Environmental Toxicology

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Environmental Toxicology

• Chemical Fate and Action in Organisms

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Contaminant Uptake

• Once internalized, there are four SITES of
  contaminant/toxicant fate within organisms
• ACTION STORAGE
• METABOLISM EXCRETION
• Transport is often the result of physiological
  fluid movement
• Blood
• Lymph
• Haemolymph (for invertebrates)

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Contaminant Sites in Organisms

• Action
• Location in the body where toxins interact with
  endogenous macromolecules or biological
  structures
• Proteins (and enzymes) and DNA act to alter
  physiological function of cellular processes
• Cellular membranes or those membranes of cellular
  organelles
• Can alter membrane (and thus cellular) integrity
• Makes cells leaky and disrupts normal function

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Contaminant Sites in Organisms

• Metabolism
• By enzyme systems within organisms
• Most are associated with detoxification, but some
  can lead to activation (increasing toxicological
  properties)
• Storage
• Incorporation within tissues in an inactive
  state

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Contaminant Sites in Organisms

• Excretion
• Removal of toxicants from the body, often in a
  transformed (altered, i.e., detoxified) state
• Elimination
• Removal in feces
• Excretion
• Filtration via kidneys, removal in urine

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DERMAL CONTACT
INGESTION
INHALATION
EXPOSURE
UPTAKE
TRANSPORT
STORAGE
METABOLISM
EXCRETION
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A Review of Biological Membranes

• All cells bounded by membranes, externally and
  internally
• Plasma membrane
• Outer cellular barrier
• Nuclear membrane (envelope)
• Double-walled membrane with nuclear pores
• Membrane-bound organelles
• Endoplasmic reticulum, mitochondria, Golgi
  apparatus

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Animal Cell
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Membrane Composition

• Phospholipid bilayer
• Hydrophilic heads
• Directed towards the cytosol and the
  extracellular environment
• Hydrophobic tails
• Long-chain lipids directed towards one another
• Fluid-mosaic model
• Not static, components within the membrane are in
  motion

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Membrane Composition

• Membranous proteins
• Transmembranous proteins
• Span the membrane from the inside to the outside
• Some with hydrophilic portions create channels
  and pores
• Embedded only interact with one surface at a
  time
• Carbohydrates (sugars)
• often attached to proteins at the external
  surface of the membrane

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Cell Membrane Structure
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Contaminant-Membrane Interactions

• For many compounds, biological membranes present
  themselves as a barrier to intracelluar migration
• There are four (4) mechanisms by which compounds
  cross cell membranes
• Passive diffusion
• Factors influencing rate and degree of diffusion
  are
• Concentration gradient across membranes
• Lipid solubility

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Passive Diffusion

• Depends on ionization state and lipid solubility
  of compound
• Stomach as main site of absorption for compounds
  that are weak acids
• These exist in diffusible, nonionic,
  lipid-soluble form, due to the acidic nature of
  the gastric environment

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Digestive Diffusion

• Stomach does not allow for absorption of weak
  bases
• Acid environment means that these compounds will
  remain in ionized state and not be absorbed
• Intestine serves as main site for absorption of
  weak bases
• These exist in non-ionized form and are allowed
  to cross intestinal membranes
• Absorption is enhanced by increased surface area
  and long contact time

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Lipid solubility

• Partitioning coefficient for lipophilic compounds
  expressed by
• log Pow octanol water coefficient
• Developed in 1963 by C. Hausch (German chemist)
• Pow log10 X octanol
• ------------------------
• X water

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Octanol-water coefficient

• log Pow gt 3, i.e., is 1000 X more soluble in
  octanol than in water
• Means that a compound has significant lipid
  solubility,and can be biomagnified by trophic
  transfer
• Examples
• log Pow for DDT 6.2
• Cypermethrin 6.3, but is rapidly hydrolyzed and
  does not persist
• Atrazine 2.7

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Contaminant-Membrane Interactions

• Filtration (bulk transport)
• Endothelial pores in renal glomeruli
• Size about 70 nm
• Allows for passage of molecules smaller than 60
  KDa (albumin _at_ 66KDa)
• Most other cellular pores are small
• Size about 4 nm
• Allows for passage of chemicals with MW only of
  100 200 Da
• Most OCs and OPs with MWs 200 - 400

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Contaminant-Membrane Interactions

• Carrier-mediated transport
• Active transport
• Facilitated diffusion
• Engulfing by cells
• Endocytosis and phagocytosis

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Xenobiotic Metabolism

• Evolution of detoxifying enzyme systems
• Ability to circumvent toxic compounds in food
  allows for inherent increase in fitness by
  exploitation of unused resources
• Fortuitous in Nature - not planned
• Those animals that posses abilities to detoxify
  noxious compounds may be favored evolutionarily

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Natural Detoxification

• Not directed
• Non-specific
• The greater the utility of detoxifying enzyme
  system, the greater the benefit in terms of
  exploiting resources

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Example

• Mixed-function oxidase system with broad chemical
  interaction capabilities
• P450 enzyme system
• Found associated with endoplasmic reticulum
• Much activity in liver, but also found in other
  organs (lungs, stomach, intestines, and kidneys)
  and somatic tissues (skin)
• NOT found in Central Nervous System
• Catalyses a broad array of oxidation reactions
• P450 enzymes are part of a superfamily of enzymes
  with many physiological functions

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Cellular metabolism of xenobiotics

• Follows a two-step process
• Phase I metabolism
• Degradative reactions
• Oxidations
• Reductions
• Hydrolysis
• Phase II metabolism
• Conjugation reactions

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Phase I Metabolism

• Biotransformation
• Adds a molecular handle to the compound to
  facilitate subsequent metabolism
• Oxidation, reduction, and hydrolysis
• Acts to increase the polarity of the original
  molecule
• Making it more water soluble and, thus, able to
  be excreted

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Phase I Metabolism

• Microsomal oxidative reactions
• Many types (mechanisms)
• Generally, oxidation is accomplished by transfer
  of eletrons bound to O2, one atom oxidizes the
  substrate (xenobiotic), the other forms H2O
• Source of electrons is often NADPH

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Phase I Oxidations

• Aliphatic oxidation
• Oxidation of aliphatic side chains of aromatic
  compounds
• Aromatic hydroxylation
• Often proceeds through the formation of epoxide
  intermediates
• Benzene ? phenol (hydroxylation)

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Phase I Oxidations

• Epoxidation forms epoxides (adds an O)
• Benzo(a)pyrene ? benzo(a)pyrene epoxide (a
  potent carcinogen)
• Aldrin ? dieldrin
• Oxidative deamination
• Dealkylations
• N-, O-, or S-
• Hydroxylation (non-aromatic)
• Sulfoxidation

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Phase I Reactions

• Desulfuration replaces S with O
• Parathion ? Paraoxon
• Malathion ? Malaoxon

Insects 10,000X AChE inhibition
Malathion
Humans - rapid carboxyesterase metabolism
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Acetylcholinesterase activity
Acetylcholine
Acetylcholinesterase
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Acetylcholinesterase activity
O
CH3 - C
HO - CH2 - CH2 - N(CH3)3
O
H2O
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Acetylcholinesterase activity
These reactions occur very rapidly Functional
AChE is restored to act on another molecule of
acetylcholine
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Organophosphate inhibition of AChE
Paraoxon
Acetylcholinesterase
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Acetylcholinesterase activity
O
Acid leaving group
FAST Reaction
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Acetylcholinesterase activity
O
C O
O
SLOW Reaction - Chemical Aging
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Phase I Oxidations

• Non-microsomal oxidations
• Amine oxidations
• Monoamine oxidase (MAO) system of mitochondria
  and diamine oxidase of cytosol
• Alcohol and aldehyde dehydrogenase enzyme activity

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Phase I Reactions

• Reductions
• Not very important in mammals and other
  vertebrates, but is important mechanism in
  environmental bacteria and some intestinal
  bacteria in higher animals
• Hydrolysis
• Generally accomplished through nonspecific
  esterases and amidases found in tissues and plasma

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Phase I Hydrolytic Reactions

• Arylesterases
• Hydrolyze aromatic esters
• Carboxylesterases
• Hydrolyze aliphatic esters
• Cholinesterases
• Hydrolyze esters in which the alcohol moiety is
  choline
• Acetylesterases
• Hydrolyze esters in which the acid moiety is
  acetic acid

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Phase II Biotransformations

• Glucuronide formation
• Sulfate conjugation
• Methylation

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Phase II - Conjucation Reactions

• Glucuronide formation
• Primarily occurs in Liver
• Most important type of conjugation reaction
• Allows for excretion in urine and faeces
• UDP-glucuronyl transferase is enzyme which
  catalyses reaction
• Uridine diphosphate
• UDPGA as coenzyme
• Uridine-5-diphospho-a-D-glucuronic acid
• Found in mitochondria

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UDPGA
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Glucuronide Formation

• Acts on
• Aliphatic or aromatic alcohols
• Carboxylic acids
• Sulphydryl compounds
• Amines
• Donates glucuronic acid residue to xenobiotic
  phenols and amines
• UDPGA R-OH R-O-glucosiduridine UDP
• Important in steroid metabolism and excretion

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A simple example - Phenol

Phenol

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Sulphate Conjugation

• Found in liver, kidney, and intestine
• Catalysed by sulphotransferases
• Coenzyme is PAPS
• 3-phosphoadenosine 5phosphosulphate
• Acts on phenols, aliphatic alcohols, and aromatic
  amines
• Interaction, again, is with hydroxyl groups (OH)

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Methylation Reactions

• Minor pathway
• UDPGA is more readily available
• This reaction does not always increase H2O
  solubility of xenobiotic substrates
• Catalysed by methyl transferases
• Coenzyme is SAM
• S-adenosylmethionine

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Amino Acid Conjugation

• Acts on
• Aromatic carboxylic acids
• Aryl acetic acids
• Aryl-substituted acryl acids
• a-amino acids
• glycine in many organisms
• glutamine in Humans
• ornithine in birds
• Catalytic reaction by amino acid conjugates and
  Coenzyme A

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Glutathione Conjugation

• A very important detoxifying pathway
• Not dependent on presence of hydroxyl groups
• Enzyme is Glutathione-S-transferase
• Cofactor is Glutathione
• Conjugates undergo cleavage and acetylation to
  form N-acetylcystiene (mercapturic acid)
• These are easily excreted
• Acts on
• Epoxides
• Aromatic halogens

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Glutathione
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Epoxide Metabolism

• As we saw with Benzoapyrene epoxide
• These compounds are highly reactive electrophiles
• Can react with cellular components
• Cause cell death
• Induce tumor formation
• Affect immune system function
• Glutathione acts to neutralise the damaging
  effects of epoxides
• High concentrations can deplete glutathione
• Leaves Phase I metabolites (including epoxides)
  free to cause cellular damage
• In humans, some compounds deplete glutathione -
  acetominophen

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Xenobiotic Metabolism