A theoretical and experimental study on meridional-facial isomerization of tris(quinolin-8-olate)aluminum (Alq3).

The rationale behind the stereospecific synthesis of a facial isomer of tris(quinolin-8-olate)aluminum (Alq3) is studied by density functional theory (DFT) calculations, which predict the favourable influence of an H3O(+) ion on the distribution ratio between a meridional and a thermodynamically unstable facial isomer.

Radical production and cytotoxic activity of tert-butyl-substituted phenols.

2,4,6-Tri-tert-butylphenol (TBP)-related compounds are used for stabilizing plastics by making them resistant to oxidation. However, the cytotoxic activity of these compounds has not yet been established. TBP produced phenoxyl radicals at pH >or= 9.0 and 2,4-di-t-butylphenol (DBP) at pH 12.5, but 3,3',5,5'-tetra-t-butyl-1,1'-biphenyl-2,2'-diol (bisDBP) did not, using ESR spectroscopy. Both superoxide anion radical (O(2)(-)) scavenging activity and reactive oxygen species (ROS) production activity declined in the order of TBP > DBP > bisDBP. The cytotoxic activity against human oral tumor cell lines (HSC-2, HSG) and human gingival fibroblast cells (HGF) declined in the order of DBP >> bisDBP = TBP = TBP-OOH (2,4,6-tri-t-butyl-4-hydroperoxy-2,5-cyclohexadiene-1-one). The cytotoxic activity of TBP, but not of DBP or bisDBP was significantly enhanced after visible light (VL)-irradiation for 10 min. The cytotoxicity of irradiated TBP was significantly higher than that of either original TBP or TBP-OOH, the oxidative metabolite of TBP, possibly due to the formation of TBP stable radical and ROS via oxidation. In contrast, the cytotoxic activity of DBP and bisDBP was independent of radical production, and therefore, may be intrinsic. A non-enzymatic oxidation decomposition of DBP or TBP was estimated from the formation of reaction enthalpy (DeltaH) using a modified neglect of diatomic overlap, parametric method 3 (MNDO-PM3) semi-empirical method, suggesting that O(2) is capable of activating DBP to a reactive quinone or dimer and that TBP phenoxyl radicals via oxidation directly affect extra- or intracellular bioactive materials, resulting in the induction of cytotoxicity.

Reactive oxygen species generation and photo-cytotoxicity of eugenol in solutions of various pH.

In order to clarify the mechanism of photo-damage caused by eugenol (4-allyl-2-methoxyphenol), we measured cell survival in the presence of eugenol at concentrations of 10(-3) - 10(-7) M, with and without VL (visible light) irradiation by a VL dental lamp and at various pHs (7.2, 7.8 and 8.2) using two different cells (HSG, a human submandibular gland tumor cell line; HGF, a human gingival fibroblast in primary culture). Also, ROS (reactive oxygen species) generation in the above adherent single cells was measured by ACAS laser cytometry combined with CDFH-DA, a peroxide probe. The survival of both HSG and HGF cells treated with eugenol was significantly decreased as the VL irradiation time and/or the pH of the medium was increased. The amount of ROS generated from eugenol was also enhanced by increasing the VL irradiation time and elevating the pH of the medium. Cytotoxicity and ROS generation of HGF cells were significantly lower than that of HSG cells. Glutathione (1 mM) or cysteine (1 mM) protected the photo damages. We conclude that the cytotoxicity of VL-irradiated eugenol possibly was caused by the generation of eugenol radicals and additionally by ROS, both of which were produced dependent on the dose of eugenol, length of irradiation time, and pH of the medium.

The production of reactive oxygen species by irradiated camphorquinone-related photosensitizers and their effect on cytotoxicity.

Camphorquinone (CQ) is widely used as an initiator in modern light-cured resin systems but there are few reports about its effects on living cells. To clarify the mechanism of photosensitizer-induced cytotoxicity, the production of initiator radicals and subsequent reactive oxygen species (ROS) by CQ, benzil (BZ), benzophenone (BP), 9-fluorenone (9-F) in the presence of the reducing agent (2-dimethylaminoethyl methacrylate or N,N-dimethyl-p-toluidine, DMT) with visible-light irradiation was examined in a cell or cell-free system. Initiator radical production was estimated by the reduction rate of 1,1-diphenyl-2-picrylhydrazyl and by the conversion of poly-triethyleneglycol dimethacrylate; the results indicated that CQ/DMT had the highest activity among them. The cytotoxic effects of the photosensitizers on both human submandibular gland (HSG) adenocarcinoma cell line and primary human gingival fibroblast (HGF) showed that the 50% toxic concentration (TC(50)) declined in the order: CQ>BP>9-F>BZ. ROS produced in HSG or HGF cells by elicited, irradiated photosensitizers were evaluated in two different assays, one using adherent cell analysis and sorting cytometry against adherent cells and the other, flow cytometry against floating cells, with fluorescent probes. ROS production was dose- and time- dependent, and declined in the order: BZ>9-F>BP>CQ. Cytotoxic activity was correlated with the amount of ROS. Cytotoxicity and ROS generation in HGF cells was significantly lower than in HSG cells. ROS induced by aliphatic ketones (CQ) were efficiently scavenged by hydroquinone and vitamin E, whereas those by aromatic ketones (9-F) were diminished by mannitol and catalase, suggesting that OH radicals were involved in ROS derived from 9-F. A possible link between the cytotoxic activity and ROS is suggested.

Cytotoxicity and radical intensity of eugenol, isoeugenol or related dimers.

To investigate the possible link between radicals and cytotoxicity of eugenol-related compounds, dimer compounds were synthesized from eugenol (4-allyl-2-methoxyphenol) or isoeugenol (4-propenyl-2-methoxyphenol): bis-eugenol (3,3'-dimethoxy-5,5'-di-2-propenyl-1,1'-biphenyl-2,2'-diol); dehydrodiisoeugenol (2-(3-methoxy-4-hydroxyphenyl)-3-methyl-5-(1-propenyl)-7-methoxy-2,3- dihydrobenzofuran) and alpha-di-isoeugenol (r-l-ethyl-5-hydroxy-t-3-(4-hydroxy-3-methoxyphenyl)-6-methoxy-c-2- methylindane). Both the cytotoxic activity and the DNA synthesis inhibitory activity of these compounds against a salivary gland tumor cell line (HSG) and normal human gingival fibroblast (HGF) were decreased in the order of: dehydrodiisoeugenol, alpha-di-isoeugenol > isoeugenol > eugenol > bis-eugenol. Electron spin resonance (ESR) spectroscopy showed that dehydrodiisoeugenol, alpha-di-isoeugenol and eugenol, but not isoeugenol and bis-eugenol, produced phenoxyl radicals under alkaline condition (pH > 9.5). However, benzyl radicals were produced during the dimerization of isoeugenol to dehydrodiisoeugenol. The radical intensity of alpha-di- and dehydrodiisoeugenol appeared at relatively later incubation time than eugenol, suggesting that their phenoxyl radical was more stable than that of eugenol. Such a phenoxyl radical is produced by scavenging free radicals, during the inhibition of lipid peroxidation. Higher cytotoxic activity of isoeugenol dimers was thought to be induced by interaction with cell membranes via the lipophilic radical. The present study supports the notion that relative cytotoxicity of chemicals can be evaluated by measuring the radical intensity using ESR.

Cytotoxic activity of low molecular weight polyphenols against human oral tumor cell lines.

A total of 150 chemically-defined natural and synthetic polyphenols (flavonoids, dibenzoylmethanes, dihydrostilbenes, dihydrophenanthrenes and 3-phenylchromen-4-ones), with molecular weights ranging from 224 to 824, were investigated for cytotoxic activity against normal, tumor and human immunodeficiency virus (HIV)-infected cells. They showed higher cytotoxic activity against human oral squamous cell carcinoma HSC-2 and salivary gland tumor HSG cell lines than against normal human gingival fibroblasts HGF. Many of the active compounds had a hydrophilic group (hydroxyl group) in the vicinity of a hydrophobic group (prenyl, phenyl, methylcyclohexene or methylbenzene moiety), similar to isoprenoid-substituted flavones. Substitution of hydrophobic group (prenyl or geranyl group) did not significantly change the cytotoxic activity of flavanones, isoflavans, chalcones or 5-hydroxy-3-phenoxychromen-4-ones. However, the prenylation(s) of an isoflavone and a 2-arylbenzofuran significantly enhanced the cytotoxic activity. Agarose gel electrophoresis showed that active components induced internucleosomal DNA fragmentation in human promyelocytic leukemic HL-60 cells, but not in HSC-2 cells. Most of the polyphenols failed to reduce the cytophathic effect of HIV infection in MT-4 cells.

T-1095, a renal Na+-glucose transporter inhibitor, improves hyperglycemia in streptozotocin-induced diabetic rats.

The effect of T-1095, an inhibitor of renal glucose reabsorption, on hyperglycemia and the expression of Na+-glucose cotransporters (SGLTs) and facilitative glucose transporter 2 (GLUT2) in streptozotocin (STZ)-induced diabetic rats was examined. There was an elevation of blood glucose, hemoglobin A1c (HbA1c), kidney weight, and urinary excretion of both glucose and albumin in STZ rats. Administration of 0.03% and 0.1% (wt/wt diet) T-1095 to STZ rats for 4 weeks improved the hyperglycemia and dose-dependently decreased HbA1c. Moreover, treatment with 0.1% (wt/wt diet) T-1095 in STZ rats for 8 weeks not only reduced blood glucose and HbA1c, levels but also prevented the elevation of urinary albumin levels and kidney weight and the development of epithelial vacuolation. The expression of renal SGLT2, a major glucose transporter in the kidney, was not different in normal, STZ, and T-1095-treated STZ rats. In contrast, the elevated renal GLUT2 level in STZ rats was suppressed by T-1095. These data suggest that T-1095 improves hyperglycemia by suppressing the renal reabsorption of glucose, which results in a suppression of the development of functional and histological changes and abnormal expression of GLUT2 in the kidney.

Cytotoxic activity of hydrolyzable tannins against human oral tumor cell lines--a possible mechanism.

Hydrolyzable tannins showed higher cytotoxic activity against human oral squamous cell carcinoma and salivary gland tumor cell lines than against normal human gingival fibroblasts, whereas gallic acid, a component unit of tannins, showed much weaker selective cytotoxicity. The cytotoxic activity of dimeric compounds was generally higher than that of monomeric compounds. Macrocyclic ellagitannin oligomers, such as oenothein B, woodfordin C and woodfordin D showed the greatest cytotoxic activity, and their activity (per given number of molecules) was one order higher than those of gallic acid and epigallocatechin gallate, a major component of green tea. These compounds induced apoptotic cell death characterized by DNA fragmentation (as demonstrated by the TUNEL method) and cleavage of cytokeratin 18 by activated caspase(s) (as demonstrated by M30 monoclonal antibody). ESR spectroscopy revealed that these macrocyclic compounds at higher concentrations produced their own radicals and significantly enhanced the radical intensity of sodium ascorbate, possibly by their prooxidant actions. Catalase failed to eliminate their apoptosis-inducing activity, reducing the possibility of the involvement of hydrogen peroxide production in the extracellular fraction. These observations suggested that the antitumor activity of macrocyclic ellagitannin oligomers reported previously might be explained by their apoptosis-inducing activity.

Induction of apoptosis by flavones, flavonols (3-hydroxyflavones) and isoprenoid-substituted flavonoids in human oral tumor cell lines.

Various flavones, flavonols (3-hydroxyflavones) and isoprenoid-substituted flavones (flavonols) were investigated for their cytotoxic activity. Most of these compounds were more cytotoxic against human oral squamous cell carcinoma and salivary gland tumor cell lines than human gingival fibroblasts. The cytotoxic activity of flavonoids was generally higher than that of tannin-related compounds. Flavonoids induced apoptotic cell death characterized by DNA fragmentation (as identified by TUNEL method) and activation of caspase(s) (as identified by degradation products of cytokeratin 18 with M30 monoclonal antibody). ESR spectroscopy revealed that higher concentrations of flavonoids produced radicals under alkaline conditions. However, not all of them enhanced the radical intensity of sodium ascorbate, suggesting that the redox potential of flavonoids differs considerably from samples to samples. Catalase failed to eliminate the cytotoxic activity of flavonoids, reducing the possibility of the involvement of hydrogen peroxide for the cytotoxicity induction by them.

Application of bis-eugenol to a zinc oxide eugenol cement.

OBJECTIVES: To assess the usefulness of dimerized eugenol (bis-eugenol) in dentistry, the physical properties of zinc oxide eugenol cement (ZOE) with bis-eugenol and the cytotoxicity of bis-eugenol were studied. METHODS: Setting time, compressive strength, solubility and disintegration of ZOE cement with bis-eugenol according to the specifications of JDMAS315 were evaluated. The cytotoxicity of bis-eugenol and eugenol toward two different cell types, HGF (a primary culture of human gingival fibroblast) and HSG (a human epidermoid carcinoma cell line derived from a salivary gland) was evaluated by the MTT test and in terms of cell survival. RESULTS: Addition of bis-eugenol to ZOE did not decrease the physical properties when employed at the ratio of 9:1 or 6:1 (liquid ND:bis-eugenol, w/w). Bis-eugenol was less toxic than eugenol in the cell culture tests. CONCLUSIONS: The results of this assay demonstrated that bis-eugenol is useful in ZOE.

Free radical scavenging activity in the nonenzymatic fraction of human saliva: a simple DPPH assay showing the effect of physical exercise.

Free-radicals and other reactive oxygen species (ROS) have been implicated as being major damaging species in pathology and they have been widely investigated. Using 1,1'-diphenyl-2-picrylhydrazyl (DPPH), we estimated total free radical scavenging activity in the low-molecular-weight nonenzymatic fraction (LMNEF) of human whole saliva. The activity of the whole saliva and serum were measured in terms of the rate of decrease in the absorbance at 517 nm in a 40% ethanol DPPH solution (pH 7.4) at room temperature. The DPPH activity of saliva and serum showed a significant linear relationship. The mean DPPH activities of saliva from 257 subjects aged 4-72 was found to be 0.389+/-.190 micromol/ml and bore no relation to age or sex. The activity in saliva of 86 subjects aged 4-11 was significantly different before and after exhaustive aerobic dance exercise for 1 hr. Physical exercise markedly decreased free radical scavenging activity in whole saliva of children. On the basis of the above results, we concluded that DPPH is useful for evaluating the total antioxidant capacity of LMNEF of human saliva.

Alkalization produced by high-dose carbachol in HSG cell line is independent of Ca2+.

OBJECTIVES: The aim of this investigation was to clarify the mechanism of alkalization induced by carbachol in HSG cells. MATERIALS AND METHODS: Cells of the HSG cell line derived from a human submandibular gland adenocarcinoma and those of the A-431 human epidermoid carcinoma cell line were loaded with a fluorescent pH indicator, BCECF/AM, and the change in the intracellular pH of adherent cells and suspended ones were measured following stimulation with various concentrations (10(-7) M to 10(-2) M) of neurotransmitters (carbachol, noradrenaline, and isoproterenol). RESULTS: Isoproterenol did not cause alkalization of either cell type, whereas, noradrenaline and carbachol alkalized both types over the concentration ranges of 10(-6) M to 3 x 10(-3) M (HSG cell by noradrenaline), 10(-7) M to 2 x 10(-4) M (A-431 cell by noradrenaline), and 7 x 10(-5) M to 10(-4) M (A-431 cell by carbachol). On the other hand, alkalization induced by carbachol in the HSG cells was recognized at concentrations higher than 6 x 10(-5) M, and it showed no upper limit in terms of carbachol concentration. This high-dose carbachol alkalization was not eliminated by preincubation with nifedipine (100 microM), a Ca2+ channel blocker, or with thapsigargin (100 microM), a microsomal Ca(2+)-ATPase inhibitor. CONCLUSIONS: The alkalization system induced by carbachol in the HSG cell was quite different from that in the A-431 cell, and that induced by high-dose carbachol in HSG cells appeared to be independent of intracellular Ca2+. These findings will be useful to clarify the mechanism of salivary secretion stimulated by neurotransmitters.