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Plant extracts as modulators of genotoxic effects.

II. Introduction

Traditional medicines based on plants have been useful in discovering new drugs from natural products, in helping to identify new biochemical loci for drug action, and in developing new classes of bioactive molecules. At present, about 75% of the human population depends on plant extracts as tools of traditional medicine. There are about 121 clinically useful prescription drugs derived from indigenous medicine. In 1985, of a total of 3500 new chemical structures discovered, 2619 were isolated from higher Plants (see Abelson, 1990; Kinghorn & Balandrin, 1993). The potential for future uses is vast, since of an estimated total of 250,000 to 300,000 species of higher living plants, only about 5000 have been studied extensively for possible medical application (see Cordell, 1977).

Interest has intensified in plant-based pharmaceuticals with the development of new methods of screening for anticarcinogenic drugs (Hartwell, 1967). One of the earliest reports is the use of the extract of Colchicum autumnale bulb (Liliaceae) in reducing uncontrolled cell division. The property of preventing carcinogenesis has been reported in many plant extracts, from cruciferous vegetables against alimentary cancers (Bjelke, 1974; Graham et al., 1978; Haenszel et al., 1972, 1976) to degradation of carcinogenic nitrosamine formation factors by extracts of onion, pepper, and ginger (Kim et al., 1987). Protective activity of total vegetable or fruit extract as dietary supplement against epithelial, ovarian, oral, and esophageal cancers has also been reported (Barone et al., 1992; Shu et al., 1989). Anticancer activity has been observed for extracts of bamboo leaf (Kuboyama et al., 1981), Chinese tea (Han & Yong, 1990), and betel leaf (Azuine et al., 1991; Bhide et al., 1991; Parma et al., 1989); for Cupressus semipervirens L., Anchusa strigosa L., Myrtus communis L., and Crataegus monogyna (Alwan et al., 1990); for Trifolium pratense L. (Cassady et al., 1988); for root, stem, and leaf of Ervatamia heynaena (Chitnis et al., 1971); and for rhizomes of turmeric (Kuttan et al., 1985).

The observation of a close association between carcinogenesis and mutagenesis has extended the survey to include plant extracts and plant products able to modify the process of mutagenesis, alteration in the genetic material.

Natural plant products may, apart from inducing mutations, modify the action of other known mutagens on the living organisms by 1) inactivating the existing mutagens within the cell (desmutagens), 2) inhibiting the production of mutagens in the cell (bioantimutagens), 3) synergising the activity of existing mutagens (comutagens), or 4) activating the promutagens within the cell into mutagens. Such activities, particularly the first two, often overlap. Some plant extracts can also act differently under different experimental conditions.

A vast amount of literature, both scientific and folkloric, is available on plants and cancer. In this review, the scope has been confined to actual data obtained from research on the modulatory effects of plant extracts on mutagenesis and clastogenesis - two genotoxic phenomena associated with carcinogenesis.

III. Plant Extracts as Inhibitors of Mutagenesis

In screening for antimutagenic effects, various test systems have been used, principally bacterial ones such as Salmonella typhimurium and Escherichia coli (Ames et al., 1973). Both mutagens and the plant products were administered in culture.

The term "antimutagen" was originally used to denote an agent that reduced the apparent yield of mutations - spontaneous or induced. These included both desmutagens and bioantimutagens. The former cause chemical and biochemical modification of mutagens before DNA damage, and the latter reduce the apparent frequencies of mutations interfering with cellular processes of mutation fixation. Environmental toxicants entering the living systems are metabolised in two stages: biotransformation (phase 1) and conjugation (phase 2). In the detoxication of toxic chemicals, the oxidative reactions of phase 1 result in the formation of metabolites that subsequently undergo conjugation in phase 2, through the action of epoxide hydrolase, glutathione transferase, UDP-glucoronyl transferase, etc., to form conjugates. The latter are eliminated from the cell and finally from the organism. In contrast, activation by oxidation results in the formation of proximate carcinogens or reactive intermediates. The latter are usually poor substrates for the conjugating enzymes. Therefore, a non-enzymatic interaction of these reactive intermediates takes place with intracellular constituents, including proteins, RNA, and DNA, leading to covalent binding and formation of neo-antigens, mutations, cancer, and cell-death. These two alternative routes of oxidative biotransformation, leading to detoxication or to activation, indicate two different modes of oxygenation and the probable existence of two families of enzymes, separately responsible for the alternative pathways. The cytochromes P-450 result mainly in detoxication, while the cytochromes P-448, flavoprotein mono-oxygenase, and non-enzymatic free radical hydroxylations are responsible for oxidative activation.

A. EXTRACTS FROM VEGETABLES

A large amount of data is available on the antimutagenic activity of plant extracts from different plant groups on bacterial test systems. These observations deal with extracts from leafy vegetables, from fruits, from underground storage organs, and from whole plants (Table I). The extracts were prepared mainly in water or organic solvents. Vegetables such as cabbage, spinach, celery, and sprouts were recorded to suppress the mutagenicity of pyrolysate mutagens derived from tryptophan (Kada et al., 1978; Morita et al., 1978). Lai et al. (1980) reported that addition of acetone extract of vegetables to the Salmonella mutagenicity assay mixture of benzo(a)pyrene (BaP) and 3-methylcholanthrene, reduced the number of revertant colonies. Some vegetables have also been found to have desmutagenic effects against products obtained from boiled fish (Yoshikawa et al., 1981). Juices of cauliflower, spinach, and lettuce have been reported to inhibit the mutagenicity of nitrite combined with nitrosable compounds in mice in vivo (Barale et al., 1983). Aqueous dialysates of 16 kinds of vegetables and fruits such as burdock, eggplant, spinach, and apple were found to be antimutagenic against a number of known mutagens in Salmonella typhimurium TA 100 strain (Shinohara et al., 1988). Investigation of the urinary mutagenicity of 3 nonsmoking healthy men using Ames Salmonella/microsome assay, showed a reduction in the number of revertants in the urine of the subjects who consumed fried salmon and parsley leaves simultaneously (Ohyama et al., 1987). Among six vegetable juices tested, parsley juice was most effective in suppressing mutagenicity (88.5%) of roasted beef extract (Nakashima, 1989). Extracts of lettuce and chard leaves reduced the mutagenicity of BaP in urine samples of Balb/c mice (Perez & Gago, 1991). Vegetables belonging to the families Compositae, Labiatae, Cruciferae, and Umbelliferae showed antimutagenic activities against Trp-P-1 in an assay using Salmonella typhimurium TA 98 strain. Sixty mushroom samples were also tested in the [TABULAR DATA FOR TABLE I OMITTED] same assay system but were found to be relatively weak in reducing mutagenicity as compared to the vegetables (Ueda et al., 1991).

B. EXTRACTS FROM FRUITS

Of fruit juices, citrus juices are effective antimutagens. Juices from ten citrus varieties reduced significantly the number of revertant colonies induced by N-nitro-o-phenylenediamine (NPD) in TA 97a and sodium azide in TA 100 strains of Salmonella typhimurium (Bala & Grover, 1989). A similar reduction in the number of his + revertants was observed with water, acetone, and chloroform extracts of Emblica officinalis Gaertn. (syn. Phyllanthus emblica L.) (Grover & Kaur, 1989). Extracts of Terminalia chebula Willd. fruit suppressed the mutagenic effects of N-methyl-N[prime]-nitro-N-nitrosoguanidine (MNNG) and UV radiation in E. coli WP-2 and S. typhimurium TA 98 strains (Jain et al., 1987). Similar action was shown by water extract of Cnidium monnieri (L.) Cuss. fruits, using Ames test (Liu et al., 1988). Water and chloroform extracts of guava (Psidium guajava L.) and myroblan (Terminalia chebula Willd.) fruits were tested against two direct-acting mutagens NPD and sodium azide and the S9-dependent mutagen 2-aminofluorene (AF-2) in Salmonella tester strains. Only the water extract was effective in reducing the mutagenic effects. Enhancement of the inhibitory activity of the water extracts on pre-incubation with the mutagens showed that desmutagens may be present in the extract itself (Grover & Bala, 1992, 1993).

C. EXTRACTS FROM UNDERGROUND STORAGE ORGANS

Amongst underground parts, aqueous extracts of garlic bulbs (Allium sativum L.) have been observed to inhibit a number of known mutagens, including ionizing radiations, peroxides, adriamycin, and MNNG in Salmonella strains and also in eukaryotic Chinese hamster cells (Knasmueller et al., 1989). In two tester strains of E. coli, this extract suppressed the mutagenicity of 4-nitroquinoline-1-oxide (4 NQO) but not that induced by UV (Zhang et al., 1989). Turmeric extract effectively decreased the level of BaP and 7,12-dimethylbenzanthracene (DMBA)-induced mutagenesis in strain TA 98 of S. typhimurium (Nagabhushan & Bhide, 1987). Carrot juice, rich in vitamin A, has been found to suppress mutagenic activities of metabolites of cyclophosphamide (Darroudi et al., 1988). Peony root extract inhibited BaP-induced mutagenicity in S. typhimurium reversion test (Sakai et al., 1990)

D. OTHERS

Several plant parts were tried against certain common mutagens. Lemon, black and green tea, extracts of guava, and Cassia fistula L. leaves and Zizyphus jujuba L. bark suppressed the mutagenicities of BaP and Trp-p-2, while Psidium guajava L., Terminalia chebula Willd., T. arjuna Willd., Zizyphus jujuba L., Eucalyptus spp., Aegilops spp. and Acacia arabica Willd. extracts were antimutagenic against UV irradiation (Jain et al., 1987).

Extracts of green and black tea leaves decreased the mutagenic activity of MNNG in E. coli WP2 in vitro and in the stomachs of rats as shown by bacterial mutagenicity in vitro assays. Priming the rats with the extracts proved to be more effective than simultaneous administration of extract and mutagen (Jain et al., 1989).

Aqueous fractions of roots and leaves of sprouted wheat selectively inhibited the mutagenicity of known mutagens requiring metabolic activation, as demonstrated by the Ames/Salmonella microsome test (Lai, 1979). Later studies on the subfractions of extracts from wheat sprout also reduced the mutagenic effects of BaP in S. typhimurium tester strains (Peryt et al., 1988, 1992).

Different aquatic plants have been screened for their antimutagenic activities. Whole plant extracts of curled pondweed (Potamogeton crispus L.), European cutgrass (Leersia japonica Makino), and smartweed (Polygonum hydropiper L.) decreased mutagenic effects of BaP, 2-nitrofluorene, and 2-aminofluorene (Fujimoto et al., 1987): Water extract of grass wrack pondweed (Potamogeton oxyphyllus Miquel) also reduced reverse mutations induced by BaP, AF-2, and 2-nitrofluorene in S. typhimurium TA 98 and TA 100 strains (Sato et al., 1984, 1990).

Ethanol extract of the fungus Craterellus cornucopioides completely inhibited the mutagenic action of aflatoxin [B.sub.1], BaP, acridine half-mustard ICR-191, and 2-nitrofluorene in a forward mutation system using Salmonella typhimurium TM 677 (Gruter et al., 1990).

IV. Mechanism of Antimutagenic Activity of Plant Extracts

A. CERTAIN SPECIFIC FACTORS OBSERVED

Studies on antimutagenic activities of plant extracts have, in some cases, indicated the involvement of certain factors, which are mainly intrinsic components of the extracts. These components range from specific compounds such as ascorbic acid to vegetable fibres, which could act as nonspecific redox agents, free radical scavengers, or ligands for binding metals or toxic principles.

Morita et al. (1978) identified a desmutagenic factor in cabbage which was sensitive to heat (100 [degrees] C) and pronase treatment, suggesting a protein character. Kada et al. (1978) suggested a similar vegetable factor in cabbage and radish, capable of reducing tryptophan-induced mutagenesis in Salmonella tester strains. In vegetables such as burdock, a heat- and enzyme-resistant desmutagenic factor was isolated with molecular weight higher than 300,000 and showing characteristics of a polyanionic substance. It reduced the action of mutagens both with and without S9-mix (Morita et al., 1984). Of the four compounds isolated from dried leaves of cabbage, nonacosane and pheophytin did not inhibit 2-aminoanthracene (2AA) or N-methyl-N-nitrosourea-induced mutagenesis, whereas 15 nonacosane was more effective than [Beta]-sitosterol. In V-79 cell mammalian mutagenicity assay, all the four fractions were active against mutagenicity of 2 AA (Lawson et al., 1989). Vegetable juices modified the mutagenic effects of beef extract and nitrosated beef extract only in the presence of S9-mix as shown by Ames test. It was therefore suggested that the constituents do not act directly upon the mutagens, but interact with the metabolic activation system (Muenzner, 1986). The antimutagenic principle of the green fruits of Momordica charantia L. was found to be an inextractable mixture of novel acylglucosylsterols. Ingestion of these compounds may result in their adsorption in the plasma membrane lipid bilayer, which could adversely affect the membrane permeability toward the known mutagen mitomycin C and disrupt cellular activity of the latter (Guevera et al., 1990).

The antimutagenic activity of some common vegetables was attributed to their chlorophyll content (Lai et al., 1980). Chlorophyll was suggested to be the major active factor in wheat sprout extract inhibiting the metabolic activation of carcinogens in vitro (Lai, 1979). However, later studies with wheat sprout gave conflicting results (Peryt et al., 1988, 1992). Two heat-resistant compounds were isolated from such extracts, located within the cell cytosol, that showed antimutagenic activity - one with low and the other with high molecular weight. The strong inhibition of BaP mutagenicity with non-chlorophyllic wheat sprout extract suggested that chlorophyll is not the main factor involved in antimutagenicity (see Sarkar et al., 1994). Among the aquatic plants, two species of Potamogeton (grasswrack pond weed and curled pond weed) reduced BaP-induced mutagenesis (Fujimoro et al., 1987; Sato et al., 1984, 1990). The factor involved in both these plants was water-soluble, heat-resistant, and with a molecular weight above 300,000. Smartweed (Polygonum sp.) also has similar desmutagenic factors, but the molecular weights were both above and below 300,000. The active factor in European cutgrass may not be a desmutagen. There is a possibility that it is a bioantimutagen or an inhibitor of S9-mix (Fujimoro et al., 1987). Whatever may be the nature of antimutagenic factor, all these studies have shown that chlorophyll does not play an important role in antimutagenesis. On the other hand, plant mucilage present throughout the body of the plants may be considered to have inhibitory properties and may act by absorbing mutagens (Sato et al., 1990). Antimutagenic effects of garlic extract have been attributed to molecules with sulfur moieties which act as scavengers of free radicals (Knasmueller et al., 1989). It has also been suggested that garlic bulbs reduce markedly the mutagenicity of 4 NQO by inactivating the electrophilic groups of the mutagen or inhibiting metabolic activation (Zhang et al., 1989).

Turmerin, a water-soluble 5-KDa peptide from turmeric, has been found to be an efficient antioxidan/DNA protectant/antimutagen (Srinivas et al., 1992). The antimutagenic action of the ethanol extract of mushroom Craterellus cornucopioides may be due to direct chemical interaction with the mutagen and/or inhibition of the activation process of promutagen (Gruter et al., 1990).

Inhibitory activity of extracts of the fruits of Emblica officinalis Gaertn., Psidium guajava L., and Terminalia chebula Willd. was enhanced when the extracts were pre-incubated with the mutagen at 37 [degrees] C for 30 minutes prior to plating, suggesting a desmutagenic action (Bala & Grover, 1989; Grover & Bala, 1992, 1993). The antimutagenic activity of citrus fruits had been attributed to the principal components, ascorbic and citric acids. However, in general, the effects of the crude extract are more than that of an equivalent amount of any single component, indicating that the different components, both major and minor, are involved in the process.

Vegetable fibres have been shown to be able to suppress the action of pyrolysates by absorbing them (Kada et al., 1984). Such fibres may be responsible for the activity of most crude plant extracts in eliminating possible mutagens from the system.

B. STEPS INVOLVED IN PROTECTING AGAINST MUTAGENESIS

In general, the ways in which inhibitors of mutagenesis can act include 1) the inhibition of interaction between genes and biochemically reactive mutagens and 2) the inhibition of metabolic activation of indirectly acting mutagens. The latter mechanism includes a) inactivation of metabolizing enzymes and b) interaction with promutagens making them unavailable for the enzymatic process (see Hayatsu et al., 1988). Inhibition of effect of mutagens can be outside the cells or inside the cells (Table II.)

The majority of inhibitors that naturally occur in many edible plants are phenols, aromatic isothiocyanates, coumarins, flavones, diterpenes, retinoids, ascorbic acid, alphatocopherol, selenium salts and plant sterols. Eighteen active chemical antimutagenic compounds were identified from 200 diverse plants, one of them being protoanemonia (Minakata et al., 1983; Ramel et al., 1986).

Deactivation of mutagens in the alimentary tract was by crude juices from various vegetables, which reduced in vitro the mutagenicity of tryptophan pyrolysis products (Kada et al., 1978, 1984). Inactivation enzymes possessing peroxidase and NADPH-oxidase activities were also isolated from cabbage and broccoli.

Intracellular inhibition of mutagenesis: The extracts of cruciferous plants - e.g., brussel sprout, cabbage, cauliflower and broccoli - are capable both of activating enzymes such as arylhydrocarbon hydroxylase and of detoxifying enzymes such as the cytosolic GSH S-transferase. The latter effect prevails. The extracts contain phenols, isothiocyanates and indole derivatives like indole 3-carbinole, 3,3[prime] diindolylmethane, and indole 3-acetonitrile. The protection afforded has been ascribed to a changed balance in the enzyme activities involved in the biotransformation of these compounds and to the effect of conjugating enzyme systems.

Blocking of reactive chemical species: Ellagic acid may protect DNA from the attack of electrophilic species such as BaP diolepoxide or free radicals by binding to its nucleophilic sites. It is a naturally occurring polyphenol from coffee, nuts, and grapes. The flavonoids myrecetin and rutin show similar effects, as also gallic acid and sulphydryl compounds.

Endogenous N-nitroso compound (NOC) formation through interaction between nitrites and nitrosable amines or amides in the stomach has been inhibited by complex mixtures of plant origin, including tea, coffee, vegetables and fruit juices, soya products, and betel nut extracts. This inhibitory action is due to the presence or vitamins A, C, and E and natural phenols, which have different functions. Caffeic, chlorogenic, ellagic, ferulic, gallic, and tannic acids and vitamin C inhibit mutagenicity of direct-acting N-nitroso compounds, and vitamin A inhibits metabolic activation of promutagenic nitrosamines (Ames, 1982; Gichner & Veleminsky, 1988).
Table II

Mechanisms for inhibition of mutagenesis by plant products
(from DeFlora & Ramel, 1988)

I. Inhibition of mutagens acting outside the cells (stage 1
inhibitors)

A. Inhibiting the uptake of mutagens or precursors by:

1. hindering their penetration into

the organism body shielding, washing

the cells fatty acids, putrescin, aromatic
 amino acids

2. favouring their removal fibres

B. Inhibiting endogenous formation of mutagens by:

1. Inhibiting nitrosation ascorbic acid, tocopherols, phenols

2. Modifying microsomal
intestinal flora fermented products

C. Reactions deactivating mutagens

1. Physical maintenance of physiological pH

2. Chemical thiols, antioxidants

3. Enzymatic vegetables with peroxidase activity

II. Inhibition of mutagens acting inside the cells (stage 2
inhibitors)

A. Modulation of metabolism by:

1. Inhibiting cell replication retinoids

2. Sequestering mutagens in thiols
non-target cells

3. Inhibiting activation extracts of Cruciferae
of promutagens

4. Inducing detoxification phenols, thiols

B. Blocking reactive molecules by:

1. Reacting with electrophiles sulphur compounds

2. Scavenging reactive oxygen antioxidants

3. Protecting nucleophilic ellagic aid, retinoids
sites of DNA

C. Modulation of DNA replication/repair by:

1. Increasing fidelity of DNA Co[Cl.sub.2], NaAs[O.sub.2]
replication

2. Increasing repair of DNA cinnamaldehydr coumarins,
damage umbelliferone, vanillin, thiols

3. Inhibiting error-prone protease inhibitors
repair


Inhibition of uptake of mutagens: Purified fibres from different vegetables can bind in vitro and irreversibly adsorb the mutagenic products of proteins and amino acids. Refined corn bran binds dinitropyrene, thus decreasing its mutagenicity (Takeuchi et al., 1988).

Vitamin E functions as a chain-breaking antioxidant for the lipid phase of biological systems. It is a mixture of four phenols called tocopherols, of which the alpha form is most biologically active. It is the most effective natural chain-breaking antioxidant due to the stereo-electronic properties of its hydroxychroman group.

Diallylsulphide, allyl methyl disulphide, and diallyl-trisulphide are components of Allium cepa and A. sativum and are able to suppress BaP-induced neoplasia in the mouse forestomach. These compounds enhance the level of glutathione-S-transferase in the forestomach, accelerating the detoxication of mutagens.

Protease inhibitors are proteins and peptides found in microbes, animals, and plants, particularly in the beans. Certain soybean proteins, Edipro A and BBI, suppress tumours induced by dimethylhydrazine (DMH). In vitro, they decrease hydrogen peroxide formation through blockage of active oxygen-mediated processes.

V. Plant Extracts as Inhibitors of Clastogenesis

The effects of toxicants can be observed at the level of chromosomes (clastogenesis) through alterations in chromosome structure (chromosomal aberrations or CA) and number (aneuploidy, polyploidy). A wide range of short-term and long-term screening procedures is available. The most common ones use higher plants (Allium cepa, Allium sativum, Vicia faba, Tradescantia virginiana) or rodents (mice, rats) in vivo as test systems for monitoring chromosomal aberrations (see Hsu, 1982; Kihlman, 1971; Levan, 1949; Naismith, 1987; Sharma & Sharma, 1989). In vitro studies using leukocytes or cell lines are relatively rare.

Such experiments with a number of crude vegetable and fruit extracts have demonstrated the anticlastogenic activities of these extracts against known clastogens (Table III). The data, however, are not always conclusive.

Ten vegetable juices (both fresh and boiled) were administered to rats exposed to DMBA and the CA screened in bone marrow cells. Both fresh and boiled juices of onion, burdock, eggplant, cabbage, and welsh onion reduced the frequency of CA to a significant level. Fresh pumpkin juice, on the other hand, enhanced the incidence of aberrant metaphases, while boiled juice reduced it to a significant extent (Ito et al., 1986). Anticlastogenic effects of carrot and spinach were observed against the chemotherapeutic drug cyclophosphamide in rat bone marrow cells in vivo following short-term micronucleus tests (Abraham et al.,1986). Both simultaneous and prior administration of the vegetable juices reduced the cyclophosphamide-induced micronuclei significantly in the treated animals.

Priming of Swiss albino mice with fresh spinach leaf extract for 7 days reduced significantly the frequency of CA induced by chromium compounds (Sarkar et al., 1995, 1996). The frequencies of CA induced by mitomycin C, cyclophosphamide, and sodium arsenite were reduced in bone marrow cells of mice that had been administered aqueous garlic extract for different periods as a dietary supplement (Das et al., 1993a, 1993b; Roychoudhury et al., 1993). The extracts of garlic bulbs and spinach did not themselves induce appreciable number of chromosome aberrations when administered alone, indicating that these extracts were non-clastogenic at the doses used.

Green tea extract suppressed chromosome aberrations induced by aflatoxin [B.sub.1], in rat bone marrow cells in vivo (Ito et al., 1989). Anticlastogenic effect was also observed with essential oil of seeds of Apium graveolens against C[Cl.sub.4] in mice in vivo (Sobti et al., 1991). Leaf extract of Apocynum venetum L. is unable itself to induce micronuclei in bone marrow polychromatic erythrocytes in mice and also prevents the increase of micronuclei formation induced by cyclophosphamide (Hao et al., 1988).

Extensive work has been done in our laboratory to test the protection afforded by crude extract of fruits of Phyllanthus emblica L. (Emblica officinalis Gaertn.) and equivalent amounts of synthetic ascorbic acid (vitamin C) against a number of known clastogens, including zinc chloride, metanil yellow, and ethyl parathion (Giri & Banerjee, 1986), nickel and lead (Agarwal et al., 1989), lead and aluminium (Dhir et al., 1990), cesium chloride (Ghosh et al., 1992), and chlordane (Sarkar, 1992). Separate sets of mice were administered orally the crude fruit extract and equivalent amount of synthetic ascorbic acid for periods from 24 hours to 60 days. The toxicants were administered as a single acute dose or a series of subtoxic doses at intervals for prolonged periods. It was observed that the crude extract reduced the cytotoxic effects to a greater extent than vitamin C alone. The protective ability of Emblica officinalis Gaertn. grated fruit extract had been confirmed against radiation-induced chromosome damage in Allium sativum root tips (Yadav, 1987).

VI. Mechanism of Anticlastogenic Activity

The mechanisms by which crude vegetable and fruit extracts reduce the cytotoxic effects of various clastogens may be different for different plants. It has been reported that intake of diets containing powdered preparations of brussels sprouts, cabbage, coffee beans, or tea leaves increased the activity of glutathione-S-transferase (GST), which catalyses the binding of electrophiles to glutathione (GSH) (Sparnins et al., 1982). Thus, GST activated by vegetable juices may be involved in the anticlastogenic activity of the vegetables. The oral administration of GSH alone reduced DMBA-induced chromosomal aberrations (Ito et al., 1984, 1986). The SH-compounds, present abundantly in onion and welsh onion, have a chemical structure analogous to SH (Whitaker, 1976). These SH compounds were presumed to be responsible for the CA-suppressing activity of onion juices.

Many vegetable and fruit juices also contain flavonic compounds such as quercetin and kaempferol. Non-carcinogenic and anti-tumor-promoting activity of quercetin is well documented (Hirono et al., 1981; Morino et al., 1982; Nishino et al., 1984). Flavone compounds of vegetables may also be involved in the anticlastogenic activity.

Crude spinach leaf extract has been found to suppress chromosomal aberrations induced by cyclophosphamide (Abraham et al., 1986) and chromium compounds (Sarkar et al., 1995, 1996) in mice bone marrow cells in vivo. It was earlier suggested that chlorophyll plays an important role in spinach anticlastogenicity (Abraham et al., 1986). However, other compounds - e.g., ascorbic acid, fibres, other vitamins present in the extract - are suspected to have an additive interaction with chlorophyll (Barale et al., 1983).

Chemical constituents of fruits of Phyllanthus emblica L. include ascorbic acid, dehydroascorbic acid, gallic acid, ellagic acid, mucic acid, citric acid, reducing sugars, and tannin (Shrivastava & Shrivastava, 1964; Soman & Pillay, 1962). Among these, the [TABULAR DATA FOR TABLE III OMITTED] major component vitamin C, due to its antioxidant and chelating effects, has been already observed to act as anticarcinogen, antimutagen, and anticlastogen, respectively, in different test systems (Mirvish, 1975; Parshad et al., 1978; Gebhart et al., 1985; Ginter et al., 1989). Ellagic acid may protect DNA from the attack of electrophilic species like BaP diol epoxide or free radicals by binding to its nucleophilic sites (Wood et al., 1982). Gallic acid, ellagic acid, tannic acid, and vitamin C inhibit mutagenicity of direct-acting N-nitroso compounds (Ames, 1982; Takeuchi et al., 1988). Crude plant extracts are complex mixtures of a number of individual components. Their antimutagenic and anticlastogenic activity cannot be attributed to any one of the individual components. They are the sum of interactions between the components and the clastogens or mutagens added (Sharma, 1990).

VII. Plant Extracts as Genotoxic Agents

Under certain conditions, plant products may induce mutagenic effects, due to the presence of multiple biological properties. Some inhibitors can stimulate simultaneously both enhancing and detoxifying mechanisms, as do, e.g., inducers of coordinated enzyme activities. Many oxidants can, depending on the redox potential, either accept or donate electrons, rendering them protective or harmful.

A. AS MUTAGENS

Ames (1983) compared mutagenic activities of 16 mutagens of plant origin including components like coumarins, eugenols, hydrazine and phorbol esters, and plant extracts of Vicia faba, alfalfa, and others, using bacterial assay. Aqueous extracts of three plant species (Achyrocline satureoides Gaertn., Baccharis anomala DC., Luchea divarticata L.) used in Brazilian popular medicine showed positive mutagenic activity in the Ames test with microsomal activation (Vargas et al., 1991). Chili extract and its pure alkaloid capsaicin induced mutations in Salmonella typhimurium histidine-deficient tester strains with metabolic activation. Capsaicin was positive in micronucleus test with V-79 CHO cells and also inhibited DNA synthesis in tests with Swiss mice (Nagabhushan & Bhide, 1985). Among the six vegetables commonly consumed in the Netherlands, cultivars of lettuce, paprika, and rhubarb were mutagenic in TA 98 strain of Salmonella typhimurium; string beans were mutagenic in TA 98 and TA 100. Spinach and brussels sprouts, however, could not induce any mutation (vander Hoeven et al., 1983). Ingredients of betel quid, which have been related to high incidence of oral cancers, were examined. Among these, extract of areca nut was found to enhance the formation of BPV DNA-induced transformed loci, though no such promoting activity was shown by chewing tobacco. A chemopreventive effect was afforded by the administration of vitamin A to the betel quid chewers (Stich & Tsang, 1989).

B. AS PROMUTAGENS

Plants play an active role in the accumulation, metabolism, and environmental distribution of xenobiotics. The property of plants to activate promutagens that may enter the food chain is of great significance in view of the large number and types of chemicals to which the plants are exposed. A promutagen is a chemical that is not mutagenic itself but can be biologically transformed by a plant system into a mutagen. Several methods for studying promutagens from plants were developed both in vivo and in vitro, including plant cell-free systems (Gentile & Gentile, 1991; Gentile & Plewa, 1988; Plewa et al., 1988).

Several categories of chemicals have been activated by crude plant extracts and have induced mutations in Salmonella systems. These range from pesticides, herbicides, and insecticides to maleic hydrazide and polyaromatic hydrocarbons. The extracts include those of wheat and corn seedlings, tobacco callus, pea apical bud, potato tuber and tulip bulb, Tradescantia leaf, and Vicia faba roots.

The extent to which plants can store mutagenic xenobiotics or convert non-mutagenic chemicals to promutagenic or mutagenic forms has not yet been fully clarified. Since most carcinogenic chemicals are also mutagenic, the mutagenic properties of xenobiotics and their metabolities are receiving increasing attention. In plants the mutagenic activation may be studied at two levels. The mutagenic damage may be caused in the plant itself. Alternatively, the mutagenic metabolite may be conjugated and stored in the plant until it is liberated and becomes active upon consumption of the plant by animal or man, e.g., after application of pesticides to food crops.

Certain plant microsomal enzymes and peroxidases have been shown to form reactive intermediates. The best-studied examples are 2-aminofluorene, BaP, and pentachlorophenol. The latter two xenobiotics are converted to quinoid derivatives, which are, in principle, able to participate in redox cycle and generate active oxygen species. Therefore, covalent binding of reactive intermediates to DNA as well as fragmentation of DNA are proposed as mechanisms of action of mutagenic plant metabolites (Sandermann, 1988).

Plants also produce mutagen precursors. A nitrosable mutagen precursor, 4-chloro-6-methoxyindole, was found in lava beans, and a relationship was suggested between the high incidence of gastric cancers and the intake of lava beans and nitrite in Central and South American countries. The precursors may be activated by fermentation as well; e.g., soybeans do not show mutagenic activity, even when treated with nitrite, unless fermented. A mutagen precursor isolated from Chinese cabbage was indole-3-acetonitrile (Wakabayashi et al., 1988).

C. AS CYTOTOXIC AND CLASTOGENIC AGENTS

Low concentrations of tobacco leaf extract exerted a stimulating effect, whereas high concentration acted as a mitodepressant, on root-tip cells of Allium sativum L. (Sopova et al., 1983). Stronger concentrations of extract of immature Solanum nigrum L. fruits reduced the intensity of mitosis in A. sativum L., whereas weaker concentrations stimulated it. The presence of a cytokinin-like substance in the extract has been suggested to be responsible (Krivokapic et al., 1970). Extracts of leaves and inflorescences of male spinach and aster plants increased the frequency of chromosomal aberrations and mutations in welsh onion and barley, respectively, whereas the female plants inhibited the processes (Sidorskii, 1984). Cellular damage including heavy pycnosis, clumping of chromosomes, fragmentation, and spindle disturbances in Allium cepa L. root meristem were induced by the leaf extract of Ricinus communis L. (George & Geethamma, 1990). Abraham and Cherian (1978) investigated the cellular changes produced by extracts of betel leaves on root tip cells of onion and demonstrated the cytotoxicity of such extracts. Chromosome-breaking activity has been exhibited by aqueous extract of mushroom (Paxillus involutus) in dry and pre-soaked seeds of Nigella damascena L. (Gilot-delhalle et al., 1991).

Extracts of Vicia faba L. roots and leaves and Zea mays L. leaves were compared for their ability to induce chromosomal aberrations and sister-chromatid exchanges in Chinese hamster ovarian cells and human lymphocytes. Both the extracts induced CAs in both systems; however, maize extract was more potent than Vicia extract (Kanaya et al., 1992). Aqueous extract of Heliotropium curassavicum L., though employed widely in therapeutics, has been found to induce chromosomal aberrations and anaphase delay in CHO cell line. This toxic effect was associated with the pyrrolidizing alkaloids and the N- oxides, which are changed into pyrrolic derivatives through a process of in vitro metabolism (Carballo et al., 1992).

D. SYNERGISTIC AND ANTAGONISTIC EFFECTS

Certain plant extracts are observed to induce both mutagenic and antimutagenic effects in different test systems. Rhizome juice of ginger was found to be antimutagenic against tryptophan pyrolysate-induced mutagenesis (Kada et al., 1978; Morita et al., 1978) and 6-gingerol (Nakamura & Yamamoto, 1982). However, when added to known mutagens such as AF-2 and MNNG, mutagenesis was increased by ginger juice, and the potent mutagen identified in this case was 6-gingerol (Nakamura & Yamamoto, 1982). It was presumed that ginger juice contains antimutagenic substances that can suppress the activity of 6-gingerol and that, in the presence of certain specific mutagens like AF-2 and MNNG, 6-gingerol is able to express its mutagenicity (Nakamura & Yamamoto, 1982).

Extracts of a desert mushroom, Al-faga (Tirmania pinoyi), in water and methanol failed to show any mutagenic activity, but the chloroform extract was mutagenic with and without metabolic activation. Moreover, the ethanol extract, combined with some known mutagens, inhibited carcinogen-induced mutagenicity. These results indicated that both mutagens and antimutagens can be extracted from the same food item using different solvents (Hannan et al., 1989). Agaricus bisporus has been reported to be carcinogenic (Toth, 1979) and mutagenic in microbial systems (Sterner et al., 1982). However, no mutagenicity or genotoxicity of the same fungal extract was detected by Pool-zobel et al. (1990) in either in vitro or in vivo studies.

Among the chemical constituents of plant extracts, vitamin C inhibits the formation of some nitrosamines but accelerates the formation of others, which might give rise to mutagens by transnitrosation. The change from protective to harmful effects may be related to the dose, the mode of administration, or even the sequence of administration. The inhibitor may have opposite effects in different tissues. For example, mixed treatment of rats with BHA and plant antioxidants such as propyl gallate and alpha tocopherol enhanced or inhibited the induction of hyperplasia at different sites of the forestomach epithelium (Hirose et al., 1987).

Interactions between inhibitors may lead to synergistic effects, e.g., the potential preventive effects of vitamins A, C, and E may need a high level of carotene and vice versa. Similarly, an optimal amount of vitamin E may be essential for protective effects of vitamin A. These combined actions may be due to the different inhibitors acting at different levels or being localised at different cellular areas. The higher protection afforded by crude plant extracts than an equivalent amount of the purified or synthetic ingredients, as observed with Phyllanthus emblica L., may be related to this phenomenon (Giri & Banerjee, 1986; Giri et al., 1988).

The administration of aqueous leaf extracts of spinach-beet (Beta vulgaris var. benghalensis Hort.) to mice as dietary supplements for prolonged periods reduced significantly the clastogenic activity of chromium (VI) oxide. Chlorophyll extracted from the leaf, in equivalent amounts, was itself clastogenic to a lower degree and did not markedly affect the genotoxicity of chromium. An equivalent amount of chlorophyllin - a synthetic derivative with Na/Cu replacing the Mg in chlorophyll molecule - was significantly anticlastogenic (Sarkar et al., 1993). In protecting against the genotoxic effects, therefore, the crude extract is significantly more effective than chlorophyll due to the interactive effects of its ingredients.

VIII. Plant Products as Modulators of Mutagenesis in Traditional Systems of Medicine

As mentioned earlier, extensive use is made of plant products in traditional systems of medicine and as part of life style. A limited screening of some of these products indicates a combination of effects. Some examples are cited below.

A. INDIAN

Chili and its pure alkaloid capsaicin, and ginger and its phenolics gingerol and shogaol are mutagenic. Turmeric (Curcuma longa L.) and its pure components are non-mutagenic and suppress the mutagenicity of chili and capsaicin and also of several mutagens and carcinogens such as tobacco, cigarette, and benzo(a)anthracene. A diet that included 1% turmeric reduced BaP-and DMBA-induced stomach tumours and spontaneous mammary tumours in mice (Nagabhushan & Bhide, 1985; Nagabhushan et al., 1987a & 1987b).

The daily intake of turmeric powder by Indian adults is between 3 and 6 g, containing 2-5% curcumin. The exposure of such populations to nitroso-compound precursors through vegetables, marine foods, and drinking water may be neutralised by the relatively high consumption of turmeric, which reduces the formation of mutagenic/carcinogenic nitroso compounds (Nagabhushan et al., 1988).

Long-term studies were made simulating betel-chewing habits in India, with and without tobacco, on Swiss mice in vivo. Crude aqueous extracts of Areca catechu L. and Nicotiana tabacum L. leaf given separately were mitogenic and also increased nuclear DNA content. Tobacco, in any combination of chewing mixture, induced duration-dependent clastogenicity. The addition of high lime and betel leaf (Piper betel L.) to the quid reduced the degree of mitogenicity and induction of aneuploidy but was ineffective when both tobacco and areca nut were added to the quid (Sen et al., 1987, 1991).

B. CHINESE

Screening of 169 Chinese medicinal herbs used orally as aqueous extracts showed antimutagenic activity. The following plants reduced the mutations induced by picrolinic acid: Pteris multifida, Actinidia chinensis Planch., Artemisia vulgaris L., Paris polyphylla S., and Ampelopsis brevipedunculata Maxim. ex Trautv. Mutagenicity of benzo(a)pyrene was completely inhibited by Smilax china L., Prunella vulgaris L., and Actinidia chinensis Planch. and moderately inhibited by Pteris polyphylla S., Ampelopsis brevipedunculata Maxim. ex Trautv., Gossypium herbaceum L., Lithospermum erythrorhizon Sleb. & Zucc., Artemisia lavendulaefolia DC., Selaginella doederleinii H., Dianthus superbus L., Centipeda minima A. Br. & Aschers., Curcuma zedoaria R., Marsdenia tenacissima Wight & Arn., and Kalopanax septemlobus K. Five of these were antimutagenic against both mutagens tested (Lee & Lin, 1988).

On the other hand, extracts from Buplei radix, Aurantii nobilis pericarpium, and Pinelliae tuber increased the mutagenicity of BaP slightly but significantly. Small doses of Angelicae radix and Cnidii rhizoma extracts enhanced the mutagenicity, but at higher doses a decreasing effect was noted. The factors isolated contained umbelliferone, protoanemonin, and plant phenols. These medicinal plants containing blocking agents for mutagenic activity are very important and are used very frequently in Chinese herbal medicines. In these cases, the effect seems to be unrelated to the part of the plant used or the family to which it belongs; rather, the substances in each extract combine to produce the effect on mutagenicity (Sakai et al., 1988).

IX. Conclusions

Specific biological action of a drug is due to its specific binding to a functional molecular receptor. In complex plant extracts, the variation in effects observed can be attributed to the many chemically reactive species that are formed during the processing and ingestion of the extract, which could act as nonspecific redox agents, scavengers of free radicals, and ligands for binding to toxicants. The final effects are obviously the outcome of interactions between the components and their individual and collective interaction with the toxicant. The specificity and efficacy of such responses will be influenced also by the physiological factors influencing the plants and the process followed for administration of the extract.

In utilizing pharmacologically active herbs, both beneficial and potential adverse effects must be taken into account. The actual dose and form of the plant also need to be worked out (see DeSmet et al., 1992).

The activation of chemicals that have entered the plant system into potential mutagens by plant enzymes is another field that needs immediate attention, particularly in view of the large number of agricultural chemicals such as fertilisers, herbicides, and pesticides that are being continuously added. Since the plants, particularly the crops, that receive most of these chemicals are at the beginning of the food chain, it is imperative to study the interaction of plants with environmental agents. The wide diversity of plants and environmental chemicals make the problem more complicated. The information available gives the mutagenic/antimutagenic action of individual components on a limited number of test systems. But this information is very limited, as is shown by contradictory activity of the same product under different conditions. The inhibitory action tends to lower the active dose of genotoxic agents and the accelerating action raises it. The ultimate load of mutations is the result of interaction between these opposing forces, modified by a large number of exogenous and endogenous factors. Therefore, a comprehensive overview is needed before arriving at conclusions regarding the environmental safety of any new chemical.

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Author:Sarkar, Debisri; Sharma, Archana
Publication:The Botanical Review
Date:Oct 1, 1996
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