Basic Toxicology PDF

8/10/2024 Basic Toxicology Ts. Dr. Eddie Tan Ti Tjih Food Science & Technology Department UiTM K...

8/10/2024 Basic Toxicology Ts. Dr. Eddie Tan Ti Tjih Food Science & Technology Department UiTM Kuala Pilah Campus Dr Siti Aimi Sarah bt Zainal Abidin Food Science & Technology Program UiTM Shah Alam 1 2 3 Able to Able to Able to describe basic explain on explain on toxicology toxicokinetics different types concepts of xenobiotics of toxicity, its level and testing. Learning outcome 1  8/10/2024 Subchapters 01 Introduction to xenobiotics Absorption, distribution and 02 storage of xenobiotics 03 Biotransformation and excretion of xenobiotics 04 Systematic and organ specific toxicity 05 Dose-response relationship 06 Toxicity testing 01 Introduction to xenobiotics 2  8/10/2024 01 Toxicology: The study of the nature and action of poisons. Poisons: Chemical/physical agents that produce adverse responses in biological organisms. Father of toxicology: Paracelsus (1493-1534) German: 'Alle Ding sind Gift und nichts ohn' Gift; allein die Dosis macht, das ein Ding kein Gift ist. English: All things are poison and nothing (is) without poison; only the dose makes that a thing is no poison. Xenobiotics: 01 Any toxin which disrupts human normal biological function. Human can be exposed to xenobiotics by inhalation, dermal absorption and ingestion. It may affect different parts of the body in different ways. It may be removed from the body, usually by hepatic metabolism (biotransformation) and renal excretion Illnesses caused by Xenobiotics: cardiac arrhythmia, sclerosis, hypertension… eczema, edema, psoriasis… autism, aggressive behavior, anxiety, insomnia, mental disorder, epilepsy, fatigue, headache… tinnitus, rheumatism, vasculitis… 3  8/10/2024 01 Toxicokinetics: Quantitation of the time course of toxicants in the body during the processes of absorption, distribution, biotransformation, and excretion (clearance of toxicants). i.e.: It is about measuring and modelling of the movement of toxin in the body. Toxicokinetics are "what the organism does with the chemical" Toxicodynamics: Molecular, biochemical, and physiological effects of toxicants or their metabolites in biological systems. Toxicodynamics are "what the chemical does to the organism“ These effects are result of the interaction of the biologically effective dose of the ultimate (active) form of the toxicant with a molecular target. 01 Molecular targets concept: The toxic action of a chemical is a consequence of the physical/chemical interaction of the active form of that chemical with a molecular target within the living organism. Examples of molecular targets: Protein Arylhydrocarbon(Ah) receptor—Dioxin Hemoglobin—CO Lipids—Carbon tetrachloride DNA—Aflatoxin Source: www.ocw.jhsph.edu 4  8/10/2024 01 Toxicokinetic-toxicodynamic models and adverse outcome pathways (AOP) Source: www.ecotoxmodels.org Toxicological paradigm 5  8/10/2024 01 The toxicological process Source: www.ocw.jhsph.edu 02 Absorption, distribution and storage of xenobiotics 6  8/10/2024 02 Four processes of toxicokinetics: Absorption: the substance enters the body. Distribution: the substance moves from the site of entry to other parts of the body. Biotransformation: the body changes (transforms) the substance into new chemical (metabolite). Excretion: the substance or its metabolites leave the body. 02 Absorption of Xenobiotics Respiratory system Skin Digestive tract 7  8/10/2024 02 Absorption of Xenobiotics- Digestive tract Absorption through digestive system Digestive system: mouth, esophagus, stomach, small intestine, large intestine and rectum. Route for entry: mouth. Passage/pathway of absorption: through wall of gastrointestinal tract (stomach, small intestine, large intestine). Site for most absorption of xenobiotics occurs in the small intestine epithelial layers (villi and microvilli) with large surface area. Xenobiotics must pass through the intestinal epithelium before they can reach the blood stream Epithelial layers of the intestine to blood, to liver (biotransformation), to systemic circulation, to organs. 02 Absorption of Xenobiotics- Digestive tract Five processes of xenobiotics transport across the membrane: Lymphatic absorption- absorption of fats and fat-soluble vitamins, and bypass the first-pass metabolism or the presystemic metabolism. Passive diffusion: major process for the absorption of xenobiotics. Active transport: require cellular energy. Pinocytosis- occurs primarily for absorption of fat droplets. Diffusion: small molecules through membrane pores. 8  8/10/2024 02 Absorption of Xenobiotics- Digestive tract Absorption of xenobiotics depends on the characteristics of the xenobiotics: Weak acid and base xenobiotics are readily absorbed and not readily eliminated from the body. Lipophilic ('lipid-loving', pass through the membrane lipid layer easily) xenobiotics are readily absorbed; pass through lipid membrane of the small intestine by passive diffusion (based on concentration gradient). Low molecular weight xenobiotics pass across the membrane through the pores (aqueous phase) of the membrane Highly dissociated xenobiotics and hydrophilic compounds are hardly absorbed unless the epithelium is damaged. (Why? ChatGPT) Degree of ionisation: strong and highly ionised acid and base are not readily absorbed; completely ionised compounds are very slowly absorbed (depends on the pH of the intestine). 02 Absorption of Xenobiotics- Respiratory system Absorption through respiratory tract: Air contaminants: gas, volatile solvents and aerosols (small solid or liquid particles suspended in air) Route of entry for xenobiotics: nose Passage/ pathway of absorption through lungs: trachea, to bronchi, to bronchioles, to alveoli and to blood vessels. Absorption site: alveoli (alveoli epithelium: exchange of gases occurs between alveoli and blood) 9  8/10/2024 02 Absorption of Xenobiotics- Respiratory system Mode of absorption for various types of xenobiotics through alveoli Low molecular weight and lipophilic xenobiotic gasses are quickly absorbed into the blood. Hydrophilic xenobiotics diffuse primarily through the water-filled pores, the rate of absorption decreasing with increasing molecular size. The smaller the size of xenobiotics the deeper will penetrate into the tissues and organ Solubility increase the chance of absorption. 02 Absorption of Xenobiotics- Skin Absorption through the skin: Commonly occurs in the chemical industry and agricultural sector. Skin consists of hard keratinized layer of dead cells (stratum corneum) with no blood supply. Living cells and blood vessels are found only in the deepest layer of the epidermis Compounds which are absorbed via the skin will first have to pass across the keratinized skin layer before they can reach the bloodstream. Passage for absorption through skin: outer surface of the skin, to keratinized layer, to epidermis, to dermis and hypodermis. Low molecular weight lipophilic xenobiotics are absorbed rapidly e.g.: lipophilic pesticides (nicotine, parathion and dinitro-ortho-creso (DNOC). 10  8/10/2024 02 Absorption of Xenobiotics- Skin Factors influence the penetration of xenobiotics through skin Degree of ionization (electrical charge): ionized xenobiotics do not pass easily through the membrane. Molecular size: the smaller the size the easier to pass through the membrane pores Hydrophilicity: diffuse through the outer surface of the hydrated keratinized layer of stratum corneum (7%). Lipophilicity/ Hydrophobicity: diffuse through the lipid material between the keratin filaments. pH of xenobiotics: acid, alkali Others: corrosive xenobiotics, skin burn, dermatitis. 02 Distribution of xenobiotics Distribution Initial distribution: by diffusion. Main avenue for distribution: blood. Others: interstitial fluid, dissolve in plasma aqueous phase, binding with plasma protein, bind to cellular component of the blood, bind to tissue protein. Depends on blood flow and characteristics of the xenobiotics. 11  8/10/2024 02 Distribution of xenobiotics Source: www.ironsportfitness.com/ 02 Distribution of xenobiotics-The fate Transported to the liver Stored in the organ The substance is activated or in-activated. Most common storage depot: Fat tissue, liver, kidney, and bone May be stored in the tissue in the form of precipitate E.g.: deposition of fluoride, lead and strontium in the bone tissue) Accumulate near the site where it Excreted induces its toxic effect Excretion routes are urine, The degree of accumulation in organ is faeces, breath, and sweat. dependent on: The chemical structure of the xenobiotics The affinity of the organs to particular xenobiotics 12  8/10/2024 02 Storage of xenobiotics Storage sites of xenobiotics Sometimes occurs. A xenobiotic may be bound to plasma proteins. Albumin is the most abundant plasma protein that binds toxicants for a relatively short time. The primary sites for toxicant storage are adipose tissue, bone, liver, and kidneys. Source: www.levitise.com.sg 02 Storage of xenobiotics- Adipose tissue Adipose Tissue Lipid-soluble toxicants are often stored in adipose tissues. Lipid-soluble toxicants can be deposited along with triglycerides in adipose tissues. The lipids are in a continual exchange with blood and thus the toxicant may be mobilized into the blood for further distribution and elimination, or redeposited in other adipose tissue cells. Source: www.azolifesciences.com 13  8/10/2024 02 Storage of xenobiotics- Bone Bone Bone is composed of proteins and the mineral salt hydroxyapatite. Bone is continually being remodeled under normal conditions, calcium and hydroxyl ions are incorporated into the hydroxyapatite-calcium matrix. Several chemicals, primarily elements, follow the same kinetics as calcium and hydroxyl ions and therefore can be substituted for them in the bone matrix. For example, strontium (Sr) or lead (Pb) may be substituted for calcium (Ca), and fluoride (F-) may be substituted for hydroxyl (OH-) ions. Calcium and other minerals are continually being resorbed and replaced, on the average about every 10 years. Thus, any toxicants stored in the matrix will eventually be released to re-enter the circulatory system. 02 Storage of xenobiotics- Liver and kidneys Liver and Kidneys The liver is a storage site for some toxicants. It has a large blood flow and its hepatocytes (that is, liver cells) contain proteins that bind to some chemicals, including toxicants. The kidneys have a high blood flow, which preferentially exposes these organs to toxicants in high concentrations. Storage in the kidneys is associated primarily with the cells of the nephron (the functional unit for urine formation). Source: www.primomedico.com 14  8/10/2024 03 Biotransformation and excretion of xenobiotics 03 Biotransformation It is the process whereby a substance is changed from one chemical form into another by a chemical reaction within the body (eg by increasing or decreasing molecular size or polarity). Products of biotransformation: metabolites. Biotransformation can result ▪ higher toxicity (bioactivation), ▪ reduce toxicity (bioinactivation) of xenobiotics ▪ or no effect of toxicity Biotransformation depends on age, nutritional status, health status. Major organ for biotransformation: liver (in endoplasmic reticulum; cytochrome P450 enzymes) Other organ: intestine (metabolizing enzyme in endoplasmic reticulum of intestine epithelial cells) : rate of metabolism lower than liver Examples of biotransforming enzymes: acetylcholinesterase, alcohol dehydrogenase, cytochrome P450, methyl transferases, sulfotransferases, lipoxygenase, prostaglandin synthase ©All rights reserved by UiTM Do not copy or upload this document into public domain 15  8/10/2024 03 Two general types of biotransformation Phase I (modification) and Phase II (conjugation) Phase I and Phase II may occur simultaneously or sequentially May result in bioactivation, bioinactivation or no change in the activity of the parent xenobiotics ©All rights reserved by UiTM Do not copy or upload this document into public domain 03 Phase I and Phase II https://www.youtube.com/watch?v=ZPLZo_GjQlE ©All rights reserved by UiTM Do not copy or upload this document into public domain 16  8/10/2024 03 Phase I Modification of the xenobiotic by the addition of functional group (OH, -NH2, -SH, -COOH) Catabolic reactions : oxidation, reduction and hydrolysis Catalyzed by: cytochrome P450 Slight increase in hydrophilicity (polar) ©All rights reserved by UiTM Do not copy or upload this document into public domain 03 Phase II Enzymatic reactions that conjugate the modified xenobiotic with another substance Polar compound (sulfate, glucuronic acid, glutathione, methyl group) added to functional group Sulfate conjugation (sulfation), methylation, acetylation, amino acid conjugation, glucuronide conjugation, glutathione (GSH) conjugation). Catalyzed by : sulfotransferase, methyltransferase, glutathione-S-transferase (GST), acetyltransferase, glucuronosyltransferase. Large increase in hydrophilicity (very water loving) result in more water soluble compound. Render the parent xenobiotics inactive (bioinactivation). Biliary excretion, renal excretion, plasma circulation. ©All rights reserved by UiTM Do not copy or upload this document into public domain 17  8/10/2024 03 Effects of biotransformation: Result in lesser availability of the toxic compound in the systemic circulation (detoxification). Facilitate excretion [converts xenobiotics from a lipophilic to hydrophilic characteristics, more water soluble] Shorten the duration of toxic effect of the xenobiotic End products of biotransformation: Usually less lipid soluble More ionized at physiological pH Less bound to tissue plasma and tissue protein Less stored in fat ©All rights reserved by UiTM Do not copy or upload this document into public domain 03 Excretion Removal of xenobiotics or metabolites out of the body. Depends on the characteristics of the xenobiotics (e.g.: size, polarity and ionic strength) Major routes of excretion: bile and urine. Renal (kidney) excretion: Kidney (glomerular filtration) in the form of urine. Usually for smaller xenobiotics. Liver (Biliary excretion): Liver to bile, to intestinal tract, to feces. For larger xenobiotics. ©All rights reserved by UiTM Do not copy or upload this document into public domain 18  8/10/2024 03 Enterohepatic circulation: The process of excretion into the intestinal tract via the bile and reabsorption and return to the liver by the portal circulation. Toxicological implication: if there is reabsorption of a metabolite which is more toxic than its parent compound. Many lipophilic xenobiotics undergo this process causing repeated liver damage. ©All rights reserved by UiTM Do not copy or upload this document into public domain ©All rights reserved by UiTM Do not copy or upload this document into public domain 19  8/10/2024 03 Other excretion routes: Excretion via the lungs (for volatile substance through exhaled air) Xenobiotics in the form of gas Blood flow to alveoli (by passive diffusion) Gases which do not dissolve well in the blood (e.g.: ethylene) are more rapidly excreted via expired air than those gases which dissolve rapidly in the blood (e.g. chloroform) Excretion via milk, placenta, eggs Excretion via nails, hair, sweat, tears, saliva ©All rights reserved by UiTM Do not copy or upload this document into public domain 03 Toxicological implication of excretion sites Excretion of toxic substances in milk mother to infant (breast feeding) in cow’s milk to human Excretion or toxic substance via sweat dermatological disorders. Excretion via saliva It will go into the mouth, swallowed and may be reabsorb. ©All rights reserved by UiTM Do not copy or upload this document into public domain 20  8/10/2024 04 Systematic and organ specific toxicity 04 Toxicity The extent to which a substance is poisonous or exerts toxic effect to the body Can be acute, subchronic or chronic The toxicity of a substance depends on:- ▪ Chemical and physical properties of the substance (eg lipophilic/hydrophilic/gas (solubility)/acid/alkaline ▪ Dosage ▪ Route of exposure ▪ Species ▪ Age ▪ Gender ▪ Frequency of exposure ▪ Rate and amount that can be absorbed ▪ Concentration of substance at the portal of entry ▪ Distribution in the body and concentration at specific body sites 21  8/10/2024 04 Efficiency of biotransformation and nature of the metabolites The ability of the substance or its metabolites to pass through cell membranes and come into contact with specific cell components or organs The amount and duration of storage of the substance or its metabolites in body tissues (bioaccumulative) The rate and sites of excretion 04 Types of toxicity Chronic Toxicity Occurs after a prolonged or Acute Toxicity repeated exposures; cause harmful effects over an Short term exposure where the effect occur extended period sometimes immediately after lasting for the entire life of the exposure (single exposure Systemic toxicity exposed organism; and large amount of two types chemical) When the toxic effect Carcinogenic occur on multiple sites Non-carcinogenic Subchronic Toxicity (many organs) Resulted after repeated Specific toxicity exposure; cause harmful When the toxic effect effects for more than one occur on one site year but less than the lifetime (specific target organ) of the exposed organism; 22  8/10/2024 04 Types of specific toxicity 1) Blood (hemotoxicity) : xenobiotics acting directly on cells in circulating blood, bone marrow and heart 2) Dermal toxicity: result from direct contact or internal distribution to the skin 3) Eye toxicity: results from direct contact or internal distribution to the eye 4) Hepatotoxicity: toxicity to the liver, bile duct, and gall bladder 5) Immunotoxicity: toxicity to immune system 04 6) Nephrotoxicity: toxicity to kidney 6) Neurotoxicity: toxicant damage to cells of the central nervous system (brain and spinal cord) and the peripheral nervous system (nerves outside the CNS). 6) Reproductive toxicity: toxicant damage to either the male or female reproductive system 6) Respiratory toxicity: effects on the upper respiratory system (nose, pharynx, larynx, and trachea) and the lower respiratory system (bronchi, bronchioles, and alveoli). 6) cardiovascular toxicity: xenobiotics acting directly on cells of the heart. 23  8/10/2024 04 Types of systemic toxicity Carcinogenicity : abnormal cell growth and differentiation which can lead to cancer. Developmental Toxicity (Teratogenicity) : adverse toxic effects to the developing embryo or fetus (structural abnormalities) Genetic Toxicity (somatic cells, Mutagenicity or Genotoxicity) : results from damage to DNA and altered genetic expression. 05 Dose-response relationship 24  8/10/2024 05 Toxicity assessment A tool to investigate the potential for a substance to cause harm and how much of the substance causes what kind of harm A quantitative toxicity assessment is based on the dose-response concept Dose –response concept: the relationship between the amount of toxin received (dose) and the observed toxic reaction (response ie the illnesses such as hypersensitivity, skin rashes, etc) 05 Dose: The amount of toxin/agent that comes into contact (exposed/ administered) with a living organism Usually expressed as mg/kg (mg of substance per kg body weight) ▪ Administered dose: dose at which experimented animal are exposed to. ▪ Internal dose: amount absorbed into the organism. ▪ Biologically effective dose: the amount that reaches the site at which the adverse effect occur. Response (effect) depends on : 1. Route of exposure 2. Age 3. Gender of the exposed individual 4. Health status 5. Nutritional status 6. Frequency of exposure 7. Duration of exposure 25  8/10/2024 05 A typical dose-response curve *NOEL/ NOAEL No observable effect/ adverse-effect level (safe exposure level) LOEL LOEL/ LOAEL Lowest observable effect/ adverse-effect NOEL level 05 LD50: ; lethal dose to 50% population 26  8/10/2024 05 Dose-response curves for 4 different chemicals 06 Toxicity testing 27  8/10/2024 06 Assessment of Acute toxicity A large amount of substance is given to the experimented animals and the results is observed within 14 days Use of animals Toxic and lethal dose are determined LD: lethal dose: level of a substance that can cause death to the animal under study TD: toxic dose that will produce sign of toxicity to the test animals LD50 (median lethal dose): the amount (dose) of a substance that will kill half (or 50%) of the test animals TD50: median toxic dose that will produce sign of toxicity to 50% of the test animals The lower the LD50, the lower the lethal dose, the more toxic the substance. unit for LD50: (mg/kg) ie milligram of chemical per kilogram of body weight of animals There are 5 acute toxicity categories under Globally Harmonized System (GHS) although some jurisdictions (i.e, EU) have not implemented category 5. The table below shows GHS classification criteria for acute toxicity for different routes. LD50 and LC50 values are needed for GHS classification. Acute toxicity category 1 represents the most severe toxicity. 28  8/10/2024 06 Categories of severity of acute toxicity ORAL Category 1: LD50 ≤ 5 mg/kg of bodyweight Category 2: LD50 ≤ 50 mg/kg of bodyweight Category 3: LD50 ≤ 300 mg/kg of bodyweight Category 4: LD50 ≤ 2000 mg/kg of bodyweight Category 5: LD50 ≤ 5000 mg/kg of bodyweight 06 Categories of severity of acute toxicity DERMAL Category 1: LD50 ≤ 50 mg/kg of bodyweight Category 2: LD50 ≤ 200 mg/kg of bodyweight Category 3: LD50 ≤ 1,000 mg/kg of bodyweight Category 4: LD50 ≤ 2,000 mg/kg of bodyweight Category 5: LD50 ≤ 5,000 mg/kg of bodyweight 29  8/10/2024 06 Categories of severity of acute toxicity INHALATION - GASES Category 1: LC50 ≤ 100 ppmV Category 2: LC50 ≤ 500 ppmV Category 3: LC50 ≤ 2,500 ppmV Category 4: LC50 ≤ 20,000 ppmV 06 Assessment of chronic toxicity 1) Non carcinogenic toxicity assessment ▪ Dose-response studies on animals ▪ Length of experiment : 24 to 30 months (rat) ▪ Smallest and largest dose are determined ⮚ To conduct these dose-response studies, scientist:- i. Administer Different Small Doses Of A Substance To Several Groups Of Test Animals Every Day Over A Lifetime. ii. Determine The Smallest Dose That Causes Any Detectable Effect. iii. Periodically Examine And Finally Autopsy The Animals To Determine If Any Effects Have Occurred. ⮚ This smallest dose is called the Lowest Observable Adverse Effect Level (LOAEL) ⮚ LOAEL unit: mg/kg ie milligrams (mg) of substance per kilogram (kg) of body weight, or in parts per million (ppm) of substance in food. 30  8/10/2024 06 ⮚ No Observable Adverse Effect Level (NOAEL) is also determined ⮚ No Observable Adverse Effect Level (NOAEL): is the highest dose or exposure level of a substance that produces no noticeable toxic effect on the test animals. ⮚ NOAEL: “safe level” for that chemical in the species studied ⮚ The NOAEL for animals is not necessarily the "safe level" for humans ⮚ divide the NOAEL by a “safety factor”, usually 100, to arrive at a presumed "safe level" for humans 06 Assessment of chronic toxicity 2) Carcinogenic assessment (carcinogenesis bioassay) ⮚ Dose-response study called a carcinogenesis bioassay ⮚ Carcinogenesis bioassay: a method of testing substances for carcinogenic effects of a compound ⮚ Not looking for the safe level but for the cancer risk of the substance ⮚ Assess carcinogenic toxicity by feeding large doses of the substance to animals 31  8/10/2024 06 To conduct carcinogenic bioassay:- ⮚ Test animals are administered with different large doses of a substance daily over a period of time (24-30 months in rats). ⮚ At the end of the study, the animals are examined to see if cancer can be found. If cancer is found, scientists use available data and mathematical models to:- 1) Estimate if the cancer incidence at the lower doses more likely to occur. 2) Estimate the effect of the size and sensitivity differences between the test animals and human beings 06 Mathematical models 1) Non-Threshold models: based on the assumption that even one molecule of a cancer-causing agent can lead to the disease (also known as “one-hit” model) 1) Threshold Models : based on the premise that repeated exposures to a chemical are needed before a threshold of exposure is reached and cancer follows. 32  8/10/2024 06 Ames assay (mutation assay) 3) Mutagenic assessment ▪ To test for mutagenic potential of a compound ▪ Mutagenic compound : compound that changes the structure of DNA ▪ Bacterial reversed mutation assay ▪ Usually employ mutant microorganisms strain : strain of salmonella typhimurium and E. Coli that carry mutated genes (unable to grow in the absence of amino acid histidine) ▪ S. Typhimurium mutant strain : TA1535, TA1537, ta97a, TA97, TA98, TA100, TA 102) ▪ E. Coli mutant strain : WP2 uvra, WP2 uvra (pkm101) 33  8/10/2024 06 Ames assay S. typhimurium + substance A S. typhimurium + substance B S. typhimurium + water Welcome!! 34