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J Dent Res 82(10): 849-853, 2003
© 2003 International and American Associations for Dental Research

RESEARCH REPORT
Clinical

Detectable Dioxins in Human Saliva and Their Effects on Gingival Epithelial Cells

T. Ogawa1,*, Y. Asai1, M. Yamashita2, and T. Takasuga2

1 Department of Oral Microbiology, Asahi University School of Dentistry, 1851-1 Hozumi, Mizuho, Gifu 501-0296, Japan; and
2 Shimadzu Techno-Research, Inc., 1 Shimoai-cho, Nishinokyo, Nakagyo-ku, Kyoto 604-8436, Japan;

*corresponding author, tomo527{at}dent.asahi-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dioxin, a powerful hormone-disrupting chemical, exhibits serious health effects when it reaches body fat. Here we analyzed coplanar polychlorinated biphenyls (PCBs) and polychlorinated-dibenzo-p-dioxins (PCDDs) in human saliva as compared with blood specimens, and examined their effects on human gingival epithelial cells (HGEC). High levels of tri- and tetrachlorinated PCBs were found in saliva, whereas we detected predominantly hexa- and heptachlorinated PCBs in blood. Among PCDDs, the saliva and blood specimens contained mainly 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin (OCDD). Among the toxic dioxins proposed by the World Health Organization (WHO) in 1998, 2,3',4,4',5-pentachlorobiphenyl (PCB 118) and OCDD, which were mainly found in saliva, significantly induced IL-8 production in HGEC. Furthermore, these two dioxins markedly augmented IL-8 production stimulated with fimbriae from Porphyromonas gingivalis, which is well-known as a pathogenic factor in periodontal diseases. These results suggest that dioxins in saliva may be a risk factor for periodontal diseases.

KEY WORDS: dioxins • coplanar polychlorinated biphenyls • saliva • human gingival epithelial cells • aryl hydrocarbon receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dioxins such as coplanar polychlorinated biphenyls (PCBs) and polychlorinated-dibenzo-p-dioxins (PCDDs) are chemically inert and lipid-soluble compounds that tend to accumulate in the food chain and animals (Svensson et al., 1994). The exceptionally dielectric and lubricating properties of PCBs resulted in their extensive use during the 1950s and 1960s as lubricants in electrical generators and transformers (Safe, 1984). However, the use of these compounds has ceased, because their presence in the environment has caused concerns related to toxicity in humans and a long half-life (Clarke et al., 1984), since 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like polyhalogenated aromatic hydrocarbons remaining in the environment have been found in animals and exposed individuals (McFarland and Clarke, 1989). As a result, the general population may be associated with a spectrum of toxic effects on reproductive, immune, and endocrine systems (Elferink and Whitlock, 1994; Kerkvliet, 1995).

Porphyromonas gingivalis, a black-pigmented anaerobic Gram-negative rod, has been associated with the development and progression of periodontal diseases (Slots and Listgarten, 1988), and has been shown to possess short and hairlike structures, fimbriae, on its cell surfaces (Ogawa et al., 1991b). Among the various bacterial cell-surface components, fimbriae are known to be a specific adherent factor in its microbial etiology (Pearce and Buchanan, 1980).

Since approximately 8-10 mg of lipid components were contained in 100 mL of saliva (Slomiany et al., 1982), it is stipulated that lipid-soluble dioxins are included in saliva. In the present study, we examined whether PCBs and PCDDs were measurable in saliva, as well as the influence of these compounds contained in saliva on human gingival epithelial cells (HGEC) and their synergistic effects with P. gingivalis fimbriae.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
2,4,4'-trichlorobiphenyl (PCB 28), 2,3',4,4'-tetrachlorobiphenyl (PCB 66), 2,4,4',5-tetrachlorobiphenyl (PCB 74), 3,3',4,4'-tetrachlorobiphenyl (PCB 77), and 2,3',4,4',5-pentachlorobiphenyl (PCB 118) were obtained from AccuStandard Inc. (New Haven, CT, USA). 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin (OCDD) came from Wellington Laboratories Inc. (Ontario, Canada). These compounds were prepared at 10-3 M in dimethyl sulfoxide (Wako, Osaka, Japan).

Bacteria and Fimbrial Preparation
Porphyromonas gingivalis strain 381 was grown anaerobically in Gifu anaerobic medium (GAM) broth (Nissui, Tokyo, Japan) supplemented with hemin and menadione for 26 hrs at 37°C. Fimbriae were isolated and purified as described previously (Ogawa et al., 1989). Briefly, bacterial cells were suspended in Tris-buffered saline and gently pipetted, before being agitated. The supernatant obtained by centrifugation was subjected to ammonium sulfate precipitation, and the precipitate was then purified by DEAE-Sepharose column chromatography.

Subjects and Sampling
We collected whole saliva from eight healthy subjects, after receiving informed consent, into a glass bottle that had been washed with toluene followed by acetone. Peripheral blood was obtained from the same healthy subjects and stored in a plastic bag (Terumo, Tokyo, Japan). The study protocols were approved by the Ethical Committee for Clinical Research of Asahi University School of Dentistry.

Sample Clean-up
Approximately 50 g of each saliva specimen was diluted 10-fold in n-hexane-washed water. After the addition of 19 13C12-labeled markers of PCDDs and 22 markers of PCBs as an internal standard (Wellington Laboratories Inc.), the sample solutions were adsorbed onto octadecyl-silica (ODS; 120 Å, 50 µm) for removal of polar lipids and other impurities, and the eluted sample solution was partitioned 3x between dichloromethane and n-hexane-washed water. The concentrated sample extracts were then applied to a multilayered silica gel column (silica, 10% AgNO3/silica, silica, 22% H2SO4/silica, 44% H2SO4/silica, silica, 2% KOH/silica, silica) with hexane used for the mobile phase. Furthermore, clean-up for the PCDD analysis was performed with the use of activated carbon (Wakimoto and Tatsukawa, 1985; Stanley et al., 1988). Lipids in the blood specimens were also extracted, determined gravimetrically, and subjected to a column chromatographic clean-up procedure (Lamparski et al., 1979; Smith et al., 1984).

High-resolution Gas Chromatography (HRGC)/High-resolution Mass Spectrometry (HRMS) Analysis
Samples were concentrated to 30-100 µL after the addition of 4 congeners of 13C12-labeled PCBs and -1,2,3,4-TCDD as a syringe spike, and then the PCBs were analyzed with the use of HRGC (HP6890; Agilent Technologies, Palo Alto, CA, USA)/HRMS (AutoSpec Ultima; Micromass, Manchester, UK) with a DB-5MS column (60 m, 0.32 mm i.d., 0.25-µm film thickness; Agilent Technologies). The following cycle was used: 150°C for 1 min, then 20°C/min up to 185°C, 2°C/min up to 245°C with a three-minute hold, and 6°C/min up to 290°C. PCDD analysis was also performed on a HRGC/HRMS with SP2331 (60 m, 0.32 mm i.d., 0.20-µm film thickness; Supelco, Bellefonte, PA, USA) and DB-17HT (60 m, 0.32 mm i.d., 0.15-µm film thickness, Agilent Technologies) columns. The following cycles were used: 170°C for 3 min, 3°C/min up to 230°C with a three-minute hold, then 3°C/min up to 260°C (SP2331); 150°C for 3 min, 20°C/min up to 200°C, then 3°C/min up to 280°C (DB-17HT). The interface temperature was set 5-10°C higher than the maximum value of each temperature program. Helium was used as the carrier gas, and the electron impact ionization energy was 35-40 eV. The MS was operated in selected ion monitoring mode (SIM) at a resolution of > 10,000 (10% valley). Two predominant ions (M+, M+2, or M+4) were monitored for each congener group.

Cells
Establishment of HGEC has been described previously (Asai et al., 2001). The cells were subsequently maintained in a long-term culture with HuMedia-KG2 (Kurabo Biomedicals, Osaka, Japan). Heparinized venous blood drawn from healthy donors was subjected to fractionation with a Histopaque-1077 (Sigma, St. Louis, MO, USA) used to obtain human peripheral blood mononuclear cells (PBMC). The cells were suspended with RPMI1640 (Sigma) supplemented with 10% FBS (Sigma). The study protocols were approved by the Ethical Committee for Clinical Research of Asahi University School of Dentistry.

RT-PCR Analysis
Total cellular RNA from HGEC and human PBMC was isolated with Trizol (Life Technologies, Rockville, MD, USA), and RNase-free DNase (Takara Biochemicals, Shiga, Japan) was used to remove genomic DNA. Extracted RNA was reverse-transcribed into first-strand cDNA, and PCR amplification of cDNA was performed with oligonucleotide primers specific for aryl hydrocarbon receptor (AhR), 5'-CTTCCAAGCGGCATAGAGAC-3' and 5'-CTACTGTCTGGGGGAGACCA-3'; and ß-actin, 5'-GTGGGGCGCCCCAGGCACCA-3' and 5'-CTCCTTAATGT CACGCACGATTTC-3'.

Cytokine Production
HGEC (1 x 105 cells per well) were seeded in a 24-well flat-bottom microtiter plate (Falcon 3072; Becton Dickinson and Co., Lincoln Park, NJ, USA). After incubation for 16 hrs at 37°C in humidified air containing 5% (v/v) CO2, the monolayers were washed 3 times with PBS (Sigma). The cells were incubated with the indicated doses of test specimen for 24 hrs at 37°C, and then centrifuged at 12,000 x g for 5 min. The production of IL-8 in the culture supernatants was determined by an ELISA. The assays were performed according to the manufacturer’s instructions (ELISA kit system, Genzyme-Techne, Minneapolis, MN, USA), and results were determined with use of a standard curve prepared for each assay. Data were analyzed by a one-way analysis of variance (ANOVA) with the Bonferroni or Dunn method, and the results are presented as the mean ± standard error of the mean (SEM).

Cytotoxicity
The release of lactate dehydrogenase (LDH) from HGEC was measured in the culture supernatants by means of a colorimetric kit (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions. Background control of LDH activity was obtained by measurement of the culture medium. Low control of LDH activity was obtained with the use of culture supernatants released from the untreated cells, and high control of LDH activity was obtained with the use of whole-cell lysates. The percent cytotoxicity was determined according to the following calculation: [(release of LDH from stimulated cells - low control) / (high control - low control)] x 100.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Detectable Dioxins in Human Saliva
We found tri- and tetrachlorinated PCBs and lesser amounts of penta-, di-, and hexachlorinated PCBs in saliva, as well as high levels of hexa- and heptachlorinated PCBs in blood (Figs. 1A, 1BGo). Among the PCBs for which the World Health Organization (WHO) provided toxicity equivalency factor (TEF) definitions in 1998 (Van den Berg et al., 1998), PCB 118 was found predominantly in both saliva and blood specimens (Figs. 1C, 1DGo). Further, we clearly detected PCB 77 and 2,3,3',4,4'-pentachlorobiphenyl (PCB 105), as well as lower amounts of 2,3,3',4,4',5-hexachlorobiphenyl (PCB 156) in saliva, and high levels of PCB 156 and 2,3',4,4',5,5'-hexachlorobiphenyl (PCB 167) followed by 2,3,3',4,4',5'-hexachlorobiphenyl (PCB 157) in blood (Fig. 1BGo). The total amount of PCBs in saliva was approximately 50% of that in blood. Among the PCDDs given TEFs in 1998 (Van den Berg et al., 1998), OCDD was detected in both saliva and blood specimens (Figs. 1E, 1FGo), while 2,3,7,8-TCDD, a strongly toxic dioxin in humans and animals (Holsapple et al., 1991), was found in some blood specimens but not in saliva. The amount of PCDDs in blood was approximately 10 times greater than that in saliva.



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  		Figure 1. Measurement of dioxins in saliva specimens. Coplanar PCBs (A,B) and those with WHO-defined TEFs (C,D) in saliva (A,C) and blood (B,D) specimens were measured with the use of an HRGC/HRMS with a DB-5MS column. Twenty-two markers of PCBs were added to these specimens from eight subjects as internal standards. PCDDs with WHO-defined TEFs found in saliva (E) and blood (F) specimens were measured by means of an HRGC/HRMS with SP2331 and DB-17HT columns. Nineteen PCDD markers were added to these specimens as internal standards. The MS was operated in selected ion monitoring mode at a resolution of > 10,000. ND: not detected.

 
Expression of AhR mRNA
Dioxins are known to stimulate the expression of a variety of genes via AhR, a ligand-activated transcription factor that belongs to the basic-helix-loop-helix (bHLH) superfamily of DNA binding proteins (Gonzalez and Fernandez-Salguero, 1998). In the present study, we examined the expression of AhR mRNA in HGEC and human PBMC by RT-PCR. HGEC and human PBMC exhibited AhR mRNA expression; however, that of the former was weaker than that of the latter (Fig. 2Go). RT-PCR analysis of ß-actin expression confirmed the quality of all RNA preparations used for RT-PCR, and no band was detected in the non-RT sample.



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  		Figure 2. AhR mRNA expression in HGEC and human PBMC. AhR mRNA expression was analyzed by RT-PCR as described in MATERIALS & METHODS. The ß-actin gene was assayed as a positive control, and PCR products of non-RT samples were examined as a negative control. Following PCR, a 10-µL quantity of the total amplified product underwent electrophoresis on ethidium-bromide-stained 1% agarose gels, and was visualized under ultraviolet fluorescence. Data presented represent three independent experiments.

 
Induction of IL-8 Production in HGEC
We estimated that approximately 10-8 M of PCBs and 10-12 M of OCDD could be detected in saliva (Figs. 1A, 1B, 1CGo); thus, we examined the IL-8-producing activity in HGEC after incubation with those amounts of the dioxins (Fig. 3Go). PCB 118 and OCDD significantly induced IL-8 production in HGEC, whereas PCB 28, PCB 66, PCB 74, and PCB 77, trichlorinated and tetrachlorinated PCBs detected in the saliva specimens, induced scarcely any IL-8 production. Further, no cytotoxic effect by any test specimens on HGEC was observed (Fig. 3Go).



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  		Figure 3. IL-8-producing activity and cytotoxicity of PCBs and OCDD. HGEC were stimulated with 10-8 M of PCBs and 10-12 M of OCDD at 37°C for 24 hrs. After incubation, the supernatants were collected, and IL-8 production was determined by ELISA. Experiments were performed at least 3 times, with representative results presented. Each assay was done in triplicate wells, and the data are expressed as the mean ± SEM of results. Significant differences were seen between the groups with and without the test specimens (*P < 0.05). Cytotoxicity of the test specimens was also measured by LDH released into the culture supernatants. Percent cytotoxicity was calculated as detailed in MATERIALS & METHODS.

 
Effects of Dioxins in Saliva on IL-8-producing Activity in HGEC Stimulated with P. gingivalis Fimbriae
We previously demonstrated that P. gingivalis fimbriae induced IL-8 production in HGEC (Asai et al., 2001). To examine the influence of dioxins in saliva on periodontal tissues, we estimated IL-8 production in HGEC after stimulation with P. gingivalis fimbriae and PCB 118 or OCDD. The addition of either with P. gingivalis fimbriae resulted in a definite increase in the induction of IL-8 production in a dose-dependent manner (Fig. 4Go).



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  		Figure 4. PCB 118 and OCDD augmented IL-8-producing activities stimulated with P. gingivalis fimbriae. HGEC were incubated with PCB 118 (A), OCDD (B), or medium alone, together with the indicated doses of P. gingivalis fimbriae (Pg fim) at 37°C for 24 hrs. After incubation, the supernatants were collected, and IL-8 production was determined by ELISA. Experiments were performed at least 3 times, with representative results presented. Each assay was done in triplicate wells, and the data are expressed as the mean ± SEM of results. Significant differences were seen between the groups with and without PCB 118 or OCDD (*P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In studies of dioxin accumulation in the human body, blood specimens have often been used (Schecter et al., 1994; Svensson et al., 1994; Ayotte et al., 1997; Falk et al., 1999; Iida et al., 1999). However, investigations of that in the elderly and infants are few, because a large amount of blood is required for dioxin analysis. Thus, the population used for analyzing dioxins in blood is generally limited, whereas it is possible to conduct epidemiological investigations with no such limits by using saliva samples as test specimens. This is the first known attempt to detect dioxins in saliva, during which we clearly found PCBs and PCDDs in both saliva and blood specimens (Fig. 1Go). Tri- and tetrachlorinated PCBs were contained mainly in saliva, while hexa- and heptachlorinated PCBs were seen primarily in blood. Among the PCBs with TEFs defined by WHO in 1998, PCB 118 was found predominantly in saliva (Fig. 1CGo). In human blood analyses for a variety of PCBs, PCB 118 was also detected more frequently than other PCBs. Further, the total amount of PCDDs in blood was 10 times greater than that in saliva, whereas we detected predominantly OCDD in both saliva and blood specimens (Figs. 1E, 1FGo).

In periodontal tissues, HGEC have been shown to secrete various cytokines, including IL-8, which attracts and activates neutrophils in the inflammatory lesion (Bickel, 1993; Huang et al., 1998). Further, IL-8 has been shown to be localized in the gingival tissues of patients with periodontal diseases, and IL-8 mRNA levels are known to correspond to the severity of periodontal diseases (Tonetti et al., 1994). In the present study, both PCB 118 and OCDD significantly induced IL-8 production in HGEC under comparable conditions and concentrations in saliva (Fig. 3Go).

HGEC and human PBMC constitutively expressed mRNA for AhR as a dioxin receptor (Gonzalez and Fernandez-Salguero, 1998), though the expression in HGEC was weaker (Fig. 2Go). In addition, the IL-8-producing activities of HGEC induced by PCB 118 or OCDD were weaker than those of PBMC in a manner that corresponded to the level of AhR mRNA expression (data not shown). These results suggested that AhR in HGEC recognized PCB 118 and OCDD, and that these dioxins induced IL-8 production in saliva, similar to PBMC in blood.

P. gingivalis fimbriae are associated with the adherence of the bacterium to host target cells (Ogawa et al., 1991a). Further, the fimbrial protein possesses various functional properties, including the production of various cytokines in host cells such as human PBMC, gingival fibroblasts, and gingival epithelial cells (Ogawa et al., 1991a, 1997; Asai et al., 2001). These findings suggest that P. gingivalis fimbriae are an important pathogenic factor involved with the inflammation of periodontal tissues seen in patients with periodontal diseases. In the present study, we demonstrated that PCB 118 and OCDD were contained mainly in saliva and clearly augmented IL-8 production in HGEC stimulated with P. gingivalis fimbriae (Fig. 4Go). Recently, we demonstrated that P. gingivalis fimbriae activated HGEC and human monocytes through Toll-like receptor (TLR) 2, which is one of the pathogen-associated molecular pattern (PAMP) receptors (Asai et al., 2001; Ogawa et al., 2002). These results suggested that AhR and TLR signaling synergistically augmented IL-8 production in HGEC.

In conclusion, we determined that dioxins in human saliva as well as blood specimens were measurable. Among them, PCB 118 and OCDD were contained predominantly in saliva, and definitely induced IL-8 production in HGEC and augmented IL-8 production induced by P. gingivalis fimbriae. These results indicate that dioxins in saliva may induce a sustained production of pro-inflammatory cytokines in periodontal tissues, and may also be a risk factor for chronic inflammatory diseases including periodontal diseases.


   ACKNOWLEDGMENTS
 
This study was supported by Grants-in-Aid for Exploratory Research from The Ministry of Education, Culture, Sports, Science, and Technology of Japan (No. 14657474) and Miyata Research Foundation (A) of Asahi University (No. 98005).

Received April 30, 2003; Last revision July 23, 2003; Accepted July 24, 2003


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 ABSTRACT
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 MATERIALS & METHODS
 RESULTS
 DISCUSSION
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