Two mechanisms of oral malodor inhibition by zinc ions

Abstract Objectives The aim of this study was to reveal the mechanisms by which zinc ions inhibit oral malodor. Material and Methods The direct binding of zinc ions to gaseous hydrogen sulfide (H2S) was assessed in comparison with other metal ions. Nine metal chlorides and six metal acetates were examined. To understand the strength of H2S volatilization inhibition, the minimum concentration needed to inhibit H2S volatilization was determined using serial dilution methods. Subsequently, the inhibitory activities of zinc ions on the growth of six oral bacterial strains related to volatile sulfur compound (VSC) production and three strains not related to VSC production were evaluated. Results Aqueous solutions of ZnCl2, CdCl2, CuCl2, (CH3COO)2Zn, (CH3COO)2Cd, (CH3COO)2Cu, and CH3COOAg inhibited H2S volatilization almost entirely. The strengths of H2S volatilization inhibition were in the order Ag+ > Cd2+ > Cu2+ > Zn2+. The effect of zinc ions on the growth of oral bacteria was strain-dependent. Fusobacterium nucleatum ATCC 25586 was the most sensitive, as it was suppressed by medium containing 0.001% zinc ions. Conclusions Zinc ions have an inhibitory effect on oral malodor involving the two mechanisms of direct binding with gaseous H2S and suppressing the growth of VSC-producing oral bacteria.


Introduction
Oral malodor is primarily the result of microbial metabolism of amino acids from local debris in the oral cavity 19 . The primary compounds of oral malodor are volatile sulfur compounds (VSCs), such as hydrogen sulfide (H 2 S), methyl mercaptan (CH 3 SH), and dimethyl sulfide (CH 3 SCH 3 ) 24 . Periodontopathic anaerobic bacteria, such as Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia, Fusobacterium nucleatum, and Eubacterium can produce large amounts of H 2 S and CH 3 SH from cysteine, methionine, or serum proteins 16,17 .
Various anti-malodor agents for oral use have been introduced and have proven to be effective in reducing VSC concentration in the oral cavity.
Antimicrobial agents such as chlorhexidine, triclosan, and cetylpyridinium chloride can reduce oral malodor by reducing the number of microorganisms present in the mouth 3 . Chlorine dioxide has also been shown to reduce oral malodor by chemically neutralizing VSCs 12 . Natural ingredients, such as hinokitiol, green tea powder, and Eucalyptus extract, also reduce oral malodor through various antibacterial mechanisms 7,11,22 .
Zinc ions are often found in commercial antimalodor mouthwashes in combination with other active ingredients. A combination of zinc ions and chlorhexidine or cetylpyridinium chloride was reported to inhibit VSC production synergistically 29 .
We considered two mechanisms of oral malodor inhibition by zinc ions. The first is that zinc ions have a strong affinity for the thiol groups present in VSCs 28 .
Zinc ions exhibit immediate inhibitory effects on VSC production compared to chlorhexidine 30 , by effectively and directly reducing the activities of VSCs. The second is that zinc ions have an antibacterial effect. and P. intermedia were cultivated in BHI medium with hemin (5 µg/mL) and vitamin K (1 µg/mL).

Bacterial cultures were incubated at 37°C
anaerobically until full growth, then suspended in fresh BHI medium or fresh BHI medium with hemin and vitamin K to an optical density at 600 nm (

Statistical analysis
The Mann-Whitney U-test was used to evaluate the direct inhibitory effects of metal ions on gaseous H 2 S compared with 10 -5 % NaHS.nH 2 S solution, and the inhibitory effects of zinc ions on the growth of oral bacteria compared with that in the 0% ZnCl 2 medium.
Differences were considered to be significant when P<0.05. Statistical evaluations were carried out using the R software package, ver. 3.4.0 (http://www.Rproject.org).
* P<0.05 compared with the 10-5% NaHS.nH 2 S solution (Mann-Whitney U-test). Overall, four ions, zinc, copper, cadmium, and silver, were considered to have excellent inhibitory effects on

Discussion
Among the nine metal ions examined in this study, calcium, iron, zinc, cadmium, copper, and silver had direct inhibitory effects on H 2 S volatilization. These metals have been widely used in dental materials,  This study determined that zinc ions inhibit oral malodor by two mechanisms, direct inhibition of H 2 S volatilization and antimicrobial activity. Both mechanisms may be caused by the strong affinity for the thiol groups characteristic of metal ions, including zinc ions. The direct reaction experiment between gaseous H 2 S and metal ions verified the first mechanism. Concerning the second mechanism, it has been reported that sulfur-containing amino acids such as cysteine and glutathione neutralized the activity of silver ions on bacteria 5,13 . This finding shows that metal ions react with sulfur-containing intracellular and extracellular amino acids, causing functional failure of proteins and resulting in damage to bacterial cells. In addition, functional failure of proteins may lead to the production of reactive oxygen species. In fact, the antimicrobial action of silver ions increased in the presence of oxygen 5 , which suggests that the generation of reactive oxygen species is related to the antimicrobial activity of metal ions.
There are several limitations to this study. First, the main VSC compounds related to odor in the oral cavity are H 2 S and CH 3 SH. Only the effect of zinc ions on H 2 S production was evaluated, and the effect on CH 3 SH should also be assessed. Second, no sustainability assessment of the inhibitory effect of zinc ions on H 2 S volatilization was performed in this study.
A previous clinical trial reported that the inhibitory effect of rinsing with 0.1% ZnCl 2 on H 2 S continued for only 1 hour, less than those of SnF 2 and CuCl 2

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. The longitudinal evaluation of the binding reaction between zinc ions and H 2 S should be performed in vitro. Finally, the antimicrobial mechanism of zinc ions has been compared to the characteristics of silver ions, which have been studied well. The reason why the effect of zinc ions is weaker than those of silver and copper ions remains unclear. Further investigations into the chemical reactivity between zinc ions and thiol groups as well as the specific active sites of antimicrobial activity of zinc ions are necessary.
In conclusion, zinc ions, which exhibit an inhibitory effect on VSCs and are incorporated into products for oral malodor prevention, employ the two mechanisms of direct binding with gaseous H 2 S and antimicrobial activity. In particular, the growth of bacteria related to VSC production was inhibited at a lower concentration of ZnCl 2 compared with bacteria not related to VSC production.