Mammalian Toxicology - University of Alabamarpitt.eng.ua.edu/Class/International urban water systems/Mammalian... · Mammalian Toxicology ENVE 569 Environmental Risk Assessment Introduction

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Mammalian Toxicology

ENVE 569Environmental 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 intothe 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.

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The study of the adverse effects of atoxicant 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

enzymesPrinciples of Toxicology: The Study of Poisons Elizabeth CasarezDepartment of Pharmacology and Toxicology, University of Arizona

What is a Poison?

All substances are poisons;there is none that is not a poison.

The right dosedifferentiates a poison and a remedy.

Paracelsus (1493-1541)

Principles of Toxicology: The Study of Poisons Elizabeth CasarezDepartment of Pharmacology and Toxicology, University of Arizona

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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


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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 nonePrinciples of Toxicology: The Study of Poisons Elizabeth CasarezDepartment of Pharmacology and Toxicology, University of Arizona

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.

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Dose-Response Relationship:As the dose of a toxicant increases, so does the response.

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3

4

0 1 DOSE

RESPONSE

0-1 NOAEL2-3 Linear Range4 Maximum Response


Example Dose-Response Relationship

1050940

530

325120

11005

No. of animals responding

Dose (mg/kg)

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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 LD50 (the lethal dose for 50% of the

animals) is a direct result of this curve. The LD50 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 LD50 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 LD50 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.

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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 LD50.

Dose-Response Relationship:As the dose of a toxicant increases, so does the response.

2

3

4

0 1 DOSE

RESPONSE

0-1 NOAEL2-3 Linear Range4 Maximum Response


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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.

LD50

• 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 LD50 can be generated from the curve

• Different toxicants can be compared: lowest dose is most potent


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LD50 Comparison

Chemical LD50 (mg/kg)Ethyl Alcohol 10,000Sodium Chloride 4,000Ferrous Sulfate 1,500Morphine Sulfate 900Strychnine Sulfate 150Nicotine 1Black Widow 0.55Curare 0.50Rattle Snake 0.24Dioxin (TCDD) 0.001Botulinum toxin 0.0001

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.

0.00001Guinea PigBotulinus

0.0006Guinea PigDioxin

0.045RatDioxin1RatNicotine100RatDDT

900RatMorphine sulfate

1,500RatFerrous sulfate

10,000MouseEthyl alcohol

LD50 (mg/kg)

AnimalAgent

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Scale of Toxicities

• Classifications are arbitrary!• A typical scale of toxicities:

Extremely toxic 50 mg/kg or lessModerately toxic 50 – 500 mg/kgSlightly toxic 0.5 – 5 g/kgRelatively 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.

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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 LD50 to the ED50 (effective dose for 50% of the animals).

Routes of Exposure

• Ingestion• Inhalation• Injection• Surface absorption

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Exposure: Pathways

• Routes and Sites of Exposure– Ingestion (Gastrointestinal Tract)– Inhalation (Lungs)– Dermal/Topical (Skin)– Injection

• intravenous, intramuscular, intraperitoneal

• Typical Effectiveness of Route of Exposureiv > inhale > ip > im > ingest > topical


Exposure: Duration

Acute < 24hr usually 1 exposureSubacute 1 month repeated dosesSubchronic 1-3mo repeated dosesChronic > 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


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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).

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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 lipophyliccompounds (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


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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


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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


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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 SalivaPrinciples of Toxicology: The Study of Poisons Elizabeth CasarezDepartment of Pharmacology and Toxicology, University of Arizona

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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 nonionizedform.

HAc ↔ H+ + Ac-

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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 receptorcauses 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 WithoutMetabolism

WithMetabolism

Ethanol 4 weeks 10mL/hr

Phenobarbital 5 months 8hrs

DDT infinity Days to weeks


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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.

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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 10-30 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


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Individual Susceptibility

• Age--old– 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

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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.

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(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

NoseSwimmingNagelaria

EyeWater, swimmingAcantamabea

RespiratoryWarmth, humidity, water coolers, air conditioners, potable water supplies

Legionnaire’s diseaseSoil, air, water, foodInanimate sources

SkinInfected animalsLeptospirosisWater (swimming)Urine

MouthDay-care centers, water, food

RotavirusStool → water, fomites, food, vomit

MouthFood, animal contactSalmonellosisStool → food, water

MouthWater, foodCholeraStool → water, foodFecal-oral

SkinBathroom floorsPapillomavirus, wartsFomites, direct contact

SkinLow socioeconomic level, tropics

Impetigo due to staph and/or strep infection

Direct contact, fomites

Nose, respiratory, skin

Hospitalization, surgeryNosocomial bacterial infections

Respiratory, direct contact, fomites

Skin squames

RespiratoryHousehold contact, carrierTuberculosisRespiratory system → droplet nucleic

RespiratoryClose contact, carrierStreptococcalRespiratory system → droplets

RespiratoryPneumonia casePlague (pneumonia)Respiratory system → air

RespiratoryDirect contactInfluenzaRespiratory system → air, fomites

Respiratory, eyeHousehold contactRhinovirusNasal discharges, fomitesRespiratory

Route of EntryFactorsExamplesRoute of TransmissionsRoute of Exit

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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


• 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

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• 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


• 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

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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


• 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

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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.

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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

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Mammalian Toxicology - University of Alabamarpitt.eng.ua.edu/Class/International urban water systems/Mammalian... · Mammalian Toxicology ENVE 569 Environmental Risk Assessment Introduction