**2. Cytogenetic analyses applied to environmental toxicology**

Plants constitute a system of great importance as bioindicators of pollution, having long been used for this end. International institutions such as the United Nations Environmental Programme (UNEP), the World Health Organization (WHO), and the US EPA approve the use of bioassays with plants to investigate toxic effects of chemical agents released into the environment [13].

ensure the quality and health of the environment, in both the short and long terms, it is fun-

Overall, research in the environmental area is based on analyses and physicochemical characteristics of pollutants. However, it has been recognized that the effects of these compounds on living organisms, as well as their toxicity mechanisms, are excellent tools to complement the obtained physicochemical data [1], being important for decision-making and in the search for

In this sense, the biological effects of pollutants can be assessed in vivo (in situ and ex situ) and in vitro via bioassays using test organisms, allowing to evaluate their toxic potential in a rapid and efficacious manner and at relatively low cost. Overall, the response in relation to toxicity can be given a different organization level, such as behavior, physiology, anatomy, cell, and DNA, among others, with each organism and test representing a certain endpoint. The integrity of the genetic material and its consequence for the proliferation and reproduction of model organisms are the most targeted outcomes and estimate the dimension of the risks of compounds to the environment and living beings in a real and functional manner [2–4].

Among the different bioassays performed in living organisms, those that use higher plants as models to evaluate the biological effects of environmental pollutants stand out. Besides being validated by the US Environmental Protection Agency (US EPA) as efficacious in the determination of toxicological risks in toxicity monitoring programs, they present important characteristics such as high sensitivity, fewer false-negative responses, low cost, not requiring approval from ethics commissions, and being as efficient as assays performed in animal models or even human cells [5–8]. In addition, they are in accordance with the Toxicology Guidelines for the twenty-first century, which calls for models that substitute animal ones to assess toxic risks [9]. Among the assays using higher plants highlighted by the Genetic Toxicology (Gene-Tox) program of the US EPA described by Ma [10], one of the most widespread is the *Allium* test. It was developed and described in 1938 by Levan [11] and consists in the evaluation of alterations in the mitotic phases of root meristem cells of *Allium cepa* [12]. In general terms, the test can be applied to any plant model that presents chromosomes of easy visualization under the microscope. It is employed to evaluate the cell cycle in meristematic root tip cells, observing disturbances in the frequency of cells in division as well as induction of alterations in the mitotic phases or in the interphase nucleus, arising from action of the tested pollutant.

In this chapter, the main characteristics of the assay based on evaluation of the cell cycle will be presented, as well as the endpoints that can be assessed and used for evaluation, determination of cyto(geno)toxicity, and understanding of the mechanisms of action of potential

Plants constitute a system of great importance as bioindicators of pollution, having long been used for this end. International institutions such as the United Nations Environmental

**2. Cytogenetic analyses applied to environmental toxicology**

environmental pollutants.

damental to obtain information about contaminating agents.

118 Cytotoxicity

preventive, mitigating measures as well as alternatives to this scenario.

The use of plants as models to evaluate the toxicity and mutagenicity of substances or pollutants enables the analysis both in the natural environment (in situ) and in the laboratory (ex situ). They are excellent tools to complement the physicochemical analyses of investigated compounds, as they allow a practical confirmation of the theory developed in studies on the physicochemical properties of the potentially dangerous materials [1, 14, 15].

Tests ex situ commonly use meristematic root tip cells as biological material for analysis. In the natural environment, the root is the first part of the plant to be exposed to toxic agents dispersed in the soil and water. Therefore, the analysis of root cells represents a rapid method for the monitoring of toxicity. Moreover, the observed damage to the DNA and/or chromosomes of plant cells can be extrapolated to further organisms based on the universality of the DNA structure and genetic code [16]. This way, if a chemical substance causes damage to the DNA of one plant, it should also be considered potentially damaging to the DNA of other organisms [17].

The assay with meristematic root tip cells is based on cytogenetic evaluations involving the movement of chromosomes during the mitotic division, which allows deriving the mechanisms of action of the pollutant. The root of a propagule (bulb, seeds, cutting, etc.) is exposed to the agent that shall be tested. By the end of the exposure interval, the meristem is separated from the root and fixed; a slide is subsequently prepared, generally by squashing technique, and the meristematic cells are stained with acetic orcein and/or Schiff's reagent (for the detailed methodology, see [18]). The slide is observed under light microscope, and various parameters of the cell cycle are evaluated. The cell cycle stages, including interphase and mitotic division (prophase, metaphase, anaphase, and telophase), are observed, and the alterations detected in each phase are recorded. Based on the results, the assessed endpoints are (1) frequency of dividing cells or mitotic index (MI), given by the sum of cells in phase M (mitosis) divided by the total number of observed cells, being expressed in number of dividing cells out of every 100 observed cells; (2) total frequency of chromosome alterations (CA), given by the sum of all observed alterations, independently of type and division phase, divided by the total number of observed cells, expressed as number of altered cells out of 100 observed cells; or (3) nuclear alterations (NA), related to the presence of abnormal interphase nuclei, with unusual form or extremely condensed appearance, also given by the sum of total observed alterations by the total number of counted cells, and expressed as the number of alterations out of 100 cells (for calculations, see [19]).

In summary, the tested agent can be characterized as cytotoxic when it alters the normal MI (increase or reduction) of the used plant model, hence causing malfunctioning of cell structures and possibly leading to cell death, and/or genotoxic if the alterations observed throughout the cycle are related to DNA breakage, including the formation of micronuclei. These bodies are considered a mutagenicity parameter as they represent damage not corrected by the cell repair system and, thus, permanent and transmissible to the subsequent cell generations. An alteration can also be classified as aneugenic, when it is related to malformation or malfunctioning of the mitotic spindle or the attachment of the chromosomes on the spindle and leads to gain or loss of one or more chromosomes, or clastogenic, when associated to breakages and rearrangements in the DNA or chromosomes [16, 20, 21]. Each of these endpoints and possible alterations that can be observed throughout the mitotic cell cycle, and their consequences, will be detailed next.
