This study of inhibitors and modulators of the effects of environmental genotoxic agents is aimed at understanding the mechanisms involved in the protection against mutagens and carcinogens and at creating an anti-risk strategy in the protection of human health. Antigenotoxic potentials of certain naturally occurring compounds extracted from plants are estimated by applying prokaryotic and eukaryotic tests.

Differently prepared extracts of wild and cultivated sage (Salvia officinalis L.), basil (Ocimum basilicum L.), osage orange (Maclura pomifera Rob.) and their pure constituents, are screened for antimutagenic potential against UV-and EtBr-induced mutations. For that purpose, the new E. coli assay system is designed and validated to detect bioantimutagens and to determine molecular mechanisms of their antimutagenic action. The results obtained with newly constructed test are also confirmed with Ames test (Salmonella typhimurium) and eukaryotic test (Saccharomyces cerevisiae).

The most significant result indicate that monoterpenoids from cultivated sage inhibit UV-induced mutagenesis by modulating DNA repair pathways (enhanced recombination, inhibited mutagenic repair). Protective effect of ethereal oils, various fractions distinguished by different content of mono-, di- and sesqui-terpenoids, as well as pure monoterpenoids, is due to modulation of DNA repair process.

Antioxidants, an integral part of plant extracts, evidenced in sage fractions with high content of diterpenoids, in extract of osage orange and its pure constituent, pomiferin, significantly inhibited EtBr-induced mutagenesis. Their protective effect is due to inhibition of metabolic activation of EtBr, one of desmutagenic mechanisms of antimutagenesis. Our study, as well as data from literature, has shown that natural antimutagens derived from medicinal and aromatic plants may be useful as anticarcinogenic agents.

Keywords: antimutagenesis/anticarcinogenesis, medicinalplantextracts, pro-/eukaryotic tests.

Because mutations play a central role in fundamental biological processes, including the initiation of cancer, the possibility of modulating the responses of particular cells to mutagens has opened new frontiers in cancer control.

Cancer is a complex set of more than 200 diseases with multiple causes and multiple stages. To a large extent, current cancer pattern reflects genetic factors and exposures to environmental factors such as radiation, industrial pollutants, pesticides, viruses and other life style factors.

In recent years the study and application of the active substances from medicinal and aromatic plants, that reduce the risk of cancer, find its place in antimutagenesis and anticarcinogenesis. Given appropriate screening and fractionating methodologies it is possible to identify a wide variety of structural types in higher plants (essential oils, flavonoids, etc.) possessing inhibiting or modulating effect on environmental genotoxic agents, which may allow the use of particular antimutagens as anticarcinogens.

Prokaryotic and eukaryotic test-systems are applied for detection of antigenotoxic potentials of plant extracts. Tests are considered to measure genetic endpoints: reverse mutation, replication fidelity, recombination, mutagenic repair, chromosome aberration, etc. (Goetz et al., 1975; Zimmermann, 1983; Simić et al., 1998).

Antigenotoxic potentials of essential oils of sage (Salvia officinalis L.), basil (Ocimum basilicum L.), osage orange (Maclura pomifera Rob.) fruit extract and their major constituents have been investigated. Detection of bioantimutagens, agents preventing mutagenesis by modulation of DNA repair and replication, was performed with the new Escherichia coli K12 assay (Simic et al., 1994; Simic et al., 1997) and Salmonella typhimurium (Ames) test (Maron and Ames, 1983). Antimutagenic potential of antioxidants from sage and osage orange was investigated with modified prokaryotic tests (Mitić et al., 1998). Moreover, comparative study of terpenoids from sage using eukaryotic tests (Saccharomyces cerevisiae, mice) is in progress.

Escherichia coli K12, Salmonella typhimurium and Saccharomyces cerevisiae strains are listed in Table 1.

Media and growth conditions

The bacteria were cultured overnight in LB medium (5 g NaCl, 10 g bacto-tryptone, 5 g yeast-extract, 1000 ml distilled water) at 37°C with aeration. The media for E. coli K12 tests were as described (Simić et al., 1997). All media for S. typhimurium and S. cerevisiae assays were as described by Maron and Ames (1983) and Zimmermann (1983), respectively. S9 fraction was isolated from the liver of Albino Wister male rats (170-180 g) induced with pheno-barbital/b-naphthoflavone (Ong et al., 1980). S9 mix contained 4% (v/v) S9 fraction, 33 mM KCl, 8 mM MgCl₂, 5 mM glucose-6-phosphate and 4 mM NADP in 0.1 M phosphate buffer pH 7.4.

Chemicals

a+b Thujone and myrcene ("Extrasynthese"), (±)-camphor (ICN Galenika), and 1,8-cineole, limonene and linalool (kindly provided by D. Brkić) were dissolved in ethanol. Ethidium bromide ("Bio-Rad") was dissolved in sterile distilled water. Butylated hydroxytoluene ("Sigma") was dissolved in DMSO and used as model antioxidant.

Preparation of plant extracts

Essential oils from sage and basil were prepared by steam distillation according to Ph. Jug. IV. Osage orange fruit extract was prepared as described (Đarmati et al., 1998). The fractions F1-F5 of sage were prepared by vacuum rectification. Preparation of sage CO₂ re-extracts was described by Đarmati et al. (1993). Plant extracts were dissolved in ethanol or DMSO and applied/plate in non-toxic concentrations.

UV-irradiation

UV-irradiation conditions were the same as described previously (Simić et al., 1985).

Detection of antimutagenic effect

Detection of antimutagenic effect and estimation of the mechanisms of mutagenesis inhibition was performed using Escherichia coli K12, Salmonella typhimurium and Saccharomyces cerevisiae tester strains, as described (Zimmermann, 1983; Simić et al., 1997; Mitić et al., 1998). Evaluation of antimutagenic potential was performed according to classification of Wall et al. (1988).

Over 25 differently prepared extracts of wild and cultivated sage, basil and osage orange and their constituents were screened for antimutagenic potential against spontaneous, as well as UV- and EtBr-induced mutagenesis.

Bioantimutagenic effect of terpenoids from sage

Our data concerning protective effect of essential oils (EO) obtained from sage cultivated by Institute for Medicinal Plant Research "Dr. Josif Pančić", as well as various fractions of EO, distinguished by different content of mono-, di- and sesqui-terpenoids are shown in Table 2.

In all tests applied (E. coli K12, S. typhimurium TA100 and TA102, S. cerevisiae D7), EO and different fractions show an antimutagenic effect on UV-induced mutagenesis. The metabolic activation has no effect on the extent of mutation inhibition. Inhibition of mutagenesis varies in the range of 30-80%, depending on screening test and fraction's content. Only exception is F5 fraction, with high content of high molecular weight terpenoids, inhibiting mutagenesis exclusively in S. typhimurium.

The most significant results obtained in our previous study indicated that monoterpenoids from cultivated sage inhibit UV-induced mutagenesis, most probably by modulation of DNA repair pathways (Simić et al., 1998).

Considering the above, we tested pure monoterpenoids from sage EO: camphor, a+b thujone, 1,8-cineole (>15% in EO) and myrcene, limonene and linalool (<1,5% in EO). We also tested EO of basil because of its high content of linalool (70%). The results are shown in Table 3, indicating the significant antimutagenic effect (35-70%). However, limonene, known from the literature as anticarcinogen (Russin et al., 1989), failed to show antimutagenic effect in E. coli.

Bioantimutagens modulate DNA repair and replication (Kuroda and Inoue, 1988). As the modulation of DNA repair mechanisms is extremely delicate and the antimutagenic effects may be the consequence of the involvement of multiple mechanisms, a demand for a rigorous and discriminating experimental approach is an imperative. The E. coli K12 assay-system (Simić et al., 1997) is highly recommended for screening of plant extracts for bioantimutagens. Our data obtained with E. coli K12 assay-system (Table 4) indicate that monoterpenoids from sage modulate the mutagenesis by enhancement of recombination (Test D) and by inhibition of the SOS induction (Test C), and may be classified as bioantimutagens.

Antigenotoxic effect of sage EO is confirmed in eukaryotic test i.e. dietary exposure suppressed in vivo mytomicin-C induced chromosome aberration in mice (to be published).

Our results support the idea that naturally occurring dietary constituents might modulate the mutagenesis and possibly carcinogenesis process. The reported results that monoterpenoids from plants inhibited or significantly extended the latency of rat mammary carcinoma development (Russin et al., 1989) suggest, together with our data, that plant terpenoids may be effective as anticarcinogens.

Antimutagenic effect of sage antioxidants

Antioxidants, an integral part of plant extracts, evidenced in sage fractions with high content of diterpenoids (E1/5, E2/5, A2/5), in extract of osage orange fruit and its pure constituent, pomiferin, significantly inhibited EtBr-induced mutagenesis, with no effect on spontaneous mutagenesis in E. coli mutT (Mitić et al., 1998; Mitić, 1999). Our results are summarized in Table 5, indicating that antioxidative fractions are less effective as bioantimutagens. Moreover, preliminary experiments indicate that protective effect of these fractions is due to the inhibition of metabolic activation of EtBr, which is one of desmutagenic mechanisms of antimutagenesis (Kuroda and Inoue, 1988).

In recent years, there have been considerable efforts to search for naturally occurring substances for intervention and prevention of carcinogenesis. Many components from dietary or medicinal plants have been identified to posses potential chemopreventive properties. Many studies, including our work with extracts derived from medicinal and aromatic plants, have shown that natural antimutagens, at least some of them, may inhibit the clastogenic effects of carcinogens and thus may be classified as anticarcinogens. Epidemiological studies indicate that many cancers are dependent on multiple mutational etiology, as well as on inherited mutator phenotype (Mac Phee, 1997). Because of that, the search for natural inhibitors of mutagenesis may be useful for discovering anticarcinogenic agents. Moreover, once that the mechanisms of inhibitory effects of antimutagens have been elucidated, their usefulness in the prevention of cancer would become more convincing.

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