Microscopy as a tool in toxicological evaluations · useful in toxicology, a relatively new field...

Microscopy as a tool in toxicological evaluations

C.S. Fontanetti, C.A. Christofoletti, T.G. Pinheiro, T.S. Souza and J. Pedro-Escher

Instituto de Biociências, UNESP- Univ Estadual Paulista, Campus de Rio Claro, Departamento de Biologia, Laboratório

de Mutagênese, Av. 24A 1515, 13506-900 Rio Claro, São Paulo, Brasil.

The microscope was created to satisfy our interest in observing objects and/or structures under higher magnification. Both

the physical structure of the microscope and the techniques used for microscopic observation in the various fields of

science have undergone great technological changes since the device was first invented. Microscopy has been particularly

useful in toxicology, a relatively new field within the Biological sciences that studies the impacts of environmental

pollution on the different levels of biological organization. In this chapter, we demonstrate the many different ways in

which microscopy can be used in ecotoxicological studies to evaluate the impact of toxic substances on bioindicator

organisms. In this context, light microscopy aids in the identification of chromosomal aberrations, micronuclei, nuclear

abnormalities and histopathological alterations caused by exposure to chemical contamination; fluorescence microscopy can help detect whether a contaminant has genotoxic and/or mutagenic effects by revealing DNA damage; electron

microscopy enables the observation of alterations in the ultra-structure of the cell. Collectively, the different microscopy

techniques can contribute to the evaluation of the toxic, cytotoxic, genotoxic and mutagenic potential of pollutants, and

provide tools for a better understanding of the issue of contamination.

Key words: bioindicators; genotoxicity; histopathology; mutagenesis; pollutant; toxicity; ultrastructure.

Introduction

The microscope was created to satisfy our interest in observing objects under higher magnification, and later, to study

the morphology and function of cell structures and organelles, among other things. The first lens with magnifying

power, known as Lanyard Lens, was made from rock crystal and is datable to 721 a.d. Despite controversies

surrounding who actually created the first microscope, credit is usually given to Zacharias Jansen, from Holland, around

the year 1595. The device contained two superimposed lenses. Centuries later, Antonie van Leeuweenhoek (1632-1723)

and Robert Hooke (1635-1703) pioneered the utilization of the microscope in the evaluation of biological materials.

Since then, great technological advances have been incorporated into the microscope. For example, the electron

microscope has scanning coils that capture images produced by an electron beam tracing over the object, as opposed to

the rock crystal lenses of the primordial microscopes that needed the sun as a light source. Advances such as these have

enabled the development of techniques that allow for refined histological and ultrastructural evaluations.

Ecotoxicology is a relatively new science that can be defined as the study of the effects of pollutants (physical,

chemical and biological) on living organisms and how the latter interact with their respective habitats. Moreover, it is

also concerned with transport mechanisms, distribution, transformation, interaction and the final fate of pollutants in the

different compartments of the environment. Therefore, ecotoxicological analyses allow scientists to evaluate the

damage caused to the different levels of biological organization of an ecosystem.

Organisms respond to pollutants mainly through biochemical, physiological, and behavioural alterations [1, 2]. The

use of microscopy has enabled researchers to include several additional parameters to evaluate toxicity on bioindicators

such as fish, earthworms, diplopods, insects, onion, fava beans, and bacteria, among others; their reactions to toxic

agents are as diverse as they are.

In this context, light microscopy helps in the identification of chromosomal aberrations, micronuclei, nuclear

abnormalities and histopathological alterations caused by exposure to a chemically altered environment; fluorescence

microscopy can help detect whether the contamination has genotoxic and/or mutagenic effects by revealing damage to

the DNA; electron microscopy allows for the observation of alterations in the cell ultra-structure.

Below we describe the main techniques employed by light and electron microscopy in ecotoxicological evaluations,

and mention some commonly observed effects on selected bioindicator organisms.

1. Light microscopy

1.1. Cytogenetic techniques

Meristematic cells of Allium cepa (onion) are an efficient cytogenetic tool in the analysis of chromosomal aberrations

caused by environmental pollution [3].

Microscopy: Science, Technology, Applications and Education A. Méndez-Vilas and J. Díaz (Eds.)

©FORMATEX 2010 1001

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The technique used to stain the roots of anion is the Feulgen reaction [4]. According to the authors, this

stoichiometric method has become the best known cytochemical procedure used in conventional, quantitative DNA

analyses.

After Feulgen reaction, the radicular meristemes are lightly crushed with the help of one drop of 2% acetic carmin;

under the light microscope, a series of morphologic and cytogenetic parameters can then be quantified, including root

morphology and growth, mitotic rate, micronuclei induction and abnormal metaphase, anaphase and telophase (see

review in [5]).

Molluscs and fish are also excellent experimental models in toxicological studies. These organisms are specially

recommended for genotoxicity studies for two reasons. First, their component cells are very responsive to genotoxic

agents, even in low concentrations; second, they are an important source of protein and other nutrients in the human

diet. Food is the most important way through which toxic substances reach humans, and fish have been recognized as an

important vehicle of contamination [6].

The blood of fish exposed to an environmental sample and/or contaminant can be tested in two basic ways: through

the micronucleus test, which detects nuclear abnormalities, or with the comet assay. In mollusks, the hemolymph and

gills can be used in these tests. Both techniques are advantageous because they do not require a karyotypic profile of the

organism being tested [7], and can be used on any cell population, proliferating or not (as with the comet assay).

The micronucleus test is a promising, fast and cheap technique for genotoxicological analyses [8-12]. Slides for the

visualization of micronuclei and nuclear abnormalities are prepared with blood swabs previously subjected to Feulgen

reaction. Abnormalities are then quantified under the light microscope (Figure 1).

Figure 1. Micronucleus and nuclear alterations in erythrocytes of Oreochromis niloticus (Pisces). (A) Micronucleated erythrocyte

(head of arrow) and blebbed nuclei (arrow); (B) Notched nuclei (arrow); (C) Blebbed nuclei (arrow); (D) Lobed nuclei (arrows)

(Photos: Anita Martins Fontes Del Guercio).


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The comet assay allows for the detection of potentially pre-mutagenic lesions, such as DNA chain ruptures, alkali-

labile sites, DNA adducts, base changes, crossed DNA-DNA and DNA-protein links and incomplete DNA repair [13-

14] in proliferating or non-proliferating in vitro cells [15]. This technique, which can be used on any tissue, has been

applied with success to erytrocytes of various species of mollusks and fish [16, 12, 17]. DNA damage is assessed using

a visual classification method on individual cells. Damaged cells, called comets, present a particular DNA migration

pattern in electrophoresis, presenting a “tail”. The severity of the DNA damage can be accessed through the length of

the tail, which is composed of DNA fragments, and classified into the following categories: 0, 1, 2 and 3; zero

representing the lowest amount of damage.

The results of the comet test can be visualised under the light microscope, after silver nitrate staining, or under the

fluorescent scope, after ethidium bromide staining (Figure 2).

1. 2. Histological and histochemical techniques

Histological alterations are sensitive tools that can be used to detect the direct toxic effects of various compounds on

different organs; therefore, they are good environmental stressor markers [18].

The main techniques used in the detection of histopathological damage caused by toxic agents include the inclusion

of the sample of interest in historesin or paraffin, followed by coloration with hematoxilin and eosin; other stains can

also be used. Histochemical techniques are specifically for the detection and distribution of certain compounds that aid

in the investigation of physiological alterations.

In addition to genotoxic alterations, particularly in the erythrocytes, fish exposed to pollutants may also present

histopathological changes in different tissues and organs such as liver, kidney, spleen and gills [19-21]. In molluscs,

alterations are observed mainly on the gills [22-23].

Morphological alterations found in the gills of fish (Figure 3) and mollusks vary, depending on the stressor and on

the intensity of the toxic agent [19-24].

Morphological alterations detected in the tissues of diplopods, particularly in the midgut and fat body, can be used to

access soil toxicity [25-27].

Figure 2. Comet assay applied in erythrocytes of Oreochromis niloticus (Pisces). (A) Class 0; (B) Class 1; (C) Class 2; (D) Class 3

(Photos: Tatiana da Silva Souza).



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Figure 3. Histological sections of Oreochromis niloticus gills (Pisces) from non-impacted environment (A) and impacted

environment (B, C and D). In (B) it is noted aneurism; In (C), lamellar fusion and in (D) detachment of the respiratory epithelium

(From Biagini et al., 2009).

2. Electronic microscopy

Ultramorphological analyses can be used to detect various tissue alterations and help in the diagnosis of symptoms of

cell intoxication [28]. Both the scanning electron microscope (SEM) and the transmission electron microscope (TEM)

are useful in such evaluations. The SEM provides a tridimentional view of the cell surface. For SEM visualization,

samples are dehydrated with increasing concentrations of acetone, dissected and covered with a thin layer of gold. By

contrast, the TEM produces a bidimentional image of the interior of the cell by detecting differences in density between

the various parts of the sample. Sample preparation for TEM comprises inclusion in resine and microslicing (20 a 400

nm slices).

The TEM allows for the observation of alterations on the surface of structures which cannot be detected under the

light microscope. In fish, for example, it is possible to observe alterations in the filaments of the gills and pavement

cells that cover them (Figure 4) [24, 29].



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The TEM allows the observation of alterations in the organization of the cell (Figure 5) such as a decrease in the

number of organelles, loss of citoplasmatic and nuclear integrity, among others [23, 25].

Figure 4. SEM micrographs of the gill of Oreochromis niloticus (Pisces) from non-impacted environment (A, C) and impacted

environment (B, D). In (A) the gill filaments (f) are narrower and the lamellae (l) are longer than in (B). In (C), pavement cells (pc)

covered with microridges (arrows) and in (D), pavement cells with considerable loss of microridges (arrows); cc=chroridre cells.

Photos: A,B-Frederico Biagini; C, D-Luciana Tendolini Brito.



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Figure 5. Electron micrographs of the gill filaments of Mytella falcata (Mollusca) from non- impacted (A, C) and impacted

environments (B, D). (A) Frontal cells; (B) note the strong presence of secretory cells (sc); (C) lateral cells; (D) note smaller and

more abundant mitochondria; c=cilia; bb=basal bodies; m=mitochondria; mv=microvilli; n=nucleus; rer=rough endoplasmic

reticulum; sr=skeletal rod. Scale bars = 5 mm (From David et al., 2008b).

The growing concern with the effects of environmental pollutants is reasonable, not only because pollution is

ubiquitous, but also because little is known about its effects on living organisms. Microscopy is an invaluable tool in the

interpretation of the parameters used to measure toxicity, and contributes to the establishment of measures that aim to

prevent or eliminate environmental contamination, a risk not only to ecosystems, but also to human health.

References

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Microscopy as a tool in toxicological evaluations · useful in toxicology, a relatively new field...

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