Mutagenesis, Vol. 18, No. 2, 113-118,
March 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Antimutagenic activity of extracts of natural substances in the Salmonella/microsome assay
Programa de Pesquisas Ambientais, Divisão de Biologia, Fundac
ão Estadual de Protecão Ambiental Henrique Luis Roessler, Av. Dr Salvador Franca 1707, CEP 90690–000 Porto Alegre-RS, Brasil
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Abstract |
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Scientific information regarding plants used in folk medicine in the form of teas and their effect on human health or on genetic material has been the subject of many different types of investigation. The antimutagenic activity of two plants Maytenus ilicifolia and Peltastes peltatus, both rich in compounds of the flavonoid and tannin groups and frequently employed in folk medicine, was studied. Antimutagenicity was determined against known mutagenic substances (4-oxide-1-nitroquinoline, sodium azide, 2-nitrofluorene, aflatoxin B1, 2-aminofluorene and 2-aminoanthracene), using the Salmonella/microsome assay. Infusions of P.peltatus showed high cytotoxicity and a co-mutagenic effect for induction of base pair substitution mutations with 4-oxide-1-nitroquinoline (–S9 mix). Infusions of M.ilicifolia produced similar effects for frameshift and base pair substitution mutations. With the mutagens 2-nitrofluorene (TA98) and sodium azide (TA100) no significant enhancement effects (co-mutagenic effects) were observed and inhibition of mutagenic activity and cytotoxicity were also diminished. In assays evaluating antimutagenic activity in the presence of metabolic activation utilizing S9 mix, high and significant inhibition of aflatoxin B1-, 2-aminofluorene- and 2-aminoanthracene-induced mutagenicity was observed in the presence of the infusions using both TA98 and TA100 and employing doses ranging from 25 to 500 mg/plate. Seventy-five percent of the doses tested exhibited a significant or suggestive decrease in induced mutagenicity with the infusion of M.ilicifolia. With the infusion of P.peltatus significant or suggestive antimutagenic responses were observed with 50% of the doses evaluated. Complexity was clearly noted in the responses observed in the interaction of aqueous extracts of M.ilicifolia and P.peltastes with the genetic material and metabolites generated by the S9 mix played an important role in the protection of DNA.
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Introduction |
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Large numbers of plant species are a great source of biologically active compounds whose effect on human health or genetic material is mostly unknown. The use of plant infusions to cure many different types of diseases is very common in Brazilian folk medicine. They frequently substitute for modern medicines. In recent years there has been greater interest in investigating compounds originating from plants and their effects on DNA. This is done with many different types of assays employing different organisms. The actions of these compounds may be involved in maintaining the balance between the consumption of mutagenic and antimutagenic substances, thus contributing to increases or reductions in the incidence of cancer in the population (Ames, 1971). Compounds from plants could act as protective agents with respect to human carcinogenesis, acting against the initiation, promotion or progression stages of this process (Edenharder et al., 1993
) or, perhaps, destroying or blocking the DNA-damaging mutagens outside the cells, thus avoiding cell mutations (Ruan, 1989). Mutagenic and antimutagenic activities have been correlated with the presence of certain phytochemical substances, such as compounds of the flavonoid group (Brown, 1980; Huang et al., 1983; Ravanel et al., 1987; Ayrton et al., 1988; MacGregor and Wilson, 1988; Vargas et al., 1989; Czeczot et al., 1990; Edenharder et al., 1993; Mitscher et al., 1996; Beudot et al., 1998; Ferreira and Vargas, 1999). A relationship has been reported between structure and activity, both for mutagenic activity (MacGregor, 1986; Ravanel et al., 1987; Beudot et al., 1998) and for protection of the genetic material (Edenharder et al., 1993). Flavonoids are consumed naturally through the intake of beverages such as beer, coffee and wine (on average 
1 mg/l flavonoids) and may reach 25 mg/l in black tea (Hertog et al., 1993). According to MacGregor and Jurd (1978) these are relatively stable compounds, resistant to heat, light and oxygen, and are moderately acidic.
Mainly antimutagenic activity has also been attributed to the tannins (Kada et al., 1985; Sakai et al., 1990; Imanishi et al., 1991; Tanaka et al., 1998). These are phenolic compounds and are widely distributed in plants. Studies in cell cultures performed by Imanishi et al.(1991) revealed that tannins have an antimutagenic effect at low concentrations in assays in the presence of S9 mix, promoting increased excision repair. In contrast, a co-mutagenic effect was observed in the absence of S9 mix and in the presence of high concentrations of tannin metabolites.
The purpose of this study was to determine the antimutagenic activity of two plants, Maytenus ilicifolia and Peltastes peltatus, rich in compounds of the flavonoid and tannin groups. They are used in folk medicine and we tested them against known mutagenic substances using the Salmonella/microsome assay. These two plants previously showed negative results for mutagenicity and cytotoxicity in the same assay (Alice et al., 1991; Vargas et al., 1989, 1991).
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Materials and methods |
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Chemical substances
The mutagenic substances used in this study were sodium azide (AZS) (CAS no. 26628–22–8) (Merck do Brasil) and 2-aminoanthracene (2AA) (CAS no. 613–13–8), 2-aminofluorene (2AF) (CAS no. 153–78–6), aflatoxin B1 (AFB1) (CAS no. 1162065–8), 2-nitrofluorene (2NF) (CAS no. 607–57–8) and 4-oxide-1-nitroquinoline (4NQO) (CAS no. 56–57–5) (all Sigma Chemical Co., St Louis, MO). A metabolic activation fraction (S9 mix), purchased from Molecular Toxicology Inc. (Boone, NC), was prepared from Sprague–Dawley rat livers pretreated with Aroclor 1254. Glucose 6-phosphate and NADP from Sigma Chemical Co. were also used.
Samples
Two plant species used as teas in folk medicine were utilized. They were M.ilicifolia C. Martius (Celastraceae family), employed as an antiinflammatory medicine, and P.peltatus (Vell.) Woods (Apocynaceae family), utilized as an antiinflammatory medicine and against venereal diseases. Among their constituents are: alkaloid photochemicals (Mayer’s reagent and Wagner’s reagent), flavonoids, sterols, saponins, tannins (M.ilicifolia); sterols, saponins, tannins (P.peltatus) (Alice et al., 1991). The phytochemical constituents are described in Table I.
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Plants dried at room temperature were used to prepare infusions. The aqueous extracts were prepared with deionized water at 80°C at 25% (w/v) concentration. Samples were filtered and sterilized with 0.45 µm Millipore filters (Vargas et al., 1989
). The assays were performed at concentrations of 100, 300, 500, 1000, 1500 and 2000 µl/plate, corresponding to 25, 75, 125, 250, 375 and 500 mg/plate dry matter, respectively. The material was collected in pollution-free areas and far from towns and plantations.
Antimutagenic assay
The antimutagenic assay used was the Salmonella/microsome assay developed by Maron and Ames (1983) and as modified by Ruan et al.(1992). Bruce M.Ames (University of California, Berkeley, CA) provided bacterial strains TA98 and TA100.
The antimutagenic assay was performed using the preincubation (20 min) procedure at 37°C, with the addition of 100 µl of a bacteria strain grown overnight (1–2x109 cells/ml), the plant extract to be tested and the mutagenic agent with or without S9 mix (500 µl) in triplicate plates. The experiments were repeated once. After plating in a minimal medium and incubation for 48 h at 37°C, the numbers of revertants per plate were counted. In all assays, negative (200 µl nutrient broth) and positive controls (AZS, 5 µg/plate; 2AA, 5 µg/plate; 2AF, 5 µg/plate; AFB1, 0.5 µg/plate; 2NF, 10 µg/plate; 4NQO, 0.5 µg/plate) were added according to the strain and treatment utilized, accompanied by a cellular viability assay (Maron and Ames, 1983; Vargas et al., 1988).
The inhibition rate for mutagenic activity was calculated according to the formula: inhibition rate (%) = (A – B)/Ax100, where A is revertants in the positive control and B is revertants in the infusion sample, having subtracted the spontaneous revertants. In the cytotoxicity assays the samples with values >80% viable cells are considered non-toxic as compared with the viability of the negative control.
Statistical analysis
The significant differences (P 
0.05) between the means of revertants per plate of the samples in relation to the mutagens were calculated using the Tuckey honest significant difference (HSD) test for unequal sample sizes (Spjotovoll and Stoline, 1973).
The final criterion used to interpret the results of significant increase (enhancement effect) or decrease (inhibition effect) in the number of Salmonella revertants was a reversion rate >50% or <50% in relation to that observed in positive controls, accompanied by a significance analysis (P 0.05) in the Tuckey HSD test. Where one of these two criteria was fulfilled, the sample was considered to present signs of the effect (co-mutagenic or antimutagenic).
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Results |
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In preliminary studies (Alice et al., 1991
; Vargas et al., 1989, 1991) no mutagenic effect or cytotoxicity was observed for any tea extracts at doses <500 mg/plate, using the Salmonella typhimurium TA98 and TA100 strains.
Antimutagenic assay without metabolic activation
As shown in Table II, the infusions of M.ilicifolia and P.peltatus had a synergistic mutagenic effect with 4NQO, increasing the mutagenic activity in both frameshift and base pair substitution mutagenic assays, and produced cytotoxicity. The synergistic effect was highest in strain TA100, with both the M.ilicifolia and P.peltatus infusion assays showing it at several concentrations (e.g. 330%, 500 mg/plate and 338%, 375 mg/plate, respectively). Low inhibition rates, mostly below the levels of significant decrease, were observed in the presence of the mutagens 2NF (TA98) and AZS (TA100). In assays performed using the mutagen 2NF, the greatest inhibition of mutagenicity was obtained using the M.ilicifolia infusion at doses of 125 or 375 mg/plate.
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In assays carried out using the mutagen AZS, only the samples of M.ilicifolia, at a dose of 500 mg/plate, presented signs of inhibition of mutagenicity.
Antimutagenic assay with metabolic activation
Table III and Figure 1 show the results of tests of antimutagenic activity using the same plant extracts against AFB1, 2AF and 2AA with metabolic activation of both strains. Significant inhibition was detected in the absence of cytotoxicity, except in the case of TA98 with 2AF and M.ilifolia (375 mg/plate, 75% survival) and TA100 with 2AF and P.peltatus (125–500 mg/plate).
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With the M.ilicifolia infusion the inhibition rates against AFB1 ranged from 66.15 to 100% for strain TA98 and from 83.54 to 100% for strain TA100. Thus, an antimutagenic effect was observed. These rates were higher at concentrations >25 mg/plate in TA98. Samples of P.peltatus could also modulate the mutagenic effect of this substance at rates ranging from 66.66 to 89.51% in TA98 and from 70.51 to 91.39% in TA100, and signs of an antimutagenic effect are present at 25 (TA98 and TA100) and 125 mg (TA98) with a significant antimutagenic effect beginning at a dose of 250 mg/plate.
Results with the M.ilicifolia infusion show a significant decrease in 2AF mutagenic activity at rates ranging from 77.86 to 100% for TA98 and from 89.90 to 100% for TA100, with signs of such effects at lower concentrations. In the study performed with P.peltatus extracts rates of 85.55–95.52% were observed for TA98 and antimutagenic effects (45.87–100%) for TA100 were also observed.
The M.ilicifolia infusion in the presence of 2AA produced inhibition of mutagenicity ranging from 56.71 to 88.20% for TA98 and from 53.98 to 93.88% for TA100. However, for the P.peltatus–2AA mixture a significant inhibition (78.06%) was only observed for frameshift mutations at a dose of 500 mg/plate. Furthermore, with these extracts co-mutagenic activity was observed for both frameshift and base pair substitution mutations. These activities ranged up to 69.20 and 99.62% (Table III
).
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Discussion |
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The interactions between the aqueous extracts of M.ilicifolia or P.peltatus and three mutagens (4NQO, 2NF and AZS) and three indirect agents (AFB1, 2AF and 2AA) presented different ranges of inhibition or even enhancement of mutagenic activity as assessed by the Salmonella/microsome assay. The different actions depended on the mutagen used, the dose of the sample infusion tested and the treatment (+/– S9 mix) and are summarized in Table IV
. It was possible to observe synergistically increased effects with or without a cytotoxic response in some assays, a clear dose–response effect for antimutagenic activity in others and also, in some cases, the presence of an optimum dose effect (Tables II and III).
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In the assays performed without metabolic activation (–S9 mix) significant or suggestive antimutagenic activity responses were found only in the assays performed with M.ilicifolia and 2NF or AZS. Considering all the concentrations of mutagen–plant extract mixture results, it can be seen that 50% showed an increase in Salmonella revertants and 18.75% of those presented a significant co-mutagenic response. Significant enhancement results were observed only in the 4NQO data in the presence or absence of cytotoxicity.
Effective inhibition of mutagenicity was observed in the assays with metabolic activation. A comparison between the dose–responses curves for antimutagenic and cytotoxic activity is present in Figure 1. None of the levels of significant antimutagenic responses to the AFB1- and 2AA–plant extract mixtures present survival responses below 80%. As compared with 2AF, similar responses could be observed except for the P.peltatus–TA100 mixture, where inhibition of mutagenic activity was associated with cytotoxic responses. These data indicate that cytotoxicity can interfere with the inhibition response observed. The interactions of the plant infusions with 2AA produced intriguing results, with responses that ranged from enhancement with different concentrations of the modulating agent and significant inhibition of a mutagenic response at higher doses to significant antimutagenic responses at different doses (Fig. 1).
Maytenus ilicifolia–mutagen mixtures exhibited a significant or suggestive decrease in induced mutagenicity at 72.2% of the doses tested with strain TA98 and at 77.8% of those performed with strain TA100. Significant antimutagenic responses were observed at 50% of the doses evaluated using the P.peltatus–mutagen mixtures and these responses were equal in both strains (50%). Therefore, the greatest inhibition for frameshift lesions and for base pair substitution was obtained with M.ilicifolia.
It is important to note that 19.4% of the plant extract concentrations mixed with AFB1, 2AF and 2AA showed an increase in Salmonella revertants at intermediate doses and 21.43% of those 19.4% produced significant enhancements or suggestive responses, all of which were associated with the mutagen 2AA. A similar response was observed for N-acetylcysteine (NAC) tested at varying concentrations. It enhanced the mutagenicity of several promutagens, including AFB1 and 2AF, at intermediate doses using strains TA98, TA100, TA1535 and TA1538 in the presence of S9 mix. However, at higher doses NAC completely inhibited the mutagenicity of these same compounds. These studies were continued with other aminothiols, suggesting that low doses of aminothiols potentiate the activity of promutagens in vitro by affecting metabolic activation, whereas high doses inhibit mutagenicity by binding electrophilic metabolites (Waters et al., 1996).
Natural substances such as flavonoids and tannins or their derivatives, present in these samples, were previously described as possessing antimutagenic properties (Kada et al., 1985; Imanishi et al., 1991; Edenharder et al., 1993; Yen and Chen, 1996) and these metabolites could be involved in mutagen deactivation. Antimutagenic activity associated with the presence of tannins was observed for the extracts of P.peltatus and M.ilicifolia in assays after metabolic activation using the mutagens AFB1 and 2AF. Epigallocatechin gallate (EGCg), extracted from the leaves of Camellia sinensis, acts as a bio-antimutagen, reducing the high level of spontaneous mutations due to altered DNA polymerase III in a mutator strain of Bacillus subtilis. This enzyme might improve the fidelity of DNA replication (Kada et al., 1985). Also, Yen and Chen (1996), studying the antimutagenic activity of plant extracts rich in EGCg, observed a correlation between inhibition of mutagenic activity against benzo[a]pyrene and AFB1. Imanishi et al.(1991), studying the effects of tannin components extracted from green and black tea on mutagen-induced sister chromatid exchange (SCEs) and chromosome aberrations in a culture of mammalian cells, suggested that tea tannin components themselves inhibited DNA excision repair and resulted in a co-mutagenic effect; while in the presence of S9 mix metabolites of tea tannin components promoted DNA excision repair activity and resulted in an antimutagenic effect. Similarly, the plant extracts of P.peltatus and M.ilicifolia in this study produced a significant direct co-mutagenic and cytotoxic effect in assays in the presence of 4NQO and protective actions when using mainly AFB1 and 2AF and for some interations with 2AA after metabolic activation. Indeed, some drugs and diet components may act as antimutagens by affecting other drugs or toxicants, by changes in the absorption rates and increased uptake, by reacting or binding with the drug, by competing with the drug for binding to plasma proteins or by affecting activation and detoxification systems (Hayatsu et al., 1988; Yang et al., 1992; Waters et al., 1996).
In the majority of results obtained in this study the extracts of M.ilicifolia, in which compounds of the flavonoid and tannin groups are present, showed significantly higher rates of mutagenic activity modulation than the P.peltatus infusions, which possess only tannins. These results imply greater chemical inactivation or prevention of damage to the DNA by the chemical compounds of the M.ilicifolia infusion. Compounds of the flavonoid group have often been identified as mutagenic, antimutagenic or both. The protective action observed with the plant infusions investigated is in accordance with the results of Edenharder et al.(1993), who, examining the effect of structurally related flavonoids and similar compounds, showed distinct structure–activity relationships and a protective effect. The total antimutagenic activity should depend not only on the structure of the flavonoid, but to some extent on the structure of the mutagen. Edenharder et al.(1993) reported that Wall et al.(1990) showed an antimutagenic effect of quercitin against the mutagen 2AA. They also reported that flavonoids have been demonstrated to be effective inhibitors of mutagenicity induced in Salmonella by AFB1. The rates of mutagenic activity modulation observed in this study may indicate a contribution of flavonoids to the deactivation of AFB1, 2AF and, for some results, 2AA, or even a protective action of the M.ilicifolia infusion or its metabolites.
The responses were more significant in the presence of metabolic activation. Edenharder et al.(1993) reported that inhibition mechanisms, including antimutagenesis, of flavonoids and structurally related compounds can be dependent or independent of concentration. The antimutagenic response is mobilized by invoking the competitive inhibition by liver glycosides of P450 isoenzymes (Edenharder et al., 1993). Buening et al.(1981) have also postulated that some flavonoids are potent inhibitors of cytochrome c (P450) reductase. This type of protective activity after metabolic activation has been related to the function of isoforms of cytochrome P450s in the detoxification system with reductase or oxygenase (Kappus, 1986), whose function in the system is antioxidant scavenging, neutralizing compounds that generate oxygen radicals, free radicals and reactive oxygen species (Parke et al., 1991). Therefore, the greatest antimutagenic potential observed in assays in the presence of a metabolic fraction may be related to the activation of a cytochrome P450 which mediates the oxidation of promutagens (Mitscher et al., 1996), indicating that this action could be due to the competitive inhibition by glycosides of cytochrome P450, thus avoiding formation of the promutagen (Edenharder et al., 1993).
The complexity of the responses observed in the interaction of aqueous extracts of the M.ilicifolia and P.peltatus infusions with the genetic material is clear. Although a co-mutagenic effect occurred, especially in direct assays against the mutagen 4NQO, the metabolites generated by the S9 mix played a significant role in the DNA-protective action. It should be stressed that there are many reactive components in plants of an unknown chemical nature. New studies are being performed attempting to assess the antimutagenic mechanisms of the phytochemical components of these extracts.
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Acknowledgments |
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The authors are very grateful to A.G.Silva and R.R.Guidobono for their assistance, Dra Jandyra M.G.Fachel for her help in statistical analyses and Dr Michael D.Waters for critical review of the manuscript. This research was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPERGS (Fundac
ão de Amparo à Pesquisa do Rio Grande do Sul). |
Notes |
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1 To whom correspondence should be addressed. Tel: +55 51 3334 6765; Fax: +55 51 334 6765; Email: rubemch{at}fepam.rs.gov.br
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Received on February 25, 2002; accepted on May 30, 2002.
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