Glutathione, glutathione S-transferase, and glutathione ... - Springer Link

Environ Sci Pollut Res (2012) 19:2007–2023 DOI 10.1007/s11356-012-0909-x

REVIEW ARTICLE

Glutathione, glutathione S-transferase, and glutathione conjugates, complementary markers of oxidative stress in aquatic biota Jocelyne Hellou & Neil W. Ross & Thomas W. Moon

Received: 26 September 2011 / Accepted: 2 April 2012 / Published online: 25 April 2012 # Springer-Verlag 2012

Abstract Contaminants are ubiquitous in the environment and their impacts are of increasing concern due to human population expansion and the generation of deleterious effects in aquatic species. Oxidative stress can result from the presence of persistent organic pollutants, metals, pesticides, toxins, pharmaceuticals, and nanomaterials, as well as changes in temperature or oxygen in water, the examined species, with differences in age, sex, or reproductive cycle of an individual. The antioxidant role of glutathione (GSH), accompanied by the formation of its disulfide dimer, GSSG, and metabolites in response to chemical stress, are highlighted Communicated by: Henner Hollert J. Hellou Bedford Institute of Oceanography, Department of Fisheries and Oceans, Dartmouth, Nova Scotia, Canada J. Hellou Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada J. Hellou (*) Department of Oceanography, Dalhousie University, Halifax, Nova Scotia, Canada e-mail: [email protected] N. W. Ross Industrial Research Assistance Program, National Research Council of Canada, Halifax, Nova Scotia, Canada N. W. Ross Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada T. W. Moon Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Canada

in this review along with, to some extent, that of glutathione S-transferase (GST). The available literature concerning the use and analysis of these markers will be discussed, focusing on studies of aquatic organisms. The inclusion of GST within the suite of biomarkers used to assess the effects of xenobiotics is recommended to complement that of lipid peroxidation and mixed function oxygenation. Combining the analysis of GSH, GSSG, and conjugates would be beneficial in pinpointing the role of contaminants within the plethora of causes that could lead to the toxic effects of reactive oxygen species. Keywords Antioxidants . Oxidative stress . Conjugates . Metabolites . Glutathione . Review . Analyses . Integration of biomarkers

Introduction Oxygen is reactive and extremely important in the metabolism of aerobic organisms. However, oxygen’s reactivity is a double-edged sword that can lead to the formation of free radicals and other reactive moieties that can oxidize biological molecules such as lipids, proteins, DNA, as well as xenobiotics present within a variety of organisms. These reactive species can also be utilized as defense agents against invading pathogens. Organisms maintain a control over the production of reactive oxygen species (ROS) so as to minimize the cellular damage that they may cause. Oxidative stress is the condition where this balance is tipped because of exposure to, e.g., pathogens or xenobiotics. Antioxidants are at the frontline of cellular defense mechanisms acting to slow down or prevent oxidative stress (Winston and DiGuilio 1991). Antioxidants act by scavenging ROS and reactive nitrogen species associated with oxidative stress experienced by organisms living in an aerobic

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Environ Sci Pollut Res (2012) 19:2007–2023

environment. These compounds can be divided into two classes, one with smaller molecular weight molecules that include vitamins A, C, and E and a second group with larger molecules including enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST). Sometimes these molecules are divided according to their lipophilicity or hydrophilicity, with the larger ones and those with many functional groups belonging to the latter group. The enzymes can also be divided as extracellular or intracellular, where the first line of defense against ROS is on the surface of a cell, such as SOD, and where GST represents a protective mechanism within cells. In aquatic animals, the water-soluble antioxidant glutathione (GSH, 1, structure in Fig. 1) is present in micromolar amounts (Dickinson and Forman 2002), similar

Cl NO2

NO2

1-chloro-2,4-dinitrobenzene (CDNB, 8)

Cl Cl

NO2 1,2-dichloro-4-nitrobenezene (DCNB, 9) OCH2COOH Cl Cl

Glutathione or γ-glutamylcysteinyl glycine (1)

COCCH2CH3 CH2 ethacrynic acid (ETHA, 10)

NH2-CH-(CH2-CH2-COOH)-COOH

Cl

Glutamic acid or Glu (2)

Cl CCl2 NH2-CH-(CH2-SH)-COOH

NH2-CH2-COOH

Cysteine or Cys(3)

Glycine or Gly (4)

Cl

O S O O

Cl Endosulfan (11)

Glu-Cys-Gly

Glu-Cys-Gly

S

Fig. 1 (continued)

S

Glutathione dimer (5)

Glu-COOH-CH-(NH2)-CH2-Gly S-R Glutathione-conjugate (6) with R representing a xenobiotic

Pyrene (7) Fig. 1 Structure of molecules cited in bold numbers in parenthesis in the text

to the levels of polyunsaturated fatty acids (Ackman 1989; Ognjanovic et al. 2008). Antioxidants represent an important molecular protective mechanism, and changes in their concentration are often used as biomarkers of environmental stress. Sometimes, these measurements are used in combination with other biological, biochemical, and chemical analyses (Ruus et al. 2002; Gagné et al. 2007a, b; Yeats et al. 2008; Guimaraes et al. 2009; Petala et al. 2009; “GST, GSH, and/or GSSG in tissues” and “Associations to levels of GSH, GSSG, and GST activity” sections). It is important to highlight that many definitions of biomarkers have appeared in the literature and have been reviewed by van Gestel and van Brummelen (1996). These authors provide a clear outline of the meaning of biomarker as a “biochemical, physiological, histological and morphological measurement of health” due to chemical


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COOH-CH-(NH-CO-CH3)-CH2-S-R

Cl Cl

Cl

Cl

Cl

Acetylated mercapturic acid GSH conjugate (16)

Cl

(CH3 )2 CHNH

Hexachlorobenzene (12)

N N

Cl N

NHCH2 CH3

Atrazine (17)

Benzo(a)pyrene (13)

CN Cl

Cl

Cl

CN Cl

Chlorothalonyl (18) OH

Cl Microcystin LR (14)

4-chlorophenol (19) Fig. 1 (continued)

COOH-CH-(NH2)-CH2-S-R mercapturic acid GSH conjugate (15) Fig. 1 (continued)

exposure. They also say that a biomarker is a measure of health performed below the organism level by sampling tissues or biofluids. In another review with a slightly broader definition, biomarkers are divided into three categories, i.e., as markers of exposure, of effects, and/or of susceptibility (NRC 1987) which has been adopted by some researchers. Many thousands of publications describe the role of GSH and its derivatives (with enzymes included in this group) in diverse topics covering environmental assessments, biochemical processes, genomics, medicine, metabolites, and toxicology, along with specific laboratory or field studies. The interest in GSH goes back to 1935 when the structure of this molecule was confirmed (Harrington and Meade 1935). Recent GSH reviews include a series of nine papers in the issue of a medical journal dedicated to “Glutathione in health and disease” that treats the key roles of GSH in humans. The first article of the series (Forman et al. 2009) covers the protective roles of GSH derivatives and parts of

this text and some of the following ones apply to animals lower in the food web. The most recent publications dealing with ROS and antioxidants are authored by Luschak (2011a, b) and examine the subject from the perspective of genomics. One manuscript is on aquatic organisms, the other compares the mechanism of ROS regulation in bacteria, fungi, plants, and animals. Both point out the need for additional physiological and biochemical understanding of these processes. Hahn and Hestermann (2008) discuss advances in gene expression based on mammalian studies, and Blanchette et al. (2007) review how molecular, immunochemical, and genomic tolls are being used to characterize the GST superfamily of genes in marine organisms. Two broad approaches are available to understand the mechanism of action of GSH in defense against oxidative damage inherent to species living in aerobic environments. One can examine the induction of total GST and/or specific forms of the superfamily of isozymes with substrate specificity, molecular, and immunochemical tools (James et al. 1979; Balabaskaran et al. 1986; Blanchette et al. 2007; Schlenk et al. 2008; “GST, GSH, and/or GSSG in tissues” and “Associations to levels of GSH, GSSG, and GST

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activity” sections) and the second deals with the structure of biotransformation products (Barron et al. 2002; Pflugmacher 2004; Limón-Pachecoa and Gonsebatt 2009; Campos and Vasconcelos 2010; “Examples of studies of GSH metabolites” and “LC-MS analyses of conjugates” sections). The present review approaches this topic from chemical and biochemical perspectives and discusses the benefits of joining these two often separate types of studies, with limited discussion of the genetic research. The second half of the publication emphasizes the less well-researched field of biotransformation, as it relates to GSH conjugates.

Glutathione and link to oxidative stress The tripeptide GSH (1) is made up of glutamic acid (2), cysteine (3), and glycine (4). GSH or, more specifically, Lγ-glutamyl-L-cysteinyl-glycine contains four types of functional groups with the sulfide moiety carrying the reactivity involved in the defensive role against ROS electrophiles. GSH is in a reduced state and acts as a catalyst in the reduction of peroxides of a biogenic or anthropogenic origin. This reaction leads to an intermediate oxidized state (GS) that undergoes the formation of a disulfide dimer, GSSG (5), and takes place with the involvement of GPx, a selenium-containing protein (Eberhardt 2001; Forman et al. 2009). Further on, the glutathione reductase (GR) enzyme reduces the disulfide back to GSH which will once again maintain the reduced cellular environment. The mechanism of detoxification involving GSH is believed to proceed through two distinct pathways, by either forming the GSSG dimer by donating hydrogen to a receptor molecule or forming a conjugate catalyzed by GST. One approach to assess the redox state examines the ratio of GSH to GSSG to determine if oxidative stress has been encountered by a cellular system (Forman et al. 2009). The availability of cysteine (3) is the limiting factor in regenerating GSH, and it is the ratio of (1) to its dimer (5) that is often used (Forman et al. 2009) as a biomarker. The second pathway involving GSH can include a nonenzymatic reaction or one catalyzed by GST, where GSH reacts with an electrophile to generate a biotransformation product (6) (Dringen 2000). Reaction with biomolecules produces proteins with GSH, while reaction with xenobiotics (R in 6) produces diverse GSH derivatives. The GSH metabolites result from a two-step process; the first one is related to the formation of the ROS, an oxidation reaction, phase I (Stegeman and Hahn 1994; Van der Oost et al. 2003) and the second, a conjugation reaction, phase II (Hellou and Payne 1988; George 1994). Phase I mixed function oxygenase (MFO) enzymes are represented by ethoxyresorufin-Odeethylase (EROD) and cytochrome P450 (Stegeman and Hahn 1994). Phase I reactions typically add functional


groups to allow conjugation to a more polar moiety through phase II enzymes and this derivative can then be pumped out or released from cells. Phase II enzymes are numerous and include GST. Phase II conjugation can take place with a number of biogenic molecules leading to the formation of new structures with one or several substituents. The conjugation products can be represented by an addition of acetate, methyl, glucoside, glucuronide, sulfate, or amino acid moieties; GSH conjugation being one more possibility (“Examples of studies of GSH metabolites” and “LC-MS analyses of conjugates” sections). The species involved and the structure of the anthropogenic chemical will play a major role in the type of conjugate that can be formed (Hughes 1996; Lu 1996). GST conjugation can also result from the displacement of a halogen. It should be noted that other GSH-related enzymes are involved in cellular function, as described in more detail elsewhere (e.g., Lu 2009; Franklin et al. 2009). Biotransformation through phase I and II reactions is seen as a detoxification process that will result in the formation of a more water-soluble derivative that facilitates the elimination of a xenobiotic. However, in some cases, phase I metabolites lead to the formation of reactive and toxic compounds, as is the case with quinones deriving from the oxidation of aromatic compounds (Kirby et al. 1990), while in rare cases, the phase II conjugates are toxic (van Bladeren 2000). When the rate of phase II conjugation does not keep up with that of oxidation, an excess of phase I intermediate is produced (Reynaud et al. 2008; Erskine et al. 2010). The reactive intermediate can then bind to macromolecules and deactivate their function, such is the case with proteins acting as enzymes. This change could then lead to observable biological effects (“Deleterious effects and biotransformation” section). The chemicals that undergo the detoxification process of phase II conjugation with GSH have diverse reactive groups that characterize them as electrophiles (“Examples of studies of GSH metabolites” and “LC-MS analyses of conjugates” sections). These molecules can have conjugated double bonds and/or an aromatic moiety, a halide, a nitro group, or an epoxide. The investigation of the nature of the conjugate(s) and/or of the presence of the free xenobiotic(s) determines if and what chemical(s) is involved in a stress response (Barron et al. 2002). The identification of a foreign compound would help pinpoint the cause of oxidative stress and hence allow remedial action to take place. The ability of vertebrates and invertebrates to biotransform, relative to bioaccumulate, xenobiotics represents a delicate balance (Ito et al. 2002; Stroomberg et al. 2003; Rust et al. 2004; Dam et al. 2006; Hellou et al. 2010; Beach and Hellou 2011). Accumulation can lead to effects on a species or its predator(s), while transformation can lead to negative effects on the organism of interest. For example, as demonstrated by our group, the lack of balance between the


bioaccumulation and biotransformation of the ubiquitous contaminant pyrene (7) is linked to behavioral effects (Erskine et al. 2010; Hellou et al. 2010). As reviewed in Hellou (2011), a broad range of behavioral changes induced by contaminants can be diagnostic of population level effects.

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reason, bile metabolites are referred to as biomarkers (Van der Oost et al. 2003). Reynaud et al. (2008) reviewed the interactions between biotransformation in fish and the immune system, which in turn interacts with the nervous and neuroendocrine systems. The authors highlighted the importance of pursuing the relationship between these two aspects of health in ecotoxicological studies.

Deleterious effects and biotransformation Historical context of aquatic research Environmental toxicology learns many facts from medicine, and it is important to highlight the diseases that are associated with variations in the concentration of GSH in humans. These include neurological disorders such as the wellknown Alzheimer’s, Huntington’s, and Parkinson’s diseases (Dringen 2000). Problems with lung inflammation and asthma (Biswas and Rahman 2009), as well as virally induced diseases, cancers, alcoholic liver diseases, cystic fibrosis, and diabetes, while strenuous exercise and psychological stress represent additional triggers for changes in the concentration of GSH and GSSG (Eberhardt 2001; Fraternale et al. 2009; Lu 2009). There are few equivalent health impacts covered in studies of aquatic animals that highlight the diversity of effects. It is important to come back to the fact that an imbalance between the production and elimination of GSH results in the oxidation of lipids as reflected by levels of lipid peroxidation (LPO), changes in proteins, DNA strand breaks, and cellular damage (DiGuilio and Meyer 2008). Effects at a higher level of complexity include physiological alterations, behavioral changes, endocrine disruption, and tumor promotion leading to cancer and death (Kleinow et al. 1987; Van den Berg et al. 2003). The importance of investigating metabolic products associated with exposure to xenobiotics becomes obvious when discussing the fate of drugs in humans. Korfmacher (2004) stated that 95 % of these compounds are metabolized. This also explains the wealth of information generated by research involving pharmaceuticals and the development of analytical approaches to characterize their biotransformation products. Livingstone (1998) provides an elegant discussion of the reasons why biotransformation is an important field of research to pursue on aquatic organisms. He points out that metabolites represent markers of exposure to specific contaminants, that there is a lack of sufficient data on metabolites to accurately model the fate of reactive contaminants, that metabolites are also markers of effects, and that biotransformation studies can help in the fields of animal ecology and evolution. In the effects context, bile metabolites have been associated with the development of DNA adducts involved in the genotoxic path of cancer and can help in the design of toxicity tests (Johnson et al. 1998, 2002). For this

It becomes obvious when searching the literature that studies involving GST activity are more numerous than those concerned with GSH conjugation products. As well, that the measurement of total GST is done more commonly than of the more specific and challenging GST isozymes. The most up-to-date discussion of biotransformation provided by Schlenk et al. (2008) advises caution when interpreting GST activity. It recommends the quantification of metabolites after ascertaining their formation. This advice is partly due to the effect of two sets of variables: endogenous, intrinsic or animal-related, and exogenous, extrinsic or environment-related, on the depletion or induction of GST (“Endogenous and exogenous variables affecting the enzymatic response” section). There is also a tendency to assume that changes in phase I enzymes detected in organisms exposed to contamination reflect the fate and effect of the xenobiotics. However, as demonstrated, especially in medical research, GST is involved in the conjugation of biogenic molecules such as steroids and bile acids without the involvement of phase I enzymes (Winston and DiGuilio 1991; Salinas and Wong 1999). These lipids are affected by the presence of anthropogenic chemicals and this is why biomarkers such as LPO are often part of the battery of tests performed to examine oxidative stress (“GST, GSH, and/or GSSG in tissues” and “Associations to levels of GSH, GSSG, and GST activity” sections). Another reason for caution is that the GST response is generally tested with the model compound 1-chloro-2,4dinitrobenzene (CDNB, 8). However, there are several GST isozymes and it has been shown that not all species react to CDNB and that other model compounds such as 1,2-dichloro-4-nitrobenzene (DCNB, 9) or ethacrynic acid (ETHA, 10) should also be used when measuring GST activity (Stenersen et al. 1987). The choice of a single substrate could limit the perception of induction or lack of GST activity in some species (Ankley et al. 1986; Van der Oost et al. 1996; Henson et al. 2000; Costa et al. 2011). Temperature, pH, and concentration of the substrate in a bioassay (test conditions) will affect the level of activity that is detected. This was nicely illustrated in a study by Vidal and Narbonne (2000) that involved the conditions needed to investigate the GST activity in clams. In the review by

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Schlenk et al. (2008), the authors also highlighted the fact that realistic levels of exposure need to be tested and that it is important to choose contaminants that are found in the field inhabited by an animal species. As with other enzymes, the induction or depletion of GST is concentrationdependent and the response towards a chemical is not always linear or predictable (Petrivalsky et al. 1997; Winters et al. 2005; Jurgella et al. 2006; Ballesteros et al. 2009). The molecular characterization of the GST superfamily in marine organisms has again followed the discoveries in mammalian systems and molecular and immunochemical methods have been used in an attempt to better classify marine GSTs (Blanchette et al. 2007). With the discovery of different GST genes in fish, it is now possible to examine changes in specific GST gene transcripts in response to environmental toxins, as demonstrated in the response by common carp and goldfish to microcystins (MC) (Fu and Xie 2006; Li et al. 2008). A recent study by Crago et al. (2011) reported a significant correlation between caged fathead minnow GST mRNA transcripts and a variety of anthropogenic pollutants across 17 different streams. The variety of stream habitats and range of parameters tested demonstrated the strength of GST mRNA as a key biomarker of exposure. Molecular or immunochemical techniques could be used to characterize the expression of specific marine GSTs in response to various xenobiotics or oxidative stress (Blanchette et al. 2007).

Endogenous and exogenous variables affecting the enzymatic response Oxidative stress can result from changes in temperature, oxygen, or salinity in the aquatic environment or exposure to contaminants. These include metals, pesticides, polycyclic aromatic hydrocarbons (PAH), nitroaromatics, toxins, and chlorinated organic compounds, such as dioxins, furans, and polychlorinated biphenyls (Kleinow et al. 1987; Rudneva et al. 2010) and emerging contaminants such as polybrominated diphenyl ethers (Roberts et al. 2011) and nanomaterials (Chae et al. 2009; Scown et al. 2010). The antioxidant status also differs with aging of individuals and their feeding, swimming, oxygen consumption, and analyzed organs, and sometimes, an adaptation is observed over time. These variables add to the complexity of parameters to be taken into account when interpreting results from field or laboratory assessments. In terms of environmental temperature, a higher sensitivity to oxidative stress was noted in invertebrates and vertebrates living in a polar environment and was reflected by an increased GST activity (Abele and Puntarulo 2004). In this review, the authors pointed out the higher water solubility of oxygen in cold water that could explain the increase in oxidative stress, although this simple explanation is not


entirely confirmed by Speers-Roesch and Ballantyne (2005). In the latter study, antioxidant enzymes (other than GST) were not enhanced in the liver of Arctic fish species relative to temperate ones. Another property associated with fish living in polar environments is the high level of unsaturated fatty acid that can lead to more oxidation and effects as expressed by higher LPO levels. Corroborating this link, when Ross et al. (2001) compared antioxidant enzymes’ activities in the liver and gill of fish collected from various environments with measured seawater characteristics, those at sites with higher dissolved oxygen had higher activity. The role of temperature on toxicity endpoints, including oxidative stress, is the subject of a book by Gordon (2005), where aquatic organisms are discussed in more detail. Studies demonstrate that an increase in age is related to changes in the level of GSH and GST metabolic activity. For example, Canesi and Viarengo (1997) examined three age groups of mussels collected in a river for their oxidative defense capacity in two tissues. Depletion was notable for GSH and GST in gills and for GSH in the digestive gland where levels of GSH were five times higher than gills. The defense capacity against oxidative damage was reduced in older individuals relative to younger individuals; hence, aging was associated with higher oxidative stress in mussels. Unlike the preceding work, antioxidant enzymes including SOD, CAT, and GST analyzed in the blood of seven fish species were not significantly associated with any consistent variation of activity related to age (Rudneva et al. 2010). The ecological and physiological states of the animals were proposed as more important in affecting the antioxidant response. It remains that a relationship between age and metabolic capacity has been observed in some species (Baumann et al. 1990; Ruus et al. 2002) and should be, as discussed by Carney Almroth et al. (2010), taken into account when interpreting results of biomarkers analysis. Radi et al. (1985) discovered that five fish species with herbivorous preferences had lower antioxidant enzyme activities (not GST) than those with omnivorous feeding. The bioaccumulation of contaminants in carnivorous and omnivorous animals is higher than that of species feeding lower in the food web (Ruus et al. 2002). A higher contaminant intake would lead to higher levels of enzyme activity. In work involving another six fish species, Filho et al. (1993) determined a correlation between metabolic activity and the swimming activity of the animals that was reflected by oxygen consumption. Although other antioxidants than GST or GSH were assayed and six tissues were examined, the levels of GSH, GSSG, and GST would also be expected to change (“Endogenous and exogenous variables affecting the enzymatic response” section). There is also seasonality in the antioxidant response, as recently highlighted by Da Rocha et al. (2009) in a study of fish species from Brazil. Enzymatic results varied across


fish species and between tissues, where muscle, liver, and gills were analyzed for biomarkers including GST and GSH. In the fall, fish were less capable to detoxify than in spring and this decrease was from nearly 5 to 30 times lower depending on the tissue examined. The lowest seasonal variation in the concentration of GSH was observed in the liver and the highest in the gills. The more consistent and significant change in GST activity was detected in the muscle of the four fish species where levels were twice as high in spring than fall. The reproductive stage of females had previously been shown to play a role in the activity of phase I enzymes involved in the mechanism of defense against xenobiotics (Snowberger et al. 1991; Elskus et al. 1992). Overall, changes in phase I enzymes generally precede changes to be observed in phase II enzymes. As well, energy invested in reproduction will limit that directed to biochemical defense (Gagné et al. 2007b, 2009). It is also important to point out that the enzymatic response, uptake, and elimination of xenobiotics can vary with exposure time. Untangling these complex interactions to assess ecosystem health necessitates an integrated set of tools.

GST, GSH, and/or GSSG in tissues As indicated in the preceding section, enzymatic activity differs between tissues. Traditionally, the hepatic system of fish has been used to examine phase I and phase II enzymes (Paetzold et al. 2009), while the presence of metabolites is investigated in the gall bladder bile or urine (McKim and Nichols 1994). The mode of entry of a xenobiotic in an organism affects the enzymatic activity detected in a tissue. Water-soluble contaminants taken up through the gills lead to a higher induction in this tissue, while particle-bound chemicals are more available from food and induction could be more pronounced in the digestive system (Van Veld et al. 1997). Overall, induction reflects the relative bioavailability of exogenous compounds in tissues and the circulation of the chemicals in organisms. Depending on the analyzed biomarker or species, the gills, visceral mass, or digestive system, hepatopancreas or liver, or nearly all tissues could be affected by oxidative stress (Yadwad 1989; Vidal and Narbonne 2000; Radi et al. 1985). It has also been shown that the brain is a highly sensitive organ to oxidative stress in fish as in humans. Unfortunately, this organ is not conventionally analyzed. The effect on brain has been explained in humans by the fact that 20 % of oxygen consumption is in the brain, while only 2 % of the body mass is in the brain (Dringen 2000). This could lead to a more apparent impact in the human brain. The higher sensitivity of the brain was shown after exposing fish, Jenynsia multidentata, to the chlorinated pesticide endosulfan (11) added to water (Ballesteros et al. 2009).

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A 24-h exposure to various levels of this compound led to a tenfold increase in GST at the lowest exposure level (0.014 μg/L) in the brain, but with a significant decrease in the muscle, gills, and liver of the animals. At higher doses of 0.072, 0.288, and 1.4 μg/L, enzymatic induction was lower. At all doses, LPO increased in the brain, while it increased in the liver only at the higher doses with no observed change with other exposure levels for the liver, gill, or intestine. These observations indicated that brain was the most sensitive organ to effects. Song et al. (2006) compared the sensitivity of the liver to the brain in the common carp exposed to hexachlorobenzene (12) over 10 days. There were significant reductions in GSH and GST in the brain on all sampling days and at exposures from 2 to 200 μg/L. However, analyses in the liver only displayed an effect at the highest exposure level. The importance of investigating biochemical activity in the brain of animals exposed to anthropogenic chemicals is apparent when one realizes that behavioral effects represent an early warning signal for more pronounced deleterious effects (Hellou 2011). Avoidance is the most effective defense available to an organism. Avoidance can occur by either physical or biochemical means, such as with the antioxidant response, since conjugation is intended to lead to the elimination of the ROS, hence preventing the phase I metabolite from reacting with biomolecules (“Introduction” section). Physical behavioral effects have been associated with metals, pesticides, and many lipophilic contaminants; these would include chemicals that elicit a change in GSH and GST.

Associations to levels of GSH, GSSG, and GST activity Biochemical processes are interconnected and a foreign compound introduced into a living cell can have a cascade of repercussions going from the chemical up to the scale of biological organization to the population, community, and ecosystem levels. A discussion of oxidative stress needs to place a broader context around the changes associated with GSH and GST involved in the conjugation of ROS. Ankley et al. (2010) recommend investigating the mechanism of action associated with toxicity to better define differences detected between species and to be able to predict impacts. These authors recognized the enormity of the task and that only with perseverance and dedication that steps leading to that goal can be accomplished. This recommendation follows on publications pointing out the shortcomings of biomarkers used in isolation to diagnose environmental health (e.g., Forbes et al. 2006). As indicated in the following studies and many, many more that are available in the literature, a battery of tests targeting biomarkers and chemicals are needed as forensic tools to assess ecosystem health.

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The following examples provide the context of research conducted to assess the state of ecosystems. The first example highlights the link between the level of GSH indicating oxidative stress and the level of accumulated contaminants in tissues of mussels. Although bivalves and especially mussels have been used since the 1970s to assess the level of chemicals in their habitat, it is only since the 1990s that they have been examined for effects using biomarkers (Hellou and Gagné 2011). Animals were exposed for 30 days after being transplanted from a reference site in Hong Kong to increasing levels of bioavailable contaminants, as was displayed by the body burden of individuals. The mussels were analyzed for oxidative stress and showed increased levels of CAT, GPx, GR, GST, GSH, and SOD (Cheung et al. 2001). The most significant correlation was observed between the increase in GSH levels and the sum of PAH in the gills followed by GSH and PAH levels in the hepatopancreas. Although some of the other biomarkers were enhanced to various levels in some of the tissues, LPO did not correlate with the body burden of contaminants. The authors commented on the limitations of short-term studies where, in some cases, a depletion of markers was detected and the importance of mimicking a realistic field exposure encountered at urbanized locations. A range of associations can be found between the ubiquitous PAH and proteins. For example, a correlation was found between PAH levels and GST activity in mussels from PAH-contaminated sites (Gowland et al. 2002). Killifish sampled from a creosote-contaminated river also demonstrated higher GST activity (Van Veld et al. 1991; Armknecht et al. 1998). Liver GST-mu expression was found to be elevated in a population of killifish from the Sydney Tar Ponds, a site having high PAH levels (Paetzold et al. 2009). In addition to elevated GST-mu, these fish also demonstrated higher hepatic expression of the phase 1 enzyme, CYP1A, and the ABC transporters, ABCC2 and ABCG2, suggesting a coordinated upregulation of cellular detoxification genes. A major experiment with green-lipped mussels varied the exposure regime and examined the same series of biomarkers as above, with the muscle, gill, and hepatopancreas analyzed (Richardson et al. 2008). Four types of dosing were attempted: continuous, two and four alternating pulses of exposure, four steps with increasing or decreasing levels of exposure, as well as a control and solvent-only exposure. Four PAH and four organochlorine pesticides were spiked in the media with sampling taking place every week, for a total of 4 weeks. Hepatic SOD and GSH displayed the highest correlation with body burden. Higher antioxidant defense was detected in the hepatopancreas than the gill, while LPO was increased in the gill only. Under the experimental conditions, GSH was induced when the body burden was >60 ng/g, lipid


weight. The type of dosing did not matter, but the final bioaccumulation level was important. A study of five deep-sea fish species collected at three stations in the NW Mediterranean used GST activity to assess exposure to contamination (Escartin and Porte 1999). When performed, this investigation was unique in targeting a remote location and in using typical tools employed in near-shore research to assess the exposure of fish to xenobiotics. Activities of phase I and phase II enzymes were analyzed in the liver and specific, more abundant glucuronide and sulfate metabolites were examined in the gall bladder bile of the fish. The analyzed metabolites were represented by five PAH derivatives. The enzymatic activity displayed a high coefficient of variation within species (10–50 %) and results differed between species. However, they were within the range of measurements observed in fish living near contamination in the coastal zone. GST activity was highest in the same species displaying the most elevated concentrations of PAH metabolites. The sum of metabolites was highest in the smallest fish species with lowest condition factor as well as largest liver somatic index. In this case, GST displayed more change than the phase I enzyme (cytochrome P450) and was as sensitive as the measurement of specific bile metabolites. A decade later, nine species of marine fish covering pelagic and benthic feeders were collected from varying depths in the NW Mediterranean and examined for stomach content and swimming capacity along with the antioxidants CAT, GR, GST, and phase I EROD enzyme in the liver (Sole et al. 2009). There were larger interspecies than intraspecies differences in enzyme activities, including that of GST. None of the biomarkers covaried consistently. There was no association between GST and sampling depth of collected fish; however, the highest activities were observed in top predators that would experience a higher exposure to contaminants. A negative correlation was observed between GST and swimming capacity; this physiological state was linked to higher consumption of oxygen and formation of ROS, hence in further defense. This investigation was part of a long-term monitoring program. It added more insight into the parameters affecting the biochemical response of wild fish. A more expanded description of this study by Sole et al. (2010) with 18 species of fish concluded that ecological variables play a significant role in controlling the biomarker response (discussed in “Endogenous and exogenous variables affecting the enzymatic response” section). Another field study sampled yellow eels over four seasons in three estuaries in Portugal, where CAT, GR, GSH, GSSG, GST, LPO, SOD, EROD enzymes, and more markers were analyzed in liver and gills, along with physiological responses (Guimaraes et al. 2009). Water-related parameters such as temperature, pH, salinity, nutrients, and PAH were measured. The results indicated variability in the


impacts with GST, GSH, and GSSG and the ratio of the last two markers being somewhat more pronounced at one location in winter. Another phase of the study regarding the bioavailability of contaminants was recommended by the authors to continue the environmental assessment and provide a better interpretation of the data. Over the past two decades, the fate and effects of nonconventional chemicals coming under the umbrella of pharmaceutical and personal care products and of new persistent organic pollutants due to their continuous discharge have been at the frontline of environmental research (Muir and Howard 2006; Ramirez et al. 2009). This concern was in part motivated by the launch of the Commission on the Registration, Evaluation, Authorization and Restriction of Chemical Substances (REACH 2007) in Europe. The aim of REACH is to be proactive, detect persistent substances, and determine if they are harmful in order to replace them when and if alternatives are available. Population expansion and the disposal of human wastes have focused social and scientific attention on our impact on aquatic ecosystems receiving sewage effluents. Many studies have examined whether aquatic inhabitants of locations receiving effluents experience biological effects, and in a large number of cases, oxidative stress was indicative of impact. Gagné et al. (2007a, b) examined oxidative status, other stress biomarkers, and endocrine disruption in freshwater mussels caged for 60 days near wastewater aeration lagoons. Different levels of effects were observed with all examined endpoints. These included phase I (several cytochrome P450) and phase II enzymes determined in the digestive system, DNA strand breaks in the gills, digestive glands, and gonads, LPO in the digestive gland, and heme oxygenase. LPO was enhanced in all samples, reflecting oxidative damage, and correlated with cytochrome P4501A. GST activity displayed a variable response between sampling sites, but also correlated with cytochrome P4501A (EROD) activities. The culprit chemicals were not investigated; however, research available in the literature that associated contaminants to the studied effects helped to provide the possible causative agents. Another investigation concerning the impact of treated sewage effluents examined toxicological effects in the liver and kidney of trout (Petala et al. 2009). Oxidative stress was measured with GPx, GSH, GST, heme peroxidase, and LPO. The level of impact differed between tissues and with secondary relative to tertiary effluent treatment, when ozonation and coagulation were used. Treatment with chlorination affected GSH in the liver and kidney of trout. As well, GSH and LPO were induced in the liver of fish exposed to secondary effluents, while GST was inhibited. A strong GSH and LPO correlation has been observed with many other animals and exposures to various contaminants and toxins (Pinho et al. 2005; Cazenava et al. 2006; Ognjanovic

2015

et al. 2008; Ballesteros et al. 2009). This is most likely because many ROS contain a hydroxyl radical (HO·) that reacts with lipids and form by-products that damage the cell membrane. The above studies demonstrate the complexity of interpreting the oxidative stress experienced by aquatic organisms. It is also undeniable that chemical stress as experienced by human population expansion and the generation of more chemical wastes lead to oxidative stress. The plethora of intrinsic and extrinsic variables that affect the enzyme measurements is often unpredictable. It can compromise the interpretation of results and unknown relationships are continuously discovered in studies (Gallagher and Sheehy 2000; Sole et al. 2010). As seen in the above examples, chemical analyses help to interpret the results of environmental investigations. They help to sort out the causes associated with effects and decide what further action can take place to enhance the health of ecosystems.

Available analytical approaches to analyze GSH, GSSG, and metabolites Two recent reviews discussed the available techniques for the analysis of GSH and GSSG in biological samples (Iwasaki et al. 2009; Monostori et al. 2009). The methods that were covered included the ones discussed later in this section and capillary electrophoresis. Liquid chromatography coupled to mass spectrometry (LC-MS) appears to be the most sensitive approach with the least manipulations needed to detect the natural products. The lowest limit of detection obtained by tandem MS-MS listed in these reviews is between 0.2 and 2 pmol (or nearly 60 and 600 ng) for GSH and GSSG, respectively (Norris et al. 2001). As pointed out by Norris et al. (2001), GSSG is present in many samples at nearly ten times lower levels than GSH and is more difficult to quantify in a sensitive and selective manner. The reviews by Iwasaki et al. (2009) and Monostori et al. (2009) point out the differences in concentrations reported in the literature and the need to adopt a validated method that would eliminate the formation of artifacts during extraction. They also noted that such a method has been developed by Rossi et al. (2006) using blood. The approach avoids the accidental formation of ROS that affect the actual levels of GSH and GSSG and GSH derivatives to be measured. The approaches described in this section can also apply to analyzing other metabolites mentioned in “Glutathione and link to oxidative stress” section. When investigations concern metabolites in fish or invertebrates, often the gall bladder bile or hemolymph is examined since these biofluids concentrate metabolites prior to their recirculation or elimination (Hellou and Payne 1988; Krahn et al. 1993; Holdway et al. 1995). In humans, urine is the biofluid of

2016

choice. The bile, hemolymph, or urine is also easier to handle in terms of analyses because the liquid media can be examined without the interference of biogenic molecules more abundant in other organs. However, conjugates can be detected in additional tissues such as the liver and digestive system (Hellou and Leonard 2004). Many previous studies employed exposures that were through a single injection, one uptake of spiked food, with a relatively short-term follow-up. This scenario represents an acute type of exposure due to an accidental encounter in the habitat of a species. This would be unlike a continuous exposure potentially leading to a steady state between uptake and elimination processes. In fish, the gall bladder bile reflects the encounter of xenobiotics in the days to a week prior to sampling (Upshall et al. 1993; Hellou et al. 1999; Jonsson et al. 2004). Some studies have used isotope-labeled molecules and differentiated metabolites from parent compounds using liquid extraction and scintillation counting to quantify the labeled chemicals (Tjeerdema and Crosby 1987; Ownby et al. 2005). The solubility and presence of counts in the organic or aqueous layer helped determine if the initial, often more lipophilic, xenobiotic was transformed into a water-soluble conjugate. In some cases, chemical or enzymatic hydrolysis helped confirm the nature of the conjugate, followed by comparing the amount of labeling in the organic and aqueous phases. Gas chromatography (GC) is used to separate volatile chemicals present in an extract and further identify them (Kellner et al. 2004). Separation depends on the size, volume, and three-dimensional structure of molecules with 60 characterized MC structures, are more extensive than those related to contaminants. Biological effects and/or fate have been investigated in many species, over a range of doses, time periods, and exposure conditions (Beattie et al. 2003; Baganz et al. 2004; Pflugmacher 2004; Wiegand and Pflugmacher 2005; Pflugmacher et al. 2005; Cazenava et al. 2006; Malbrouck and Kestemont 2006). An abundant enzymatically formed MC (specifically MC-LR, 14) GSH conjugate produced by a freshwater plant, crustacean, mollusk, fish eggs, and adult fish was identified by Pflugmacher et al. (1998). Matrix-assisted laser desorption/ionization time-of-flight MS was used for the identification of the major biotransformation product, along with the LC fingerprinting of minor metabolites. A decade later, with the development of more sensitive MS techniques, tandem MS-MS was available to detect the parent MC-LR toxin and its GSH conjugate. This monocyclic heptapeptide toxin of the MC family produced by cyanobacteria was investigated in carp that received an intraperitoneal injection (Dai et al. 2008). Fish weighing 265 g were exposed to one dose of 100 μg/kg and the liver and kidney of three animals were analyzed on five occasions over the following 48 h. The two compounds were detected and the concentration of the parent compound diminished from the first hour of sampling after exposure going from 0.59 to nearly 0.22 μg/g (dry weight), with 0.25 μg/g remaining after 24 h. The level of conjugate went from about 0.04 to 0.08 μg/g and maximizing after 24 h. However, at the end of the experiment, it was lower than the initially measured level. This indicated that, in this acute exposure, on a sampling occasion, at maximum (assuming the liver represents 5 % the fish body mass with 80 % water) 5.3 % of the dose was present in liver, where MC was three times higher than the GSH conjugate. On other sampling times, the proportion was around 10–15 to 1 for the parent to conjugate. The method detection limit was 7 ng/g of dry tissue and the limit of quantification was 20 ng/g. Some research has also indicated that GSH conjugates produce mercapturic acid products (15) from the GSH conjugate or the N-acetylated form of these (16) (George 1994; Hinchman and Ballatori 1994; Sciuto 1997; Carney Almroth et al. 2008; Ballesteros et al. 2009). For example, Zhang et al. (2009) sampled snail, shrimp, and fish, the silver carp in lake Tahui, China, where cyanobacteria regularly bloom in summer. The relative proportion of MC-LR (14) and metabolites was investigated in the hepatopancreas of invertebrates and liver, kidney, and intestinal content of fish. The GSH conjugate was generally not detected (limit of

2018

detection of 5 ng/g dry weight) in the three species, but a smaller derivative, the cysteine conjugate, and the parent compound were present in extracts. The concentration of the cysteine conjugate was from 4 to 5 times lower up to 100 times higher than MC-LR. It peaked in July–August reaching nearly 1.5 and 8 μg/g, dry weight for MC-LR-Cys and MC-LR, respectively, in snail, 4 and 0.6 μg/g in shrimp, 25 and 0.1 μg/g in liver, 10 and 0.1 μg/g in kidney, and 15 and 60 μg/g in intestinal extracts of carp. Atrazine (17), a chlorinated heteroaromatic herbicide with two amine groups, is persistent and toxic. Fertilized eggs of zebrafish were exposed to this pesticide at 0, 0.1, 1, 5, and 10 mg/L for 48 h, at four stages during their development. After 1.5, 8, 24, and 48 h, GST activity was measured. Physiological endpoints (deformities) were examined microscopically after 12, 24, 36, and 48 h. As well, the ability of the 24-h embryonic stage to metabolize the pesticide was studied. An inverted U-shaped dose–response curve was detected for microsomal and soluble GST activities that maximized at 5 and 1 mg/L, respectively (Wiegand et al. 2001). Exposure to 0.1 mg/L had a negligible effect on either enzymatic response, while at 5 mg/L, soluble GST activity was decreased relative to the control. Embryonic development was affected at 5 mg/L after 48 h for the number of stages completed, with more visible effects detected earlier in time at higher exposure levels going up to 40 mg/L. The GSH conjugation was examined with in vitro incubation using atrazine at 5 mg/L for the 24h embryos. It displayed a linear increase in conjugation ability when sampled after 0.5, 1, 2, 6, and 12 h, reaching a plateau after 24 h. The metabolite was analyzed by LC-MS using positive electrospray and had GSH substitution at C-1. The above study is unique in covering a series of toxic endpoints and emphasized the generation of numerous developmental effects over the 48 h of experimentation. It would have benefited from additional resources to examine in more detail the relationship between enzyme activity, conjugation, and the generation of physiological effects. This is because conjugation ability is also dose-dependent, with a balance between bioaccumulation and biotransformation capacity. Biotransformation is also accomplished by microorganisms. For example, the chlorinated fungicide chlorothalonil (18) was transformed into three metabolites by a soil bacteria, where the mono-, di-, and tri-glutathione derivatives were tentatively identified using relative retention times and positive atmospheric pressure ionization MS (YoungMog et al. 2004). With this pesticide, conjugation replaced chlorine atoms in the molecule by GSH. This detoxification process started within seconds of exposure by forming a diconjugate. This derivative was more abundant than the other two over the 24-h period of sampling. Another publication concerning synthetic GSH derivatives describes the advantage of using negative ionization


electrospray LC-MS to characterize the presence of GSH conjugates (Dieckhaus et al. 2005) relative to the earlier approaches of positive ionization. The difference between these two approaches is that, by focusing on a common fragment generated by GSH (1) and GSH conjugates (6), a scan for the precursor ions of m/z 272 (GSH−), representing the molecular ion and fragment ion of (6), would identify the presence of monoconjugate or multiconjugate without pinpointing the target compound. This would permit the detection of unknown conjugates of a biogenic or exogenous origin in a sample. An additional step represented by positive ionization would confirm the mass of the metabolites. Various phenols were used in the exposure of duckweed where several types of conjugates were produced, with glucoside representing the major compound (Fusijawa et al. 2010). Exposure to 4-chlorophenol (19) led to the formation of a relatively small amount of GSH metabolite compared to the parent compound (1:4) over a 24-h period, with half as much glucoside (0.5) produced as well. Structural proof was ascertained by negative electrospray LC-MS and using cellulase and β-glucosidase enzymatic hydrolyses. As outlined in the above examples, the availability of MS instrumentation opens the door to pursuing the presence of various forms of a xenobiotic in an extract. The analytical targets are determined by the researcher and can easily include the natural products GSH and GSSG. The broad range of fates that indicate effects has led to development of a new field of research, i.e., metabolomics (van Ravenzway et al. 2007). Metabolomics consists of studying the fingerprint of metabolites regarded as molecules of