Genotoxicity induced by medicinal plants

The use of medicinal plants in curing diseases is an ancient culture still in use in many parts of the world. Many plants have been proven to have precise ethno-pharmacological relevance. On the contrary, many folkloric plants have also been found to possess DNA damaging effects. Hence, assessing the safety profile of medicinal herbs before being approved for use must be undertaken. This review focuses on medicinal plants exerting genotoxicity effect within through in vivo studies on the bone marrow, erythrocyte or other organs on animal models and in vitro studies on bacterial cells or mammalian cell lines such as mammalian lymphocytes, human hepatoma cell line or HepG2, mouse lung fibroblast cell lines or human adenocarcinoma cell lines. This review has found that several medicinal plants possess genotoxic potentials and are not safe to use. The common methodologies several authors have used include the comet assay, micronucleus assay, bacterial reverse mutation assay, Ames test or Salmonella/microsome assay. Plants that have been proven to be genotoxic are not reduced to a particular family, while groups including Fabaceae, Asteraceae, Euphorbiaceae, Rosaceae, Lamiaceae and Apocynaceae appear to be frequent. To avoid any mutation in its users, genotoxicity assessment of therapeutic plants appears to be required.


Background
The ethno-social lives of different tribes of people throughout the world have seen to be inevitably reliant on plants and their products for therapeutic purposes. In many indigenous cultures, knowledge and practice of using herbal medicine has been passed from generation to generation. According to the World Health Organization (WHO), 70-80% of the world population depends exclusively on herbs for their primary health care (Chan 2003;Muhammad et al. 2011;Sponchiado et al. 2016). In India, about 7000-7500 species of plants have medicinal usage in folk and documented systems of medicine (Pandey et al. 2013). It is believed that as many as 25,000 different formulations are prepared from these plants as a cure against various diseases and disorders (Pandey et al. 2013). Even in the process of developing new drugs, plant constituents constitute an important component as in vitro molecular syntheses are difficult, and most can be used as a prototype for the synthesis of new drugs.
It is a common belief that the herbal products are safe and more efficient than their allopathic counterparts which make its uses more prominent. However, since this assumption is false and harmful, toxicological research on herbal medications should be conducted (Kahaliw et al. 2018). Studies have revealed that some plants frequently used in folk medicine were potentially genotoxic (Marques et al. 2003;Ananthi et al. 2010;Melo-Reis et al. 2011;Regner et al. 2011;Shin et al. 2011;Sponchiado et al. 2016). The interaction of any toxic material may lead to numerous chromosomal aberrations including chromatid breaks, isochromatid breaks, gaps, chromosomal fragments, exchanges, and sister chromatid unions; even in case of the change in DNA structure. The consequences of such DNA impairment could be the establishment of and/or predisposition to diseases, increased morbidity/mortality, changes in heritable characteristics, and impaired reproductive capacity (Lázaro et al. 2010;Sponchiado et al. 2016).
Page 2 of 11 Bardoloi and Soren Bulletin of the National Research Centre (2022) 46:119 The leap in the biophysical techniques has made studies on toxicity diverse and advance. There were many assays following different methodologies and strategies that have been developed to make the toxicity study efficient. The establishment of these protocols has been based on studies demonstrating the correlation between carcinogenicity and mutagenicity, and the correlation of both parameters with genotoxicity (Dearfield et al. 1991;Waters et al. 1999). Since then, established processes for assessing the genotoxic risk associated with the use of medications, food additives, pesticides, industrial and environmental chemicals, as well as natural items such as medicinal plants and their oils, have been employed (Waters et al. 1999;Chen-Chen and Sena 2002;Costa et al. 2018).
There are several different species of plants that are being used to cure various diseases such as headache, respiratory infection, inflammation, gastrointestinal disorders, memory loss, stress, insomnia, anemia, diminishing eyesight and sexual impotency, dermatitis, diabetes, wounds and pains, and cancers. The genotoxic potentials have been recorded here. Any plant may have potential genotoxic capability in a limited region or throughout, and the chemical employed to make the extract, as well as the model organism utilised, have a significant impact on this conclusion in vivo or in vitro. Through this study, an effort has been made to find out the assays used and where and how they have been experimented on along with families where these plants belong.

Medicinal plants and genotoxic assays
The inherent knowledge of folkloric herbal medicine is a pivotal aspect of ethnicity which should be scientifically tested upon. Many familiar plants have been found to contain compounds bearing DNA damaging impact (Fig. 1). There are specific procedures and protocols known as "genotoxic assay" to ensure the safety profile of any suspected candidate (Fig. 2). Genotoxic assays have different endpoints, such as single-and double-strand breaks, point mutations, deletions, chromosomal aberration, micronuclei formation, DNA repair, and cellcycle interactions . Different assays which have been found to be used while studying these plants are in vivo or in vitro comet assay (CA), micronucleus test (MN), Ames test or Salmonella/microsome assay or Bacterial reverse mutation assay (BRM), Allium cepa test (ACT), and chromosomal aberration assay (ChA). Vitotox assay (VA), yH2AX In-Cell Western Assay (WA), mouse lymphoma tk assay (MLtk), and lysogenic induction assay (LI) have also been used to find out genotoxic medicinal plants.
The choice of assay to be performed is a crucial decision in geno-toxicological studies. It's worth noting that genotoxicity should be assessed using a series of assays because no one test can identify total genotoxicity (Saravanan et al. 2020). Specificity in detection even comes along with models and extracts. An aqueous extract of nine medicinal plants known in natural Korean medicine under the name "Gumi ganghwaltang" demonstrated genotoxicity in the Ames test and Chinese hamster lung cell culture (CHL cells) but was inactive for the micronuclei count in polychromatophylic mice erythrocytes (Shin et al. 2011;Durnev and Lapitskaia 2013). An aqueous extract of six plants "Pyungwisan, " which belong to the same group of natural drugs, induced chromosome aberrations in a test with CHL cells in vitro (Shin et al. 2011;Durnev and Lapitskaia 2013).
Preparation of the extract from the desired parts of the plant is a major determining step in any scientific study. Different solvents were used to prepare them based on the physical and chemical properties of the constituents (Fig. 3). Solvents commonly used in extraction of medicinal plants are polar solvent (e.g., water, alcohols), intermediate polar (e.g., acetone, dichloromethane), and nonpolar (e.g., n-hexane, ether, chloroform) (Abubakar and Haque 2020). In general, extraction procedures include maceration, digestion, decoction, infusion, percolation, Soxhlet extraction, superficial extraction, ultrasound-assisted, and microwave-assisted extractions (Abubakar and Haque 2020). When the manner of extracts utilized in research is quantified, alcoholic extracts are shown to be the most popular.

In vitro studies
In vitro genotoxic assays represent simple, robust and time and cost-effective testing of targeted toxicity and underlying mechanisms (Dusinka et al. 2012). In vitro studies have been done using Ames test, in vitro comet and micronucleus test, in vitro chromosomal aberration assay, vitotox assay, mouse lymphoma tk assay on bacterial strains or mammalian cell lines such as HepG2 cell line, lymphocytes or peripheral mononuclear blood cell line following Organisation for Economic Co-operation and Development (OECD) guideline 473 (2016). Several workers have worked using both in vitro and in vivo assays. Ames test or Salmonella/microsome assay. It was based on induction of reverse mutation in the histidine gene, which enables the bacteria to synthesize histidine and form visible colonies in minimal histidine medium (Dusinka et al. 2012). The protocols that are followed are given by Maron and Ames (1983) and Mortelmans and Zeiger (2000).
The first ever toxicity (genotoxicity and cytotoxicity) study using Vitotox assay which was also a bacterial genotoxicity test was performed by Chichico-Hernandez et al. (2011) with 100% methanolic extracts of Cassia fistula, Derris elliptica, Ficus elastica, Gliciridia sepium, Michelia alba, Morus alba, Pogostemon cablin and Ricinus communis on two different strains of Salmonella typhimurium (TA 104) based on their SOS response and found genotoxic effect only on P. cablin and R. communis. One has a luciferase gene under the control of the recN promoter, which leads to light production when DNA is damaged (TA 104-recN2-4 strain or Genox strain) while the second one contains lux-genes under the control of a constitutive promoter so that the light production is not influenced by genotoxic compounds (pr1 or Cytox strain). It serves as an internal control wherein, if the light production goes up, the test compounds affect the lux gene in a different way than damaging the DNA. On the other hand, a decrease in light production would indicate a toxic response (Chichico-Hernandez et al. 2011).
The in vitro chromosomal aberration test identifies agents that cause structural chromosomal aberrations in cultured media. Kulkarni et al. (2010) used this technique with the methanolic extracts made from fruit and leaf of Persea Americana on human peripheral blood cells and found the occurrence of acrocentric associations and premature centromeric separation. Cell cultures are exposed to the test substance both with and without metabolic activation and are treated with a metaphase-arresting substance (colcemid or colchicine) before harvest (Dusinka et al. 2012) which then can detect the presence of aberrant chromosome microscopically.
Comet assay is found to be the most repeated assay. This method represents a rapid, sensitive, reliable, robust and relatively inexpensive way to study DNA damage (including DNA oxidation), and repair in different cell types both in vitro as well as in vivo (Dusinka et al. 2012). Sassi et al. (2016) 2021) with the extract of Ziziphora clinopodioides on PBMC. Any breakage in DNA strand can be observed as that of comets when put under electrophoresis. The more is the damage in DNA; more is the number of comets formed and hence is a proof of more genotoxicity impact.

In vivo study
Compared with in vitro genotoxicity assay, in vivo genotoxicity assay has been used to verify in vitro assay result and definitely provide biological significance for certain organs or cell types (Kang et al. 2013). In vivo genotoxicity tests using tissues can be used when obtaining in vitro positive results that can reflect absorption, excretion, distribution and metabolism of chemicals but the in vitro test does not (Sasaki et al. 2002;Benigni et al. 2012;Kang et al. 2013). Most of the studies are anyway based on in vivo protocols of comet and micronucleus assay.
In vivo comet assay and micronucleus test can be performed on any type of animal tissue and even blood sample after the proper treatment on the model organism. Tsuboy et al. (2010) performed in vivo comet assay by withdrawing peripheral blood puncturing caudal vein following the guideline given by Tice et al. (2000) in Coccoloba mollis. Whether several other workers followed the protocols given by Singh et al. (1988) which was modified by Hartmann and Speit (1997), except for Boiera et al. (2010) who followed the modified protocol of Da Silva et al. (2000). One of the most popular protocol of micronucleus (MN) test is to observe the presence of micronucleus on bone marrow after flushing out the femurs as done by Regner et al. (2011)

Geno-toxicological findings
Genotoxicity study to explore the toxicity measures of different medicinal plants used in folkloric medicine culture have led quite a prominent way till now (Table 1). The detailed study of the literature mentioned is a proof of that. There were many in vitro and in vivo studies that  (Durnev and Lapitskaia 2013) are found to have genotoxic compounds. Allyl thiocyanate is contained in the amount of 50-100 ppm from Brassicacae family (Durnev and Lapitskaia 2013) which is observed to bear mutagenic properties at the light of the genotoxicity study. Aristolchic acid, a major compound in Aristolochiaceae family has recently been classified in the International Agency for Research on Cancer (IARC) as a human carcinogen (Durnev and Lapitskaia 2013). Other compounds such as propenyl benzene, hydrazine, antheraquinones and their derivatives, allyl isothiocyanates and flavonoides as found in Ashitaba chalcone, coumarines and psoralens have already been tested and proven to possess genotoxicity inducing property. Genotoxicity study depends on the mode of extract being used. For example, Jatropha gossypiifolia has found to be genotoxic at other extracts except aqueous. Cecropia pachystachyca and Pterocaulon polystachycum showed genotoxicity only to mice brain and kidney tissues, respectively. Even each part of a plant might not bear similar genotoxic phenomenon, such as in Opuntia ficus-indica, where only the fruit and seed extracts were found to be genotoxic, whereas other parts were found to be negative of this aspect.
It is a habitual aspect of people to believe in the naturally obtained products more than that of synthesized ones. But while setting to this aspect, its inherent toxicological properties tend to be ignored. Several workers have already done many experiments with the natural compounds obtained from plants yet many are still unknown. Many regular culinary ingredients have found to contain toxic compounds after experimentation. Regular studies have been adding new evidences on this aspect. Such as saponins, which were not mentioned among geno-toxicants before, are considered to be responsible in Chinese hamster ovary (CHO) cell cultures for the mutagenic activity of water and the organic extracts of the cortex of the medicinal plant Nauclea (Rubiaceae) (Liu et al. 2000). There are many controversies related to the extract used. An aqueous extract of nine medicinal plants known in natural Korean medicine under the name "Gumiganghwaltang" demonstrated genotoxicity in the Ames test and Chinese hamster lung cell culture (CHL cells) but was inactive for the micronuclei count in polychromatophylic mice erythrocytes (Shin et al. 2011). The study of 19 different extracts of 11 plants (Arctium minus (Hill) Bernh., Ecballium elaterium L., Momordica charantia L., Plantago major L., Urtica dioica L., Viscum album L., Salvia triloba, Euphorbia rigida, Stachys lavandulifolia, Acteoside and Abies nordmannia) that are considered as phyto-immunomodulators did not reveal a mutagenic effect using the strains TA98 and TA100 in the Ames test but indicated clear DNA damaging action toward lymphocytes of human peripheral blood in vitro (Durnev and Lapitskaia 2013). Even in case of in vitro and in vivo studies, some extracts that induce genotoxicity in vitro show negative result in vivo. According to Durnev and Lapitskaia (2013), in vivo studies suffer from a number of drawbacks due to the imperfection of their design from the point of view of the evaluation of real genotoxic risks for humans. First of all, the expressed discrepancy of the compound dosage used in an experiment and that that is actually consumed by a person is relevant. The second issue is the discordance of the regimes of introducing a compound into organism.

Conclusions
There might be many controversies and uncertainty lying down behind each experiment, but each toxicological experiment brings the safety of humans a step closer. Therefore, identifying the geno-toxicants and then taking the required measures for their sanitation is always necessary before switching onto any herbal products.