Basic Concepts in Toxicology

Ref: Casarertt & Doull’s

Essentials of Toxicology
 Definition of Toxicology

Old Definition-
- the basic science of poisons
or the study of the effect of poisons on the function of
living systems

New Definition-
- the study of the adverse effects of chemical agents on
biological systems
so
Toxicology is the study of the adverse effects of xenobiotics on
living systems
 History and scope
• Toxicology assimilates knowledge and techniques from
biochemistry, biology, chemistry, genetics, mathematics,
medicine, pharmacology, physiology and physics

• Toxicology applies safety evaluation and risk

assessment to the disciplines
 WHAT TOXICOLOGISTS DO

- involved in the recognition, identification, and

quantitation of hazard
- develops standards and regulations to protect health and
the environment
- involved in safety assessment and use of data as a
basis for regulatory control of hazards

- determines risk associated with use of chemicals

AREAS OF TOXICOLOGY (FIELDS
OF SPECIALTY)
-Descriptive
-Environmental
-Mechanistic
-Regulatory
-Forensic
-Clinical
 General terms in toxicology
• Allergic reactions: antigen-antibody reaction.
• Idiosyncreatic reactions: genetically determined
abnormal reactivity of an individual to a chemical.
• Immediate versus Delayed toxicity
• Reversible versus Irreversible toxicity
• Local versus Systemic toxicity
• Tolerance: a state of decreased responsiveness to a
toxic effect of a chemical resulting from prior exposure to
that chemical or to a structurally related chemical.
The science of Toxicology helps people make
informed decisions and balance
RISKS vs. BENEFITS

The study AND Spinach.

found the
highest levels
of pesticide
residues in
peaches,
apples,
pears…….
 RISK AND SAFETY

RISK: the probability that harm will occur

under specified conditions

SAFETY: the probability that harm will not

occur under specified conditions
 RISK ASSESSMENT

- Hazard identification
- Dose Response Assessment
- Exposure Assessment
- Risk Characterization
 FACTORS CONSIDERED IN
DETERMINING ACCEPTABLE RISK

- benefits
- availability of substitutes
- anticipated public use
- employment considerations
- economic considerations
- effects on environmental quality
- conservation of natural resources
 MAJOR FACTORS THAT
INFLUENCE TOXICITY

-route of administration
-exposure : source, duration and frequency
-dose or concentration
 Routes of Exposure
• The route (site) of exposure is an
important determinant of the ultimate dose
—different routes may result in different
rates of absorption.
-intravenous -intradermal
-inhalation -topical
-intraperitoneally -subcutaneous
-intramuscular -oral

• The route of exposure may be important if

there are tissue-specific toxic responses.

• Toxic effects may be local or systemic

 Exposure
Sources of exposure to
chemicals
• Environmental, including
home and school
• Occupational
• Therapeutic
• Dietary
• Accidental
• Deliberate
 Exposure
• In order for a chemical to produce a biological effect, it
must first reach a target individual (exposure pathway).

• Then the chemical must reach a target site within the

body (toxicokinetics)

• Toxicity is a function of the effective dose (how much) of

a foreign chemical (xenobiotic) at its target site, integrated
over time (how long)

• Individual factors such as body weight will influence the

dose at the target site
 Time of Exposure
• How long an organism is
exposed to a chemical is
important
• Duration and frequency
contribute to dose. Both may alter
toxic effects.
– Acute Exposure = usually entails a
single or short term exposure < 24 h
– Chronic Exposures = multiple or
repeated exposures over time
(frequency) > 3 months
– Sub-acute Exposure = usually
entails between acute and chronic
exposure < 3-4 weeks
 Dose
THE KEY CONCEPT in Toxicology

Father of Modern Toxicology

Paracelsus—1564

• “All things are poisonous, only the dose makes it non-

poisonous.”
• Dose alone determines toxicity
• All chemicals—synthetic or natural—have toxic effects
 Dose

All Interactions between

chemicals and biological systems
follow a
Dose-Response Relationship
 Dose
Woman Dies after Water-drinking Contest: Water
Intoxication eyed in ‘Hold Your Wee for a Wii’ contest Death

SACRAMENTO, California—A
woman who competed in a radio
station’s contest to see how much
water she could drink without going to
the bathroom died of water
intoxication, the coroner’s office said
Saturday.

Updated: 10:24 p.m. ET Jan 13, 2007

 Dose-Response Relationship
• A key concept in Toxicology is the quantitative
relationship between the concentration of a xenobiotic
in the body and the magnitude of the biological effect it
produces.

• The magnitude of the effect of a xenobiotic is usually a

function of the amount of xenobiotic to which a person is
exposed (i.e., “The Dose Makes the Poison”).

• In any given population, there will be a range of

sensitivities to a xenobiotic. It is extremely useful to know
what is the average sensitivity of a population to a
xenobiotic, and what the average dose required to elicit a
toxic response will be.
 Dose
• The magnitude of the toxic response is
proportional to the concentration of the
chemical at the target site
• The concentration of a chemical at the
target site is proportional to the dose.
• Four important processes control the
amount of a chemical that reaches the
target site.
– Absorption
– Tissue distribution
– Metabolism
– Excretion
 Dose
Determines Whether a Chemical Will Be
Beneficial or Poisonous
Beneficial Dose Toxic Dose

Aspirin 300 – 1,000 mg 1,000 – 30,000 mg

Vitamin A 5000 units/day 50,000 units/day
Paracetamol 4gm/day 10gm – more/day
 Spectrum of toxic dose
• A poison can be defined as any agent capable of
producing a deleterious response in a biological system.

• Measures of acute lethality such as LD50 may not reflect

accurately the full spectrum of toxicity.
• Some chemicals with low acute toxicity may have
carcinogenic or teratogenic effects at doses that produce
no evidence of acute toxicity.
 Spectrum of undesired effects
• The spectrum of undesired effects of chemicals is broad

• Each drug produces a number of effects, but usually only

one effect is associated with the primary objective of the
therapy; all other effects are called as undesirable
effects or side effects.
• However, some side effects may be desired for another
therapeutic indication. Some side effects of drugs are
always deleterious to the well being of human. They are
referred as the adverse or toxic effects.
 Interaction of chemicals
• Additive effects: When the combined effect of two
chemicals is equal to the sum of the effects of each
agent given alone (e.g. 2 + 3 = 5). The effects most
commonly observed when two chemicals are given
together.

• Synergistic effect: When the combined effects of two

chemicals are much greater than the sum of the effects
of each agents given alone (e.g. 2 + 2 = 20). CCl4 and
ethanol both are hepatotoxic compounds, but together
they show such effect on liver.
 Interaction of chemicals
• Potentiation: When one substance does
not have a toxic effect on a certain organ
or system but when added to another
chemical makes that chemical much more
toxic (e.g. 0 + 2 = 10). Isopropanol is not
hepatotoxic, it shows such effect when
added with CCl4.
 Interaction of chemicals
• Antagonism: When two chemicals
administered together interfere with each
other's actions (e.g. 4 + 6 = 8, 4 + 4 = 0).
 Antagonism
• Functional antagonism: When two
chemicals counterbalance each other by
producing opposite effects on the same
physiologic function.
• The marked fall in BP during severe
barbiturate intoxication can be
antagonized effectively by the IV
administration of a vasopressor agent
such as norepenephrine or metaraminol.
 Antagonism
• Chemical antagonism: A chemical reaction
between two compounds that produces a
less toxic product.
• Chelators of metal ions decrease metal
toxicity and antitoxins antagonize the
actions of various animal toxins.
 Antagonism
• Dispositional antagonism: When the absorption,
biotransformation, distribution, or excretion of a
chemical is altered so that the concentration
and/or duration of the chemical at the target
organ are diminished.
• The prevention of absorption of a toxicant by
epecac or charcoal, increased activity of
metabolizing enzymes with enzyme inducers,
and the increased excretion of a chemical
caused by the administration of a diuretic.
 Antagonism
• Receptor antagonism: When two
chemicals that bind to the same receptor
produce less of an effect when given
together than the addition of their separate
effects or when one chemical antagonizes
the effect of the second chemical.
Receptor antagonists often are called
blockers.
 Dose-response
• Individual, or Graded Dose-response:
Dose related increase in the severity of the
response.

• Quantal Dose-response:
At any given dose, an individual in the
population is classified as either a “responder” or
a “non-responder”.
 Dose-Response Curves
“The Dose Makes the Poison”
Maximum Response Maximum Response
1.0 1.0
Rate
Approx.
0.5 Linear
0.5 Range
Threshold
EC50
EC50
0 0
0 20 40 60
80 100 0.1 1.0 10 100
Concentration Concentration
Arithmetic Scale Logarithmic Scale
 Dose-Response Relationship
“The Dose Makes the Poison”
Effective Dose Lethal Dose
100 100
Animals Sleeping (%)

Animals Killed (%)

80 80

60 60
ED50 40
LD50
40
20 20

1 2 3 5 7 10 10 20 30 50 100
Phenobarbital (mg/kg) Log Scale
 What is 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
 How is a Response?
The degree and spectra of responses
depend upon the dose and the organism–
need to describe exposure conditions with
description of dose
• Change from normal state
– could be on the molecular, cellular, organ, or
organism level--the symptoms
• Local vs. Systemic
• Reversible vs. Irreversible
• Immediate vs. Delayed
• Graded vs. Quantal
– degrees of the same damage vs. all or none
 Importance

• Studying dose response, and

developing dose response models, is
central to determining "safe" and
"hazardous" levels and dosages for
drugs, potential pollutants, and other
substances that humans are exposed
to.
 Dose-response curve
• A dose-response curve
defines the relationship
between dose and

 
response based on the
following assumptions:
                            
1) response increases as
dose increases
2) there is a threshold
dose- a dose below which
there is no effect.
 Dose-Response Graphs

• There are two types:

1. Frequency-Response
Graphs

2. Cumulative-Response
Graphs
 Frequency-Response Graphs
• Dose response to a chemical will likely vary
from one species to the next. Likewise,
response will vary within a group of test
organisms of the same species.
• Intraspecies variation typically follows a normal
Gaussian distribution (Figures 1.1a and 1.1b)
Figure 1.1 (a) & (b)
 Population Dose-Response
Many
Number of Individuals

Resistant Sensitive
Individuals Majority of Individuals
Individuals

Average Effect
Minimal Maximal
Effect Effect

Few

Mild Response to SAME dose Extreme

 Some chemicals have both therapeutic
and toxic effects: Vitamin A

Too low:
Too high: Anorexia,
Blindness,
Adverse response

anemia, nose bleeds,

dry skin,
muscle and joint pain
increased
infections
Threshold

Dose
 Cumulative-Response Graphs

• Typically, cumulative response curves are

used instead of frequency response to
depict the summation of several
frequency response curves for a range of
doses (see Figure 1.1c)
• In addition, the logarithm of the dose is
usually used to yield a more linear plot
(see Figure 1.1d)
Figure 1.1 (c)

Figure 1.1 (d)

Regions of Dose-Response Curves
• No-observable-effect levels (NOELs) –
No Effect
• Highest NOEL = Threshold Dose
• 100% Effect Region – Maximum efficacy
 No Observed Effect Level
(NOEL)
• The NOEL is the greatest
concentration or amount of
an agent, found by study or
observation, that causes
no detectable, usually
adverse alteration of
morphology, functional
capacity, growth,
development or lifespan of
the target.
Dose-Response Relationship for a Typical
Chemical
 Potency vs. Efficacy
• Potency – the range of doses over which a
chemical produces increasing responses
• Efficacy – the limit of the dose-response
relationship on the response axis
Response

Efficacy

Potency

Dose
 Using Dose-Response Data
• Lethal Dose (LD) - when the response is death
• Effective Dose (ED) - when the response is a
desirable effect
• Toxic Dose (TD) - when the response is an
undesirable toxicity other than death
• Sentinel Dose (SD) – minimal adverse effects
– Ex. - minor irritation, headache, drowsiness (may serve
as a warning indicator for more serious effects at
greater doses)
• The cumulative response curves reveals doses
that affect a given percentage of the exposed
population.
– Examples: LD50, LD10, LD01
 Measures of Toxicity:
The Median Lethal Dose

LD50
The amount (dose) of a chemical which produces
death in 50% of a population of test animals to
which it is administered by any of a variety of
methods

mg/kg
Normally expressed as milligrams of substance
per kilogram of animal body weight
 Measures of Toxicity:
The Median Lethal Concentration
LC50
The concentration of a chemical in an environment
(generally air or water) which produces death in
50% of an exposed population of test animals in
a specified time frame

mg/L
Normally expressed as milligrams of substance
per liter of air or water (or as ppm)
 Lethal Dose (LD)
• LD50 - is the calculated dose of a substance that is
expected to kill 50 percent of a defined experimental
animal population, as determined from the exposure
to the substance by any route other than inhalation.
- The more potent or toxic the chemical, the lower the
LD50 and the smaller the dose needed to cause death.

• LC50 (Lethal Concentration, 50% kill) – refers to the

concentration of inhaled material that kills 50% of the
treated population. Expressed as either parts per
million (ppm) or mg/m3 of inhaled material.
 The LD50
LD50 Dose-Response Results
 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--10-30 fold variation w/in a population
• 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
 Acute LD50 Comparisons

Agent LD50 (mg/kg)

• Ethyl alcohol (ethanol) 10,000
• Sodium chloride (salt) 4,000
• Caffeine 75
• Nicotine 1
• Botulinum Toxin 0.00001
 Margin of Safety
• Sometimes warning signs can be used to
establish a margin of safety for a substance by
comparing SD, TD, and LD curves (See Figure
1.5)
• Typically the margin of safety is calculated by
dividing the higher lethal dose (e.g., LD50) by a
lower innocuous dose (e.g., ED50) - Therapeutic
Index (LD50/ED50)
• The higher the margin of safety, the safer it is to
use.
 Therapeutic Indices
The therapeutic index (TI) is defined as the ratio of the dose required to produce a
toxic response. The TI of a drug expressed as the ratio of the lethal or toxic dose
to the therapeutic dose:

TD50 LD50
Therapeutic Index = =
ED50 ED50

Thus, the larger the ratio, the greater the relative safety.

Limitation of TI
The median doses indicate nothing about the slopes of the dose-
response curves for therapeutic and toxic effects.
Does not represent the true data, only animal data.
 Therapeutic and Toxic Effects

100

Therapeutic
80 Toxic

60
% Responding

40

20 ED99
TD1 TD50
ED50
0
70 80 90100 200 300
Dose
In this dose-effect plot, the therapeutic and toxic
effects are plotted.
This study illustrates an important principle when a
drug is administered to an organism - there is no
single dose-effect curve relationship that can
adequately characterize the full spectrum of activity of
a drug. All drugs produce at least 2 effects and
therefore have at least 2 quantal dose-response
curves - one for the therapeutic effect and one or
more for the toxic effect.
The safety of a drug depends on the degree of
separation between the doses producing a
therapeutic effect and the doses which produce side
effects, both of which can be characterized by quantal
dose-effect curves.
Therapeutic indices are calculated from dose-
effect curves in order to quantify the relative
safety of a drug.
Using these curves we can calculate a
therapeutic ratio using the TD50 and ED50. The
TD50/ED50 ratio for the drug shown on the
previous graph is about 2.5, which means that
about 2.5 times as much drug will cause a toxic
effect in half the subjects as is needed to
produce a therapeutic effect in the same
proportion of subjects. However this ratio of
toxic to therapeutic dose may not hold across
the entire dose range if the dose-effect curves
are not parallel.
 Margins of Safety and Exposure
Way to overcome
One way to overcome this deficiency is to use the ED99 for the desired effect and the
LD1 for the undesired effect. These parameters are used to calculate the margin of
safety:
LD1
Margin of safety =
ED99

For non-drug chemicals, the term margin of safety is an indicator of the magnitude of
the difference between an estimated “exposed dose” to a human population and the
NOAEL (no observable adverse effect level) determined in experimental animals.

TD1 - ED99
Standard Safety Margin = X 100
ED99
 Limitation of Dose-Response
• Single values such as LD50 provide no
information about the slope of the dose-response
curve or toxicity at low doses (e.g., LD10 or TD10)
(Fig. 1.6)
• Information most often generated in these studies
is acute toxicity and may not reflect chronic
toxicity dose-response relationships.
– Benzene vs. Toluene – Acute exposure…toluene
more potent CNS depressant; Chronic exposure…
benzene is carcinogenic, toluene is not
• Does animal data mimic human response? Test
species more sensitive or less sensitive than
humans? (See Table 1.6)
 Species Differences
• Basal metabolic rates
• Anatomy and Structure
• Physiology and cellular biochemistry
• Distribution of chemicals to certain tissues
• Metabolism, bioactiviation, and detoxification
of the chemical
• The cellular, tissue, or organ response to
actions of the chemical at the biochemical,
cellular, tissue, or organ level
 Variables Influencing Dose-Response
Curves
• Route of Exposure
– How a substance enters the body determines how
much of it enters (rate of absorption) and which organs
are exposed to the largest concentration of the
substance.
– These variables can have an effect on the rates of
metabolism and excretion
• The amount of chemical that is orally toxic may be more toxic,
or less toxic, when applied to the skin (First-Pass Effect)
– Ingestion - not only eating and drinking - substances
inhaled can be trapped by mucous in the respiratory
tract and brought back up by the Mucociliary Escalator
and swallowed.
Variables Influencing Dose-Response
Curves
– Skin Absorption - about 2m2 or 20ft2 of surface area
- absorption dependent on polarity of the
compound, hydration of the skin, number of portals
(hairy or hairless), and physical condition of the skin
– Inhalation - about 70m2 or 700ft2 of surface area for
gas exchange, voluntary response (we must
breathe), effective transfer to the blood by the
alveolus (thin membrane in contact with capillaries)
• Breathing Rate - Average 15 breaths/min, 500-750
ml/breath at rest - Moderate Work (1450 ml/breath), Heavy
Work (2150 ml/breath)
– Injection - IV, IM, IP
 Variables Influencing Dose-Response
Curves
• Sex (Gender Characteristics)
– On average, females have larger percent of fat in their
total body weight than men
– Women also have different susceptibilities to
reproduction-system disorders or teratogenic effects
– Capacity for metabolism (Ex. male rodents more P450)
• Age
– Young children
• Lower total body weight
• Differences in metabolism and elimination (liver not fully
developed)
• Higher respiration rates
• Different organ susceptibilities (sensitive to CNS depressants)
 Variables Influencing Dose-Response
Curves
• Age (continued)
– Older People
• Lower total body weight
• Slower metabolism
• Deterioration of liver
• Differences in musculature
 Variables Influencing Dose-Response
Curves
• Effects of Chemical Interaction
– Additive
• Chemicals with the same toxicity combine to give an additive
effect
– Synergism
• Chemicals with the same toxicity (i.e., same target organ)
that, when combined, cause a greater than additive effect
– Potentiation
• When a chemical that does not produce a specific toxicity
nevertheless increases the toxicity caused by another
chemical
– Antagonism
• Chemical that, when combined, diminish each other’s
measured effect (i.e., one has an antidote effect)
 Variables Influencing Dose-Response
Curves

• Modes of Chemical Interaction

• Four ways in which chemical interactions can
be increased or decreased
– Functional: two chemicals affect the same
physiological function (e.g., additive, synergism,
antagonism)
– Chemical: chemical interaction between the two
compounds affect the toxicity of one of the chemicals
– Dispositional: absorption, metabolism, distribution, or
excretion of one chemical is altered by another (e.g.,
methanol and ethanol)
 Variables Influencing Dose-Response
Curves

• Modes of Chemical Interaction (continued)

– Receptor-mediated: when two chemicals bind to the
same tissue receptor, the second chemical, which differs
in activity, competes for the receptor and thereby alters
the effect produced by the first chemical (e.g., oxygen
and carbon monoxide - CO has 200 times the affinity for
hemoglobin as O2)
• Genetic Variation
– Substances that normally don’t cause problems can be
toxic without certain enzymes present, and vice versa
• Species Variation
• Health Status
 Factors Influencing the Toxicity of a
Chemical
• Physical and chemical properties:
– Chemical composition (e.g., salt, free
radical, anion)
– Physical characteristics (e.g., state of matter,
particle size)
– Chemical properties (e.g., volatility,
solubility, polarity)
– Presence of impurities (e.g., may alter
absorption)
– Stability in mixtures
 Factors Influencing the Toxicity of a
Chemical
• Exposure Conditions (see Fig. 2-1 Casarett &
Doull’s)
– Concentration
– Amount or dose
– Type of exposure (e.g., skin, oral, inhalation, injection)
– Duration (acute or chronic)
• Environmental conditions
– Media (i.e., air, water, soil, food)
– Presence of additional chemicals (e.g., synergism)
– Temperature and Pressure (volatility, ventilation)
 Assumptions in deriving the Dose-
response relationship
• The response is due to the chemical
administered, a cause and effect relationship
• The magnitude of the response is in fact related
to the dose. This assumes that there is a
molecular target site (or sites) with which the
chemical interacts to initiate the response, which
is related to the concentration of the agent at the
target site.
• There exists both a quantifiable method of
measuring and a precise means of expressing
the toxicity.
Organs Respond to Chemicals in
Various Ways

Desired Effects
Nutritive
Blood Organs Therapeutic

Undesired Effects
Toxic
 Toxicological evaluation in an
animal model
• With a new substance, the customary
starting point in toxicological evaluation
utilizes lethality as an index.
• Lethality provides an unequivocal measure
of comparison among many substances
whose mechanisms and sites of action are
different.
 Toxicological evaluation in an
animal model
• Detailed observation of the intact animal
while it is alive
• Histologic examination of major tissues
and organs for abnormalities after death
 Some Chemicals Are Transformed by
the Body (Metabolized) to Aid Excretion

Liver and other Organs

Detoxication

Less Toxic Metabolic Product

Kidney Liver Lung

Urine Feces / Bile Expired Air

Some Chemicals are Partially
Converted to Products that are
More Toxic than the Parent
Substance
Liver and other Organs
Activation

More Toxic Metabolic Product

 Toxicological Paradigm
Toxicokinetics Toxicodynamics
What We do to the Chemical What the Chemical Does to Us

Biologically Early Altered

Internal
Exposure Effective Biological Structure & Disease
Dose
Dose Effect Function

Absorption Susceptibility and

Distribution Modifying Factors
Metabolism (Genetics and Nutritional Status)
Excretion
Storage
 Biotransformation
• Metabolism
• major mechanism for
terminating the biological
activity of chemicals
• frequently the single most Liver

important determinant of the

duration and intensity of the
pharmacological response to a
chemical
The LIVER is the
Biotransformation occurs in the primary site of
Liver, kidney, lung, gastrointestinal metabolism
track, and other organs
Pharmacogenetics of Metabolism
25
Fast Metabolizers
Response Frequency

20
Harmful Side Effects
(# of individuals)

15
Slow Metabolizers
10
(elevated plasma levels)

0
0 2 4 6 8 10 12
Drug Concentration (g/mL)
Plasma levels 6 hrs after oral dose
 Typical Population
The emerging field of
“Pharmacogenomics” or
“Toxicogenomics” offers
the potential to identify and
protect subsets of people
predisposed to toxicity from
chemicals or drugs
Id
en Less
tif
Identify People with “normal” responses y
Sensitive
ch dif peo
e fe p
se mic ren le w
ns al/ t ith
iti dr
vi ug
ty

More
Sensitive
Tools of Modern Molecular Toxicology:
Genomics and Proteomics

R
+TOF MS: 24 MCA scans from Myo_tryptic.wiff Max. 5191.0 counts.

-COOH
1360.7892
5191

NH2
5000

4500 1606.8892

4000

K -COOH
3500

NH2 In te n s ity , c o u n t s
3000

2500
1938.0629

2000 1815.9397

K -COOH
1378.8696
1500

NH2
1000 2316.3092
1506.9692
1271.6925 1661.8925 1886.0672
500 1001.4584 1983.1071
1589.8688
1343.7703 1798.9216
1071.6147 1959.0339 2298.2643 2505.3460 2602.5045
0
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
m/z, amu