Summary human toxicology_Updated.docx

Contents {#contents.TOCHeading} ======== [**Lecture 1 Introduction** 5](#lecture-1-introduction) [History 5](#history) [Ecotoxicology 5](#ecotoxicology) [One health approach: 5](#one-health-approach) [Environmental toxicology 6](#environmental-toxicology) [Ecology 6](#ecology) [Difference between environmental chemistry and environmental toxicology 6](#difference-between-environmental-chemistry-and-environmental-toxicology) [Generic principles in toxicology 7](#generic-principles-in-toxicology) [Routes of exposure 7](#routes-of-exposure) [Phases of toxic response 7](#phases-of-toxic-response) [Classification in toxic response 7](#classification-in-toxic-response) [**Lecture 2 Chemical properties and fate** 7](#lecture-2-chemical-properties-and-fate) [Processes in chemical fate 8](#processes-in-chemical-fate) [Physico-chemical properties and partitioning coefficients 8](#physico-chemical-properties-and-partitioning-coefficients) [Henry-constant (water air) (K**~H~**) 8](#henry-constant-water-air-kh) [Octanol-water-partition coefficient (organic-polar distribution of compound) (K**~OW~**) 8](#octanol-water-partition-coefficient-organic-polar-distribution-of-compound-kow) [Acid dissociation constant pKa 9](#acid-dissociation-constant-pka) [Sorption coefficient (water -- solid phase) (K**~p~**) 9](#sorption-coefficient-water-solid-phase-kp) [General rules on biodegradation resistance: 10](#general-rules-on-biodegradation-resistance) [Global Ocean Conveyer System 10](#global-ocean-conveyer-system) [Grashopper effect 10](#grashopper-effect) [**Lecture 3 ADME** 10](#lecture-3-adme) [Absorption = uptake 11](#absorption-uptake) [Distribution 11](#distribution) [Accumulation 11](#accumulation) [Barriers in distribution 12](#barriers-in-distribution) [Metabolism 13](#metabolism) [Phase I 13](#_Toc170312829) [*Negative effect of biotransformation:* 13](#negative-effect-of-biotransformation) [Phase II 14](#_Toc170312831) [Excretion 15](#excretion) [Phase III 15](#_Toc170312833) [**Lecture 4 dose-response curve** 15](#lecture-4-dose-response-curve) [Dose response relationships 15](#dose-response-relationships) [Toxicological endpoints 15](#toxicological-endpoints) [Use of dose-response curves 16](#use-of-dose-response-curves) [**Lecture 5 bioaccumulation** 17](#lecture-5-bioaccumulation) [Bioaccumulation: 17](#_Toc170312839) [Factors related to bioaccumulation 17](#factors-related-to-bioaccumulation) [Bioconcentration factor (BCF): 18](#_Toc170312841) [Biomagnification (BMF): 18](#_Toc170312842) [Bioaccumulation factor (BAF): 18](#_Toc170312843) [Bioconcentration test 19](#_Toc170312844) [Key Components of OECD 305 BCF Test: 19](#key-components-of-oecd-305-bcf-test) [Trophic magnification (TMF) 19](#trophic-magnification-tmf) [Key differences and relationships: 19](#key-differences-and-relationships) [Relationship bioaccumulation and physical-chemical properties 20](#relationship-bioaccumulation-and-physical-chemical-properties) [Biomagnification (BMF) 20](#_Toc170312849) [**Lecture 6 Adverse outcome pathways** 21](#_Toc170312850) [What is an AOP? 21](#what-is-an-aop) [Important definitions related to AOPs: 22](#important-definitions-related-to-aops) [Five principles of AOP development 22](#five-principles-of-aop-development) [**Lecture 7 Mixture toxicity** 23](#lecture-7-mixture-toxicity) [Effect summation 23](#effect-summation) [Independent action 23](#independent-action) [Concentration addition 24](#concentration-addition) [Isobologram method 24](#isobologram-method) [How to calculate toxic dose 25](#how-to-calculate-toxic-dose) [**Lecture 8 dioxin-like compound and TEF concept** 27](#lecture-8-dioxin-like-compound-and-tef-concept) [Definition 27](#definition) [Properties 27](#properties) [Sources PCBs: 27](#sources-pcbs) [Background levels (**adverse effects**) 28](#background-levels-adverse-effects) [Know differences in species sensitivity and congener potencies 28](#know-differences-in-species-sensitivity-and-congener-potencies) [Mechanism of action of dioxin-like compounds 29](#mechanism-of-action-of-dioxin-like-compounds) [Ah Receptor (AhR) theory: effects on gene expression and phosphorylation 29](#ah-receptor-ahr-theory-effects-on-gene-expression-and-phosphorylation) [TEF & TEQ 30](#tef-teq) [How to determine TEQ values using reporter gene assay 30](#how-to-determine-teq-values-using-reporter-gene-assay) [**Lecture 9 Ecotoxicology studies** 31](#lecture-9-ecotoxicology-studies) [Definition 31](#definition-1) [Approaches in ecotoxicity testing 31](#approaches-in-ecotoxicity-testing) [Animal exposure routes: 31](#animal-exposure-routes) [Criteria for selecting ecotoxicity test 31](#criteria-for-selecting-ecotoxicity-test) [Test organisms in ecotoxicology 32](#test-organisms-in-ecotoxicology) [**Lecture 10 in vitro toxicity testing** 33](#lecture-10-in-vitro-toxicity-testing) [Definition 33](#definition-2) [Protein based bioassay 33](#protein-based-bioassay) [Cell-based techniques 34](#cell-based-techniques) [Primary cell culture 34](#primary-cell-culture) [Cell lines 35](#cell-lines) [Immortal cell line = continuous cell line 35](#immortal-cell-line-continuous-cell-line) [Reporter gene bioassay 36](#reporter-gene-bioassay) [Differentiation models 36](#differentiation-models) [Stem cell differentiation models 36](#stem-cell-differentiation-models) [Induced pluripotent stem cell differentiation (iPSC) 37](#induced-pluripotent-stem-cell-differentiation-ipsc) [transdifferentiation models 37](#transdifferentiation-models) [Complex in vitro models 37](#complex-in-vitro-models) [2D monolayer to 3D culturing 37](#d-monolayer-to-3d-culturing) [Cell co-culturing 37](#cell-co-culturing) [Organ-on-a-chip or body-on-a-chip 37](#organ-on-a-chip-or-body-on-a-chip) [Body-on-a-chip 37](#body-on-a-chip) [**Lecture 11 Endocrine distruptors** 38](#lecture-11-endocrine-distruptors) [EDC background 38](#edc-background) [Classes of hormones 38](#classes-of-hormones) [Biological classification of hormones: Role 39](#_Toc170312896) [Biological classifications of hormones : receptors 39](#biological-classifications-of-hormones-receptors) [Mode of action EDCs 39](#mode-of-action-edcs) [**Lecture 12 Metabolomics** 41](#lecture-12-metabolomics) [Definition 41](#definition-3) [Application metabolomics 41](#application-metabolomics) [Workflow of metabolomics 42](#workflow-of-metabolomics) [Extraction steps of metabolites 42](#extraction-steps-of-metabolites) [Quenching methods 42](#quenching-methods) [Metabolomic analysis 42](#metabolomic-analysis) [Visualization of metabolomics data 44](#visualization-of-metabolomics-data) [Challenges in metabolomics 44](#challenges-in-metabolomics) [**Lecture 13 Epidemiology** 45](#lecture-13-epidemiology) [Definitions 45](#definitions) [Study designs 45](#study-designs) [Cross sectional 45](#cross-sectional) [case-control 46](#case-control) [Cohort study 46](#cohort-study) [Summary: 47](#summary) [**Lecture 14 Animal models in toxicology** 48](#lecture-14-animal-models-in-toxicology) [History 48](#history-1) [EU legislation on animal testing 48](#eu-legislation-on-animal-testing) [How to perform an animal toxicity experiment: 49](#how-to-perform-an-animal-toxicity-experiment) [**Lecture 15 Carcinogenicity** 49](#lecture-15-carcinogenicity) [Definition 49](#definition-4) [Stages of cancer 50](#stages-of-cancer) [Initiation stage 50](#initiation-stage) [Types of mutations 50](#types-of-mutations) [**Lecture 16 effect-based monitoring** 54](#lecture-16-effect-based-monitoring) [Definition 54](#definition-5) [Types of biomarkers 54](#types-of-biomarkers) [Biomarkers examples 55](#_Toc170312927) [In vivo 55](#in-vivo) [In vitro 56](#in-vitro) [**Lecture 17 Human biomonitoring and the exposome** 57](#lecture-17-human-biomonitoring-and-the-exposome) [**Lecture 18 Effect directed analysis** 60](#lecture-18-effect-directed-analysis) [Lecture 19 Alternative animal models: Nematodes 65](#lecture-19-alternative-animal-models-nematodes) [Lecture 20 Alternative animal models: Zebrafish 65](#lecture-20-alternative-animal-models-zebrafish) [**Lecture 21 Risk assessment ecotoxicological** 66](#lecture-21-risk-assessment-ecotoxicological) [Definitions 66](#definitions-1) [Legislation of chemicals: Main requirements for ecotoxicological data in regulatory frameworks 67](#legislation-of-chemicals-main-requirements-for-ecotoxicological-data-in-regulatory-frameworks) [1. New chemical substances 67](#new-chemical-substances) [2. Pesticides (Registration only when no unacceptable risk) 68](#pesticides-registration-only-when-no-unacceptable-risk) [Estimation of exposure 69](#estimation-of-exposure) [How to derive safe levels of chemicals from available toxicity data 69](#how-to-derive-safe-levels-of-chemicals-from-available-toxicity-data) [**Lecture 22 Risk Assessment humans** 72](#lecture-22-risk-assessment-humans) [Disability Adjusted Life Years (DALY) 72](#disability-adjusted-life-years-daly) [Terms used in risk assessment 72](#terms-used-in-risk-assessment) **Lecture 1 Introduction** ========================== Geel: kennen Groen: half kennen Blauw: ken ik niet History ------- - Paracelsus: Everything is poisonous and nothing is without poison; only the dose makes that something is a poison (dose-dependency). - Industrial revolution more and more chemicals registered - 20th century "Chemical age" - More environmental pollution - Chemical warfare (weapons and holocaust) - Scandals and disasters: for example DDT, pesticides, radioactivity - DDT - a pesticide affecting the nervous system and motor coordination of the insects. After malaria outbreaks, entire islands were sprayed with DDT. - Rachel Carson (wrote the book silent spring) discovered a dangerous effect on the wildlife. DDT is very persistent and will concentrate when moving through the food chain. - This results in **Bioaccumulation** in birds causing eggshell thinning with as a result a decrease in bird population Ecotoxicology ------------- ***Definition** = The branch of toxicology concerned with the study of toxic effects, caused by natural or synthetic pollutants, to the constituents of ecosystems, animal (including human), vegetable and microbial, in an integral context* - Essential to Ecotoxicology is indirect relationship between effects on individual organisms and effects on populations in the natural environment ### One health approach: - health of people connected with health of animals and the environment **Different levels of biological organization:** studies not only effects individuals but also on populations and communities: - structural effects (pattern description) - functional effects (process characterization) Environmental toxicology ------------------------ *Definition = The science that studies the fate and effects of potentially hazardous chemicals in the environment.* ![](media/image2.png)Environmental toxicology has as endpoint the human health, whereas ecotoxicology is restricted to ecological endpoints Ecology ------- *Definition = the study of interactions determining the distribution and occurrence of organisms effects on populations, communities and ecosystems* Difference between environmental chemistry and environmental toxicology ----------------------------------------------------------------------- **Environmental chemistry** **Environmental toxicology** -------------------------------------------------------------------------- --------------------------------------------- Occurrence of chemical in the environment Biotransformation Pathways of chemicals in the environment Distribution of chemical over organs/tissue Effects of physio-chemical properties on environmental fate of chemicals Effects at the individual level Deposition and degradation processes Mechanisms of detoxification Distribution in environmental compartments Mode of action Intake in organisms Toxicokinetic & toxicodynamic Mixture toxicity Generic principles in toxicology -------------------------------- ### Routes of exposure - **Oral** exposure: absorption through gastrointestinal tract (GI tract) - **Dermal** exposure: absorption through skin, cuticle, exoskeleton - **Respiratory exposure**: absorption through lungs, gills - **Special routes**: direct penetration of epithelial layer through injection, infusion etc. ### Phases of toxic response - **Exposure** (environmental chemistry) *How does an organism get into contact with compound?* - Routes of exposure - **To what extend does an organism get into contact with compound?** - External (concentration in air, water, soil, food) - Dose (intake, quantity per bodyweight per day) - Internal (concentration in body or body compartment) - **Toxicokinetics** *what does body do to the compound?* - ADME - Quantitatively or qualitatively - **Toxicodynamics** *what does the compound do to the body?* - Irritation, inflammation, corrosion, narcosis, suffocation, sensitization etc. - Quantitatively: dose-response relationships - Qualitatively: what type of effect? ### Classification in toxic response - **Possibility for repair** - Reversible - Irreversible - **Rate** - Acute toxicity occurs short after a single exposure to a compound - Chronic toxicity occurs after prolonged and repeated exposure to a compound - **Site of action** - Local (at the site of exposure) - Systemic (after systemic distribution of the compound) - e.g. Softenon baby: Thalidomide from mother to child which causes absence of limbs or reduction of long bones of limbs **Lecture 2 Chemical properties and fate** ========================================== Fate of the chemical in the environment depends on: - compound and environment-properties - emission path (e.g. to air or water) *is the net of a suite of transport, transfer and degradation processes that start to act on the chemical directly after its emission and during the subsequent environmental distribution.* Transfer and the exchange between compartments (air-water-sediment/soil-biota) and degradation determines the concentration in each of the compartments. Chemicals in these compartments can evaporate or sorb to sediment and accumulate into biota. These differences are based on: hydrophobicity, volatility and degree of ionization ![](media/image4.png)Processes in chemical fate ----------------------------------------------- **Emission to air** When exposing benzene and PCBs to air, most benzene will be present in the air whereas the PCB will be in the sediment & soil **Emission to water** When exposing benzene and PCBs to water, most benzene will be present in the water whereas the PCB will be in the sediment Physico-chemical properties and partitioning coefficients --------------------------------------------------------- ### Henry-constant (water air) (K**~H~**) - ![](media/image6.png)Measure of volatility - K~H~ = V~p~ / S~w~ (1) - Benzene has a high henry's constant which means that it will volatize readily from water - **The higher the henry's constant, the faster it volatizes from water to air** - K~air-water~ = C~air~ / C~aq~ **Units** V~p~ = vapour pressure (Pa)\ S~w~ = water solubility (mol/m3)\ K~H~ = Pa m3/mol\ C~air~ = concentration in air (mol/m3)\ C~aq~ = aqueous concentration (mol/m3) ### Octanol-water-partition coefficient (organic-polar distribution of compound) (K**~OW~**) - Measure of lipophilicity, hydrophobicity - N-octanol is a good surrogate for lipids in organisms - K~OW~ = C~octanol~ / C~water~ (2) - The lower the log K~ow~, the better soluble in water (polar) - The more hydrophobic, the higher the K~OW~ and the [higher the chance of bioaccumulation] - If \>7 very difficult to measure it exactly in water **Hydrophobicity (tendency of a chemical to 'escape' from water)** - depends on molecular size & polarity (hydrogen bonding) - Bigger molecules are more hydrophobic (exception when polar groups are attached) - Water solubility low (ng/L or mg/L) - Cl and Br make molecule more hydrophobic **Hydrophilicity:** Polar groups such as OH and NH units reduce hydrophobicity of organic contaminants because of the interaction between water molecules **Determination of KOW in three methods:** - **Shake flask method** (only reliable for compounds with logK~OW~ up to 5) (can use eq. 2) - **Chromatographic parameters** = retention time indicative of lipophilicity (only reliable for compounds till logK~OW~ 6-7) - **Prediction from chemical structure using computer models (EPISUITE**) (only reliable for compounds of very high logK~OW~ ### Acid dissociation constant pKa - measure of polarity, ionisation - Only non-dissociated forms of acids or base will be taken up in organisms - pH\>pKa ionization ### Sorption coefficient (water -- solid phase) (K**~p~**) - From soil/sediment to (pore)water = desorption - From (pore)water to soil/sediment = adsorption - K~p~ = C~s~ / C~a~ - K~p~ depends on: chemical properties of chemical & physio-chemical properties of soil or sediment - Because of differences in carbon contents of solid, the K~OC~ is preferred because it is corrected for organic carbon - K~OC~ = sorption coefficient (K~p~) / fraction organic carbon (f~OC~) - The higher the log K~OW~ (more hydrophobic), the higher the log K~OC~ (better sorption) The pore water is the main route of exposure for soil or sediment in living organisms. Pore water concentrations can be predicted from total concentration using K~OC~. However, there is often no equilibrium between compartments, because of the presence of dissolved organic carbon (DOC) in pore water. **Units** K~P~ = sorption coefficient\ C~S~ = concentration in soil/sediment\ C~a~ = concentration in water\ f~OC~ = fraction organic carbon General rules on biodegradation resistance: ------------------------------------------- - **[Aromatic structures] more persistent** than aliphatic structures - **[Saturated aliphatic] more persistent** than non-saturated ones - **[Branched long-chain aliphatic] more persistent** than non-branched ones - **[Highly chlorinated] compounds more persistent (persistency increases with degree of chlorination)** Global Ocean Conveyer System ---------------------------- - The ocean is constantly in motion, functioning like a global conveyor belt. - This motion results from a combination of thermohaline currents (temperature and salinity-driven) and wind-driven currents. - Dense, cold, salty water sinks to the ocean bottom, while warmer, less dense water stays on the surface. - The conveyor belt starts in the Norwegian Sea, where the Gulf Stream\'s warm water cools and sinks. - This sinking water moves south, replaced by more warm water transported north. - The cold bottom water flows south past the equator to Antarctica. - Eventually, this cold water returns to the surface through mixing and wind-driven upwelling, perpetuating the global conveyor belt cycle. Grashopper effect ----------------- - Chemicals (POPs) travel in multiple cycles of evaporation, transport by air and condensation. - **Transported from warmer to colder regions of the earth** - Explains why high concentrations of POPs have been found in the arctic environment and in bodies of animals/people even though most chemical have not been used in this region - In warm temperatures VOCs evaporate - VOCs move in air by winds to colder places - In cold temperatures VOCs condense and fall to earth POP = persistent organic pollutant\ PBT = persistent, bio accumulative, and toxic compound ![Afbeelding met tekst, schermopname, grafische vormgeving, ontwerp Automatisch gegenereerde beschrijving](media/image8.png) **Lecture 3 ADME** ================== Toxicokinetics: fate of a compound after exposure Absorption = uptake ------------------- [Absorption after oral intake ] **Enterohepatic circulation:** repeat of absorption, conjugation (=metabolism), elimination, deconjugation, absorption etc. *Circulation of bilirubin, drugs or other substances from the liver to the bile, which is followed by entry into the small intestine, absorption by the enterocyte and transport back to the liver* **Pre-systemic elimination**: elimination before systemic distribution can take place (first pass effect!) Drugs are carried through the *portal vein* into the liver. The liver metabolizes the drug and ensures that the drug is excreted [Absorption after intake by inhalation: gasses] - Can be very fast depending on air: water partitioning constant (Henry coefficient) -\> Passive diffusion - Intake is not similar as the uptake - Intake is what you inhale or oral intake - Uptake is the actual part that is ultimately absorbed from the full intake - Oral intake: passing the gastro-intestinal track (GI track) without absorption - **Absorption = f \* intake** - **total absorption = Σ(f~i~ x I~i~)**\ - f = absorption efficiency\ \> example of calculation: - **Example** *Smoking : cigarette contains 0.16 ug per cigarette + 25 cigarettes per day\ f~lung~ = 30% and f~GI\_track~ = 6%* - *Calculation: 0.3 \* 25 \* 0.16 = 1.2 ug/day* Distribution ------------ ### Accumulation Accumulation can have selective binding or uptake into specific tissues: - In an accumulation organ (POPs) - In the target organ (Sr^++^ and F^-^) However accumulation is in most cases not toxic and can be mobilized from deposit (sometimes has dynamic equilibrium with concentration blood) Lipophilic compounds (e.g. PCB) can accumulate (ophoping) in fat tissue. this will be excreted through blood -\> urine (slow) or breastmilk (fast) [Losing weight] - Losing weight increases bioavailability of lipophilic compounds! - fat tissue decreases so it can happen that some lipophilic compounds transfer to the hydrophilic part -\> it gets not excreted but this part just gets more dense - lipophilic compound (high KOW) can be releases during fat mobilization (losing weight) and when women is pregnant it can give small fat deposits to the foetus/infant (mothermilk) ![](media/image10.png) [Pb^++^, Sr^++^ and F^-^ in bones (Target organ)] - Exchange with Ca++ or OH- - Sr^++^ and F^-^ have effect whereas Pb^++^ has no effect - Released during Ca-mobilization - Cadmium is located in the liver and kidneys and can bind to metallothionein. This complex itself is nephrotoxic! ### ### Barriers in distribution #### **Blood-Brain Barrier** The blood-brain barrier (BBB) is a highly selective semi-permeable border that separates the circulating blood from the brain and extracellular fluid in the central nervous system (CNS). Its primary function is to protect the brain from harmful substances in the bloodstream while allowing essential nutrients to reach the brain. [The BBB consists of: ] - Endothelial cells: These cells line the brain capillaries and are connected by tight junctions, which prevent the passive diffusion of most molecules. - Tight junctions: These specialized connections between endothelial cells create a physical barrier, limiting paracellular transport. - Efflux transporters: The BBB expresses various efflux transporter proteins, including multidrug-resistant proteins (MDR) and multidrug resistance-associated proteins (MRP), which actively pump out a wide range of drugs and toxins, contributing to multidrug resistance. - No pinocytosis: Unlike other blood vessels, brain endothelial cells lack pinocytotic vesicles, further preventing the non-specific uptake of substances. (small molecules with dissolved molecules (sugars and proteins) are taken up from the extracellular fluid into the cytoplasm (it can complex with cysteine) - Biotransformation capacity: The BBB possesses some enzymatic activity, capable of metabolizing certain substances before they reach the brain. - Astrocytes: These glial cells provide physical support to the BBB and may also contribute to efflux transport and biotransformation. - Efflux is the degree of rate that the chemical is pumped out of the cell using active transport. In the BBB this is done by efflux transporter proteins - Exceptions: Certain substances, like methylmercury, can bypass the BBB due to their lipophilic nature or specific transport mechanisms. #### Placenta - Many lipophilic compounds will get through so it's actually not a true 'barrier' - Defence by: - 1\) active transport (mdr/oct protein) - Active transport: biological process that allows cells to move molecules from low concentration to a high concentration using ATP - 2\) biotransformation capacity - Passive transport: biological process that allows cells to move molecules from high concentration to a low concentration without needing energy Metabolism ---------- Phase Reactions Enzymes Purpose Outcome ------- ------------------- -- ---------------------------------- ---------------------------------------------------------------------- ------------------------------------------------ I Functionalization CYP450s Increase polarity, prepare for phase II Metabolites may be active or toxic II Conjugation Transferases (UGTs, SULTs, etc.) Further increase polarity, decrease reactivity, facilitate excretion Metabolites usually less active and less toxic III Transport ABC and SLC transporters Remove conjugated metabolites from the body Excretion of metabolites Metabolism = Biotransformation =\> biochemical transformation AFTER uptake Goal from biotransformation (toxic compounds): - accelerated excretion & detoxification - Side effect: activation of compounds into more toxic compounds! ![](media/image12.png)[]{#_Toc170312829.anchor}Phase I - creating a reactive 'handle' to the compound (addition (-OH), (-=O), (-COOH)) [Add a functional group] - function: to increase water solubility of the substance or introduce functional groups that are more amenable to certain modifications in phase II - Phase I reactions: - Oxidation: Oxygen and NAD(P) as electron acceptor - Hydrolysis (breaking of the bond by H2O) - Reduction **Example:** Oxygenase (incorportating oxygen from O2 in substrate) by cytochrome P450 or flavin monooxygenase (FMO) NAD(P)+ as electron acceptor Afbeelding met tekst, diagram, schermopname, lijn Automatisch gegenereerde beschrijving #### Negative effect of biotransformation: - **1)** product of phase I can be very reactive (bio-activation) → reacts with macromolecule before it can be detoxified in phase II - **2)** reaction with DNA (chemical mutagenesis) → basis for carcinogenicity of polycyclic aromatic hydrocarbons ![](media/image14.png) - **Example** : Benzopyrene (BaP) is an example of an DNA reacting molecule. It can form into three metabolites, but only when modifying the bay region it can give toxic compound (diol-epoxides - Local distortion in DNA double helix structure & hampering proper DNA replication - Leads to change in nucleotide order in the DNA chain, inherited by daughter cells (mutation) []{#_Toc170312831.anchor}Phase II [Conjugation = coupling of a water soluble molecule to the handle of the compound ] Type Enzyme Co-substrate ---------------------- -------- -------------- Glucuronidation UGT UDPGA Sulfonation SULT PAPS Glutathione coupling GST Glutathione Methylation MT SAM co-substrate is a molecule that binds to the metabolite. In figure below is the blue part the part that conjugates to the metabolite. In glutathione is the whole tripeptide conjugated. Conjugated part is only bound to the metabolite! With this conjugated part it makes the molecule more water-soluble and better for excretion. #### Enzyme properties: ##### Specificity (Lock and key) The substrate in this model is the key and the active site in the receptor is the lock. They both represent the key-lock principle. E.g. the acetylcholine binds in the specific pocket of the enzyme acetylcholinesterase ##### Saturation (rate of enzyme) When the substrate concentration increases, so does the rate of enzyme activity. However, the rate of enzyme activity will reach an end point where the enzyme is fully saturated and no more substrates can fit even though enough substrates are available ##### Inhibition - [Non-competitive enzyme inhibition:] the inhibitor (another molecule) will bind in different pocket than the substrate. Once bound, the inhibitor will adjust the shape of the pocket of the substrate to ensure this cannot not bind anymore - [Competitive inhibition:] inhibitor binds in the same pocket as from the substrate to ensure the substrate can not bind ##### Induction Induction of an enzyme is the capacity of an organism to increase the activity and/or synthesis of an enzyme as a consequence of exposure to a substrate of the enzyme **Consequences:** - accelerated biotransformation after repeated exposure - tolerance (alcohol, drugs) #### Biotransformation of enzymes Many xenobiotics induce their own biotransformation: - ![](media/image16.png)Xenobiotic: is a compound that is foreign to a living organism. E.j. drugs, carcinogens and various compounds that have been introduced into the environment by artificial means How the molecule will metabolize its very dependent on the reaction that will occur. **For example**, if malathion (inactive) will be oxidized it can convert to the active form (malaoxon), this can to breakdown into products (so become inactive). However, when it is hydrolysed it can breakdown into their products and becomes inactive. This Is the reason why differences in metabolism can lead to differences in sensitivity. Differences in metabolism can lead to differences in sensitivity (within and between species) - Differences in metabolism can contribute to variations in sensitivity to substances or drugs both within and between species - Individuals or species with different enzyme variants may have different sensitivities to certain compounds - Variations in the enzymes involved in biotransformation can impact speed and efficiency of this process affecting sensitivity. **For example**, some individuals can faster eliminate certain drugs and reduced sensitivity (also example of alcohol when there is intraspecies ( species from another country e.g.) then certain types can better eliminate the alcohol than others because of the certain enzyme variants) Excretion --------- Done by active transport (e.g. kidney or liver) []{#_Toc170312833.anchor}Phase III Definition= [excretion/elimination: removal of the conjugated product from the body] Excretion referred to as phase III This is because it's the final step in the process of eliminating substances from the body after they have undergone biotransformation. The transformed and conjugated substances are transported from the cells or tissues to the body's excretory organs for removal. Excretion takes place in kidneys, liver, lungs and intestines. Kidneys filter the water-soluble substances from the blood into the urine & contributes to excretion by eliminating substances into the bile, which is then excreted through the intestines. Excretion by the lungs can be done with volatilization. **Lecture 4 dose-response curve** ================================= Dose response relationships --------------------------- Relationships between amount of exposure (dose) and the resulting effect or response in the body. ### Toxicological endpoints - LC50/LD50; median lethal concentration/dose in water (umol/L or % alcohol) (for human; LD50) - Acute oral toxicity LD50 in ug/kg body weight - EC50/ED50; concentration/dose causing 50% effect (for human; ED50) - EC10/ED10; concentration/dose causing 10% effect - LOEC/NOEC; lowest/no observed effect concentration - LOEC lowest concentration tested with significant difference in response compared with control - NOEC highest concentration tested with no significant difference in response with control - **! LC50/EC50/EC10 determine by fitting dose-response model** and **LOEC/NOEC determined by statistical test !** - student t-test (simple way) (comparing concentrations one-by-one with control) - ANOVA combined with posthoc test (Williams) - **THE LOWER THE VALUE, THE MORE TOXIC THE CHEMICAL!!** - Obtained by statistical test - Equal to one of the concentrations - Sensitive for the number of replicates - Sensitive for variation in response - Depends on the statistical test chosen **Disadvantages NOEC** - Inefficient use of test data (most data points ignored) - Level of effect at NOEC regularly \>20% - Poor testing leads to high (unprotective) NOECs Alternative approach **[Curve fitting (ECx)]** - This approach estimates applying a logistic model. - Three parameters are determined: - Ymax, b (slope) and EC50. These are estimated by fitting equation to data using **sum-of-squares method** (SEE PRACTICAL). - The statistical programme (SPSS/R)needed to give 95% confidence intervals for these parameters **Advantages ECx approach** - Obtained via estimation (regression based) - Not restricted to one of the test concentrations (cj's) - Uses all information from test - Depend on the model chosen - Confidence intervals - Makes use of all available data from the test ### Use of dose-response curves **Forward use** predict effect caused by toxicant concentration (reading the dose-response curve as knowing the % response that they want to obtain and figuring out what dose this includes) **Backward use** estimate exposure concentration that triggered certain response (reading the dose-response curve as knowing the dose that they want to give to the patient and figuring out what effect this gives) - From this you can derive chemical properties (ED50) and trigger values (NOAEL, LOAEL) - NOAEL: no observed adverse effect level - LOAEL: lowest observed adverse effect level - Critical effect dose (CED) - Benchmark dose (BMD) - Critical effect size (CES) - Confidence interval (CI) - Benchmark dose lower confidence bound (BMDL) - response to unknown mixtures Different types of dose-response relationships **Linear dose-response**; in a linear dose-response relationship, the response increases or decreases in a proportional manner with increasing dose or concentration (straight line) **Sigmoidal (S-shaped**); initial slow response -\> rapid increase/decrease -\> plateau phase **Non-monotonic**; they show a complex pattern with multiple peaks, valleys, or reversals in the response **Threshold-like response**; there is a sharp transition from no response to a complete response once a certain threshold dose or concentration is reached. **U-shaped**; response pattern where moderate doses or concentrations produce the highest response, while both low and high doses result in lower responses **Hormesis\*** is a phenomenon observed in dose-response relationships where exposure to low doses of a substance or stressor produces a beneficial or stimulatory effect, while higher doses result in toxic or inhibitory effects. In other words, hormesis is a biphasic dose-response relationship characterized by a low-dose beneficial response and a high-dose detrimental response. **Lecture 5 bioaccumulation** ============================= - Biotransformation (metabolisation) is a key factor in bioaccumulation - Persistent lipophilic chemicals with low biotransformation can undergo biomagnification - Water-soluble and readily biodegradable chemicals cause usually less problems with bioaccumulation []{#_Toc170312839.anchor}Bioaccumulation: - describes the transfer and accumulation of a chemical from the environment into an organism - Chemical concentration in the organism achieves a level that exceeds that in the environment (water) ### Factors related to bioaccumulation - fat content, - age - sex - weight (body mass of organism relative to the surface area across which exchange with water phase takes place) - difference in uptake route - metabolic activity (biotransformation) Examples: - the levels of DDT (dichlorodiphenyltrichloroethane) (pesticide) and its metabolites vary based on the age and sex. It appears that when the female and male dolphins have the same age they also have similar DDT levels. As they become older the female dolphins increase in DDT in comparison to males who have decreased levels. This is because the female dolphins contain more fat and DDT is lipophilic, and female may accumulate higher levels of fat due to lactation and pregnancy. Also higher levels of DDT can happen due to lactation. Because of this newborns can have higher levels. - Dietary, dolphins higher in food chain consume larger fish so probably higher levels of DDT due to biomagnification ![](media/image18.png) []{#_Toc170312841.anchor}Bioconcentration factor (BCF): - concentration in organism higher than in its **surrounding environment** - Environment is main route of uptake (e.g. water) - BCF = Corg / Caq = Kw / Ke - Corg: concentration in organism (lipid weight) (mg/kg) - Caq: concentration in water (volume basis) (mg/L) - Eu Legislation: - BCF ≥ 2000 -\> bio accumulative - BCF ≥ 2=5000 -\> very bio accumulative []{#_Toc170312842.anchor}Biomagnification (BMF): - concentration in organism is higher than that in its **food** - food is main route of uptake - BMF = Corg / Cf - Cf: concentration in food - Best determined under laboratory conditions - Accumulation of a chemical in organism **both** from food and the environment (Caq) under field conditions. - BAF = Corg from the field / Caq from the field []{#_Toc170312844.anchor}Bioconcentration test - OECD 305 BCF Test protocol - Fish test of 28 days - exposure of two concentrations of chemical - [uptake phase] (process where contaminants enter organism's body) -\> Uptake phase is the parabola shown in graphs whereas the depuration phase is when the line becomes constant - [depuration phase] (process where elimination or clearance phase follows) ### Key Components of OECD 305 BCF Test: - **Test Organisms:** Typically, juvenile fish of a standard species - [Exposure Phase:] Fish are exposed to a constant concentration of the test substance in a flow-through system for a predetermined period (usually 28 days, but can be extended to 60 days). - [Depuration Phase:] fish are transferred to clean water (free of the test substance) to allow for the elimination of the accumulated chemical from their tissues. This phase typically lasts until the chemical concentration in the fish reaches a steady state or a predetermined level. - [Sampling and Analysis:] Fish samples are collected at regular intervals during both phases, and the concentration of the test substance in their tissues (whole body or specific organs) is measured using analytical techniques. - [Data Analysis]: The bioconcentration factor (BCF) is calculated as the ratio of the chemical concentration in the fish tissue to the concentration in the water at steady state. Kinetic parameters like uptake and elimination rate constants can also be determined. - Validity Criteria: The test results are considered valid only if certain criteria are met, such as maintaining stable test conditions, achieving a steady state in the fish tissues, and ensuring adequate analytical quality control. Trophic magnification (TMF) --------------------------- Process in which concentration of certain substances increase at higher levels of the food chain. E.g. when organisms at lower trophic levels are exposed to substance that can be absorbed and accumulated in their tissues it can transfer to next trophic level when predators consume lower-levels. Organisms at higher levels have slower metabolic rates which results in in not easily metabolized tissue so high concentration in the food-chain. - Primary producers = algae - Grazers and filter feeder = cockles, worms, shrimps, plankton - First level predators = fish - Second level predator = bird Key differences and relationships: ---------------------------------- BAF is a more comprehensive measure of bioaccumulation, accounting for all exposure routes, while BCF focuses solely on uptake from water. BMF and TMF both assess biomagnification, but BMF is typically calculated from laboratory studies with specific predator-prey pairs, while TMF is derived from field data and represents an average across multiple trophic levels. BCF and BAF can be used to predict the potential for biomagnification (BMF or TMF). If a chemical has a high BCF or BAF, it is more likely to biomagnify in the food web. Different factors can influence the bioaccumulation: fat content, sex, weight (body mass of organism relative to the surface area across which exchange with water phase takes place), difference in uptake route, metabolic activity ### Relationship bioaccumulation and physical-chemical properties #### octanol-water-partitioning coefficient (water lipid) (see lecture 2) - Compounds with high Kow -\> - 1\) pass cell membranes easily - 2\) accumulate in fatty tissues (so more fattier fish means more accumulation) - High Kow = high bioconcentration factor - high Kow = high soil sorption - high Kow = low lethal concentration - !!! for very high lipophilic chemicals is the BCF no longer correlated with Kow !!! #### Koa = octanol-air-partitioning coefficient (biota-air) - Measure of retention of a compound in air-breathing organisms - Koa = Coctanol / Cair - Compounds with high Koa - pass respiratory surface relatively easily - accumulate in air-breathing organisms [Bioavailability = fraction of the total concentration, in a specific medium, that can be absorbed by an organism] [Bioavailable fraction = fraction of a chemical 'freely' dissolved in the water phase] [Total water concentrations: chemical bound to particles + free dissolved] []{#_Toc170312849.anchor}Biomagnification (BMF)\ **\ **due to biomagnification it can occur that higher levels of DDT are detected because of the food chain\ ![A picture containing text, screenshot, graphic design, design Description automatically generated](media/image20.png) [Key factors in BMF are:] - concentration in food & organism, lipid or protein content of diet & biotransformation - When calculating BMF calculation you need prey-predator relationships and diet composition\ \ Biotransformation (metabolisation) is a key factor in bioaccumulation Bioaccumulation refers to buildup of substances (chemicals) in the tissues of living organisms over time. Biotransformation is the process by living organisms chemically modify foreign substances or toxic compounds in order to make them more water-soluble and easier to eliminate. Enzymes in the body catalyse and can either activate or detoxify the compounds, depending on transformation pathway. Biotransformation can influence the rate at which substance is eliminated from the body. If a substance is poorly metabolized or transformed into more persistent and bioactive metabolite it can lead to its accumulation in tissues which could have adverse effects on organisms. Biomagnification typically occurs with chemicals that have the following characteristics: - [High persistence:] The chemicals are resistant to degradation or breakdown processes in the environment or within organisms. - [High bioaccumulation potential:] The chemicals have a tendency to accumulate in organisms at a rate faster than they can be eliminated. - [Biomagnification potential:] The chemicals can transfer and increase in concentration as organisms at different trophic levels consume one another. Water-soluble and readily biodegradable chemicals cause usually less problems with bioaccumulation Water-soluble chemicals have a higher affinity for water rather than fats or lipids. As a result, they are less likely to accumulate in the fatty tissues of organisms. Instead, they tend to remain dissolved in water and can be easily excreted or eliminated from the body through processes like urination or defecation. These chemicals have lower potential for bioaccumulation because they do not have a strong tendency to accumulate and persist in the tissues of organisms. Readily biodegradable chemicals are substances that can be broken down by natural processes, such as microbial action or enzymatic reactions, into simpler and less harmful compounds. These chemicals undergo biodegradation relatively quickly, which reduces their persistence in the environment and within organisms. As a result, they have a reduced ability to accumulate in organisms over time. The combination of water solubility and biodegradability makes chemicals less likely to persist in the environment and accumulate in living organisms. This decreases their potential for bioaccumulation and subsequent biomagnification through []{#_Toc170312850.anchor}**Lecture 6 Adverse outcome pathways** What is an AOP? --------------- AOPs are a conceptual framework that describes the key biological events that lead to an adverse outcome, such as a disease or a toxicological effect, caused by exposure to a stressor, such as a chemical or physical agent. AOPs are intended to provide a structured, transparent, and mechanistic understanding of the relationship between exposure to a stressor and an adverse outcome. AOPs consist of a series of key events, starting with the initial interaction between the stressor and the biological system and ending with the adverse outcome. Each key event is connected by a series of causal relationships that link the events together. AOPs are intended to serve as a tool for risk assessment, regulatory decision-making, and the development of alternative testing methods. They provide a mechanistic understanding of how a stressor can lead to an adverse outcome, which can help in identifying potential hazards, predicting the likelihood of adverse outcomes, and assessing the effectiveness of interventions. AOPs can also be used to identify data gaps and research needs, as well as to guide the development of new testing methods that are more predictive of human health effects. Overall, AOPs provide a valuable tool for understanding the mechanisms of toxicity and improving the safety of chemicals and products. Important definitions related to AOPs: -------------------------------------- - **MIE (Molecular initiating event):** This type of key event is the starting point of the AOP, where a chemical on molecular level in an organism results in biological perturbations. This could be enzyme binding, free radicals, phototoxicity, physical effects like corrosion, etc. - **KE (Key event):** A change in biological or physiological state that is both measurable and essential to the progression of a defined biological perturbation leading to an adverse outcome. - **KER (Key Event Relationship):** Defines a causal and predictive relationship between the upstream and downstream event. - **AO (Adverse Outcome):** This type of key event is the endpoint of the AOP, it could be disease on organism level or on population level. Five principles of AOP development ---------------------------------- - 1.AOPs are NOT chemical-specific - AOPs do not describe what a single chemical does, but what ANY chemical can do that triggers the MIE. - 2.AOPs are MODULAR - Building blocks: Key Events and Key Event Relationships - 3.AOPs are a pragmatic functional unit of development and evaluation - AOPs are a pragmatic simplification of complex biology. It helps you to focus on a specific aspect of toxicology, for development and evaluation. - 4.AOP networks are the functional unit of prediction - Multiple linked AOPs, better representative of biological complexity and functional unit of prediction of what happens in the real world. - 5.AOPs are living documents - AOPs are a way of organising existing knowlegde **What are AOPs useful for?** \- [Test prioritization:] AOP can be used to prioritize chemistry tests and focus limited resources on the issues that matter most. \- [Improved transparency and public participation]: AOPS provide a clear system for identifying potential toxins in substances. \- [Identification of KE and mechanisms] \- [Improving risk assessment:] Risk assessments can be improved by incorporating AOPs into a comprehensive and evidence-based framework for evaluating the potential toxicity of substances. \- [Improved understanding of toxicity mechanisms] \- [Increased efficiency in toxicity testing] \- [Provide a framework for regulatory decision-making:] AOPs can be used by regulatory agencies to inform risk assessment and regulatory decision-making. They provide a transparent and scientifically rigorous framework for evaluating the potential risks of chemicals, and can be used to identify **data gaps and prioritize further testing.** **Lecture 7 Mixture toxicity** ============================== Effect summation ---------------- You would assume that if you have an effect of compound A which is 30% and the other compound B is 10% together it is 40% effect, but actually it is 37%. This is called effect summation! It is an overestimation because for example you cannot have 110% effect because you have one life which is 100% Distinguish between independent action and concentration addition 4 classes joint effect No interaction (additive) Interaction (non-additive) ------------------------ --------------------------- ---------------------------- Similar action Simple similar action Complex similar action Dissimilar action Independent action Dependent action Independent action ------------------ - there is no interaction between the compounds in the mixture - A does not influence the amount of B reaching B's site of action and reversely - Chemicals in a mixture act independent, i.e. they act on different target sites (have different mode of action) - It assumes that each chemical acts independently and contributes to the total effect based on its own potency or efficacy, regardless of the presence of other chemicals [**Example** Compound A has 40% effect and compound B has 70% effect ] Exposure only com. A: 60% chance of not being affected Since there is only 60% left, only 70% of the remaining 60% can be affected. Which means 30% remains non-affected Pnon-affected = 0.6 x 0.3 = 0,18 Pafffected = 1 -- 0,18 = 0,82 ALWAYS USE CHANCES (0-1), NOT PERCENTAGES Concentration addition ---------------------- - **(simple similar action): there is interaction between the compounds in the mixture** - Compounds have the same mode of action - One chemical can be replaced totally or in part by an equal fraction of an equi-effective concentration (EC50) of another, without changing the overall combined effect! - The fractions of the substance concentrations are also called toxic units (TU) \> overall toxic unit of the mixture - It assumes that the chemicals act independently but contribute to the total effect in proportion to their concentrations or doses. [EC50A = 2 um; EC50B = 10 um] To calculate by what combination of Ca and Cb causes 50% effect, you need the formula: (CA / EC50A) + (CB / EC50B) = 1 (mixture) CA / EC50A = 1 (single compound) You need the concentration of one compound to calculate the other concentration that's needed Mixture concentration ECxmix can be calculated for any effect level x ECxmix = (pi / ECxi ) + (pi / ECxi ) etc. \* HOWEVER, the CA-expected mixture effect cannot be calculated directly \> direct calculations are restricted to the level of effect concentrations (ECx-values) Isobologram method ------------------ **Interaction** refers to the combined effect of two or more chemicals that is different from the effects that would be predicted based on the individual effects of each chemical. Interactions can be synergistic, antagonistic, or additive. See on the right the isobologram. - ![](media/image22.png)**Synergistic**; A synergistic interaction occurs when the combined effect of two or more chemicals is greater than the sum of their individual effects - **Antagonistic**; An antagonistic interaction occurs when the combined effect of two or more chemicals is less than the sum of their individual effects - **Additive**; An additive interaction occurs when the combined effect of two or more chemicals is equal to the sum of their individual effects ![](media/image24.png) You can perform a full factorial design to obtain an isobologram. You take different amount of every individual compound in a mixtures. In the end you have a lot of calculated points and can make met those a good equitoxic curve. When interpretating the below isobologram 1, you can say that all three lines act similar and concentration addition (this case all acetylcholine inhibitors so expected). Isobologram 2 needs less TU to have the same effect; synergistic. Isobologram 3 needs more TU to have same effect; antagonistic A picture containing line, plot, diagram, slope Description automatically generated ![A picture containing line, diagram, plot, text Description automatically generated](media/image26.png) A picture containing text, screenshot, diagram, line Description automatically generated How to calculate toxic dose --------------------------- The EC50 can be established on 1 TU. So for example if you want to calculate a concentration series you can do the calculation below: ![A picture containing text, font, screenshot, electric blue Description automatically generated](media/image28.png) This is only applicable if you consider individual solutions. Lets say if you consider a mixture design, you can have 1:1 ratio's were the TU consists of 0.5 TU A and 0.5 TU B. but in ratio 1:3 consists the TU of 0.25 TU A and 0.75 TU B. you need to recalculate for the specific concentrations. TU = CONC / EC50 If you want to make a conclusion about an interaction of compounds in a mixture; you can calculate the TU for every single compound and then sum them up. if they are 1 together it is concentration addition otherwise synergistic or antagonistic. Calculate mixture effects for compounds with parallel dose-response curves using relative potency factors [Parallel dose-response curves! -\> for different receptor affinities] At every point there is the same distance. Set the most toxic compound as reference compound (I). to calculate the relative potency (REP) you can use below formula: REPi = ECxreference compound / ECxi The most left curve is the most toxic (potent) -\> needs the least amount of compound to be toxic Below is not concentration addition, because the blue one need less concentration to obtain the same effect, so different TU than the other. ![A picture containing line, diagram, plot, text Description automatically generated](media/image30.png) **Lecture 8 dioxin-like compound and TEF concept** ================================================== Definition ---------- Persistent Organic Pollutants (POPs) are organic chemical substances, that is, they are carbon-based. They possess a particular combination of physical and chemical properties. Properties ---------- - Remain intact for long periods of time (Very persistent against breakdown) - 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 (Bio accumulative (hydrophobic)) - Are toxic to both humans an wildlife - Same mode of action - Same clinical symptoms - Toxicity is additive: can be summed together Dioxin and dioxin-like compounds are highly toxic chemicals that belong to the class of persistent organic pollutants (POPs). - They are formed as side products of various combustion processes, organochlorine production, leaded fuel and metal industry. - They share similar chemical structures and exhibit similar toxicological properties. Dioxins refers to a group of halogenated compounds, that are polychlorinated dibenzo-p-dioxins (PCDDs), but also PCDFs and PCBs are included. General structure PCDD; two benzene rings connected by oxygen atom with chlorine substituents attached. ![](media/image32.png)General structure PCDFs; dibenzofuran General structure PCBs; biphenyl with chlorine substituents Sources PCBs: ------------- - Produced on purpose 1930-1980 - Very stable compounds (resistant against electrical, thermal, chemical breakdown) - Used in closed applications (thermal conductors) - Used in open applications (plasticizers, solvents) - Biomagnification in the food chain - Toxic properties - Ban on production - STILL ENVIRONMENTAL PROBLEM! -\> 'leak' from existing applications and waste ![](media/image34.png) Different substitution patterns can be produced but not all are stable enough or dioxin like. For example the di-ortho PCB is not a stable compound, because both chlorine atoms are placed at the ortho position. This creates more steric hindrance and the is compound not planar (cannot rotate). **Properties** **Exposure** ---------------------------------------------- ------------------------------------------------------- very persistent against breakdown Natural origin (use of marl clay in potato selection) bioaccumulative (hydrophobic) Intentional poisoning (Yushchenko) same mode of action Disaster (Vietnam war) same clinical symptoms Illegal activities toxicity is additive; can be summed together Background levels\* Background levels (**adverse effects**) --------------------------------------- - **Grasshopper effect:** move from hotter regions to colder regions -\> alternates processes of volatilisation and condensation - **Food chain accumulation:** PCB available in water + plankton etc which comes in fish and humans eat fish. Food is the main source of human TEQ exposure! (fish have the highest levels) - These levels of PCB in humans are excreted through breast milk and is surrendered to the baby Know differences in species sensitivity and congener potencies -------------------------------------------------------------- - **Species sensitivity** refers to the variation in sensitivity to toxic substances among different species. Different species can exhibit varying degrees of sensitivity to specific toxic compounds due to differences in physiological, biochemical, and metabolic processes. - **Metabolism**: Species can differ in their ability to metabolize or detoxify specific compounds. Some species may have more efficient detoxification mechanisms, such as enzyme systems that can break down or eliminate toxic substances more effectively than others. - **Receptor Sensitivity**: Variations in the sensitivity of target receptors or biological pathways can influence species sensitivity. Differences in receptor structure or affinity for a toxic compound can lead to varying responses among species. - **Absorption and Distribution**: Variations in the absorption and distribution of toxic compounds within an organism can affect sensitivity. Species may differ in the ability to absorb or distribute a compound to its target sites or organs, which can influence the magnitude of the response. - **Elimination**: Variations in the efficiency of elimination mechanisms, such as metabolism, excretion, or biotransformation, can impact species sensitivity. Some species may eliminate toxic compounds more efficiently than others, reducing their potential for adverse effects Congener potencies, on the other hand, refer to the relative toxicities or potencies of different congeners within a group of chemicals, such as dioxins or PCBs. Congeners are different chemical variations or compounds within the same family or group that share similar chemical structures but have slight differences, usually in the number or position of chlorine atoms. The toxicological potency of congeners can vary, with some congeners being more toxic or potent than others. For example, within the group of dioxins, the congener 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is considered the most toxic and potent, while other congeners have lower toxicities. Similarly, within the group of PCBs, different congeners can have varying toxicities and potencies depending on factors such as the degree of chlorination and the position of chlorine substitutions. Mechanism of action of dioxin-like compounds -------------------------------------------- ### Ah Receptor (AhR) theory: effects on gene expression and phosphorylation Dioxin like compounds are binding to the Ah-receptor -\> conformational change -\> translocates into the nucleus -\> forms complex with protein -\> complex binds to specific DNA sequence (xenobiotic response elements (XREs) -\> activates gene transcription -\> induction of enzymes particularly cytochrome P450 Induced gene expression The binding of dioxin to AhR can result in downregulation of certain CYP enzymes. When AhR is activated by dioxins, it leads to the increased expression of enzymes involved in the metabolism and elimination of dioxins themselves, such as CYP1A1 and CYP1B1. The increased expression of CYP1A1 and CYP1B1 as a response to AhR activation by dioxins is accompanied by the downregulation of other CYP enzymes, including some of the CYP450 enzymes responsible for the metabolism of drugs and other xenobiotics. This downregulation can result in decreased metabolic activity of these CYP enzymes, leading to altered drug metabolism and potentially affecting the efficacy and safety of drugs that are substrates for these enzymes. ![A diagram of a cell Description automatically generated with medium confidence](media/image36.png) TEF & TEQ --------- [Toxic equivalence factor (TEF):] instrument to express the relative potency of individual toxin congeners compared to the most potent TCDD. [TCDD Equivalent Concentration (TEQ):] potency of a compound or mixture expressed as a TCDD concentration with equivalent potency - Indirect: calculate TEQ concentration based on results from GC-HRMS analysis *TEF = ED50~2,3,7,8-TCDD~ / ED50~x~* *TEQ (total) = (TEF1 x concentration) + (TEF1 x concentration) etc.* TEQ = (TU X EC50TCDD) + (TU X EC50TCDD) etc [Can only use this method as:] - Very persistent against breakdown - Bioaccumulative - Toxicity; - Same mechanism of action - Same clinical symptoms - Toxicity is additive; can be summed together ### How to determine TEQ values using reporter gene assay Normally, the TEQ values are calculated based on ability of the ligands to active the AhR pathway. In this assay is a reporter gene that is response to AhR activation used, such as luciferase known TEF values are exposed to the receptor and measured afterwards to determine the amount of luminescence. ![A picture containing text, diagram, screenshot, font Description automatically generated](media/image38.png) TEQ assessment can be done via DR-CALUX assessment **Lecture 9 Ecotoxicology studies** =================================== Definition ---------- Ecotoxicology = study of the effects of toxic chemicals on biological organisms, especially at the population, community, ecosystem and biosphere levels **Aim:** predicting effects of chemical pollutants on populations, communities and ecosystems from knowledge on effect on individual species (so first you test the individuals by toxicity testing and then the population by ecotoxicity testing) Approaches in ecotoxicity testing --------------------------------- - Predictive: start in the lab, extrapolate to field (estimating future outcomes) - Follows paradigms of human toxicology & indicator/model species cultured in laboratory & test protocol & models for dose-response analysis & for new chemicals - Diagnostic: assess the field directly (identifying and classifying existing conditions) - Ecology and environmental chemistry crucial & validation of laboratory approach & more suitable for existing chemicals & more complex & no negative control Feature Diagnostic Testing Prognostic Testing ---------- -------------------------------------------- --------------------------------------------------- -- -- Purpose Identify or confirm a disease or condition Predict the likely course or outcome of a disease Focus Present Future Examples Blood tests, imaging scans, biopsies Genetic tests, tumor markers, imaging scans Outcome Diagnosis Information about expected health outcomes Animal exposure routes: ----------------------- - Oral via GI track (all animals) - via food and water - Contact (topical) - via skin (dermal): vertebrates - via cuticle: arthropods - via body wall (slugs, worms) - Respiratory - via lung or gills (vertebrates) - via trachea (invertebrates) - Special routes, e.g. - via injection - via suppository Criteria for selecting ecotoxicity test --------------------------------------- - Determine dose-response relationships - Using single species (so one type of species), surrogate for the environment - Testing single chemicals - Controlled test conditions - End-points; survival, growth, reproduction, biochemical parameters etc **What type of information required**? Descriptive (what are the effects of a compound at what dose?) or mechanistic (how does compound cause this type of toxicity?) **For what purpose is the information used**? prognosis (certain dose/concentration, what is the risk?) or diagnosis (is my sample toxic?) - **Acute toxicity** : effects upon short-term exposure (hours-days), focused on mortality - **Chronic toxicity**: effects upon long-term exposure (weeks-months) Understand why testing a positive control is needed It is a verification that the test methods used is sensitive enough to detect the intended target, it can also help by determining the reproducibility (routinely including positive controls by monitoring). Also for differentiation of false positives & confirmation of method performance. A lot of standardization is done by specific protocols that need to be performed by every researcher Test organisms in ecotoxicology ------------------------------- Models for human: - rat, - mouse, - guinea pig Most used test organism in ecosystem: - bacteria, - algae, - aquatic plant, - invertebrate, - zebrafish Describe the reasons why an effect-based assessment using bioassays is to be preferred over a chemical analysis of contaminated soil or water samples Bioassays are experimental procedures used to assess the biological activity or effects of a substance or mixture. They involve measuring and quantifying the response of living organisms or cells to determine the potency, toxicity, or other biological properties of the tested sample. Toxicity tests on environmental sample Performed in lab under controlled conditions Using laboratory-cultures test organisms Response on test sample compared with ; non-polluted reference sample or least contaminated sample or dilution required to obtain no effect Aim: detection pollution or enable selection of samples for chemical analysis or to set priorities for remediation Reasons bioassays preferred over chemical analysis - Chemical analysis usually restricted to a limited selected set of chemical pollutants - Chemical analysis does not provide insight into: bioavailability and presence of metabolites - Standards (threshold values) only developed for single chemicals **Examples bioassyas;** - Bacterial tests (enzyme inhibition) - Test with cell lines modifies to specifically respond to different chemical - Microtox using luminiscent bacteria - Mutatox - Algae - Daphnis and other crustaceans conclusions 1. Laboratory (single-species) toxicity tests available with several soil, sediment and aquatic organisms 2. Species mainly selected for ease of culturing in lab, but: 3. Should also be representative of community/ecosystem to be protected 4. Standardization important à reproducibility of responses 5. Tests applicable for predictive and diagnostic testing 6. Diagnosis requires effect-based assessment (bioassays) to overcome problems not solved by chemical analysis only **Lecture 10 in vitro toxicity testing** ======================================== Definition ---------- 2 types of Information can be requested: - **descriptive**: what are the effects of a compound at what dose? - In other words: what is the HAZARD? - **Mechanistic:** how does the compound cause this type of toxicity? - From mechanism of action (molecular) to mode of action For wat purpose information used: - **prognosis**: if I have this dose/concentration, what are the risks? - **diagnosis**: is my sample toxic? Is it polluted? Protein based bioassay ---------------------- Protein is isolated from an organism and expressed in transformed bacteria or transfected cells (recombinant) **Two types of assays:** - Binding assays - Radiolabelled ligand (RLBA) - Fluorescent-labeled ligand (FLBA) - Enzyme inhibition assay - Measure reaction rate - Decrease in substrate - Increase in product **[Example binding assay:]** Competitive radioligandbinding assay [Estrogen receptor binding assay (anticonceptiepil)] Mechanism of action: You have estrogen receptors which bind to (xeno-)estrogens -\> these come in the nucleus of the cell and bind to the ERE gene -\> certain mRNA makes the specific protein and gives signal, in this case feminizing effects The ER is isolated from a rat cytosol and the specific estrogen + (anti)-estrogen and the radioactive estradiol is added to the mixture -\> in the end the amount of unbound radioactivity is measured (competitive radioligand) **[Example binding assay:]** Fluorescent-labelled ligand (FLBA) [Acetylcholineesterase inhibition assay] Mechanism of action (neurotransmission): normally the acetylcholine is binding to the acetylcholinesterase and is then broken down to acetate and choline in the neuron. You can also inhibit this enzyme by using inhibitor in order to prevent producing acetate and choline By determining the amount of acetate and choline you can use DTNB which binds to thiocholine. The amount of resting TNB is determined by fluorescent. ![A screenshot of a computer Description automatically generated](media/image40.png) Cell-based techniques --------------------- ### Primary cell culture A picture containing diagram, line, screenshot, design Description automatically generated\ primary cells are derived from living tissues and isolated from donor organism by cutting -\> enzymes are added and added to a suitable culture medium -\> reproduction in the sterile system at appropriate temperature under certain conditions. Primary cells retain many of the characteristics and functions of the tissues or organ that the derived from. They eventually stop diving and die off so they have a limited lifespan More accurate representation of in vivo conditions compared to cell lines Powerful tool for research of cell production, reproduction and metabolism Limited, cell-type specific number of cell doublings ### Cell lines ![A picture containing text, diagram, screenshot, line Description automatically generated](media/image42.png) single cell isolation from living tissues from the rat are dissected and cut by specific separation method -\> enzymes are added (proteases) -\> added to a suitable culture medium -\> reproduction in the sterile system at appropriate temperature under certain conditions -\> each medium is again used for further reproduction Cell lines are derived from primary cells but have undergo immortalization -\> can divide indefinitely Immortalization can occur in cancer cells or genetic modifications Continuous supply of cells for experiments because they can propagate for extend of time For investigate drug development and disease studies ### Immortal cell line = continuous cell line A picture containing text, diagram, screenshot, line Description automatically generated a typical virus with it specific genes is introduced to the target cells through viral infection -\> integration can disrupt normal cellular processes and leads to alteration of cell cycle control mechanisms -\> introduced genes (immortalizing genes) override natural cellular mechanisms (transformation) -\> cells grown in culture allowing them to multiply -\> individual transformed cells are isolated and expanded as separate clones (ensures the cell line consists of a homogeneous population) -\> characterization Derived from cancer cells For long-term studies and investigations cellular process Example is HeLa-cells: sample of cervical cancer from a women was taken and were the first human cells to be successfully cultured and grown in lab **Examples of cell-based toxicity assays** - Cell viability (cytotoxicity) - Mitochondrial functioning, membrane leakage, energy levels - Cell growth - Uptake, biotransformation, elimination - Cell metabolism - Cell-type dependent functioning - Omics - Transcriptomics: gene expression (mRNA) - Proteomics; protein progile - Metabolomics: products and intermediates of metabolism - Reporter gene assay - Differentiation Reporter gene bioassay![](media/image44.png) -------------------------------------------- Introducing a reporter gene into cells of interest, which produces a signal in response to the activation of a specific pathway or the presence of a particular compound. Ligand binds to receptor and activates reporter gene + responsive gene -\> mRNA + reporter gene mRNA are produced and the reporter gene's transcription and translation is activated into reporter protein -\> reporter protein produces detectable signal such as fluorescent or luminescent Convert substrate into coloured product or into luminescent product Differentiation models ---------------------- Goal: to create differentiated cells in vitro for further toxicity testing ### Stem cell differentiation models Pluripotent cells are used (stem cells derived from embryos which do not have any cell type in the body yet -\> can still differentiate). Researchers induce differentiation into specific cell types or tissues of interest by manipulating the culture conditions and providing signalling cues. Stem cells can divide indefinitely while remaining undifferentiated Totipotent = present in fertizilized egg (zygote)\ pluripotent = cells derived from human blastocyst: can rise to any type of cell except embryonic cells in placenta\ multipotent = germ layer endoderm, mesoderm, ectoderm ECS are derived from inner cell mass of blastocysts (obtained from IVF procedures) -\> ECSs are pluripotent -\> cultured in lab -\> -\> embryonic stem cell line -\> manipulation by researchers to get one type ### Induced pluripotent stem cell differentiation (iPSC) Reprogramming adult somatic cells into a pluripotent state so that they can make any type of cell (mesoderm, endoderm and ectoderm) Bypass ethical concerns iPSCs from patients with specific disease can be analysed in a personalized manner use of iPSC in medicine: adult cells are taken from human -\> reprogramming factors make iPSC cells -\> gene correction can be done in vitro -\> bad cells adding in organism and give cure with gene correction -\> transplantation of corrected cells in human ### transdifferentiation models direct (lineage) programming of conversion of one somatic cell type into another without passing through pluripotent state. Fibroblast (wound healing, produce collagen etc) can be transdifferentiated into neurons and adipocyte (fat cells) Classify stem cells as pluripotent and multipotent Pluripotent: differentiate into cells representing all three germ layers ESCs: already pluripotent and can also differentiate in any cell iPSCs: reprogramming somatic cells into pluripotent state multipotent: can only differentiate into related cell types within a specific tissue or organ Complex in vitro models ----------------------- Increase the complexity of the in vitro system needs to be to take into account: - cell-cell interactions - extracellular matrix (ECM) -- cell interactions - physiology and inter-tissue communication ### 2D monolayer to 3D culturing More realistic type of cell growth, including cell-cell interactions, polarization, differentiation ### ![](media/image46.png)Cell co-culturing Growing multiple cell types together in a culture system. ### Organ-on-a-chip or body-on-a-chip Multichannel 3D microfluid cell culture chip which functions as an artificial organ ### Body-on-a-chip Multi-compartmental perfused systems **Lecture 11 Endocrine distruptors** ==================================== **Endocrine disruptors:** exogenous substance or mixture that alters function(s) of the endocrine (hormonal) system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub-)populations. **Example** :DDT, Bisphenol, PCB, PAHs, dioxins, phenols, pesticides, herbicides, fungicides, metals, insecticides or industrial chemicals Ideal concentration space (see below) **Endocrine disruption hypothesis**: exposure to certain chemicals (endocrine disruptors) can interfere with the normal functioning of the endocrine system leading to adverse health effects in both humans and wildlife. Book global assessment of the state-of-the-science of endocrine disruptors: concluded that scientific knowledge provided evidence that certain effects observed in wild life can be attributed to chemicals that function as EDCs Effects are endocrine related, not species dependent. Effects shown in wildlife can also occur in human if they are exposed to EDCs at vulnerable time and at concentrations Early development effects are of high concern considering the irreversibility EDC background -------------- The endocrine system is very conserved among all vertebrates (gewervelde dieren). The endocrine systems is a system of glands (klieren) producing and transporting chemical signals (hormones) throughout the body. The biological response is taking place in the target cell (receptor) **Major targets:** - Gonads (reproductive disorders) - Thyroid (development and physiological disorders) **Endocrine system** - system of glands producing and transporting chemical signals (hormones)throughout the body - Biological response is taking place in the target cell (receptor) Classes of hormones ------------------- - Amino acid derivatives (e.g. catecholamines, thyroid hormones) - Peptide hormones (e.g. pituitary tropic hormones or insulin) - Steroid hormones (e.g. testosterone and estradiol) []{#_Toc170312896.anchor}Biological classification of hormones: Role - 'releasing hormones' - Hypothalamus -\> pituitary - 'stimulating hormones' - Pituitary -\> endocrine tissue - 'non-tropic hormones' - Endocrine tissue -\> target cell ### Biological classifications of hormones : receptors - Membrane receptors - Non-steroid receptors - Extracellular - Hormone -receptor binding -\> activates second messenger system (signalling cascade) in target cell - Nuclear receptors - Steroid and thyroid hormones - Intracellular - Hormone-receptor complex -\> activates cell itself ![A picture containing text, circle, diagram, screenshot Description automatically generated](media/image48.png) Mode of action EDCs ------------------- - Agonism: binding and activation of hormone receptors ("hormone mimic") - Antagonism: binding and inactivation of hormone receptors ("hormone blocking") - Indirect: by disrupting the normal hormonal balance **The effect of EDCs in human could be:** - the sperm density - fertility or cancer forming in the gonads - Depends on: - Dose , age, sex and timing of exposure - In utero exposure vs at a later age **The effect of EDCs in wildlife could be**: - Adverse effects on reproduction due to egg shell thinning and feminization of males in birds by DDT and other pesticides - Impairment of reproduction and immune function in seals by PCB/DDT metabolites and dioxins - Distorted sex organ development in alligators linked to a DDT spill - Feminization of male fish populations by natural estrogens and xeno-estrogens - Masculinization of female marine snails by TST from antifouling paints **Diseases in wildlife observations**: - Gonad changes: - feminized/intersex gonads in fish and amphibia - reduced phallus size in alligators & imposex in molluscs - Physiological changes: - altered sex hormone levels, - elevated vitellogenin levels in male fish, - reduced T3/T4 levels in gulls, seals and polar bears, - altered parental care behaviour in fish - reduced immune response **In vitro models** : isolated hepatocytes from rainbow trout **In vivo models**: zebrafish, medaka, fathead minnow (puffy fish -\> toxic) End points: vitellogenin (protein & mRNA) & chloriogenin H (mRNA) & zona pellucia (mRNA) End points (population-relevant): survival rate, growth, sex ratio, teratogenicity, reproductive behaviour Aquatic laboratory test organisms: African clawed frog Amphibian metamorphosis assay used to assess the effects of chemical substances on the development and metamorphosis of amphibians. The assay focuses on the sensitive period of amphibian development known as metamorphosis, during which aquatic larvae undergo dramatic physiological and morphological changes to transition into terrestrial adults. Some outcomes can be different bodyweight, snout-vent length or hind limb length Zebrafish To asses are flow-through exposure experiments done 'fish short-term reproduction test' & '21-day fish assay' (21 days of chemical exposure) Example: vinclozolin has negative effect on the number of eggs Other tests:\ - 'fish sexual development test' (FSDT) (60 days) Example: expose certain hormones to fish will impact the gender of the fish. E.g. testosterone, ethinylestradiol or certain steroids 'medaka extended one-generation reproduction test' (MEOGRT) [Biomarker vitellogenin; ] Steps: estrogen receptor bind to regulatory regions in the VTG gene promoter -\> transcription into mRNA -\> transport to cytoplasm for translation -\> forming VTG protein -\> glycosylation/phosphorylation occurs -\> VTG protein released from liver into bloodstream -\> once ovulation starts the VTG is taken up by developing oocytes (eggs) Can be stimulated with estrogen receptor agonist Can be inhibited with estrogen receptor antagonist [Thyroid end points in fish tests] Important functions: metabolism, reproduction, stress response, immune system, fin formation etc. Thyroid of a fish could be an important biomarker **Lecture 12 Metabolomics** =========================== Definition ---------- **Metabolomics:** Systematic study of small organic molecules (\ performance more variable than targeted) Workflow of metabolomics ------------------------ A diagram of a scientific experiment Description automatically generated ### Extraction steps of metabolites 1. sampling 2. quenching & extraction 3. sample extract 4. analysis Sample quenching and extraction are very crucial factors in metabolite analysis because they can cause variability in the results!\ quenching: sample handling to stop all biochemical processes -\> stop activity of enzymes (proteins). WHY? -\> Metabolites have very fast turn over times ### Quenching methods - Liquid nitrogen - Acid treatment - Cold methanol Metabolomic analysis -------------------- - Targeted metabolomics - Analytical method designed to analyse specific target metabolites - Untargeted metabolomics - Analytical method should extract a wide range of different "types" of metabolites - General extraction methods - Performance (recoveries) is more variable than targeted metabolite analysis. The quenching from untargeted metabolites can be done with sample with cold solvents. ![A diagram of a blue and green column Description automatically generated with medium confidence](media/image52.png) Know data visualization of untargeted metabolomics data Data visualization is done with chromatography combined with mass spectrometry or NMR **Chromatography** **NMR** ------------------------------------------ ---------------------------------------- Targeted and untargeted metabolomics Targeted metabolomics More sensitive Less sensitive Large range of metabolites detected Limited number of metabolites detected Combined with GC or LC Little sample handling Difficult to quantify amount metabolites Quantification easy Reproducible Untargeted analysis : - LC/HRMS ToFMS or orbitrap - Direct MS (DIMS) - GC-MS with derivatisation Alignment of peaks from chromatograms of different samples -\> peak picking (based on Rt and m/z) -\> peak table -\> peak annotation/identification (most difficult) Provides retention time, m/z value, isotope ratio and MS/MS spectra If you have runned the MS you obtain exact masses with potential elemental compositions. Then you do an isotope filtering that filters all the potential isotopes. The data can be compared with other MS spectra until you have a potential candidate metabolite **Annotation** = tentative identification of a metabolite **Identification** = exact structure of the metabolite based on confirmation with an analytical standard Quality control (QC) and quality assurance (QA) also very important! To monitor the performance of metabolomics workflows With QC are the samples randomly analysed during sequence with regular analysis of a quality control sample. Evaluation of coefficients of variation of each metabolite Correction for inter-batch systematic error Visualization of metabolomics data ---------------------------------- - Box whisker plot - Fold change - FC = increase (upregulated) or decrease (downregulated) of a metabolite level between a specific group and reference group - FC = Average level of a metabolite / average of reference group - Heat map with FC - Graphical presentation of data showing the fold changes of each metabolite. The fold changes are displaced on a colour scale - Principal component analysis (PCA) (used for untargeted metabolomics) ![](media/image54.png)Statistical technique used to simplify and extract most important information from high-dimensional dataset. It transforms a dataset with potentially correlated variables into a new set of uncorrelated variables (principal components). These directions are linear combinations of the original variables. **PCA score plot** is a graphical representation of the PCA data. Each data point represents its scores on the selected principal components. Each axis is a principal component. (conc. of metabolites) The score allows observing clustering of data points and identify patterns or groupings. It also highlights similarities or differences between data points Interpretation of a PCA score plot involves examining the position of data points in relation to each other and any predefined groups or categories. Data points that are close together on the score plot indicate similar patterns or characteristics, while points that are farther apart represent greater dissimilarity. **PCA loading plot:** relationships between the original variables and the principal components The loading plot helps interpret the underlying structure of the data and understand the contribution of each variable to the principal components. It allows you to identify variables that have the strongest influence on each principal component and visualize the patterns of association between variables and components. Challenges in metabolomics -------------------------- - Metabolome is very dynamic, therefore, very time sensitive - Metabolites have a wide range of physiochemical properties and vary widely in concentration in cells and tissues - Some metabolites are very unstable during sample collection and sample treatment - Identification of chemical structure of unknown metabolites is still the biggest challenge - Different analytical techniques are needed to determine the metabolome - Sample handling is the most crucial factor in metabolomics analysis - Some metabolites are very unstable during sample collection and sample treatment - Crucial is the identification/annotation of the metabolites **Lecture 13 Epidemiology** =========================== Definitions ----------- **Epidemiology:** study and analysis of the distribution (who, when, and where), patterns and determinants (risk factors) of health and disease conditions in a defined population. **Determinant**: refers to any factor or variable that influences the occurrence or distribution of a health-related event or outcome. Determinants can be individual-level factors (e.g., age, gender, genetics) or broader social, environmental, or behavioural factors (e.g., socioeconomic status, access to healthcare, lifestyle choices) that affect health outcomes and disease occurrence. **Outcome:** refers to a measurable event or condition that is of interest in the study. It can be a health-related event, such as a disease, illness, death, or a specific health indicator, such as blood pressure, cholesterol levels, or quality of life. Outcomes are often used to assess the impact of exposures, interventions, or risk factors on health. **Sample**: refers to a subset of individuals or units from a larger population that is selected for study ![](media/image56.png)Study designs ----------------------------------- ### Cross sectional Cross sectional = measure [determinant] and [outcome] at **same point** (high exposed vs no exposed)\ -\> present because you are going to expose the people Start with population where : - no/low exposure and - high exposure. You don't know if they have the disease! - Non-smokers vs smokers Assessment relationship between exposure and outcome? ![](media/image58.png)Odds ratio : odds on the outcome in the exposed group (disease) / odds on the outcome in the unexposed group (no disease) Example: OR = (10/30) / (4/36) = 3\ -\> The odds on having lung cancer in smokers is 3 times higher compared to non‐smokers **PROS** **CONS** ------------------------------------ ---------------------------------------------------------------------- Quick and cheap No causality: measurement determinant and outcome at same time point Hypothesis generating Only odds ratios can be used Looking at more than 1 determinant ### case-control Start with the disease - select cases with the disease - ![](media/image60.png)select controls: people who are no affected by the disease, but come from the population that is representative for the cases as well Determinant: exposure, usually in the past (questionaires) **Example**: same odds ratio but in another point of few (now we look at the disease instead of the exposure): OR = (10/30) / (4/36) = 3\ -\> People with lung cancer (cases) had 3 times higher odds on being a smoker (determinant), compared to people without lung cancer **PROS** **CONS** ------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Rare diseases No causality: measurement determinant and outcome at same time point (you do not know the entire exposed population, because you did not start with a population without the outcome) Long retention period: time between determinant and disease Recall bias: people might not remember exposure in the past Quick and cheap Number of participants Looking at more than 1 determinant ### Cohort study Cohort study: want to know if they develop - Prospective or longitudinal, follow participants over time - Retrospective, ask back about the past (e.g. registry) - start with healthy population, at risk - define determinant: e.g. high (A) versus low (B) exposure - Look at incidence of disease Risk ratio/relative risk (RR): ratio of the incidence of risk in the exposed group to the incidence of risk in the unexposed group ![](media/image62.png)RR = (A / (A+B)) / (C / (C + D)) = ((6 / (6+14)) / (2 / (2+18)) = 3\ -\> People with higher exposure to chemical X have a 3 times higher risk on developing disease Y, compared to people lower exposure to chemical X. Risk difference (RD); difference of the risk in the exposed group to the risk in the unexposed group RD = (A/A+B) -- (C/C+D) = (6/6+14) -- (2/2+18) = 0,2\ -\> People with higher exposure to chemical X have 0.2 higher risk on developing disease Y, compared to people lower exposure to chemical X. **PROS** **CONS** ------------------------------------------------------------------------------------ ---------------------------------------------------------------------- Causality Not suitable for rare diseases or disease with long retention period We can use RR, because we start with a healthy population who will develop disease Duration, costs, drop-out Behaviour participant (stop smoking e.g.) Summary: -------- **Odds ratio**: odds on that event happens (having disease) / odds on that event does not happen (not having disease) **Risk ratio** (change to become ill): risk on event (having disease) / risk on that the event does not happen (not having disease) [Differences in study designs] case-control vs prospective cohort: in case-control is the selection already based on the disease (outcome). In the prospective cohort is there no selection at the beginning, every group can be chosen **[Confounding and effect modification (third variable)]** [Confounding]: extraneous variable that is related to both the exposure and the outcome, independently of their relationship (influences both)\ -\> alcohol consumption is associated with both smoking (exposure) and cancer (outcome) [Effect modification]: association between an exposure and the outcome is modified because of the presence of another factor (modifies relationship between exposure and outcome)\ -\> associations different for men and women Solution: split data for men/women (EM) or adjust for confounder in analysis **ETHICS!** - Vulnerable people (children, pregnant women) - Dependency (students) - Type of research: - Invasive: collection of blood - Non-invasive: collection of dust at a home - Studies that concern human should always be approved by a medical ethics committee **Lecture 14 Animal models in toxicology** ========================================== History ------- Testing with humans is previously done: - Edward Jenner's experiment (vaccination): - He injected a 8 year old boy with pus from a cowpox sore. The boy recovered a couple weeks later and again got injected by the same pus. However, now he didn't develop any systems what indicated that the boy got immune - Nazi's experiment: - Included involuntary sterilization, inducing hypothermia and exposing subjects to diseases - Tuskegee syphilis trials: - Performed by the CDC observed the effects of untreated syphilis --even thought it was treatable! - PortonDown Chemical experiments - ![](media/image64.png)Military personnel were exposed to mustard and nerve gas EU legislation on animal testing -------------------------------- Since 1986, the EU has had in place specific legislation covering the use of animals for scientific purposes (**directive 86/609/EEC**) On 1 January 2013, EU introduced **directive 2010/63/EU**. This was to strengthen the

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