1. 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)

2. Introduction to Detoxification Enzymes

4. 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)

5. 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

6. 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

7. Cytochrome P450s

8. 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

9. 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

10. 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.

11. 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.

12. 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)

13. 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

14. 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)
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)
CYP27C Unknown function (1 subfamily member)
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 demethylase

15. Some CYP enzymes involved in Drug Metabolism

16. 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

17. Various CYP alleles in Caucasians

18. Extra copy of CYP 2D6 (gene duplication)

19. 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

20. 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

21. 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)

22. The number of different types of chemicals in the environment has been increasing over time

23. CHEMICALS & THE ENVIRONMENT
11,000,000 Chemicals are known
100,000 Chemicals are produced deliberately
90,000 Registered Chemicals in the US
New Chemicals are registered in the US/year
Only ~50 Organic toxins with legally enforceable
environmental standards in drinking water
See

24. Evolution of Insecticide Resistance
Example:
Evolution of Insecticide Resistance

26. 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

27. Pesticides Atrazine PCB 4-nonylphenol DDT Malathion Kepone Dioxin
Note that many of the polychlorinated biphenyls (PCBs) look like eyeglasses. Two 6-sided carbon rings (the lenses) are joined by a bond (the nose bridge) that connects one carbon from each ring. Different PCBs vary in the placement and number of chlorine atoms attached to the basic eyeglass structure. PCBs were used for a variety of purposes including electrical transformers and capacitors. Although their use is now severely restricted, PCBs are still found in older electrical stations and other machinery. The 209 different PCBs differ greatly in their estrogenic potency. Most PCBs do not readily degrade, and they accumulate in marine animals, birds, and mammals (Zhu and Norstrom, 1993).
The structural differences between natural hormones and environmental estrogens may lead to functional differences. Natural sex hormones (estrogens or androgens) travel in the bloodstream searching out compatible receptor sites located in the nucleus of a cell. The hormones enter the cell, lock onto a specific receptor, and turn on specific genes on the DNA. The genes tell the cell to make new proteins or other substances that can change cell functions (grow, divide or make more enzyme).
Although natural steroid hormones generally function by binding to specific receptor sites, synthetic environmental estrogens can affect the hormonal system in a number of different ways. They can interact directly with hormone receptors where they can have some, little, or very different effects than natural estrogens. The chemicals can even block normal hormone action.
Malathion
Kepone
Dioxin

28. Evolution at the Targets of Pesticide Action OR
Evolution of Detoxification Capacity (CYP450s)

29. Evolution at the Targets of Pesticide Action
In Response to Neurotoxins
• Evolution of Ion Channels
• Evolution of Acetylcholinesterase

30. 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 --> 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)

31. Parallel evolution of insectide resistance conferring mutations across species
Point mutations within the Rdl allele in different species replace the same amino acid

32. 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

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

34. 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

35. 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

36. Daborn et al Science 297:
Example:
Examined Drosophila populations worldwide, and examined the genome of insecticide resistant populations

37. 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

38. 10-100 times mRNA synthesis in the resistant strains
The individuals that overtranscribed the CYP6g1 gene possessed the DDT-R allele
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

40. “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

41. “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