Tox 300
Introduction to Toxicology
- Xenobiotics are anything that’s foreign to the body. Any biological active chemical that’s
foreign to the body. It’s an exogenous chemical
- Xenobiotics can be industrial chemicals, pharmaceuticals, plant/animal toxins; essentially
any exogenous agent that is foreign to the body.
- In the 1970’s, the dilution paradigm was replaced by the boomerang paradigm
- Dilution paradigm: dilution is the solution to pollution
- In the 1970’s with the publication of Rachel Carson’s silent spring that warned us about
the insectides. The boomerang paradigm was implemented
- DDT & Methylmercury cause toxicity to both wildlife and humans and are rather
insidious as they increase in concentration or bio magnify through food webs.
Modern Toxicology
- More rigorous toxicity testing of pharmaceuticals, pesticides and other industrial
chemicals (1960s) – we have invented over 100,000 chemicals and vast majority of these
chemicals haven’t been tested properly for their toxic effects
- Technological advances in analytical chemistry – we can easily measure in parts per
billion in a sample.
- Increasing importance of social sciences – involved in areas such as risk communication
and risk perception.
- Legislation to clean up contaminated sites
- Technological advances in molecular biology
Toxicokinetic & Toxicodynamic
Toxicokinetics
- What the body does the xenobiotic
- The process involved in toxicokinetics are absorption, distribution, metabolism, and
excretion of a xenobiotic
- Toxicokinetics determines the delivery or dose of a xenobiotic at its site of action
Toxicodynamic
- What the xenobiotic does to the body
- Toxicodynamic is the effects of the xenobiotic on cellular and physiological process
- Toxicodynamics determines the response to the xenobiotic
Absorption
- Toxicokinetics: determination of the time course of disposition (ADME) of xenobiotics
in the body
- It determines the concentration of xenobiotic at its sites of action, and thus is linked to the
intensity of biological effect
- The exposure routes that absorption relevant to are ingestion through the gastrointestinal
tract, inhalation through the lungs, dermal through the skin and different injections
Absorption
- Animal cell membranes are phospholipid bilayers
- Because it is a lipid membrane, the lipophilicity of a xenobiotic is the most important
factor allowing it to diffuse across cell membranes. Molecular size is also important
- Passive transport (Simple diffusion): chemical follows a concentration gradient across
cell membrane, this is termed transcellular diffusion. The key is the chemical must be
lipophilic, must be lipid soluble, no charge on it.
, - Filtration: passive transport through cell junctions due to pressure gradient, mainly due
to blood pressure this pushes small xenobiotic molecules through the cell, it is also
termed paracellular transport
- Facilitated diffusion: also passive transport following a concentration gradient, but
requires a transporter to assist movement across membrane
- Active transport: movement against concentration gradient, requires ATP
Absorption across cell membranes
1. Passive transport
- Most common absorption pathway for all xenobiotics
- Simply follows its concentration gradient across membrane. If there’s a higher
concentration on one side of the cell membrane than the other and if that chemical is lipid
soluble it will diffuse across the membrane
a. Organic lipophilic chemicals: diffusion across lipid membrane (transcellular
diffusion)
- Lipophilicity of chemicals is measures using the octanol: water partition coefficient
(KOW)
- High log KOW value (>4) indicates significant potential for bioaccumulation and toxicity.
b. Weak Organic Acid Bases: only the non-ionized form of the xenobiotic can
passively diffuse across cell membranes
- such chemicals exist in both ionized and non-ionized forms in solution, and the relative
proportion of ionized vs. non-ionized form depends on the pKa of the xenobiotic and the
pH solution
- The ionized form of weak organic acids or bases usually have low lipid solubility and
does not cross readily through the lipid membrane
- Non-ionized form is more lipid soluble and diffuses across membranes at a rate that is
proportional to its lipid solubulity.
2. Filtration
- Xenobiotic moves with water through gap junctions between cells due to pressure
gradient
- Most cells have gaps of 4nm, most blood vessel are 2nm, glomerulus 70nm, the CNS
have tight junctions they have no gaps and serve as a protective function in the brain to
prevent xenobiotics from going in.
3. Facilitated diffusion
- Important for nutrients and electrolytes; certain xenobiotics can compete for these
systems
- Major families are organic anion transports and organic cation transporters
- Important for excretion
4. Active transport
- All are ATP-binding cassette (ABC proteins); major families are the multi-drug
resistance proteins and breast cancer resistance protein
- Names originated from the high expression in certain cancer cells, severely limiting
chemotherapeutic treatments
- Referred to as lipophilic vacuum cleaners
Octanol: Water Partition Coefficient
- Log KOW is a standard method to determine lipophilicity of xenobiotics
, - Log KOW values >4 indicate high potential for accumulation and toxicity, e.g.,
organochlorine pesticides (DDT)
Weak Organic Acid & Bases
- The Henderson-Hasselbach equation can be used to determine the ratio of non-ionized vs.
ionized of the drug
- Log ([pronated]/ [non-protonated]) = pKa – pH
Routes of Exposure
GI absorption
- Very important route of absorption for many xenobiotics
- Most absorption occurs in small intestine due to high surface area
Pulmonary absorption
- Important for gases, vapors and particulates
Dermal
- Skin of mammals is essentially a non-permeable barrier to most chemicals, unless
chemical is highly lipophilic or skin is damaged
Distribution
- Different locations in body where a xenobiotic is distributed are referred to theoretically
as compartments
- Initial absorption to central compartment is followed by distribution to peripheral
compartments
- Thus, after absorption and entry into systemic circulation there is an immediate rapid
distribution throughout the body, especially to well-perfused tissues
Factors influencing distribution
1. Blood flow: depending on tissue, can range from 3-30% of cardiac output
2. Physiochemical properties of xenobiotic: lipid solubility, pKa, molecular size
3. Binding of xenobiotic to plasma proteins and cellular binding proteins
4. Barriers to distribution
Plasma Protein Binding
- Albumin is the most abundant; xenobiotics have varying affinities
- Free: bound equilibrium in blood plasma due to reversible binding
- Key point: only free xenobiotic can diffuse out of bloodstream into tissues because
plasma proteins are huge and cannot cross capillary walls
- Consider dynamic nature – as free xenobiotic diffuses from blood to tissue; more is
released from plasma proteins until tissue sites become saturated. Also, as free xenobiotic
is excreted, more is released from plasma proteins
Tissue Protein Binding
- Liver and kidney have high binding capacity for certain xenobiotics
- Adipose tissue is an important storage depot for highly lipophilic xenobiotics (e.g.,
persistent organic pollutants)
- Consider lactation; breast milk is high in fat and can accumulate lipophilic xenobiotics,
where they can become a route of exposure to neonate
- Bone binds certain xenobiotics (e.g, heavy metals such as lead)
Barriers to Distribution
Blood-Brain Barrier
- Major barrier to many xenobiotics because of tightly joined endothelial cells that
surround the CNS and active transporters for removal (MDR, MRP, BCRP proteins)
, - Not fully developed in embryo and at birth, hence important toxicological implications as
these protective systems between cells and active transporters are not fully operational
Placental Barrier
- Must assume that any xenobiotic entering maternal circulation is capable of crossing
placenta unless proven otherwise
- Xenobiotics are tested extensively for their ability to cross placenta and cause teratogenic
effects in offspring
Volume of Distribution (VD)
- The apparent fluid volume in which a xenobiotic appears to be dissolved (how widely a
xenobiotic is distributed throughout the body)
- VD is a proportionality constant used to compare distribution of xenobiotics, especially
pharmaceuticals
- VD = total xenobiotic dose (mg) / plasma xenobiotic conc (mg/L)
- High VD means extensive distribution of xenobiotic and high affinity for tissues
- Low VD means xenobiotic is restricted mainly to blood plasma, mainly due to high
plasma protein binding
Metabolism
- Bio transformation: the enzyme catalyzed conversion of one xenobiotic into another
- Most important determinant of the duration of action of xenobiotics in the body
- Detoxification: biotransformation results in a less toxic metabolite
- Bioactivation: biotransformation results in a more toxic metabolite
- Biotransformation of xenobiotics to more water-soluble metabolites is essential to
terminate their biological activity and eliminate them from the body
- Biotransformation enzymes convert lipophilic xenobiotics into highly water-soluble
metabolites that are easily excreted from the body
Bio transformation Reactions
- Phase 1: biotransformation enzymes modify the xenobiotic molecule mainly by
oxidation – addition of an OH group to the xenobiotic
- Phase 2: synthetic reactions that conjugate the xenobiotic with a highly polar endogenous
compound in the cell (e.g, carbohydrate, sulphate, or acetate)
- a xenobiotic is often bio transformed sequentially through both Phase 1 & Phase 2
reactions
- the liver is the most important site of xenobiotic bio transformation
Phase 1 biotransformation
- Cytochrome P450-dependent monooxygenases are the major Phase 1 oxidative
enzymes
- Reactions usually involve adding or exposing a polar function group (-OH, -COOH, -
NH2) to lipophilic xenobiotics molecule
- CYP enzyme are located on the smooth ER
- Monooxygenase indicates that they catalyze the insertion of an oxygen atom into the
xenobiotic molecule, for example the simple hydroxylation reaction: Xenobiotic (R-H) +
O2 + NADPH = Metabolite (R-OH) + H2O + NADP+
- CYPS are not only involved in xenobiotic biotransformation’s but also in a wide variety
of catabolic and anabolic reactions involving endogenous compounds (steroid hormones)
- >100 different CYP enzymes have been identified: CYP1A2, CYP2E1, CYP3A4 based
on DNA sequence similarity of genes coding for enzymes
Introduction to Toxicology
- Xenobiotics are anything that’s foreign to the body. Any biological active chemical that’s
foreign to the body. It’s an exogenous chemical
- Xenobiotics can be industrial chemicals, pharmaceuticals, plant/animal toxins; essentially
any exogenous agent that is foreign to the body.
- In the 1970’s, the dilution paradigm was replaced by the boomerang paradigm
- Dilution paradigm: dilution is the solution to pollution
- In the 1970’s with the publication of Rachel Carson’s silent spring that warned us about
the insectides. The boomerang paradigm was implemented
- DDT & Methylmercury cause toxicity to both wildlife and humans and are rather
insidious as they increase in concentration or bio magnify through food webs.
Modern Toxicology
- More rigorous toxicity testing of pharmaceuticals, pesticides and other industrial
chemicals (1960s) – we have invented over 100,000 chemicals and vast majority of these
chemicals haven’t been tested properly for their toxic effects
- Technological advances in analytical chemistry – we can easily measure in parts per
billion in a sample.
- Increasing importance of social sciences – involved in areas such as risk communication
and risk perception.
- Legislation to clean up contaminated sites
- Technological advances in molecular biology
Toxicokinetic & Toxicodynamic
Toxicokinetics
- What the body does the xenobiotic
- The process involved in toxicokinetics are absorption, distribution, metabolism, and
excretion of a xenobiotic
- Toxicokinetics determines the delivery or dose of a xenobiotic at its site of action
Toxicodynamic
- What the xenobiotic does to the body
- Toxicodynamic is the effects of the xenobiotic on cellular and physiological process
- Toxicodynamics determines the response to the xenobiotic
Absorption
- Toxicokinetics: determination of the time course of disposition (ADME) of xenobiotics
in the body
- It determines the concentration of xenobiotic at its sites of action, and thus is linked to the
intensity of biological effect
- The exposure routes that absorption relevant to are ingestion through the gastrointestinal
tract, inhalation through the lungs, dermal through the skin and different injections
Absorption
- Animal cell membranes are phospholipid bilayers
- Because it is a lipid membrane, the lipophilicity of a xenobiotic is the most important
factor allowing it to diffuse across cell membranes. Molecular size is also important
- Passive transport (Simple diffusion): chemical follows a concentration gradient across
cell membrane, this is termed transcellular diffusion. The key is the chemical must be
lipophilic, must be lipid soluble, no charge on it.
, - Filtration: passive transport through cell junctions due to pressure gradient, mainly due
to blood pressure this pushes small xenobiotic molecules through the cell, it is also
termed paracellular transport
- Facilitated diffusion: also passive transport following a concentration gradient, but
requires a transporter to assist movement across membrane
- Active transport: movement against concentration gradient, requires ATP
Absorption across cell membranes
1. Passive transport
- Most common absorption pathway for all xenobiotics
- Simply follows its concentration gradient across membrane. If there’s a higher
concentration on one side of the cell membrane than the other and if that chemical is lipid
soluble it will diffuse across the membrane
a. Organic lipophilic chemicals: diffusion across lipid membrane (transcellular
diffusion)
- Lipophilicity of chemicals is measures using the octanol: water partition coefficient
(KOW)
- High log KOW value (>4) indicates significant potential for bioaccumulation and toxicity.
b. Weak Organic Acid Bases: only the non-ionized form of the xenobiotic can
passively diffuse across cell membranes
- such chemicals exist in both ionized and non-ionized forms in solution, and the relative
proportion of ionized vs. non-ionized form depends on the pKa of the xenobiotic and the
pH solution
- The ionized form of weak organic acids or bases usually have low lipid solubility and
does not cross readily through the lipid membrane
- Non-ionized form is more lipid soluble and diffuses across membranes at a rate that is
proportional to its lipid solubulity.
2. Filtration
- Xenobiotic moves with water through gap junctions between cells due to pressure
gradient
- Most cells have gaps of 4nm, most blood vessel are 2nm, glomerulus 70nm, the CNS
have tight junctions they have no gaps and serve as a protective function in the brain to
prevent xenobiotics from going in.
3. Facilitated diffusion
- Important for nutrients and electrolytes; certain xenobiotics can compete for these
systems
- Major families are organic anion transports and organic cation transporters
- Important for excretion
4. Active transport
- All are ATP-binding cassette (ABC proteins); major families are the multi-drug
resistance proteins and breast cancer resistance protein
- Names originated from the high expression in certain cancer cells, severely limiting
chemotherapeutic treatments
- Referred to as lipophilic vacuum cleaners
Octanol: Water Partition Coefficient
- Log KOW is a standard method to determine lipophilicity of xenobiotics
, - Log KOW values >4 indicate high potential for accumulation and toxicity, e.g.,
organochlorine pesticides (DDT)
Weak Organic Acid & Bases
- The Henderson-Hasselbach equation can be used to determine the ratio of non-ionized vs.
ionized of the drug
- Log ([pronated]/ [non-protonated]) = pKa – pH
Routes of Exposure
GI absorption
- Very important route of absorption for many xenobiotics
- Most absorption occurs in small intestine due to high surface area
Pulmonary absorption
- Important for gases, vapors and particulates
Dermal
- Skin of mammals is essentially a non-permeable barrier to most chemicals, unless
chemical is highly lipophilic or skin is damaged
Distribution
- Different locations in body where a xenobiotic is distributed are referred to theoretically
as compartments
- Initial absorption to central compartment is followed by distribution to peripheral
compartments
- Thus, after absorption and entry into systemic circulation there is an immediate rapid
distribution throughout the body, especially to well-perfused tissues
Factors influencing distribution
1. Blood flow: depending on tissue, can range from 3-30% of cardiac output
2. Physiochemical properties of xenobiotic: lipid solubility, pKa, molecular size
3. Binding of xenobiotic to plasma proteins and cellular binding proteins
4. Barriers to distribution
Plasma Protein Binding
- Albumin is the most abundant; xenobiotics have varying affinities
- Free: bound equilibrium in blood plasma due to reversible binding
- Key point: only free xenobiotic can diffuse out of bloodstream into tissues because
plasma proteins are huge and cannot cross capillary walls
- Consider dynamic nature – as free xenobiotic diffuses from blood to tissue; more is
released from plasma proteins until tissue sites become saturated. Also, as free xenobiotic
is excreted, more is released from plasma proteins
Tissue Protein Binding
- Liver and kidney have high binding capacity for certain xenobiotics
- Adipose tissue is an important storage depot for highly lipophilic xenobiotics (e.g.,
persistent organic pollutants)
- Consider lactation; breast milk is high in fat and can accumulate lipophilic xenobiotics,
where they can become a route of exposure to neonate
- Bone binds certain xenobiotics (e.g, heavy metals such as lead)
Barriers to Distribution
Blood-Brain Barrier
- Major barrier to many xenobiotics because of tightly joined endothelial cells that
surround the CNS and active transporters for removal (MDR, MRP, BCRP proteins)
, - Not fully developed in embryo and at birth, hence important toxicological implications as
these protective systems between cells and active transporters are not fully operational
Placental Barrier
- Must assume that any xenobiotic entering maternal circulation is capable of crossing
placenta unless proven otherwise
- Xenobiotics are tested extensively for their ability to cross placenta and cause teratogenic
effects in offspring
Volume of Distribution (VD)
- The apparent fluid volume in which a xenobiotic appears to be dissolved (how widely a
xenobiotic is distributed throughout the body)
- VD is a proportionality constant used to compare distribution of xenobiotics, especially
pharmaceuticals
- VD = total xenobiotic dose (mg) / plasma xenobiotic conc (mg/L)
- High VD means extensive distribution of xenobiotic and high affinity for tissues
- Low VD means xenobiotic is restricted mainly to blood plasma, mainly due to high
plasma protein binding
Metabolism
- Bio transformation: the enzyme catalyzed conversion of one xenobiotic into another
- Most important determinant of the duration of action of xenobiotics in the body
- Detoxification: biotransformation results in a less toxic metabolite
- Bioactivation: biotransformation results in a more toxic metabolite
- Biotransformation of xenobiotics to more water-soluble metabolites is essential to
terminate their biological activity and eliminate them from the body
- Biotransformation enzymes convert lipophilic xenobiotics into highly water-soluble
metabolites that are easily excreted from the body
Bio transformation Reactions
- Phase 1: biotransformation enzymes modify the xenobiotic molecule mainly by
oxidation – addition of an OH group to the xenobiotic
- Phase 2: synthetic reactions that conjugate the xenobiotic with a highly polar endogenous
compound in the cell (e.g, carbohydrate, sulphate, or acetate)
- a xenobiotic is often bio transformed sequentially through both Phase 1 & Phase 2
reactions
- the liver is the most important site of xenobiotic bio transformation
Phase 1 biotransformation
- Cytochrome P450-dependent monooxygenases are the major Phase 1 oxidative
enzymes
- Reactions usually involve adding or exposing a polar function group (-OH, -COOH, -
NH2) to lipophilic xenobiotics molecule
- CYP enzyme are located on the smooth ER
- Monooxygenase indicates that they catalyze the insertion of an oxygen atom into the
xenobiotic molecule, for example the simple hydroxylation reaction: Xenobiotic (R-H) +
O2 + NADPH = Metabolite (R-OH) + H2O + NADP+
- CYPS are not only involved in xenobiotic biotransformation’s but also in a wide variety
of catabolic and anabolic reactions involving endogenous compounds (steroid hormones)
- >100 different CYP enzymes have been identified: CYP1A2, CYP2E1, CYP3A4 based
on DNA sequence similarity of genes coding for enzymes
