## 4.1.4. Xenobiotic defence and metabolism

Author: Nico M. van Straalen

Reviewers: Timo Hamers, Cristina Fossi

Learning objectives:

You should be able to:

- recapitulate the phase I, II and III mechanisms for xenobiotic metabolism, and the most important molecular systems involved.
- describe the fate and the chemical changes of an organic compound that is metabolized by the human body, from absorption to excretion.
- explain the principle of metabolic activation and why some compounds can become very reactive upon xenobiotic metabolism.
- develop a hypothesis on the ecological effects of xenobiotic compounds that require metabolic activation.

Keywords: biotransformation; phase 1; phase II; phase III; excretion; metabolic activation; cytochrome P450

Synopsis

All organisms are equipped with metabolic defence mechanisms to deal with foreign compounds. The reactions involved, jointly called biotransformation, can be divided into three phases, and usually aim to increase water solubility and excretion. The first step (phase I) is catalyzed by cytochrome P450, which is followed by a variety of conjugation reactions (phase II) and excretion (phase III). The enzymes and transporters involved are often highly inducible, i.e. the amount of protein is greatly enhanced by the xenobiotic compounds themselves. The induction involves binding of the compound to cytoplasmic receptor proteins, such as the arylhydrocarbon receptor (AhR), or the constitutive androstane receptor (CAR). In some cases the intermediate metabolites, produced in phase I are extremely reactive and a main cause of toxicity, a well-known example being the metabolic activation of polycyclic aromatic hydrocarbons such as benzo(a)pyrene, which readily forms DNA adducts and causes cancer. In addition, some compounds greatly induce metabolizing enzymes but are hardly degraded by them and cause chronic cellular stress. The various biotransformation reactions are a crucial aspect of both toxicokinetics and toxicodynamics of xenobiotics.

Introduction

The term “xenobiotic” (“foreign to biology”) is generally used to indicate a chemical compound that does not normally have a metabolic function. We will use the term extensively in this module, despite the fact that it is somewhat problematic (e.g., can a compound be considered “foreign” if it circulates in the body, is metabolized or degraded in the body?, and: what is “foreign” for one species is not necessarily “foreign” for another species).

The body has an extensive defence system to deal with xenobiotic compounds, loosely designated as biotransformation. The ultimate result of this system is excretion the compound in some form or another. However, many xenobiotics are quite lipophilic, tend to accumulate and are not easily excreted due to low water solubility. Molecular modifications are usually required before such compounds can be removed from the body, as the main circulatory and excretory systems (blood, urine) are water-based. By introducing hydrophilic groups in the molecule (-OH, =O, -COOH) and by conjugating it to an endogenous compound with good water-solubility, excretion is usually accomplished. However, as we will see below, intermediate metabolites may have enhanced reactivity and it often happens that a compound becomes more toxic while being metabolized. In the case of pesticides, deliberate use is made of such responses, to increase the toxicity of an insecticide once it is in the target organism.

The study of xenobiotic metabolism is a classical subject not only in toxicology but also in pharmacology. The mode of action of a drug often depends critically on the rate and mode of metabolism. Also, many drugs show toxic side-effects as a consequence of metabolism. Finally, xenobiotic metabolism is also studied extensively in entomology, as both toxicity and resistance of pesticides are often mediated by metabolism.

The most problematic xenobiotics are those with a high octanol-water partition coefficient (Kow) that are strongly lipophilic and very hydrophobic. They tend to accumulate, in proportion to their Log Kow, in tissues with a high lipid content such as the subcutis of vertebrates, and may cause tissue damage due to disturbance of membrane functions. This mode of action is called “minimum toxicity”. Well-known are low-molecular weight aliphatic petroleum compounds and chlorinated alkanes such as chloroform. These compounds cause their primary damage to cell membranes; especially neurons are sensitive to this effect, hence minimum toxicity is also called narcotic toxicity. Lipophilic chemicals with high Log Kow do not reach concentrations high enough to cause minimum toxicity because they induce biotransformation at lower concentrations. The toxicity is then usually due to a reactive metabolite.

Xenobiotic metabolism involves three subsequent phases (Figure 1):

1. Activation (usually oxidation) of the compound by an enzyme known as cytochrome P450, which acts in cooperation with NADPH cytochrome P450 reductase and other factors.
2. Conjugation of the activated product of phase I to an endogenous compound. A host of different enzymes is available for this task, depending on the compound, the tissue and the species. There are also (slightly polar) compounds that enter phase II directly, without being activated in phase I.
3. Excretion of the compound into circulation, urine, or other media, usually by means of membrane-spanning transporters belonging to the class of ATP-binding cassette (ABC) transporters, including the infamous multidrug resistance proteins. Hydrophilic compounds may pass on directly to phase III, without being activated or conjugated.

Figure 1. The various phases of xenobiotic metabolism. Redrawn by Wilma Ijzerman.

Phase I reactions

Cytochrome P450 is a membrane-bound enzyme, associated with the smooth endoplasmic reticulum. It carries a porphyrin ring containing an Fe atom, which is the active center of the molecule. The designation P450 is derived from the fact that it shows an absorption maximum at 450 nm when inhibited by carbon monoxide, a now outdated method to demonstrate its presence. Other (outdated) namings are MFO (mixed function oxygenase) and drug metabolizing enzyme complex. Cytochrome P450 is encoded by a gene called CYP, of which there are many paralogs in the genome, all slightly differing from each other in terms of inducibility and substrate specificity. Three classes of CYP genes are involved with biotransformation, designated CYP1, CYP2 and CYP3 in vertebrates. Each class has several isoforms; the human genome has 57 different CYP genes in total. The CYP complement of invertebrates and plants often involves even more genes; many evolutionary lineages have their own set, arising from extensive gene duplications within that lineage. In humans, the genetic complement of a person’s CYP genes is highly relevant as to its drug metabolizing profile (see the section on Genetic variation in toxicant metabolism).

Cytochrome P450 operates in conjunction with an enzyme called NADPH cytochrome P450 reductase, which consists of two flavoproteins, one containing Flavin adenine dinucleotide (FAD), the other flavin mononucleotide (FMN). The reduced Fe2+ atom in cytochrome P450 binds molecular oxygen, and is oxidized to Fe3+ while splitting O2; one O atom is introduced in the substrate, the other reacts with hydrogen to form water. Then the enzyme is reduced by accepting an electron from cytochrome P450 reductase. The overall reaction can be written as:

RH + O2 + NADPH + H+ → ROH + H2O + NADP+

where R is an arbitrary substrate.

Cytochrome P450 is expressed to a great extent in hepatocytes (liver cells), the liver being the main organ for xenobiotic metabolism in vertebrates (Figure 2), but it is also present in epithelia of the lung and the intestine. In insects the activity is particularly high in the Malpighian tubules in addition to the gut and the fat body. In mollusks and crustaceans the main metabolic organ is the hepatopancreas.

Phase II reactions

After activation by cytochrome P450 the oxidized substrate is ready to be conjugated to an endogenous compound, e.g. a sulphate, glucose, glucuronic acid or glutathione group. These reactions are conducted by a variety of different enzymes, of which some reside in the sER like P450, while others are located in the cytoplasm of the cell (Figure 2). Most of them transfer a hydrophilic group, available from intermediate metabolism, to the substrate, hence the enzymes are called transferases. Usually the compound becomes more polar in phase II, however, not all phase II reactions increase water solubility; for example, methylation (by methyl transferase) decreases reactivity but increases apolarity. Other phase II reactions are conjugation with glutathione, conducted by glutathione-S-transferase (GST) and with glucuronic acid, conducted by UDP-glucuronyl transferase. In invertebrates other conjugations may dominate, e.g. in arthropods and plants, conjugation with malonyl glucose is a common reaction, which is not seen in vertebrates.

Conjugation with glutathione in the human body is often followed by splitting off glutamic acid and glycine, leaving only the cysteine residue on the substrate. Cysteine is subsequently acetylated, thus forming a so-called mercapturic acid. This is the most common type of metabolite for many xenobiotics excreted in urine by humans.

Like cytochrome P450, the phase II enzymes consist in various isoforms, encoded by different paralogs in the genome. Especially the GST family is quite extensive and polymorphisms in these genes contribute significantly to the personal metabolic profile (see the section on Genetic variation in toxicant metabolism).

Figure 2. Schematic view of xenobiotic metabolism (phase I and phase II) in human liver. P450 = cytochrome P450, FP = flavoprotein, RED = cytochrome P450 reductase, EH = epoxide hydrolase, UDP-GT = uridyldiphospho-glucuronyl transferase, GSH-T = glutathione-S-transferase, ST = sulphotransferase. Reproduced from Vermeulen & Van den Broek (1984) by Wilma Ijzerman.

Phase III reactions

In the human body, there are two main pathways for excretion, one from the liver into the bile (and further into the gut and the faeces), the other through the kidney and urine. These two pathways are used by different classes of xenobiotics: very hydrophobic compounds such as high-molecular weight polycyclic aromatic hydrocarbons are still not readily soluble in water even after metabolism but can be emulsified by bile salt and excreted in this way. It sometimes happens that such compounds, once arriving in the gut, are assimilated again, transported to the liver by the portal vein and metabolized again. This is called “entero-hepatic circulation”. Lower molecular weight compounds and hydrophilic compounds are excreted through urine. Volatile compounds can leave the body through the skin and exhaled air.

Excretion of activated and conjugated compounds from tissues out of the cell usually requires active transport, which is mediated by ABC (ATP binding cassette) transporters, a very large and diverse family of membrane proteins which have in common a binding cassette for ATP. Different subgroups of ATP transporters transport different types of chemicals, e.g. positively charged hydrophobic molecules, neutral molecules and water-soluble anionic compounds. One well-known group consists of multidrug resistance proteins or P-glycoproteins. These transporters export drugs aiming to attack tumor cells. Because their activity is highly inducible, these proteins can enhance excretion enormously, making the cell effectively resistant and thus case major problems for cancer treatment.

Induction

All enzymes of xenobiotic metabolism are highly inducible: their activity is normally on a low level but is greatly enhanced in the presence of xenobiotics. This is achieved through a classic case of transcriptional regulation upon CYP and other genes, leading to de novo synthesis of protein. In addition, extensive proliferation of the endoplasmic reticulum may occur, and in extreme cases even swelling of the liver (hepatomegaly).

The best investigated pathway for transcriptional activation of CYP genes is due to the arylhydrocarbon receptor (AhR). Under normal conditions, this peptide is stabilized in the cytoplasm by heat-shock proteins, however, when a xenobiotic compound binds to AhR, it is activated and can join with another protein called Ah receptor nuclear translocator (ARNT) to translocate to the nucleus and bind to DNA elements present in the promotor of CYP and other genes. It thus acts as a transcriptional activator or transcription factor on these genes (Figure 3). The DNA motifs to which AhR binds are called xenobiotic responsive elements (XRE) or dioxin-responsive elements (DRE). The compounds acting in this manner are called 3-MC-type inducers, after the (highly carcinogenic) model compound 3-methylcholanthrene. The inducing capacity of a compound is related to its binding affinity to the AhR, which in itself is determined by the spatial structure of the molecule. The lock-and key fitting between AhR and xenobiotics explains why induction of biotransformation by xenobiotics shows a very strong stereospecificity. For example, among the chlorinated biphenyls and chlorinated dibenzodioxins, some compounds are extremely strong inducers of CYP1 genes, while others, even with the same number of chlorine atoms, are no inducers at all. The precise position of the chlorine atoms determines the molecular “fit” in the Ah receptor (see Section on Receptor interaction).

In addition to 3MC-type of induction there are other modes in which biotransformation enzymes are induced, but these are less well-known. A common class is PB-type induction (named after another model compound, phenobarbital). PB-type induction is not AhR-dependent, but acts through activation of another nuclear receptor, called constitutive androstane receptor (CAR). This receptor activates CYP2 genes and some CYP3 genes.

The high inducibility of biotransformation can be exploited in a reverse manner: if biotransformation is seen to be highly upregulated in a species living in the environment, this indicates that that species is being exposed to xenobiotic compounds. Assays addressing cytochrome P450 activity can therefore be exploited in bioindication and biomonitoring systems. The EROD (ethoxyresorufin-O-deethylase) assay is often used for this purpose, although it is not 100% specific to the isoform of P450. Another approach is to address CYP expression directly, e.g. through reverse transcription-quantitative PCR, a method to quantify the amount of CYP mRNA.

Figure 3. Scheme illustrating how AhR-reactive compounds induce biotransformation activity. 1 xenobiotic compounds enter the cell through diffusion, 2 compound binds to AhR, releasing it from Hsp90, 3 AhR complex is activated by ARNT, 4 translocation to nucleus, 5 binding to XRE, 6 enhanced transcription, 7 translocation of mRNA to cytoplasm and translation, 8 incorporation of P450 enzyme in sER and biotransformation of compounds. AhR = arylhydrocarbon receptor, Hsp90 = heat shock protein 90, ARNT = aryl hydrocarbon nuclear translocator, XRE = xenobiotic responsive element, CYP1 = gene encoding cytochrome P450 1, sER = smooth endoplasmic reticulum. Drawn by Wilma Ijzerman.

Secondary effects of biotransformation

Although the main aim of xenobiotic metabolism is to detoxify and excrete foreign compounds, some pathways of biotransformation actually enhance toxicity. This is mostly due to the first step, activation by cytochrome P450. The activation may lead to intermediate metabolites which are highly reactive and the actual cause of toxicity. The best investigated examples are due to bioactivation of polycyclic aromatic hydrocarbons (PAHs), a group of chemicals present in diesel, soot, cigarette smoke and charred food products. Many of these compounds, e.g. benzo(a)pyrene, benz(a)anthracene and 3-methylcholanthrene, are not reactive or toxic as such but are activated by cytochrome P450 to extremely reactive molecules. Benzo(a)pyrene, for instance, is activated to a diol-epoxide, which readily binds to DNA, especially to the free amino-group of guanine (Figure 4). The complex is called a DNA adduct, the double helix is locally disrupted and this results in a mutation. If this happens in an oncogene, a tumor may develop (see the Section on Carcinogenesis and genotoxicity).

Not all PAHs are carcinogenic. Their activity critically depends on the spatial structure of the molecule, which again determines its “fit” in the Ah receptor. PAHs with a “notch” (often called bay-region) in the molecule tend to be stronger carcinogens than compounds with a symmetric (round or linear) molecular structure.

Figure 4. Metabolism of benzo(a)pyrene (B[a]P) to a mutagenic and carcinogenic metabolite, BaP-diol epoxide. Cytochrome P450 activity (CYP1A1 and CYP1B1) introduces an epoxide on the 7,8 position of the molecule, which is then hydrolyzed by epoxide hydrolase to a dihydrodiol (two OH groups next to each other, which can be in trans position, pointing in different directions relative to the plane of the molecule, or in cis, pointing in the same direction). The trans metabolite is preferably formed. Subsequently another epoxide is introduced on the 9,10 position. The diolepoxide is highly reactive, and may bind proteins and to guanine in DNA, causing a DNA adduct. Modified from Bui et al. (2009) by Steven Droge.

Another mechanism for biotransformation-induced toxicity is due to some very recalcitrant organochlorine compounds such as polychlorinated dibenzodioxins (PCDDs, or dioxins for short) and polychlorinated biphenyls (PCBs). Some of these compounds are very potent inducers of biotransformation, but they are hardly degraded themselves. The consequence is that the highly upregulated cytochrome P450 activity continues to generate a large amount of reactive oxygen (ROS), causing oxidative stress and damage to cellular constituents. It is assumed that the chronic toxicity of 2,3,7,8-tetrachlorodibenzo(para)dioxin (TCDD), one of the most toxic compounds emitted by human activity, is due to its high capacity to induce prolonged oxidative stress. On the molecular level, there is a close link between oxidative stress and biotransformation activity. Many toxicants that primarily induce oxidative stress (e.g. cadmium) also upregulate CYP enzymes. Two defence mechanisms, oxidative stress defence and biotransformation are part of the same integrated stress defence system of the cell.

References

Bui, P.H., Hsu, E.L., Hankinson, O. (2009), Fatty acid hydroperoxides support cytochrome P450 2S1-mediated bioactivation of benzo[a]pyrene-7-8-dihydrodiol. Molecular Pharmacology 76, 1044-1052.

Stroomberg, G.J., Zappey, H., Steen, R.J.C.A., Van Gestel, C.A.M., Ariese, F., Velthorst, N.H., Van Straalen, N.M. (2004). PAH biotransformation in terrestrial invertebrates – a new phase II metabolite in isopods and springtails. Comparative Biochemistry and Physiology Part C 138, 129-137.

Timbrell, J.A. (1982). Principles of Biochemical Toxicology. Taylor & Francis Ltd, London.

Van Straalen, N.M., Roelofs, D. (2012). An Introduction to Ecological Genomics, 2nd Ed. Oxford University Press, Oxford.

Vermeulen, N.P.E., Van den Broek, J.M. (1984). Opname en verwerking van chemicaliën in de mens. Chemisch Magazine. Maart: 167-171.