# Chapter 2: Biotransformations of Xenobiotics
## Introduction

Fish and other aquatic organisms are exposed life-long to the combined effluents of human sources, erosion runoff, and natural excretions from plants and animals. Exposure of aquatic organisms to the chemical mixture is very different from that of terrestrial species like humans. For example, while both humans and aquatic organisms might be exposed to the water-soluble herbicide atrazine, in humans the exposure would most likely be through ingestion of contaminated food or drinking water. Fish would be exposed through their skin and gills. Ingested atrazine first travels to the liver whereas atrazine taken up through gills go directly to the bloodstream. In this way, the environment of terrestrial and aquatic organisms plays a significant role in exposure to various environmental chemicals.
All organisms have defenses to help them deal with and survive potentially harmful chemicals originating from outside the body known as xenobiotics. A large group of these defenses take the form of enzymes that transform a xenobiotic into a different molecule, ideally one that will no longer pose a threat to the host organism. This process is called biotransformation (Figure 1). Biotransformation is the method for metabolic detoxification of xenobiotics. Aquatic organisms have evolved an array of methods to perform biotransformation when they encounter a potent mix of chemicals dissolved in their aquatic environment.

## ADME: Absorption, Distribution, Metabolism and Excretion

Absorption, Distribution, Metabolism, and Excretion (ADME) are the four steps used to describe the ways a toxicant will interact with an organism to allow or disallow it from inducing a toxic outcome. ADME describes where, when, and how much of a potential toxicant is present (the toxicokinetics), but it does not describe how the toxicant causes harm (the toxicodynamics). Many of the principles of ADME are very similar between related terrestrial and aquatic organisms; however, absorption can be very different due to varying potential exposures when a toxicant crosses an aqueous medium. This differential absorption--based on the physical properties of the xenobiotic compounds--determines which chemicals will be engaged in the third part of the acronym: metabolism.
One physical property that largely influences how a potential toxicant will interact with a body is its propensity to associate with water or lipids. A measure that describes this physical property is the partition coefficient (P) which is often reported as log P. Log P is sometimes written as log K$_{ow}$ where P = K$_{ow}$ = the octanol/water partition coefficient as the measurement is taken while performing a liquid/liquid separation with octanol and water. Higher values of log P mean that a given molecule will spend more time associated with non-polar conditions such as octanol over a highly polar aqueous environment. This has major implications for fish and other aquatic organisms that have fat rich tissues separated from the water-based environment by complex membranes having both non-polar and polar characteristics.
Physical properties of gills: The gills of fish are highly specialized oxygen and ion exchange tissues that include high surface area lamella and extensive vasculature. Even with these specializations, some of the properties of fish gills (e.g. thin membrane size and partial permeability to water, gases, and solutes) allow general comparison to membranes of other taxa such as insect gills that do not have pulmonary vasculature. The gill cells in direct contact with the environment are epithelial cells of various kinds, including lamellar cells that facilitate oxygen exchange and chloride cells that are essential for ion balance (Evans, 1987). Fluids near a solid surface create a slow-moving layer called a boundary layer. When a xenobiotic interacts with the boundary layer of water around a gill, it is called an aqueous diffusion layer. The xenobiotic must cross this layer via diffusion rather than being carried by the flow turbulence due to the relative stillness of this boundary layer (Figure 2). Erickson, et al. (1990) explored how potential xenobiotics cross the aqueous diffusion layer and the cell membrane of the gills in rainbow trout. They found that chemicals of high lipophilicity (log K$_{OW}$ > 3) had low uptake due to an inability to readily diffuse across the aqueous diffusion layer. Chemicals of particularly low lipophilicity (log K$_{OW}$ less than 1) also had lower uptake because it is more difficult to cross the cell membrane which contains lipids. Uptake of chemicals peaked in a Goldilocks zone of moderate lipophilicity (log K$_{OW}$ between 1 and 3), as they could cross both barriers at higher rates. It is likely that other taxa of organisms with gills or permeable skin would have similar chemical uptake profiles in relation to the compound’s log K$_{OW}$.
The excretion of xenobiotics in aquatic life and land dwelling counterparts is very different. Terrestrial animals typically have some mechanism to withdraw excess water from fecal material to reduce water lost during defecation. Nitrogen waste in the urine depends on the common availability of water to a species. Bursell (1967) describes several different strategies for nitrogen waste removal in insects. Terrestrial insects that reside in dry climates such as the migratory locust (Locusta migratoria), pack their nitrogen waste in the form of insoluble crystals of uric acid and excrete a mostly dry waste. Insects that spend part of their life history in water such as Aeshna cyanea larva, a species of the hawker dragonfly, are more similar to fish in that they produce the potentially toxic nitrogen product, ammonia, but allow it to diffuse into the surrounding water before it builds to harmful levels. Humans take a middle route. We have no sink that allows ammonia to simply diffuse away when it occurs, and we require a relatively high water intake to partially eliminate the need to spend energy packing nitrogen into uric acid. Instead, humans and many other species make urea: a compound with lower toxicity and solubility in-between ammonia and uric acid (OpenStax, 2013). The general excretion strategy used by aquatic organisms is to make use of the surrounding environment by creating a sufficiently water-soluble molecule that will diffuse away from the body.

## Biotransformations, Phase I

The major metabolic pathways responsible for biotransformation of potential toxicants are called phase I and phase II, although they do not necessarily occur in that order or in sequence at all. Phase I bio-transformations typically employ water or oxygen as an enzymatic factor in reactions such as oxidation, hydrolysis, and reductions. Phase II bio-transformations often form covalent bonds to existing biomolecules in the body. Bio-transformations typically lead to detoxification of a xenobiotic, most commonly by making it more readily excretable. Once a chemical is excreted from the body it is much less likely to cause harm. Changes to the chemical structure can also make a xenobiotic less toxic, even if it is retained in the body.
Oxidation: Many phase I bio-transformations are performed by mixed-function oxidases (MFOs) which are common in the liver as well as in other tissues. A major family of MFOs is called cytochrome P450 (Klaassen, 2013). Cytochrome P450 enzymes perform oxidations (the number in the name refers to the wavelength of light absorbed by the enzyme and helps in their characterization). Many different cytochrome P450’s have been discovered and have a wide range of specificity and substrate compatibility (Figure 3). Stegeman et al. (1991) explored how several carcinogenic toxicants are metabolized by different Cytochrome P-450’s in fish. Distribution of these enzymes were spread out over the locations, liver, kidney, and gills in Scup fish. Despite subtle differences in the specific enzyme used, cytochrome P-450’s are highly conserved. Moktali et al. (2012) trace the evolution of these detoxification enzymes and show that it is more ancient than any terrestrial lineage.
Reductions: Xenobiotics of an elevated oxidation state (certain metals and functional groups such as carbonyls, disulfides, quinones, azo, and nitro groups) can be reduced directly or enzymatically using reducing agents often referred to in consumer foods as “anti-oxidants.” These reducing agents or cofactors include glutathione, FAD (Figure 4), FMN, and NAD(P) (Klaassen, 2013). Many of these cofactors are modifications of basic essential molecules: glutathione is a modified polypeptide and NAD(P) are modifications of dinucleotides. Some enzymes considered oxidases can perform reductions: cytochromes P450 can sometimes use a xenobiotic as the oxidizing cofactor which will effectively perform a reduction on the xenobiotic to oxidize something else. Reductases can vary in specificity. Nitrate reductases are important for the nitrogen cycle and allow plants to utilize nitrate fertilizers. Timmermans, et al. (1994) investigated the effects of available iron on nitrate reductases in phytoplankton and found that the ability to produce a functional enzyme requires the presence of dissolved iron in the environment.
Hydrolysis: Hydrolysis is the process of adding water across a chemical bond. The most generic example is breaking a C-O bond, and making a new C-O bond and an O-H bond (Figure 5). Other bonds can be hydrolyzed as well. The defining characteristic is the consumption of water to split a bond, as shown in blue in Figure D. The process leads to a net zero change in oxidation state: it is neither an oxidation nor reduction reaction. Some compounds undergo hydrolysis spontaneously in water or in plasma at appreciable rates, but living organisms have evolved enzymes that can speed up this reaction. Some of these enzymes include esterases which hydrolyze esters, peptidases which hydrolyze protein peptide bonds, and phosphatases which hydrolyze phosphoester bonds.
Chlorine metabolism: While humans are the main generators of organic chlorines in terrestrial environments, producing chemicals and legacy pollutants such as polychlorinated biphenyls (PCB’s) and insecticides (Figure 6), some living organisms produce chlorinated organic compounds as well, particularly those in aquatic environments. Gribble (1996) discussed “the diversity of natural organochlorines in living organisms” and offered examples, including a chemical produced by the freshwater fungus Kirschsteiniothelia sp., 3,3’-oxybis(2,4-dichloro-5-methylphenol) (Figure 7).
Organochlorines or chlorinated organic molecules are chemicals containing a C-Cl bond. The presence of chlorine on the molecule tends to make it more lipophilic and more difficult to degrade via phase I biotransformations or energy metabolism by microorganisms. These two characteristics together, high lipophilicity and persistence, promote retention of these chemicals within biological tissues and can lead to toxic buildup. Removing chlorines from organic molecules, dehalogenation, is energetically less favorable than removing common heteroatoms such as sulfur or nitrogen (Dugat-Bony, 2016). The enzymes that detoxify organochlorines are rare in life; they are usually relegated to microorganisms that use compounds for energy metabolism. Dugat-Bony, et al. (2016) described various methods used to dehalogenate the organocholrines which overlap with other detoxification processes described above. Monooxygenases such as cytochromes P-450 can both oxidatively or reductively eliminate chlorine. Glutatione S-transferase (see next section, phase II biotransformations) can reductively eliminate chlorine. Detoxification of organochlorines are undoubtedly useful for the host microbe, but they have also been studied as potential solutions to human environmental contamination involving persistent organochlorine pollutants (Jugder, 2016).

## Biotransformations, Phase II

Phase II biotranasformations are characterized by additions, called conjugations, of small bio-molecules to the xenobiotic such as sugars and amino acids. These additions, larger in size than hydroxyl additions from phase I oxidations, both potentially increase the hydrophilicity of the toxicant and potentially block the molecule from its site of toxic action by changing how it moves through tissues and cell membranes. While Phase II does not always follow phase I, the functional groups created in phase I transformations such as a hydroxyl –OH group, can be used as a target for conjugation reactions of phase II biotransforamtions. Conjugations are usually more effective than phase I biotransformations in regards to increasing hydrophilicity and decreasing toxic effects. Many examples of conjugations can be found including methylations and acetylation (Figure 8); however, this chapter focuses on the most well studied: glucuronidation, sulfonation, and glutathione conjugation.
Glutathione: The glutathione S-transferases (GST) is a highly conserved conjugation enzyme family. Glutathione, a tripeptide capable of acting as a reducing agent for phase I biotransformations on its own, can be used with a GST to bind with a xenobiotic via the sulfur group on the cysteine moiety of a reduced glutathione molecule. This greatly increases the polarity and molecular weight of most target substrates, allowing them to be excreted with water soluble waste. Stenersen et al. (1986) investigated nine different animal phyla, examining both terrestrial and aquatic organisms for the presence and activity of GST. They studied aquatic and non-aquatic vertebrates, insects, crustaceans, and mollusks and found that nearly all the animals had GST activity, though activity was significantly higher for terrestrial organisms than for aquatic life. As described by Edwards, et al. (2005), plants have a small subset of water-soluble GST enzymes. Glutathione is very important for the glutathione-ascorbate cycle and reduction of hydrogen peroxide in plants, and it can also play a part in plant defense against pathogens.
Glucuronidation: Sugars are a common biomolecule and are highly water soluble, making them ideal conjugates to increase the solubility of a xenobiotic when carbohydrates are in abundance. Glucuronic acid, a derivative of glucose, is a major cofactor of phase II biotransformations and are used by the enzyme family: uridine 5’-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT). Glucuronic acid itself is initially transformed into uridine diphosphate glucuronic acid which is the sugar derivative covalently bonded to another biomolecule through a diphosphate linkage. The diphosphate raises the energy of the molecule (similar to why Adenosine triphosphate is used as energy by the cell) which allows the UDP-glucuronosyltransferase enzyme to proceed with the conjugation by releasing the energy stored in the phosphate linkage of the cofactor. In this way the cofactor acts as both conjugate and energy source (Klaassen, 2013). Enzymes without active sources of energy drastically slowdown in response to temperature decline. UDP-glucuronosyltransferase is also affected by temperature, with an optimal range dependent on the organism and its environment, but not to the same extent due to energetics. As such, UDP-glucuronosyltransferase is helpful to aquatic organisms that experience temperature changes in the water. HÄNNINEN, et al. (1984) describe how rainbow trout lose CYP-450 enzyme functionality at colder stream temperatures, but maintain the use of UDP-glucuronosyltransferase to allow continued detoxification processes in colder climates.
Sulfonation: Sulfates can be conjugated to open hydroxyl groups on xenobiotics. These are performed by sulfotransferases which take a sulfonate group (SO$_3$-) from the cofactor 3’-phosphoadenosine-5’-phosphosulfate, which is a modified nucleotide similar to ATP (adenosine triphosphate) but with a sulfate instead of a phosphate (Figure 9). Sulfonation is an important way to biotransform the active estrogen, 17β-estradiol. Exposure to exogenous estrogens can lead to deformations of the gonads in aquatic organisms. Wang et al. (2007) explored what can happen when the sulfonation of these estrogens is blocked by other environmental contaminants. The authors found that polychlorinated biphenyls (PCBs) with hydroxyl groups (OH-PCBs) inhibited the activity of sulfotransferases to sulfonate 17β-estradiol. In this case, products of phase I biotransformations (OH-PCBs) were detrimental to the biotransformation of other xenobiotics (estrogen), essentially making the PCB more toxic or toxic in a different way. See the following section for more examples of biotransformations increasing the toxicity of a xenobiotic.

## Biotransformations that Increase Toxicity

Biotransformations typically result in changing a xenobiotic so that it will not harm the organism. The range of xenobiotics that can affect an organism are diverse however, and the same biotransformation that causes one xenobiotic to become harmless could transform a different xenobiotic into something much more harmful. A human example is alcohol dehydrogenase. Ethanol, which can have harmful effects on the body or behavior on its own, undergoes an oxidative biotransformation catalyzed by alcohol dehydrogenase or other enzymes (Figure 10). The ethanol becomes ethyl aldehyde (or acetaldehyde) which is toxic to the liver and other organs (NIH NIAAA, 2007). The aldehyde is further oxidized to acetic acid (the tangy part of vinegar) which is much less toxic. A xenobiotic that becomes more toxic after a biotransformation is called a pro-toxin. The above example illustrates that alcohol is both a toxin and pro-toxin for humans.
An endangered fish species found in Mexico’s freshwater environments, Chirostoma riojai, is an example. Vega-Lo´pez et al. (2011) did a study on how naturally occurring halomethanes could be affecting this species. Halomethanes are single carbon molecules with some number of halogens, and occur naturally in water. The halomethanes underwent biotransformation with a subset of cytochrome P450s and were oxidized (see section on chlorine metabolism). The oxidized halomethanes caused oxidative stress to the fish, which can further lead to oxidative damage. The oxidation caused the xenobiotic to become more reactive and increased the exposure risk. This is an occasional downside to the broad specificity of some enzymes such as mix function oxidases.

## Enzyme Induction

Types of pollutants and xenobiotics can vary according to location and time. Keeping a suit of enzymes to answer every potentially harmful xenobiotic would be energetically expensive and potentially counterproductive when there is only a finite subset of xenobiotics to address at any one time. The key to surviving and thriving is to have the correct enzymes in place when needed. Enzymes are proteins which are produced constantly by reading DNA. Cells self-regulate which DNA genes are being read and how much protein to create, depending on signals that tell the cell what is currently needed. Genes corresponding to detoxification enzymes can be turned on or “upregulated” by a process called enzyme induction. When an organism encounters a particular xenobiotic it signals a response to induce the production of detoxification enzymes to address that specific xenobiotic or closely related group. This process is not instantaneous and means that when encountering a new xenobiotic, it will likely be harmed. If an organism survives the initial encounter it will induce the production of enzymes to protect itself from subsequent encounters, similar to the immune system’s reaction to a virus. This could result from switching food sources where a new plant food is high in secondary metabolites--in this case the enzymes induced may be of general detoxifying function. A review of biotransformation systems in fish by Chambers et al. (1976) compared the prevalence of enzyme induction of mixed function oxidases between mammals and fish. Fish were found to upregulate these enzymes following a change in diet or pesticide exposure, but at lower levels than that found in mammals.
Many of the enzyme groups discussed in phase I and II biotransformations have some members that act broadly on a wide range of molecules. Mixed function oxidases are named for their broad specificity and have more than one function. More specific enzymes can be induced once a xenobiotic is encountered, and they may possess more potent activity due to their specialization. Hence, organisms can balance the tradeoff between specificity and activity while maintaining the highest chance of evolutionary success.

## References

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## Glossary

Aquatic organism – An organism that spends a part of its life-cycle (e.g. larval stage) or a whole life-cycle in the water. For this chapter it includes both freshwater and saltwater environments. Some literature uses the term “marine organism” for saltwater environments.
Xenobiotic – A molecule that originates from outside an organism that is not naturally produced (synthetic) or expected to be present in the organism. They are not always toxic (many are beneficial) but this chapter will use the term to refer to a potentially toxic substance.
Biotransformation – The process of a living organism changing a chemical. A biotransformation is often carried out by an enzyme.
Enzyme – A protein that catalyzes a chemical reaction. This means it facilitates and speeds up chemical reactions. Enzymes are unchanged after the reaction, allowing them to repeat the process; but, may be limited by the availability of cofactors.
Cofactor – A biomolecule that is required by an enzyme to function. In biotransformations the cofactor is used up by the enzyme to change the xenobiotic.
Oxidation/Reduction – Terms for chemical reactions in which electrons are transferred from one molecule to another. These reactions go in pairs because if one molecule is gaining electrons another must lose them. The names in Phase I biotransformations refers to the effect on the xenobiotic. I.e. “oxidation reactions” are where the xenobiotic is oxidized (looses electrons); the cofactor is reduced and the enzyme stays the same.
Enzyme Induction – The upregulation of transcription for a certain protein due to internal signaling in an organism. In this chapter it refers to the upregulation of detoxifying enzymes after a poisoning to counteract further poisonings.
Conjugation – The chemical process of joining two molecules together.
Toxicokinetics – Movement of a toxic chemical through a living organism. This includes absorption, distribution, metabolism and excretion. Toxicokinetics is sometimes referred to as, “How the body acts on the chemical.”
Toxicodynamics – The way a toxic chemical exerts its toxic effect. How the toxin interacts with the target site of action. Toxicodynamics is sometimes referred to as, “How the chemical acts on the body.”
Pollutant – A substance in the environment at least in part as a result of human origin or activity which has deleterious effect on living organisms.
Toxin – A chemical that causes harm to an organism. Any chemical can be a toxin depending on the dose/concentration and exposure.
Metabolism – The breaking down or conjoining of molecules by an organism.
Energy metabolism – Metabolism involved in generating energy for an organism to live; it is also called “catabolism.”
Co-metabolism – Metabolic processes used to break down molecules, but do not yield energy for the organism.
Conserved (evolution) – A characteristic of a gene or morphological feature. Conserved genes/morphologies stay the same or relatively unchanged across different branches of the evolutionary tree. For example, if there is an enzyme that is the same in rainbow trout and humans, that enzyme and the corresponding gene is “conserved between rainbow trout and humans.” If the same enzyme is present in many other taxa as well, it would be called “generally conserved” or “highly conserved.”