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Mutagenic and antimutagenic potential of essential oil (EO) of cultivated sage (S. officinalis L.) and its monoterpenes: thujone, 1,8-cineole, camphor and limonene against UVC-induced mutations was studied with Salmonella/microsome, E. coli WP2, E. coli K12 [Simić, D., Vuković-Gacić, B., Knezević-Vukcević, J., 1998. Detection of natural bioantimutagens and their mechanisms of action with bacterial assay-system. Mutat. Res. 402, 51-57] and S. cerevisiae D7 reversion assays. The toxicity of EO differed, depending on the strain used. The most sensitive were permeable strains TA100, TA102, E. coli K12 IB112 and non-permeable WP2. Mutagenic potential of EO and monoterpenes was not detected, with or without S9. EO reduced the number of UV-induced revertants in a concentration-dependent manner, reaching 50-70% of inhibition at the maximum non-toxic concentrations: 3 microl/plate (TA102), 5 microl/plate (WP2), 7.5 microl/plate (IB112), 30 microl/plate (E. coli K12 SY252) and 60 microl/plate (D7). The metabolic activation had no effect on antimutagenic potential of EO. Similar toxicity of monoterpenes was observed in TA100, E. coli SY252 and D7, with the exception of limonene (less toxic to D7). Reduction of UV-induced revertants by non-toxic concentrations of monoterpenes, tested with SY252 and D7, reached 40-50% at 15-20 microl/plate of thujone, 10 microl/plate of cineole and 1-10 microg/plate of camphor. Limonene showed antimutagenic effect only in D7. Our data recommend sage monoterpenes for further chemoprevention studies.

The inhibition of spontaneous and UV-induced mutations by essential oil (EO) of sage (Salvia officinalis L.) and its fractions F1-F5 containing different proportions of mono- and sesquiterpenes was studied with the Salmonella/microsome, E. coli K12, and S. cerevisiae D7 reversion assays. The EO, F1, and F2 exhibited antimutagenic potential against UV-induced mutations in all tests. Fractions F3 and F4 produced a toxic, mutagenic, or antimutagenic response, depend?ing on the test organism used. Reduction of spontaneous and UV-induced mutations by F5 was detected only in permeable strains of E. coli. The obtained results demonstrate antimutagenic activity of volatile sage terpenes and recommend them for further antimutagenesis and anticarcinogenesis studies.

Antibacterial activity of volatiles from sage against Gram-positive and Gram-negative bacteria from the ATCC collection was screened with the disk diffusion test. The essential oil and its fractions showed a significant antibacterial effect against S. aureus and B. subtilis. The minimum inhibitory concentrations were 1.25-2.5 ?L/mL for S. aureus and 0.15-2.5 ?L/mL for B. subtilis. The effect on S. aureus was bactericidal, while initial bactericidal effect on B. subtilis was impaired by the presence of a resistant fraction of the population, probably endospores. The results obtained with wild type and permeable strains of E. coli and S. typhimurium indicate that transport through the cell wall limits the antibacterial effect of sage volatiles.

Escherichia coli K12 assay-system is designed in order to detect bioantimutagens, agents preventing mutagenesis by modulation of DNA repair and replication. The assay is composed of four tests aimed at the detection of inhibition of spontaneous and induced mutations (Tests A and B) and the estimation whether the anti-mutagenic agent acts by increasing the fidelity of DNA replication (Test B), by inhibition of SOS error prone repair (Test C), or by favoring error-free recombinational repair (Test D). In Test A, repair proficient strain and its uvrA counterpart are used for detection of spontaneous and UV-induced mutations, while in Test B mismatch repair deficient strains ( mutH, mutS, mutL and uvrD ) are used for amplified detection of spontaneous mutations caused by replication errors. In Test C, repair proficient strain carrying sfiA::lacZ fusion is used for measuring the level of SOS induction by monitoring the level of β-galactosidase. In TestD, the strains carrying different recA alleles ( recA + , rcA730 and Δ recA ) are used for measuring intrachromosomal recombination between nonoverlapping deletions in duplicated lac operon, by monitoring Lac + recombinants. The assay-system is validated with model bioantimutagens and used for detection of anti-mutagenic potential of different terpenoid fractions from sage ( Salvia officinalis L.). Extract E1/3 of cultivated sage, distinguished from others by its high content of monoterpernoid camphor, reduces UV-induced mutagenesis in Test A, while it has no effect in Test B and C. In Test D, it enhances intrachromosomal recombination in untreated and UV-irradiated recA + and recA730 strains. The results suggest that the protective effect is due to stimulation of recombinational repair, similarly to coumarin. We speculate that monoterpenoids from sage enhance genetic recombination by intervening in a formation of RecA-DNA complex and channeling it into recombination reaction.

The effect of the recombinational deficiency on W-reactivation of UV-damaged phage lambda was explored. In this paper we show that W-reactivation is reduced by the recB21 and recF143 mutations after bleomycin (BM) and UV treatment. Combination of these mutations in the recB21recF143 double mutant blocks W-reactivation completely after BM induction, but leaves residual W-reactivation ability after UV-irradiation, which is abolished by the introduction of uvrB deficiency (delta(uvrB-chlA]. W-reactivation has been rendered constitutive in recB21C22sbcB15, but the efficiency of reactivation remained virtually constant over the range of BM and UV doses, indicating the role of the RecBC(D) enzyme in W-reactivation.

A collection of 212 gram-positive bacilli isolated from natural habitats was screened for the presence of intervening sequences (introns and intein-coding sequences) in the SPbeta prophage-related ribonucleotide reductase genes bnrdE and bnrdF. Three novel configurations were identified on the basis of the presence of (i) intervening sequences in bnrdE and bnrdF, and (ii) an ORF in the bnrdE-bnrdF spacer. Analysis of the cell wall genetic determinants as well as of the incorporation of radio-labelled glycerol into cell wall allowed newly and previously identified B. subtilis strains with different configurations of bnrdE/bnrdF intervening sequences to be assigned to one of two subspecies. Strains apparently belonging to the subsp. subtilis contain three intervening sequences many of which are associated with the putative homing endonuclease activity. Strains of the subsp. spizizenii contain only one or two ORF-less group I introns. Introns occupying bnrdF are confined to the subspecies subtilis.

The antimutagenic potential of Vitamin E due to its antioxidative properties was studied. The new Escherichia coli K12 assay-system designed in our laboratory was employed in order to detect the antimutagenic potential of Vitamin E and to determine its molecular mechanisms of action. The assay is composed of three tests. In Test A, we examine the influence of the antioxidant on induced oxidative mutagenesis in a repair-proficient strain. Spontaneous mutagenesis is monitored in Test B, which is performed with two mutator strains, one mismatch repair-deficient (mutS) and another deficient in 8-oxo-dGTP-ase activity (mutT). In Test M, a repair-proficient strain and its mismatch repair-deficient counterpart (mutH), both carrying a plasmid with microsatellite sequences, are used to measure the level of microsatellite instability. To examine the antimutagenic potential of Vitamin E we also used the WP2 antimutagenicity test. Protective properties of Vitamin E against oxidative mutagenesis were detected in all tests with the E. coli K12 assay-system as well as in the WP2 antimutagenicity test. This study confirms that mismatch repair is essential for repair of oxidative DNA damage. The results obtained indicate that Vitamin E prevents the formation of DNA adducts by lipid peroxidation products rather than those formed by direct oxidation of DNA bases. Moreover, it can reduce microsatellite instability. After further validation, the new E. coli K12 assay-system can be used to test the antimutagenic potential of antioxidants.

The results of a study of bioantimutagenesis, with emphasis on natural antimutagens from plant extracts with modulating effects on DNA repair in Escherichia coli bacteria are presented in this chapter. Comparative screening for spontaneous or induced mutagenesis, as well as expression of the SOS gene, sfiA was accomplished.

Activation of the RecA protein following UV-irradiation or bleomycin (BM) treatment was measured in rec mutants of E. coli by monitoring β-galactosidase activity. We provide evidence here that the defect in the recN mutant results in high constitutive and induced levels of activated RecA protein. In all rec mutants studied, with the exception of the recN mutant, induction of enzyme activity, following DNA-damaging treatments, was reduced relative to the wild type. The kinetics of induced sfiA expression indicates that the DNA-unwinding activity of the RecBCD enzyme plays a major role in SOS-signal formation. The RecF protein is not needed for BM induction in strains with a functional RecBCD pathway of recombination. However, a functional product of recF gene is implied in the formation of an efficient inducing signal after UV-irradiation, as well as in the additional processing of BM-induced lesions after exposure to the drug. A fully expressed RecF pathway of recombination does not provide a high level of activated RecA protein following DNA-damaging treatments.