 Independent Action  cA causes 40% effect; cB causes 70% effect If only exposed to A: 60% chance of not being affected Additional exposure to B: only 70% of the remaining 60% can be affected So only 30% of remaining 60% has chance of being non- affected pnon-affected = 0.6 x 0.3 = (1-0.4) x (1-0.7) paffected = 1 - pnon-affected = 1 - 0.6 x 0.3 = 1 - (1-0.4) x (1-0.7) = 0.82 NB: multiply chances (0-1), not percentages (0-100)! 10 10 5  6/6/2024 In our example  EC50A = 2 µM; EC50B = 10 µM  Consider a mixture with ratio cA:cB=1:3 11 11 Independent action  No interaction between the compounds in the mixture, i.e. A does not influence the amount of B reaching B’s site of action; A does not influence the effect of B at B’s site of action; and/or reversely with A and B interchanged (Plackett & Hewlett, 1952)  Chemicals in a mixture act independent, i.e. they act on different target sites (Bliss, 1939) (have different mode of action) A1 A2 A3 A4 A5 A6 Active sites Organism’s response 12 12 6  6/6/2024 Example interaction: Prochloraz enhances malathion toxicity to birds Prochloraz (fungicide) induces P450 system that malathion in mixture activates or detoxifies with prochloraz more chemicals toxic to birds than expected from toxicity Malathion (insecticide) of single compound is oxidized by P450 to form (Walker & Johnston, 1993) the more toxic malaoxon P450 malathion malaoxon 13 13 Example independent action: algal growth (Faust et al. 2003) 14 14 7  6/6/2024 Toxicological Mixture Theory: The Basics The 4 classes No interaction Interaction of joint effect (additive) (non-additive) Simple similar Complex Similar action action similar action Dissimilar Independent Dependent action action action (Hewlett and Plackett, 1959) 15 15 Mixtures in practice 16 16 8  6/6/2024 Simple similar action (= concentration addition)  Imagine two compounds A and B, with same mode of action  EC50A = 2 µM; EC50B = 10 µM What combination of cA and cB causes 50% effect? cA=2 µM; cB=0 µM cA=0 µM; cB=10 µM cA=1 µM; cB=??? µM cA=??? µM; cB=2 µM 17 17 Concentration addition  Imagine two compounds A and B, with same mode of action  EC50A = 2 µM; EC50B = 10 µM What combination of cA and cB causes 50% effect? cA= ½ x EC50A ; cB= ½ x EC50B cA= ⅓ x EC50A ; cB= ⅔ x EC50B cA= ⅔ x EC50A ; cB= ⅓ x EC50B cA= ⅝ x EC50A ; cB= ⅜ x EC50B (Faust et al. 2003) 18 18 9  6/6/2024 Concentration addition Starting points:  one chemical can be replaced totally or in part by an equal fraction of an equi-effective concentration (e.g. an EC50) of another, without changing the overall combined effect.  These fractions of equi-effective single substances concentrations – also called toxic units (TU) – simply sum up to an overall toxic unit of the mixture.  CA is also known as “Toxic Unit Summation”. 𝑇𝑈𝑥 = 1 19 19 Concentration addition in general  For any effect level x holds: ∑ =∑ 𝑇𝑈𝑥 = 1  At the same effect level x holds 𝑐 = 𝐸𝐶𝑥 or =1  Combining both equations yields ∑ =  For each compound in the mixture, its concentration is a fraction of cmix, i.e. 𝑝 = ×  Reshuffling these equations yields ∑ = ∑ = so 𝐸𝐶𝑥 = ∑  So now the mixture concentration ECxmix can be calculated for any effect level x 20 20 10  6/6/2024 In our example  EC50A = 2 µM; EC50B = 10 µM  Consider a mixture with ratio cA:cB=1:3, i.e. pA=¼ and pB=¾ 21 21 Example concentration addition: uncoupling oxidative phosphorylation (luminescent bacteria) Cmix = Σ(1/16)EC50i Cmix = ΣEC1i Altenburger et al. 2000 22 22 11  6/6/2024 Concentration addition in summary  𝐸𝐶𝑥 = ∑  So now the mixture concentration ECxmix can be calculated for any effect level x  BUT: The CA-expected mixture effect cannot be calculated directly > So relationship E=ƒ(cmixture) does not exist Direct calculations are restricted to the level of effect concentrations (ECx-values) 23 23 Isobologram method 𝑐 = 𝑇𝑈𝑥 = 1 Performance unaffected fraction 𝐸𝐶𝑥 Conc B 1 EC50B EC50 0.8 EC50 B 0.6 0.4 0.2 0.5xEC50 B 0 200 400 8 10 Conc A 600 6 0.5 xEC50 A 800 4 [tox 1] 1000 0 2 EC50 A [tox 2] EC50A 24 24 12  6/6/2024 Isobologram method Conc B 1. Concentration addition ECx-B 2. Synergism 3 3. Antagonism 1 2 ECx-A Conc A 25 25 Same mode of action – Different affinities + I + II + III + IV 26 26 13  6/6/2024 Different receptor affinities? Parallel dose-response curves!  Which compound is most toxic? 2 20 62.5 27 27 Parallel dose-response curves  Which compound is most toxic? Independent of the effect level! I 2x more toxic than II II 20x more toxic than III III 62.5x more toxic than IV 2 20 62.5 28 28 14  6/6/2024 Parallel dose-response curves Independent of the effect level! I 2x more toxic than II II 20x more toxic than III III 62.5x more toxic than IV 2 20 62.5 29 29 Different receptor affinities? Parallel dose-response curves!  Which compound is most toxic? Set the most toxic compound (I) as reference compound II 2x less toxic than I III 40x less toxic than I IV 2500x less toxic than I 2 20 62.5 30 30 15  6/6/2024 Different receptor affinities? Parallel dose-response curves!  Which compound is most toxic? Set the most toxic compound (I) as reference compound Relative potency of II (REPII) = 0.5 REPIII = 0.025 REPIV = 0.0004 SO: 𝑅𝐸𝑃 = 2 20 62.5 31 31 Simple similar action Compound Concentration (nM) REP value I-EQ concentration (nM) I (reference) 2 1 2 II 8 0.5 4 III 360 0.025 9 IV 30 0.0004 0.012 Mixture 15.012 Cmix = 400 nM 32 32 16  6/6/2024 Different receptor affinities? Parallel dose-response curves! 33 33 Simple similar action 15.012 nM 400 nM 34 34 17  6/6/2024 Non-parallel dose-response curves  Which compound is most toxic? Depends on the effect level! 80%: B more toxic than A REP values cannot be determined! 35 35 Goals: after this lecture, you can…  Distinguish between independent action and concentration addition  Explain what interaction means  Interpret results from isobologram method  Calculate toxic units and explain what they mean  Calculate mixture effects for compounds with parallel dose- response curves using relative potency factors  Prepare for tomorrow’s working group 36 36 18  6/6/2024 Right or wrong?  Independent action is a model used for compounds acting by the same mechanism of action  In case of concentration addition, 10% effect is found if the sum of toxic units corresponding to 10% effect is equal to 1  In an isobologram, a synergistic mixture shows an outward bending iso-effect line  Relative potencies are only useful in case of parallel dose- response curves  A mixture in which compounds are present at concentrations below NOEC (or below EC1) cannot cause a significant effect 37 37 19