Final exam prep toxicology

Everything is poisonous and nothing is without poison; only the dose makes that something is a poison (dose-dependency).

environmental toxicology: The science that studies the fate and effects of potentially hazardous chemicals in the environment. ecotoxicology: 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

• 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.

1. Exposure (How does an organism get into contact with compound?)
2. Toxicokinetics (what does body do to the compound? )
3. Toxicodynamics (what does the compound do to the body?)

1. Possibility for repair o Reversible o Irreversible
2. Rate o Acute toxicity occurs short after a single exposure to a compound o Chronic toxicity occurs after prolonged and repeated exposure to a compound
3. Site of action o Local (at the site of exposure) o The whole body

5 processes in chemical fate:

1. 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

2. 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

3. Advection

4. Diffusion

5. Degradation

Kh: Henry-constant (water -> air) is a measure of volatility Kow: Octanol-water-partition coefficient Kp: IV. Sorption coefficient (water – solid phase) Koc: sorption coefficient (Kp) / fraction organic carbon (fOC)

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

1. In warm temperatures VOCs evaporate
2. VOCs move in air by winds to colder places
3. In cold temperatures VOCs condense and fall to earth

• 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.

DT50, also known as the half-life, is the time required for half of the initial amount of a substance (such as a chemical) to degrade or disappear in a given environment. It is a key parameter used to assess the persistence of a substance in the environment and its potential for long-term impact.

• 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
• Inhalation: exhaling of non-absorbed gasses

Lipophilic compounds (e.g. PCB) can accumulate in fat tissue -> this will be excreted through blood , urine or breastmilk

1. Blood brain barrier: consists of the tight junctions, endothelial cells and astrocytes (gliacells) . (methylmercury can pass the BBB because it can complex with cysteine)
2. pLAcenta: - Many lipophilic compounds will get through so it’s actually not a true ‘barrier’

• Defence by: 1) active transport (mdr/oct protein) 2) biotransformation capacity

Phase I: Reactions: Primarily involves functionalization reactions, which expose or introduce a functional group on the parent compound. Common reactions include ox., red., and hydrolysis. Enzymes: Mostly carried out by cytochrome P450 (CYP450) enzymes Purpose: increase the polarity and water solubility of the compound, making it easier to excrete or to prepare it for phase II reactions. Outcome: Often results in metabolites that are still biologically active, and sometimes even more active or toxic than the parent compound. Phase II: Reactions: Involves conjugation reactions, where an endogenous substance (e.g sulfate, or amino acids) is attached to the functional group introduced or exposed in phase I. Enzymes: Catalyzed by various transferases, such as sulfotransferases and N-acetyltransferases Purpose: To further increase the water solubility and decrease the reactivity of the compound, facilitating its excretion. Outcome: Typically results in metabolites that are less active and less toxic than the parent compound. Phase III: Reactions: Involves the transport of the phase II metabolites across cell membranes for excretion. This can be either facilitated diffusion or active transport. Transporters: Several families of transporters are involved, including ATP-binding cassette (ABC) transporters and solute carrier (SLC) transporters. Purpose: To remove the conjugated metabolites from the body, primarily through urine or bile.

A. Specificity

• 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 B. Saturation
• 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 C. 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 D. 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)

1. Enzyme Variations:
2. Metabolic Rate:
3. Biotransformation Differences: Within Species: Variations in the expression and activity of biotransformation enzymes can result in different metabolic pathways and product formation. This can lead to individuals metabolizing chemicals into more or less toxic forms, affecting their sensitivity. Between Species: Species may lack certain enzymes or possess unique ones, leading to the production of different metabolites with varying toxicity. This is a major factor in species-specific sensitivities to chemicals.
4. Excretion Differences: Within Species: Genetic variations and physiological factors can affect the efficiency of excretion pathways, such as renal or biliary excretion. This can lead to differences in the accumulation of chemicals and their subsequent toxicity. Between Species: Excretion pathways vary widely across species, influencing the ability to eliminate specific chemicals. For example, some species have specialized mechanisms for excreting certain toxins, rendering them less sensitive.
5. Target Site Sensitivity: Within Species: Genetic variations in the target sites of chemicals (e.g., receptors, enzymes) can affect their binding affinity and subsequent response. Between Species: The structure and function of target sites can differ substantially between species, resulting in variations in the sensitivity and nature of the response to chemicals.

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.

Relationships between amount of exposure (dose) and the resulting effect or response in the body. In simpler terms, it examines how the effect of a substance changes as the exposure level changes.

1. Potency (EC50 or ED50): This is the dose or concentration of a substance required to produce 50% of the maximum response. A lower EC50/ED50 indicates higher potency, meaning a smaller amount of the substance is needed to produce a given effect.

2. Efficacy (Emax): This is the maximum response achievable by a substance. It represents the upper limit of the dose-response curve, indicating the maximum biological effect that can be elicited, regardless of the dose.

3. Slope: This describes the steepness of the dose-response curve. A steeper slope indicates a more rapid increase in the response with increasing dose, while a shallower slope suggests a more gradual change in the response. The slope can provide insights into the mechanism of action and the therapeutic window of a substance.

NOEC = highest concentration tested with no significant difference in response with control LOEC = lowest concentration tested with significant difference in response with control

NOEC/LOEC approach has been traditionally used, the ECx approach offers several advantages in terms of information content, statistical robustness, and flexibility. It provides a more precise and comprehensive assessment of the dose-response relationship and is increasingly preferred in ecotoxicological studies and regulatory decision-making.

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 fig-uring out what effect this gives)

• From this you can derive chemical properties (ED50) and trigger values (NOAEL, LOAEL)

1. Linear dose-response; in a linear dose-response relationship, the response increases or de-creases in a proportional manner with increasing dose or concentration (straight line)
2. Sigmoidal (S-shaped); initial slow response -> rapid increase/decrease -> plateau phase
3. Non-monotonic; they show a complex pattern with multiple peaks, valleys, or reversals in the response
4. Threshold-like response; there is a sharp transition from no response to a complete re-sponse once a certain threshold dose or concentration is reached.
5. U-shaped; response pattern where moderate doses or concentrations produce the highest response, while both low and high doses result in lower responses
6. 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 det-rimental response.

BAF (Bioaccumulation Factor): Definition: The ratio of the concentration of a chemical in an organism to its concentration in the surrounding environment (water, sediment, or soil) at a steady state. Calculation: BAF = Concentration in organism / Concentration in environment Significance: Indicates the overall accumulation of a chemical in an organism from all exposure routes (water, food, sediment). A higher BAF suggests a greater potential for bioaccumulation.

BCF (Bioconcentration Factor): Definition: The ratio of the concentration of a chemical in an organism to its concentration in the surrounding water at a steady state, considering uptake only from water. Calculation: BCF = Concentration in organism / Concentration in water Significance: Specifically measures the accumulation of a chemical in an organism due to direct uptake from water. It is useful for assessing the potential for bioconcentration in aquatic organisms.

BMF (Biomagnification Factor): Definition: The ratio of the concentration of a chemical in a predator to its concentration in its prey. Calculation: BMF = Concentration in predator / Concentration in prey Significance: Measures the increase in concentration of a chemical as it moves up the food chain. A BMF greater than 1 indicates biomagnification, meaning the chemical is accumulating at higher levels in predators.

TMF (Trophic Magnification Factor): Definition: Similar to BMF, but typically determined through field studies and represents the average increase in concentration across multiple trophic levels. Calculation: TMF is usually calculated using statistical models based on field data. Significance: Provides a broader assessment of biomagnification across entire food webs, rather than just individual predator-prey relationships.

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 up-take route, metabolic activity Example: 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 preg-nancy. Also higher levels of DDT can happen due to lactation. Because of this newborns can have higher levels.

1. Lipophilicity (Hydrophobicity): The tendency of a chemical to dissolve in fats and oils rather than water. It is often quantified by the octanol-water partition coefficient (Kow).
2. Molecular Size and Shape: The size and shape of a molecule affect its ability to pass through biological membranes and interact with biological targets.
3. Polarity and Ionization: Polarity refers to the distribution of electrical charge within a molecule. Ionization is the process of gaining or losing electrons, resulting in a charged molecule.
4. Volatility: The tendency of a chemical to vaporize or become a gas.
5. Degradability: The susceptibility of a chemical to breakdown or transformation through biological or chemical processes.
6. Water Solubility: The ability of a chemical to dissolve in water.

Bioconcentration: The process by which a chemical concentration in an organism becomes higher than its concentration in the surrounding environment (water, soil, or air) due to direct uptake from the environment. Mechanism: Occurs primarily through direct absorption of the chemical from the surrounding medium, typically water in aquatic organisms. Location: Happens within a single organism and does not involve the food chain. Example: A fish absorbing a pesticide directly from contaminated water. Biomagnification: The increasing concentration of a chemical in organisms at successively higher trophic levels of a food chain. Mechanism: Occurs as a result of an organism consuming other organisms that contain the chemical, leading to a progressive increase in concentration. Location: Happens across multiple levels of a food chain. Example: A bird of prey accumulating high levels of mercury by eating fish that have accumulated mercury by eating smaller contaminated organisms.

Bioavailability refers to the fraction of a chemical that is available for uptake by an organism, whereas bioaccumulation refers to the process by which a chemical accumulates in an organism

The OECD 305 BCF test protocol is a bioconcentration test that involves exposing fish to two concentrations of a chemical for 28 days. The uptake and depuration phases are monitored to determine the BCF. . (It aims to determine the extent to which a chemical substance can accumulate in fish tissues over time when exposed to a constant concentration in water.)

Independent Action (IA): Assumption: Each chemical in the mixture acts independently of the others, affecting different biological targets or pathways. The effects of each chemical are not influenced by the presence of the others. Mechanism: The chemicals exert their effects through distinct mechanisms, and their combined effect is the sum of the individual effects. Mathematical Model: The combined effect is calculated as the product of the probabilities of each individual chemical not causing the effect. This assumes that the individual dose-response curves are known. Applicability: Suitable for mixtures of chemicals with dissimilar modes of action or that act on different target sites. Concentration Addition (CA):

Assumption: Chemicals in the mixture have a similar mode of action, meaning they affect the same biological target or pathway. They can be considered as dilutions or concentrations of each other. Mechanism: The chemicals contribute to the overall effect in proportion to their individual potencies and concentrations. Mathematical Model: The combined effect is calculated by summing the concentrations of the individual chemicals, each weighted by their relative potency. Applicability: Suitable for mixtures of chemicals with the same or similar modes of action.

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.

Dioxins and dioxin-like (DL) compounds are a group of persistent organic pollutants (POPs) that are highly toxic and can cause a range of adverse health effects in humans and animals. They are not intentionally produced but are mainly byproducts of industrial processes and combustion.

Dioxins are a group of 75 chemically related compounds known as polychlorinated dibenzo-p-dioxins (PCDDs). The most toxic dioxin is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Dioxin-like compounds (DLCs) include: Polychlorinated dibenzofurans (PCDFs): A group of 135 compounds similar in structure and toxicity to PCDDs. Polychlorinated biphenyls (PCBs): A group of 209 compounds, some of which have dioxin-like properties.

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

1. 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

2. 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

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.

Bioassays are experimental procedures used to assess the biological activity or effects of a sub-stance 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 contami-nated sample or dilution required to obtain no effect

Protein is isolated from an organism and expressed in transformed bacteria or transfected cells (re-combinant)

Introducing a reporter gene into cells of interest, which produces a signal in response to the activa-tion 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

Toxicity profiling is an essential component of chemical safety assessment and drug discovery processes. It involved evaluating the potential toxic effects of compounds and prioritizing them base don their toxicity and modes of action. Group: compounds with similar toxicity profiles Rank: compounds based on: number of ‘red flags’ & mode of action Identify: most important modes of action & relevant structural characteristics & representative com-pounds for each class Prioritize: compounds for further research or quality standard setting the food chain

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.

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)

The effect of EDCs in human could be the sperm density, fertility or cancer forming in the gonads 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 di-oxins
• 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

Metabolomics: Systematic study of small organic molecules (<2000 Da) within cells, body fluids, tis-sues, organisms

• small molecules can be endogenous metabolites / exogenous chemicals (drugs, food additives)

Targeted metabolomics: to focus on changes in the abundances of specific metabolites in a biologi-cal system (analytical method designed to analyse specific target metabolites) Untargeted metabolomics: to provide a complete overview of changes in abundances of all detect-able metabolites in a biological system (analytical method should extract a wide range of different types of metabolites -> performance more variable than targeted)

• Metabolome is very dynamic, therefore, very time sensitive
• Metabolites have a wide range of physiochemical properties and vary widely in concentra-tion 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

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.

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 1) no/low exposure and 2) high exposure. You don’t know if they have the disease! Non-smokers vs smokers
• Assessment relationship between exposure and outcome?

Case-control: start with people with disease (disease vs no disease)  Present and past because the people already developed the disease

• They know that they have the disease! 1, select cases with the disease 2, 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 Example: same odds ratio but in another point of few (now we look at the disease instead of the exposure):

Cohort study: want to know if they develop  Can be prospective and retrospective 1, start with healthy population, at risk 2, 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 in-cidence of risk in the unexposed group

Confounding: extraneous variable that is related to both the exposure and the outcome, inde-pendently 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

• Use of existing toxicity data from previous animal studies
• Conduct new studies as a last report to fill data gaps in the core data essential for registra-tion
• Information combined together from various resources (WoE)
• Studies using cells, tissues or organs (in vitro)
• Computer modelling
• Use of information on similar substances

1. Initiation: This is the first and irreversible step, where a normal cell's DNA is damaged by a carcinogen (e.g., chemical, radiation, virus). This damage causes a mutation in one or more genes, altering the cell's normal function and potentially leading to uncontrolled growth.

2. Promotion: This stage involves the selective growth and proliferation of initiated cells. Promoters are agents that do not directly damage DNA but create a favorable environment for the growth of mutated cells. They may stimulate cell division, inhibit apoptosis (programmed cell death), or induce inflammation.

3. Progression: This final stage is marked by the conversion of precancerous cells into malignant cancer cells. These cells acquire additional mutations that confer invasive and metastatic properties, allowing them to invade surrounding tissues and spread to distant organs.

I. Silent mutations

• Do not result in any change in the amino acid sequence of the corresponding protein
• Substitution of a nucleotide in a codon can differ but has no affects on the protein II. Missense mutations
• Lead to a change in the amino acid sequence of the resulting protein
• Substitution of a nucleotide in a codon resulting in a difference amino acid being incorpo-rated into the protein during translation III. Nonsense mutations
• Introduce a premature stop codon into the DNA sequence, resulting in the truncation of the protein
• Single nucleotide substitution leads to the creation of a stop codon

Mutagen = agent that permanently changes genetic material (DNA) in an organism and increases mutations

• Direct mutagens = substances that directly interact with DNA, leading to changes in the nu-cleotide sequence (e.g. alkylation agents)
• Indirect mutagens = substances that do not directly interact with DNA but instead act through intermediary processes to cause genetic mutations (PAHs)

A. Oncogenes: normally, they are known as proto-oncogenes that help control cell growth and regulation. However, when mutations, duplication or chromosomal exchange occurs they become oncogenes. This can result in uncontrolled cell growth (activation in cancer cells) B. Tumor suppressor gene: normally, regulate cell growth an prevent formation tumors. They can also repair DNA damage. If there is a mutation, the suppressor gene becomes inactive and loses its function. (inactivation)

Oxidative stress is the imbalance between the production of reactive oxygen species (ROS) and the ability to neutralize. ROS contain reactive molecules such as oxygen/hydroxyl radicals, hydrogen per-oxide and produced by cellular metabolism. Excessive production of ROS leads to oxidative stress, accumulation.

Types of biomarkers I. Biomarkers of exposure (indicate whether individual has been exposed to specific agent and can help assess the dose of the exposure) (onderzoeken het specificeke stofje zelf of aanwezig is in bijvoorbeeld bloed)

• Indicate exposure to chemicals
• No information on the degree of adverse effect caused by the biomarker response
• Example; blood levels or urinary levels II. Biomarker of toxic effect (These biomarkers provide information about the adverse effects or changes that occur in biological systems as a result of exposure to toxic substances. They reflect the physiological, biochemical, or molecular alterations induced by the expo-sure) (onderzoeken de bijkomige effecten wanneer die stofje aanwezig is)
• Demonstrate an adverse effect
• Example; DNA or protein adducts

EDA: A combination of bioassay and fractionation methods and chemical analysis. Here, the bioas-say is directing the identification process, where cause-effect relationships between the bioassay response and toxicant are researched. The analytical chemistry here is used for reducing the com-plexity of the sample and to identify the responsible toxicants.

Explain the difference between hazard and risk Hazard = something that has the potential to harm you Risk = the likelihood of a hazard causing harm (actually dangerous) Risk = hazard x exposure

• Risk = probability a negative effect will occur

Human Biomonitoring (HBM) involves the analysis of natural and synthetic substances and their me-tabolites in the human body. It’s an established method in occupational and environmental expo-sure assessment. HBM studies are important for the link of human health and the environment.

Phase I metabolism: Reduction, oxidation, or hydrolysis of chemicals. These reactions serve to con-vert them into more polar molecules by adding or exposing a polar functional group such as -NH2 or -OH. Phase II metabolism: Addition hydrophilic groups to the original molecule, a toxic intermediate or a nontoxic metabolite formed in phase I, that requires further transformation to increase its polarity. These reactions include conjugation reactions, glucuronidation, acetylation, and sulfation. Microbial metabolism: The gut microbiome interacts directly with drugs to break them down into metabolic products. Phase III metabolism: Extracellular mobilization and excretion Sample matrices; blood, urine, saliva, tissues, CSF , breast milk

The exposome is composed of every exposure to which an individual is subjected from conception to death. Therefore, it requires consideration of both the nature of those exposures and their changes over time.

• In other words: the exposome is everything that we are exposed to starting from birth to death. We cannot do analysis for every day, so we have to choose e.g. in utero, newborn, child, adulthood.

1. External General: social capital, education, financial status, psychological and mental stress, urban-rural environment, climate, etc.
2. External Specific: radiation, infectious agents, chemical contaminants and environmental pollutants, diet, lifestyle factors (tobacco, etc.) occupation, medical interventions, etc.
3. Internal: metabolism, endogenous hormones, body morphology, physical activity, gut micro-flora, inflammation, lipid peroxidation, oxidative stress, ageing, etc.