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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.

Pretreatment of mice with lyophilized hot water extracts of five poly-herbal formula protected them from lethal infection by E. coli. ESR spectroscopy shows that these extracts produced radicals under alkaline condition, and scavenged radicals such as superoxide anion, hydroxyl radical and nitric oxide (NO) radical. There was a positive relationship between their radical intensity and radical scavenging activity. Among the extracts, HD-02 efficiently inhibited the production of NO and citrulline, and the expression of inducible NO synthase (iNOS) mRNA by LPS-stimulated mouse macrophage-like cells Raw 264.7. DLH-3073 not only inhibited the LPS-stimulated NO production at lower concentration, but also induced NO production at higher concentrations, suggesting the presence of two different antagonizing components in the DLH-3073 extract. These data suggest that poly-herbal extracts may alleviate radical-mediated diseases.

Lignins, tannins and flavonoids are commonly found polyphenols. Among these polyphenols, lignins, polymers of phenylpropenoids complexed with polysaccharides, were the least cytotoxic and most potently stimulated the production of nitric oxide (NO), citrulline and asparagine by mouse macrophage-like Raw 264.7 cells. The maximum production of these substances reached the level attained by lipopolysaccharide (LPS). However, epigallocatechin gallate, phenylpropenoid monomers (ferulic acid, caffeic acid) and gallic acid (component unit of tannin) were inactive. These data suggest that the macrophage-stimulation activity of polyphenols depends, at least in part, on their molecular weight or structural configuration. There was a positive relationship between the extent of asparagine production and that of NO or citrulline. Western blot analysis demonstrated that both lignins and LPS elevated the cellular level of asparagine synthetase. The present study suggests the possible link between the stimulated asparagine production and macrophage activation.

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.

We investigated the effect of 2 flavanones and 8 chemically-defined prenylflavanones on the growth and activation of mouse macrophage-like Raw 264.7 cells. Amino acid analysis in the culture medium demonstrated the rapid consumption of serine and glutamine by Raw264.7 cells, suggesting the necessity to supplement these amino acids for the prolonged culture. Naringenin and hesperetin showed little or no cytotoxic activity. However, addition of the isoprenyl group (sophoraflavanone B, euchrestaflavanone A) or the lavandulyl and hydroxyl group (sophoraflavanone G) significantly enhanced the cytotoxic activity. The cytotoxic activity of these compounds was significantly influenced by both log P value and ionization potential. These compounds slightly, but significantly, reduced both nitric oxide (NO) and tumor necrosis factor (TNF) production by lipopolysaccharide (LPS)-stimulated Raw 264.7 cells, regardless of their cytotoxic activity. These data suggest that the macrophage inhibitory effect of prenylflavanones might not be related to their cytotoxic activity.

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.