Evolutionary Response to Chemicals in the Environment

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Evolutionary Response to Chemicals in the
Environment

1. Introduction to Detoxification enzymes (focusing
   on cytochrome P450s)
2. Evolutionary response to toxins (pesticides)
3. Evolution at the pesticide target
4. Evolution of generalized detoxification
   mechanisms (e.g. cytochrome P450s)

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Introduction to Detoxification Enzymes
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Detoxification Enzymes(or Drug Metabolizing
Enzymes, Effector-Metabolizing Enzymes)

• Involved in detoxification of plant metabolites,
  dietary products, drugs, toxins, pesticides,
  carcinogens
• All DMEs have endogenous compounds as natural
  substrates (used in natural process of breaking
  down compounds)
• Located in every eukaryotic cell, most
  prokaryotes
• Many different types, many families, many
  alleles each individual has a unique set of
  enzymes
• Selection result from variation in diet, climate,
  geography, toxins (pesticides)

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Detoxification Enzymes

• Exogenous compounds (toxins, pesticides) compete
  with endogenous ligands (estrogen, other
  hormones)
• for binding to receptors (estrogen,
  glucocorticoid)
• channels (ion or other ligand)
• acting as agonists or antagonists.
• Such binding to receptors could result in
  toxicitiy, abnormal development, or cancer
• Detoxification enzymes act to break down these
  chemicals before they bind to receptors or
  channels

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• Partial list of detoxification enzymes
• Phase I (functionalization) reactions
  oxidations and reductions
• Cytochrome P450s, flavin-containing
  monooxygenases (FMOs), hydroxylases,
  lipooxygenases, cyclooxygenases, peroxidases,
  mononamine oxidases (MAOs)and various other
  oxidases, dioxygenases, quinone reductases,
  dihydrodiol reductases, and various other
  reductases, aldoketoreductases, NAD-and
  NADP-dependent alcohol dehydrogenases, aldehyde
  dehydrogenases, steroid dehydrogenases,
  dehalogenases.
• Phase II (conjugation) reactions transfer
  chemical moieties to water-soluble derivatives
• UDP glucuronosyltransferases,GSH S transferases,
  sulfotransferases, acyltransferases,glycosyltransf
  erases, glucosyltransferases, transaminases,
  acetyltransferases, methyltransferases
• Hydrolytic enzymes
• Glycosylases, glycosidases, amidases,glucuronidase
  s, paraoxonases, carboxylesterases, epoxide
  hydrolase and various other hydrolases,
  acetylcholinesterases and various other esterases

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Cytochrome P450s
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CYPs (cytochrome P450s)

• At least 74 gene families
• 14 ubiquitous in all mammals
• CYP1, 2, 3, involved in detoxification of
  lipophilic, or nonpolar substances
• Other CYP families involved in metabolism of
  endogenous substances, such as fatty acids,
  prostaglandins, steroids, and thyroid hormones

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CYP450

• CYP catalyses a variety of reactions including
  epoxidation, N-dealkylation, O-dealkylation,
  S-oxidation and hydroxylation.
• A typical cytochrome P450 catalysed reaction is
• NADPH H O2 RH gt NADP H2O R-OH

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Evolutionary History of CYP450s

• Different types arose through gene duplication
  and differentiation
• The first CYP450s likely evolved in response to
  an increase in oxygen in the atmosphere (along
  with CAT and SOD)
• The massive diversity of these CYP is thought to
  reflect the coevolutionary history between plants
  and animals.
• Plants develop new alkaloids to limit their
  consumption by animals - the animals develop new
  enzymes to metabolize the plant toxins, and so
  on.

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CYP Evolution duplication and differentiation
The number of CYP2 genes appear to have exploded
after animals invaded land, 400 million years
ago (50 gene duplications) and began eating plants
The start of the invasion of land

• Phylogenetic tree of 34 CYP450 proteins.
• Black diamonds gene-duplication events.
• Unmarked branch points speciation events.

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Human population variation in DME allele
frequencies

• Many different alleles (amino acid differences)
  at many DME genes
• Differences among populations might arise due to
  natural selection arising from Dietary
  differences, or differences in Climate and
  Geography
• There might also be differences arising from
  genetic drift (random loss of alleles in small
  populations)
• Maintenance of genetic variation might be
  explained by balancing selection (such as
  heterozygote advantage)

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Human population variation in DME allele
frequencies

• Implications of genetic variation
• Differences in dietary capacities
• Many drugs are plant derivatives, such that
  differences in response to plant compounds would
  affect drug responses
• Differences in drug metabolism, drug excretion
  rates and final serum drug concentrations

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Humans have 18 gene families of cytochrome P450
genes and 43 subfamilies

• CYP1 drug metabolism (3 subfamilies, 3 genes, 1
  pseudogene)
• CYP2 drug and steroid metabolism (13 subfamilies,
  16 genes, 16 pseudogenes)
• CYP3 drug metabolism (1 subfamily, 4 genes, 2
  pseudogenes)
• CYP4 arachidonic acid or fatty acid metabolism (5
  subfamilies, 11 genes, 10
• pseudogenes)
• CYP5 Thromboxane A2 synthase (1 subfamily, 1
  gene)
• CYP7A bile acid biosynthesis 7-alpha hydroxylase
  of steroid nucleus (1
• subfamily member)
• CYP7B brain specific form of 7-alpha hydroxylase
  (1 subfamily member)
• CYP8A prostacyclin synthase (1 subfamily member)
• CYP8B bile acid biosynthesis (1 subfamily member)
• CYP11 steroid biosynthesis (2 subfamilies, 3
  genes)
• CYP17 steroid biosynthesis (1 subfamily, 1 gene)
  17-alpha hydroxylase
• CYP19 steroid biosynthesis (1 subfamily, 1 gene)
  aromatase forms estrogen
• CYP20 Unknown function (1 subfamily, 1 gene)
• CYP21 steroid biosynthesis (1 subfamily, 1 gene,
  1 pseudogene)
• CYP24 vitamin D degradation (1 subfamily, 1 gene)
• CYP26A retinoic acid hydroxylase important in
  development (1 subfamily member)
• CYP26B probable retinoic acid hydroxylase (1
  subfamily member)

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Some CYP enzymes involved in Drug Metabolism
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Human Polymorphism at CYP2D6

• Oxidative metabolism of over 40 common drugs
• More than 50 different alleles have been
  identified
• 5-10 Caucasians have null alleles, and no
  function
• 7 Caucasians have duplication causing excessive
  function due to excessive expression of the
  enzyme
• Many intermediate levels of functioning

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Various CYP alleles in Caucasians
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Extra copy of CYP 2D6 (gene duplication)
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Pharmacological consequences of genetic variation
at CYP

• Individual differences in the ability to
  breakdown different chemicals
• Inefficient drug metabolism higher serum drug
  concentration, increase risk of
  concentration-dependent side-effects
• Over-efficient metabolism failure to attain
  therapeutic doses

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Can the response to toxins in the environment
evolve?

• Do cytochrome P450s play a role in some cases?
• In the case of CYP450s, there is genetic
  variation

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Evolutionary Response to Chemicals in the
Environment

• Introduction to Detoxification enzymes (focusing
  on cytochrome P450s)
• Evolutionary response to toxins (pesticides)
• Evolution at the pesticide target
• Evolution of generalized detoxification
  mechanisms (e.g. cytochrome P450s)

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The number of different types of chemicals in the
environment has been increasing over time
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CHEMICALS THE ENVIRONMENT 11,000,000
Chemicals are known 100,000 Chemicals are
produced deliberately 90,000 Registered
Chemicals in the US 1200-1500 New Chemicals
are registered in the US/year Only 50 Organic
toxins with legally enforceable environmental
standards in drinking water http//www.epa.
gov/safewater/mcl.html
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Evolution of Insecticide Resistance
Example
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Mechanism of Action (on the pests) of some Major
Classes

• Chlorinated hydrocarbons (DDT, Lindane, dioxin)
  Accumulate in fatty tissue, causing chronic
  disease
• Organophosphates (Malathion) Inhibit
  acetylcholinesterase
• Carbamates (NHRCOOR) Inhibit acetylcholinesteras
  e
• Pyrethroids (modeled after natural products)
  neurotoxin
• Growth regulators Block juvenile hormone
  receptors (Methoprene), block chitin synthesis,
  formation of cuticle
• Triazines (Atrazine) Inhibit photosynthesis
• Phenoxy herbicides Mimic plant hormone auxin,
  causing abnormal growth

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Pesticides
Atrazine
PCB
4-nonylphenol
DDT
Malathion
Kepone
Dioxin
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Evolution at the Targets of Pesticide
Action OR Evolution of Detoxification Capacity
(CYP450s)
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Evolution at the Targets of Pesticide Action In
Response to Neurotoxins Evolution of Ion
Channels Evolution of Acetylcholinesterase
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Evolution at the targets of pesticide action
Ligand-gated Ion Channels (bind to
neurotransmitters, e.g. GABA, acetylcholine) Site
of action of pesticides cyclodiene,
neonicotinoids, ivermectin, etc.

• Amino Acid substitution in the GABA receptor
• The Rdl allele codes for a GABA-receptor
  subunit that is resistant to cyclodiene
  pesticides
• This allele has an amino acid substitution of
  alanine --gt serine (or glycine) at position 302,
  that is crucial for insecticide binding (Zhang et
  al. 1994)
• This amino acid substitution occurs across many
  different taxa, and is a striking case of
  parallel evolution
• Duplications of Rdl allele (Anthony et al. 1998)
• Up to 4 copies of the Rdl allele, with different
  amino acid compositions (allowing different
  response to different toxins)

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Parallel evolution of insectide resistance
conferring mutations across species
Point mutations within the Rdl allele in
different species replace the same amino acid
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Evolution at the targets of pesticide action
Voltage-gated Ion Channels (important for
neuronal signaling) Site of action of DDT and
pyrethroids

• Changes in target receptor or channel
• Point mutation in neuronal Na channel (Martin et
  al 2000)
• Single Amino Acid substitution in Chloride
  channel (ffrench-Constant et al 2000)
• Equivalent mutations at similar positions in the
  channel have been found across a wide variety of
  insect species

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Evolution at the targets of pesticide action
Acetylcholinesterase Breaks down
acetylcholine Target of organophosphate and
carbamate pesticides

• Amino acid substitution in the acetylcholinesteras
  e (Ace) gene
• All resistant strains (different subspecies) of
  the mosquito Culex pipiens have the same amino
  acid substitution
• glycine --gt serine at position 119 within the
  active site of the enzyme

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Evolution at the Targets of Pesticide Action

• Single amino acid substitutions in single genes
  can confer resistance
• Only a limited number of amino acid substitutions
  can be tolerated and still maintain receptor or
  enzyme function
• Become the possible mutations are limited, often
  end up with Parallel Evolution Identical
  replacements occur across a wide range of taxa

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Evolution of Detoxification Capacity(Cytochrome
P450s)

• Pesticide resistance could be gained through the
  action of cytochrome P450s
• Their evolution is not as restricted as the
  targets of action (channels, etc.), which have to
  retain function (unlike the previous cases, here
  we have functional redundancy)
• The multigene families could detoxify a wide
  range of toxins

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Daborn et al. 2002. Science 297 2253-2256

• Example
• Examined Drosophila populations worldwide, and
  examined the genome of insecticide resistant
  populations

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Result

• Insecticide resistant populations of Drosophila
  exhibited an overtranscription of a single
  cytochrome P450 gene Cyp6g1 (regulatory shift)
• Cyp6g1 is an enzyme responsible for breaking
  down DDT and other toxins

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The individuals that overtranscribed the CYP6g1
gene possessed the DDT-R allele
10-100 times mRNA synthesis in the resistant
strains
The DDT-R allele has an insertion of the Accord
element into the 5 end of the Cyp6g1 gene, via a
transposon
Daborn et al. 2002
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• Pesticide Treadmill
• A few years after a pesticide is introduced,
  insects evolve resistance
• So another chemical is used
• Then another chemical is used
• Then another
• Then another

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• Pesticide Treadmill
• We cannot evolve as quickly, so potentially the
  accumulated pesticides in the environment could
  have a more detrimental effect on us than on the
  organisms we are trying to kill