**3. What can cell cycle analyses reveal?**

Evaluation of the cell cycle, which comprises the interphase (G1, S, and G2) and the M phase (mitosis-prophase, metaphase, anaphase, and telophase), allows gaining knowledge about the organizational structure of the chromosomes and how they behave during the cell division. As mentioned previously in this chapter, such assessment can be employed to determine the toxicity of a chemical compound. Alterations in the mitotic index help determine the degree of cytotoxicity of an agent, whereas chromosome alterations observed in the cell cycle define the genotoxicity of the agents and their capacity of causing damage to the DNA, which may or may not be repaired by the cellular repair mechanisms. Together, the cell cycle alterations express the cyto(geno)toxicity of chemical compounds and environmental pollutants and are used to investigate their toxic potential.

Several endpoints can be monitored in the division of meristematic cells, such as the chromosomal and nuclear aberrations previously described, besides the formation of micronuclei.

To better understand the alterations observed in the cell cycle, it is necessary to remember that the movement of chromosomes for segregation of the DNA into the daughter cells relies on the mitotic spindle, formed by microtubules. The whole dynamics of the mitotic process thus depend on the binding of the microtubules to the chromosome centromeres, besides microtubule polymerization and depolymerization mechanisms. In this sense, alterations in these dynamics affect the segregation of chromosomes to the daughter cells and may be considered the first origin of alterations observed in the cell cycle. Hence, as consequences of alterations in the spindle and correct attachment of the chromosomes, we can cite the interruption of the cell cycle in metaphase, originating c-metaphases (**Figure 1A**) and formation of polyploid cells (**Figure 1B**), as consequence, and multipolar anaphase (**Figure 1C**), non-oriented chromosomes at the equatorial plan (**Figure 1D**) or delayed segregation of the chromosomes/chromatids in anaphase/telophase (**Figure 1E** and **F**) [21–24].

When interference in the polymerization and depolymerization of the microtubules occurs, the cell cycle may be paralyzed in metaphase, and the chromosomes are visualized as well condensed, with well-defined centromere and spread inside the cell [22]. In the laboratory, this situation is caused with substances' denominated blockers, such as colchicine, which gives this alteration its name: colchicine metaphase or c-metaphase (**Figure 1A**). These extremely condensed and separated chromosomes are used in karyotype studies of the species, as they allow observing the morphology of each chromosome individually.

nucleus, each chromatid of the chromosome starts representing one DNA molecule of the cell that will be replicated in the S phase. Upon initiating a new mitotic cycle, after G2, the proteins of the chromosome's protein scaffold keep the sister chromatids united, and the cell starts mitosis with a duplicated number of chromosomes, characterizing polyploidy. Under light microscope, a cell is characterized as polyploid when an excess number of chromosomes and/or cell volume larger than usual can be observed at the end of prophase or beginning of metaphase (**Figure 1B**).

**Figure 1.** Example of cell cycle alterations observed in meristematic cells of *Allium cepa* (onion) and *Lactuca sativa* (lettuce) root tips. (A) C-metaphasis in lettuce exposure to methyl methanesulfonate (MMS); (B) polyploidy metaphasis in lettuce exposure to cadmium; (C) multipolar anaphases in onion exposure to atrazine herbicide; (D) non-oriented chromosome (black arrow) in onion metaphasis exposure to cadmium; (E) not normal/laggard segregation (black arrow) in lettuce anaphase exposure to cadmium; (F) not normal/laggard segregation (black arrow) in onion telophase exposure to MMS; (G) micronuclei in onion exposure to MMS; (H) anaphase bridge (black arrow) in lettuce exposure to spent Potliner (SPL); (I) anaphase bridge (red arrow) in onion exposure to MMS with a fragment (black arrow) and a micronucleus (green arrow); (J) chromosome fragments (black arrow) in onion exposure to MMS; (K) condensed nuclei (black arrow) in lettuce exposure to atrazine herbicide; and (L) stickiness chromosome in lettuce exposure to SPL. Images obtained in a light microscope at oil objective (100×). Bars 10 μm.

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Polyploidy emerges as a consequence of the prolonged effect of a substance or toxic compound in the cells. In the absence of the spindle, the cell with duplicated DNA, represented by the chromosome with two chromatids, returns to interphase, initiating a new cell cycle. In the G1 Cyto(Geno)Toxic Endpoints Assessed via Cell Cycle Bioassays in Plant Models http://dx.doi.org/10.5772/intechopen.72997 121

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

Evaluation of the cell cycle, which comprises the interphase (G1, S, and G2) and the M phase (mitosis-prophase, metaphase, anaphase, and telophase), allows gaining knowledge about the organizational structure of the chromosomes and how they behave during the cell division. As mentioned previously in this chapter, such assessment can be employed to determine the toxicity of a chemical compound. Alterations in the mitotic index help determine the degree of cytotoxicity of an agent, whereas chromosome alterations observed in the cell cycle define the genotoxicity of the agents and their capacity of causing damage to the DNA, which may or may not be repaired by the cellular repair mechanisms. Together, the cell cycle alterations express the cyto(geno)toxicity of chemical compounds and environmental pollutants

Several endpoints can be monitored in the division of meristematic cells, such as the chromosomal and nuclear aberrations previously described, besides the formation of micronuclei.

To better understand the alterations observed in the cell cycle, it is necessary to remember that the movement of chromosomes for segregation of the DNA into the daughter cells relies on the mitotic spindle, formed by microtubules. The whole dynamics of the mitotic process thus depend on the binding of the microtubules to the chromosome centromeres, besides microtubule polymerization and depolymerization mechanisms. In this sense, alterations in these dynamics affect the segregation of chromosomes to the daughter cells and may be considered the first origin of alterations observed in the cell cycle. Hence, as consequences of alterations in the spindle and correct attachment of the chromosomes, we can cite the interruption of the cell cycle in metaphase, originating c-metaphases (**Figure 1A**) and formation of polyploid cells (**Figure 1B**), as consequence, and multipolar anaphase (**Figure 1C**), non-oriented chromosomes at the equatorial plan (**Figure 1D**) or delayed segregation of the chromosomes/chro-

When interference in the polymerization and depolymerization of the microtubules occurs, the cell cycle may be paralyzed in metaphase, and the chromosomes are visualized as well condensed, with well-defined centromere and spread inside the cell [22]. In the laboratory, this situation is caused with substances' denominated blockers, such as colchicine, which gives this alteration its name: colchicine metaphase or c-metaphase (**Figure 1A**). These extremely condensed and separated chromosomes are used in karyotype studies of the species, as they

Polyploidy emerges as a consequence of the prolonged effect of a substance or toxic compound in the cells. In the absence of the spindle, the cell with duplicated DNA, represented by the chromosome with two chromatids, returns to interphase, initiating a new cell cycle. In the G1

their consequences, will be detailed next.

120 Cytotoxicity

**3. What can cell cycle analyses reveal?**

and are used to investigate their toxic potential.

matids in anaphase/telophase (**Figure 1E** and **F**) [21–24].

allow observing the morphology of each chromosome individually.

**Figure 1.** Example of cell cycle alterations observed in meristematic cells of *Allium cepa* (onion) and *Lactuca sativa* (lettuce) root tips. (A) C-metaphasis in lettuce exposure to methyl methanesulfonate (MMS); (B) polyploidy metaphasis in lettuce exposure to cadmium; (C) multipolar anaphases in onion exposure to atrazine herbicide; (D) non-oriented chromosome (black arrow) in onion metaphasis exposure to cadmium; (E) not normal/laggard segregation (black arrow) in lettuce anaphase exposure to cadmium; (F) not normal/laggard segregation (black arrow) in onion telophase exposure to MMS; (G) micronuclei in onion exposure to MMS; (H) anaphase bridge (black arrow) in lettuce exposure to spent Potliner (SPL); (I) anaphase bridge (red arrow) in onion exposure to MMS with a fragment (black arrow) and a micronucleus (green arrow); (J) chromosome fragments (black arrow) in onion exposure to MMS; (K) condensed nuclei (black arrow) in lettuce exposure to atrazine herbicide; and (L) stickiness chromosome in lettuce exposure to SPL. Images obtained in a light microscope at oil objective (100×). Bars 10 μm.

nucleus, each chromatid of the chromosome starts representing one DNA molecule of the cell that will be replicated in the S phase. Upon initiating a new mitotic cycle, after G2, the proteins of the chromosome's protein scaffold keep the sister chromatids united, and the cell starts mitosis with a duplicated number of chromosomes, characterizing polyploidy. Under light microscope, a cell is characterized as polyploid when an excess number of chromosomes and/or cell volume larger than usual can be observed at the end of prophase or beginning of metaphase (**Figure 1B**).

Multipolar anaphase and abnormal segregation of chromatids in anaphase/telophase also arise from the action of chemical substances on the organization of the microtubules. These alterations are observed as a consequence of incorrect binding of the mitotic spindle to the centromere of the chromosomes [24] or from the shortening and elongation of some microtubules of the mitotic spindle out of synchrony with the other microtubules. Unequal disjunction of the chromosomes may thus occur (non-oriented chromosomes, **Figure 1D**), giving rise to micronuclei (**Figure 1G**) when these chromosomes cannot be reincorporated into the main nucleus along with the other chromosomes [25].

All these reported alterations, if persistent and deleterious, activate the cell death mechanisms. Under light microscope, the evidence for occurrence of cell death is the observation of highly condensed interphase nuclei (**Figure 1K**), with very heterochromatic chromatin, appearing well rounded, darker, and smaller than the normal interphase nuclei [26, 27].

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The cell death process due to abiotic stress is cytologically characterized by condensed nuclei and molecularly by DNA fragmentation [26]. This death mechanism is related to destruction

Toxic substances can also trigger the formation of sticky chromosomes (**Figure 1L**). Overall, they are characterized by alterations in the physicochemical structure of the DNA, proteins, or both, formed from complexes with phosphate groups of the DNA, inter- and intrachromatid linkages, and DNA condensation [22, 29]. These factors promote loss of the normal characteristics of condensation, causing the formation of agglomerates [22, 30]. Chromosome stickiness is considered a highly toxic alteration [31] that hinders the segregation of the chromatids and the normal continuation of the cell division, which may trigger the cell death

Of the observed alterations, chromosome stickiness is considered the most intriguing as regards the classification in aneugenic or clastogenic, in function of the mechanisms involved in their occurrence in the cell cycle. Here, it is considered a complex cyto(geno)toxic effect arising from previous events, for instance, polyploidy or excessive breakages and bridges in the DNA molecule, present at different levels. As regards the consequences of stickiness to the cell, some authors like Andrade et al. [31] cite that the high frequency of stickiness may activate the cell death mechanisms. Thus, the induction of severe stickiness cannot be repaired by

process, avoiding that the toxic effect be passed onto the following generation.

the cells, having as consequence the heterochromatinization of the whole nucleus.

tion of techniques available as kits containing a marker for fragmentation.

**4. Investigation of cell death mechanisms and DNA damage applied** 

DNA fragmentation, previously reported as the clastogenic effect of a toxic agent in the cell, is one of the mechanisms related to the cell death process. It can be evaluated through applica-

The Terminal d-UTP Nick End Labeling (TUNEL) assay is one of the tests used for the analysis of DNA fragmentation and investigation of the cell death mechanisms. It is based on incorporation of nucleotides (d-UTP = 2′-deoxyuridine, 5′-triphosphate) marked with a fluorochrome (fluorescein isothiocyanate, FITC) in the free 3'OH region of the breakages in the DNA chain by the enzyme terminal deoxynucleotidyl transferase (TdT) [26, 32]. This reaction relies on the capacity of the enzyme TdT of coupling a deoxy-uracyl-fluorescein (d-UTP) conjugated to the 3'OH end of the broken DNA [32, 33]. The incorporation of fluorescein-12-d-UTP is then amplified by various enzymatic reactions [34]. These nucleotides can be marked with a fluorescent dye and detected by fluorescence microscopy or the laser

and subsequent elimination of damaged cells [28].

**to environmental toxicology**

of a cytometer [35].

So far, some alterations of the cell cycle have been demonstrated which arise from effect of the toxic agent on the malformation of a cellular structure. Together, these alterations characterize an aneugenic effect and mechanism of the toxic agent, as they can have as consequences the increase or decrease in the number of chromosomes of the species. All these aneugenic alterations represent the cytotoxicity of a given substance, as they relate to a cell structure.

The action of toxic substances can also occur directly on the DNA. In this case, they are observed in the cell cycle as alterations in chromosome structure. Since the effects occur on the genetic material in this case, the mechanism of action of the substance is clastogenic and represents its genotoxicity.

The most evident clastogenic effect when observing the cell cycle under the microscope is the presence of bridges (**Figure 1H** and **I**) and chromosome fragments (**Figure 1I**) arising from breakages in the DNA molecule. Overall, one of the consequences of the breakage is the loss of telomeres, a region in the terminal extremity of the chromosomes that has the function of ensuring the chromosome protection and stability. With the loss of the chromosome stability, fusion of the terminal portion of two chromosomes may occur. Upon division, chromosomes with two centromeres are observed as bridges in anaphase/telophase (**Figure 1H** and **I**), where each of the centromeres is linked to the spindle of one of the cell poles.

The chromosome region originated from the breakage that is devoid of centromere is denominated acentric fragment (**Figure 1J**). These chromosome fragments, due to containing parts of the genetic material, are recognized by the cell and involved in membrane during cell division, giving rise to micronuclei, which are easily observed in cells of the F1 generation [7].

Several authors highlight and affirm that micronuclei are the most effective and simple endpoint for the analysis of mutagenic effects caused by chemical compounds, owing to their arising from non- or incorrectly repaired damage in the parental cells. They are easily observed in daughter cells as a structure similar to the main nucleus but with smaller size (**Figure 1G**). Indeed, the micronucleus is easily recognized in the cell visualized under the microscope, particularly if the preparation was accomplished using a DNA-specific dye. In several cytological study models, including human blood cells in culture, the micronuclei assay is applied as a marker of mutagenicity. However, as explained here, it can originate from both acentric fragments and entire chromosomes that were not bound to the spindle. Since each of these causes of micronuclei formation originates from a distinct mechanism of action, assessment of the entire cell cycle, if possible, together with evaluation of micronuclei induction is seen as the cheapest strategy to determine the mechanism of action of the studied substance or compound.

All these reported alterations, if persistent and deleterious, activate the cell death mechanisms. Under light microscope, the evidence for occurrence of cell death is the observation of highly condensed interphase nuclei (**Figure 1K**), with very heterochromatic chromatin, appearing well rounded, darker, and smaller than the normal interphase nuclei [26, 27].

Multipolar anaphase and abnormal segregation of chromatids in anaphase/telophase also arise from the action of chemical substances on the organization of the microtubules. These alterations are observed as a consequence of incorrect binding of the mitotic spindle to the centromere of the chromosomes [24] or from the shortening and elongation of some microtubules of the mitotic spindle out of synchrony with the other microtubules. Unequal disjunction of the chromosomes may thus occur (non-oriented chromosomes, **Figure 1D**), giving rise to micronuclei (**Figure 1G**) when these chromosomes cannot be reincorporated into the main

So far, some alterations of the cell cycle have been demonstrated which arise from effect of the toxic agent on the malformation of a cellular structure. Together, these alterations characterize an aneugenic effect and mechanism of the toxic agent, as they can have as consequences the increase or decrease in the number of chromosomes of the species. All these aneugenic alterations represent the cytotoxicity of a given substance, as they relate to a cell structure.

The action of toxic substances can also occur directly on the DNA. In this case, they are observed in the cell cycle as alterations in chromosome structure. Since the effects occur on the genetic material in this case, the mechanism of action of the substance is clastogenic and

The most evident clastogenic effect when observing the cell cycle under the microscope is the presence of bridges (**Figure 1H** and **I**) and chromosome fragments (**Figure 1I**) arising from breakages in the DNA molecule. Overall, one of the consequences of the breakage is the loss of telomeres, a region in the terminal extremity of the chromosomes that has the function of ensuring the chromosome protection and stability. With the loss of the chromosome stability, fusion of the terminal portion of two chromosomes may occur. Upon division, chromosomes with two centromeres are observed as bridges in anaphase/telophase (**Figure 1H** and **I**), where

The chromosome region originated from the breakage that is devoid of centromere is denominated acentric fragment (**Figure 1J**). These chromosome fragments, due to containing parts of the genetic material, are recognized by the cell and involved in membrane during cell division, giving rise to micronuclei, which are easily observed in cells of the F1 generation [7].

Several authors highlight and affirm that micronuclei are the most effective and simple endpoint for the analysis of mutagenic effects caused by chemical compounds, owing to their arising from non- or incorrectly repaired damage in the parental cells. They are easily observed in daughter cells as a structure similar to the main nucleus but with smaller size (**Figure 1G**). Indeed, the micronucleus is easily recognized in the cell visualized under the microscope, particularly if the preparation was accomplished using a DNA-specific dye. In several cytological study models, including human blood cells in culture, the micronuclei assay is applied as a marker of mutagenicity. However, as explained here, it can originate from both acentric fragments and entire chromosomes that were not bound to the spindle. Since each of these causes of micronuclei formation originates from a distinct mechanism of action, assessment of the entire cell cycle, if possible, together with evaluation of micronuclei induction is seen as the cheapest strategy to determine the mechanism of action of the studied substance or compound.

each of the centromeres is linked to the spindle of one of the cell poles.

nucleus along with the other chromosomes [25].

represents its genotoxicity.

122 Cytotoxicity

The cell death process due to abiotic stress is cytologically characterized by condensed nuclei and molecularly by DNA fragmentation [26]. This death mechanism is related to destruction and subsequent elimination of damaged cells [28].

Toxic substances can also trigger the formation of sticky chromosomes (**Figure 1L**). Overall, they are characterized by alterations in the physicochemical structure of the DNA, proteins, or both, formed from complexes with phosphate groups of the DNA, inter- and intrachromatid linkages, and DNA condensation [22, 29]. These factors promote loss of the normal characteristics of condensation, causing the formation of agglomerates [22, 30]. Chromosome stickiness is considered a highly toxic alteration [31] that hinders the segregation of the chromatids and the normal continuation of the cell division, which may trigger the cell death process, avoiding that the toxic effect be passed onto the following generation.

Of the observed alterations, chromosome stickiness is considered the most intriguing as regards the classification in aneugenic or clastogenic, in function of the mechanisms involved in their occurrence in the cell cycle. Here, it is considered a complex cyto(geno)toxic effect arising from previous events, for instance, polyploidy or excessive breakages and bridges in the DNA molecule, present at different levels. As regards the consequences of stickiness to the cell, some authors like Andrade et al. [31] cite that the high frequency of stickiness may activate the cell death mechanisms. Thus, the induction of severe stickiness cannot be repaired by the cells, having as consequence the heterochromatinization of the whole nucleus.
