Biotransformation

CHAPTER 12

Biotransformation 12.1

METABOLIC TRANSFORMATION AND ELIMINATION OF POLLUTANTS

Living organisms possess different means of getting rid of foreign chemicals, termed xenobiotics, bearing potential toxicity. This set of biochemical pathways for the elimination of xenobiotics are frequently referred to as biotransformation processes. The strategy may consist of immobilization and neutralization of the potential toxicity of the chemical (sequestration), removal of the toxic molecules or radicals (scavenging), or enzymatic transformation to facilitate excretion. A more formal definition for biotransformation is the in vivo metabolic pathways intended to prevent toxic effects of xenobiotic substances and eventually eliminate them from the organism.

Biotransformation: the metabolism of foreign chemicals

Most biotransformation processes take place in the liver of vertebrates, and in the hepatopancreas or digestive gland in mollusks and crustaceans (or the anterior intestine in echinoderms), while the excretion of the biotransformed metabolites may take place in the kidney via urine (or alternative excretory system of invertebrates) or through the bile. When a xenobiotic chemical is taken up by an organism it may be readily excreted if its chemical properties allow so, i.e., if its polar nature allows it to be eliminated through the excretory system. Alternatively, it can be bound to endogenous molecules or cellular structures (granules, membranes) where it is retained and cannot exert toxicity, a process termed sequestration. Extracellular insoluble granules rich in otherwise toxic trace metals have been described for instance in bivalves.1 Exposure to heavy metals also induce the synthesis of low molecular weight proteins called metallothioneins (see Section 12.6), rich in cysteine residues whose thiol groups bind divalent cations (Cd2þ, Hg2þ, Cu2þ, etc.) preventing toxicity by inappropriate binding of the metal ion to enzymatic or structural proteins.

Biotransformation may be achieved by sequestration, scavenging, or reaction catalyzed by detoxification enzymes

Some toxic molecules can be removed just by nonenzymatic reaction with endogenous chemicals present in high concentrations, which is termed 205 Marine Pollution. https://doi.org/10.1016/B978-0-12-813736-9.00012-X Copyright © 2018 Elsevier Inc. All rights reserved.

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scavenging. This is the case of the tripeptide glutathione (GSH), which contains cysteine and acts as scavenger of otherwise highly toxic reactive oxygen species (ROS). Oxidative stress can also be fought, though, via enzymatic pathways such as those involving catalase and peroxidases (see Section 12.5). Detoxification enzymes are induced in the presence of xenobiotics and may thus be used as biomarkers of pollution

Highly lipophilic xenobiotics tend to bioaccumulate in the body fat. However, they can also be metabolized through biotransformation routes catalyzed by detoxification enzymes that normally proceed in a stepwise manner as described in the next section. The synthesis of the enzymes and other molecules (stress proteins, cofactors) involved in the detoxification metabolism is induced by the presence in the environment of chemical pollutants. This has been used as a biological tool for monitoring chemical pollution, and the inducible molecules involved can be used as biomarkers. This issue will be treated in depth in Chapter 16.

12.2 Biotransformation of xenobiotics proceeds through two steps: oxidation plus conjugation

PHASES OF THE ENZYMATIC BIOTRANSFORMATION

Enzymatically mediated biotransformation of organic xenobiotics frequently proceeds through the steps summarized in Fig. 12.1. In Phase I reactions the molecule is normally oxidized to increase polarity and to provide more reactive groups for further transformation. If the substance is highly lipophilic Phase I can involve several oxidative steps. This is the case of the degradation of polycyclic aromatic hydrocarbons (PAHs) (see Fig. 7.2, Section 7.3). The first oxidation of organohalogenated compounds is frequently the limiting reaction for

FIGURE 12.1 Schematic representation of a typical biotransformation process of a lipophilic organic xenobiotic. In Phase I the chemical is oxidized by a cytochrome p450-dependent monooxygenase (CYP). In Phase II the resulting metabolite, frequently more toxic than the parental compound, is conjugated to an endogenous nontoxic polar molecule. The resulting conjugate is suitable for elimination by the excretory system.

12.3 Phase I: Cytochrome P450 Dependent Oxidations

the overall elimination process. In Phase II reactions, the oxidized metabolite is conjugated with an endogenous nontoxic metabolite to form a water-soluble higher molecular weight product that can be excreted through the bile, the kidney, or equivalent excretory system. Biotransformation affects toxicity. Oxidized derivatives of xenobiotics can be highly reactive and even more toxic than the parent compound. This is, for example, the case of paracetamol, which is normally conjugated with nontoxic endogenous metabolites but can also be oxidized by a Phase I reaction into a toxic quinone that must be conjugated to suppress its toxicity to the organism. Therefore, Phase 2 reactions, to increase the polarity of the molecule, have also the function of buffering the potential toxicity of the degradation product formed after Phase I. The chemical nature of the xenobiotic determines the biotransformation pathway. If it is a very hydrosoluble substance it can be readily excreted in the urine or directly undergo Phase II by conjugation through one polar group. The excretion rate is related to the polarity of the molecule. This is again illustrated by the case of paracetamol, whose conjugates are 10e20 times more rapidly excreted than the parental compound.2

12.3

PHASE I: CYTOCHROME P450 DEPENDENT OXIDATIONS

Phase I reactions (Table 12.1) include different kinds of oxidation (hydroxylation, epoxidation, dealkylation, desulphuration) but can also consist of hydrolysis or in some cases (for instance DDT) even reduction. These reactions are catalyzed by enzymes called cytochrome P450 monooxygenases (CYP monooxygenase or simply CYP), also termed in the past mixed function oxidases (MFOs) (see Section 7.3). These enzymes are located in the smooth endoplasmic reticulum (SER) of hepatocytes and some other cellular types. Induced activity of detoxification enzymes is used as biomarker of chemical

Table 12.1 Main Types of Phase I Biotransformation Reactions, Enzymes Involved, and Examples of Substrates Phase I Reaction

Enzymes

OXIDATIONS Hydroxylation

CYP monooxygenases

Epoxidation Desulphuration Dealkylation HYDROLYSIS REDUCTION

Esterases Reductases

Examples of Substrates Benzene, aliphatic hydrocarbons, 4-MBC,3 BaP and other PAHs, PCB Parathion Ethoxyresorufin Carbamates DDT, PCB

Most Phase I oxidations take place in the smooth endoplasmic reticulum (SER) of hepatocytes.

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pollution. Invertebrates show in general much lower CYP activities than aquatic vertebrates, and this contributes to explain higher BCF values for many xenobiotics. For biochemical assay, the SER of hepatocytes or other cells is isolated by differential ultracentrifugation in the so-called microsomal fraction, and so the associated enzymes like CYP are called microsomal, in contrast to the fewer oxidation enzymes present in the cytosol.a .because they involve several membrane-bound electron transport proteins The overall enzymatic complex is termed cytochrome P450 monooxygenase system

Indeed, the apparently simple Phase I scheme, from the lipophilic xenobiotic, ReH to the oxidized metabolite ReOH in the presence of oxygen (see Fig. 12.1), is in fact a quite complex process, and CYP mediated reactions take place in the SER because they involve a number of membrane-bound proteins responsible for electron transport. Those include cytochrome p450, a hemeprotein with an iron protoporphyrin group that forms a complex with the substrate and the oxygen, an electron donner -the NADPH-, a NADPH cytochrome P450 reductase, a second electron donor -the NADH-, a NADH cytochrome b5 reductase, and its coenzyme, the cytochrome b5. The whole system (for details, see4) is loosely termed the cytochrome P450 monooxygenase system, and the global reaction catalyzed by this system is: R e H D NADPH D H D D O2 / R e OH D NADP D D H2 O:

Different CYP families catalyze the biotransformation of different kinds of chemicals

(Eq. 12.1)

The CYP are in fact a series of isoenzymes that, in theory, specifically catalyze the oxidation of different types of organic molecules. In mammals there are three main families of CYP implicated in biotransformation of xenobiotics: CYP1, CYP2, and CYP3. Each family may present subfamilies termed CYP2A, CYP2B, etc., and each subfamily may be further divided into distinct proteins coded by different genes. For instance, cytochromes CYP1A1 and CYP1A2 are coded by the corresponding CYP1A1 and CYP1A2 genes (note the italics for genes). The families and subfamilies are defined on the basis of similarity in DNA sequence. Other phylogenetic groups greatly differ in CYP variability. For example CYP 1 to 3 families are present in sea urchins but absent in bivalves and other protostomates.5 The induction of the synthesis of a certain CYP is mediated by the specific binding of the chemical inducer to a protein receptor that interacts with the regulatory element of the gene and triggers its transcription. For example, coplanar polyaromatic molecules such as the TCDD dioxin bind to the Aryl hydrocarbon receptor (AhR) in the cytoplasm. The TCDD-AhR complex enters the nucleus and, mediated by a specific nuclear translocator protein, interacts with the CYP1A1 to promote its transcription into mRNA. The latter travels back to

a

One of the few nonmicrosomal oxidation enzymes are dehydrogenases that transform primary and secondary alcohols, including ethanol into aldehydes and ketones, and the latter into organic acids. These enzymes are cytosolic and require NAD or NADP.

12.4 Phase II: Conjugation With Glutathione or Glucuronic Acid

the cytoplasm, and activates CYP1A1 synthesis in the rough endoplasmic reticulum.6 The binding strength of a ligand to the AhR is thought to be directly proportional to the enhanced gene transcription and associated toxicity. TCDD is the agonist with the strongest affinity to AhR. Other less potent inducers via AhR binding are other dioxins, dibenzofurans, coplanar PCBs and PAHs, and thus toxicity equivalents may be calculated for all those molecules on the basis of their relative affinity to AhR compared to TCDD (see also Section 8.1). In mammals, similar roles have been proposed for the constitutive androstane receptor (CAR) in relation to CYP2 induction, and the pregnane-X-receptor in relation to CYP3 induction. The CAR receptor is absent in fish, which may explain the lack of CYP2 inducibility despite fish do have the CYP2 gene.7 Therefore, the induction of biotransformation pathways caused by exposition to chemical pollutants can be monitored at transcriptional level, by quantifying the amount of the specific mRNA that results from the expression of the gen, or at posttranscriptional level by quantifying the amount (or activity, if the protein is an enzyme) of the protein coded by the mRNA. The earlier may provide a better indication of exposure, if additional pollutants interfere with protein synthesis or enzyme activity at posttranscriptional level.

12.4

Induction of biotransformation caused by exposition to organics can be monitored at mRNA or protein levels

PHASE II: CONJUGATION WITH GLUTATHIONE OR GLUCURONIC ACID

Phase II reactions consist of the addition to the foreign compound of an endogenous metabolite readily available in vivo and added to a suitable functional group present on the original xenobiotic or introduced by Phase I metabolism. The resulting conjugate is more polar, thus facilitating excretion, and less toxic. The most common endogenous metabolites used in Phase II reactions are the glucuronic acid, the sulfate, and the glutathione (GSH), a tripeptide formed by glutamic acid, cysteine, and glycine. Glucuronic acid and sulfate show more affinity for hydroxyl radicals (see Fig. 12.2), whereas GSH is frequently conjugated to epoxide and ketone groups (see Fig. 7.2).

Phase II aims at increasing polarity and buffering toxicity .

Many Phase II reactions involve the transfer of glucuronic acid in its activated form, bound to the energy storing nucleotide uridine diphosphate (UDP) (Fig. 12.2). The reaction is catalyzed by the UDP-glucuronosyltransferase (UDPGT), an enzyme present in the SER of hepatocytes and other tissues. Unlike the GSH conjugation, where the activated compound is the xenobiotic (type II conjugations), here the break-down of the endogenous metabolite provides the energy for the reaction (type I conjugations).

.by conjugation mediated by glucuronyltransferase .

GSH is a tripeptide with cysteine very abundant in hepatocytes. The thiol group of the cysteine confers GSH a double role in cellular defense against toxicants. First, the GS chemically reacts with electrophiles protecting cells by removing reactive metabolites. Second, GSH is the substrate for energy demanding Phase

.or glutathione-Stransferases

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CYP

OH + H2O

+ O2

COOH O OH

COOH O + UDP + H2O

OH Glucuronosyl transferase

OH

O

OH Ether glucuronide

OH

O UDP OH UDP-glucuronic acid

FIGURE 12.2 Biotransformation of benzene, including an oxidation by a cytochrome P450-dependent monooxygenase (CYP) (Phase I), and a conjugation with glucuronid acid (Phase II). The conjugation is catalyzed by a transferase, and the energy needed is provided by the cleavage of the bond with UDP.

II conjugation reactions catalyzed by the glutathione-S-transferases (GST), which mediates the formation of conjugates with the potent electrophilic metabolites originated in Phase I biotransformation of PAHs, PCBs,8 and other xenobiotics. GST is particularly relevant in the detoxification of the carcinogenic metabolite 7,8-dihydrodiol-9,10-epoxide formed in the Phase I biotransformation of BaP (see Fig. 7.2), since diol epoxides are poor substrates for epoxide hydrolases and cannot be detoxified by this enzyme.9 GST are very abundant (up to 0.7 percent of total soluble proteins) and ubiquitous enzymes primarily found in the cytosol of many tissues. Again, like for CYP, there is a number of different GST isoenzymes theoretically substratespecific. This time the different functional isoenzymes result from the different arrangements of the dimer subunits than constitute the GST.10 Phase II enzymes are also inducible in the presence of environmental xenobiotics, and thus susceptible to be used as biomarkers of chemical pollution. However, the inducibility of both GST and UDPGT in fish has been reported to be moderate (up to twofold) in comparison with the inducibility of EROD (up to 100 fold).11 The use of GST activity as chemical pollution biomarker in bivalves and other invertebrates has shown more promising results (see Section 16.2). Phase III: catabolism and/or excretion of the conjugates

After Phase II, the resulting conjugates may either be excreted, usually into the bile, or catabolized through further degradative steps. These processes are sometimes termed phase III metabolism. GSH conjugates are degraded to mercapturic acids present in the urine.12 This is the case of Naphthalene biotransformation products.13 Excretion of conjugates out from hepatocytes involves a transmembrane pump and requires energy provided by ATP. The common endogenous metabolite conjugated during Phase II allows that a single type of pump can excrete many different biotransformed xenobiotics.14

12.5 Oxidative Stress Metabolism

12.5

OXIDATIVE STRESS METABOLISM

Although we usually think of oxygen as the essential element for life, the release of uncoupled high-energy electrons from its molecule may also cause serious injury to cells and tissues. That is why those electrons are handled with inside special cell compartments such as mitochondria and chloroplasts. Many chemicals (e.g., paraquat, cytotoxic quinones, transition metals) produce ROS, which are strong electron donors. This electrons are donated to molecular oxygen to yield superoxide (•O2−), which may then be metabolized to hydrogen peroxide by the enzyme superoxide dismutase (SOD). 2•O2− + 2H+ → H2O2 + O2

Reactive oxygen species (ROS) may cause cellular injury if not counteracted by antioxidant enzymes

(Eq. 12.2)

The hydrogen peroxide is then removed by catalase (CAT) 2H2 O2 / 2H2 O D O2

(Eq. 12.3)

Reduced GSH can also play a role reducing hydrogen peroxide to water, a reaction catalyzed by glutathione peroxidase (GPx). 2GSH D H2 O2 /GSSG D 2H2 O

(Eq. 12.4)

When the amount of superoxide formed overwhelms the capacity of available SOD, then hydroxyl radicals (OH) are formed. •O2– + H2O2 → •OH + OH– + O2

(Eq. 12.5)

Metals such as iron can also be responsible for the formation of hydroxyl radicals through the Fenton reaction: H2 O2 D Fe2 D D H D / OH D Fe3 D D H2 O

(Eq. 12.6)

Hydroxyl radicals can also be involved in the production of further active oxygen species such as singlet oxygen (1O2), an electronically excited state of molecular oxygen. Superoxide anions (•O2−), hydroxyl radicals (OH), and singlet oxygen (1O2) are termed ROS, and they are cytotoxic because they cause membrane damage by peroxidation of its lipids. The lysosome membrane is affected by this mechanism of toxicity and its permeability becomes increased. ROS may also cause damage to proteins (including inactivation of enzymes) and DNA. The metabolic condition caused by all these ROS is known as oxidative stress, and the enzymes SOD, CAT, and GPx are known as antioxidant enzymes. Antioxidant enzymes, along with low molecular weight ROS scavengers such as GSH or vitamin E, constitute the antioxidant defense system of the organisms. The synthesis of antioxidant enzymes can be induced in the presence of oxidative stress conditions, and hence their activity serves as biomarker of environmental oxidative stress. Increased permeability due to impaired membrane stability in

antioxidant enzymes are induced upon exposure to oxidative stress

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the lysosomes is also used as a biomarker, particularly in organisms prone to accumulation of chemicals, such as bivalves.15 GSH is the main cytosolic ROS scavenger

Scavengers neutralize ROS by direct reaction with them, thus being temporarily oxidized before being reconverted by specific reductases to the active form. Polar scavengers act as antioxidants in the cytoplasm, while liposoluble scavengers arrest the propagation of lipid peroxidation reactions on the membranes. The most abundant cytosolic scavenger is reduced GSH, a tripeptide that directly neutralizes several reactive species through its oxidation to the oxidized form of glutathione (GSSG). The main lipid soluble scavengers are carotenoids and vitamin E.16

12.6 The synthesis of stress proteins and metallothioneins is induced by many sources of cellular stress

METALLOTHIONEINS AND STRESS PROTEINS

The induction of proteins synthesis as a response to any kind of environmental stress (temperature, salinity, hypoxia, chemicals, etc.), has been demonstrated in all organisms examined to date, from prokaryotes to humans. These stress proteins were also termed heat shock proteins because the first one was discovered in Drosophila flies submitted to heat shock.17 Stress proteins are classified according to their molecular weights that cluster around 7e8, 16e24, 60, 70 and 90 kDa, resulting the names Stress90, Stress70, and so on. Proteins around 7e8 kDa are called ubiquitin, and the 60 KDa family has been renamed chaperon 60 (cpn60). Their roles are related with the homeostasis of protein levels, structure, and function, including facilitation of three-dimensional folding needed for normal protein function, and preservation and continuous repair of protein structure upon adverse conditions.18 Despite their ubiquity, for some of them there appears to be a great deal of homology in their aminoacid sequence across taxonomic groups. Thus, ubiquitin, Stress70, and cpn60 seem good candidates as general cellular stress biomarkers because they are inducible, their background levels are low, and their sequence is highly conserved among taxonomic groups.19 Metallothioneins (MT) are heat-stable soluble proteins of low molecular weight (below 7 KDa) containing about 30 percent of cysteine residues preferentially placed in Cys-Cys, Cys-X-Cys, or Cys-X-X-Cys sequence. They are involved in the transport of essential metals present in dissolution as divalent cations such as Cu2þ and Zn2þ. Due to this regulatory role basal levels of MTs are present in unpolluted environments. However, MTs are inducible upon exposure to xenobiotic metals such as Cd, Hg, and Ag in both vertebrates and invertebrates. The divalent cations of these metals are sequestered by the MTs, thus preventing metal toxicity. The MT-metal complexes are synthesized in the liver and transported to the kidney, and show a rapid turnover in the lysosomes. The protease degradation of the complex in the lysosomes of the kidney glomerulal cells releases free Cd and explains the nephrotoxicity that this metal causes in vertebrates.

Endnotes

The induction of MT synthesis upon metal exposure has been used as biomarker of metal pollution. However, unfortunately many other chemical and even physiological sources of stress induce MT synthesis, and they are considered as general stress proteins. Since other chemicals, oxidative stress, and physiological stimuli can also induce MT synthesis, this induction is not by itself diagnostic of metal exposure.20

KEY IDEAS n

Biotransformation of organic xenobiotics normally proceed in a first step of oxidation (Phase I) that introduces a reactive group in the xenobiotic, and a second step of conjugation (Phase II) with a nontoxic endogenous metabolite that yields an excretable product.

n

Phase I of biotransformation is mediated by inducible cytochrome P450 dependent monooxygenases (CYP).

n

In principle, different CYP forms, coded by different genes, are specifically responsible for the degradation of certain families of chemicals.

n

The synthesis of the Phase I and Phase II enzymes involved in the detoxification metabolism is induced by the presence in the environment of chemical pollutants. This can be used as a biological tool for monitoring chemical pollution, and the inducible molecules are considered biomarkers.

n

Exposure to some organics and transition metals cause oxidative stress through the formation of ROS that affect the lysosomes membrane increasing its permeability. This can be used as a quantitative and rather unspecific biomarker of chemical pollution.

n

The antioxidant defense of the organism is composed by inducible antioxidant enzymes (SOD, CAT, and GPx) that metabolize cytotoxic ROS and low molecular weight oxidable molecules (GSH, vitamin E) that directly react with ROS.

Endnotes 1. e.g. Darriba S, Sánchez-Marín P. Lead accumulation in extracellular granules detected in the kidney of the bivalve Dosinia exoleta. Aquat Living Resour 2012;26(1):11e17. 2. Marris ME, Levy G. Renal clearance and serum protein binding of acetaminophen and its major conjugates in humans. J Pharm Sci 1984;73(8):1038e1041; Landrum PF, Lydy MJ, Lee H. Toxicokinetics in aquatic systems: Model comparisons and use in hazard assessment. Environ Toxicol Chem 1992;11:1709e1725. 3. EC. Opinion on 4-methylbenzylidene camphor (4-MBC); 2008. 4. Timbrell J. Principles of biochemical toxicology. 3rd ed. London: Taylor & Francis; 2000. 5. Hahn ME. In: Newman MC, editor. Fundamentals of ecotoxicology. 4th ed. 2015. pp 186e192.

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6. Whitlock JP. Induction of cytochrome P4501A1. Annu Rev Pharmacol Toxicol 1999;39: 103e125. 7. Hahn (2015) op. cit. 8. Bakke JE, Bergman ÅL, Larsen GL. Metabolism of 2,4’,5-Trichlorobiphenyl by the mercapturic acid pathway. Science 1982;217(13):645e647. 9. Foureman GL. Enzymes involved in metabolism of PAH by fishes and other aquatic animals: hydrolysis and conjugation enzymes (os Phase II enzymes). In: Varanasi U, editor, Metabolism of polycyclic aromatic hydrocarbons in the aquatic environment. Boca Raton: CRC Press; 1989. pp. 185e202. 10.

Sheehan D, Meade G, Foley VM, et al. Structure, function and evolution of glutathione transferases: implications for classificatuon of non-mammalian members of an ancient enzyme superfamily. Biochem J 2001;360:1e16.

11.

Foureman GL. Enzymes involved in metabolism of PAH by fishes and other aquatic animals: hydrolysis and conjugation enzymes (os Phase II enzymes). In: Varanasi U, editor. Metabolism of polycyclic aromatic hydrocarbons in the aquatic environment. Boca Raton: CRC Press; 1989. pp. 185e202.

12.

Foureman (1989) op. cit.

13.

Timbrell (2000) op. cit. (p. 99).

14.

Sheehan D. Applications of in vitro techniques in studies of biomarkers and ecotoxicology. In: Mothersill C, Austin B. In vitro methods in aquatic toxicology. Chichester: Springer; 2005. pp. 55e76.

15.

Regoli F. Total oxyradical scavenging capacity (TOSC) in polluted and translocated mussels: a predictive biomarker of oxidative stress. Aquat Toxicol 2000;50:351e361.

16.

Regoli F, Giuliani ME. Oxidative pathways of chemical toxicity and oxidative stress biomarkers in marine organisms. Mar Enviorn Res 2014;93:106e117.

17.

Wright DA, Welbourn P. Environmental toxicology. Cambridge: Cambridge University Press; 2002. (pp. 123-125).

18.

Schüürmann G, Markert B. editors. Ecotoxicology. Ecological fundamentals, chemical exposure, and biological effects. New York: Wiley; 1998. (pp. 543e546).

19.

Newman MC. Fundamentals of ecotoxicology. 4th ed. Boca Raton: CRC Press; 2015.

20.

Roesijadi G. Metallothioneins. In: Newman MC. Fundamentals of ecotoxicology. 4th ed. Boca Raton: CRC Press. 2015; pp. 195e200.

Suggested Further Reading 

Regoli F, Giuliani ME. Oxidative pathways of chemical toxicity and oxidative stress biomarkers in marine organisms. Mar Environ Res 2014;93:106e17.



Sheehan D, Meade G, Foley VM, Dowd CA. Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem J 2001;360:1e16.



Timbrell J. Principles of biochemical toxicology. 4rd ed. London: CRC Press; 2008.



Whyte JJ, Jung RE, Schmitt CJ, Tillit DE. Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure. Crit Rev Toxicol 2000;30(4):347e570.