Clements: “3357_c003” — 2007/11/9 — 12:42 — page 23 — #1 3 Biochemistry of Toxicants All chemical pollutants must initially act by changing structural and/or functional properties of molecules essential to cellular activities. (Jagoe 1996) 3.1 OVERVIEW Two themes are often explored in expositions of biochemical toxicology: the nature of the biochemical change and the mode of toxic action. Relative to the nature of the change, biochem- ical changes such as those associated with cytochrome P450 monooxygenases, metallothioneins, or stress proteins are considered in the context of general toxicant detoxification or sequestration phenomena. Other changes such as DNA adduct formation, enzyme inhibition, or lipid peroxidation might be viewed as evidence of a particular mode of action resulting in damage. Consequently, tox- icants sharing a common mode of action are discussed together, such as the coplanar polychlorinated biphenyls (PCBs), dioxins, and furans whose common mode of action involves the aryl hydrocarbon receptor (Lucier et al. 1993). The discussion here will adopt these organizing themes because doing so facilitates integration of the chapter’s content with the rich mammalian toxicology literature that is similarly organized. But, in keeping with the series emphasis on interlinking phenomena, chapter topics will also be described in an information transfer context (Figure 3.1 and also Figure 36.1 in Chapter 36). The fields describing relevant levels of information transfer and complexity are genomics → transcriptomics → proteomics → metabolomics → bioenergetics or biochemical physiology → molecular toxicology. All these areas of study explore different, yet linked, levels of organiza- tion relative to biological information flow and complexity. Genomics explores the entire nuclear DNA complement and variations within it. 1 Toxicogenomics specifically focuses on the influence of toxicants on the nuclear DNA. The next level of the biochemical information flow emerges at transcription. Transcription initiation occurs when RNA polymerase attaches to promoter regions of DNA. Nucleotides are added according to the DNA base sequence to produce mRNA during the elongation step of translation that ends with mRNA release. Transcriptomics attempts to describe and explain the complement of mRNA transcripts and their abundances present in cells or tissues under various conditions. Through translation, pools of various proteins are created in the cytoplasm. Proteomics is the study of the full complement of these proteins, their relative abundances, changes, and interactions. Finally, metabolomics attempts to explain the metabolite complement in cells or tissues under various conditions, including toxicant exposure. Repeating an important theme in this book, the greatest insight is gained by applying combinations of these approaches to a research question. 1 Despite the focus here on nuclear DNA, mitochondrial DNA can also provide valuable information about contaminant effects. Baker et al. (1999) quantified genetic damage in voles from the contaminated area surrounding the Chernobyl reactor using a portion of the mitochondrial cytochrome b gene. They measured heteroplasmy (DNA sequence variation within an individual) to suggest increased rates of somatic mutation in the liver of irradiated voles. 23 © 2008 by Taylor & Francis Group, LLC Clements: “3357_c003” — 2007/11/9 — 12:42 — page 24 — #2 24 Ecotoxicology: A Comprehensive Treatment DNA RNA Proteins Metabolites Energy currency, structural and storage molecules By-products and dysfunctional molecules Function or purpose Associated process Metabolism (anabolism and catabolism) Translation TranscriptionExcretion, respiration, detoxification, and sequestration Maintain soma, control aging Maintain and increase soma, reproduction Cellular information processing FIGURE 3.1 Hierarchical organization of biochemical effects discussed in this chapter. The genome contains the instructions for growing and maintaining the soma. Although genomics often focuses on consequences to the germ line, somatic risks are also created by toxicant-induced changes to the genome. Carcinogenesis gives rise to the most obvious somatic risk (see Burdon (1999) for a fuller treatment of this topic). Changes in the genome will be discussed below relative to toxicant-induced modification of the DNA molecule. Transcription and translation activities can provide evidence of response to a toxicant. As an example, El-Alfy and Schlenk (1998) discovered that up-regulation of a monooxygenase in Japanese medaka (Oryzias latipes) explained salinity-enhanced toxicity of aldicarb. In another study, differ- ences in cytochrome P450 1A induction for chub (Leuciscus cephalus) populations with different contaminant exposure histories was taken as evidence of pollutant-induced changes in population genomics (Larno et al. 2001). Shifts in metabolites can also suggest effects of, or responses to, toxicants. Kramer et al. (1992) measured glycolysis and Krebs cycle metabolites in mosquitofish (Gambusia holbrooki) exposed to mercury, finding decreased Krebs cycle flux during exposure. De Coen et al. (2001) noted increased Krebs cycle activity during Daphnia magna exposure to lindane, suggesting that biochemical assays be used to define the metabolic state of daphnids under stress. Proteomics also has diverse applications in biochemical toxicology. Examples range from indu- cible detoxification proteins to evidence of effects at higher levels of organization.Aspecific example of evidence of potential effect at a higher level of biological organization is the abnormal induction of the egg protein, vitellogenin, in male fish exposed to methoxychlor (Schlenk et al. 1997) or synthetic estrogens (Schultz 2003). This induction will be discussed again in the following chapters in the context of endocrine dysfunction. Processes ensuing at higher levels of biological organization can manifest as shifts in biochemical pools. Stressor-induced changes in bioenergetics can be detected with shifts in energy storage or pools of high-energy molecules. Biochemical by-products can also be assessed in cells, tissues, and physiological fluids. These types of biochemical shifts (e.g., shifts in heme biosynthesis) will also be © 2008 by Taylor & Francis Group, LLC Clements: “3357_c003” — 2007/11/9 — 12:42 — page 25 — #3 Biochemistry of Toxicants 25 discussed. The discussion of cellular, tissue, and bioenergetic effects detected with biochemical qualities will be addressed again in chapters exploring these higher levels of biological organization (i.e., Chapters 4–6). 3.2 DNA MODIFICATION Damage to DNA occurs in several ways. It can result from strand breakage and subsequent imperfect repair. Damage can also result from chemical bonding directly to the DNA or by some similar DNA modification. Although cancer is a paramount concern relative to somatic risk following toxicant-induced DNA modification, some DNA changes to the germ line have population consequences, and in some cases, these germ line-associated changes affect an exposed individual’s Darwinian fitness. The population ecotoxicology section describes such changes and their consequences. As an example, men working in certain conditions or occupations can have elevated risks of teratogenic effects in their children or of their children developing cancer (Gardner et al. 1990, Stone 1992). In an even broader context, the mutation accumulation theory proposes that the accumulation of genetic damage determines the rate of aging for individuals (see Medvedev (1990) for details). Somatic longevity may be determined by DNA modifications accrued during an individual’s life. DNA can be damaged by contaminants or their metabolites that are free radicals or can facilitate free radical 2 generation. Free radicals can break one or both strands of the DNA molecule, or can oxidize bases in the DNA molecule. As an example of manifest breakage, Shugart (1996) noted elevated levels of double-strand breaks in DNA of sunfish from contaminated reaches of East Poplar Creek (Tennessee).As an example of base modification, Malins (1993) reported high concentrations of the guanine product, 2,6-diamino-4-hydroxy-5-formaminidopyrimidine, in tumors of English sole exposed to carcinogens in the field. Contaminants or their metabolites can also bind covalently to DNAto form adducts. For example, Ericson and Larsson (2000) found DNA adducts in perch caught below a Kraft pulp mill. As another important example, metabolites of the carcinogen benzo[a]pyrene combined with guanine to form a guanosine adduct. Still other modes of DNA damage are possible. Mercury cross-links DNA with proteins. Some metals bind to phosphate groups and heterocyclic bases of DNA. This changes the stability of the molecule and increases the incidence of mismatched bases. Damage, modification, and imperfect repair of protooncogenes or tumor suppressor genes can initiate carcinogenesis (Burdon 1999). It can also accelerate the rate at which somatic mutations accumulate, and in doing so, accelerate the rate of aging. Genomic damage changes cell functioning and ultimately influences individual fitness. 3.3 DETOXIFICATION OF ORGANIC COMPOUNDS A wide range of organic contaminants are transformed within organisms. The design behind such transformations is to render the toxic chemical more amenable to elimination; however, this is not always achieved without adverse consequences. The products of detoxification reactions can sometimes be more toxic or reactive than the original compound. Such a transformation that makes an inactive compound bioactive or an active compound more bioactive is called activation. In the case of cancer-producing agents, the original compound is a procarcinogen and the cancer-causing metabolite is called the carcinogen. Detoxifying reactions are often classified as Phase I or II reactions. Phase I reactions produce a more reactive, and sometimes more hydrophilic, metabolite from the original compound; the product 2 Free radicals are extremely reactive molecules possessing an unshared electron. © 2008 by Taylor & Francis Group, LLC Clements: “3357_c003” — 2007/11/9 — 12:42 — page 26 — #4 26 Ecotoxicology: A Comprehensive Treatment is more amenable to further reaction and, in some cases, elimination. The reactive groups −− OH, −− NH 2 , −− SH, and −− COOH are added or made available by oxidation, hydrolysis, or reduction. Products of a Phase I reaction can be eliminated directly, be subject to additional Phase I trans- formations, or undergo Phase II transformations. Phase II reactions conjugate the compound or its Phase I metabolite(s) with some compound such as acetate, cysteine, glucuronic acid, sulfate, gly- cine, glutamine, or glutathione. The conjugate is more hydrophilic and readily eliminated than the compound was before conjugation. 3.3.1 PHASE IREACTIONS In Phase I, reactive groups are added or existing sites are made more readily available to further reactions. This can be illustrated with the metabolism of the dioxin benzo[a]pyrene (Figure 3.2). The addition of oxygen by the microsomal mixed function oxidase system (MFO, also referred to as the cytochrome P450 monooxygenase system) is the most prominent Phase I reaction. The cyto- chrome P450 system is present in diverse species from bacteria to vertebrates, and functions in the metabolism of endogenous (e.g., steroids and fatty acids) as well as xenobiotic compounds (Synder 2000). Associated Phase I oxidations involve two membrane-bound enzymes (cytochrome P450 isozymes and NADPH–cytochrome P450 reductase), NADPH, and molecular oxygen. The epoxida- tions of benzo[a]pyrene to benzo[a]-4,5-oxide, benzo[a]-7,8-oxide, and benzo[a]-9,10-oxide shown in Figure3.2 areachieved bythe MFO system. The MFOsystem isalso responsible for the conversion of benzo[a]pyrene-7,8-dihydrodiol to benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide. Phase I enzymes also include epoxide hydrolases, esterases, and amidases that expose existing functional groups on compounds (George 1994). For example, epoxide hydrolase is responsible for Bay region K Region O O Benzo[a]pyrene-9,10-oxide Benzo[a]pyrene Benzo[a]pyrene-7,8-dihydrodiolBenzo[a]pyrene-7,8-oxide Benzo[a]pyrene-7,8-dihydrodiol-9,10-oxideBenzo[a]pyrene-4,5-oxide O O O HO HO HO HO FIGURE 3.2 Phase I reactions for benzo[a]pyrene. © 2008 by Taylor & Francis Group, LLC Clements: “3357_c003” — 2007/11/9 — 12:42 — page 27 — #5 Biochemistry of Toxicants 27 the Phase I conversion of benzo[a]pyrene-7,8-oxide to benzo[a]pyrene-7,8-dihydrodiol, shown in Figure 3.2. Epoxide hydrolase catalyzes the addition of water to MFO-generated epoxides. Other enzymes such as alcohol and aldehyde dehydrogenases, aldehyde oxidases, and carbonyl reductase generate products that are more rapidly eliminated than the original compound (George 1994, Parkinson 1996). As an example, ethanol is oxidized to acetaldehyde by alcohol dehydrogenase. This aldehyde is then oxidized by aldehyde dehydrogenase to acetic acid. Type I reactions can also activate compounds to produce more poisonous or carcinogenic ones (Figure 3.2). The epoxide formed at the K region of benzo[a]pyrene (e.g., the epoxide in benzo[a]pyrene-4,5-oxide) and bay region dihydrodiols (e.g., benzo[a]pyrene-7,8-dihydrodiol) of polycyclic aromatic hydrocarbons are potent carcinogens (Timbrell 2000). These products of benzo[a]pyrene metabolism are strong electrophiles that bind to guanosine in the DNA molecule. Formation of such adducts within protooncogenes can result in cancer. Another example of Phase I activation is MFO-mediated epoxidation of the organochlorine pesticide aldrin to produce the more toxic dieldrin (Chambers and Yarbrough 1976). 3.3.2 PHASE II (CONJUGATIVE)REACTIONS In Phase II reactions, endogenous compounds are conjugated with contaminants or their metabolites to detoxify them or to accelerate their elimination. Phase II conjugation can occur without any Phase I reactions if the appropriate groups are already available. A compound is made more polar by binding it to some amino acid, carbohydrate derivative, glutathione, or sulfate. However, Phase II reactions can also involve methylation or acetylation that does not generally increase hydrophilicity. Many Phase II reactions produce hydrophilic compounds readily eliminated from the indi- vidual. Conjugates are commonly organic anions that are eliminated by glomerular filtration and tubular transport in vertebrates (James 1987). Conjugation with glucuronic acid by UDP- glucuronosyltransferases involves generation of a polar, hydrophilic glucuronide by combining the compound with uridine diphosphate-glucuronic acid. As a relevant example, stimulated by con- cern about birth control compounds released from sewage treatment plants into waterways, Schultz (2003) studied the conjugation of the synthetic estrogen 17α-ethynylestradiol after its injection into trout. Sulfate conjugation by sulfotransferases produces hydrophilic conjugates of polyaromatic compounds, aliphatic alcohols, aromatic amines, and hydroxylamines. Xenobiotics with aromatic or aliphatic hydroxyl groups are prone to such sulfation (James 1987). Amino acids may be con- jugated to carboxylic acid or aromatic hydroxylamine groups of contaminants or their metabolites. The amino acids most often involved are glycine, glutamine, and taurine (Jones 1987). Glutathione (i.e., glycine–cysteine–glutamic acid) can be conjugated by glutathione S-transferases with a wide array of electrophilic compounds. As examples, the benzo[a]-9,10-oxide and benzo[a]-4,5-oxides shown in Figure 3.2 can undergo further Phase I transformations and the products of these reactions conjugated with glutathione. In contrast to the Phase II reactions just described, Phase II methylation and acetylation are reactions thatdo notgenerally producemore hydrophilicproducts. The reader is directed to Parkinson (1996) for more details about such reactions. Box 3.1 There Is More to It Than Phase I and II Reactions Our understanding ofreactions associated withxenobiotic conversion andelimination has grown to include those outside the conventional Phase I and II reactions. The associated mechanisms have been referred to as Phase III reactions (Zimniak et al. 1993). The ATP-dependent gluta- thione S-conjugate export pump described byIshikawa (1992) facilitates a Phase III reactionthat removes xenobiotic Phase II metabolites from the cell. Probably the best Phase III example is the membrane-associated P-glycoprotein (P-gp) that acts as an energy-requiring efflux pump for © 2008 by Taylor & Francis Group, LLC Clements: “3357_c003” — 2007/11/9 — 12:42 — page 28 — #6 28 Ecotoxicology: A Comprehensive Treatment xenobiotics and is described by Bard (2000) as the cell’s first line of defense. It also eliminates metabolites from Phase I and II reactions from cells. The P-gp mechanism for xenobiotic removal is similar to the multidrug resistance (MDR) transporter protein discovered first in cancer cells that had become resistant to chemotherapeutic agents. The cancer cell resistance results from reduced intracellular concentrations of these chemotherapeutic agents due to the overexpression of an efficient ATP-dependent membrane- bound pump, P-gp. This 170-kDa protein not only increases resistance to the original anticancer drug, but also improves resistance to unrelated chemotherapy agents. The P-gp acts as a bar- rier to xenobiotic absorption and accelerates their removal if they gained entry into the cell (Abou-Donia et al. 2002). The mammalian P-gp is expressed at high levels in the kidney, adrenal glands, liver, and lungs. Expression in mammalian brain capillary endothelial cells has also been shown to reduce neurotoxicity of the pesticide ivermectin (Sckinkel et al. 1994). The multixenobiotic resistance (MXR) mechanism is similar to MDR, involving a membrane-associated transport P-gp that removes moderately hydrophobic, planar compounds (Segner and Braunbeck 1998). Bard (2000) defines its substrates as “moderately hydrophobic, amphipathic (i.e., somewhat soluble in both lipid and water), low molecular weight, planar molecules with a basic nitrogen atom, cationic or neutral but never anionic, and natural products.” P-gp can be induced during exposure to xenobiotics and has regulatory genes in com- mon with the cytochrome P450 system. It has been found in mussel (Mytilus galloprovincialis) cell membranes, leading Kurelec and Piv ˇ cevi ´ c (1991) to speculate that this mechanism could account for the relatively high tolerance of these mussels to contaminants. The MXR gene was also found recently in marine fish (Anoplarchus purpurescens) (Bard et al. 2002), Mytilus edulis (Luedeking and Koehler 2004), and the Asiatic clam, Corbicula fluminea (Achard et al. 2004). Their levels have been correlated with elevated concentrations of a variety of toxicants ranging from crude oil (Hamdoun et al. 2002) to metals (Achard et al. 2004). Induction by metals likely reflects the fact that protein-damaging chemicals induce several systems simultaneously, including stress proteins, MXR, and cytochrome P450. How does the P-gp work? A “flippase” model was proposed by Higgins and Gottesman (1992) in which the xenobiotic binds to the P-gp at the inner surface of the cell membrane and is “flipped” via an energy-requiring mechanism to the outside surface of the cell membrane. The MXR’s presence in many taxonomic groups and its role in detoxification of many con- taminants led Smital and Kurelec (1998) to define a new group of pollutants, that is, those that modify the MXR response. In the laboratory, MXR can be readily inhibited with verapamil, so there is potential for some environmental chemicals doing the same. A water-soluble fraction of weathered crude oil, for example, appears to competitively inhibit MXR in larvae of the marine worm, Urechis caupo (Hamdoun et al. 2002). Bard (2000) reviewed reports of such chemo- sensitizers (Smital and Kurelec 1998), listing the following contaminants: pentchlorophenol, 2-acetylaminofluorene, diesel oil, and several pesticides (chlorbenside, sulfallate, and dacthal). 3.4 METAL DETOXIFICATION, REGULATION, AND SEQUESTRATION Predicting the consequences of metal exposure is complicated because metals may be essential or nonessential. Very low concentrations of essential metals 3 can be as harmful as high concentrations (Figure 3.3, upper panel). Nonessential metals display more conventional toxicity curves, showing 3 The essential metals are currently believed to be Co, Cr, Cu, Fe, Mn, Mo, Ni, Se, V, and Zn (Fraústo da Silva and Williams 1991, Mertz 1981). © 2008 by Taylor & Francis Group, LLC Clements: “3357_c003” — 2007/11/9 — 12:42 — page 29 — #7 Biochemistry of Toxicants 29 Tox icity Metal concentration Mortality (proportion dying) Optimal Deficiency Toxicity Sublethal FIGURE 3.3 Mortality versus concentration for essential (upper panel) and nonessential (lower panel) metals. A deficiency occurs if an essential metal is present below a certain concentration. This is not the case for a nonessential metal. An essential metal will have an optimal range above and below which mortality begins to be expressed. Increasing concentrations of the nonessential metal will increase the level of mortality experienced in a group of exposed individuals. There might or might not be an apparent threshold concentration below which no effect is expressed. a sigmoidal increase in proportion of exposed individuals dying with an increase in metal concentra- tion (Figure 3.3, lower panel). Essential metal deficiencies manifest in many ways other than death. For example, insufficient intake of copper or zinc causes immunodeficiencies in mice (Beach et al. 1982, Prohaska and Lukasewycz 1981). Understanding this dichotomy of essential and nonessential metal concentration–effect curves can still be insufficient for sound prediction of metal effects. For example, chronic exposure to the nonessential element cadmium can cause symptoms of zinc deficiency because cadmium displaces zinc in metalloenzymes. Excessive amounts of nonessential tungsten can cause an apparent defi- ciency of molybdenum, an essential and chemically similar element (Mertz 1981). Such an effect would appear as a shift to the left for the curve shown in the upper panel of Figure 3.3 (x-axis being the essential metal concentration). The bioactivity of some nonessential elements can also be affected by another element. For example, mercury toxicity is lowered if sufficient concentrations of selenium are also present. This would cause the curve in the lower panel of Figure 3.3 to shift to the right. Excess metals are dealt with in two ways, elimination or sequestration. Sequestration can involve metal complexation with proteins or incorporation into granules. Sequestration in granules will be discussed in the next chapter. Biomolecules involved in lessening metal intoxication will be described here. Metallothioneins are low-molecular-weight, cytosolic proteins that take up and facilitate trans- port, sequestration, and excretion of metals such as cadmium, copper, silver, mercury, and zinc. They © 2008 by Taylor & Francis Group, LLC Clements: “3357_c003” — 2007/11/9 — 12:42 — page 30 — #8 30 Ecotoxicology: A Comprehensive Treatment have high cysteine content, giving them the ability to form metal–thiolate clusters. Elevated metal concentrations induce the production of metallothioneins to levels above those needed for normal metal homeostasis. Metallothioneins bind metals, lowering the concentrations of metal available to interact with sites of adverse action. Titers of metallothionein-coding mRNA or metallothionein itself are often used as biomarkers of response to elevated metal concentrations. Phytochelatin serves a similar protective role in plants. Phytochelatins are peptides of the form (γ-glutamic acid–cysteine) n -glycine where n = 3, 5, 6, or 7 (Grill et al. 1985). Elevated concen- trations of other phytochelatin-like peptides have recently been found in zinc-tolerant green algae (Pawlik-Skowro ´ nska 2003). 3.5 STRESS PROTEINS AND PROTEOTOXICITY The adverse effects of some agents result from protein damage (proteotoxicity). Indeed, this mode of action is so pervasive that a general cellular stress response has evolved in most animal, plant, or microbial species. Early studies of the stress-induced synthesis of protective proteins involved the heat shock reaction—the organisms’ response to an abrupt change in temperature (Craig 1985). Consequently, the proteins involved were first referred to as heat shock proteins. However, we now know that a wide range of agents stimulate their production, including metals, metalloids, ultraviolet (UV) radiation, and diverse organic compounds such as amino acid analogs, puromycin, and ethanol (Hightower 1991, Sanders and Dyer 1994, Vedel and DePledge 1995). Because of their induction by stressors other than heat, these proteins are now referred to as stress proteins. They function to facilitate normal protein folding, protection of proteins under conditions that might lead to denaturation, repair of denatured proteins, and movement of irreparably denatured protein to lysosomes (Sanders and Dyer 1994). 4 Some stress proteins are present at basal levels but others are present only after induction by some agent. Regardless of whether they were present under normal conditions or induced by proteotoxic conditions, they collectively function to maintain homeostasis by fostering essential protein levels, structure, and function. The stress proteins are classified and named based on their molecular size. Stress70 and Stress90 are 70 and 90 kDa stress proteins, respectively. Smaller (60 kDa) stress proteins are called chaperons owing to their role in mediating proper protein folding. Chaperons are abbreviated cpn60 (Di Giulio et al. 1995). Stress70, Stress90, and cpn60 are present at basal levels that increase to reduce pro- teotoxicity on appropriate induction. Another group of stress proteins (20–30 kDa) are the Low Molecular Weight (LMW) stress proteins that are present only after induction. Proteomic analysis of stress proteins is advocated by Sanders and Dyer (1994) for potentially identifying agents responsible for adverse impact on species in the field. Their argument was based on the observation that different chemicals induce different stress proteins to varying degrees. Comparison of stress protein expression in field organisms to those of organisms exposed to each candidate toxicant individually in the laboratory could provide causal insight. For example, Vedel and DePledge (1995) measured Stress70 increase in crabs (Carcinus maenas) after laboratory copper exposure. Currie and Tufts (1997) explored the combination of anoxia and heat stress on Stress70 induction in trout (Oncorhychus mykiss) red blood cells. Still other researchers focus on stress protein genomics. Hightower (1991) made the novel suggestion that we could use the change in heat shock protein genomes of various species to track the consequences of global warming. He hypothesized that, as suggested by laboratory studies and field studies of desert species, the heat shock genes will move in the direction of overexpression with adaptation to rapid warming. 4 Because our focus is chemical toxicology, other stress proteins will be ignored here. However, it should be mentioned for the sake of completeness that glucose-regulated proteins (GRPs), metallothionein, hemeoxygenase, and the multidrug- resistant p-glycoprotein are considered by many to be stress proteins (Di Giulio et al. 1995, Hightower 1991, Sander and Dyer 1994). © 2008 by Taylor & Francis Group, LLC Clements: “3357_c003” — 2007/11/9 — 12:42 — page 31 — #9 Biochemistry of Toxicants 31 3.6 OXIDATIVE STRESS Molecular oxygen is both benign and malign. On the one hand it provides enormous advantages and on the other it imposes a universal toxicity. This toxicity is largely due to the intermediates of oxygen reduction, that is, O • − 2 ,H 2 O 2 , and OH • , and any organism that avails itself of the benefits of oxygen does so at the cost of maintaining an elaborate system of defenses against these intermediates. (Fridovich 1983) A price was levied when much of the life on Earth took on the energetic advantage of using molecular oxygen as a terminal electron acceptor for respiration. Very reactive, free oxyradicals 5 and oxyradical-producing molecules suchashydrogen peroxide aregeneratedduring aerobic metabolism. Oxyradicals oxidize lipids, proteins, and DNA, causing diverse effects ranging from membrane damage to enzymedysfunction tocancer to accelerated aging. Consequently, organisms usingaerobic respiration had to develop ways of coping with oxidative stress. Oxidative stress is reduced in two ways. Antioxidant molecules are produced that react with oxyradicals and enzymes are synthesized that consume oxyradicals or oxyradical-generating chem- icals.Antioxidantsinclude catecholamines, glutathione, uric acid, andVitaminsA, C,andE. Enzymes include superoxide dismutase, catalase, and glutathione peroxidase that catalyze the reactions shown in Equations 3.1–3.3, respectively. (The unpaired electron in free radicals is designated as a dot by convention. GSH and GSSG in these equations are reduced and oxidized glutathione, respectively.) 2O • − 2 +2H + → H 2 O 2 +O 2 (3.1) 2H 2 O 2 → 2H 2 O + O 2 (3.2) 2GSH +H 2 O 2 → GSSG + 2H 2 O (3.3) The removal of hydrogen peroxide, which is not itself an oxyradical, is crucial because it produces the hydroxyl radical (OH • ). This is accomplished through the Fenton reaction which, catalyzed by a transition metal ion, generates OH • and OH − from H 2 O 2 (Equation 3.4). The transition metal ion can be Cu(I), Cr(V), Fe(II), Mn(II), or Ni(II) (Gregus and Klaassen 1996). H 2 O 2 +Fe 2+ → Fe 3+ +HO − +HO • (3.4) Why is this discussion relevant to environmental toxicants? Many organic chemicals become free radicals during biochemical reactions or can generate oxyradicals. For example, paraquat reacting within the MFO system becomes a charged free radical that reacts with molecular oxygen to produce the superoxide anion, O • − 2 . After reacting with molecular oxygen, the paraquat becomes available again to enter the same reactions, producing more superoxide anions each time it passes through the redox cycle. Another example is carbon tetrachloride, which is converted to the trichloromethyl radical (CCl 4 + e − → CCl • − 3 + Cl − ) during Phase I reactions (Slater 1984). As a final example, enhanced oxidative damage at high metal concentrations occurs due to hydroxyl radical formation. In such a case, more metal ion is available to catalyze the Fenton reaction and more oxyradicals are formed as a consequence. Responses to oxidative stress are used with field and laboratory exposures as evidence for xeno- biotic hazard (Livingston et al. 1990, Winston and Di Giulio 1991). As an example, glutathione and antioxidant enzymes shifted in mussels (M. galloprovincialis) transplanted from clean to metal- contaminated conditions (Regoli and Principato 1995). Regoli (2000) later used the total oxyradical scavenging capacity of mussels to indicate adverse effect of field exposure to metals. 5 A free radical is a charged or uncharged molecule or molecular fragment that has an unpaired electron (Slater 1984). An oxyradical is a free radical in which the unpaired electron is associated with an oxygen atom. © 2008 by Taylor & Francis Group, LLC Clements: “3357_c003” — 2007/11/9 — 12:42 — page 32 — #10 32 Ecotoxicology: A Comprehensive Treatment 3.7 ENZYME DYSFUNCTION Metals inhibit many types of enzymes that range in function from facilitating digestion (Chen et al. 2002) toheme synthesis (Dwyer et al. 1988). Eichhorn (1975) and, more extensively, Fraústo da Silva and Williams (1991) provide details about metal binding to, and modifying the activity of, enzymes. Ametal can displace another metal from an enzyme’s active site or otherwise interact with the enzyme to change its secondary or tertiary structure. Metal ions can produce dysfunction by either increasing or decreasing enzyme activity (Brown 1976, Eichhorn 1975). Organic contaminants can also modify enzyme activity and, in so doing, modify an exposed individual’s fitness. For example, brain cholinesterase activity was depressed for individuals of several bird species found dying after organophosphorus or carbamate insecticide spraying (Hill and Fleming 1982). More global examples exist such as the population consequences of DDT or DDE inhibition of Ca–ATPase in the eggshell gland of birds. Its inhibition resulted in thin-shelled eggs that broke before full development and hatching (e.g., Kolaja and Hinton 1979). Inhibition of this one enzyme resulted in abrupt decreases in population size for osprey, Pandion haliaetus (Ambrose 2001, Spitzer et al. 1978), bald eagle, Haliaeetus leucocephalus (Bowerman et al. 1995), falcon, Falco peregrinus (Ratcliffe 1967, 1970), and brown pelican, Pelecanus occidentalis (Hall 1987). 3.8 HEME BIOSYNTHESIS INHIBITION Porphyrin and heme synthesis (Figure 3.4) is central to producing hemoglobin, myoglobin, cyto- chromes, tryptophan pyrrolase, catalase, and peroxidase.Although all cells produce heme, mammals produce most heme in the liver and erythroid cells (Marks 1985). In the mitochondria, where Porphobilinogen Linear tetrapyrrole δ-Aminolevulinic acid Succinyl CoA + Glycine δ-Aminolevulinic acid 3 Porphobilinogen Uroporphyrinogen III Coproporphyrinogen III Protoporphyrin IX Heme Protoporphyrinogen IX FIGURE 3.4 Steps in heme synthesis. © 2008 by Taylor & Francis Group, LLC [...]... hydrophilic Reactive groups are added or existing sites are made more readily available to further reactions In Phase II (conjugative) reactions, endogenous compounds are conjugated with contaminants or their metabolites to accelerate their elimination Phase II conjugation can occur without any Phase I reactions if the appropriate groups are already available Toxic metals can bind with metallothioneins... Japanese medaka, Oryzias latipes, at high salinity, Toxicol Appl Pharmacol., 152, 175–1 83, 1998 Ericson, G and Larsson, A. , DNA adducts in perch (Perca fluviatilis) living in coastal water polluted with bleached pulp mill effluents Ecotoxicol Environ Saf., 46, 167–1 73, 2000 Franks, N.P and Lieb, W.R., Where do general anaesthetics act? Nature, 274, 33 9 34 2, 1978 Franks, N.P and Lieb, W.R., Do general... cadmium on the hematology and on the activity of δ-aminolevulinic acid dehydratase (ALA-D) in blood and hematopoietic tissues of the flounder, Pleuronectes flesus L., Environ Res., 17, 191–204, 1978 Johansson-Sjöbeck, M.-L and Larsson, Å., Effects of inorganic lead on delta-aminolevulinic acid dehydratase activity and hematological variables in the rainbow trout, Salmo gairdnerii, Arch Environ Contam... few chapters in discussions such as that addressing cellular accumulation of degradation products from oxidative damage Others such as the important MXR transporter (Hamdoun et al 2002) are relevant to discussions of contaminant uptake and elimination Together, they provide strong causal insights and sensitive biomarkers of contaminant exposure or effect 3. 11.1 SUMMARY OF FOUNDATION CONCEPTS AND PARADIGMS... nonpolar, and lipophility was adequate to predict trends in potency for them Almost any nonelectrolyte organic compound that can become associated with the cell membrane can express a nonspecific narcosis, but chemicals commonly categorized as nonelectrolyte narcotics are ethers, alcohols, and chlorinated alkanes Other narcotics are weak acids The most important of these polar narcotics have already been... Clements: 33 57_c0 03 — 2007/11/9 — 12:42 — page 39 — #17 Ecotoxicology: A Comprehensive Treatment 40 Medvedev, Z .A. , An attempt at a rational classification of theories of ageing, Biol Rev Camp Philos Soc., 65, 37 5 39 8, 1990 Mertz, W., The essential trace elements, Science, 2 13, 133 2– 133 8, 1981 Pawlik-Skowro´ ska, B., When adapted to high zinc concentrations the periphytic green alga Stigeoclonium n... to elimination In some cases, the transformation products can be more toxic or reactive than the original compound A transformation in which an inactive compound becomes bioactive or an active compound becomes more bioactive is called activation A series of Phase I and II reactions can occur, which render a toxicant more amenable to elimination Phase I reactions make compounds more reactive and sometimes... F., Total oxyradical scavenging capacity (TOSC) in polluted and translocated mussels: A predictive biomarker of oxidative stress, Aquat Toxicol., 50, 35 1 36 1, 2000 Regoli, F and Principato, G., Glutathione, glutathione-dependent and antioxidant enzymes in mussel, Mytilus galloprovincialis, exposed to metals under field and laboratory conditions: Implications for use of biochemical biomarkers, Aquat Toxicol.,... Toxicants 33 the tricarboxylic acid cycle generates ample succinyl CoA, succinyl CoA and glycine are converted to δ-aminolevulinic acid by δ-aminolevulinic acid synthetase The δ-aminolevulinic acid then passes into the cytoplasm where two molecules of δ-aminolevulinic acid are then combined by δ-aminolevulinic acid dehydratase to form porphobilinogen Four molecules of porphobilinogen are then acted... cross-linking Consequent effects to the © 2008 by Taylor & Francis Group, LLC Clements: 33 57_c0 03 — 2007/11/9 — 12:42 — page 36 — #14 Biochemistry of Toxicants • • • • • • • • • 37 soma include cancer and perhaps accelerated aging (i.e., the mutation accumulation theory of aging) Many organic contaminants are subject to transformation within organisms that renders the toxic chemical more amenable . translation activities can provide evidence of response to a toxicant. As an example, El-Alfy and Schlenk (1998) discovered that up-regulation of a monooxygenase in Japanese medaka (Oryzias latipes). two ways. Antioxidant molecules are produced that react with oxyradicals and enzymes are synthesized that consume oxyradicals or oxyradical-generating chem- icals.Antioxidantsinclude catecholamines,. longevity may be determined by DNA modifications accrued during an individual’s life. DNA can be damaged by contaminants or their metabolites that are free radicals or can facilitate free radical 2 generation.