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Home , Toxicant, Toxicology

Introduction to

Presented by: Dr. Marjan Shariatpanahi Department of Toxicology and , School of , International Campus,, Iran Department of Toxicology and Pharmacology, School of Pharmacy, International Campus, Iran University of Medical Sciences, Tehran, Iran

IUMS Nanosafety Summer School - are naturally produced by plants, animals, or bacteria.

Xenobiotic - man-made substance and/or produced by but not normally found in the body. Terminology of Definitions • The literal meaning of the word ‘toxicology’ is the study of toxins. • The root of the word is from the Latin word Toxicus (meaning ), which came into English around 1655 and is derived from the word Toxikon.

• Toxikon is an ancient Greek word, and at that time it was called the that impregnated the spears.

• Exposure:

• Any toxic compound for causing an , must first contact the live being. It means that the live being is Exposed to chemicals through a particular path (Air, soil, food). • : Any substance that causes harmful effects on a living at a specified concentration is called a toxicant. • Hazard: The qualitative of adverse effects arising from exposure to a physical agent or a particular toxicant. For example, suffocation is a danger caused by severe exposure to carbon monoxide. • Safety: The extent or likelihood that a material with a specified and specific exposure conditions will not produce any toxic effect. • Risk: The extent or likelihood that a substance with a certain dose and specific exposure conditions will cause a toxic effect. • Risk Assessment: The process by which the potential for causing adverse effects on health is recognized after exposure to toxins .

• Toxicology fields

• Mechanistic toxicology: • Analytical Toxicology • • : Investigates the toxic effects of chemical compounds in the environment on living , especially non-human beings. • Ecosystem Toxicology • Industrial Toxicology • Military Toxicology • Food Toxicology • Evolutionary Toxicology - • Epidemiological Toxicology • Regulatory Toxicology • Pharmaceutical Toxicology • Toxicovegillance • Clinical Toxicology • Oncologic or Carcinogenicity • Immunotoxicology • Toxic compound: Any agent that causes harmful effects on a biological system. Depending on the concentration, these harmful effects can range from a simple, reversible disorder to the death.

• The severity of toxic effects varies depending on the duration of exposure, dose, and type of exposure.

• The classification of toxic compounds can vary depending on the field of toxicology. Classification of toxic compounds by toxicity severity

The experimental animals: Rat Exposure Features Mice Hindi pigs • Routs of administration Rabbit • Oral Dog • Cutaneous Monkey • Inhale Squirrel • Intravenous Chinese hamster • Intramuscular Syrian hamster • Subcutaneous Mini pig • Inside the brain ventricle Micro pig Factors affecting the toxicity of a chemical compound

• Dose of chemical compound • Duration of Administration • Species, subspecies, age, sex, nutritional status, health status, individual allergy, presence of other chemicals • Physicochemical properties of such as solubility, melting rate, boiling point, vapor pressure, purity. If the chemical is a volatile solution and has high vapor pressure, it must be tested for toxicity by inhalation and skin absorption Criteria for choosing the suitable Species for toxicological studies

• Best species to study • Animal sex used • How to keep the animal • Animal diet • Animal health • Metabolic similarity of animal to human • The way of taking poison • Duration of study • Number of animals needed Toxicology Definitions

• Tolerance: Decrease in response to the toxic effect of a chemical compound resulting from previous exposure to that chemical or structure-dependent chemical compound. • Reversible and irreversible complications • Idiosyncratic Reactions Example: Succinylcholine, a genetic polymorphism in plasma butyrylcholinesterase

• Other than dose, factor that influence the body response to : idiosyncratic (occurring for no known reason)

• Allergic complications Interaction of chemicals with one another

• The interaction of toxic agents can be studied at both kinetic and dynamic levels. • The effect of two chemicals administered concurrently may manifest as one of the followings:

• Additive effects: The effect obtained from the administration of two compounds at the same time is equal to the sum of the effects of each compound alone. • Synergistic effects: The effect of the two compounds being administered simultaneously more than the sum of the effects of each combination alone. • Potentiation: When a substance does not have a known toxicity effect, but if co-administered with another compound, it can exacerbate the toxicity effects of that compound.

• : If two different substances counteract the effects of each other. Additive Effects

A + B

Response A B

Time

The effect of two chemicals is equal to sum of the effect of two chemicals taken separately. Synergistic Effects

A + B

Response A B

Time

The effect of two chemicals taken together is greater than the sum of their separate effect at the same doses, e.g., and other drugs Antagonistic Effects

A + B

Response A B

Time

The effect of two chemicals taken together is less than the sum of their separate effect at the same doses Important principles in toxicology tests • If the dose is calculated on the basis of body weight, the human dose is equal to one tenth of the animal dose to observe a toxic effect that had previously occurred in the animal.

• All carcinogenic compounds in animals are assumed to be carcinogenic in humans.

• Exposure of experimental animals to high doses of a toxic compound is a prerequisite and valid method of detecting possible hazards in humans. Toxicology experiments in animal studies

( test) •

(Subacute toxicity test) •

(Subchronic toxicity test) •

(Chronic toxicity test) • Acute toxicity test

• Duration of study 24 hours after administration of the specified dose of the substance • The purpose of acute toxicity tests : • Calculating LD50 • Identification of target organs and other clinical signs of acute toxicity • Reversibility of Toxic Responses • Determination of dose range for other toxicological studies • Evaluating the ability of a toxin to irritate skin or eyes What is LD50?

• LD50 is equal to the amount of toxic compound that causes 50% death in the animals. • LD50 is an indicator to compare the toxicity of a substance with other substances in a specified condition. In order to evaluate the therapeutic effects of a substance (known as an ) on the toxicity of a toxicant, an LD50 calculation of that toxin is necessary. • It requires the use of two different administration routs and in two different animal species (mice and rats). • At least 3 different doses are required to calculate this index. • The mortality rate is assessed 24 hours after administration, and the remaining animals will be monitored for up to 14 days. Draize test

• Draize Test: The ability of a chemical to induce skin and eye irritation after an acute exposure is usually determined in rabbits.

• In order to evaluate the potential of a chemical compound to cause skin sensitization, the effect of other accompanying compounds should also be studied in addition to the desired compound. Subacute toxicity test

• Purpose: To obtain information on the toxicity of a chemical compound after repeated doses. • The results of this study can be used to determine the doses required in subchronic studies. • The maximum duration of the study is one month. • A typical protocol is to give three to four different dosages of the chemicals to the animals by mixing it in their feed. For rats, 10 animals per sex per dose are often used; for dogs, three dosages and 3 to 4 animals per sex are used. Clinical and histopathology are performed after 14 days of exposure. Subchronic toxicity test

• The duration of contact in this study is usually between 1 - 3 months. • Usually two animal species (rat and dog) and three different doses are used. • The rout of administration depends on the method of use or contact with the studied chemical compound.

• The purpose of subchronic studies:  Determination of NOAEL (No observed adverse effect level)  Determination of LOAEL (Lowest observed adverse effect level)  Identifying the tissues that are specifically affected by the toxic compound.  The possibility of predicting appropriate doses for chronic studies Chronic toxicity test

• The purpose of the chronic toxicity test is to evaluate the cumulative toxicity of the substance, along with its potential for . • Study duration is 3 months to 2 years. • The animal species is determined by subchronic studies. • The study should be carried out in at least 3 animal species. • The depends on how the toxic compound is used or contacted. • Investigating the effects of the toxic compounds on the fetus, genetic toxicity, and mutagenesis are among other things that are being evaluated for completing the test. Chronic toxicity is in 2 category:

1. Chronic toxicity requiring prolonged or repeated exposure to a compound. Example: Long exposure to low concentrations of such as arsenic, mercury, and cadmium, which can cause a variety of .

2. Chronic toxicity that occurs after a prolonged period following exposure to only one or more limited doses of the chemical compound. Example: Tumor formation in the kidney by dimethyl nitrosamine or delayed neuropathy due to intoxication with organophosphorus compounds. Dose THE KEY CONCEPT in Toxicology

Father of Modern Toxicology —1564

“All things are poisonous, only the dose makes it non-poisonous.” Dose alone determines toxicity All chemicals—synthetic or natural—have the capacity to be toxic The relationship between drug dose and effect

Relationship between dose and response

O All chemicals at a specified dose cause adverse effects.

O (Toxic chemicals) are factors that can have adverse effects on the biological system. O A relatively safe chemical can cause toxicity if the dose and amount are sufficiently high.

O On the other hand a highly toxic chemical can be safe if it has low concentration in contact. O The main components of experiments that generate dose-response information:

O Selection of the appropriate experimental animal in the laboratory O Selecting a response to measure O Duration of exposure O Experiment period (observation period) O Considering toxin values ​​for experiments Dose response curve: O The toxicologist should try to deduce the following information from dose-response curves in order to have a safe assessment of any particular chemical:

O 1.Threshold O 2. LD50 O 3.LOAEL (Low observed adverse effect level) O 4.NOAEL (No observed adverse effect level) O 5.MED )Minimum effective dose) O 6.MTD (Minimum toxic dose) O 7. O 8.Margine of safety

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Dose-Response Relationship “The Dose Makes the Poison”

Effective Dose 100 100 80 80 60 60 ED LD50 40 50 40 20 20

1 2 3 5 7 10 10 20 30 50 100 Phenobarbital (mg/kg) Log Scale

What is the therapeutic range?

O There is a range of concentration of the drug in the serum that in most people has therapeutic effects and the risk of drug toxicity is low. The lower from this range, the drugs are ineffective. The higher from this range, toxic side effects are observed. O In fact, concentrations known as ‘therapeutic’ ranges between "lowest effective concentration" or MEC and "lowest toxic concentration" or MTC. Therapeutic Index

• Effective dose (ED50) = dose at which 50% population shows response

• Lethal dose (LD50) =dose at which 50% population dies

• TI = LD50/ED50, an indication of safety of a drug (higher is better)

ED50 LD50 Therapeutic Index

O Margin of safety O % population responding

O Compare effective dose (ED50) to

O Lethal dose (LD50)

O Or toxic dose (TD50) LD50

Therapeutic index (TI)= LD50/ED50 The lower TI, the smaller the margin of ED50 safety, e.g. digoxin TI = LD50/ ED50 ED LD 100

% Population 50

0 0 X DRUG DOSE

O Relatively safe ~ TI = LD50/ ED50 ED LD 100

% Population 50

0 0 X DRUG DOSE

O Less safe drug ~ Dose-response curve

Indicates the relationship between the amount of drug administered (or the resulting plasma concentration) and the pharmacological effect.

They are distinguished by features such as: power, effect, slope, efficiency and individual responses.

Maximum B 100 A

% Maximum Response 50

0 0 X DRUG DOSE

O B has greater max efficacy than A ~ B 100 A % Maximum Response 50

0 0 X DRUG DOSE

O A is more potent than B ~

Relative toxicity also be evaluated by comparing the slope of each dose- response curve. A steep dose-response curve is indicative of a chemical that is rapidly and extensively absorbed and slowly detoxified: therefore, the chemical is more toxic.

MECHANISMS of TOXICITY

Dr. Marjan Shariatpanahi

53 TOXICANT

Toxicant is any toxic substance.

Note: In popular usage, the term is often used to denote substances made by humans or introduced into the environment by human activity, in contrast to toxins, which are toxicants produced naturally by a living organism.

54 Toxicity pathway

1. Poison access to the target tissue. 2. Ultimate toxicant reaction with endogenous target molecules. 3. Creating sequential disruptions in cell function and structure. 4. Initiation of compensatory mechanisms at the cell, tissue or molecule level. 5. Toxicity occurs when toxicant-induced disorders exceed the compensatory capacity or when there are some defects in compensatory mechanisms.

55  Theoretically, the severity of a toxic effect depends primarily on the concentration and stability of the ultimate toxin at its site of action.  The ultimate toxin is a chemical compound that react with endogenous target molecules (Including receptors, enzymes, DNA, small stranded proteins, and fats) and lead to toxicity by making structural and practical changes. In most cases, the ultimate toxin is the main chemical compound that the live being is in contact with (the parent compound).

 In other cases, the ultimate toxin is metabolite of the main constituent or reactive oxygen species formed during biological deformation.

 Sometimes, the ultimate toxin is an endogenous molecule.  Accumulation of the ultimate toxin in the target tissue is facilitated by absorption, distribution to the site of action, reabsorption and toxicogenesis (metabolic activation).

 In contrast, presystemic , dislocation from the target tissues, and detoxification prevent from accumulation of the ultimate toxin in the target tissue. 56 57 58 MECHANISMS of TOXICITY 1. Delivery: Site of Exposure to the Target

2. Reaction of the Ultimate Toxicant with the Target Molecule

3. Cellular Dysfunction and Resultant Toxicity

4. Repair or Dysrepair

59 WHAT ARE the TARGET MOLECULES

• Nucleic acids (esp. DNA) • Proteins ( e.g. Enzymes) • Membranes

60 TYPES of REACTION with TARGET MOLECULES

 Non-covalent binding (hydrogen bond, ionic bond) Reversible, membranes receptors, intracellular receptors, ion channels, some enzymes  Covalent binding Irreversible, free radical bond to proteins , nucleic acids  Hydrogen abstraction Free radicals bond to H and CH2 groups of amino acids  Electron transfer Exchange electrons to oxidize or reduce other molecules, e.g. Fe II to Fe III in hemoglobin → methemoglobinemia by benzocaine, dapsone, and nitrates.

61 EFFECTS of TOXICANTS on TARGET MOLECULES Toxicant

Target molecules

Dysfunction Destruction

Neo-antigen

62 EFFECTS of TOXICANTS on TARGET MOLECULES

1) Dysfunction of target molecules

2) Destruction of target molecules

3) Neo-antigen formation

63 DYSFUNCTION of TARGET MOLECULES

Cellular dysfunction

Cellular maintenance Cellular dysregulation impairment اختالل در عملکرد سلولی اختالل در تنظیمات سلولی

64 65 CELLULAR DYSREGULATION اختالل در تظیمات داخل سلولی • Dysregulation of gene expression 1) Dysregulation of transcription (promoter, TFs,…) 2) Dysregulation of signal transduction 3) Dysregulation of extracellular signal production

• Dysregulation of ongoing cellular activity 1) Dysregulation of electrically excitable cells: neurons, skeletal, cardiac, smooth muscles 2) Dysregulation of the activity of non-excitable cells: endocrine cells, kupffer cells...

66 67 Cellular maintenance impairment )اختالل در عملکرد سلولی)

Sustained rise of Depletion of ATP intracellular Ca 2+

Overproduction of ROS and RNS

68 Sustained rise of Depletion intracellular of ATP Ca 2+

↑ROS, RNS

69 ATP PRODUCTION in MITOCHONDRIA

70 TOXICANT CLASSES in ATP DEPLETION

1) Class A: interfere with the delivery of H to ETC سمومی که مانع از در اختیار قرار دادن هیدروژن توسط NADH به زنجیره انتقال الکترون میشود. 2. Class B: inhibition of electron transfer along ETC سمومی که مانع از انتقال الکترون در مسیر زنجیره انتقال می شوند. 1) Class C: interfere with O2 delivery to terminal ETC سمومی که مانع رسیدن اکسیژن به انتهای زنجیره میشود.)جلوگیری ازاکسیداسیون( 1) Class D: inhibition of oxidative phosphorylation سمومی که از تبدیل ADP به ATP جلوگیری میکنند.

71 CONSEQUENCES of ATP DEPLETION ATP Depletion

Compromised ion pumps (e.g. Na/K ATPase and Ca2+-ATPases)

1. loss of ionic and volume regulatory controls 2. Ca2+/Na+ levels rise intracellularly

cell swelling (water influx)

cell lysis → necrosis

72 AGENTS IMPAIRING ATP SYNTHESIS

• Inhibitors of electron transport 1. Cyanide inhibits cytochrome oxidase 2. Rotenone inhibits complex I—insecticide 3. Paraquat inhibits complex I—herbicide, but also causes lung damage

• Inhibitors of oxygen delivery 1. Ischemic agents such as ergot alkaloids, cocaine 2. Carbon monoxide—displaces oxygen from hemoglobin

• Inhibitors of ADP phosphorylation – DDT • Chemicals causing mitochondrial DNA damage - chronic ethanol

73 CONSEQUENCE of SUSTAINED RISE of INTRACELLULAR Ca 2+ 1. Depletion of energy reserves—decreased mitochondrial ATP production and increased loss of ATP by activation of Ca 2+ -ATPase. مصرف ATP و تخلیه ذخایر ATP سلولی 2. Dysfunction of microfilaments—impaired cell motility, disruption in cell morphology, cellular functions مستهلک شدن میکروفیالمانتها بخصوص اکتین که به دنبال ان تخریب غشای سلولی رخ میدهد. 3. Activation of enzymes, including phospholipases, endonucleases, and proteases—disintegration of membranes, proteins, DNA, etc. فعال شدن آنزیمهای هیدرولیز و کاتالیز کننده 4. Generation of ROS/RNS—disintegration of membranes, proteins, DNA, etc ایجاد رادیکالهای آزاد

74 OXIDATIVE STRESS

Imbalance of cellular oxidants and antioxidants in favor of oxidants

1) ↑Free radicals production 2) Ineffective Scavenger systems

75 CONSEQUENCE of ROS/RNS

1. ROS can directly oxidize and affect protein function and can mutate DNA leading to cellular dysfunction 2. ROS/RNS oxidatively inactivate Ca2+ /ATPases and elevate Ca2+ 3. ROS and RNS also drain ATP reserves: a. NO. is a reversible inhibitor of cytochrome oxidase. b. ONOO- (Peroxynitrite) irreversibly inactivates complexes I/II/III . c. ROS can disrupt mitochondrial membranes and dissipate the electrochemical gradient needed for ATP synthase. 4. ONOO- induces DNA single-strand breaks. 5. Lipid peroxidation, cell swelling, and cell rupture

76 MECHANISMS of ACUTE TOXICITY

77 TOXICANT EXPOSURE and EFFECT

78 DOSE & TOXICITY

All chemicals elicit acute toxicity at a sufficiently high dose, whereas all chemicals do not elicit chronic toxicity

chronic toxicity typically occurs at dosages below those that elicit acute toxicity, toxicity observed at the higher dosage may simply reflect acute, and not chronic toxicity

79 ACUTE TOXICITY Toxicity elicited immediately following short-term exposure to a chemical

80 MECHANISMS OF ACUTE TOXICITY

1) Narcosis 2) Acetylcholinesterase Inhibition 3) Ion Channel Modulation 4) Inhibition of Cellular Respiration

81 NARCOSIS

 Toxicity resulting from chemicals associating with disrupting the lipid bilayer of cell membrane.

 Narcotics Nonpolar( class1): aliphalic hydrocarbone, CH4 Polar( class2): ethanol  The central is the prime target of chemical narcosis and symptoms initially include disorientation, euphoria, dizziness, and progress to unconsciousness, convulsion, and death. Acetylcholinesterase inhibitors

 Acetylcholine is an important at the central nerve synapse, in the autonomic system and at the nerve-muscle junction.  Cholinesterase enzyme hydrolyzes and destroys this neurotransmitter. Toxins such as organophosphates, carbamates and nerve gases such as sarin bind to the acetylcholine binding site in the enzyme.  This binding can occur either weakly or temporarily (carbamates) or strongly (by covalent bonding) such as organophosphates.  Toxic symptoms: exacerbated cholinergic symptoms

83 ACETYLCHOLINESTRASE INHIBITOR ACETYLCHOLINESTRASE INHIBITOR

• The inhibition of acetylcholineasterase results in prolonged, uncoordinated nerve or muscle stimulation.

Toxic effects of cholinesterase inhibition typically are evident when the enzyme activity is inhibited by about 50%. Symptoms: nausea and vomiting, increased salivation and sweating, blurred vision, weakness, chest pains. Convulsions typically occur between 50% and 80% enzyme inhibition with death at 80– 90% inhibition. Death is most commonly due to respiratory failure. ION CHANNEL MODULATION • Ion exchange plays an important role in the transmission of nerve impulses along the axons. Some directly block these channels. • Inhibition of the Na / K ATPase Pump: Inhibition of Potential Action Transfer in the Cell Membrane: DDT, Pyrethroids • Inhibition of GABAergic Receptors: Closure of the Channel and Over-stimulation of nerves : Dieldrin, Pyrethroids • DDT: Effective on sodium and calcium channels. ION CHANNEL MODULATION

• The GABAA is associated with chloride channels on the postsynaptic region of the neuron. • binding of GABA to the receptor → open chloride channel. • Thus activation of GABAA serves to prevent excessive excitation of the postsynaptic neuron. • Many neurotoxicants inhibit the GABAA receptor, resulting in prolonged closure of the chloride channel and excess nerve excitation: – Cyclodiene insecticides (i.e., dieldrin) – Organochlorine insecticide lindane – pyrethroid insecticides

• Symptoms of GABAA inhibition include dizziness, headache, nausea, vomiting, fatigue, tremors, convulsions, and death.

• Barbituates (i.e., phenobarbital) and ethanol enhance the ability of GABA to bind the receptor and open the chloride channel. These compounds suppress nerve transmission which contributes to the sedative action of the chemicals. CELLULAR RESPIRATION

• Cellular respiration is the process whereby energy, in the form of ATP, is generated in the cell while molecular oxygen is consumed INHIBITION of CELLULAR RESPIRATION

Many chemicals can interfere with cellular respiration by binding to the cytochromes that constitute the electron transport chain and inhibiting the flow of electrons along this protein complex.

Symptoms of toxicity from the inhibition of respiratory chain include excess salivation, giddiness, headache, palpitations, respiratory distress, and loss of consciousness. Potent inhibitors such as cyanide can cause death due to respiratory arrest immediately following  Questions?  Comments?

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