Mechanisms of cytotoxicity of 2- or 2,6-di-tert-butylphenols and 2-methoxyphenols in terms of inhibition rate constant and a theoretical parameter.

To clarify the mechanism of phenol toxicity, the radical-scavenging activity of 2- or 2,6-di-tert-butyl- and 2-methoxy-substituted phenols was investigated by combining two distinct approaches: first, the induction period method for methacrylate polymerization initiated by benzoyl peroxide or 2,2'-azobisisobutyronitrile; and secondly, 1,1'-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging test. The homolytic bond dissociation enthalpy (BDE) and ionization potential (IP(koopman)) were calculated by the DFT/B3LYP method. The cytotoxicity was investigated using tumor cells (human submandibular gland carcinoma cells, HSG; human promyelocytic leukemia cells, HL-60) and primary cells (human gingival fibroblasts, HGF; human periodontal ligament fibroblasts, HPLF; human pulp fibroblasts, HPF) derived from oral tissues. The cytotoxicity between tumor and primary cells was similar, except for eugenol dimer showing less toxicity for primary cells. The DPPH assay was not useful for tert-butylphenols due to their steric hindrance. For both HSG and HGF cells, a linear relationship was found between 50% cytotoxic concentration (CC(50)) and inhibition rate constant (k(inh)), but not BDE, IP, or logP. The acceptable quantitative structure-activity relationships (QSAR) obtained for cytotoxicity vs. k(inh) suggested that the cytotoxicity of phenols may be dependent on radical reactions. The cytotoxicity of vanillin and 3,5-di-tert-butyl-4-hydroxy-benzaldehyde with large k(inh) values, weak antioxidants was markedly less than that of 2,6-di-tert-butyl-4-methylphenol, 2,4,6-tri-tert-butylphenol and curcumin with small k(inh) values, potent antioxidants.

Radical-scavenging activity of natural methoxyphenols vs. synthetic ones using the induction period method.

The radical-scavenging activities of the synthetic antioxidants 2-allyl-4-X-phenol (X = NO2, Cl, Br, OCH3, COCH3, CH3, t-(CH3)3, C6H5) and 2,4-dimethoxyphenol, and the natural antioxidants eugenol and isoeugenol, were investigated using differential scanning calorimetry (DSC) by measuring their anti-1,1-diphenyl-2-picrylhydrazyl (DPPH) radical activity and the induction period for polymerization of methyl methacrylate (MMA) initiated by thermal decomposition of 2,2'-azobisisobutyronitrile (AIBN) and benzoyl peroxide (BPO). 2-Allyl-4-methoxyphenol and 2,4-dimethoxy-phenol scavenged not only oxygen-centered radicals (PhCOO*) derived from BPO, but also carbon-centered radicals (R*) derived from the AIBN and DPPH radical much more efficiently, in comparison with eugenol and isoeugenol. 2-Allyl-4-methoxyphenol may be useful for its lower prooxidative activity.

Predicting the biological activities of 2-methoxyphenol antioxidants: effects of dimers.

UNLABELLED: Selective cyclooxygenase (COX)-2 inhibitors have attracted much attention in relation to the design of non-steroidal anti-inflammatory agents (NSAIDs). The relationship between experimentally derived data on the antioxidant capacity, cytotoxicity and COX-2 inhibition for a range of 2-methoxyphenols and their calculated descriptors was investigated. MATERIALS AND METHODS: Quantitative structure-activity relationship (QSAR) studies were performed on a series of 2-methoxyphenols that act as COX-2 inhibitors using electronic descriptors, such as the highest occupied molecular orbital (HOMO), the lowest unoccupied molecular orbital (LUMO), ionization potential (IP), chemical hardness (q), and electronegativity (chi), which were calculated by the CONFLEXIPM3 method. The antioxidant capacity of a range of 2-methoxyphenols was evaluated by 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity, and the anti-peroxy radical activity (stoichiometric factor, n) was determined by the induction period method in the polymerization of methyl methacrylate (MMA) initiated by thermal decomposition of benzoyl peroxide (BPO). The 50% cytotoxic concentration (CC50) against human submandibular gland tumor cell line (HSG) was determined by the MTT method. RESULTS: Cytotoxicity declined in the order of curcumin > dehydrodiisoeugenol > isoeugenol >bis-MMP > eugenol > ferulic acid > 2-methoxy-4-methylphenol (MMP) > bis-eugenol > bis-ferulic acid. The inhibitory effects on LPS-induced COX-2 gene expression in RAW 264.7 cells were determined by Northern blot assay. The majority of 2-methoxyphenols studied were COX-2 inhibitors. In particular, dehydrodiisoeugenol was a potent inhibitor, followed by bis-ferulic acid and curcumin. A linear relationship between anti-DPPH radical activity (log 1/IC50) and IP for 2-methoxyphenols except for dehydrodiisoeugenol was observed (r2=0.768.) The n for methoxyphenols was less than 2 in most cases. A linear relationship (r(2)=0.713) between the log (1/CC50) and the r1-term except for ferulic acid was observed. COX-2 inhibition, except for hesperetin, was related to the chi-term (r(2)=0.685). CONCLUSION: It may be possible to predict the mechanism responsible for the biological activities of 2-methoxyphenols.

Comparative cytotoxicity and ROS generation by curcumin and tetrahydrocurcumin following visible-light irradiation or treatment with horseradish peroxidase.

In order to clarify the cytotoxic mechanism of curcumin, a well-known chemopreventive agent, the cytotoxicity (by MTT method), intracellular glutathione (using GSH detection kit) and intracellular reactive oxygen species (ROS) levels (with a flow cytometer), were measured in curcumin- and tetrahydrocurcumin (TH-curcumin)-treated cancer (HSG) and normal (HGF) cells under two different oxidation conditions: irradiation with visible light (VL) and enzymatic oxidation with horseradish peroxidase (HRP)/H2O2. The cytotoxicity of curcumin was highly enhanced by VL-irradiation, whereas that of TH-curcumin was enhanced by HRP/H2O2 treatment. The cytotoxicity of curcumin against HGF cells was greater than that against HSG cells. Curcumin significantly reduced the intracellular GSH level significantly under VL-irradiation, and increased it under HRP/H2O2, whereas TH-curcumin had no effect with either oxidation treatment. HRP/H2O2 treatment of TH-curcumin enhanced generation of ROS; in contrast, VL-irradiation of curcumin was considered to produce ROS preferably. In conclusion, curcumin was highly photo-toxic, caused a decrease in GSH and mediated ROS generation. In contrast, the cytotoxicity of TH-curcumin was enhanced by enzymatic oxidation. A low-level pro-oxidant intracellular milieu induced by TH-curcumin could be effectively useful for cancer prevention.

Smelling lavender and rosemary increases free radical scavenging activity and decreases cortisol level in saliva.

Free radicals/reactive oxygen species are related to many biological phenomena such as inflammation, aging, and carcinogenesis. The body possesses various antioxidative systems (free radical scavenging activity, FRSA) for preventing oxidative stress, and saliva contains such activity. In the present study, we measured the total salivary FRSA induced after the smelling of lavender and rosemary essential oils that are widely used in aromatherapy. Various physiologically active substances in saliva such as cortisol, secretory IgA, and alpha-amylase activity were found to be correlated with aroma-induced FRSA. The subjects (22 healthy volunteers) sniffed aroma for 5 min, and each subject's saliva was collected immediately. FRSA was measured using 1,1-diphenyl-2-picrylhydrazyl. The FRSA values were increased by stimulation with low concentrations (1000 times dilution) of lavender or by high-concentrations (10 times dilution) of rosemary. In contrast, both lavender and rosemary stimulations decreased cortisol levels. A significant inverse correlation was observed between the FRSA values and the cortisol levels with each concentration of rosemary stimulation. No significant changes were noted in sIgA or alpha-amylase. These findings clarify that lavender and rosemary enhance FRSA and decrease the stress hormone, cortisol, which protects the body from oxidative stress.

A quantitative approach to the free radical interaction between alpha-tocopherol or ascorbate and flavonoids.

Despite numerous previous studies, the mechanism of the free radical interaction between alpha-tocopherol (VE), or ascorbate and flavonoids, as coantioxidants remains unclear. The synergistic antioxidant effects of VE or L-ascorbyl 2,6-dibutyrate (ASDB, an ascorbate derivative) with the flavonoids (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG) and methyl gallate (MG), were investigated by the induction period method in the polymerization of methyl methacrylate (MMA), initiated by thermal decomposition of 2,2'-azobis(isobutyronitrile) (an alkyl radical, R *), under nearly anaerobic conditions. For VE, a synergistic antioxidant effect was observed with MG, EC, EGC and ECG, whereas this activity was decreased by the addition of EGCG. For ASDB, a synergistic antioxidant effect was observed with EGC and ECG, whereas this activity was decreased by the addition of EGCG or MG. A synergistic antioxidant effect (regeneration of VE) appears to be feasible even though the BDE (phenolic O-H bond dissociation entropy) of the coantioxidants is significantly higher than that of VE. The driving force for the regeneration process may be the removal of the semiquinone radical from the flavonoids MG, EC, EGC and ECG by the VE radical. In the ASDB/flavonoid mixture, flavonoid radicals are scavenged by ASDB. The partial regeneration of flavonoids by ASDB may follow a similar recycling mechanism to that of the well-known VE/ascorbate mixture. The free radical interaction between EGCG and VE or ASDB decreased the antioxidant effect. Such enhancement of prooxidation in EGCG/VE or EGCG/ASDB mixtures oxidized by R * may increase their cytotoxic effects.

(Meth)acrylate monomer-induced cytotoxicity and intracellular Ca(2+) mobilization in human salivary gland carcinoma cells and human gingival fibroblast cells related to monomer hydrophobicity.

To elucidate a possible link between the cytotoxicity and Ca(2+) mobilization by (meth)acrylates, we investigated the cell survival of and change in [Ca(2+)](i) in human salivary gland (HSG) cells (salivary gland carcinoma cell line) and human gingival fibroblasts (HGF) cells treated separately with 9 (meth)acrylate monomers used in dentistry. The cell survival was determined by the MTT method, and the [Ca(2+)](i) changes after the stimulation with the (meth)acrylate monomers were measured in floating indo-1/AM-loaded cells in Ca(2+)-free medium. For both HSG and HGF cells, the cytotoxicity of the monomers was approximately proportional to their hydrophobicity (logP). No increase in [Ca(2+)](i) was found with hydrophilic monomers, even with 10mm stimulation. [Ca(2+)](i) elevation by hydrophobic monomers occurred in a dose- and hydrophobic-dependent manner. The [Ca(2+)](i) change in HSG cells appeared as twin peaks, i.e., an initial sharp peak followed by a delayed broad one; whereas with the HGF cells only a single broad peak was seen, possibly dependent on their membrane quality. Pretreatment with n-butanol or methylmethacrylate enhanced the butylmethacrylate-induced [Ca(2+)](i) elevation, suggesting the [Ca(2+)](i) elevation by (meth)acrylate may be related to monomer hydrophobicity and cell type. The causal link between the cytotoxicity and [Ca(2+)](i) mobilization of monomers is discussed.

Induction of early apoptosis and ROS-generation activity in human gingival fibroblasts (HGF) and human submandibular gland carcinoma (HSG) cells treated with curcumin.

OBJECTIVE: Curcumin [1] is well known to possess apoptosis-inducing activity in some cancer cells, but little is known about its activity in normal cells of oral origin, such as HGF. The aim of the present study was to clarify the relationship between early apoptosis in HGF and the induction of reactive oxygen species (ROS) generation by curcumin. DESIGN: We treated HGF and HSG cells with curcumin [1] and the related compounds biseugenol [2], eugenol [3], alpha-diisoeugenol [4], and isoeugenol [5] and measured cell survival (MTT method), ROS generation (DCFH-DA staining), and induction of early apoptosis. Early apoptosis was detected by monitoring loss of mitochondrial membrane potential (DeltaPsi(m)) by JC-1 staining and externalization of phosphatidylserine (PS) on the cell surface by annexin V-FITC/PI staining combined with flow cytometry. RESULTS: The cytotoxic activities of curcumin [1] and [4] were similar and were nearly 10- to 100-fold stronger than those of the other compounds. Only curcumin was able to induce ROS generation and early apoptosis. Loss of DeltaPsi(m), PS externalization and ROS generation were significantly more pronounced in HGF cells than in HSG cells at curcumin concentrations lower than about 15microM, and were inhibited by the addition of the antioxidants N-acetyl-l-cysteine and glutathione. CONCLUSION: The potent PS externalization and loss of DeltaPsi(m) in curcumin-treated HGF cells appears to be mediated by ROS generation.

Induction of cytotoxicity and apoptosis and inhibition of cyclooxygenase-2 gene expression, by curcumin and its analog, alpha-diisoeugenol.

Cytotoxici and alpha-diisoeugenol were investigated. The cytotoxicity of curcumin and a-diisoeugenol against human promyelocytic leukemia cells (HL-60 cells) and human submandibular cancer cells (HSG cells) was similar (CC50 1-3 microM). However, curcumin induced much more apoptosis, particularly in HL-60 cells compared with HSG cells, as revealed by measurement of the sub-G1/G0 DNA fraction in flow cytometric histograms. Treatment with 15 microM curcumin increased the number of cells with a sub-G1/G0 DNA fraction from control levels of <5% to 55% in HL-60 cells and 30% in HSG cells. Flow cytometry, after staining with annexin V-FITC/PI (the exposure of phosphatidylserine (PS) on the surface of apoptotic cells), showed a dose-dependent induction of early apoptosis by curcumin, which reached about 65% in HL-60 cells and about 20% in HSG cells after treatment with 10 microM curcumin. In contrast, alpha-diisoeugenol failed to induce apoptosis in either cell type. For both cell types, the proportion of late apoptotic/necrotic cells increased rapidly at concentrations of curcumin and a-diisoeugenol greater than 10 microM. The generation of intracellular reactive oxygen species (ROS) in curcumin-treated HL-60 cells was greater than that in HSG cells, as judged by CDFH-DA staining. In both cell types, ROS generation by a-diisoeugenol was at control levels. ROS generation by curcumin was suppressed by antioxidants such as N-acetyl-L-cysteine (NAC) and glutathione (GSH) and by scavengers of hydroxy radicals such as mannitol, but, conversely, was promoted by prooxidants such as the transition metal ions Cu(II) and Zn(II). ROS generation may play a part in the exposure of PS. Curcumin, but not a-diisoeugenol, at 10 microM inhibited LPS (lipopolysaccharide)-induced COX-2 gene expression in RAW 264.7 cells. Semiempirical PM 3 calculations suggested that this activity of curcumin, in which it behaves as a non-steroidal anti-inflammatory drug (NSAID)-like compound, is dependent on its phenolic function, which is more pronounced than that of alpha-diisoeugenol. Taken together, our results suggest that the bioactivity of curcumin is a result of its ability to act as both a prooxidant and an antioxidant.

Theoretical prediction of the relationship between phenol function and COX-2/AP-1 inhibition for ferulic acid-related compounds.

Ferulic acid-related compounds possess antioxidant activity. Dehydrodiisoeugenol and ferulic acid dimer (bis-FA), but not the parent monomers isoeugenol and ferulic acid, inhibit lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2) gene expression in RAW 264.7 cells. To clarify the mechanism of their inhibitory effects on COX-2 expression, the phenolic O-H bond dissociation enthalpy (BDE) and ionization potential (IP) of 8 ferulic acid-related compounds were calculated by both semi-empirical molecular orbital (AM1, PM3) and ab initio (3-21G* 6-31G*) and density function theory (DFT) (B3LYP) methods. COX-2 inhibition appeared in compounds with phenolic O-H BDE higher than 85.76 kcal/mol, as calculated by the density function theory (DFT) approach. The phenolic O-H BDEs of the most potent compounds, dehydrodiisoeugenol and bis-FA, were 85.99 and 85.76 kcal/mol, respectively. No causal relationship between COX-2 inhibition and IP was found. Neither dehydrodiisoeugenol nor bis-FA possessed significant scavenging activity against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The NSAID-like activity of dehydrodiisoeugenol and bis-FA appears to be related to their phenol function. Binding of activator protein-1 (AP-1) to the 12-tetradecanoylphorbol-13-acetate-responsive element (TRE) sequence in LPS-stimulated cells was inhibited by bis-FA at 1 microM and dehydrodiisoeugenol at 0.1 microM, but not by the parent monomers isoeugenol and ferulic acid.

Ferulic acid dimer inhibits lipopolysaccharide-stimulated cyclooxygenase-2 expression in macrophages.

Phenylpropanoids may act as nonsteroidal anti-inflammatory drug (NSAID)-like compounds. 4-cis, 8-cis-Bis (4-hydroxy-3-methoxyphenyl)-3, 7-dioxabicyclo-[3.3.0]octane-2,6-dione (bis-FA, compound 2), a dimer of ferulic acid, was synthesized from ferulic acid (1), and its effect on lipopolysaccharide (LPS)-stimulated cyclooxygenase-2 (COX-2) expression in RAW 264.7 cells was compared with those of the parent ferulic acid (1) and of iso-ferulic acid (3-hydroxy-4-methoxycinnamic acid) (3). LPS-induced gene expression of COX-2 was markedly inhibited by compound 2 at a concentration of 10 microM and by compound 3 at 100 microM, but was not inhibited by compound 1 at 100 microM. This observation suggests that compound 2 may possess potent anti-inflammatory activity. These ferulic acid-related compounds were able to scavenge the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical. The 50% inhibitory concentration for DPPH radicals declined in the order 3 (40.20 mM) > 2 (3.16 mM) > 1 (0.145 mM). Compound 1 possessed potent anti-radical activity, but no COX-2 inhibitory activity, which may be a result of enhancement of its conjugate properties by abstraction of an H atom from the phenolic OH group, causing loss of phenolic function. In contrast, inhibition of COX-2 expression by compounds 2 and 3 could be caused by their increased phenolic function, which is associated with decreased anti-radical activity. Compounds 2 and 3, particularly 2, may have potential as NSAID-like compounds.

Dimerization, ROS formation, and biological activity of o-methoxyphenols.

o-Methoxyphenols are antioxidants widely used in the cosmetic and food industries. Dimers from 1, 2, or 3 were synthesized and their radical-scavenging and biological activities were compared with those of the original or other phenols. Radical-scavenging was evaluated from a kinetic induction period method (IPM). To simulate biomimetic thiolcooxidation with antioxidants, the behavior of mixtures of 1, 2, 3, 4, or catechin with mercaptomethylimidazole (MMI), a thiol was investigated using IPM. Polyphenols 4 and catechin was accompanied by extensive oxygen uptake, suggesting the formation of thiyl radicals from MMI and their reaction with molecular oxygen. In contrast,1 markedly enhanced radical-scavenging without oxygen uptake, probably because of the formation of EUGQM/MMI-conjugates. 2 showed relatively small oxygen uptake, probably resulting from the predominant formation of benzyl radicals. Intracellular reactive oxygen species (ROS) in cancer cells by 4 ,but not by compounds 1, 2, 6, 7, 8, 9, and 10 was found, suggesting a possible link between physicochemical oxygen-uptake and intracellular ROS. The induction of apoptosis by 4 in HL-60 cells was accompanied by intracellular ROS. Dimers 6 and 7 inhibited nuclear factor (NF)-kappaB, activation stimulated by lipopolysaccharide (LPS) in RAW 264.7 cells. Also,6 ,7,and 9 inhibited LPS-induced cyclooxygenase-2 expression in RAW 264.7 cells in a dose-dependent manner, whereas 1, 2, and 3 did not. Dimerization of o-methoxyphenols may be a useful tool for the design of drugs to act as potent chemopreventive and anticancer agents.

Kinetic radical scavenging activity and cytotoxicity of 2-methoxy- and 2-t-butyl-substituted phenols and their dimers.

The dimers bis-EUG, bis-MMP, bis-BHA, bis-BMP and bis-DBP were synthesized from the monomers 4-allyl-2-methoxyphenol (EUG), 2-methoxy-4-methylphenol (MMP), 2-t-butyl-4-methoxyphenol (BHA), 2-t-butyl-4-methylphenol (BMP) and 2,4-di-t-butylphenol (DBP), respectively. The stoichiometric factors (n; number of free radicals trapped by one mole of phenolic moiety) of these compounds were determined by induction period methods with a kinetic approach in the 2'2-azobisisobutyronitrile (AIBN) and benzoyl peroxide (BPO) systems at 70 degrees C. The n values for bis-EUG, bis-MMP and bis-BHA were approximately two-fold greater than those for their monomers in both the AIBN and BPO systems, whereas the n values for bis-BMP and bis-DBP were identical to those of their monomers. bis-EUG, bis-MMP and bis-BHA, containing methoxy groups, were potent antioxidants. The n values (1.3-1.6) for EUG and MMP were considerably less than 2, as is commonly observed for the stoichiometric factors of phenolic compounds. The antiradical efficiencies against DPPH (diphenylpicrylhydrazyl) of the monomers and their dimers were also investigated, likewise indicating that bis-EUG, bis-MMP and bis-BHA were potent antioxidants. DBP and bis-DBP were less effective radical scavengers because of the steric factor of their bulky t-butyl groups. On the basis of cytotoxic activity against a human submandibular gland carcinoma cell line (HSG) and human gingival fibroblasts (HGF), these compounds could be classified into a high-activity group (DBP, bis-DBP and bis-BMP, with butylated hydroxytoluene (BHT) as a positive control) and a low-activity group (MMP, EUG, BHA, BMP, bis-BHA and bis-EUG). The cytotoxicity of EUG and BHA was markedly reduced by dimerization, whereas that of MMP was enhanced. The sensitivity index (ratio of 50% cytotoxic concentration for HGF cells to that for HSG cells) of EUG, MMP, bis-MMP and bis-BHA was approximately 9, 5, 7 and 2, respectively, whereas that of the other compounds was approximately 1. Potential mechanisms of cytotoxicity were assessed by PM3 semiempirical molecular orbital (MO) calculations. Tumor cells were highly sensitive to 2-methoxy-4-alkylphenols such as EUG and MMP, possibly due to the formation of cytotoxic quinone methide intermediates. In contrast, the high sensitivity index of bis-MMP may be related to the production of a highly reactive substance, CH3+, via oxidation. Structure-activity relationship (SAR ) models using PM3 calculations may be useful to predict biological activity during the development of potential anticancer drugs.

Dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol liposomes as predictors of the cytotoxicity of bis-GMA related compounds.

In light of recent development, dental materials such as 2, 2-bis [4-2(-hydroxy-3-methacryloyloxypropoxy)phenyl] propane, ( bis-GMA); 2, 2-bis [4-(1-hydroxymethyl-2-methacryloxy)phenyl] propane, (iso-bis-GMA); and triethyleneglycol dimethacrylate, (TEGDMA) were investigated to determine whether their phase transition properties (phase transition temperature, temperature width, cooperativity) could be induced in samples of DPPC or DPPC/cholesterol (CHOL) liposomes using differential scanning calorimetry (DSC). The changes in phase transition properties of DPPC liposomes caused by addition of TEGDMA were greater than those caused by addition of bis-GMA or iso-bis-GMA, but the extent of changes in the properties of DPPC/CHOL (10:1 or 4:1) liposomes declined in the order of bis-GMA > iso-bis-GMA > TEGDMA. The degree of alteration was related to the cytotoxicity of these compounds. DPPC/CHOL liposomes were found to be better predictors of cytotoxicity than DPPC liposomes. Whether the computational approach to studying the molecular mechanism of alteration is applicable using descriptors such as reactivity of energy of the highest occupied molecular orbital (HOMO) and/or lowest unoccupied molecular orbital (LUMO) was investigated, and the data suggested that these descriptors are useful for studying the interactive roles of dental materials.

Cytotoxicities of a 4-META/MMA-TBBO resin against human pulp fibroblasts.

This study was designed to investigate the cytotoxicity of MMA (methyl methacrylate) and 4-META (4-methacryloyloxyethoxycarbonylphthalic anhydride)/MMA with or without TBBO (tri-n-butylborane partially oxide), an initiator and TBBO alone against human pulp fibroblasts (HPF). Cell viabilities were measured by MTT assay. The cytotoxicity of 4-META/MMA-TBBO was comparable with that of MMA-TBBO. TBBO showed higher cytotoxicity than 4-META/MMA. The cytotoxicity induction of a 4-META/MMA-TBBO resin may be preferably associated with TBBO.

Cytotoxicity, ROS-generation activity and radical-scavenging activity of curcumin and related compounds.

The cytotoxicity, ROS (reactive oxygen species)-generation activity and radical-scavenging activity of curcumin and related compounds such as eugenol, eugenol orthodimer (bis-eugenol; 3,3'-dimethoxy-5,5'-di-2-propenyl-1,1'-biphenyl-2,2'-diol) and isoeugenol were investigated. Their cytotoxicity against a human submandibular gland adenocarcinoma cell line (HSG) declined in the order curcumin > isoeugenol > bis-eugenol > eugenol. Since the hydrophobicity (log P) of curcumin, isoeugenol and eugenol is about 2.5, whereas that of bis-eugenol is 4.8, there was no relationship between cytotoxicity and log P. Generation of intracellular ROS in HSG cells was observed for curcumin alone in an assay using 5- (and -6)-carboxy-2',7'-dichlorofluorescein diacetate (CDFH-DA). The cytotoxicity of, and ROS generation by, curcumin were reduced by the addition of N-acetyl-L-cysteine (NAC) and glutathione, suggesting a possible link between cytotoxicity and ROS. The radical-scavenging (antioxidant) activity of curcumin and related compounds was determined quantitatively by the induction period method for polymerization of methyl methacrylate (MMA) initiated by peroxy radicals derived from benzoyl peroxide (BPO) under nearly anaerobic conditions. The length of the induction (inhibition) period for curcumin was significantly greater than that of the other compounds. This suggests that curcumin is an efficient scavenger of peroxy radicals. The curcumin radical possibly reacts with itself or with other radicals to yield polymeric stable products such as curcumin dimer. Such polyphenolic behavior of curcumin was considerably different from that of bis-eugenol, which, like curcumin, has two hydroxy groups, or of other compounds with one hydroxy group. The radical-scavenging activity was also investigated with 2,2-diphenyl-1-picrylhydrazyl (DPPH). Curcumin scavenged approximately one DPPH free radical, suggesting the formation of curcumin dimer. The possible formation of curcumin dimer was explored with a PM3 semiempirical molecular orbital method. A molecular mechanism of cancer prevention by curcumin is proposed, based on its high reactivity with peroxy radicals at low oxygen pressure and on ROS generation induced by curcumin radicals.

Antioxidant and prooxidant action of eugenol-related compounds and their cytotoxicity.

To clarify the possible link between radicals and the cytotoxicity of eugenol-related compounds, 2-allyl-4-X-phenols (2-allyl-4-chlorophenol (1), 2-allyl-4-phenylphenol (2), 2-allyl-4-methoxyphenol (3), 2-allyl-4-acetylphenol (4), 2-allyl-4-nitrophenol (5), 2-allyl-4-t-butylphenol (6), 2-allyl-4-methyphenol (7), 2-allyl-4-bromophenol (8), 2,4-dimethoxyphenol (9)), and dimeric compounds from eugenol (4-allyl-2-methoxyphenol), BHA (2-t-butyl-4-methoxyphenol) or MMP (2-methoxy-4-methylphenol); bis-EUG (3,3'-dimethoxy-5,5'-di-2-propenyl-1, 1'-biphenyl-2,2'-diol) (10), bis-MMP (3,3'-dimethoxy-5,5'-dimethyl-1,1'-biphenyl-2,2'-diol) (11) bis-BHA (3,3'-di-t-butyl-5,5'-dimethoxy-1,1'-biphenyl-2,2'-diol) (12) were synthesized. The radical production, radical-scavenging activity and the cytotoxicity of these synthetic compounds and conventional antioxidants (i.e. butylhydroxytoluine, BHT; butylhydroxyanisole, BHA; alpha-tocopherol (alpha-Toc); eugenol, phenol) were studied. Erectron spin resonance (ESR) spectroscopy suggested that compounds of 3, 6, 9, eugenol and BHA, but not compounds of 10, 11, and 12 produced radicals in alkaline solutions (pH>9.5) and compounds, 3, eugenol and 9 most efficiently scavenged reactive oxygen species (ROS, O(2)(-)). The cytotoxic activity of 6 toward human submandibular gland carcinoma (HSG) cells was the highest and was 1000-fold greater than that of eugenol and 100-fold greater than that of BHA, possibly due to the high hydrophobicity and stable phenoxy radicals of this compound. The kinetic polymerization method in the presence of methyl methacrylate (MMA), an antioxidant, and 2,2'-azobisisobutyronitrile (AIBN) was developed for the measurements of the number of moles of peroxy radicals trapped by moles of the relative phenols (stoichiometric factors, n), the inhibition rate of polymerization (R(inh)), and the inhibition rate constants (k(inh), the rate constants for scavenging of radicals by an antioxidant). The n values of conventional phenolic antioxidants decreased in the order: alpha-Toc>BHT>eugenol>phenol. Those for eugenol and phenol, less hindered phenols, were much less than two, whereas those for alpha-Toc and BHT, hindered phenols, were approximately two. The R(inh) of alpha-Toc significantly increased tcompared with that of BHT, eugenol and phenol. The k(inh) of the polymer radicals of the MMA reaction with conventional phenolic antioxidants was a low value of 1-2x10(2) M(-1) s(-1), suggesting that the antioxidants trapped radicals quickly. The comparative cytotoxicity of methoxyphenols against HSG cells was investigated. The cytotoxic activity of dimers of 10 and 12 was markedly lower than that of their corresponding monomers, whereas that of the dimer of MMP, 11 was not reduced even after the dimerization. In particular, visible-light (VL) exposure enhanced the cytotoxicity of 11 similar to the monomers of eugenol, BHA and MMP. Changes in BDE (ph(O-H)) (homolytic bond dissociation energy) for phenols is well known to be associated with the n and k(inh) values, and consequently the cytotoxic activity. Thus, the BDE was calculated using a PM3 semiempirical method. The n and k(inh) values for monophenols, but not for dimers were correlated to the BDE, possibly due to the steric hindrance of orthosubstituents of dimers. The quantitative structure-activity relationship (QSAR) of eugenol-related compounds was investigated, indicating that logP (octanol-water partition coefficients), the redox potential measured in culture medium, was effective as a term for QSAR. A parabolic relation between the cytotoxic activity and the logP or the redox potential, but not the BDE was observed with an optimum value. In conclusion, the cytotocity of eugenol-related compounds was significantly associated with the activity of the production of phenoxyl radicals, their stability of the subsequent quinonemethide (QM) and the hydrophobicity.

Radical scavenging activity and cytotoxicity of ferulic acid.

Ferulic acid and eugenol were examined for their superoxide (O2-), hydroxyl radical (.OH) and nitric oxide (NO)-scavenging ability, using ESR spectroscopy with spin trap agents DMPO and carboxy-PTIO/NOC-7. Ferulic acid more efficiently scavenged .OH and NO than eugenol. The O2- scavenging activity of ferulic acid was comparable with that of eugenol. Ferulic acid significantly reduced the NO production by lipopolysaccharide (LPS)-stimulated mouse macrophage-like cells (Raw 264.7 cells) compared to eugenol. The cytotoxic activity of ferulic acid against Raw 264.7 cells was comparable with that against human submandibular gland carcinoma (HSG) cells and the cytotoxicity of ferulic acid was about 10-fold smaller than that of eugenol. The stoichiometric factor (n) (number of moles of peroxy radical trapped by moles of the relevant phenol) of ferulic acid and eugenol was investigated, using the induction period methods of the methyl methacrylate polymerization system. The n-value of ferulic acid (1.5) was higher than that of eugenol (1.0) and was similar to that of 2, 6-di-t-butyl-4-methylphenol (BHT). Ferulic acid as well as eugenol may produce a dimer during the induction period due to an n-value less than 2. These results suggested that ferulic acid may be useful for preventing cell damage perhaps caused by O2-, and in particular by .OH and NO, in living systems.