Clements: “3357_c004” — 2007/11/9 — 12:42 — page 43 — #1 4 Cells and Tissues Cells are the site[s] of primary interaction between chemicals and biological systems. (Segner and Braunbeck 1998) 4.1 OVERVIEW The biochemistry explored in Chapter 3 is central to all life. Also essential are the spatial differences in the distribution of biochemical activities and moieties within cells and tissues. Examples include essential differences in respiratory activities within the mitochondria versus nucleus, or glycogen synthesis differences in liver versus kidney cells. Macromolecular complexes forming membranes, organelles, cell junctions, and extracellular matrices facilitate this spatial heterogeneity. Such differences, emergingat thelevels of themembrane, organelle, cell, andtissue, alsoproduce spatial differences in effects of, and responses to, toxicants. Cyanide inhibits mitochondrial elec- tron transport reactions by interfering with cytochrome a function. Methylated forms of arsenic can damage chromosomes localized in the nucleus and, in so doing, provide a mechanism for arsenic’s carcinogenicity. Differences in the biochemical processes and moieties in various cell organelles determine the site of action for poisons such as cyanide and arsenic, and spatial separation of cell types into different tissues determines which tissue is most affected by a toxicant or is most responsive to toxicant damage. High microsomal mixed function oxidase (MFO) activity in hepatocytes make liver tissue a major site of Phase I reactions. It also makes hepatocytes particularly prone to can- cers initiated by strongly electrophilic metabolites of toxicants. High levels of metallothionein and lysosomal activity in vertebrate proximal tubules make renal damage an unfortunate consequence of acute cadmium poisoning. The extent of toxicant-induced cell death within a tissue and the tissue’s regenerative capacity determine whether or not that tissue can support the proper functioning of the associated organ. Histopathology is the science that focuses on cellular and tissue changes resulting from infectious and noninfectious diseases. This brief chapter explores histopathology of toxicants by building on the previous chapter. Hopefully, it also provides a bridge to the organ and organ system effects discussed next. 4.2 CYTOTOXICITY 4.2.1 N ECROSIS AND APOPTOSIS Pathological changes (lesions) in cells, tissues, or organs occur at sites of toxic action. Some lesions reflect failures to maintain a viable cellular state while others reflect only partially successfulattempts to maintain optimal cellular homeostasis. Cells die if stress is insurmountable or injury irreparable. Necrosis, cell death resulting from disease or injury, is apparent in many kinds of lesions. Necrotic cells are characteristically swollen, with swollen mitochondria and disintegrating cell membranes (Gregus and Klaassen 1996). Swollen mitochondria take in calcium with consequent and often pervasive internal precipitation of calcium phosphate. This leads to eventual breakdown of the mitochondria’s inner membrane and loss of its 43 © 2008 by Taylor & Francis Group, LLC Clements: “3357_c004” — 2007/11/9 — 12:42 — page 44 — #2 44 Ecotoxicology: A Comprehensive Treatment capacity for oxidative phosphorylation (La Via and Hill 1971). Pyknosis, the condensation of the nuclear material into a dark staining mass, is also characteristic of necrotic cells. Diffuse strands of chromatin condense during cell death to form these darkly staining masses. Also karyolysis can be seen in necrotic cells. Karyolysis is the dissolution of the nucleus and its lost ability to be stained with basic stains such as hematoxylin. The nuclear membrane remains intact with karyolysis. The loss of staining qualities results from DNAase and lysosomal cathepsin destruction of the DNA (La Via and Hill 1971). Karyorrhexis might occur later with the disruption of the nuclear membrane, fragmentation of the nucleus, and breaking apart of the chromatin into small granules. Necrotic cells can be dislocated from their normal position within tissues. Often inflammation accompanies necrosis. Necrosis can occur in distinct zones (Figure 4.1, middle panel) or diffusely (Figure 4.1, lower panel) within tissues. Necrosis would seem at first consideration to be the only kind of cell death relevant to chemical intoxication. What other kind could there be? Apoptosis, programmed cell death (PCD), can also occur by a genetically controlled series of cellular events. Cells undergoing apoptosis character- istically shrink, their nuclear material condenses, and they break into membrane-bound fragments called apoptotic bodies. Inflammation is characteristically absent. (The dead cells in the lower panel of Figure 4.1 appear like cells that have undergone apoptosis.) Remnants of cells experiencing apoptosis can be engulfed by phagocytic cells or shed from the gut lining or skin surface. Apoptosis occurs in normal and toxicant-exposed cells. In fact, a balance between cellular mitosis and apoptosis is essential in development and maintenance of tissue homeostasis (Roberts et al. 1997). As one example involving development, some cells must die away between the developing fingers of a human fetus to facilitate normal development of the hand. Toxicant-induced imbalance between mitosis and apoptosis can produce developmental abnormalities. As another example, apoptosis is essential for developing the appropriate gaps between connecting neurons. Human neutrophils formed in and released from the bone marrow also undergo apoptosis after a brief time in circulation. 1 Apoptosis may also remove cells that become a threat to tissues, e.g., cells infected with a virus or damaged by a toxicant. For example, cadmium-induced oxidative stress in trout hepatocytes results in apoptosis of damaged cells (Risso-de Faverney et al. 2004). Similarly, apoptosis removes cells in snails (Helix pomatia) after exposure to cadmium-enriched food (Chabicovsky et al. 2004). As a contrasting illustration, cadmium’s adverse effect on mammalian male fertility is a consequence of testicular necrosis, not apoptosis (Fowler et al. 1982). Clearly, the relative importance of necrosis and apoptosis varies with the particular toxicant and tissue. 4.2.2 T YPES OF NECROSIS Four major categories of necrosis exist: coagulative, liquefactive, caseous, and fat. Other less general classes mentioned in the histopathology literature are Zenker’s, fibrinoid, and gangrenous necrosis. Coagulative, or coagulation, necrosis involves extensive protein coagulation throughout the dead cell. This coagulation makes the cell appear opaque, having a cloudy and weakly eosinophilic appearance. Cells might maintain their relative positions within tissues for days after coagulative necrosis occurred: cell ghosts, a term applied to the opaque dead cells, are characteristic of this type of necrosis. Coagulative necrosis can be expected under a variety of situations, including poisonings. Inges- tion of phenol or inorganic mercury by mammals produces coagulation necrosis in the intestinal lining because both toxicants rapidly denature proteins (Sparks 1972). Accordingly, this type of 1 The term necrobiosis, coined first by Rudolf Virchow, is synonymous with apoptosis (Sparks 1972). It was used specifically for the natural aging and death of cells, such as epithelial cells, that are then replaced by new cells (Sparks 1972). © 2008 by Taylor & Francis Group, LLC Clements: “3357_c004” — 2007/11/9 — 12:42 — page 45 — #3 Cells and Tissues 45 FIGURE 4.1 Liver necrosis. The upper panel is a section through a normal F. heteroclitus liver with branching hepatic tubules lined with hepatic sinusoids. Note that the hepatocytes are relatively uniform in size and shape. The middle panel is an example of necrosis in the liver. Notice the difference in staining between the living and dead cells. Dead cells show nuclear pyknosis and karyolysis, and loss of cell adherence. The lower panel shows necrosis of individual cells, not a localized area as seen with the necrosis shown in the middle panel. Three necrotic cells are at the tips of the dark arrows. They are round or oval remnants that stain strongly with eosin. The basophilic chromatin remnants are visible in dead cells identified by the white arrows. Such a scattering of single necrotic cells in the liver suggests the effect of a chemical toxicant (Roberts et al. 2000). (Photomicrographs and general descriptions provided by W. Vogelbein, Virginia Institute of Marine Science.) © 2008 by Taylor & Francis Group, LLC Clements: “3357_c004” — 2007/11/9 — 12:42 — page 46 — #4 46 Ecotoxicology: A Comprehensive Treatment necrosis is favored by Hinton and Laurén (1990) as a biomarker 2 of environmental toxicant expos- ure. Heat also produces coagulative necrosis. Ischemia, the sudden loss of oxygen supply as might occur with a myocardial infarct or a puncture wound, can also induce this type of necrosis by shifting metabolism to glycolysis and decreasing cellular pH by production of lactic acid. With liquefactive (cytolytic or liquefaction) necrosis, the cell contents are liquefied by the cell’s proteolytic enzymes, and perhaps also enzymes from leukocytes that move into the injured area. Relative to coagulative necrosis, cell liquefaction tends to be rapid and extensive. Liquefactive necrosis in tissues possessing considerable enzymatic activity can produce fluid-filled spaces in tissues. This type of necrosis is often associated with bacterial or fungal infections, and can produce cell debris-filled abscesses. It can also be associated with a brain infarct. Given these characteristics, especially its frequent association in infectious disease, this type of necrosis is a less useful indicator of toxicant effects than coagulative necrosis. The two other common forms of necrosis, caseous and fat necrosis, are also not useful as general biomarkers of toxicant exposure. Caseous (caseation or cheesy) necrosis, named for its milk casein or soft cheese appearance, involves the complete disintegration of cells into a mass of fat and protein. It is often associated with mycobacterial infections such as the lung necrosis characteristic of tuberculosis. Fat necrosis involves deposition of calcium with released fatty acids, which imparts a white color to lesions. Fat necrosis can result from lipase and other enzyme activities (enzymatic fat necrosis) or from physical trauma to fat cells (traumatic fat necrosis). The mammalian pancreas, which can release high levels of lipases and other pertinent enzymes, is a common site of fat necrosis. Other types of necrosis exist. Gangrenous necrosis occurs with ischemia and consequent bacterial infection. As such, gangrenous necrosis will have characteristics of liquefactive and coagulative necrosis. Fibroid necrosis is another form of necrosis that is associated with autoimmune disease (e.g., lupus erthematosis) or vessel wall necrosis with extreme hypertension. Zenker’s (hyaline or waxy) necrosis is a specific condition in striated muscle that is associated with acute infections such as typhoid infections and is similar to coagulative necrosis. Although reported in goat heart muscle tissue with chronic mercury poisoning (Pathak and Bhowmik 1998), Zenker’s necrosis is not generally useful as an ecotoxicological biomarker. Box 4.1 Death by Trichloroethylene: Intentional and Otherwise Several themes discussed to this point can be illustrated using a recent study by Lash et al. (2003). These toxicologists were interested in the effects on humans from exposure to tri- chloroethylene, a metal degreaser and solvent. This chemical enjoys very widespread use but has been classified by EPA as a probable carcinogen. As such, it is the subject of much justified interest. Trichloroethylene undergoes a variety of Phase I and II reactions. It can be acted on by cytochrome P450 monooxygenase with subsequent glutathione conjugation. S-(1,2-dichlorovinyl)-l-cysteine (DCVC) is produced via β-lyase activity after cysteine con- jugation to a cytochrome P450 monooxygenase metabolite of trichloroethylene. The β-lyase activity is primarily a result of glutamine transaminase K that is localized in the kidney’s proximal tubules (Lash and Parker 2001). The DCVC causes necrosis in the human kidney. Relatively high doses of DCVC were found to be nephrotoxic to cultured proximal tubular cells of rats, inducing apoptosis. 2 This term was used loosely in Chapter 3 but now needs to be defined more precisely. A biomarker is a cellular, tissue, body fluid, physiological, or biochemical change in living organisms used quantitatively to imply the presence of significant pollutant exposure (Newman and Unger 2003). © 2008 by Taylor & Francis Group, LLC Clements: “3357_c004” — 2007/11/9 — 12:42 — page 47 — #5 Cells and Tissues 47 Necrosis (% LDH release) Apoptotic cells (%) 5 0 0 10 10 15 20 20 0 100 200 300 400 500 30 Necrosis: 2 h Necrosis: 4 h Apoptosis: 2 h Apoptosis: 4 h FIGURE 4.2 Necrosis and apoptosis occur- ring in primary cultures of human proximal tubular cells exposed to DCVCS, a nephro- toxic metabolite of trichloroethylene. Data for concentration-dependent necrosis (squares) and apoptosis (circles) are shown for DCVCS exposure durations of 2 h (open symbols) and 4 h (filled symbols). (Data extracted from Figures 2 and 5 of Lash et al. 2003.) A flavin-containing monooxygenase can produce S-(1,2-dichlorovinyl)-l -cysteine sulfoxide (DCVCS) from DCVC. The potency of DCVCS was much higher than DCVC in rat proximal tubular cell cultures, leading Lash et al. (2003) to be concerned that DCVCS might also be responsible for the nephrotoxic effects of trichloroethylene exposure of humans. To assess this hypothesis, they examined injury resulting from DCVCS exposure of cultured human proximal tubular cells. Necrosis and apoptosis were measured at different DCVCS concentrations and exposure durations; however, only results for 2- and 4-h exposures are discussed here. Necrosis was quantified in this study by measuring the amount of lactate dehydrogenase (LDH) in the cultured cells and the amount released from cells into the culture media. The more LDH measured in the media, the more necrosis. The percentage of LDH metric was simply 100 times the amount in the media divided by the sum of the LDH in the media plus the amount in the cells. LDH (%) = 100 LDH media LDH cells +LDH media The amount of necrosis present in cultures increased with DCVCS dose and exposure dura- tion (Figure 4.2). This was also the case for results from other exposure durations (1, 8, 24, and 48 h) not shown here. In contrast, apoptosis increased at the lowest exposure concentration and remained at that elevated level at all DCVCS concentrations. This induction of apoptosis by DCVCS was consistent with apoptosis induced by DCVC. A set level of apoptosis appeared to be triggered by DCVCS but necrosis increased steadily with any increase in DCVCS. Regard- less, both contributed to the net loss of cells due to DCVCS exposure. The authors concluded that flavin-containing monooxygenase activation and subsequent sulfoxidation of DCVC play important roles in human kidney damage after exposure to trichloro- ethylene. Both necrosis and apoptosis contribute to kidney cell death due to trichloroethylene exposure but the pattern of response differs for necrosis and apoptosis. Within the hierarchical framework of this book, the study illustrates that Phase I and II bio- chemical reactions activate xenobiotics in cells. Beyond a certain stress level, cells are unable to recuperate and death occurs due to necrosis and apoptosis. Sufficient levels of cell death within kidney tissues can result in renal failure and death of the individual. 4.2.3 INFLAMMATION AND OTHER RESPONSES Inflammation is a general response to damage or infection. It is characterized by “infiltration of leucocytes into the peripheral tissues, followed by the release of various mediators eliciting non- specific physiological defense mechanisms” (House and Thomas 2002) (Figure 4.3). The intended © 2008 by Taylor & Francis Group, LLC Clements: “3357_c004” — 2007/11/9 — 12:42 — page 48 — #6 48 Ecotoxicology: A Comprehensive Treatment EP MA MA MA MA MA MA FIGURE 4.3 Inflammation in the liver of the estuarine fish, F. heteroclitus. At the top center of the top photomicrograph is a focus of inflammation. The bottom photomicrograph shows macrophage aggregates (MA) produced during inflammation in Fundulus liver. (EP is exocrinic pancreas tissue.) (Photomicrographs and general descriptions provided by W. Vogelbein, Virginia Institute of Marine Science.) result is tissue repair with a return to a healthy state; however, chronic inflammation or inflammation after extensive damage can produce compromised tissue structure and function. With toxicant- induced injury, inflammation isolates, removes, and replaces damaged cells. Consequently, ongoing inflammation or telltale signs of past inflammation can be evidence of cell poisoning. Classic work by Elie Metchnikoff established the scientific foundation of inflammation theory. Taking advantage of the transparency of minute invertebrates, he explored phagocytic responses in injured or infected individuals. In one set of experiments, he closely observed the cellular response of Daphnia to infection with Monospora bicuspidata. In others, he studied responses to mechanical injury. Bibel (1982) describes one of Metchnikoff’s initial experiments, done while staying in a Sicilian seaport with his family. Whiling away time after resigning from the University of Odessa, Metchnikoff gazed through his microscope and hypothesized that all organisms, even the simplest, will exhibit inflammation. We had a few days previously organized a Christmas tree for the children on a little tangerine tree: I fetched from it a few thorns and introduced them at once under the skin of some beautiful starfish larvae as transparent as water . I was so excited to sleep that night in the expectation of the result of my experiment and very early the next morning I ascertained that it had fully succeeded. (Metchnikoff 1921) © 2008 by Taylor & Francis Group, LLC Clements: “3357_c004” — 2007/11/9 — 12:42 — page 49 — #7 Cells and Tissues 49 Although Metchnikoff’s experiment and early morning anticipations were not those normally expec- ted during a Christmas with one’s family, his experiment did demonstrate phagocyte infiltration into the area of injury and, combined with similar experiments, established the universal nature of this response to injurious or infectious agents. Much of this pioneering experimentation with invertebrates took place more than a century ago. But our understanding of symptoms of inflammation goes back still further. Most introductory discussions describe four cardinal signs of inflammation for humans: heat, redness, swelling, and pain. Cornelius Celsus identified these signs millennia ago and they were further detailed by Virchow (see footnote 1) and Metchnikoff a century ago (Plytycz and Seljelid 2003). The area of damage reddens as blood vessels dilate. Swelling of surrounding tissueswith fluids (edema) occurs, imparting a feeling of heat and painful pressure. Obviously, some of these signs are relevant only to red-blooded poikilotherms; however, the underlying processes are relevant to all animals. Typical of a tissue experiencing inflammation is leukocyte movement into the involved tissues. Diapedesis occurs when leukocytes, responding to chemotactic factors released from the damaged tissue, adhere to the vascular endothelium and then migrate through it into the involved tissues. The clumping of leukocytes at the endothelium is called margination. The cells in the area retract to facilitate leukocyte passage through interendothelial cell junctions. The leukocytes phagocytize cellular debris and remove it from the area. Starting as a mass called the granulation tissue, new vessels and connective tissue will eventually begin to grow back as the process continues. Scar tissue or collagenous connective tissue can form to cause tissue dysfunction in the case of chronic inflammation. Diverse examples of inflammation are easy to find because inflammation is such a universal cellular response to injury. The human autoimmune disease rheumatoid arthritis involves chronic inflammation at the synovial membrane of joints. This inflammation gradually damages joint tissues. Inhalation of zinc-rich particulate matter can produce metal-fume fever, a condition arising from pulmonary inflammation and injury (Kodavanti et al. 2002). Exposure of freshwater fish to a water- soluble fraction of crude oil results in gill and liver necrosis, and consequent inflammation (Akaishi et al. 2004). Other cellular changes such as hyperplasia and hypertrophy can indicate response to toxicants. Hyperplasia is the increase in the number of cells in a tissue. Hypertrophy is an increase in cell size (and function) that is often part of a compensatory response. Fish gill hyperplasia is evident in Figure 4.4. The upper panel of that figure shows a section through a normal gill from the estuarine fish, Fundulus heteroclitus. The axis of the primary lamellae is denoted with a black line and the letter “P,” and one of the many secondary lamellae projecting out from the primary lamellae is denoted by the letter “S.” The lower panel is a lower magnification image of a Fundulus gill that has undergone extensive hyperplasia. One of the primary lamellae in the image is shown with a dark line and “P,” and one secondary lamella with a “S.” Notice that extensive hyperplasia of epithelial cells has filled in the gaps between secondary lamellae of the labeled primary gill lamella and also of the primary lamella at the bottom right hand corner of the photomicrograph. The hyperplasia is so extensive that the primary lamellae at the center of the photograph have fused together with no discernable secondary lamellae. This can be seen easily by noting the filament cartilage (C) in the normal primary lamella (upper panel) and then locating the filament cartilage in the lower panel (C) where two of the primary lamellae have fused into one single mass of tissue. Available respiratory surface has decreased considerably because these secondary lamellae are the structures where most gas exchange occurs. Figure 4.5 shows gills of the freshwater mosquitofish, Gambusia holbrooki, which exhibit chlor- ide cell (ionocytes) hypertrophy in addition to hyperplasia as a consequence of inorganic mercury exposure. The upper panel of that figure is a gill from an unexposed fish with an arrow pointing to one of several lightly staining chloride cells on the primary lamellae. Notice in the lower panel that, in addition to chloride cell proliferation between and onto the secondary lamellae (hyperplasia), © 2008 by Taylor & Francis Group, LLC Clements: “3357_c004” — 2007/11/9 — 12:42 — page 50 — #8 50 Ecotoxicology: A Comprehensive Treatment C C S C S P S C C 115 µm 300 µm FIGURE 4.4 Normal gill (upper panel) and gill with extensive hyperplasia (lower panel) from the estuarine fish, F. heteroclitus. The epithelial cells have filled the gaps between secondary lamellae, causing fusion in the primary lamellae shown in the center of the bottom photomicrograph. Often such hyperplasia is accompanied by inflammation. (Photomicrographs and general descriptions provided by W. Vogelbein, Virginia Institute of Marine Science.) the chloride cells have become enlarged (hypertrophy) (three arrows). Chloride cells function in ion transport and this hypertrophy is seen as an attempt to compensate for a loss of ion transport capabilities due to mercury damage (Jagoe et al. 1996). Other toxicants produce such compensatory hypertrophy in other tissues. The trichloroethylene metabolite DCVC, which we discussed previ- ously, results in hypertrophy in primary cultures of rat proximal tubule epithelial cells (Kays and Schnellmann 1995). Heptocytes also display hypertrophy when zebrafish (Danio rerio) are injected with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Zodrow et al. 2004). 4.3 GENOTOXICITY 4.3.1 S OMATIC AND GENETIC RISK Genotoxicity is damage to the cell’s genetic material by a physical or chemical agent. The individual organism is the focus of most genotoxicity studies although implications about risk factors are often © 2008 by Taylor & Francis Group, LLC Clements: “3357_c004” — 2007/11/9 — 12:42 — page 51 — #9 Cells and Tissues 51 FIGURE 4.5 Mosquitofish (G. holbrooki) normal gills (upper panel) and gills from mercury-exposed mos- quitofish (lower panel). The gill from the fish exposed to inorganic mercury shows hyperplasia and chloride cell hypertrophy. (See Jagoe et al., 1996. Courtesy C. Jagoe, Savannah River Ecology Laboratory.) framed in a population context. By convention, genetic damage is discussed relative to somatic and genetic risk. Somatic risk is the risk to the somatic cells (soma) (e.g., genetic modifications resulting in cancer). Genetic risk involves risk to offspring of exposed individuals. Such genetic risk was mentioned briefly in Section 3.2 where examples were given of possible consequences to offspring of occupational exposure. More attention is paid to somatic than genetic risk in the field of genotoxicology primarily because of concern about cancer. In the ecotoxicological context of this book, one could argue that population risk should be considered too. Population risk would be defined as risk of decreased population viability due to genetic damage to germ cells by a physical or chemical agent. An admittedly contrived and extreme example of such a population effect would be those intended in tsetse fly, screwfly, or medfly control programs that aim to dramatically impact population size by γ irradiation and release of large numbers of sterile males (Sterile Insect Technique, SIT) (Knipling 1955, Lindquist 1955, Lux et al. 2002). But such intense exposures are not common outside of pest control programs. Perhaps, elevated cancer incidences in small, slow-growing wildlife populations could result in population risk. Such a scenario might develop for the Beluga whales in the St. Lawrence estu- ary, which have high levels of cancer deaths (18% of all deaths) (Martineau et al. 2002). These whales are exposed to polycyclic aromatic hydrocarbons (PAH) and display annual cancer rates (163 in 100,000 animals) considerably higher than those of other cetacean populations. (The link between cancer and PAH genotoxicity was reinforced by Shugart (1990) who reported elevated DNA adducts in tissues of St. Lawrence Beluga whales.) Regardless, to our knowledge, few © 2008 by Taylor & Francis Group, LLC Clements: “3357_c004” — 2007/11/9 — 12:42 — page 52 — #10 52 Ecotoxicology: A Comprehensive Treatment examples of immediate and significant population risk due to direct genetic damage to germ cells have emerged. 4.3.2 DNA DAMAGE DNA damage in cells is measured in a variety of ways. Jenner et al. (1990) applied flow cytometry to quantify differences in DNA content in individual hepatocytes of English sole (Parophrys vetulus), showing more DNAdamage in sole from contaminated areas than those from reference sites. Shugart (1988) used an alkaline unwinding assay to get a relative measure of DNAstrand breakage in bluegill (Lepomis macrochirus) and fathead minnow (Pimephales promelas) exposed to benzo[a]pyrene. In this alkaline unwinding assay, the ease with which DNA unwinds under alkaline conditions suggests the amount of strand breakage in the DNA: a DNA strand unwinds more readily as the number of breaks within it increases. More recently, a comet, or single cell electrophoresis, assay has been applied widely to reflect DNA damage (Dixon et al. 2002). For the ecotoxicologist, this method has several advantages relative to the conventional karyotyping or sister chromatid exchange (SCE) techniques described below. For example, karyotyping and SCE assays can be difficult for species with many small chromosomes. Also both methods require that cell division occur (Pastor et al. 2001). In an ecotoxicological application of the comet technique, neutrophilic coelomocytes from nickel-exposed earthworms (Eisenia fetida) were embedded in agarose, lysed in place with detergent, placed under alkaline conditions that unwound their DNA, and then subjected to electrophoresis. After electrophoresis and staining with ethidium bromide, the length of the “comet tails” extending from the original cell position in the gel to the furthest point to which the DNA migrated in the electric field was used as a measure of the extent of DNA strand breakage. Relative tail lengths derived from many coelomocytes of control and exposed worms suggested genotoxic effect of nickel. The comet assay was recently applied to hemocytes of the mussel, Perna viridis, after exposure to benzo[a]pyrene (Siu et al. 2004). It also provided evidence of genotoxic effect to white storks born near an acid and heavy metal toxic spill in Spain’s Doñana National Park (Pastor et al. 2001). 4.3.3 CHROMATIDS AND CHROMOSOMES Section 4.3.2 describes some direct effects of toxicants on DNA including cross-linking DNA with proteins, single or double strand breaks, adduct formation, base mismatching, and point mutations. Here, the topic is addressed again but at a higher scale—that of chromatids and chromosomes. Dixon et al. (2002) use the discriminating term macrolesions for these chromatid or chromosome- level genotoxic effects in order to distinguish them from the microlesions discussed previously, which occur at the molecular DNA level. Several macrolesion assays require cells that are dividing and include SCE, chromosomal aberration, and micronuclei assays. Macrolesion-associated methods are quickly becoming valuable genotoxicity monitoring tools (Hayashi et al. 1998, Jha et al. 2000a). Mutagenic or genotoxic effects are often correlated with rates of SCE (Dixon et al. 2002, Tucker et al. 1993). SCE involves DNAbreakage followed by homologous DNAsegment exchange between sister chromatids during the S phase of the cell cycle 3 (Tucker et al. 1993). To measure SCE, one chromatid in each pair comprising a chromosome is first stained with 5-bromodeoxyuridine. Cells are examined two cell cycles later under a fluorescent microscope for evidence of DNA exchange between chromatids. Each of the paired sister chromatids remains either completely stained or unstained if no exchange occurred. If exchange occurred, each chromatid will have segments that are stained and others that are not. The number of SCEs per metaphase or per chromosome is used as a metric of exchange. DNA damage is generally correlated with the level of SCE. SCE techniques are widely applied to study human exposure to mutagens or genotoxicants, and occasionally used in ecotoxicological studies. Examples of use relative to humans include exposure 3 S phase is the “synthesis” stage in which the DNA is replicated. © 2008 by Taylor & Francis Group, LLC [...]... chromosomal aberrations in rodents inhabiting a petrochemical waste site These same methods were used to prove that women exposed in the workplace to elevated lead concentrations have elevated levels of chromosomal aberrations (Forni et al 1980) and that methylated trivalent arsenicals are clastogenic (Kligerman et al 2003) 4. 4 CANCER 4. 4.1 CARCINOGENESIS Cancer results from a hyperplasia unlike that discussed... Toxicant-induced imbalance between mitosis and apoptosis can lead to deviations in tissue homeostasis, developmental abnormalities, or cancer promotion • Inflammation, a general response to damage or infection, aims to isolate, remove, and replace damaged cells • Toxicants can also produce hyperplasia and hypertrophy in tissues • Genotoxicity, damage to the cell’s DNA by a physical or chemical agent, can... the soma, they are also relatively unavailable for trophic transfer to grazers, predators, or parasites (Nott and Nicolaiduo 1993, Wallace and Luoma 2003, Wallace et al 2003) Let us examine details of metal sequestration in intracellular granules as one example Several kinds of granules occur in invertebrate tissues (Beeby 1991) In molluscs, calcium carbonate granules can be present outside and within... elevated urinary glutamine transaminase K (Trevisan et al 1996) Proteinuria, abnormally high levels of protein in the urine, can indicate tubule damage due to cadmium exposure (Nogawa et al 1978) As we saw in Chapter 3 (Section 3.8), elevated porphyrins in urine suggests exposure to several toxicants Elevated guanine in urine of rats indicated that lead disrupted guanine aminohydrolase and crystalline... action on carbon tetrachloride produces a free radical that causes lipid peroxidation of hepatocyte membranes (Snyder and Andrews 1996) Hepatocytes of mullet (Liza ramada) exposed to the striazine herbicide atrazine display large lipofuscin granules, indicating increased lipid peroxidation (Biagianti-Risbourg and Bastide 1995) Peroxisomes, vacuoles containing peroxidative enzymes, also increased in these... threshold relationship between dose and cancer 4. 5 SEQUESTRATION AND ACCUMULATION 4. 5.1 TOXICANTS OR PRODUCTS OF TOXICANTS Often toxicants sequestered or accumulated in cells are taken as evidence of exposure For example, elevated metals associated with metallothionein in kidney cells and granules of hepatopancreas cells reflect metals sequestered away from sites of action Those associated with granules are... discussed above The hyperplasia discussed earlier relative to tissue repair is referred to as physiologic hyperplasia There are also two pathological types of hyperplasia, compensatory and neoplastic The former is an excessive cell proliferation in response to damage or irritation such as that shown in Figure 4. 4 Neoplastic hyperplasia results when hereditary material of a cell is changed and the cell... Ecotoxicology: A Comprehensive Treatment 58 Beeby, A. , Toxic metal uptake and essential metal regulation in terrestrial invertebrates: A review, In Metal Ecotoxicology Concepts & Applications, Newman, M.C and McIntosh, A. W (eds.), Lewis Publishers, Boca Raton, FL, pp 65–89, 1991 Biagianti-Risbourg, S and Bastide, J., Hepatic perturbations induced by a herbicide (atrazine) in juvenile grey mullet Liza ramada (Mugilidae,... Ferrari, M., Artuso, M., Bonassi, S., Cavalieri, Z., Pescatore, D., Marchini, E., Pisano, V., and Abbondandolo, A. , Cytogenetic biomonitoring of an Italian population exposed to pesticides: Chromosome aberration and sister-chromatid exchange analysis in peripheral blood lymphocytes, Mutat Res., 260, 105–113, 1991 Dewanji, A. , Venzon, D.J., and Moolgavkar, S.H., A stochastic two-stage model for cancer... Boca Raton, FL, 2003 Nogawa, K., Ishizake, A. , and Kawano, S., Statistical observations of the dose–response relationships of cadmium based on epidemiological studies in the Kakehashi River Basin, Environ Res., 15, 185–198, 1978 Nott, J .A and Nicolaiduo, A. , Bioreduction of zinc and manganese along a molluscan food chain, Comp Biochem Physiol., 10 4A, 235–238, 1993 Pastor, N., López-Lázaro, M., Tella, . soma, they are also relatively unavailable for trophic transfer to grazers, predators, or parasites (Nott and Nicolaiduo 1993, Wallace and Luoma 2003, Wallace et al. 2003). Let us examine details. capacity to resist oxidative damage and accrues DNA damage above a certain dose. The cell’s ability to resist damage at low dose results in a threshold relationship between dose and cancer. 4. 5. chronic inflammation at the synovial membrane of joints. This inflammation gradually damages joint tissues. Inhalation of zinc-rich particulate matter can produce metal-fume fever, a condition arising