Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Inhibition of spontaneous mutagenesis by vanillin and cinnamaldehyde in Escherichia coli: Dependence on recombinational repair
Introduction
Vanillin (VAN) and cinnamaldehyde (CIN) are natural food flavorings that inhibit mutations induced by chemical and physical mutagens in bacterial test systems [1], [2], [3], [4], [5] and mammalian cell culture [6], [7]. Both VAN and CIN inhibited micronuclei formation in V79 cells treated with heterocyclic amines [8] and reduced chromosome aberrations in CHO cells exposed to UV-light and X-rays [9]. In addition, VAN and CIN reduced the frequency of spontaneous mutations in Salmonella TA98 and TA104 [10], [11], [12].
VAN and CIN have been described as bioantimutagens, a term coined by Kada to describe agents that inhibit mutagenesis by modulating cellular processes of DNA replication and repair [13]. In addition to antimutagenic effects observed in WP2s strains of E. coli, CIN also increased survival of cells exposed to 4-nitroquinoline-1-oxide. This survival-enhancing effect was dependent on recA gene function [1]. Likewise, co-treatment with VAN increased survival in UV-exposed cells except in recA strains [4]. In addition, recombination between plasmids was enhanced with VAN treatment [4]. In Drosophila, VAN modestly inhibited mutations induced by mitomycin C but dramatically increased recombination in mitomycin C-treated flies [14]. Co-treatment with VAN and either ethyl methanesulfonate (EMS) or bleomycin also produced a synergistic effect on recombination in Drosophila [15]. Observations of the recombinogenic effects of VAN, together with the requirement of recA gene function for the survival-enhancing effects of VAN and CIN, have lead to the hypothesis that VAN and CIN may inhibit mutagenesis by increasing repair of DNA damage through the recombinational repair pathway [4].
In previous work examining the effects of VAN and CIN on spontaneous mutations in Salmonella TA104, we showed that both compounds significantly reduced mutations at GC sites but not AT sites and that the antimutagenic effect was dependent on the presence of the pKM101 plasmid in homologues of TA104 [12]. To further elucidate the role of DNA repair pathways on antimutagenesis by VAN and CIN, we have investigated the effects of these compounds on spontaneous mutation in five strains of E. coli that have defects in different DNA repair pathways, including nucleotide excision repair (NR11634), mismatch repair (NR9139), and recombinational repair (NR11317). To investigate whether VAN or CIN reduces spontaneous mutations by inhibiting the error-prone SOS pathway, we tested these compounds in strains that were deficient in SOS response but retained recombination function (NR11475) or were deficient in both SOS response and recombination (NR11317).
Section snippets
Chemicals and media
Vanillin (VAN) and cinnamaldehyde (CIN) (Sigma–Aldrich, Milwaukee, WI) were dissolved in dimethyl sulfoxide (DMSO, Burdick and Jackson, Muskegon, MI) and evaluated for survival and antimutagenesis using a modified version of the standard plate incorporation assay [16] in the absence of S-9 mix.
Media used were Vogel–Bonner Medium E (VBME) agar plates containing 0.2% glucose and phenyl-β-d-galactopyranoside (P-gal) agar plates (1 × VB salts, 750 μg/ml P-gal, and 1 μg/ml thiamine). Overnight cultures
Results
To investigate the antimutagenic effects of VAN and CIN in E. coli, we used the lacI forward-mutation system, in which the mutational target is the lacI gene, which encodes the repressor of the lac operon. lacI mutants, lacking a functional repressor, can be detected based on their constitutive expression of the lac operon. Because the sugar analog P-gal is a substrate for the lac enzymes but not an inducer of the operon, such lacI mutants can be isolated and scored by their ability to form
Discussion
Both VAN and CIN were antimutagenic for spontaneous mutation in the wild-type strain NR9102. At the highest nontoxic doses tested, VAN produced a more dramatic effect than CIN, reducing the MF by 51%, compared to a 30% reduction by CIN (Table 2, Table 3). In the NER- strain NR11634, both VAN and CIN were antimutagenic. However, in this strain, the inhibitory effect of CIN was much more dramatic, reducing the MF by nearly 96% at 20 μmol/plate, a dose that was cytotoxic in all other strains
Acknowledgements
This research was supported in part by the NIH Intramural Research Program of the National Institute of Environmental Health Sciences. In particular, D.T. Shaughnessy acknowledges support from an Intramural Research Training Award. We thank Peggy Mathews (US EPA) and Sarah Warren (US EPA) for their assistance and laboratory support in this work. We thank D.A. Bell (NIEHS), K. Witt (NIEHS), L. Recio (ILS), and W. Ward (US EPA) for their helpful comments on the manuscript. This manuscript was
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