ECOTOXICOLOGY: A Comprehensive Treatment - Chapter 4 pdf

Necrosis can occur in distinct zones Figure 4.1, middle panel or diffusely Figure 4.1, lower panel within tissues.. The dead cells in the lower panel of Figure 4.1 appear like cells that

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 inChapter 3is 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, emerging at the levels of the membrane, organelle, cell, and tissue, also produce 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

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 successful attempts

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

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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

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 coagulaInges-tion 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).

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.)

necrosis is favored by Hinton and Laurén (1990) as a biomarker2of 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).

Necrosis (%

Apoptotic cells

5

10

10

15 20

20

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 LDHmedia

LDHcells+ LDHmedia 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 duradura-tions (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

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

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)

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 (seefootnote 1) and Metchnikoff a century ago (Plytycz and Seljelid 2003) The area of damage reddens as blood vessels dilate Swelling of surrounding tissues with 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 4.5shows 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),

C S

P S

C

C

115 µ m

300 µ m

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

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

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 (SeeJagoeet 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 byγ 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

examples of immediate and significant population risk due to direct genetic damage to germ cells have emerged

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 DNA damage in sole from contaminated areas than those from reference sites Shugart (1988) used an alkaline unwinding assay to get a relative measure of DNA strand 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)

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 DNA breakage followed by homologous DNA segment exchange between sister chromatids during the S phase of the cell cycle3(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.