Effects of eight antibacterial agents on cell survival and expression of epithelial-cell- or cell-adhesion-related genes in human gingival epithelial cells.
OBJECTIVE
Our previous studies suggest that little adverse effect on the growth of the periodontal ligament would be expected, if tetracycline, minocycline, ofloxacin, roxithromycin, clarithromycin, and azithromycin were topically administered to the periodontal pocket at their MIC90 doses required to inhibit the growth of 90% of periodontopathic bacteria, including Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans. In the present study, we investigated the cytocidal effects of eight antibacterial agents on the human gingival epithelial cell line NDUSD-1. We also used NDUSD-1 cells to examine the effects of these agents on the mRNA and protein expressions of genes associated with the proliferation, differentiation, or cellular adhesion important to the epithelial regeneration of the periodontal attachment.


METHODS
The cytocidal effect of the test agents was measured as a decrease in cell survival. To obtain a quantitative measure of the cytocidal effect, the LD50, i.e. the concentration which results in a 50% decrease in cell survival relative to the controls, was extrapolated from the concentration-response curves. The effects of the agents on the mRNA and protein expressions in NDUSD-1 cells were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses, respectively.


RESULTS
The cytocidal effect increased in a concentration-dependent manner as the concentration of each of the eight test agents increased. The order of the agents according to their cytocidal effects (LD50) was minocycline > tetracycline > enoxacin > clarithromycin > roxythromycin approximately ofloxacin > azithromycin > erythromycin. The cytocidal effects of minocycline, tetracycline, enoxacin, clarithromycin, roxythromycin, ofloxacin, and azithromycin ranged from 1.2 to 23.2 times greater than that of erythromycin. The maximum non-cytocidal concentrations (MNCCs) of these agents for NDUSD-1 cells were: 0.3 microm for minocycline, 1 microm for tetracycline, 3 microm for ofloxacin and erythromycin, 10 microm for enoxacin, clarithromycin, and azithromycin, and 100 microm for roxythromycin. The MNCCs of ofloxacin, azithromycin, clarithromycin, and roxythromycin were greater than their MIC90 concentrations for periodontopathic bacteria described above. The effects on the mRNA and protein expressions of epithelial-cell- or cell-adhesion-related genes were examined in NDUSD-1 cells exposed to clarithromycin, roxythromycin, ofloxacin, and azithromycin at their MNCCs. None of the agents affected the mRNA expressions of five genes: keratinocyte growth factor receptor, keratin 18, integrin beta1, integrin beta4, and laminin 5gamma2. Clarithromycin and ofloxacin slightly decreased the protein expression of integrin beta4. Roxythromycin markedly decreased the protein expressions of integrin beta4 and laminin 5gamma2. Azithromycin had little inhibitory effects on the protein expressions of any of the five genes.


CONCLUSIONS
These results suggest that little, if any, adverse effects on growth, differentiation, and adhesion of basal epithelial cells would be expected with topical administration of clarithromycin, ofloxacin or azithromycin to the periodontal pocket at a dose equivalent to the MIC90. It is important to note, however, that the extrapolation of these findings to in vivo conditions has yet to be undertaken.
Inoue
Kiichiro
K
Oral and Maxillofacial Surgery, The Nippon Dental University Hospital at Tokyo, Japan.
Kumakura
Shin-Ichi
S
Uchida
Minoru
M
Tsutsui
Takeki
T
D000900:Anti-Bacterial Agents / Q000633:toxicity*; D017963:Azithromycin / Q000633:toxicity; D002448:Cell Adhesion / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics; D017291:Clarithromycin / Q000633:toxicity; D015365:Enoxacin / Q000633:toxicity; D004847:Epithelial Cells / Q000187:drug effects; D004917:Erythromycin / Q000633:toxicity; D005346:Fibroblast Growth Factors / Q000235:genetics; D005881:Gingiva / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D016023:Integrins / Q000235:genetics; D007633:Keratins / Q000235:genetics; D007797:Laminin / Q000235:genetics; D007928:Lethal Dose 50; D008911:Minocycline / Q000633:toxicity; D015242:Ofloxacin / Q000633:toxicity; D011506:Proteins / Q000187:drug effects / Q000235:genetics*; D015575:Roxithromycin / Q000633:toxicity; D013752:Tetracycline / Q000633:toxicity
D000900:Anti-Bacterial Agents; D016023:Integrins; D007797:Laminin; D011506:Proteins; D015575:Roxithromycin; D015365:Enoxacin; D005346:Fibroblast Growth Factors; D004917:Erythromycin; D007633:Keratins; D017963:Azithromycin; D015242:Ofloxacin; D013752:Tetracycline; D008911:Minocycline; D017291:Clarithromycin