Evolutionary Response to Chemicals in the Environment 1.Introduction to Detoxification enzymes (focusing on cytochrome P450s) 2.Evolutionary response to toxins (pesticides) 1. Evolution at the pesticide target 2. Evolution of generalized detoxification mechanisms (e.g. cytochrome P450s)
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Evolutionary Response to Chemicals in the Environment 1.Introduction to Detoxification enzymes (focusing on cytochrome P450s) 2.Evolutionary response to.
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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)1. Evolution at the pesticide target
2. Evolution of generalized detoxification mechanisms (e.g. cytochrome P450s)
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)
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
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,glycosyltransferases, glucosyltransferases, transaminases, acetyltransferases, methyltransferases
Hydrolytic enzymes
Glycosylases, glycosidases, amidases,glucuronidases, paraoxonases, carboxylesterases, epoxide hydrolase and various other hydrolases, acetylcholinesterases and various other esterases
Cytochrome P450s
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
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 ==> NADP+ + H2O + R-OH
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.
Evolution at the Targets of Pesticide ActionIn Response to Neurotoxins
• Evolution of Ion Channels• Evolution of Acetylcholinesterase
Evolution at the targets of pesticide action
• 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 --> 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)
Ligand-gated Ion Channels(bind to neurotransmitters, e.g. GABA, acetylcholine)
Site of action of pesticides cyclodiene, neonicotinoids, ivermectin, etc.
Parallel evolution of insectide resistance conferring mutations across species
Point mutations within the Rdl allele in different species replace the same amino acid
• 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
Voltage-gated Ion Channels(important for neuronal signaling)Site of action of DDT and pyrethroids
Evolution at the targets of pesticide action
• Amino acid substitution in the acetylcholinesterase (Ace) gene– All resistant strains (different subspecies) of the
mosquito Culex pipiens have the same amino acid substitution
– glycine --> serine at position 119 within the active site of the enzyme
AcetylcholinesteraseBreaks down acetylcholineTarget of organophosphate and carbamate pesticides
Evolution at the targets of pesticide action
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
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
Example:
Examined Drosophila populations worldwide, and examined the genome of insecticide resistant populations
Daborn et al. 2002. Science 297: 2253-2256
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
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
The individuals that overtranscribed the CYP6g1 gene possessed the DDT-R allele
“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
“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