Abstract
Background: We recently reported that eugenol exerted comparable cytotoxicity towards human normal and tumor cells. In the present study, we investigated the effect of eugenol on interleukin-8 (IL-8) production by IL-1β-stimulated oral cells. Materials and Methods: The viable cell number was determined by direct cell counting with a hemocytometer after trypsinization. IL-8 released into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). Results: IL-1β (5 ng/ml) induced two orders of magnitude higher production of IL-8 by human cultured cells than unstimulated cells. Upon IL-1β stimulation, both gingival fibroblasts (HGF) and periodontal ligament fibroblasts (HPLF) produced the greatest amounts of IL-8 (approximately 200-300 ng/ml), followed by pulp cells (HPCs) (approximately 40-50 ng/ml), whereas skin keratinocyte (HaCat) and oral squamous cell carcinoma cells (HSC-2, HSC-4) produced much less IL-8 (less than 15 ng/ml). The production of IL-8 depended on growth factor(s), since the omission of fetal bovine serum from the culture medium resulted in an approximately 90% decline of IL-8 production. Eugenol (5-500 μM) significantly stimulated IL-8 production in HGF cells, but had bi-modal effects on HPCs, causing slight stimulation at lower concentration (5 μM) and a significant inhibition at higher concentration (500 μM), regardless of the presence or absence of serum. Eugenol exerted similar effects on lipopolysaccharide-stimulated HGFs and HPCs. Conclusion: These results demonstrate that an anti-inflammatory effect of eugenol is observed in HPCs, but not in HGFs. The narrow therapeutic range of eugenol suggests the importance of careful usage of this compound for dental treatment.
Eugenol (Figure 1) is a component of dental cements, sealers and dental impression materials, and has antiseptic, analgesic and sedative actions. It has been reported that eugenol induced apoptosis of human promyelocytic leukemia (1), colon cancer (2) and breast cancer cells (3), possibly by elevating intracellular reactive oxygen species (ROS). However, studies on the cytotoxicity of eugenol against oral cells are limited (4-8). We recently reported that treatment of normal oral human cells (gingival fibroblast, HGF; pulp cells, HPC; periodontal ligament fibroblast, HPLF cells) and oral squamous cell carcinoma cell lines (HSC-2, HSC-4) with eugenol for more than 4 h, induced irreversible non-apoptotic cell death. Eugenol did not exhibit any apparent tumor specificity, nor hormetic growth stimulation, nor did it protect cells from UV-induced damage (9). For the safe use of eugenol in dentistry, it is crucial to investigate the effect of this dental compound on oral cells.
We have reported that treatment of HGFs with interleukin (IL)-1β resulted in two orders of magnitude higher production of IL-6, IL-8, Monocyte Chemoattractant Protein-1 (MCP-1) and prostaglandin (PG)E2 compared with unstimulated cells, but not of tumor necrosis factor (TNF)-α and nitric oxide (NO) (10). Using this in vitro gingivatitis model, the anti-inflammatory effect of eugenol on IL-8 production was re-evaluated.
Materials and Methods
Materials. The following chemicals and materials were obtained from the indicated companies: Dulbecco's modified Eagle's medium (DMEM) from Gibco BRL, Grand Island, NY, USA; fetal bovine serum (FBS), eugenol (MW=164), dimethylsulfoxide (DMSO) from Wako Pure Chemical, Osaka, Japan; lipopolysaccharide (LPS) from Escherichia coli (serotype 0111:B4) from Sigma Chem. Ind., St. Louis, MO, USA; IL-1β was purchased from R&D Systems, Minneapolis, MN, USA; 6-well plates from Becton Dickinson, Franklin Lakes, NJ, USA; HuMedia-KG2 from Kurabo, Osaka, Japan. Eugenol was dissolved in DMSO at 200 mM before use, and diluted with medium.
Cell culture. HaCat cells were provided by Deutsches Krebsforschungszentrum (German Cancer Research Center), Heidelberg, Germany. Human OSCC cell lines (HSC-2, HSC-4) were kindly provided by Professor Nagumo, Showa University, Japan. Normal human oral cells, HGF, HPC and HPLF, were prepared from periodontal tissues, as previously reported (9), and used at 8-15 population doubling levels (PDL). All these adherent cells were cultured in DMEM supplemented with 10% heat-inactivated FBS. Human skin keratinocytes (HEK-a) were purchased from Kurabo Ind. Ltd., Osaka, Japan and cultured in HuMedia-KG2 supplemented with insulin, human recombinant epidermal growth factor (EGF), hydrocortisone, gentamicin, amphotericin B and bovine pituitary extract (BPE), as instructed by the supplier. DMSO used at concentrations below 0.25% did not affect the viability of the cells.
Assay for cytotoxic activity. All of the cells were inoculated at 6×103 cells/well in 6-well plates (Becton Dickinson Labware, NJ, USA), unless otherwise stated. After 48 h, the medium was removed by suction with an aspirator, and replaced with 2 ml of fresh medium containing different concentrations of eugenol. After 30 min, IL-1β (final, 5 ng/mL) was added. The cells were incubated for 24 h, and the relative viable cell number was then determined by direct cell counting with a hemocytometer, after trypsinization
IL-8 determination. The IL-8 in the culture medium was determined by ELISA, according to the manufacturer's instruction (Quantikine ELISA kit; R&D Systems) (10).
Statistical analysis. The difference between two groups was evaluated by Student's t-test. The value of statistical significance was set at the 0.01 level.
Results
Optimal conditions for IL-8 production. IL-1β dose-dependently and significantly (p<0.01) stimulated the IL-8 production by HGFs, reaching a plateau level at 5 ng/ml (Figure 2). A higher concentration of IL-1β rather slightly reduced IL-8 production. Based on these data, 5 ng/ml of IL-1β were used for subsequent experiments.
Preferential IL-8 production by fibroblasts. Upon IL-1β stimulation, two fibroblast cell lines (HGF, HPLF) produced the greatest amounts of IL-8 (approximately 200-300 ng/ml), followed by HPCs (approximately 40-50 ng/ml), whereas epithelial cells such as human skin keratinocyte (HaCat) and oral squamous cell carcinoma cell lines (HSC-2, HSC-4) produced much less amounts (less than 15 ng/ml) (Figure 3A). We also found that human skin keratinocytes (HEKa) also had a low level of IL-8 production upon IL-1β stimulation (HaCaT<HEKa<HPC) (data not shown). Similar trends were observed when the IL-8 production was expressed on a per cell basis (Figure 3B), where the difference between fibroblasts (high IL-8 production) and non-fibroblasts (low IL-8 production) was more dramatic.
The production of IL-8 depended on growth factor(s), since the omission of FBS from the culture medium resulted in an approximately 90% decline of IL-8 production in all tested cells (comparison of A and B, C and D in Figure 4).
Evaluation of the anti-inflammatory effect of eugenol. Eugenol (5-500 μM) significantly (p<0.01) enhanced IL-1β-stimulated IL-8 production in HGFs, regardless of the presence or absence of FBS (Figure 4). At 500 μM, IL-8 production nearly doubled (Figure 4A, B). Eugenol (50 μM)-alone did not induce IL-8 production. These data suggest that eugenol may exert a pro-inflammatory, but not an anti-inflammatory effect on HGFs.
On the other hand, eugenol had bi-modal actions, depending on its concentration, on HPCs. Eugenol slightly stimulated IL-8 production at lower concentration (5 μM), but rather significantly (p<0.01) inhibited it at a higher concentration (500 μM), regardless of the presence or absence of FBS (Figure 4). Since this concentration was only slightly lower than its 50% cytotoxic concentration (763 μM), the apparent decline of IL-8 production at higher eugenol concentration may be due, at least in part, to its cytotoxicity (9).
As compared with IL-1β, LPS had a much lower ability to induce IL-8 production by HGFs and HPCs, confirming our previous findings (10). Eugenol slightly enhanced LPS-stimulated IL-8 production in HGFs (A), but rather inhibited IL-8 production in HPCs (Figure 5).
Discussion
The present study demonstrated, to our knowledge for the first time, that eugenol did not inhibit, but rather enhanced IL-1β-stimulated IL-8 production by HGFs. This suggests that eugenol may in fact aggravate gingivatitis. However, the chance that this happens may not be that great, considering that eugenol is not treated directly to gingival tissue. On the other hand, we found that eugenol showed bi-modal actions, by stimulating or inhibiting the IL-8 production at lower and higher concentrations. The reason for this bi-modal action of eugenol is not clear; however, it may be due to the heterogeneous populations of HPCs that are comprised of fibroblasts, undifferentiated mesenchymal cells and defense cells (macrophages and lymphocytes). In the early stages of culture, fibroblasts, which are most reactive to IL-1β, comprise a significant proportion of HPCs, but with subculturing, the proportion of fibroblasts declines, accompanied by the decline of IL-8 production (data not shown). We found that immortalized HPCs that lack fibroblasts lost the ability to produce IL-8 upon IL-1β stimulation (data not shown), further confirming that fibroblasts are the major producer of IL-8. At present, it is not clear whethert the slight decline of IL-8 production at higher concentrations of eugenol is due to its anti-inflammatory effects, which have been reported in other systems (12, 13), or is a secondary result of growth inhibition. There is another possibility that this activity of eugenol may be due to its antioxidant action, since this compound exhibited one order higher superoxide anion scavenging activity than 2-t-butyl-4-methoxyphenol and 2-methoxy-4-methylphenol (5). Further study is needed to test these possibilities. At present, whether eugenol acts extracellularly or intracellularly is not clear. Metabolomic analysis may be useful to identify the target molecules of eugenol.
In conclusion, the present study demonstrated that non-cytotoxic concentrations of eugenol (5-50 μM) do not inhibit IL-8 production by gingival fibroblast and pulp cells, whereas a near-cytotoxic concentration of eugenol slightly (only 30%) reduced IL-8 production by pulp cells. The narrow therapeutic range of eugenol suggests the importance of careful usage of this compound for dental treatment.
- Received November 29, 2012.
- Revision received January 15, 2013.
- Accepted January 16, 2013.
- Copyright © 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved