PLH 419 General Principles of Toxicology is the

PLH - 419

General Principles of Toxicology is the study of the adverse effects of chemicals on living organisms. Toxicology also includes the study of harmful effects caused by physical phenomena, such as radiation and noise. The purposes of toxicology are protection of organisms and biological systems from deleterious effects of toxicants and development of selective toxicants.

Historical Development of Toxicology It is one of the oldest practical sciences which began with early cave dwellers who recognized poisonous plants and animals and used their extracts for hunting or in warfare. By 1500 BC, hemlock, opium, arrow poisons, and certain metals were used to poison enemies or for executions (Notable poisoning victims include Socrates, Cleopatra, and Claudius) The Death of Socrates, 1787 Jacques-Louis David By the time certain concepts fundamental to toxicology began to take shape especially by the studies of Paracelsus (~1500 AD) and Orfila (~1800 AD). Paracelsus (1493 -1541): His famous words were : "All substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy. " 1 -He determined that specific chemicals were actually responsible for the toxicity of a plant or animal poison. 2 -He also documented that the body's response to those chemicals depended on the dose received Orfila (founder of toxicology -19 th century) Spanish physician who first correlated between the chemical and biological properties of poisons. )

The 20 th century is marked by an advanced level of understanding of toxicology especially after the widespread use of anaesthetics and disinfectants. DNA (the molecule of life) and various biochemicals that maintain body functions were discovered. The discovery of radioactivity and the vitamins, led to the use of the first large-scale bioassays (multiple animal studies) to determine whether those new chemicals were beneficial or harmful to laboratory animals. The 1960 s started with the tragic of thalidomide incident, in which several thousand children were born with serious birth defects. Attempts to understand the effects of chemicals on the embryo and fetus and on the environment as a whole gained momentum. New legislation was passed, and new journals were founded. The variety of potential adverse effects and the diversity of chemicals in the environment make toxicology a very broad science. Therefore, toxicologists often specialize in one area of toxicology. A toxicologist is trained to examine the nature of those effects (including their cellular, biochemical and molecular mechanisms of action) and assess the probability of their occurrence.

Branches of toxicology Toxicology is multidisciplinary as it entails: 1 -Descriptive toxicology It is concerned directly with toxicity testing, which provides information for safety evaluation and regulatory requirements. The concern may be limited to effects on humans, as in the case of drugs and food additives, or to potential effects on fish, birds and plants, as well as other factors that might disturb the balance of the ecosystem. 2 -Mechanistic toxicology It s concerned with identifying and understanding the mechanisms by which chemicals exert toxic effects on living organisms. In risk assessment, mechanistic data may be very useful in demonstrating that an adverse outcome observed in laboratory animals is or is not directly relevant to humans. Mechanistic data are also useful in the design and production of safer alternative chemicals and in rational therapy for chemical poisoning and treatment of disease. An understanding of the mechanisms of toxic action contributes to the knowledge of basic physiology, pharmacology, cell biology and biochemistry. 3 -Regulatory toxicology has the responsibility for deciding, on the basis of data provided by descriptive and mechanistic toxicologists, whether a drug or another chemical poses a sufficiently low risk to be marketed for a stated purpose. Regulatory toxicologists also assist in the establishment of standards for the amount of chemicals permitted in ambient air, industrial atmospheres and drinking water, often integrating scientific information from basic descriptive and mechanistic toxicology studies with the principles and approaches used for risk assessment.

4 -Occupational (Industrial) toxicology is concerned with the protection of workers from toxic substances and makes their work environment safe. 5 -Environmental toxicology focuses on the impact of chemical pollutants in the environment on biological organisms, most commonly on nonhuman organisms such as fish, birds and terrestrial animals. 6 -Ecotoxicology is a specialized area within environmental toxicology that focuses more specifically on the impact of toxic substances on population at the community and/or ecosystem level. 7 -Forensic toxicology is concerned primarily with the medico-legal aspects of the harmful effects of chemicals on humans and animals, in establishing causes of death and in determining their circumstances in a postmortem investigation. 8 -Analytical toxicology is a specialized area to identify the toxicant through analysis of body fluids, stomach content, excrement, skin, or suspected containers. 9 -Clinical toxicology is concerned with disease caused by or uniquely associated with toxic substances. Efforts are directed at treating patients poisoned with drugs or other chemicals and at the development of new techniques to treat those intoxications. Clinical toxicology deals with emergency cases such as overdoses, poisonings, attempted suicides by: emergency care for patients, management of sign and symptom, identification and quantification of the drug , poisons, chemicals…etc

Toxicity is the degree to which a substance can nimal Different xenobiotics cause many types of toxicity by a variety of mechanisms. So, we have to take an idea about: -Different types of toxic agents -Different type of toxicity -Different mechanisms of toxic response

Toxic Agents Toxic agent: is the agent that can produce an adverse biological effect toliving organism. The most common terms used to describe a toxic agent are toxicant, toxin, poison.

Classification of Toxic Agents Toxic agents can be classified according to their nature: Chemicals: (as alcohols, phenols & heavy metals, Physical (radiation), and Biological (Snake & scorpion venoms). Toxic agents can be also classified in terms of their target organs (liver, kidney), use (pesticide, solvent), and effects (cancer, mutation). Toxic agents may also be classified in terms of their physical state (gas, dust, liquid), their chemical stability (explosive, flammable), general chemical structure (aromatic amine, halogenated hydrocarbon) or poisoning potential (extremely toxic, very toxic, slightly toxic). Classification of toxic agents on the basis of their biochemical mechanisms of action (e. g. alkylating agent, methaemoglobin producer) is usually more informative than classification by general terms such as irritants and corrosives.

Types of Toxicity 1 -Systemic Toxicity : Toxicity may occur at multiple sites. This is referred as systemic toxicity. The following are types of systemic toxicity a-Acute Toxicity: It occurs almost immediately (hours/days) after an exposure to single dose or a series of doses received within a 24 hour period. Death is a major concern in cases of acute exposures. Examples are: -In 1989, 5, 000 people died and 30, 000 were permanently disabled due to exposure to methyl isocyanate from an industrial accident in Bhopal, India. Many people die each year from inhaling carbon monoxide from faulty heaters. b-Subchronic Toxicity (reversible) It results from repeated exposure for several weeks or months. This is a common human exposure pattern for some pharmaceuticals and environmental agents. Examples are: -Ingestion of coumadin tablets (blood thinners) for several weeks as a treatment for venous thrombosis can cause internal bleeding. -Workplace exposure to lead over a period of several weeks can result in nemia.

c-Chronic Toxicity (irreversible) : It is a cumulative damage to specific organ or system and it takes many months or years to become a recognizable clinical disease. This damage is so severe that the organ can no longer function normally (irreversible) and a variety of chronic toxic effects may result. Examples are: -Cirrhosis in alcoholics who have ingested ethanol for several years -Chronic bronchitis in long-term cigarette smokers -Pulmonary fibrosis in coal miners (black lung disease) d-Carcinogenicity: Carcinogenicity is a complex multistage process of abnormal cell growth and differentiation which can lead to cancer. e-Developmental Toxicity: Developmental Toxicity result from toxicant exposure to either parent before conception or to the mother and her developing embryo-fetus. f-Genetic Toxicity: Genetic Toxicity results from damage to DNA and altered genetic expression. This process is known as mutagenesis. The genetic change is referred to as a mutation and the agent causing the change as a mutagen.

2 -Organ Specific Toxicity : Blood and Cardiovascular Toxicity Hypoxia due to carbon monoxide binding of hemoglobin preventing transport of oxygen Anaemia is produced by benzene and aniline. Haemolysis may be due to saponins. Leukopenia is caused by benzene. Hepatotoxicity CCl 4……. . metabolized by HME……. CCl 3 (causes lipid peroxidation in liver & lead to liver. necrosis. ) Also chloroform is hepatotoxic. Nephrotoxicity kidney damage and nephritis are produced by poisons such as phenol and sulphonamides. Mercury & gentamycin are nepherotoxic. Neurotoxicity Antimony and arsenic cause neurotoxicity Organophosphorus compounds (insecticides)………damage to sensory fibers. CNS excitation and convulsions are produced by strychnine, ephedrine and picrotoxin. CNS depression may be caused by substances such as barbiturates, ether and chloralhydrate. Delirium may indicate alcohol, atropine and related drugs. Respiratory Toxicity Aluminum…. . emphysema……inflated lung ……. fibrosis(aluminosis). Dermal Toxicity some poisons such as atropine and aconite produce dry skin. Skin rash is produced by poisons such as arsenic and antimony. Some poisons produce increase in sweating e. g. pilocarpine and eserine. Strong acids and alkalies cause tissue damage upon contact with the skin. Cyanosis is produced by poisons such as aniline, acetanilide and phenacetin.

Cardiovascular system Toxicity Myocardial depression is produced by compounds such as quinine and quinidine. Digitalis and strophanthus toxicity may lead to arrhythmia and ventricular fibrillation. Arise in blood pressure is produced by sympathomimetic agents such as ephedrine. Hypotension is caused by reserpine and nitrites. Skeletal muscle. Toxicity: Muscle paralysis is produced by lead, curare and flaxedil. . Eye Toxicity Acids and strong alkalis may cause severe corneal corrosion corticosteroids may cause cataracts , methanol (wood alcohol) may damage the optic nerve leading to blindness. Poisons such as ergot and lead salts impair general vision. The contracted pupil may be from morphine, opium, nicotine and pilocarpine while the dilated pupil may be from atropine, acotine and cocaine. Sudden death; some poisons act quickly and produce sudden death e. g. aconitine, cyanide and barium compounds.

Mechanism of Cellular Injury Transported, dispersed, and possibly altered Toxin emitted Ingested Contacts human Metabolized and/or stored Physiological chain of events Reaches an organ

Mechanism of Cellular Injury When the cell is exposed to an injurious agent or stress, a sequence of events follows that is loosely termed cell injury. Cell injury is reversible up to a certain point. If the stimulus persists or is severe enough from the beginning, the cell reaches a point of no return and suffers irreversible cell injury and ultimately cell death. Cell death, is the ultimate result of cell injury. There are two principal patterns of cell death: Necrosis (occurs after ischemia and chemical injury , it is always pathologic). Apoptosis (occurs when a cell dies through activation of an internally controlled suicide program). It is designed to eliminate unwanted cells during embryogenesis and in various physiologic processes and certain pathologic conditions.

Mechanism of Cellular Injury Toxicity can result from adverse cellular, biochemical, or macromolecular changes. Examples are: 1 -Alteration of a cell membrane permeability: Toxic agents could change cell membrane permeability through interaction with its component as; a-SH-containing proteins Heavy metals as As or Hg react with them……… change in protein structure ……… change membrane permeability. b-Lipids -Free radicals attack fatty acids in the lipid layer of biological membrane causing lipid peroxidation , these peroxides are toxic to the cell and alter membrane permeability. E. g. : CCl 4……. . metabolized by HME……CCl 3 (Trichloromethyl radical causes lipid peroxidation and finally lead to liver necrosis. ) -This is why antioxidants should be used frequently by humans where it act as a protective measure against many diseases(e. g. ) Vit. E & Vit. C. c-Na-K ATPase pump Many toxicants can inhibit these pumps which are essential for transport of major amino acids and calcium across the cell membrane. E. g. : Hg, Cu, Pb , As and alcohol.

2 -Chang in enzyme activity: a-Inhibition E. g. 1: Carbamate esters (insecticides) reversibly inhibits cholineserase leading to increase in A. Ch. Level Toxicity (SLUD are the most characteristic symptoms of toxicity). E. g. 2: Cyanide inhibits cytochrome oxidase enzyme no aerobic respiration and finally cell death. b-Activation E. g. : Barbiturates induce hepatic microsomal enzymes increase the conversion of some non carcinogenic agents (in cigarette smoke) into carcinogenic ones. 3 -Interferance with co-enzymes: E. g. : CN- binds to essential metals as Fe 3+ needed for the activity of cyochrome oxidase. 4 -Modification of carriers: E. g. 1: CO binds with hemoglobin instead of O 2 (affinity to Hb to CO is 210 times that for O 2)……… carboxyhemoglobin……. hypoxia……death. E. g. 2: Nitrates , aspirin and sulfonamides oxidize Fe 2+ in Hb into Fe 3+ Hb (methemoglobin) which can not carry oxygen H-dependent Hypoxia Me. Hb reductase & Vit. C

5 -Formation of reactive metabolites: , 8 - dihydrodibenzo(α) pyrene Non-carcinogenic Carcinogenic In cigarette smoke 6 -Reactions causing depletion of GSH: Glutathione (GSH) is an antioxidant which protects the cell from the harmful effect of oxidants. Reduction of GSH level into 20 -30% causes impairment of cell defense mechanism. E. g. : N-acetyl-P-benzoquinone imine (NABQI) , a toxic metabolite of paracetamol it is conjugated with GSH depletion of reduced form of GSH leading to NABQI (Strong electrophilic agent) attack liver tissues causing liver necrosis. -We can increase the level of GSH or overcome its depletion by; methionin (a precursor of GSH) & N-acetylcysteine (contains –SH). 7 - Action on nucleic acids: E. g. : SO 2 (air pollutant) + H 2 O HSO 3(causing damage to DNA & mutation). E. g. : Benzidine Metabolism by HME N-hydroxybenzidine. Non-carcinogenic Mutagenic & Carcinogenic In cigarette smoke

8 - Disruption of protein synthesis: Some toxicants either increase or decrease protein synthesis leading to cellular injury. E. g. : Ricin is a poison found naturally in castor beans. It works by getting inside the cells of a person's body and preventing the cells from making the proteins they need. Without the proteins, cells die. Eventually this is harmful to the whole body, and may cause death. 9 -Lysosomal changes: a-Toxicants which causes labialization of lysosomal enzymes: E. g. : Hg , Cu , silica , nicotine , bee venom , hypervitaminosis A , monosodium ureate crystals deposited in gout increase lysosomal membrane permeability release of hydrolases cell death. b-Toxicants which causes stabilization of lysosomal enzymes: E. g. : Corticosteroids causes indirect toxicity by decreasing the response of the body defense mechanism.

Factors Influencing Toxicity There are many factors which can enhance, increase or decrease toxicity. These factors are divided into : I-Factors related to the host: A-The species -Selective toxicity: refers to species differences in toxicity response between two species simultaneously exposed. Rats cannot vomit and expel toxicants before they cause severe irritation, whereas humans and dogs are capable of vomiting. B-Sex: . Men traditionally weigh more than women. Therefore, doses of a chemical in a male would be expected to produce lower blood and tissue levels than the same in females, simply because of the male's larger blood volume and greater tissue mass which dilute the chemical. · For substances that are injected intramuscularly, lower blood levels can be expected with those drugs in individuals (usually men) with a greater muscle mass. · Also, drugs with a high lipid coefficient that normally partition into fat may produce different toxicological responses in different sexes, based on the individual's ratio of body fat/total weight.

– For example, some studies report that a sex-related difference exists for absorption of erythromycin, resulting in less of the drug being absorbed by women, following oral administration. C-Age: -In geriatric patients the toxic effects of an injected drug may be reduced because of a generalized physiological reduction of blood supply into tissues. Similarly, the toxic response from an orally ingested drug or chemical may also be reduced, because once absorbed less of the substance will be delivered to a particular tissue site. Elderly people may have a greater incidence of debilitating diseases (e. g. , hepatic, renal, and cardiovascular) which may further reduce their ability to detoxify, excrete, or distribute the drug or chemical. Some chemicals are more toxic to infants or the elderly than to adults. Example: 1)-Bounded bilirubin with p. p. +Sulfonamides replacement from P. P. binding sites. conjugated with glucoronyl transferase Free bilirubin excreted in adults (Low activity of GT + Immature B. B. B in neonates) Kernikterus (in newborn) 2)-Nitates(in well , s water) due to stomach p. H is high in newborn Nitrite (oxidant) Oxidation Hb Met. Hb Met. Hb reductase

3)-Chloramphenicol conjugation by GT is low in neonates accumulation of it , and it oxidizes Hb into Met. Hb Grey baby syndrome (hypoxia, cyanosis , collapse and death). D-Genetics: I-Pharmacogentices (Idiosyncratic reaction ): An odd response to a given normal dose of a drug on hereditary bases 1) Succinylcholine apnea in individuals deficient in pseudo cholinesterase …. ? 2) Individuals deficient in glucose-6 -phosphate dehydrogenase suffer from hemolytic anemia upon using sulfa drugs , aspirin or naphthalene (oxidants) glucose-6 -phosphate + NADP 2 GSH (protect RBCs from hemolysis by oxidants) G 6 PD GSH reductase 6 -phosphogluconic acid + NADPH GSSG In case of G 6 PD deficiency NADPH GSH Oxidants attack RBCs hemolysis II-Toxicogentices: An odd response to a given toxicant on hereditary basis * smoking causes emphysema in certain individuals deficient in α 1 - antitrypsin. III-Hypersensitivity (allergic reactions): e. g. Some people suffer from an anaphylactic reaction when given penicillin.

Pharmacogentices (Idiosyncratic reaction)

E-Dietary factors 1) Heavy metal absorption is influenced by diet. : -Calcium, iron, fats, and protein are all reported to enhance lead absorption. -Deficiency of calcium, iron, or protein, on the other hand, enhances cadmium absorption. 2)Low protein in diet : P. P. level decrease free drug toxicity. Also, a low dietary protein intake may result in a decreased level of hepatic microsomal enzymes and, thus, the metabolic processes may proceed less readily. 3)Food containing tyramine as old cheese , salted dried fish , banana, Beer, Canned figs, Chicken liver , Chocolate, Sherry & wines increase MAOIs (e. g. , pargyline, phenelzine) toxicity which is severe symptoms of hypertensive crisis and even death may occur. 4) Calcium in milk, which may bind to tetracyclines, and thus reduce its absorption. 5) Foods rich in pyridoxine may significantly lower the pharmacological action of levodopa 6) Fatty foods, on the other hand, enhance griseofulvin absorption F. Occupation Individuals working in industries where organic compounds such as chlorinated hydrocarbon pesticides or volatile substances are used may have an enhanced ability to metabolize drugs and chemicals. The reason for this is related to the chemical's presence in the environment, which may have enhanced the worker's liver microsomal enzyme activity. His expected reaction to a toxic dose of any subsequent chemical, normally detoxified by the liver microsomal system, would be less than normal.

G-Health factors: 1)Acidosis: insulin activity decrease leading to hyperglycemia 2)Hypertensive patients may respond more intensely to chemicals that have sympathomimetic activity. 3)Opiates and other chemicals that cause respiratory depression are more hazardous in persons with head injuries. 4)Asthma: patient is more liable to the effect of air pollutants as SO 2. 5)Kidney & liver diseases: toxicity of many drugs increase. H. Living Conditions The living conditions of an individual could be an extremely important factor to consider. At present, we should consider that factors such as crowding in living conditions, noise, and social pressures are important areas for research. End of factors related to Host

II-Factors releated to the poison: a-Dose Anything can be toxic if enough is taken (and, conversely, even the most toxic of substances may not be harmful when low concentrations are taken). Also, the number of doses, frequency, and total time of the exposure also very important. Example: b-Routes of exposure can influence the time of onset, intensity, and duration of the toxic effects. -The route of administration may also predict the degree of toxicity and possibly the target systems which will most readily be affected. -Ingested chemicals, when absorbed from the intestine, distribute first to the liver and may be immediately detoxified. -Inhaled toxicants immediately enter the general blood circulation and can distribute throughout the body prior to being detoxified by the liver. Inhalation › Intravenous › Interapertoneal › Intramuscular › Subcutaneous ›

c-Chemical structure The physicochemical composition of the toxicant can sometimes be helpful in predicting the risk involved in exposure to a particular compound. -Silica (amorphous) after it is heated silica (crystalline) has little effect on health serious lung damage. -Cr 3+ is relatively nontoxic whereas Cr 6+ causes skin or nasal corrosion and lung cancer. d-Composition and formulation: ( mainly › absorption) -Concentration. -Lipid solubility. In general, solids are less easily swallowed than liquids, and bulky solids are more difficult to consume than light, more fluffy compounds, especially for a small child -Chemicals in liquid form are more toxic than those in solid form -Coloring agents as tartazin yellow cause allergy. -Micronization increase toxicity. -Vehicles as alcohols increase CNS depressant action of hypnotics. -Impurities; some herbicide as 2, 4, 5 -trichlorophenoxy acetic acid may contain the toxic impurity DIOXIN which is mutagenic , teratogenic and carcinogenic. -p. H of the preparation (high acidity or alkalinity) cause local sever effect. -Low stability of the compound + bad storage condition increase toxicity as food contaminants aflatoxin ( it is a product of certain molds) -The particle size: Only particles having a small diameter (1 m or less) will effectively reach the alveoli and be available for pulmonary absorption. Larger particles may be

F-Temperature -The response of a biological system to a toxic agent decreases as the environmental temperature is lowered ( but the duration of the overall response may be prolonged)· The reasons for these are: a. Decreased rate of absorption occurring in the colder environment; b. Lowered rate of metabolic degradation and excretion. Example 1 -Atropine-like compounds may produce significantly greater toxicity in a warm environment than in a colder one. Because anticholinergic agents inhibit sweating, the body temperature becomes elevated because the absence of perspiration prevents cooling of the body; so the toxic effects are from hyperthermia. 2 -Alternatively, drugs such as chlorpromazine that suppress the body's thermal regulatory center may be more toxic at certain temperatures. . End of factors related to Toxicant

III-Toxicokinetic factors: i-Factors affecting absorption: * GIT : Gastric content: -Empty stomach has higher emptying rate Toxicity. -Carbonated beverages increase G. E. Toxicity. -A full stomach with proteins & fats Toxicity. Secretion : Pepsin & HCl digest peptide poisons. GIT flora: migration of intestinal flora into the stomach in newborns due to their high gastric p. H , this flora can convert nitrates into nitrite, oxidizing Hb into met. Hb Hypoxia. *Skin (Thickness & Keratin layer protect the skin ): -Newborn (thin delicate skin). -Lipophilicity of insecticides. Toxicity -Cutting , abrasions & dryness of skin *Pulmonary: -Conc. Of toxicants in air. -Solubility of the toxin in blood & tissues. -Respiration rate. -Exposure time.

ii-Factors affecting metabolism (biotransformation): There are two types of metabolism : detoxification and bioactivation. Detoxification is the process by which a xenobiotic is converted to a less toxic form. Bioactivation is the process by which a xenobiotic may be converted to more reactive or toxic forms. Examples of chemicals known to be metabolized to more toxic compounds are; Acetanilid, Acetaminophen, Aniline, Carbon tetrachloride, Chloral hydrate, Codeine, Ethylene glycol, Isopropanol, Methanol, 2 -Naphthylamine, Parathion, Pyridine and Sulfanilamid.

iii-Factors affecting distribution: Main mechanisms opposing distribution of the toxicants are: a-Plasma proteins: -Bilirubin in neonates? ? ? . -High affinity for binding to P. P. may cause toxicity due to drug interaction as sulfonamide displace tolbutamide from P. P. binding site causing hypoglycemia. b-Storage: -DDT is stored in fat tissues and upon short term diet , mobilization of fats occur leading to release of DDT and toxicity. -Fluoride bind to calcium in bones flurosis. -Pb is stored in bones (non toxic to it) , osteoporosis mobilize Pb leading to toxicity. c-Special barriers (B. B. B. ): -Mainly depends on lipid solubility

iv-Factors affecting excretion: -The kidney is the primary excretory organ, followed by the gastrointestinal tract, and the lungs (for gases). Xenobiotics may also be excreted in sweat, tears, and milk. -Impaired cardiac , kidney or liver function causes slower elimination of toxicants and increases their toxic potential. End of Toxicokintics factors And Factors related to poison

IV-Chemical Interactions Humans are normally exposed to several chemicals at one time rather than to an individual chemical. Examples are: -Hospital patients on the average receive 6 drugs daily -Home influenza treatment consists of aspirin, antihistamines, and cough syrup taken simultaneously -Drinking water may contain small amounts of pesticides, heavy metals, solvents, and other organic chemical -Air often contains mixtures of hundreds of chemicals such as automobile exhaust and cigarette smoke -Gasoline vapor at service stations is a mixture of 40 -50 chemicals -There are four basic types of interactions.

Additivity -A tranquilizer and alcohol, often cause depression equal to the sum of that caused by each drug. -Chlorinated insecticides and halogenated solvents (which are often used together in insecticide formulations) can produce liver toxicity with the interaction being additive. P. S. : this same combination of chemicals produces a different type of interaction on CNS. Chlorinated insecticides stimulate CNS whereas halogenated solvents cause its depression. So, the effect of simultaneous exposure on CNS is an antagonistic interaction. Potentiation It occurs when a chemical that does not have a specific toxic effect makes another chemical more toxic. Example: -Warfarin (a widely used anticoagulant in cardiac disease) is bound to plasma albumin so that only 2% of the warfarin is active (FREE). Drugs which compete for binding sites on albumin increase the level of free warfarin to 4% causing fatal hemorrhage. -Phenobarbitone pre-treatment induces toxicity of paracetamol -Proniazid “MAOI” induces CVS toxicity by tyramine.

Antagonism It is often a desirable effect in toxicology and it is the basis for most antidotes. Examples include: Synergism -Exposure to both cigarette smoke and asbestos results in a significantly greater risk for lung cancer -The hepatotoxicity of a combination of ethanol and carbon tetrachloride is much greater than the sum of the hepatotoxicity of each. End of Factors affecting Toxicity

Sources of information on safety 1. Experimental studies Experimental toxicology is a branch of toxicology, which deals with toxicity studies in experimental animals to evaluate the safety of a new chemical (drugs, food additives, pesticides and industrial chemicals). 2. Controlled clinical studies Drug is tested on small number (50– 60) of healthy volunteers in a controlled dose for specified time 3. Epidemiological studies Thalidomide: its teratogenic activity was discovered in the 1960 s. Sulphonamide elixir (1930): its vehicle was ethylene glycol (metabolize to oxalic acid) that resulted in titanic convulsion

Goals of toxicity studies · Predict the toxicity of chemical in human · Give information about mechanism of toxicity · Give information about toxicity of dosage used in humans oxicity studies indicate therapeutic index which gives information about safety. Experimental toxicity studies Advantages of Experimental toxicity tests We can perform different experimental protocols using different routes of administration. And from this we can determine the following doses: 1. No-effect dose: it is the maximum dose that produces no observable toxic effect on the animals. 2. Minimal toxic dose: it is the dose that produces the least toxicity 3. Median lethal dose (LD 50 & LC 50): This is the dose that kills 50% of the animals.

Disadvantages of toxicity tests using experimental animals 1 -We can not extrapolate results obtained from experimental studies to humans: e. g. : insulin in experimental animals is toxic and causes teratogenic effects in pregnant animals, but in pregnant women it shows no teratogenic effects. 2 -Toxicity related to genetic factors can not be detected in animals: e. g. : Sulphonamides and aspirin in glucose-6 -PO 4 dehydrogenase deficient patients. 3 -Side effects of low occurrence appear only in humans but not in animal (because it needs large scale & more time). Characteristics of ideal animal species Non expensive Available Easily breaded Of short gestation period Of short life span for multigenerational life studies Physiology should be similar to human physiology Strains of rats 1 -Specific pathogen free animals (SPF) They are animals which are free from pathogenic organisms, so if used, toxicity is due to chemical not due to infection. 2 -Germ free animals. They are free from pathogenic and non-pathogenic organism. High cost Long life span 3 -Dirty animals. They contain pathogenic and non- pathogenic microorganisms Low cost Short life span 4 -Rats for specific purposes Insulin dependent rats Hypertensive rats

Ú There is a priority to carry out toxicity tests according to the exposure of humans A. Industrial chemicals: People subjected to industrial chemicals are liable to Accidental toxicity by inhalation or by oral route. So acute toxicity tests by inhalation & oral route should be carried out In case of biocides we should carry out acute, subchronic and chronic toxicity tests in addition to carcinogenicity, mutagenicity, and reproduction toxicity test. B. Food additives All types of toxicity tests should be performed C. Cosmetics: Dermal toxicity : Local allergy Oral toxicity: if taken orally by mistake in children Cosmetics carry label not tested on animals this is because they are tested on: Isolated eyes (obtained from slaughterhouse) Chorio-allantoin membrane: it is obtained from eggs before hatching and it is very rich in blood vessels.

General toxicity studies 1 -Acute toxicity tests A. Toxicometric studies These studies utilize dose response curve to determine LD 50 and/or LC 50 The dose-response correlates exposures and the spectrum of induced effects. -Generally, the higher the dose, the more severe the response. LD 50: Median lethal dose i. e. : It is the dose of a compound that causes 50% mortality in a population LC 50: Median lethal concentration (inhaled drugs).

Factors affecting LD 50 -Species, age, sex, body weight, general health condition, strain, diet, nutritional status & number of animals used in the test -Route of administration (oral route differ from parental route) -Environmental conditions (lab conditions) i. e. intra & inter laboratory conditions -Experimental procedure, stress, dosage formulation We cannot say that LD 50 of drug X is 50 mg/Kg absolutely but we must specify the route of administration e. g. oral LD 50 of drug X is 50 mg/Kg. Importance of LD 50: In spite of the many variables affecting the LD 50 determination, many governmental agencies still regard the LD 50 as the sole measurement of the acute toxicity of all materials. Many agencies classify chemicals according to LD 50 of drugs given orally to rats into the following groups. Super toxic chemicals: LD 50 > 5 mg/kg Extremely toxic chemicals: LD 50 = 5 – 50 mg/kg Very toxic chemicals: LD 50 = 50 – 500 mg/kg Moderately toxic chemicals: LD 50 = 0. 5 – 5 g/kg Slightly toxic chemical: LD 50 = 5 – 15 g/kg Practically non- toxic compound: LD 50 > 15 g/kg

Other dose estimates also may be determined from dose responce curve: Threshold dose level : The point at which toxicity first appears , and below which no toxic effect occur. -In the hypothetical curve above, no toxicity occurs at 10 mg whereas at 35 mg 100% of the individuals experience toxic effects. -A threshold for toxic effects occurs at the point where the body's ability to detoxify a xenobiotic or repair toxic injury has been exceeded. For most organs there is a reserve capacity so that loss of some organ function does not cause decreased performance. For example, the development of cirrhosis in the liver may not result in a clinical effect until over 50% of the liver has been replaced by fibrous tissue.

-Dose-response curves are also used to derive dose estimates of chemical(drug): EDs & TDs. Toxic Doses (TDs) : the doses that cause adverse toxic effects. Effective Doses (EDs) are used to indicate the effectiveness of substance. Normally, an effective dose refers to a beneficial effect (relief of pain). It might also stand for a harmful effect (paralysis). Thus the specific endpoint must be indicated. -The knowledge of the effective and toxic dose levels aides the toxicologist and clinician in determining the relative safety of pharmaceuticals. -As shown above, two dose-response curves are presented for the same drug, one for effectiveness and the other for toxicity. In this case, a dose that is 50 -75% effective does not cause toxicity whereas a 90% effective dose may result in a small amount of toxicity.

-So, dose-response curves are also used to relative safety of pharmaceuticals utilizing a known term which is : Therapeutic Index (TI) is used to compare therapeutically effective dose to the toxic dose Therapeutic Index (TI) It is the ratio of the dose producing 50% toxicity to the dose needed to produce the 50% therapeutic response. The TI is a statement of relative safety of a drug. - For example, if the LD 50 is 200 and the ED 50 is 20 mg, the TI would be 10 (200/20). A clinician would consider a drug safer if it had a TI of 10 than if it had a TI of 3.

-The use of the ED 50 and LD 50 doses to derive the TI may be misleading to safety, depending on the slope of the dose-response curves for therapeutic and lethal effects. -Knowledge of the shape and slope of the dose-response curve is extremely important in predicting the toxicity of a substance at specific dose levels. -Major differences among toxicants may exist not only in the point at which the threshold is reached but also in the percent of population responding per unit change in dose (i. e. , the slope). -As illustrated above, Toxicant A has a higher threshold but a steeper slope than Toxicant B.

To overcome this deficiency, toxicologists often use another term to determine the safety of a drug. The Margin of Safety (MOS). It is the ratio of the dose that is just within the lethal range (LD 01) to the dose that is 99% effective (ED 99). The MOS = LD 01/ED 99. -A physician must take care when prescribing a drug with MOS less than 1.

NOAEL and LOAEL: -No Observed Adversed Effect Level (NOAEL) -Lowest Observed Adverse Effect Level (LOAEL) They do not necessarily imply toxic or harmful effects , and may be used to describe beneficial effects of chemicals as well. Disadvantages of Toxicometrics -Needs large number of animals of at least 3 different species -LD 50 is not constant, it is affected by many factors (age, sex, species environmental conditions & route of administration) Toxicity of chemicals due to genetic abnormalities cannot be detected.

B. Dermal (skin) toxicity studies -It provide information on the adverse effects resulting from a dermal application of a single dose of a test substance. -The acute dermal test also provides the initial toxicity data for regulatory purposes, labeling, classification & subsequent subchronic & chronic dermal toxicity studies. Eye irritation It can be defined as reversible inflammatory changes in the eye and its surrounding mucus membranes following direct exposure to a material on the surface of the anterior portion of the eye. Draize test It is a simple test developed to study eye irritation in rabbits. It is used as the animal test to identify human eye irritants. The Draize test can adequately identify most of the moderate to severe human eye irritants

Pyramidal single dose test This test is carried out on dogs. Large number of dogs ≈ 100 are given a single daily X dose of a compound under test (X mg/Kg) At the end of the day, the number of dogs which died & those which survived is observed. The procedure is continued, till all dogs die, then plotting is done. Example: 1 st day 20 died & 80 living At the second day, double the X dose for living dogs i. e. 2 X mg/kg. This resulted in 20 died & 60 lived i. e. the dose starts small and then increases. Uses of pyramidal single dose test It helps in studying the mechanism of drug toxicity. Used to determine any pathological changes by examination of the animal after death. The effect of the drug on all body organs can be examined. . Biochemical tests can be performed on living animals (hematology, and detection of different biotransformation and excretion product, and determination of t½ of the compound).

2 -Prolonged toxicity studies (Repeated Dose Toxicity Studies) A. Sub-acute toxicity studies -They predict any cumulative effect of the drug & play a role in the safety assessment of pharmaceuticals, pesticides, food additives, and other chemicals. In this test, at least 3 species of animals are used: rats, mice and rabbits. Compound under test is given daily in 3 dose levels for 2 – 4 weeks. Animals are observed for different parameters: physiological, clinical and chemical tests, behaviour, CNS & autonomic profiles. The no-effect dose is determined & used it in the following tests. If oral route is intended, these doses are mixed with food or water. In addition, there is a fourth group of animals: control group it is used to test the vehicle of the drug. Animals are observed during the period of test for: mood symptoms, CNS effects, reflexes (corneal, righting) & autonomic responses. At the end of the test, animals are subjected to these tests and then are killed. Hematological studies: hemoglobin, RBCs, WBCs, platelets Clinical chemistry studies: serum creatinine, ALT, AST Histopathological studies: for different organs (spinal cord, heart, kidney, muscle)

B. Subchronic Toxicity Test It is performed for compounds administered for a long period. The compound is given for 90 days by the intended route. Animals are observed all over the specified time. For dead animals autopsy is performed by taking samples from different organs to be examined, chemically, microscopically & macroscopically C. Chronic Toxicity Test It is performed for compounds administered for a longer period. The compound is given for more than 90 days by the intended route. D. Life – Span Toxicity Test The same previous procedures are applied but treatment with chemicals starts after weaning of offsprings. Administration of the chemical is continued till death of animals. When animals die spontaneously, the same parameters are determined.

Specific Toxicity Studies Reproductive Toxicity It involves toxicant damage to either the male or female reproductive system. Toxic effects may cause: -Decreased libido and impotence -Infertility -Interrupted pregnancy (abortion, fetal death, or premature delivery) -Infant death or childhood morbidity -Altered sex ratio and multiple births -Chromosome abnormalities and birth defects -Childhood cancer

Developmental Toxicity pertains to adverse toxic effects to the developing embryo or fetus. This can result from toxicant exposure to either parent before conception or to the mother and her developing embryo-fetus. The three basic types of developmental toxicity are: Embryolethality Failure to conceive, spontaneous abortion Embryotoxicity Growth retardation or delayed growth of specific organ systems Teratogenicity Irreversible conditions that leave permanent birth defects in live offspring (cleft palate, missing limbs) Chemicals cause developmental toxicity by two methods. They can act directly on cells of the embryo causing cell death or cell damage, leading to abnormal organ development. A chemical might also induce a mutation in a parent's germ cell, which is transmitted to the fertilized ovum. Some mutated fertilized ova develop into abnormal embryos.

Carcinogenicity It is a complex multistage process of abnormal cell growth and differentiation which can lead to cancer. The initial neoplastic transformation results from the mutation of the cellular genes that control normal cell functions. Mutation may lead to abnormal cell growth. It may involve loss of suppresser genes that usually restrict abnormal cell growth. Many other factors are involved (e. g. , growth factors, immune suppression, and hormones). A tumor (neoplasm) is simply an uncontrolled growth of cells. Benign tumors grow at the site of origin; do not invade adjacent tissues or metastasize; and generally are treatable. Malignant tumors (cancer) invade adjacent tissues or migrate to distant sites (metastasis). They are more difficult to treat and often cause death. Mutation results from any change in DNA structure. If the mutation occurs in a germ cell the effect is heritable. There is no effect on the exposed person; rather the effect is passed on to future generations. If the mutation occurs in a somatic cell, it can cause altered cell growth (e. g. cancer)

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