**5. Final considerations**

Under fluorescence microscope, the cells can be visualized with different fluorescence intensities in function of the marker, fluorescein. They are then classified as (A) not marked (**Figure 2**), thus without fragmentation; (B) weakly marked (**Figure 2**), therefore presenting light damage that can still be recovered, since the cell death process involves several steps and only the final ones represent a "one-way road"; and (C) strongly marked (**Figure 2**), associated to cells with

The comet assay or single cell gel electrophoresis (SCGE) is another technique very useful to identify DNA damage. It allows the detection of damage to the genetic material caused by rupture of chains, alkali-labile sites (ALS), incomplete excision repair sites, and reticulations, induced by alkylating or intercalating agents and oxidative damage, even before the cell repair system acts. Further, it allows verifying the damage present after the cell repair process.

In plants, it is broadly used in ecotoxicological studies of environmental pollutants and is characterized by its high sensitivity and specificity, low cost, and rapidness in detecting the genotoxic effects, requiring small sample size, and allowing for simple analysis. The evaluation can be performed at individual cell level or applied to any cell population, without requirement of cell division. In summary, it can be executed in three versions and detect a

The three possible versions of the comet assay differ with regard to the pH of the electrophoresis buffer, which can be neutral, slightly alkaline, and alkaline-alkaline. In the neutral method, ruptures of the DNA double strand are detected. In the moderately alkaline version, simple breakages in the DNA and the double helix are observed. In turn, in the alkaline-alkaline approach, used in the majority of the studies owing to its greater sensitivity, breakages of single and double strands as well as alkali-labile sites and crosslinks are quantified. The choice

of the comet assay version depends on the type of damage that shall be observed [38].

The comet assay can be used to complement the cytogenetic data obtained from the cell cycle analyses, as it detects genomic lesions caused to the DNA arising from the action of mutagens. Unlike mutations, the lesions identified by the comet assay are prone to repair. The technique

**Figure 2.** Meristematic cells of *Lactuca sativa* L. (lettuce) treated with MMS submitted to the TUNEL test. (A) Image captured with filter at wavelength of 345–358 nm (for DAPI). (B) Image captured with filter at wavelength of 488– 495 nm. (C) Result of overlapping of images A and B made through the AxioVision program, where it is possible to observe unmarked nuclei, without damage (white arrow); weakly marked nuclei, with slight damage (yellow arrow); and strongly marked nuclei, with severe damage (red arrow) to DNA. Images obtained in a microscope of fluorescence

high frequency of fragmented DNA and in advanced stage of cell death [18].

broad spectrum of damage to the genetic material [36, 37].

124 Cytotoxicity

(Olympus BX 60) on 40× objective. Bars 50 μm.

Bioassays with plants usually comprise macroscopic tests, which involve evaluations of germination and initial plantlet development (growth of root and aerial part), as well as microscopic evaluations, including the observation of alterations during the cell cycle in meristematic cells exposed to the tested chemical agent. The results of these bioassays allow determining the phytotoxicity, cytotoxicity, genotoxicity, and mutagenicity of the pollutant or chemical compound in question.

Both in the macroscopic and microscopic evaluations, the root is the plant organ used in the tests. It is particularly useful in these eco(geno)toxicological tests, as it is the first part of the plant to be exposed to environmental pollutants. For macroscopic assessment, the observation of root growth is a rapid and sensitive method for environmental monitoring, but does not contribute to the understanding of toxicity mechanisms.

Root tips contain meristematic cells that present intensive cell division, allowing a rapid and adequate evaluation of the cell cycle, constituting the microscopic assessments whose measurable parameters have been described here. Besides providing important information to determine the mode of action of a given agent, the described microscopic evaluations are directly related to the growth parameter assessed in the macroscopic assay. In plants, as sessile organisms, the growth of an organ is closely related to the increase in the number of cells in the tissue composing it. This way, alterations in the endpoint mitotic index, assessed in the microscopic assay, explain what can be seen with the naked eye. In turn, the endpoint associated to the malfunctioning of cell structures like the mitotic spindle explains alterations in the mitotic index as well as cell death, which is an endpoint characterized by increase in condensed nuclei and which contributes to the reduction of the mitotic index. Nevertheless, the endpoint associated to DNA fragmentation can be assessed not only by direct observation of the cell cycle but also through more specific techniques such as the TUNEL or comet assays. These methods ultimately explain and confirm the induction of cell death, which, as already reported, contributes for a mitodepressive and phytotoxic effect on root growth.

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In conclusion, based on the endpoints that can be assessed through cell cycle analyses, cytotoxicity as well as genotoxicity and mutagenicity of environmental pollutants can be determined in a rapid and reliable manner. Such tests are highly useful to monitor and assess the risk of potentially toxic substances that are released into the environment.
