Protection against copper-induced cytotoxicity by inclusion of gold.

BACKGROUND: We previously reported that contact with copper (Cu) induced immediate cell death via an oxidation-involved mechanism, and the Cu-induced oxidation and cell death were effectively alleviated under hypoxic conditions. In order to explore alternative strategies for the protection from the Cu-induced cytotoxicity, we investigated whether the inclusion of gold (Au) in the Cu plate, as alloy,has a protective effect. MATERIALS AND METHODS: Human gingival fibroblast (HGF) cells, established from periodontal tissues, were inoculated on Au/Cu alloy of different Au ratios. After incubation at 37°C for different times under normoxic conditions, cellular viability and amino acid consumption were determined. Changes in the elemental composition of the alloy and in the culture medium were chemically analyzed by X-ray photoelectron spectroscopy and by inductively coupled plasma-optical emission spectrometry. RESULTS: Contact with the Cu plate induced cytotoxicity and cystine oxidation in time-dependent manners. Inclusion of Au at more than 10% in the alloy, completely abrogated the cytotoxicity and reduced the oxidation of Cu and the elution of Cu from the alloy. CONCLUSION: Inclusion of Au as a component of alloy reduces the cytotoxicity of the Cu plate, possibly by reducing its oxidation.

Changes in salivary amino acid composition during aging.

BACKGROUND: it has been suggested that the features of saliva reflect the physiological and psychological state of primates as well as subprimates, however, studies revealing the relationship between aging and the concentrations of salivary amino acids are limited. In order to better understand their physiological role, age-related changes of salivary amino acids were investigated. MATERIALS AND METHODS: forty-five participants including 5 children [6.60 ± 1.67 (5-9) years old], 20 adults [46.55 ± 14.68 (23-64) years old), and 20 senior citizens [71.60 ± 4.27 (66-82) years old] took part in this study. Whole saliva (one sample per each person) was collected in the daytime (10:00-11:00 or 14:00-15:00). Salivary amino acids were recovered after deproteinization with 5% trichloroacetic acid and determined by an amino acid analyzer. RESULTS: glycine was the most abundant amino acid in the saliva. Glycine and lysine levels increased significantly (p<0.05) with aging, regardless of gender difference. When the glycine and lysine levels were plotted, much higher correlation (p<0.001) was observed. On the other hand, there was no significant correlation between the salivary concentration of glutamic acid or histidine and age. CONCLUSION: salivary amino acid levels may be regarded as markers of aging.

Changes in amino acid metabolism during activation of mouse macrophage-like cell lines.

BACKGROUND: recent reports have suggested the applicability of salivary amino acids as markers for various diseases. In order to understand the role of macrophages in the age-related changes of salivary amino acid composition, we compared the amino acid production and consumption between control and activated macrophages. MATERIALS AND METHODS: mouse macrophage-like cells (RAW264.7, J774.1) were activated by Escherichia coli lipopolysaccharide (LPS). Concentrations of nitric oxide (NO) and amino acids in the culture medium during culture were determined by Griess method and amino acid analyzer, respectively, to delineate their metabolic rates. RESULTS: LPS activated these macrophages, as judged by the enhanced production of NO and citrulline. The activated macrophages produced greater amounts of glycine, glutamic acid, alanine and histidine as compared with control cells, and consumed serine and glutamine at the highest rates. CONCLUSION: The present study suggests the possible role of activated macrophages in age-related changes in salivary amino acid composition.

Diurnal changes in salivary amino acid concentrations.

It has been suggested that the features of saliva (e.g. fluidity, secretion and amino acid concentration) reflect physiological and psychological state of primates as well as subprimates, however, studies which revealed the relationship between the circadian rhythm and the concentrations of salivary amino acids have been limited. In order to better understand their physiological role, diurnal changes of salivary amino acids were investigated in three undergraduate students. Salivary amino acids were recovered after deproteinization with 5% trichloroacetic acid and determined by an amino acid analyzer. Most amino acids, except for methionine, cysteine and asparagine, were detected in the saliva. The intake of lunch or amino acid supplement transiently increased the salivary amino acids, and in the latter case, the amino acid levels returned to baseline within 10 minutes. Physical exercise also slightly elevated the salivary amino acid levels. During the university examination period, the secretion of saliva was slightly, but not significantly, increased, accompanied by the elevation of glycine, alanine, ornithine, histidine and threonine, and the decline of lysine, leucine, aspartic acid and hydroxyproline. Salivary amino acid levels may be useful to evaluate stressful conditions.

Quantification of enhanced osteoblastic adhesion to ultraviolet-treated titanium plate.

Although the advantage of ultraviolet (UV) irradiation of titanium plates for the attachment of osteoblast is known, the details of the experimental conditions have not been described in previous literature. We established optimal conditions of UV irradiation of titanium plate for the adhesion of mouse osteoblast MC3T3-E1 cells. The viable cell number was determined by MTT method. UV irradiation at two different wavelengths (253.7 and 365 nm) enhanced the cell attachment on titanium plate to comparable extents. The optimal UV exposure duration was 20 minutes and prolonged irradiation slightly reduced cell attachment. The attached cells proliferated during 24 hours, accompanied by the enhanced consumption of extracellular glutamine and arginine. The present study supports the previous reports of the efficacy of UV irradiation, and this simple and rapid assay system may be applicable for the study of the interaction of osteoblast and UV-activated titanium plates.

Effect of contact with titanium alloys on the proliferation of mouse osteoblastic cells in culture.

This study was aimed at studying the effect of contact with titanium alloy plates of different surface textures on the proliferative capability of mouse osteoblastic MC3T3-E1 cells. First, the proliferation characteristics of MC3T3-E1 cells were investigated. MC3T3-E1 cells showed a high capacity for proliferation and survived for a long period even under nutritionally starved conditions. During logarithmic cell growth, the consumption of Ser, Gln, Val, Ile and Leu increased time-dependently. Contact with an hydoxyapatite (HA)-coated titanium alloy plate resulted in the increase in the recovery of cells from the plate by trypsin, and an increase in the consumption of these amino acids, suggesting enhanced cell proliferation. On the contrary, contact with the sandblasted and anodized titanium alloy plates resulted in the reduction of the recovery of the cells from the plate, but a slight increase in the amino acid consumption, suggesting the tight adhesion of the cells to the plates. This study demonstrates that the present method, based on the amino acid consumption of the cells, is useful for monitoring the cell proliferative capability, without detachment of the cells from the plate. This method may be applicable to the study of the interaction between cells and metal plates.

Effect of Sasa senanensis Rehder extract on NO and PGE2 production by activated mouse macrophage-like RAW264.7 cells.

Alkaline extract of Sasa senanensis Rehder (SE) has shown diverse biological activity. As an extension, whether SE affects the function of activated macrophages was investigated. SE inhibited the nitric oxide (NO) production by lipopolysaccharide (LPS)-activated mouse macrophage-like RAW264.7 cells. Western blot and RT-PCR analyses demonstrated that this was due to the inhibition of inducible NO synthase (iNOS) expression at both protein and mRNA levels. ESR spectroscopy shows that SE dose-dependently scavenged the NO radical produced by NOC-7. In order to confirm the anti-inflammatory potency, possible effects on prostaglandin (PG) E(2) production and expression of enzymes involved in the arachidonic acid pathway were next investigated. It was found that SE effectively inhibited the PGE(2) production by LPS-stimulated RAW264.7 cells, although the extent of inhibition of PGE(2) was slightly less than that of NO production. SE inhibited cyclooxygenase (COX)-2 expression at both protein and mRNA levels, but to much lesser extents as compared with those for iNOS expression. SE contained much lower concentration of arginine, precursor of NO, as compared with the culture medium. These data suggest that SE exerts a weak anti-inflammatory activity.

Cell death induced by nutritional starvation in mouse macrophage-like RAW264.7 cells.

Amino acid utilization of mouse macrophage-like RAW264.7 cells was investigated. During the logarithmic growth stage, RAW264.7 cells grew very fast, with an approximate doubling time of 11 hours, in DMEM supplemented with 10% heat-inactivated fetal bovine serum. RAW264.7 cells consumed glutamine at the fastest rate, followed by serine, leucine, isoleucine, arginine, lysine, valine and other amino acids. When the cell density reached a critical threshold level, cells began to suffer non-apoptotic cell death characterized by mitochondrial damage (revealed by transmission electron microscopy) and a smear pattern of DNA fragmentation (revealed by agarose gel electrophoresis). At this point, glutamine, serine and glucose in the medium were almost completely exhausted, whereas other amino acids remained at more than 40% of their initial concentrations. Based on these data, it is recommended that glutamine, serine and glucose should be supplemented for the long culture of RAW264.7 cells.

Non-apoptotic cell death induced by nutritional starvation in J774.1 mouse macrophage-like cell line.

The growth and amino acid utilization of a mouse macrophage-like cell line J774.1 was investigated in two different culture media supplemented with 10% fetal bovine serum (FBS). The J774.1 cells grew faster, and consumed glutamine and serine at higher rates in DMEM than in RPMI1640 medium. The consumption of other amino acids was much less, while considerable quantities of alanine, glutamic acid and glycine were produced by the J774.1 cells. When the cells became confluent, serine, but not glutamine, was nearly depleted from the culture medium, followed by cell death characterized by smear DNA fragmentation, slight caspase-3 activation and structural damage of the mitochondria. Serine is required for the growth of mouse macrophage-like cell lines, and DMEM is superior to RPMI1640 for long-term cell culture.

Rapid changes in amino acid and polyamine metabolism during copper-induced cell death of human gingival fibroblast.

There are very few studies on the interaction between dental alloys and oral tissues. The effect of direct contact with copper (Cu) on the cellular function of human gingival fibroblast (HGF) derived from the periodontal tissues was investigated. When HGF cells were inoculated onto a Cu plate, the viability of HGF cells immediately declined. This was accompanied by vacuolization and chromatin condensation near the nuclear membrane. The intracellular concentration of spermidine and spermine declined, whereas that of putrescine slightly increased. Amino acid analysis of the medium revealed that glutamine was consumed at the greatest rate, amounting to more than half of the total amino acid consumption. Contact with the Cu plate resulted in the complete elimination of glutamine utilization and a simultaneous increase in the production of most amino acids, possibly due to enhanced proteolysis. This was accompanied by a time-dependent increase in the consumption of cystine, possibly due to oxidative reactions, and the enhanced production of glycine and glutamic acid. These data suggest that the contact with the Cu plate induced non-apoptotic cell death in HGF cells, which was tightly coupled with a rapid dysfunction of amino acid and polyamine metabolism.

Disruption of amino acid metabolism in human myelogenous leukemic cell lines destined to die after contact with metal plates.

Changes in amino acid metabolism during cell death of human myelogenous leukemic cell lines (HL-1, ML-1, KG-1) induced by contact with gold (Au), silver (Ag) or palladium (Pd) were investigated. All three leukemic cell lines consumed glutamine and serine at the highest rate (amounting to 50%-58% and 12%-16% of the total amino acid consumption, respectively). HL-60 cell growth was slightly stimulated by contact with any metal plate. Contact with Ag or Pd, but not Au plates occasionally induced cytotoxicity against ML-1 and KG-1 cells. In such cases, glutamine consumption was inhibited by 88%-90% and consumption of other amino acids completely ceased. This was accompanied by the enhanced production of arginine, glycine and glutamic acid. These data suggest the tight association of the disruption of amino acid metabolism with the cell death induced in human myelogenous leukemic cell lines by contact with metal plates.

Effect of saliva, epigallocatechin gallate and hypoxia on Cu-induced oxidation and cytotoxicity.

We have previously reported that contact with copper (Cu) induced immediate cell death via an oxidation-involved mechanism in human promyelocytic leukemic HL-60 cells, whereas contact with other metals (Au, Ag, Pd) produced no discernible effect. In the present study, we investigated the conditions under which Cu-induced oxidative stress can be reduced. Contact with a Cu plate in the absence of cells enhanced the rate of consumption of cystine to the greatest extent, followed by that of methionine and histidine. Under hypoxic conditions, the consumption of all these amino acids was significantly reduced. On the other hand, the addition of saliva slightly, but not significantly, reduced the amino acid oxidation. The addition of epigallocatechin gallate (EGCG) slightly, but significantly reduced the consumption of cystine and histidine. The inhibitory effect of EGCG on the methionine consumption was more prominent, especially at higher concentrations. The Cu-induced cell death was significantly inhibited when freshly-prepared human gingival fibroblasts were incubated under hypoxic conditions. The present study demonstrates for the first time that the Cu-induced oxidation and cell death were effectively alleviated under hypoxic conditions.

Biological impact of contact with metals on cells.

In order to investigate the in vivo effect of metals used in dentistry, we investigated the effect of direct contact with metal plates (20 x 20 x 0.5 mm3) made of gold (Au), silver (Ag), copper (Cu) or palladium (Pd) on human promyelocytic leukemic HL-60 cells grown in RPMI1640 medium supplemented with 10% fetal bovine serum. When 0.5 mL of cell suspension was applied to the metal plates, cells were precipitated on the surface of the metal plate within 10 min. Contact with Cu induced a rapid decline of cell viability, the smear pattern of DNA fragmentation, and only minor activation of caspase-3. These effects were accompanied by a progressive decrease in the extracellular concentration of methionine, cysteine and histidine, with a corresponding increase in the concentration of methionine sulfoxide. Electron microscopy showed that contact with Cu induced vacuolization and cytoplasmic damage, prior to nuclear damage, without affecting the cell surface microvilli or mitochondrial integrity. Contact with the other metals did not induce such changes during the 3 h incubation, nor was any hormetic response (beneficial action at lower concentration) observed in the cells with any metals. Addition of N-acetyl-L-cysteine (4-5 mM) almost completely abrogated the Cu-induced cytotoxicity, whereas sodium ascorbate (0.1-0.5 mM) and catalase (6,000(1)-30,000 units/mL) were ineffective. Numerous serum proteins were adsorbed to the Ag plate, while bovine serum albumin was the major protein adsorbed to other metal plates. The present study suggests that direct contact with Cu induced non-apoptotic cell death by an oxidation-involved mechanism. The present model system may be applicable to the study of the interaction between cells and dental restorative materials.

Quantitative structure-cytotoxicity relationship analysis of coumarin and its derivatives by semiempirical molecular orbital method.

A semiempirical molecular orbital method (CAChe) was applied to delineate the relationship between cytotoxicity against the human squamous cell carcinoma line HSC-2 (evaluated by 50% cytotoxic concentration, CC50) of 20 coumarin (2H-pyran-2-one) derivatives and twelve physical parameters (descriptors) calculated by the CONFLEX/PM3 method. There was a highly significant correlation between the CC50 and ionization potential, highest occupied molecular orbital (HOMO) energy, difference between electron energy of HOMO and electron energy of lowest unoccupied molecular orbital (LUMO), or absolute hardness (r2=0.756 - 0.802). On the other hand, there was no significant correlation between the CC50 and heat of formation, stability of hydration, dipole moment, electron affinity, or LUMO energy (r2=0.13.- 0.36). When the CC50 was plotted vs. log P, a parabolic curve was produced, with a maximum cytotoxicity (or the least CC50 value) at log P of 2.5. The present study demonstrated that hardness and softness, other than the electron accepting and donating properties, are important factors in estimating the cytotoxic activity of coumarin derivatives.

Interfacial behavior of fatty-acylated sericin prepared by lipase-catalyzed solid-phase synthesis.

Fatty-acylated sericin {1:0.7 molar ratio of sericin (Mr 18,700) to oleic acid} was prepared by lipase-catalyzed solid-phase synthesis in n-hexane containing oleic acid to endow sericin with interfacial properties. Acylation with oleic acid was confirmed by 1H-NMR. The fatty-acylated sericin exhibited superior emulsifying activity index and emulsion stability in the presence of 0-0.5 M NaCl, in a temperature range of 30-80 degrees C and pH range of 2-7, as compared with the control sericin. The fatty-acylated sericin (1:0.4 molar ratio) prepared by using low-molecular-weight sericin (Mr 5,000) also exhibited superior emulsifying properties. The affinity of the fatty-acylated sericin to a hydrophobic surface as evaluated by a biomolecular interaction analyzer was about twice as much as that of the control sericin. The fatty-acylated sericin showed retarded water vaporization, similar to the control sericin, indicating good retention of moistness, and was adsorbed four times as much to defatted wool with little desorption as compared with the control sericin.

Thermal behavior of fowl feather keratin.

Differential scanning calorimetry (DSC) was applied to elucidate the thermal behavior of fowl feather keratins (barbs, rachis, and calamus) with different morphological features. The DSC curves exhibited a clear and relatively large endothermic peak at about 110-160 degrees C in the wet condition. A considerable decrease in transition temperature with urea and its helical structure content estimated by Fourier transform infrared spectroscopy (FT-IR), and the disappearance of one of the diffraction peaks with heating at 160 degrees C for 30 min, indicated that DSC could be used to evaluate the thermal behavior of keratin. Barbs showed a lower denaturation temperature than rachis and calamus. The pulverized samples showed a slightly higher denaturation temperature than the native samples. In the dry condition, thermal transition occurred in a markedly higher temperature region close to 170-200 degrees C. It is hence concluded that fowl feather keratins have very high thermal stability, and that the elimination of water brings about even greater thermal stability.

Effect of antioxidants, oxidants, metals and saliva on cytotoxicity induction by sodium fluoride.

We have recently found that millimolar concentrations of sodium fluoride (NaF) induced apoptotic cell death, characterized by caspase activation and DNA fragmentation, in tumor cell lines. This finding paved the way to investigating the interaction between NaF and the oral environment. As an initial step, we investigated redox compounds, metals and saliva, which may modify the cytotoxic activity of NaF against a human oral squamous cell carcinoma cell line (HSC-2). The minimum exposure time to NaF required for cytotoxicity induction was 8 hours. Noncytotoxic concentrations of antioxidants (sodium ascorbate, gallic acid, epigallocatechin gallate, chlorogenic acid, curcumin, superoxide dismutase, catalase), oxidants (hydrogen peroxide, sodium hypochlorite), metals (CuCl, CuCl2, FeCl2, FeCl3, CoCl2) or saliva neither protected against, nor enhanced the cytotoxic activity of NaF. Cytotoxic concentrations of these compounds produced somewhat additive, but not synergistic, effects on the cytotoxicity of NaF. ESR analysis demonstrated that NaF did not apparently change the radical intensity of sodium ascorbate and gallic acid, measured under alkaline conditions. During the cell death induction in human promyelocytic leukemia HL-60 cells by NaF, the consumption of glucose rapidly declined, followed by a decline in the consumption of major amino acids. The present study suggests that the cytotoxic activity of NaF is not regulated by the redox mechanism, but rather linked to the rapid decline in glucose consumption at early stage.

Effect of endodontic agents on cytotoxicity induction by sodium fluoride.

We investigated six endodontic agents for their ability to induce apoptosis and modify the cytotoxic activity of NaF against human squamous cell carcinoma (HSC-2) and human promyelocytic leukemia (HL-60) cell lines. Four Group I agents (Form Cresol, Cam Phenic, Eucaly Soft, GC Fuji Varnish), but not two Group II agents (Caviton, Canals-N), induced internucleosomal DNA fragmentation and activated caspases 3, 8 and 9 in HL-60 cells. Only Cam Phenic among these agents additively enhanced the cytotoxic activity of NaF in HSC-2 and HL-60 cells. Form Cresol and Cam Phenic reduced the glucose consumption at early stage, possibly due to their toxic effect. Amino acid analysis suggests that the higher cytotoxicity of Form Cresol may be derived, at least in part, from its oxidizing action.

Effects of isoflavones from Sophora species on the growth and activation of a mouse macrophage-like cell line.

We investigated the effect of eleven isoflavones on the growth and activation of mouse macrophage-like Raw 264.7 cells. The study of structure-activity relationship suggests that both hydrophilic (hydroxyl) and hydrophobic (prenyl) groups within isoflavone molecules are the determinants for the induction of cytotoxic activity. When hydrophobicity was assessed by octanol-water partition coefficient (log P), the maximum cytotoxic activity was observed at a log P value above 2.5. All isoflavones did not significantly stimulate the nitric oxide (NO) production by Raw 264.7 cells, but reduced the NO production by lipopolysaccharide (LPS)-stimulated Raw 264.7 cells, at cytotoxic concentrations. Amino acid analysis in the culture medium demonstrated that isoflavones significantly inhibited the LPS-stimulated production of citrulline and asparagine. Isoflavones inhibited the LPS-stimulated NO production more efficiently than citrulline and asparagine production, possibly due to their NO scavenging activity. These data suggest that the inhibiton of LPS action by isoflavones may be coupled with their cytotoxic activity.

Stimulation of arginine consumption and asparagine production in LPS-activated macrophages.

Changes in amino acid utilization during lipopolysaccharide (LPS)-induced activation of mouse macrophage-like cells Raw264.7 were investigated. Amino acids in the medium and cell fractions were extracted by 5% trichloroacetic acid and quantitated by amino acid analyzer. Glutamine was utilized by cells at the highest rate, followed by serine and arginine, a precursor of nitric oxide (NO). When Raw264.7 cells were incubated with 10 or 100 ng/mL LPS, the consumption of arginine and the production of citrulline, nitric oxide (NO) and asparagine were significantly increased. The intracellular amino acid concentration was not significantly changed. These data suggest that arginine consumption and asparagine production might be possible markers of macrophage activation.