Article pubs.acs.org/jnp
α‑Hexylcinnamaldehyde Inhibits the Genotoxicity of Environmental Pollutants in the Bacterial Reverse Mutation Assay Silvia Di Giacomo,† Gabriela Mazzanti,† Maria Grazia Sarpietro,‡ and Antonella Di Sotto*,† †
Department of Physiology and Pharmacology “V. Erspamer”, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy Department of Drug Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy
‡
ABSTRACT: The antimutagenicity of α-hexylcinnamaldehyde (1), a semisynthetic and more stable derivative of cinnamaldehyde, was evaluated against common environmental pollutants in the bacterial reverse mutation assay. The pre-, co-, and post-treatment protocols were applied to assess the involvement of desmutagenic and/or bioantimutagenic mechanisms. Compound 1 (9−900 μM) produced a strong antimutagenicity (>40% inhibition) in the Salmonella typhimurium TA98 strain against the nitroarenes 2-nitrofluorene and 1-nitropyrene in almost all experimental conditions. A strong inhibition was also reached against the nitroarene 1,8-dinitropyrene and the arylamine 2-aminoanthracene in the cotreatment at the highest concentrations tested. In order to evaluate if an inhibition of bacterial nitroreductase (NR) and O-acetyltransferase (OAT) could be involved in the antimutagenicity of 1 against nitroarenes, the substance was further tested against 1-nitropyrene (activated by both NR and OAT) in TA98NR and TA98 1,8-DNP strains (lacking the NR and OAT enzymes, respectively). Although both desmutagenic and bioantimutagenic mechanisms appear mostly involved in the antimutagenicity of 1, based on data obtained in the TA98NR strain, applying the pretreatment protocol, compound 1 seems to act as an inhibitor of the OAT-mediated mutagen bioactivation. These results provide justification for further studies on 1 as a possible chemopreventive agent.
Drugs of natural origin include natural substances, molecules derived semisynthetically from natural products, or synthetic compounds based on natural product models.1 Compared to synthetic compounds, they have better safety profiles, are well accepted by the public opinion, and are usually relatively inexpensive.2 However, natural substances are often characterized by several factors (including chemical instability, poor water solubility, and lack of in vivo bioavailability) that limit their use in clinical practice.3 In this context, developing semisynthetic derivatives may overcome these limits. As an example, diflomotecan, a promising antitumor compound, has been developed from the natural alkaloid camptothecin in order to enhance its plasma stability.4 Likewise, to improve the bioavailability of the antitumor alkaloid paclitaxel, its semisynthetic analogue docetaxel has been successfully employed in chemotherapy.5 α-Hexylcinnamaldehyde (1; MW 216.32) is a floral-scented compound, used as an ingredient in common personal care items (perfumes, creams, shampoos, etc.) and household products and as an additive in food and pharmaceuticals.6 From a structural point of view, it is an α,β-unsaturated aldehyde, due to the presence of a polarized carbon−oxygen double bond and an additional double bond between carbons 2 and 3 (α and β, respectively), which makes the compound more reactive than a simple aldehyde.7−9 Compound 1 is a semisynthetic derivative of cinnamaldehyde (cinnamic aldehyde or 3-phenyl-2-propenal), an aromatic © 2014 American Chemical Society and American Society of Pharmacognosy
aldehyde found to be the major component (60−75%) of cinnamon bark essential oil (Cinnamomum spp., Lauraceae) and an important dietary factor. Various studies highlighted the beneficial properties of cinnamaldehyde, including antiproliferative and pro-apoptotic activities and antimutagenic effects, especially against different quinoline and nitrofuran derivatives.10−12 The reactivity of the “Michael acceptor” moiety and the lipophilicity have been proposed as structural features that make cinnamal-containing compounds potential anticancer alkylating agents.13 In spite of the protective effects, the clinical application of cinnamaldehyde is limited due to its poor chemical stability. In cinnamaldehyde (structure PhCH CHCHO), the double bond between the α and β carbons is subject to Michael addition. Including an R group at the double bond (structure PhCHCRCHO) should lessen the molecule’s reactivity due to the inductive effect of the alkyl group, which stabilizes the negative charge in the intermediate carbanion.14 Therefore, taking into account that including an Received: July 15, 2014 Published: December 10, 2014 2664
dx.doi.org/10.1021/np500567d | J. Nat. Prod. 2014, 77, 2664−2670
Journal of Natural Products
Article
alkyl chain at the double bond can stabilize the compound,14 some alkyl derivatives (e.g., α-butyl-, α-amyl-, and α-hexylcinnamaldehyde) are being currently developed for use as food and cosmetic additives, because of their lower reactivity and sensitizing power.14,15 On the basis of these data and to develop a more stable derivative of cinnamaldehyde that retains its chemopreventive properties, we evaluated the potential beneficial properties of 1 in terms of its ability to inhibit the genotoxicity induced by common environmental pollutants in the bacterial reverse mutation assay (Ames test).16 To this end, different mutagens (i.e., nitroarenes, aromatic amines, DNA-alkylating agents) were tested, according to the bacteria sensitivity and to the type of mutations induced. As bacterial strains, Salmonella typhimurium TA98 and TA100 and Escherichia coli WP2uvrA strains, which are sensitive to different mutational events due to their specific genotypes, were used.17 The isogenic strains TA98NR and TA98 1,8-DNP were included to more thoroughly investigate antimutagenic activity. By using three different treatment protocols, we were able to ask if the test substance acts by desmutagenic (pre- and cotreatments) and/or bioantimutagenic (post-treatment) mechanisms of antimutagenesis. Desmutagenic agents directly interfere with the mutagen in intra- or extracellular compartment, thereby preventing DNA damage; in contrast, bioantimutagens can block DNA replication and/or repair.18 Finally, in order to evaluate if the test substance interfered with CYP450-mediated bioactivation of promutagens, an exogenous mammalian metabolic activation system (S9) was included in the assay.17
Figure 1. Effect of compound 1 on the number of revertant colonies induced by 2-aminoanthracene (2AA) in Salmonella typhimurium TA98. Values are expressed as mean ± SEM (n = 6). (A) Cell survival. (B) Percentage of inhibition. Strong: inhibition >40%; moderate: inhibition between 25% and 40%; weak: inhibition 40%; moderate: inhibition between 25% and 40%; weak: inhibition 40%; moderate: inhibition between 25% and 40%; weak: inhibition 40%; moderate: inhibition between 25% and 40%; weak: inhibition 40%; moderate: inhibition between 25% and 40%; weak: inhibition 99.5%), methylmethanesulfonate (MMS; CAS 66-27-3; purity 99%), 4-nitroquinoline N-oxide (4NQO; CAS 56-57-5; purity ≥98%), 1-nitropyrene (1-NP; CAS 5522-43-0; purity 99%), and 1,8-dinitropyrene (1,8-DNP; CAS 4239765-9; purity 98%), were obtained from Sigma-Aldrich Co (St. Louis, MO, USA). Compounds 1, 2NF, 4NQO, 1-NP, 1,8-DNP, and 2AA were dissolved in DMSO, while SA and MMS were dissolved in deionized water. Rat liver postmitochondrial supernatant (S9 fraction) from rats treated with phenobarbital/β-naphthoflavone was purchased from Moltox (Molecular Toxicology, Boone, NC, USA) and prepared just before use by adding the following: phosphate buffer (0.2 M) 500 μL, deionized water 130 μL, KCl (0.33 M) 100 μL, MgCl2 (0.1 M) 80 μL, S9 fraction 100 μL, glucose-6-phosphate (0.1 M) 50 μL, and NADP (0.1 M) 40 μL. The final mixture was kept on ice during testing. When manipulating mutagens in the laboratory, the following 2668
dx.doi.org/10.1021/np500567d | J. Nat. Prod. 2014, 77, 2664−2670
Journal of Natural Products
Article
Figure 6. Effect of compound 1 on the number of revertant colonies induced by 1-nitropyrene (1NP) in Salmonella typhimurium TA98NR. Values are expressed as mean ± SEM (n = 6). (A) Cell survival. (B) Percentage of inhibition. Strong: inhibition >40%; moderate: inhibition between 25% and 40%; weak: inhibition 40%; moderate: inhibition between 25% and 40%; weak: inhibition
