## 4.2.14. Genetic variation in toxicant metabolism

Author: Nico M van Straalen

Reviewers: Andrew Whitehead, Frank van Belleghem

Learning objectives:

You should be able to

- explain four different classes of CYP gene variation and expression, contributing to species differences
- explain the associations between biotransformation activity and specific ecologies
- explain how genetic variation in biotransformation enzymes may lead to evolution of toxicant tolerance
- describe the relevance of human genetic polymorphisms for personalized medicine

Keywords: toxicant susceptibility; genetic variation; biotransformation evolution of toxicant tolerance

Assumed prior knowledge and related modules

- Biotransformation and internal processing of chemicals
- Defence mechanisms
- Genetic erosion

In addition, a basic knowledge of genetics and evolutionary biology is needed to understand this module.

Synopsis

Susceptibility to toxicants often shows inter-individual differences associated with genetic variation. While such differences are considered a nuisance in laboratory toxicity testing, they are an inextricable aspect of toxicant effects in the environment. Variation may be due to polymorphisms in the target site of toxicant action, but more often differences in metabolic enzymes and rates of excretion contribute to inter-individual variation. The structure of genes encoding metabolic enzymes, as well as polymorphisms in promoter regions of such genes are common sources of genetic variation. Under strong selection pressure species may evolve toxicant-tolerant populations, for example insects to insecticides and bacteria to antibiotics. In human populations, polymorphisms in drug metabolizing enzymes are mapped to provide a basis for personal therapies. This module aims to illustrate some of the genetic principles explaining inter-individual variation of toxicant susceptibility and its evolutionary consequences.

Introduction

For a long time it has been known that human subjects may differ markedly in their responses to drugs: while some patients hardly respond to a certain dosage, others react vehemently. Similar differences exist between the sexes and between ethnic groups. To avoid failure of treatment on the one hand and overdosing on the other, such personal differences have attracted the interest of pharmacological scientists. Also the tendency to develop cancer upon exposure to mutagenic chemicals is partly due to genetics. Since the rise of molecular ecology in the 1990s ecotoxicologists have noted that inter-individual differences in toxicant responses also exists in the environment.

Due to genetic variation environmental pollution may trigger evolutionary change in the wild. From quantitative genetics we know that when a trait is due to many genes, each with an independent additive effect on the trait value, the response to selection R, is linearly related to the selection differential S according to the formula: R = h2S, where h2 is a measure of the heritability of the selected trait (fraction of additive genetic variance relative to total phenotypic variance). Since anthropogenic toxicants can act as very strong selective agents (large S) it is expected that whenever h2 > 0 there will be adaptation. However, the effectiveness of “evolutionary rescue” from pollution is limited to those species that have the appropriate genetic variation and the ability to quickly increase in population size.

Polymorphisms of drug metabolizing enzymes in humans

One of the most important enzyme systems contributing to metabolism of xenobiotic chemicals is the cytochrome P450 family, a class of proteins located in the smooth endoplasmic reticulum of the cell and acting in co-operation with several other proteins. Cytochrome P450 will oxidize the substrate and enhance its water-solubility (called phase-I reaction), and in many cases activate it for further reactions involving conjugation with an endogenous compound (phase II reactions). These processes generally lead to detoxification and increased excretion of toxic substances. The biochemistry of drug metabolism is discussed in detail in the section on Xenobiotic metabolism and defence.

The human genome has 57 genes encoding a P450 protein. The genes are commonly designated as “CYP”. Other organisms, especially insects and plants have many more CYPs. For example, the Drosophila genome encodes 83 functional P450 genes and the genome of the model plant Arabidopsis has 244 CYPs. Based on sequence similarity, CYPs are classified in 18 families and 43 subfamilies, but there is no agreement yet about the position of various CYP genes in lower invertebrates. The complexity is enhanced by duplications specific to certain evolutionary lineages, creating a complicated pattern of orthologs (homologs by descent from a common ancestor) and paralogs (homologs due to duplication in the same genome). In addition to functional enzymes it is also common to find many CYP pseudogenes in a genome. Pseudogenes are DNA-sequences that resemble functional genes, but are mutated and they do not result in functional proteins).

The expression of CYP enzymes is markedly tissue-specific. Often CYP expression is high in epithelial tissues (lung, intestine) and organs with designated metabolic activity (liver, kidney). In the human body, the liver is the main metabolic organ and is known for its extensive CYP expression. P450 enzymes also differ in their inducibility by classes of chemicals and in their substrate specificity.

It is often assumed that the versatility of an organism’s CYP genes is a reflection of its ecology. For example, herbivorous insects that consume plants of different kinds with many different feeding repellents must avail of a wide diversity of CYP genes. It has also been shown that activity of CYP enzymes among terrestrial organisms is, in general, higher than among aquatic organisms and that plant-eating birds have higher biotransformation activities than predatory birds.

One of the best-investigated CYP genes, especially due to its strong inducibility and involvement in xenobiotic metabolism, is mammalian CYP1A1. In humans induction of this gene is associated with increased lung cancer risk from smoking, and with other cancers, such as breast cancer and prostrate cancer. Human CYP1A1 is located on chromosome 15 and encodes 251 amino acids in seven exons (Figure 1). About 133 single-nucleotide polymorphisms (SNPs, variations in a single nucleotide that occur at a specific position in the genome) have been described for this gene, of which 23 are non-synonymous (causing a substitution of an amino acid in the protein).

Figure 1. Non-synonymous substitutions in the human CYP1A1 gene. The figure shows the intron-exon structure of the gene with 23 non-synonymous SNP positions (with nucleotide substitutions indicated) and one insertion. Redrawn from Zhou et al. (2009) by Evelin Karsten-Meessen.

Many of these SNPs have a medical relevance. For example, a rather common SNP in exon 7 changes codon 462 from isoleucine into valine. The substituted allele is called CYP1A1*2A, and this occurs at a frequency of 19% in the Caucasian part of the human population. The allelic variant of the enzyme has a higher activity towards 17β-estradiol and is a risk factor for several types of cancer. However, the expression of such traits may vary from one population to another, and may also interact with other risk factors. For example, CYP1A1*2A is a risk factor for cervical cancer in women with a history of smoking in the Polish population, but the same SNP may not be a risk factor in another population or among people with a non-smoking lifestyle. In genetics these effects are known as epistasis: the phenotypic effect of genetic variation at one locus depends on the genotype of another locus. This is also an example of a genotype-by-environment interaction, where the phenotypic effect of a genetic variant depends on the environment (smoking habit). In toxicology it is known that polymorphisms of phase II biotransformation enzymes may significantly contribute to epistatic interaction with CYP genes. Unraveling all these complicated interactions is a very active field of research in human medical genetics.

Cytochrome P450 variation across species

Comparison of CYP genes in different species has revealed an enormously rapid evolution of this gene family, with many lineage-specific duplications. This indicates strong selective pressures imposed by the need to detoxify substances ingested with the diet. Especially herbivorous animals are constantly exposed to such compounds, synthesized by plants to deter feeding. We also see profound changes in CYP genes associated with evolutionary transitions such as colonization of terrestrial habitats by the various lineages of arthropods. Such natural variation, induced by plant toxins and habitat requirements, is also relevant in the responses to toxicants.

In general, variation of biotransformation enzymes can be classified in four main categories

1. Variation in the structure of the genes, e.g. substitutions that alter the binding affinity to substrates; such variation discriminates the various CYP genes.
2. Copy number variation; duplication usually leads to an increase in enzymatic capacity; this process has been enormously important in CYP evolution. Because CYP gene duplications are often specific to the evolutionary lineage, a complicated pattern of paralogs (duplicates within the same genome) and orthologs (genes common by descent, shared with other species) arises.
3. Promoter variation, e.g. due to insertion of transposons or changes in the number or arrangement of transcription factor binding sites. This changes the amount of protein produced from one gene copy by altered transcriptional regulation.
4. Variation in the structure, action or activation of transcriptional regulators. The transcription of biotransformation enzymes is usually induced by a signaling pathway activated by the compound to be metabolized (see the section on Xenobiotic metabolism and defence), and this pathway may show genetic variation.

To illustrate the complicated evolution of biotransformation genes, we shortly discuss the CYPs of common cormorant, Phalacrocorax carbo. This is a bird known for its narrow diet (fish) and extraordinary potential for accumulation of dioxin-related compounds (PCBs, PCDDs and PCDFs). Environmental toxicologists have identified two CYP1A genes in the cormorant, called CYP1A4 and CYP1A5. It turns out that CYP1A4 is homologous by descent (orthologous) to mammalian CYP1A1 while CYP1A5 is an ortholog of mammalian CYP1A2. However, the orthologies are not revealed by common phylogenetic analysis if the whole coding sequence is used in the alignment (see Figure 2a). This is a consequence of a process called interparalog gene conversion, which tends to homogenize DNA sequences of gene copies located on the same chromosome. This diminishes sequence variation between the paralogs, and creates chimeric gene structures, that are more similar to each other than expected from their phylogenetic relations. If a phylogenetic tree is made using a section of the gene that remained outside the gene conversion, the true phylogenetic relations are revealed (see Figure 2b).

Figure 2. Phylogenetic trees for CYP1A genes in chicken, cormorant, mouse and human, using zebrafish and killifish as outgroups. Two trees are shown, one using a full-length alignment of the protein sequence (a), the other using only positions 721 to 970 of the coding sequence (b). The fact that the two trees are different is indicative of interparalog gene conversion. Reproduced from Kubota et al. (2006) by Evelin Karsten-Meessen.

Cytochrome P450-mediated resistances

Cytochrome P450 polymorphisms are also implicated in certain types of insecticide resistance. There are many ways in which insects and other arthropods can become resistant and several mechanisms may even be present in the same resistant strain. Target site alteration (making the target less susceptible to the insecticide, e.g. altered acetylcholinesterase, substitutions in the GABA-receptor, etc.) seems to be the most likely mechanism for resistance, however, such changes often come with substantial costs as they may diminish the natural function of the target (in genetics this is called pleiotropy). Increased metabolism does not usually contribute metabolic costs and this is where cytochromes P450 come into play. A model system for investigating the genetics of such mechanisms is DDT resistance in the fruit fly, Drosophila melanogaster.

In a DDT-resistant Drosophila strain, all CYP genes were screened for enhanced expression and it was shown that DDT resistance was due to a highly upregulated variant of only a single gene, Cyp6g1. Further analysis showed that the gene’s promoter carried an insertion with strong similarity to a transposable element of the Accord family. The insertion of this element causes a significant overexpression and a high rate of protein synthesis that allows the fly to quickly degrade a DDT dose. The fact that a simple change, in only one allele, can underlie such a distinctive phenotype as pesticide resistance is a remarkable lesson for molecular toxicology.

A recent study on killifish, Fundulus heteroclitus, along the East coast of the United States has revealed a much more complicated pattern of resistance. Populations of these fish live in estuaries, some with severely polluted sediments, containing high concentrations of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). Killifish from the polluted environments are much more resistant to toxicity from the model compounds PCB126 and benzo(a)pyrene. This resistance is related to mutations in the gene encoding aryl hydrocarbon receptor (AHR), the protein that binds PAHs and certain PCB metabolites and activates CYP expression. Also mutations in a protein called aryl-hydrocarbon receptor-interacting protein (AIP), a protein that combines with AHR to ensure binding of the ligand, contribute to down-regulation of the CY1A1 pathway. The net result is that killifish CYP1A1 shows only moderate induction by PCBs and PAHs and the damaging effects of reactive metabolites are avoided. However, since direct knockdown of CYP1A1 does not provide resistance it is still unclear whether the beneficial effects of the mutations in AHR actually act through an effect on CYP1A1.

Figure 3. Showing the genetic variation among sensitive (S1 to S4) and tolerant (T1 to T4) populations of killifish, Fundulus heteroclitus along the East coast of the United States. Sensitivity and tolerance is towards sediments with high loads of PCBs and/or PAHs. The genome of Fundulus encodes four AHR (aryl hydrocarbon receptor) paralogs of which two are positioned in tandem, AHR2a and AHR1a, which carry long deletions (three different ones), indicated by black bars in the left figure. In addition, the populations have variable number of duplications of the CYP1A1 genes (right figure), not present to the same degree in the sensitive populations. Knock-out of AHR2a is protective of PCB and PAH toxicity, while duplication of CYP1A1 ensures a basal gene dose even when induction is less strong. Redrawn from Reid et al. (2016) by Wilma Ijzerman.

Interestingly, the various killifish populations show at least three different deletions in the AHR genes (Figure 3). In addition, the tolerant populations show various degrees of CYP1A1 duplication; in one population even eight paralogs are present. This can be interpreted as compensatory adaptations ensuring a basal constitutive level of CYP1A1 protein to conduct routine metabolic activities. The killifish example shows a wonderful case of interplay between genetic tinkering, and strong selection emanating from a polluted environment.

Conclusion

In this module we have focused on genetic variation in the phase I enzyme, cytochrome P450. A similar complexity lies behind the phase II enzymes and the various xenobiotic-induced transporters (phase III). Still the P450 examples suffice to demonstrate that the machinery of xenobiotic metabolism shows a very large degree of genetic variation, as well as species differences due to duplications, deletions, gene conversion and lineage-specific selection. The variation resides both in copy number variation, alteration of coding sequences and in promoter or enhancer sequences affecting the expression of the enzymes. Such genetic variation is the template for evolution. In polluted environments enhanced expression is sometimes selected for (to neutralize toxic compounds), but sometimes also attenuated expression is selected (to avoid production of toxic intermediates). In the human genome, many of the polymorphisms have a medical significance, determining a personal profile of drug metabolism and tendencies to develop cancer.

References

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