Relationship between phenol-induced cytotoxicity and experimental inhibition rate constant or a theoretical parameter.

We synthesized various dimer forms of 2-methoxyphenols and 2-tert-butylphenols, as dimers such as curcumin exhibit potent antioxidant and anti-inflammatory activity. We investigated the QSARs between the cytotoxicity and independent variables; kinetic parameters (inhibition rate constant (kinh/kp), stoichiometric factor (n)) or DFT-based theoretical parameters (i.e. phenolic O-H bond dissociation enthalpy (BDE), ionization potential according to Koopman's theorem (IP), LUMO, absolute hardness (η), electronegativity (χ) and electrophilicity (ω)) for 2-methoxyphenols and 2- tert- or 2,6-di-tert-butylphenols. The cytotoxicity of these phenols against human tumor cells (HSG, HL60) and/or human gingival fibroblasts (HGF) showed a marked negative linear relationship to kinh/kp, suggesting that the cytotoxicity of phenols may be related to radical reactions. By contrast, a linear relationship between the cytotoxicity and η-term was demonstrated; 2-methoxyphenols showed a negative slope, whereas 2-tert- or 2,6-di-tert-butylphenols showed a positive slope. Also, the cytotoxicity of tert-butylphenols was linearly dependent on the LUMO-term, showing a positive slope. The cytotoxicity of methoxy-substituted monophenols toward both HSG and HGF cells was related to both log P and η- terms. Also, that of X-phenols toward murine L-1210 cells was related to both log P and η or IP-terms, determined from a dataset reported by Zhang et al., 1998. It was concluded that the phenol-induced cytotoxicity was attributable to radical reactions resulting from the terms (kinh/kp, IP, η, and LUMO) in QSAR. The LUMO-dependent cytotoxicity of 2-tert- or 2,6-di-tert-butylphenols may be related to their quinone oxidation products. Experimental and theoretical parameters provide a useful approach for analysis of the cytotoxicity for phenolic compounds.

Comparative radical production and cytotoxicity induced by camphorquinone and 9-fluorenone against human pulp fibroblasts.

Camphorquinone (CQ) is widely used as a photo-initiator in dental materials; however, its cytotoxicity against human pulp fibroblasts (HPF) and particularly the effects of 2-dimethylaminoethyl methacrylate (DMA), a reducing agent and visible light (VL) irradiation on it remain unknown. So we investigated the cytotoxic and reactive oxygen species (ROS)-producing effects of CQ with or without DMA, in the presence or absence of VL on HPF cells. The free-radical production activity of CQ was measured by two different methods [using diphenylpicryl hydrazyl and galvinoxyl]. The phase-transition properties of dipalmitoylphosphatidyl choline (DPPC) liposomes, as a model for biomembranes, induced by CQ were investigated by differential scanning calorimetry. These findings were compared with those of 9-fluorenone (9F), an aromatic photo-initiator with long conjugated groups. Camphorquinone with VL irradiation increased the radical production, whereas 9F with VL irradiation increased ROS production, as well as effecting changes in the DPPC phase-transition properties. The cytotoxicity of CQ towards HPF cells was smaller than that of 9F despite greater radical production. The addition of DMA to the photosensitizer enhanced the free-radical production without increasing the ROS level or the cytotoxicity. Camphorquinone/DMA is a valuable combination for the polymerization of dental resins.

Kinetics of the radical scavenging activity of beta-carotene-related compounds.

To clarify the non-enzymatic radical-scavenging activity of beta-carotene-related compounds and other polyenes, we used differential scanning calorimetry to study the kinetics of radical polymerization of methyl methacrylate (MMA) by 2,2'-azobisisobutyronitrile (AIBN) or benzoyl peroxide (BPO) in the absence or presence of polyenes under nearly anaerobic conditions at 70 degrees C, and analyzed the results with an SAR approach. The polyenes studied were all-trans retinol, retinol palmitate, calciferol, beta-carotene and lycopene. Polyenes produced a small induction period. The stoichiometric factor (n) (i.e. the number of radicals trapped by each inhibitor molecule) of polyenes was close to 0. Tetraterpenes (beta-carotene, lycopene) suppressed significantly more of the initial rate of polymerization (R(inh)) than did diterpenes (retinol, retinol palmitate). The inhibition rate constants (k(inh)) for the reaction of beta-carotene with AIBN- or BPO-derived radicals were determined to be 1.2-1.6x10(5) l/mol s, similar to published values. A linear relationship between (k(inh)) and the kinetic chain length (KCL) for polyenes was observed; as (k(inh)) increased, KCL decreased. KCL also decreased significantly as the number of conjugated double bonds in the polyenes increased. Polyenes, particularly beta-carotene and lycopene, acted as interceptors of growing poly-MMA radicals.

Kinetic evaluation of the reactivity of flavonoids as radical scavengers.

The reactivity of flavonoids as radical scavengers was investigated under kinetic considerations using radical polymerization of methyl methacrylates initiated by benzoyl peroxide. The number of radicals which are trapped by each molecule of phenol (the stoichiometric factors, n values) decreased in the order of epigallocatechin-3-O-gallate (ECG) (5.5) > catechin (3.5) > resveratrol (2.4) > quercetin (1.9) > n-propylgallate (1.5) > hesperetin (1.0). The inhibition rate constants (k(inh)) (1-3 x 10(3) 1/(mol s)) for the flavonoids were not different from each other, and, therefore, the radical scavenging activity depend on n values. The n values of the fully oxidized flavonoids were estimated from the frontier orbital theory, using PM3 semiempirical molecular orbital calculation. The experimental n values were consistent with the calculated values.

Cytotoxicity and phospholipid-liposome phase-transition properties of 2-hydroxyethyl methacrylate (HEMA).

To elucidate the cytotoxic induction mechanisms of the hydrophilic HEMA, the comparative cytotoxic activities of HEMA and the hydrophobic monomers TEGDMA and MMA were studied, using erythrocytes, gingival fibroblasts and a salivary gland carcinoma cell line. Also, the gel-to-fluid phase transition properties (i.e. temperature, Tm; cooperativity, H/HHW; enthalpy, deltaH) of dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol (CS) liposomes (as a model for biological membranes) induced by methacrylates were investigated, using differential scanning calorimetry (DSC). In addition, the methacrylate-chemical-shifts in DPPC liposomes were assayed using NMR spectroscopy. Both the hemo lytic and cytotoxic activity declined in the order: TEGDMA> HEMA>MMA. The changes in Tm increased in the order: HEMA >TEGDMA, while in contrast, that of HEMA was slightly increased without changes in the deltaH. The DSC changes in DPPC/CS liposomes with HEMA were the largest of those recorded. The cytotoxicity of HEMA may be induced by the hydrophobic interaction derived from the molecular association of OH groups of HEMA and, in addition, by the preferential interaction with CS.

Kinetic evaluation of reactivity of bisphenol A derivatives as radical scavengers for methacrylate polymerization.

The reactivity of bisphenol A (BPA), diethylstilbestrol (DEST) 2,2'-biphenol (22'BP), 4,4'-biphenol (44'BP) and hydroquinone (HQ) as radical scavengers was examined in 2,2'-azobisisobutyronitrile (AIBN)- and benzoyl peroxide (BPO)-induced methyl methacrylate (MMA) polymerization with respect to kinetic considerations. The initial rate of polymerization (IRP) was found to decrease in the order: 44'BP > BPA, DEST > 22'BP >> HQ, while the stoichiometric factor (n) of free radicals trapped by phenolic moiety decreased in the order: 44'BP (2.3) > HQ (2.0) > BPA, DEST (1.8) >> 22'BP (0.8). It was found that BPA was a more highly efficient inhibitor than HQ and that HQ acts as a retarder at higher concentrations in the BPO system. The high activity of BPA indicated that BPA is probably oxidized by a radical interaction in the dental resin system.

Cytotoxicity of methyl methacrylate (MMA) and related compounds and their interaction with dipalmitoylphosphatidylcholine (DPPC) liposomes as a model for biomembranes.

OBJECTIVE: To clarify the potential mechanism of action of methyl methacrylate (MMA) and related compounds to membranes of living cells, compared with their interaction with dipalmitoylphosphatidylcholine (DPPC) liposomes as a model for biological membranes. MATERIALS AND METHODS: For (meth)acrylates, MMA, ethyl acrylate(EA), n-butyl acrylate (BA) and n-butyl methacrylate (BMA) and for living cells, primary human gingival fibroblast (HGF), human submandibular gland adenocarcinoma cell line (HSG) and human erythrocytes were used. The physicochemical changes in DPPC liposomes induced by (meth)acrylates were studied using differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR). RESULTS: Cytotoxicity decreased as follows: BA > BMA > EA > MMA. Changes in phase transition properties (temperature Tm, enthalpy delta H and Height/Half-Height Width (H/HHW) of DSC peak were decreased as follows: BA > EA > MMA. BMA enhanced H/HHW and increased Tm slightly. NMR-shielding effect decreased as follows: BMA > MMA > BA, EA. CONCLUSION: BA and BMA exhibited large cytotoxicity and high DPPC-interaction due to their lipophilicity, compared to EA or MMA. MMA showed little cytotoxicity and small changes in DPPC liposomes, whereas BA showed large cytotoxicity and large changes in the liposomes characterized by the membrane disturbance. Haemolytic activity and cytotoxicity of acrylates were higher than those of methacrylates. The physico-chemical properties (Log P or Q sigma) of (meth)acrylates affect the lipid bilayer in biological membranes.

Comparative reduction of enamel demineralization by calcium and phosphate in vitro.

In theory, calcium and phosphate in the plaque fluid exert a large influence on the demineralization of enamel surface. In order to know the effect of increasing the concentration of either of these factors, the following in vitro experiment was conducted. Three thin sections, about 150 Im thick, were cut out from each of 13 human premolars. All surfaces of the sections, except for the original enamel surface, were coated with nail varnish. These sections were immersed into one of two sets of demineralizing solutions for 1 week at 25 degrees C. Each set, the 'calcium set' and the 'phosphate set', contained three solutions. The composition of these solutions differed mainly in calcium or phosphate concentrations. After 1 week, the degree of demineralization was determined by image analysis of contact microradiograms from each section. The subsurface demineralization in enamel was reduced by 95% by increasing the calcium concentration of the demineralizing solution from 7 to 21 mmol/l. A similar reduction (87%) was observed by increasing the phosphate concentration. However, the amount of phosphate needed was approximately 20 times more than that of calcium. The larger inhibitory effect that calcium has on enamel demineralization was related to the larger effect it has on the degree of saturation of the solution. Even though no statistically significant difference was found between the effect of calcium and phosphate on the demineralization of enamel (when the solutions had the same degree of saturation), the difference in the standard deviation of demineralization suggests the existence of some other factors which have an influence on the demineralization reaction.

Synthesis of the fatty sterol bound protein for a new sterol antibody.

For the purpose of applying the particular antibodies as a new diagnostic procedure for atherosclerosis and related diseases, we successfully achieved the synthesis of the fatty sterol with a linker, then linked the target protein to this sterol. Synthesis was started from pregnenolone and achieved by the Grignard reaction with pentenyl magnesium bromide, regioselective photoaddition of thiolacetic acid toward the 25-double bond, esterification of 3-OH with linoleic anhydride, in situ conjunction of the cross-linker (MBS) to the thiol group after selective deprotection from its acetyl ester, and finally by the reaction with protein such as KLH or albumin through this linker.

Acid and base-catalyzed hydrolysis of bisphenol A-related compounds.

In order to ascertain whether an estrogenic bisphenol A is produced from bisphenol A-related monomers by chemical-induced hydrolysis and to clarify their hydrolytic mechanisms, bisphenol A dimethacrylate (Bis-DMA) and bisphenol A bis(glycidyl methacrylate) (Bis-GMA) were reacted with phosphoric acid, hydrochloric acid, and sodium hydroxide in methanol or methanol/water mixed media at 37 degrees C. Amounts of monomethacrylate intermediates as well as bisphenol A (BPA) were determined by the use of high-performance liquid chromatography (HPLC), and time-conversion curves of hydrolytic products were prepared. BPA and bisphenol A monomethacrylate were produced by acid-catalyzed hydrolysis of Bis-DMA. Bis-GMA was partly converted into monomethacrylate by phosphoric acid and into monomethacrylate and 2,2-bis[4-(2,3-dihydroxypropoxy) phenyl]propane (BHP) by hydrochloric acid. Hydrolytic reactions by sodium hydroxide were completed almost within 1 day, resulting in the production of BPA from Bis-DMA, and BHP from Bis-GMA. No BPA was formed from Bis-GMA by chemical-induced hydrolysis. The hydrolytic behaviors of these monomers were discussed.

Use of fluoride-containing sealant on proximal surfaces.

A visible light curing sealant was developed for the prevention of caries on the smooth surfaces especially on the proximal surfaces. The sealant is mainly composed of dimethacrylate monomers. It contains a total amount of 2.28 mass% fluoride as poly(methyl methacrylate-co-methacryloyl fluoride) and sodium fluoride. The fluoride-containing sealant could be successfully applied to the proximal surfaces between the first and second primary molars by making access to the interproximal space with an orthodontic separating module. Availability of fluoride was assessed by the concentration of fluoride released into 1 ml of distilled water as a function of time from disks 5 mm in diameter and 0.2 mm in thickness. Ten microg of fluoride was measured on the first day; after 3 days the rate remained fairly constant at about 1 pg/day for a period of 28 days. The fluoride uptake and the improved acid solubility were investigated in vitro using bovine incisors. The fluoride of the control, that is enamel without sealant treatment, was not detectable. The fluoride incorporated into enamel after 37 hours application was 2,100 ppm at 2 pm from the enamel surface. The enamel solubility to acids decreased by the fluoride incorporation.

Correlation in inorganic ion concentration between saliva and plaque fluid.

The composition and the concentration of inorganic ions in dental plaque significantly influence the initiation and the development of dental caries through altering the degree of saturation of the aqueous phase surrounding the dental enamel. In order to know how plaque is affected by saliva, the composition and the concentration of inorganic ions in saliva and plaque fluid were investigated. The ionic concentrations of sodium and chlorine had similar values between plaque fluid and saliva. However, the concentrations of the inorganic ions such as ammonium, potassium, magnesium, calcium, and phosphate were significantly different between plaque fluid and saliva. This meant that the saliva and plaque fluid were different in its inorganic composition presumably reflected by the metabolic activity of bacteria in the plaque. On the other hand, as for the correlation coefficients between plaque fluid and saliva composition, statistically significant correlation was observed in ions such as sodium, ammonium, potassium, magnesium, and chlorine but not in calcium, phosphate, or in pH values. This was possibly due to the fact that saliva was the main source of supply of these ions. However as for calcium and phosphate, no close correlation was found possibly because they could be supplied also through tooth enamel dissolution. The discrepancy of the results with former studies on this point was speculatively explained by,the difference of the plaque age used. It was considered reasonable that the pH value was independent, as it is mainly decided by the activity of the bacteria in the plaque.

Radical generation, radical-scavenging activity, and cytotoxicity of eugenol-related compounds.

To clarify the possible link between radicals and cytotoxicity of eugenol-related compounds, dimeric compounds were synthesized from eugenol (4-allyl-2-methoxy-phenol), butylated hydroxyanisole (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), bis-BHA (3,3'-di-t-butyl-5,5'-dimethoxy-1,1'-biphenyl-2,2'-diol), and bis-MMP (3,3'-di-methoxy-5,5'-dimethyl-1,1'-biphenyl-2,2'-diol). The cytotoxic activity of these compounds was determined using a salivary gland tumor cell line (HSG), oral squamous cell carcinoma cell line (HSC-2) and human promyelocytic leukemia cell line (HL-60). A parabolic relationship between the cytotoxicity and log P (the octanol-water partition coefficient) was observed, showing that both BHA and bis-MMP, with a log P of 3-4, were the most cytotoxic. The cytotoxic activity of the 2-methoxy derivatives, eugenol, MMP and bis-MMP, against HSG cells was significantly enhanced by visible-light irradiation, possibly due to their high redox potential. Electron spin resonance (ESR) spectroscopy indicated that eugenol and BHA alone produced radicals under alkaline conditions (pH > 9.5), and eugenol most efficiently scavenges reactive oxygen species (O2-). Antioxidative reactivity of eugenol-related compounds was determined by measuring the inhibiting periods of the AIBN (2,2'-azobisisobutyronitrile)/MMA (methyl methacrylate) polymerization system, and the number of moles of peroxy radical trapped by moles of the relevant phenols (stoichiometric factor, n). It was found that the n values of eugenol and MMP were approximately 1, whereas those of BHA >2, suggesting that eugenol and MMP undergo dimerization through radical-radical couplings through quinone methides, whereas BHA undergoes the competitive interaction with poly-MMA radicals after oxidation by AIBN-peroxy radicals. BHA was an efficient peroxy radical-scavenger, but possibly reacted with polymer radicals of the lipid, thus mediating the cytotoxicity. The n value of bis-BHA was two, whereas those of bis-EUG and bis-MMP were 1.6-1.7, suggesting that the latter were further oxidized. The enthalpies of phenoxyl radical formation were determined using the semi-empirical PM3 quantum-mechanical method and the possible link to redox potential was discussed.

A study on the adsorption structure of an adhesive monomer for precious metals by surface-enhanced Raman scattering spectroscopy.

In order to clarify the role of a primer on the adhesion between dental precious metals and resin, surface-enhanced Raman scattering (SERS) technique has been applied to the structural analysis of the adhesive monomer, 6-(N-(4-vinylbenzyl)propylamino)-1,3,5-triazine-2, 4-dithione (VBATDT) adsorbed on colloidal Au surfaces. VBATDT is one of the major components of commercial primers. A mixture of a methanol solution of VBATDT and aqueous Au colloid was illuminated by the 647.1 nm line from a Kr ion laser to obtain the SERS. The most intense peak at 458 cm-1 due to C = S stretching in the Raman spectrum of solid VBATDT disappears completely in the SERS, while all the other peaks due to vinylbenzyl group vibrations remain unchanged. It suggests that some structural change occurs in VBATDT upon adsorption on Au which involves the C = S bonds and that the molecule undergoes thione-thiol-type tautomerization in the adsorption process. The SERS spectrum of a commercial primer containing VBATDT exhibits a similar spectral pattern. The present results lead us to conclude that some specific interactions exist between the sulfur atoms of VBATDT and Au surface to form chemical bond likes, which are effective to improve the bond strength between dental precious metals and resin. The ambivalent properties of the primer, chemical stability in storage and chemical affinity to Au, are also explained by the thione-thiol-type tautomerization of VBATDT on the basis of the spectroscopic evidence.

Effect of rinse with calcium enriched milk on plaque fluid.

Previous research has shown that rinsing the mouth with milk significantly diminished the pH in dental plaque fluid; however, the degree of saturation with respect to the dental enamel (DS) was not significantly decreased because of an increase in the calcium ion concentration in plaque fluid. The aim of this study was to investigate the cariostatic effect of adding calcium to milk on the DS value of the plaque fluid after rinsing. Plaque samples were collected from 8 Japanese male dental students. Prior to plaque collection, all subjects refrained from practicing oral hygiene for 48 hr and fasted overnight. Supragingival plaque samples were collected from one side of the mouth of each subject, and then collected from the other side, following a 30-second rinse with 15 mL of calcium-enriched milk, which was prepared by adding calcium carbonate to ordinary milk, and a 10-minute waiting period. The samples were cleared by centrifugation, and the plaque fluid was analyzed for inorganic ions and pH, using an ion chromatograph and pH microelectrode, respectively. The calcium ion concentration of the milk was 6.4 mM, which was about 36% higher than that of ordinary milk. The pH decreased significantly (p<5%) from 6.4 to 6.1 following the rinse with calcium enriched milk, as tested by the paired t-test. The decrease in pH might have caused a reduction of the DS value; however, it was compensated for by a significant (p<0.5%) increase in the calcium ion concentration of plaque fluid.

DSC and NMR spectroscopic studies of the interaction between camphorated phenol and phospholipid liposomes.

To clarify the interaction mechanism of biological activities induced by camphorated phenol (CP), the interactions between CP and phospholipid liposomes [dipalmitoyl phosphatidylcholine (DPPC) liposomes, dimyristoyl phosphatidylcholine (DMPC) liposomes and DMPC/dilauloyl phosphatidylethanolamine (DLEA) liposomes] were studies by DSC and NMR spectroscopy. CP exhibited a larger DSC phase transition properties [shift of phase transition temperature to a lower temperature and decrease in Height/Half-Height Width (H/HHW) of DSC peak)] than phenol in the various liposome systems. It was concluded from the NMR studies that CP is highly incorporated into the DPPC bilayer, the 1H and 13C signals of phenol in a complex between phenol and camphor being markedly broadened but shielded in the presence of DPPC liposomes. It was clear that CP is incorporated as a complex into the lipid bilayers.

Action of eugenol as a retarder against polymerization of methyl methacrylate by benzoyl peroxide.

This study was undertaken to examine an effect of eugenol on polymerization of methyl methacrylate (MMA) by benzoyl peroxide (BPO) and 2,2'-azobisisobutyronitrile (AIBN) in the presence of eugenol. The induction period and initial rate of polymerization (IRP) were determined from polymerization curves of MMA using differential scanning calorimetry (DSC). The induction period increased with increasing concentration of eugenol in both BPO and AIBN systems. The IRP decreased as the concentration of eugenol in both BPO and AIBN systems. The IRP decreased as the concentration of eugenol increased. Its decreasing rate in the BPO system was higher than that in the AIBN system. In the BPO system, the IRP was reduced to zero at 0.5 mol% of eugenol. Eugenol in the BPO system was an efficient retarder, although the decrease in IRP was relatively small, below 0.05 mol%. Eugenol was an inhibitor even at high concentration in the AIBN system. The suppressible effect of eugenol appears to be due to the interaction between free radicals from BPO and eugenol.

Formyl-ended heterobifunctional poly(ethylene oxide): synthesis of poly(ethylene oxide) with a formyl group at one end and a hydroxyl group at the other end.

Well-defined poly(ethylene oxide) (PEO) with a formyl group at one end and a hydroxyl group at the other terminus was synthesized by the anionic ring opening polymerization of ethylene oxide (EO) with a new organometallic initiator possessing an acetal moiety, potassium 3,3-diethoxypropyl alkoxide. Hydrolysis of the acetal moiety produced a formyl group-terminated heterobifunctional PEO with a hydroxyl group at the other end.

Effect of phenolic compounds on the polymerization of methyl methacrylate.

The inhibitory effects of six phenolic compounds and two analogues on the polymerization of methyl methacrylate (MMA) by azobisisobutyronitrile (AIBM) were examined. Differential scanning calorimetry was used to determine the induction period (IP) and initial rate of polymerization (IRP). The IP values (minutes) decreased in the order: eugenol > thymol > hydroquinone > cresol > guaiacol > phenol >>> safrole > methol. The IRP values of all compounds tested ranged from 0.8 to 1.2 percent per minute, except for hydroquinone (0.2 percent per minute). Phenolic compounds inhibited the polymerization of MMA by scavenging radicals, and eugenol was the most potent inhibitor.

Hemolytic activity of a dental adhesive monomer (N-methacryloyloxy-5-aminosalicylic acid, MASA) and its interaction with phospholipid liposomes as studied by NMR and DSC.

N-methacryloyloxy-5-aminosalicylic acid (MASA) has recently been used as an adhesive primer in restorative resin systems. To monitor the biological activity of MASA, we studied changes in NMR-chemical shifts (delta H) and the differential scanning calorimetry (DSC) phase transition temperature (Tm) of dipalmitoylphosphatidylcholine (DPPC)/MASA liposomes with or without the presence of albumin and collagen. The delta H and the Tm did not alter significantly and the interaction of MASA with DPPC was found to be small. Hemolytic activity of MASA was markedly smaller than that of the phosphate monomer (MDP) in bonding agents widely used. These findings suggest that using a MASA primer in resin systems has an acceptable biocompatibility for dentin-pulp, involving its adsorption and adhesion to hard tooth tissues.