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Vanillin (VAN) and cinnamaldehyde (CIN) are dietary flavorings that exhibit antimutagenic activity against mutagen-induced and spontaneous mutations in bacteria. Although these compounds were antimutagenic against chromosomal mutations in mammalian cells, they have not been studied for antimutagenesis against spontaneous gene mutations in mammalian cells. Thus, we initiated studies with VAN and CIN in human mismatch repair-deficient (hMLH1 − ) HCT116 colon cancer cells, which exhibit high spontaneous mutation rates (mutations/cell/generation) at the HPRT locus, permitting analysis of antimutagenic effects of agents against spontaneous mutation. Long-term (1-3-week) treatments of HCT116 cells with VAN at minimally toxic concentrations (0.5-2.5 mM) reduced the spontaneous HPRT mutant fraction (MF, mutants/10 6 survivors) in a concentrationrelated manner by 19% to 73%. A similar treatment with CIN at 2.5-7.5 μM yielded a 13% to 56% reduction of the spontaneous MF. Short-term (4-h) treatments also reduced the spontaneous MF by 64% (VAN) and 31% (CIN). To investigate the mechanisms of antimutagenesis, we evaluated the ability of VAN and CIN to induce DNA damage (comet assay) and to alter global gene expression (Affymetrix GeneChip) after 4-h treatments. Both VAN and CIN induced DNA damage in both mismatch repair-proficient (HCT116 + chr3) and deficient (HCT116) cells at concentrations that were antimutagenic in HCT116 cells. There were 64 genes in common whose expression was changed similarly by both VAN and CIN; these included genes related to DNA damage, stress responses, oxidative damage, apoptosis, and cell growth. RT-PCR results paralleled the Affymetrix results for 4 selected genes (HMOX1, DDIT4, GCLM, and CLK4). Our results show for the first time that VAN and CIN Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. These and other data lead us to propose that VAN and CIN may induce DNA damage that elicits recombinational DNA repair and, consequently, reduces spontaneous mutation. NIH Public Access

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The Escherichia coli lacZ gene was stably introduced into phi C31-based phage cloning vectors in Streptomyces lividans. However, lacZ could not be stably introduced into S. lividans on the plasmid vectors pIJ702 or pIJ41. Studies of the expression of lacZ in S. lividans and S. coelicolor were facilitated by the use of mutants and/or growth conditions in which endogenous beta-galactosidase activity was low or absent. Plaques and lysogens of phi C31::lacZ constructs involving transcriptional fusions to the pBR322 tet gene contained beta-galactosidase activity. Activities were markedly higher in S. lividans than in S. coelicolor. Insertion of the major transcriptional terminator of coliphage fd between the tet promoter and lacZ reduced lacZ expression more in lysogens than in lytically infected cultures, suggesting the existence of a phage-specified anti-termination function. Several phi C31 derivatives suitable for the construction of different kinds of transcriptional and translational fusions in Streptomyces were derived. The expression of lacZ in one transcriptional fusion vector (KC659) was increased both in plaques and in lysogens by the appropriately positioned insertion of a previously characterized promoter from the aph gene of S. fradiae. When phi C31 DNA fragments were inserted into KC659, at least one recombinant phage gave high beta-galactosidase activity in lytic infections, but not in lysogens. The potential usefulness of lacZ fusions in Streptomyces is discussed.