Silvany de S. Araújo, Thaís C.C. Fernandes, Maria A. Marin-Morales, Ana C. Brasileiro-Vidal and
Ana M. Benko-Iseppon*
Introduction
Over the centuries, medicinal plants have been the basis for the treatment of different diseases, considered as the main therapeutic source for innumerous communities and ethnic groups (Borges et al. 2013).
Furthermore, their great chemical diversity offers unlimited opportunities for drug discovery (Sasidharan et al. 2011).
The systematic search for new substances useful for therapeutic purposes can be accomplished through various processes. Most of them regard the synthesis of new molecules, molecular modifi cation of natural or synthetic substances with defi ned pharmacological properties, as well as extraction, isolation and purifi cation of novel compounds from medicinal plants. Research work on medicinal plants, as a rule, give rise to drugs in a shorter time, with lower costs, being therefore more accessible to the population (Brito and Brito 1993, Ugaz et al. 1994).
Despite the therapeutic benefi ts, it is known that medicinal plants and their derivatives may exhibit toxic properties, including carcinogenic and mutagenic nature or, still, can cause changes in the DNA. Such changes may affect vital processes as replication and gene transcription, sometimes also chromosomal alterations that lead to cancer and cell death processes (Belcavello et al. 2012, Ping et al. 2012). They may also cause chromatin modifi cations associated to epigenetic reprogramming, often leading to drastic consequences to the affected organism (Csokaa and Szyf 2009).
Due to increasing reports on adverse effects of such products, regulatory and supervisory agencies from many countries require prior fulfi llment of safety tests for medicinal plants, including in vitro and
Universidade Federal de Pernambuco, Recife, 50.670-420, Recife, Brazil.
* Corresponding author: ana.iseppon@gmail.com
in vivo toxicology testing, regarding cytotoxicity, genotoxicity and mutagenicity (Nunes et al. 2012). These are the fi rst tests for the evaluation of potential clinical application of a new material, due to its greater reproducibility, reduced experimental period and need of small amounts of target compounds.
Therefore, it is clearly important to evaluate the cytotoxic potential of medicinal plants and their compounds using in vitro and in vivo systems. Throughout this chapter the main tests and model organisms adopted for such evaluations including cytotoxic, mutagenic and genotoxic potential of medicinal plants for the development of drugs will be presented.
Test organisms: their importance and application in the investigation of toxic effects of medicinal plants
The test systems may be divided into groups based on the used biological system and in the genetic endpoint detected. Bioassays with prokaryotes allow the detection of agents that induce gene mutation and primary damage in the DNA. Moreover, analyses with eukaryotes allow the detection of a greater extent of damages, ranging from gene mutations to chromosomal damage and aneuploidy (Houk 1992). In this context, genotoxicity and toxicity tests using microorganisms, plant and mammalian cells have been used alone or in combination to obtain more reliable results for the pharmaceutical industry (Zegura et al. 2009).
Microorganisms
The development and standardization of toxicity tests based on prokaryotes (bacteria) and eukaryotes, such as protozoa, unicellular algae and yeast—instead of higher organisms—allowed a rapid and inexpensive screening of samples against toxic and genotoxic effects. The fi rst generation of bioassays was based on diverse naturally sensitive microbes, whereas the second generation included genetically modifi ed microorganisms to achieve better sensitivity and/or specifi city. Most parameters measured by microbial toxicity trials are population growth, substrate consumption, respiration, ATP luminescence and bioluminescence inhibition (Logar and Vodovnik 2007).
Bacteria such as Salmonella typhimurium, Escherichia coli and Bacillus subtilis are used in the most widely applied methods for detection of gene mutations. The rapid cell division and the relative ease with which large amounts of data can be generated (approximately 108 bacteria per test pate) turned bacterial assays to be the most widely used in routine tests for mutagenicity of chemical compounds.
The mutagenicity assay using S. typhimurium—also known as Salmonella/microsome, or Ames test—is actually the most common screening method to detect genotoxic carcinogenic substances, being validated in scale by several laboratories (Umbuzeiro and Vargas 2003). The Ames test is characterized by the use of indicator strains of S. typhimurium sensitive to substances able to induce various kinds of mutations. In the presence of mutagenic agents, these strains reverse their auxotrophic character to synthesize histidine, forming colonies in a medium deprived of this amino acid. Thus, by counting colonies per plate, the mutagenic effect of a given compound may be established in association to sampled concentrations (Mortelmans and Zeiger 2000). However, bacteria are evolutionarily distant from the human model, lacking true nuclei and also enzymatic pathways to activate most promutagenic intermediates necessary to form mutagenic compounds (Teaf and Middendorf 2000).
Some carcinogenic chemicals such as aromatic amines and polycyclic aromatic hydrocarbons, for example, are biologically inactive unless they are metabolized to active forms. In humans and lower animals, the metabolic oxidation of cytochrome P450 is able to metabolize a large number of these chemicals to electrophilic forms that can react with DNA (Mortelmans and Zeiger 2000). Since bacteria do not have the cytochrome P450 oxidation system used by vertebrates in biotransformation of exogenous compounds, it is important to mimic this system in the tests, so that the responses are close to those found in eukaryotes.
This procedure is performed by adding rat liver cells homogenate pre-treated with the enzyme-inducer Aroclor 1254 (S9 fraction). Thus, substances that exert their mutagenic activity after metabolism via cytochrome P450 can be detected by the addition of S9, whereas those—who do not need this oxidation system to exert their mutagenic effects—are identifi ed in the absence of S9 (Jarvis et al. 1996).
Besides bacteria, also some fungi have been used in genotoxicity trials. Yeasts as Saccharomyces, Schizosaccharomyces, as well as Neurospora and Aspergillus have been used in mutation assays, which are similar in design to the testing of reverse histidine mutation in Salmonella (Teaf and Middendorf 2000).
Plants
Plants are also recognized as excellent indicators of genotoxic and cytotoxic effects, being applicable in the evaluation of mutagenesis generated by chemical factors. Different types of assays are available in plant systems, including locus-specifi c testing in maize (Zea mays L., Poaceae) and multilocus assay systems in Arabidopsis. Cytogenetic tests were developed for Tradescantia (Commelinaceae family), especially the micronucleus assay, whereas assays for chromosomal aberrations in root tips of onion (Allium cepa L., Amaryllidaceae) and beans (Vicia faba L., Fabaceae). Finally, analysis of DNA adducts is applicable to somatic and reproductive plant cell systems (Teaf and Middendorf 2000).
Among the higher plants used as test models, A. cepa has been considered an excellent in vivo model, in which the roots grow in direct contact with the substance of interest (effl uent, toxin, chemical compound, etc.), allowing prediction of possible damage to eukaryotic DNA (Tesdesco and Laughinghouse 2012). This test is of great value, since it has a high sensitivity to detect changes in cellular cycle and in chromosomes, from point mutations up to chromosomal aberrations (Leme and Marin-Morales 2008).
The A. cepa test shows good correlation and high sensitivity when compared to other test systems, especially with tests using mammals. It presented a correlation of 82% when compared to the carcinogenicity test in rodents. Moreover, the A. cepa test system presents almost the same sensitivity as the test system of algae and human lymphocytes (Fiskesjo 1985, Rank and Nielsen 1994, Leme and Marin-Morales 2007).
Another advantage is that A. cepa exhibits eight pairs of relatively large chromosomes (2n = 16) that allows easy detection of chromosomal aberrations. In addition, the plant material is easy to obtain and is available throughout the year at low cost, presents easy handling, high sensitivity, possibility of using the tissue without pretreatment, reliability, aiding studies of prevention of damage to human health (Bagatini et al. 2007, Leme and Marin-Morales 2009, Abdelmigid 2013). Added to these factors, it presents rapidly growing roots, large numbers of dividing cells and is tolerant to different natural conditions (Matsumoto et al. 2006). That is why it has been considered a useful tool for research on genotoxicity and cytotoxicity of chemicals, complex substances, such as plant extracts (Cuchiara et al. 2012).
The main advantage of the ‘Allium cepa assay’ concerns its simple and fast protocol, as described in the Table 7.1, demanding only a standard optical microscope with few methodological steps, as illustrated in Fig. 7.1.
Additionally the root tips are relatively large and present a tip with a large meristem that is easily spread in the slides, with large cell nuclei and chromosomes with easily recognizable aberrant and/or normal mitotic phases (Fig. 7.2).
Rodents
Rodents are commonly used in tests with animals, especially rats and mice, but also guinea pigs, hamsters, mice, desert rats and others. Two rodent species stand out as used models for carcinogenicity testing and by the pharmaceutical industry: the mouse and the rat. The derived strain Sprague-Dawley rat is more commonly used in American Pharmaceutical toxicology laboratories, followed by the Wistar and Fischer 344 strain, while strains Evans and CFE (Carworth) are used less frequently. Considering mice, strain CD-1 is the most frequently used in the pharmaceutical industry. Other strains are less frequently used, as B6C3F1, NMRI, C57B1, BALB/c and Swiss (Gad 2008). In vivo assays using rodents have the advantage of exhibiting greater similarity to the human system and, consequently, to important mutations in the etiology of degenerative diseases such as cancer and other genetic diseases (Lima and Ribeiro 2003).
The micronucleus assay (MN) in the bone marrow of rodents is widely used for the detection of clastogenic agents (that chromosomes break) and aneugenic (which interfere with the spindle fi bers) and
Figure 7.1. Methodological steps of the Allium cepa (onion) assay. For further details see Table 7.1.
Table 7.1. Allium cepa procedure (as described by Marin-Morales 2009).
1. Collect the germinating root tips from the Petri dish (as shown in Fig. 7.1) after exposition to the test substance and fi x in ethanol: acetic acid (3:1).
2. Wash the root tips three times in distilled water for 5 minutes each and remove excess water from the roots touching them gently on fi lter paper.
3. Hydrolyze roots in vials containing 1N HCl at 60°C in a water bath for 10 minutes.
4. Wash the root tips again three times in distilled water, removing excess water on fi lter paper (taking great care because the meristem is even more fragile).
5. Transfer the root tips to vials (brown glass, due to their sensitivity to light) containing Schiff´s reagent (Merk), where they should remain for 2 hours in the dark.
6. Wash the root tips in distilled water with the aid of a Pasteur pipette, until all excess stain is removed.
7. Using a clean and identifi ed slide, place an intact root meristem (with the terminal portion stained purple, standing out from the rest of the root stained in pink) over the slide.
8. Remove excess of water with the aid of a fi lter paper.
9. Cut meristem with the aid of a razor blade and discard the rest of the root (stained in pink).
10. Add a drop of 2% acetic carmine to the meristem in the slide and cover with a cover slip.
11. Hold the cover slip with a fi lter paper and spread the material with slightly tapping, using a whisk with a blunt tip until the meristem turns into a small patch of scattered cells.
12. Remove excess acetic carmine, carefully pressing the slide and the coverslip involved in a fi lter paper, being careful not to drag the material.
13. Heat the slide in fl aming lamp, monitoring its temperature to avoid burning of the cells.
14. Freeze the slides in liquid nitrogen for a time varying from 40 seconds to 3 minutes.
15. After immersion in liquid nitrogen, remove the coverslip quickly with the aid of a scalpel blade controlling if the material remained fi xed to the slide.
16. After drying the slides, add a drop of mounting medium (Entellan) and cover the material with a dry, clean coverslip.
17. Dry slides overnight and store them in a dry place until the time of analysis.
has been widely used to measure both in vitro and in vivo genotoxicity. The assay is characterized by observing the effect of the agent tested in immature enucleated erythrocytes, which have a short life, so that any MN observed represent recent chromosomal damage (Ribeiro 2003).
In vivo MN assay is especially relevant because it allows obtaining of additional information on experimental conditions, such as metabolism, pharmacokinetics and DNA repair processes. It is used fi rst to assess the ability of the tested substance to induce structural or numerical chromosomal damage, both associated with the onset and/or progression of tumors (Krishna and Hayashi 2000). Because it is easy to identify and has a well-defi ned distribution, the positive results obtained with the in vivo MN test provide strong evidence of systemic genotoxicity of the evaluated chemical compound assessed under appropriate experimental conditions. On the other hand, the negative results support the conclusion that the tested substance is not genotoxic in vivo (Salvadori et al. 2003).
Because of its advantages, the in vivo MN assay in rodent bone marrow is widely accepted by international agencies and government institutions, as part of the battery of tests recommended to establish an evaluation and registration of new chemicals and pharmaceuticals entering annually on the world market (Ribeiro 2003, Tagliati et al. 2008), since the results are considered highly signifi cant in the context of human health (Morita et al. 1997).
Figure 7.2. Main chromosome aberrations observable in Allium cepa meristematic cells. (A) Micronucleated cell. (B) Polyploid metaphase. (C) Anaphase with chromosomal breakage. (D) Metaphase with chromosomal loss. (E) Anaphase with chromosomal bridge. (F) Multipolar anaphase with chromosomal bridges. (G) Chromosomal adherence. (H) Nucleus with nuclear bud. (I) C-metaphase. Bar: 10 μm (for all pictures).
However, the in vivo toxicity tests are being mainly criticized due to the large number of animals required and suffering caused to them during some types of experiments. In this scenario, industries, government regulators and actors of quality control are under increasing pressure to replace in vivo testing by alternative methods which do not use living animals. Concern about the use of animals in experiments for toxicity tests has been a widely discussed topic (Bednarczuk et al. 2010). Therefore, in vitro studies have been increasingly used, since they limit the number of experimental variables, present simpler and faster implementation than the in vivo tests and can replace animals or, at least, serve as a previous screening prior to in vivo tests (Rogero et al. 2003).
Cell culture
In vitro cell culture method is the most widely used in pharmacology and toxicology for drug development and toxicological investigations, being considered the starting point for such studies (Spielmann et al.
2008). In vitro assays become more advantageous compared to in vivo testing since they provide better control over experimental conditions, allowing better reproducibility of testing conditions and associated results. They also present lower costs, higher speed, and simplicity with reduction of the number of animals used for overall assays.
Another signifi cant advantage is the current availability of a variety of strains in cell banks, allowing increased access and use of in vitro models, becoming an alternative to toxicity assessments of many chemical substances. Thus, in vitro toxicity testing has been also proposed as substitutes for acute toxicity test (Bernauer et al. 2005).
Cell culture tests evaluate the potential of a substance to induce point mutations, clastogenesis and/
or aneugenesis using mammalian cell lines or human primary cell cultures (Eastmond et al. 2009). With aid of these tests it is possible to evaluate the recombinogenic potential of any substance with ease, speed, security and good correlation to in vivo results (Pinhatti 2009). Results obtained in such in vitro systems direct in vivo testing, generating a rational basis for evaluating the real need for testing, the choice of the most suitable animal model as well as the type of test to be applied for the necessary prediction of the results considering the probable mechanism involved (Zucco et al. 2004).
Most cell lines correspond to cells that were initially derived from tumors. Because of their proliferative capacity, tumor cells remain in constant division, which permits their in vitro maintenance (Knasmuller et al. 1998). The use of tumor cell lines contributes to the discovery of new anticancer drugs with antineoplasic activity as well as for understanding the biological events involved in cancer. Most frequently used cell strains in cytotoxicity assays include L5178Y mouse lymphoma cells, CHO, CHO-AS52, Chinese hamster V79, human TK6 linphoblastonoids, as well as blood and bone marrow cells of different mammal species (Wang et al. 2009).
The human cell lines have a particular importance in research on the establishment of in vitro models of human diseases and also in investigating pharmacodynamic mechanisms of potentially active molecules (Gottfried et al. 2006). For example, tests with the cell lineage HepG2 (human-derived hepatoma cell line) derived from human hepatoma, have been considered important for assessing mutagenic and pro-mutagenic materials (Valentin-Severin et al. 2003, Knasmüller et al. 2004). These cells exhibit most of the morphological characteristics of liver cells and contain many enzymes responsible for the activation of various compounds (Valentin-Severin et al. 2003). They also express the activities of Phase I and Phase II metabolism of various xenobiotic enzymes that play key roles in the activation and/or detoxifi cation of reactive carcinogens. They refl ect the xenobiotic mechanisms in the human body in a most effective manner than other mammal cell lines as CHO, SHE or V79 (Hu et al. 2006). In this way, they have been considered an important tool for evaluating the genotoxicity because several genotoxic compounds are indirect mutagens (Valentin-Severin et al. 2003, Knasmüller et al. 2004). Therefore, HepG2 is useful to predict the metabolism, cytotoxicity and genotoxicity of chemicals in the human liver (Song et al. 2012).
Despite all above described advantages, cell culture methods also exhibit some disadvantages, such as maintenance of cultures in sterile conditions, gradual loss in the degree of differentiation of cells and the inability to reproduce the conditions of the organism, which turn responses only partially representative of the in vivo situation (O’Brien et al. 2000). Therefore, the use of in vivo and in vitro models—simultaneously
or subsequently—as well as the use of different test organisms are necessary when the pharmacological action of a particular extract and/or active ingredient should be proved undoubtedly. Above all, they are important to elucidate the mechanism of action of whole extracts or of active ingredients isolated from plants aiming to manufacture of medicinal extracts.
Main assays used for the assessment of genotoxic and mutagenic effects of medicinal plants
Cells possess different protection mechanisms that allow them to withstand various environmental stresses to which they may be exposed. Among these mechanisms are those that prevent the entry of toxic compounds into the cell, until their inactivation or transformation into less harmful molecules to their biological systems. However, some compounds may directly or indirectly reach the DNA and promote changes in the genetic material, but—thanks to a system of extremely effi cient DNA repair—most damages are repaired.
As a last resort, the cell triggers a process of cell death in an attempt to prevent the spread of damage to the organism. Despite all these self-protective strategies to maintain cell viability, some substances are able to circumvent all defense mechanisms, penetrating into the cells and promoting non-repairable DNA damage, which, in turn, may lead to cell death and also initiate a process of immortality. Cells that carry mutations are more susceptible to further damage to the genetic material, that enhances the development of a carcinogenic process. The main steps that may (or not) reveal the cytotoxic, genotoxic, mutagenic and carcinogenic processes of a given compound are summarized in Fig. 7.3.
Figure 7.3. Flowchart illustrating the main toxic and genetic effects that a compound may present associated with a response (or its failure) in a given organism.
Does