Mammalian Toxicology. Introduction

Transcription

1 Mammalian Toxicology ENVE 569 Environmental Risk Assessment Introduction This concept of dividing chemicals into two categories has persisted to the present day. It is a useful idea as harmful materials can be readily placed into a category that is accorded due respect. Recognizing degrees of harm and safety is a more acceptable approach. Even the most innocuous of substances, when taken into the body in sufficient amounts, may lead to an undesirable response. In contrast, the most harmful of chemical agents if ingested in sufficiently small amounts will have no observable effects. To compare the effects of chemicals on animals of different size the amount is expressed as mass per unit weight of the animal. As the dose is increased from a minimum to a maximum level, a graded response is observed rather than a sharp demarcation from no response to the ultimate response. A fundamental observation in the field of toxicology is the relationship that exists between the dose and the response of a chemical. 1

2 The study of the adverse effects of a toxicant on living organisms Adverse effects any change from an organism s normal state dependent upon the concentration of active compound at the target site for a sufficient time. Toxicant (Poison) any agent capable of producing a deleterious response in a biological system Living organism a sac of water with target sites, storage depots and enzymes What is a Poison? All substances are poisons; there is none that is not a poison. The right dose differentiates a poison and a remedy. Paracelsus ( ) 2

3 Numbers in Toxicology No chemical agent is entirely safe and no agent is entirely harmful. This idea is based on the premise that any chemical capable of causing a biological response will be inactive when the concentration or dose is below a minimal effective level. Consequently, there must be a range of concentrations that give a graded response between the two extremes of no observable effect and 100% response. Dose The amount of chemical entering the body This is usually given as mg of chemical/kg of body weight = mg/kg The dose is dependent upon * The environmental concentration * The properties of the toxicant * The frequency of exposure * The length of exposure * The exposure pathway 3

4 What is a Response? The degree and spectra of responses depend upon the dose and the organism Describe exposure conditions with description of dose Change from normal state Response (symptoms) could be on the molecular, cellular, organ, or organism level Local vs. Systemic Reversible vs. Irreversible Immediate vs. Delayed Graded vs. Quantal degrees of the same damage vs. all or none Numbers in Toxicology Dose-Response Relationship When a group of experimental animals is examined, differences between individual members of what is normally considered a homogeneous population are seldom obvious. Differences only become evident when the group of animals is challenged by an exposure or some other type of treatment. 4

5 Dose-Response Relationship: As the dose of a toxicant increases, so does the response. RESPONSE NOAEL 2-3 Linear Range 4 Maximum Response DOSE Example Dose-Response Relationship Dose (mg/kg) No. of animals responding

6 Dose-Response Relationship The shape is the curve is typically an elongated S and is normally called S-shaped. Several important (and interesting) characteristics of this type of curve: Most of the curve is linear where the incidence of the response is directly related to the concentration. The concept of LD 50 (the lethal dose for 50% of the animals) is a direct result of this curve. The LD 50 is a statistical value and should be accompanied by some means of estimating the uncertainty in the value. Dose-Response Relationship Several important (and interesting) characteristics of this type of curve (cont.): The LD 50 from the curve is obtained by drawing a horizontal line from the 50% mortality (or response) point on the y-axis to the point where it intersects the curve. From that point a vertical line is drawn to the x axis. The LD 50 is the concentration represented by the point where the abscissa is intersected. The lethal dose for any predetermined percentage of the animals may be obtained in a similar manner. 6

7 More about the Dose-Response Curve The dose-response curve is, in effect, a cumulative probability distribution. Could re-graph curve looking at the number of new animals responding at each dose. Note bell-shaped response and maximum sensitivity at the LD 50. Dose-Response Relationship: As the dose of a toxicant increases, so does the response. RESPONSE NOAEL 2-3 Linear Range 4 Maximum Response DOSE 7

8 More about the Dose-Response Curve In addition, the slope of the linear region is related to the variance in the sensitivities. A steep slope represents a homogeneous sample of animals where the change in response to a change in dose is great. By contrast, a shallow slope represents a group of animals that are less homogeneous. The lack of homogeneity is reflected in the absence of sensitivity and response to a change in dose. Calculations identical to the generic statistical discussion of Chapter 2. The median dose ± 1 s.d. includes 69% of the animals, while the median dose ± 1.96(s.d.) includes 95% of the results. The standard deviation may be estimated by drawing lines parallel to the x axis from the 50% ± 34.5% points on the y-axis. LD 50 Quantal responses can be treated as gradient when data from a population is used. The cumulative proportion of the population responding to a certain dose is plotted per dose If Mortality is the response, the dose that is lethal to 50% of the population LD 50 can be generated from the curve Different toxicants can be compared: lowest dose is most potent 8

9 LD 50 Comparison Chemical LD 50 (mg/kg) Ethyl Alcohol 10,000 Sodium Chloride 4,000 Ferrous Sulfate 1,500 Morphine Sulfate 900 Strychnine Sulfate 150 Nicotine 1 Black Widow 0.55 Curare 0.50 Rattle Snake 0.24 Dioxin (TCDD) Botulinum toxin Scales of Toxicities Potency of a chemical is related to the dose required to achieve the toxic effect under study. In the extreme, toxic response is expressed in terms of lethality. It is also related to the animal used to perform the toxicity experiments. Agent Ethyl alcohol Ferrous sulfate Morphine sulfate DDT Nicotine Dioxin Dioxin Animal Mouse Rat Rat Rat Rat Rat Guinea Pig LD50 (mg/kg) 10,000 1, Botulinus Guinea Pig

10 Scale of Toxicities Classifications are arbitrary! A typical scale of toxicities: Extremely toxic 50 mg/kg or less Moderately toxic mg/kg Slightly toxic g/kg Relatively harmless 5 g/kg or more Margin of Safety Result where dose-response curve is: Parallel to the y-axis means that dose that has no effect also has 100% effect Parallel to the x-axis means that there is a constant effect at no chemical exposure and at infinite chemical exposure Slope must lie between two parallel lines. Slope becomes an index of the margin of safety of a compound. Margin of Safety is the range of doses involved in progressing from a noneffective dose to a lethal dose. 10

11 Margin of Safety As the slope becomes more shallow, the margin of safety increases. Range of Effects Although the ultimate response resulting from a chemical agent is death, there are many other responses some of which may be desirable! For a drug developer, the margin of safety is the spread between the dose producing a lethal or undesirable effect and the dose producing the desired effect. This margin is referred to as the therapeutic index and is the ratio of the LD 50 to the ED 50 (effective dose for 50% of the animals). Routes of Exposure Ingestion Inhalation Injection Surface absorption 11

12 Exposure: Pathways Routes and Sites of Exposure Ingestion (Gastrointestinal Tract) Inhalation (Lungs) Dermal/Topical (Skin) Injection intravenous, intramuscular, intraperitoneal Typical Effectiveness of Route of Exposure iv > inhale > ip > im > ingest > topical Exposure: Duration Acute < 24hr usually 1 exposure Subacute 1 month repeated doses Subchronic 1-3mo repeated doses Chronic > 3mo repeated doses Over time, the amount of chemical in the body can build up, it can redistribute, or it can overwhelm repair and removal mechanisms 12

13 Biological Factors in Toxicology Before a chemical can produce an effect on a biological organism, a reaction must occur. The agent must contact some biological site and react. Therefore, the first task is to bring the chemical from outside to the inside of the animal. Once inside, the chemical is transported to the key site where reaction takes place. Frequently, these reactions are under the control of natural catalysts (enzymes proteins synthesized by the organism for this purpose) which speed up or slow down the processes. Translocation of Chemicals Animals are protected from the outside environment by specialized covering. Protect the organism not only from temperature extremes, but also block the free transfer of chemicals from the outside to the inside and the reverse. Coverings range in their degree of permeability from the skin (least permeable and the most protective of the layers) to the interior of the stomach and small intestine (which are designed to be more permeable so food constituents once they are broken down can pass into the organism). 13

14 Storage of Chemicals If there is a constant exposure, then eventually an equilibrium will be reached where the amount entering is equal to the amount being eliminated. The rate at which the body clears the chemical after the termination of the exposure depends on how fast the various sites will release the agent. From an environmental point of view, an important storage area is fat. Chlorinated hydrocarbons such as DDT and PCB are very fat soluble. Distribution: Storage and Binding Storage in Adipose tissue--very lipophylic compounds (DDT) will store in fat. Rapid mobilization of the fat (starvation) can rapidly increase blood concentration Storage in Bone--Chemicals analogous to Calcium--Fluoride, Lead, Strontium Binding to Plasma proteins--can displace endogenous compounds. Only free is available for adverse effects or excretion 14

15 ADME: Absorption, Distribution, Metabolism, and Excretion Once a living organism has been exposed to a toxicant, the compound must get into the body and to its target site in an active form in order to cause an adverse effect. The body has defenses: Membrane barriers passive and facilitated diffusion, active transport Biotransformation enzymes, antioxidants Elimination mechanisms Absorption: ability of a chemical to enter the blood(blood is in equilibrium with tissues) Inhalation--readily absorb gases into the blood stream via the alveoli. (Large alveolar surface, high blood flow, and proximity of blood to alveolar air) Ingestion--absorption through GI tract stomach (acids), small intestine (long contact time, large surface area Dermal--absorption through epidermis (stratum corneum), then dermis; site and condition of skin 15

16 Distribution: the process in which a chemical agent translocates throughout the body Blood carries the agent to and from its site of action, storage depots, organs of transformation, and organs of elimination Rate of distribution (rapid) dependent upon blood flow characteristics of toxicant (affinity for the tissue, and the partition coefficient) Distribution may change over time Target Organs: adverse effect is dependent upon the concentration of active compound at the target site for enough time Not all organs are affected equally greater susceptibility of the target organ higher concentration of active compound Liver--high blood flow, oxidative reactions Kidney--high blood flow, concentrates chemicals Lung--high blood flow, site of exposure Neurons--oxygen dependent, irreversible damage Myocardium--oxygen dependent Bone marrow, intestinal mucosa--rapid divide 16

17 Target Sites: Mechanisms of Action Adverse effects can occur at the level of the molecule, cell, organ, or organism Molecularly, chemical can interact with Proteins Lipids DNA Cellularly, chemical can interfere with receptor-ligand binding interfere with membrane function interfere with cellular energy production bind to biomolecules perturb homeostasis (Ca) Excretion: Toxicants are eliminated from the body by several routes Urinary excretion water soluble products are filtered out of the blood by the kidney and excreted into the urine Exhalation Volatile compounds are exhaled by breathing Biliary Excretion via Fecal Excretion Compounds can be extracted by the liver and excreted into the bile. The bile drains into the small intestine and is eliminated in the feces. Milk Sweat Saliva 17

18 Metabolism: adverse effect depends on the concentration of active compound at the target site over time The process by which the administered chemical (parent compounds) are modified by the organism by enzymatic reactions. objective--make chemical agents more water soluble and easier to excrete decrease lipid solubility --> decrease amount at target increase ionization --> increase excretion rate --> decrease toxicity Bioactivation--Biotransformation can result in the formation of reactive metabolites Chemical Factors in Toxicology Two areas of importance. Transport across the cell membrane: Crossing the cell barrier depends on chemical properties such as lipid solubility and ionization. Current evidence suggests that the nonionizable, lipid-soluble form of an organic molecule is the predominant form capable of passing through the lipophilic membrane of the organism. Recall that many chemicals exist in both ionized and nonionized form. HAc H + + Ac - 18

19 Chemical Factors in Toxicology Two areas of importance. Reaction at a key site in the cell: If the reaction between the chemical and the biological receptor causes a chemical change, the reaction is termed a biotransformation. These changes are quite common; consequently, in the determination of toxicity of an agent, the effects of both the parent material and all fragments that might result from biotransformation reactions need to be assessed. Microsomal enzymes catalyze many of the biotransformation reactions. These are present in a variety of tissues, but are particularly abundant in the liver, making this a key organ for transforming different chemicals. The total quantity of microsomal enzymes can be increased in humans and other higher animals by prior exposure to a large variety of agents. Biotransformation (Metabolism) Can drastically effect the rate of clearance of compounds Can occur at any point during the compound s journey from absorption to excretion Compound Without With Metabolism Metabolism Ethanol 4 weeks 10mL/hr Phenobarbital 5 months 8hrs DDT infinity Days to weeks 19

20 Biotransformation Key organs in biotransformation LIVER (high) Lung, Kidney, Intestine (medium) Others (low) Biotransformation Pathways * Phase I--make the toxicant more water soluble * Phase II--Links with a soluble endogenous agent (conjugation) Tolerance and Comparative Toxicology Tolerance When repeated exposure to the same dose causes a decline in the response, the organism is said to have developed a tolerance to the chemical. Tolerance to chemicals has significance in toxicology for it represents a mechanism whereby certain organisms are protected against the harmful effects of chemicals. 20

21 Tolerance and Comparative Toxicology Comparative Toxicology The increased awareness of comparative toxicology is due to the recognition that toxicity testing is done primarily in rodents and the information needs to be translated to humans. While not a great deal is known about the subject, the fact that rats are not humans must be remembered; as a consequence, testing uses many different animals. By covering a wide spectrum of species the hope is that the distribution will include organisms that behave similarly to people in their response to chemicals. Individual Susceptibility --there can be fold difference in response to a toxicant in a population Genetics-species, strain variation, interindividual variations (yet still can extrapolate between mammals--similar biological mechanisms) Gender (gasoline nephrotox in male mice only) Age--young (old too) underdeveloped excretory mechanisms underdeveloped biotransformation enzymes 21

22 Age--old Individual Susceptibility changes in excretion and metabolism rates, body fat Nutritional status Health conditions Previous or Concurrent Exposures additive --antagonistic synergistic Route of Entry The route by which materials in the environment gain entry into the organism will depend to a large extent on the chemical and physical properties of the agent. (e.g., volatile agents are inhaled while chemicals dissolved in water or food entered orally through ingestion). Four routes: Percutaneous Inhalation Oral Parenteral 22

23 Routes of Entry Percutaneous Route Simplest and most common exposure to foreign chemicals is by exposure through accidental or incidental contact with the skin. This is terms percutaneous absorption and is the transfer from the outer surface of the skin through the horny layer and into the blood circulatory system. Most often the skin is not a very efficient route for administering a chemical. Example: LD50 for DDT in rats: oral 118 mg/kg; dermal 2510 mg/kg. A variety of factors such as ionization, molecular size, and water solubility are all involved in determining the ease with which a chemical can penetrate the dermal barrier. Inhalation Route For chemicals to reach the respiratory tract they must be either gaseous or sufficiently small so that they are not removed in the airway passages of the lung. The inhalation route is obviously the main route of entry for all airborne materials. Because of the adverse effects of some exposures, standards establishing the concentration to which workers can be exposed during an 8-hour working day have been prepared by OSHA. The resulting document specifies the maximal allowable concentration in the workplace air for humans exposed in an 8-hour day and a 40-hour week. Permissible exposure limit (PEL) is the name of the standards. PELs are not intended for use in evaluating community air pollution; however, they are often used/cited. Routes of Entry Oral Route The oral route is the third most common means by which chemicals enter the body. This occurs when food or water contaminated with chemicals such as pesticides are consumed. While the chemical can be absorbed any place along the GI tract, the major site is the stomach. After absorption, translocation to the liver occurs; there the chemical is transformed (metabolized) either into harmless metabolites and excreted or into more toxic materials. Through these types of reactions the oral route can enhance the toxicity of a chemical compared to other routes of administration. Parenteral Route This route involves injecting chemicals directly into the body and bypassing the natural openings such as the mouth, nose and skin. The most rapid means to achieve a high concentration of a chemical at a specific site. Not a common exposure route for environmental risk assessment. 23

24 (Microbial) Transmission Routes Many organisms have more than one route of transmission. Sequence of spread usually involves the exit of the organisms from the infected host and transport through the environment until they come in contact with another susceptible host. Organisms transmitted through the environment must be able to survive long enough to be transmitted from one host to another. Many organisms can only survive short periods of time and are unlikely to be transmitted through the environment. Often, the respiratory route requires fewer organisms for infection than does ingestion. Transmission routes may occur over prolonged distances. Transmission routes may be simple and direct or more complex and involve several media. Routes of Transmission Route of Exit Route of Transmissions Examples Factors Route of Entry Respiratory Nasal discharges, fomites Rhinovirus Household contact Respiratory, eye Respiratory system air, fomites Influenza Direct contact Respiratory Respiratory system air Plague (pneumonia) Pneumonia case Respiratory Respiratory system droplets Streptococcal Close contact, carrier Respiratory Respiratory system droplet nucleic Tuberculosis Household contact, carrier Respiratory Skin squames Respiratory, direct contact, fomites Nosocomial bacterial infections Hospitalization, surgery Nose, respiratory, skin Direct contact, fomites Impetigo due to staph and/or strep infection Low socioeconomic level, tropics Skin Fomites, direct contact Papillomavirus, warts Bathroom floors Skin Fecal-oral Stool water, food Cholera Water, food Mouth Stool food, water Salmonellosis Food, animal contact Mouth Stool water, fomites, food, vomit Rotavirus Day-care centers, water, food Mouth Urine Water (swimming) Leptospirosis Infected animals Skin Inanimate sources Soil, air, water, food Legionnaire s disease Warmth, humidity, water coolers, air conditioners, potable water supplies Respiratory Acantamabea Water, swimming Eye Nagelaria Swimming Nose 24

25 Routes of Transmission Inhalation Airborne transmission can occur via release from the host in droplets or through natural or human-made activities. Their success in reaching a susceptible host depends on: Droplet size Force with which they are propelled into the air Resistance to drying Temperature Humidity Air currents UV light Distance to host. The most important determinant of the probability that a disease will be transmitted by aerosols is the ability of the microorganism to survive in aerosolized droplets or particulates. Controlling variables include: Nature of the suspending solution (mucus, water) Air temperature Relative humidity Sunlight/UV light Routes of Transmission Inhalation Sources of microbial aerosols Wastewater treatment plants Compost operations Domestic waste transfer stations Sewage sludge application Spray irrigation with sewage Cooling towers Earthmoving during construction Sneezing Coughing Release from clothing and skin during walking Bathing Talking Faucets Toilet flushing Air humidifiers Water fountains Showers Waves breaking on a beach Spills of infectious materials Medical devices (suction, syringes) Dental instruments 25

26 Routes of Transmission Dermal Skin is a point of entry and exit for viral and bacterial infections. May occur when skin is intact but is more likely to occur where skin integrity is broken. Examples of microbial skin infections: Staphylococcus aureus Plantar warts Hepatitis B Routes of Transmission Oral Microorganisms transmitted by the fecal-oral route usually referred to as enteric pathogens because they infect the gastrointestinal tract. Enteric pathogens characteristically stable in water and food. (and bacteria may be capable of growth) Waterborne pathogens those excreted or secreted by man or other animals. Water-based pathogens those whose natural environment is a water environment (or which are capable of growth in water). Legionella Pseudomonas aeruginosa 26

27 Routes of Transmission Oral Origin of enteric pathogens in the environment may result from many sources: Direct release of feces Sewage discharges Septic tanks Land application of sewage sludge or wastewater Solid waste disposal Transmission routes in the environment: Land runoff Leachate Contacting medium: Oceans, estuaries, rivers, lakes Groundwater Irrigation water Contact by: Shellfish Recreation Water supply Crops Aerosol Routes of Transmission Soil and fomites Ingestion of soil Mouth contact directly with fomites Indirect contamination of fingers from fomites Survival of microorganisms on fomites depends on: Humidity Suspending media Type of organism Temperature Type of surface Moisture at surface 27

28 Testing Procedures Three main categories of testing: Acute administration of one dose of the test chemical and waiting for a response (e.g., acute lethal test) Subacute (prolonged) chemical administered daily for up to 90 days. Chronic exposes the animal daily to a regular dose for most of the animal s lifetime. Responses: Teratogenicity study of the effect of physical, chemical, and infectious agents on the developing embryo and fetus. Mutagenicity concerned with the ability of a chemical to alter the genetic material of the cell. The insidious part is the effects will only appear in future generations. Carcinogenicity induction of malignant neoplasms by the interaction of an outside chemical on a cell. Testing Procedures Epidemiology study of human populations to establish correlations between environmental exposure of chemicals and specific health effects. 28

29 Types of Toxic Effects A wide variety of effects are looked for during a toxicological investigation. A few that are important for environmental exposure are: Allergic agents itching, rashes, sneezing (watery eyes). Asphyxiants cause displacement of oxygen and thus suffocation. Irritants cause pulmonary edema (fluid in the lungs) when inhaled at high concentrations and rashes when spilled onto the skin. Necrotic agents cause cell death. Cancer, mutations, and deformed embryos typically result from chronic exposure to low levels of carcinogens, mutagens and teratogens, respectively. Systemic poisons can have an adverse effect on the whole body when taken internally. Types of Toxic Effects Other effects: Synergism one contaminant enhances the effect of another Antagonism one contaminant reduces the effect of another 29