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Home , CYP2D6, CYP6G1

Evolutionary Response to Introduction to Chemicals in the Environment Detoxification

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)

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 ; each individual has a unique set of enzymes

• Selection result from variation in diet, climate, geography, toxins (pesticides)

Partial list of detoxification enzymes Detoxification Enzymes Phase I (functionalization) reactions: oxidations and reductions Cytochrome P450s, flavin-containing (FMOs), hydroxylases, lipooxygenases, , peroxidases, mononamine oxidases (MAOs)and • Exogenous compounds (toxins, pesticides) compete various other oxidases, dioxygenases, quinone reductases, dihydrodiol reductases, with endogenous ligands (estrogen, other hormones) and various other reductases, aldoketoreductases, NAD-and NADP-dependent – for binding to receptors (estrogen, ) alcohol dehydrogenases, aldehyde dehydrogenases, steroid dehydrogenases, dehalogenases. – channels (ion or other ligand) acting as agonists or antagonists. Phase II (conjugation) reactions: transfer chemical moieties to water-soluble derivatives UDP glucuronosyltransferases,GSH S , sulfotransferases, • Such binding to receptors could result in toxicitiy, acyltransferases,glycosyltransferases, glucosyltransferases, transaminases, abnormal development, or cancer acetyltransferases, methyltransferases

Hydrolytic enzymes • Detoxification enzymes act to break down these Glycosylases, glycosidases, amidases,glucuronidases, paraoxonases, chemicals before they bind to receptors or channels carboxylesterases, epoxide and various other , acetylcholinesterases and various other esterases

1 Cytochrome P450s CYPs (cytochrome P450s)

• At least 74 families • 14 ubiquitous in all mammals

• CYP1, 2, 3, involved in detoxification of lipophilic, or nonpolar substances

• Other CYP families involved in of endogenous substances, such as fatty acids, prostaglandins, steroids, and thyroid hormones

Evolutionary History of CYP450s CYP450

• Different types arose through gene duplication and • CYP catalyses a variety of differentiation reactions including epoxidation, N- dealkylation, O-dealkylation, S- • The first CYP450s likely evolved in response to an increase oxidation and . in oxygen in the atmosphere (along with CAT and SOD)

• A typical • The massive diversity of these CYP is thought to reflect the catalysed reaction is: coevolutionary history between plants and animals. • NADPH + H+ + O2 + RH ==> NADP+ + H2O + R-OH • Plants develop new to limit their consumption by animals - the animals develop new enzymes to metabolize the plant toxins, and so on.

CYP Evolution: duplication and differentiation Human population variation in The number of CYP2 appear to have exploded after DME frequencies animals invaded land, ~400 million years ago (50 gene • Many different alleles ( differences) at many DME duplications) and began eating genes plants • Differences among populations might arise due to natural selection arising from Dietary differences, or differences in Climate and Geography The start of the invasion of land

• Phylogenetic tree of 34 • There might also be differences arising from genetic drift CYP450 proteins. (random loss of alleles in small populations) – Black diamonds = gene- duplication events. – Unmarked branch points = speciation events. • Maintenance of genetic variation might be explained by balancing selection (such as heterozygote advantage)

2 Humans have 18 gene families of cytochrome P450 genes and Human population variation in 43 subfamilies • CYP1 drug metabolism (3 subfamilies, 3 genes, 1 ) DME allele frequencies • CYP2 drug and steroid metabolism (13 subfamilies, 16 genes, 16 ) • 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) Implications of genetic variation: • 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 (1 subfamily member) • Differences in dietary capacities • 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) forms estrogen • Many drugs are plant derivatives, such that differences • CYP20 Unknown function (1 subfamily, 1 gene) • CYP21 steroid biosynthesis (1 subfamily, 1 gene, 1 pseudogene) in response to plant compounds would affect drug • CYP24 vitamin D degradation (1 subfamily, 1 gene) • CYP26A retinoic acid hydroxylase important in development (1 subfamily member) responses • CYP26B probable retinoic acid hydroxylase (1 subfamily member) • CYP26C probabvle retinoic acid hydroxylase (1 subfamily member) • CYP27A bile acid biosynthesis (1 subfamily member) • CYP27B Vitamin D3 1-alpha hydroxylase activates vitamin D3 (1 subfamily member) • Differences in drug metabolism, drug excretion rates • CYP27C Unknown function (1 subfamily member) and final serum drug concentrations • CYP39 7 alpha hydroxylation of 24 hydroxy cholesterol (1 subfamily member) • CYP46 cholesterol 24-hydroxylase (1 subfamily member) • CYP51 cholesterol biosynthesis (1 subfamily, 1 gene, 3 pseudogenes) lanosterol 14-alpha

Some CYP enzymes involved in Drug Metabolism Human 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

• Many intermediate levels of functioning

Various CYP alleles in Caucasians Extra copy of CYP 2D6 (gene duplication)

3 Pharmacological consequences Can the response to toxins in of genetic variation at CYP the environment evolve? • Individual differences in the ability to breakdown different chemicals

• Do cytochrome P450s play a role in some • Inefficient drug metabolism: higher serum cases? drug concentration, increase risk of concentration-dependent side-effects • In the case of CYP450s, there is genetic variation • Over-efficient metabolism: failure to attain therapeutic doses

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 The number of different types of chemicals in – Evolution of generalized detoxification the environment has been increasing over time! mechanisms (e.g. cytochrome P450s)

Example:! 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 Evolution of Insecticide Resistance

4 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 acetylcholinesterase

• 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

Pesticides Atrazine

PCB

4-nonylphenol DDT Evolution at the Targets of Pesticide Action

OR

Malathion Evolution of Detoxification Capacity (CYP450s) Kepone Dioxin

Evolution at the targets of pesticide action Ligand-gated Ion Channels (bind to , 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 --> serine Evolution at the Targets of Pesticide Action (or glycine) at position 302, that is crucial for insecticide binding In Response to Neurotoxins (Zhang et al. 1994) • This amino acid substitution occurs across many different taxa, and is a striking case of parallel evolution

• Evolution of Ion Channels • Duplications of Rdl allele (Anthony et al. 1998) • Evolution of Acetylcholinesterase – Up to 4 copies of the Rdl allele, with different amino acid compositions (allowing different response to different toxins)

5 Parallel evolution of insectide resistance conferring mutations across species Evolution at the targets of pesticide action Point mutations within the Rdl allele in different species replace the same amino acid 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

Evolution at the targets of pesticide action Evolution at the Targets of Pesticide Action

Acetylcholinesterase Breaks down acetylcholine • Single amino acid substitutions in single genes can Target of organophosphate and carbamate pesticides confer resistance

• Only a limited number of amino acid substitutions can • Amino acid substitution in the acetylcholinesterase be tolerated and still maintain receptor or enzyme (Ace) gene function – All resistant strains (different subspecies) of the mosquito Culex pipiens have the same amino acid • Become the possible mutations are limited, often end substitution up with Parallel Evolution: Identical replacements – glycine --> serine at position 119 within the occur across a wide range of taxa of the enzyme

Evolution of Detoxification Capacity Daborn et al. 2002. (Cytochrome P450s) Science 297: 2253-2256

• 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) Example:

• The multigene families could detoxify a wide range of toxins Examined Drosophila populations worldwide, and examined the genome of insecticide resistant populations

6 The individuals that overtranscribed the CYP6g1 gene possessed the DDT- Result R allele

10-100 times mRNA synthesis in the • Insecticide resistant resistant strains populations of Drosophila exhibited an overtranscription of a single cytochrome P450 The DDT-R allele gene Cyp6g1 (regulatory has an insertion shift) of the “Accord” element into the • Cyp6g1 is an enzyme 5’ end of the responsible for breaking Cyp6g1 gene, via a transposon down DDT and other toxins

Daborn et al. 2002

“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

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