Food and Chemical Toxicology 74 (2014) 139–148
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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x
Characterizing the immunological effects of oral healthcare ingredients using an in vitro reconstructed human epithelial model E. Hagi-Pavli a,*, D.M. Williams a, J.L. Rowland b, M. Thornhill c, A.T. Cruchley a a b c
Bart’s and the London School of Medicine and Dentistry, Queen Mary University, UK GSK Oral Healthcare R & D, Weybridge, Surrey, UK School of Clinical Dentistry, University of Sheffield, UK
A R T I C L E
I N F O
Article history: Received 24 March 2014 Accepted 18 September 2014 Available online 5 October 2014 Keywords: Irritant Inflammatory cytokines Sodium lauryl sulphate (SLS) Human oral mucosal model Sensitizer Cinnamic aldehyde
A B S T R A C T
Oral healthcare products are well tolerated and while adverse occurrences are rare there is still a need to explore the interaction between these products and the oral mucosa. This study assessed the effects of oral healthcare ingredients: sodium lauryl sulphate (SLS), a detergent; cinnamic aldehyde (CA), a flavouring agent; and cetylpyridinium chloride (CPC), an antiseptic, using a reconstructed human oral mucosal model (OMM). Differential release of inflammatory cytokines IL-1α, IL-8 and cytotoxicity was compared with other known irritants and sensitizers to identify a signature response profile that could be associated with oral mucosal irritation. Response profiles differed with irritants being more cytotoxic. CA and control sensitizers nickel sulphate (NiSO4) and 1-chloro-2,4-dinitrochlorobenzene (DNCB) released lower levels of IL-1α than CPC and control irritant benzalkonium chloride (BC), whereas the opposite was observed for IL-8. Significant levels of IL-8 and IL-1α were released with 5–15 mg/ml (0.5–1.5% w/v) SLS. Quantitative PCR indicated that cytokine release at lower SLS concentrations is not entirely due to cell necrosis but in part due to de novo synthesis. These findings suggest that the OMM can be used to predict oral irritation thus making it a potentially valuable model for screening new oral healthcare ingredients prior to clinical release. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction The use and range of oral health products available to the consumer has increased dramatically over the last 50 years. On the whole, they are well-tolerated by the majority of users; however, some individuals appear to show adverse reactions to some of these products. Although these reactions occur rarely, there is a need to explore the interaction between oral health products and the oral mucosa. In vitro screening methods for assessing oral mucosal irritation and sensitization are valuable in assessing cytotoxicity and the determination of ingredient concentrations for the development of dentifrice formulations. Monolayer gingival fibroblasts have been used to assess the cytotoxic effects of alcohol containing mouth rinses (Poggi et al., 2003), TERT-1 cells to test a panel of dentifrice detergents (Moore et al., 2008) and gingival epithelial and fibroblast cells to assess the cytotoxicity of the antimicrobial agent triclosan, a component of both dentifrices and mouth-rinses (Babich and Babich, 1997). While monolayer culture experiments can offer an insight
* Corresponding author. Bart’s and the London School of Medicine and Dentistry, Queen Mary University, UK. Tel.: +44207 882 7141. E-mail address: [email protected] (E. Hagi-Pavli). http://dx.doi.org/10.1016/j.fct.2014.09.007 0278-6915/© 2014 Elsevier Ltd. All rights reserved.
into possible toxicological effects of oral care ingredients, they are not representative of the organizational complexity of the multilayered oral mucosa. Several in vitro 3D oral mucosal models have been engineered (Moharamzadeh et al., 2012). The commercial SkinEthic Oral Mucosal Model (OMM) is a 3D in vitro model that is based on the TR146 buccal carcinoma cell line grown on polycarbonate filters and has been extensively used to study the effects of Candida albicans (Schaller et al., 2006; Whiley et al., 2012) and surfactants (Hagi-Pavli et al., 2004; Lundqvist et al., 2002). The aim of this study was to investigate the in vitro effects of the oral healthcare ingredients sodium lauryl sulphate (SLS), cinnamic aldehyde (CA) and cetylpyridinium chloride (CPC), in concordance with other known irritants and sensitizers and in parallel with their known clinical tolerance profile. SLS is an anionic surfactant and detergent found in many oral healthcare products and cosmetics and is the most commonlyused detergent in dentifrice formulations in Europe and North America. The concentration of SLS in dentifrice typically ranges from 0.5% to 2% (Barkvoll and Rolla, 1994). Although widely tolerated in the general population for use in dentifrices, it is recognized that SLS can have potential adverse effects on the oral soft tissues particularly where the oral mucosa is compromised, sensitive or affected by erosive or ulcerative disease (Healy et al., 2000; Herlofson and Barkvoll, 1993; Skaare et al., 1997). In vitro work previously
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published from our laboratory has shown that SLS damages the permeability barrier of human oral mucosa (Healy et al., 2000); moreover, there are clinical reports of SLS eliciting inflammatory responses in oral soft tissues (Herlofson and Barkvoll, 1994, 1996a, 1996b, 1996c; Rantanen et al., 2002; Skaare et al., 1996). The U.S. Expert Panel of the Cosmetic, Toiletry and Fragrance Association, concluded in their Cosmetic Ingredient Review (CIR) for SLS that this ingredient is safe when used within cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing from the surface of the skin. In products designed for prolonged contact with the skin, concentrations must not exceed 1% since at higher concentrations irritation can occur (NICNAS, Scheme NICNaA, 2007). The cytotoxicity of SLS has been shown in vitro in gingival (Babich and Babich, 1997) and OKF6/TERT-1 oral mucosal epithelial cells derived from the floor of the mouth (Moore et al., 2008), as well as in a reconstructed human oral mucosal model derived from oral keratinocytes and fibroblasts (Neppelberg et al., 2007). The in vivo effects of a single exposure of SLS on CD4+and CD8+ T cell phenotypes and tissue levels of T-cell cytokines IL-2 and IFN-γ in murine oral mucosa have also been reported (Ahlfors et al., 2012). However, while there have been some clinical reports of SLS causing inflammation to date (Herlofson and Barkvoll, 1994, 1996b), there have been no detailed in vitro studies assessing whether SLS induces a cytokine response in human oral mucosa. CA is the main component of cinnamon bark oil or cassia oil that is commonly used as a flavouring agent in toothpaste. It is reported in the literature as a fragrance allergen, with case reports of delayed type sensitization from toothpastes containing cassia oil appearing as far back as the early 1950s and continuing to the present time (Gerberick et al., 2001; Johansen et al., 1996). This aside, the overall incidence of allergy associated with CA is low (Buckley et al., 2000). CPC can cause irritation when applied to the skin in ‘leave on’ applications (Watanabe et al., 2002) but it is also commonly found in oral care mouth rinses (Haps et al., 2008). Benzalkonium chloride (BC) is a skin irritant that has been extensively used as a model irritant in understanding skin irritation (Basketter et al., 2004; Willis et al., 1988). 1-chloro-2,4dinitrochlorobenzene (DNCB) is well-documented in the dermatological literature as a strong sensitizer (Dearman and Kimber, 1991; Poumay et al., 2004; Van Och et al., 2002), as is nickel sulphate (NiSO4) (Allenby and Basketter, 1994; Dou et al., 2003). The SkinEthic OMM was used to identify whether these groups of compounds exhibited a particular response profile that could be used to predict oral mucosal irritation with the purpose of using the OMM as part of the safety assessment of new oral care ingredients. Therefore, to assess the potential sensitivity of the in vitro model, we compared the cellular responses of the OMM to our panel of oral care ingredients with other known irritants and sensitizers. Irritants, BC, CPC and SLS and sensitizers, CA, 1- DNCB and NiSO4 were evaluated using cell viability, release of cytosolic lactate dehydrogenase (LDH); and protein and mRNA levels of interleukin-1α (IL1α) and interleukin-8 (IL-8) as markers of irritation. While measurement of cell viability and extracellular LDH release gives an indication of cytotoxicity, the release of IL-1α and IL-8 are associated with activation of early inflammatory responses essential to local defence at sites of tissue damage and infection (Dinarello, 2000; Gabay et al., 2010; Hirsiger et al., 2012; Lie et al., 2012). Epithelial cells constitutively express IL-1α thus IL-1α and cytosolic LDH release indicates that cells were undergoing necrosis. On the other hand, IL-8 release requires de novo synthesis and is important in recruitment of leukocytes to the damaged tissue (Hoffmann et al., 2002). Therefore, we hypothesized that we would observe distinct differences in response of the OMM between the classes of compounds and that these differences in viability, LDH, IL-1α and IL-8 release would allow us to see a response pattern that would permit predictions regarding oral irritation. Hence, the purpose of
this study was to characterize the interaction between a human reconstructed OMM and oral care ingredients in order to develop a model for predicting oral irritation. 2. Materials and methods 2.1. Chemicals DNCB was purchased from Fisher Scientific UK Limited (Loughborough, Leicester, UK). BC, CPC, NiSO 4 and MTT (3-[4, 5-Dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) were purchased from Sigma Aldrich (Poole, Dorset, UK). CPC, SLS, BC and NiSO4 were diluted in sterile phosphate buffered saline (PBS, Oxoid™). DNCB was first diluted in dimethyl sulphoxide (DMSO) and then further diluted with sterile PBS. SLS (Empicol 0303) and CA were a gift from GlaxoSmithKline (GSK). 2.2. Tissue culture The reconstituted human oral mucosal model (OMM) supplied by SkinEthic Laboratories, Lyon, France, is a three dimensional tissue culture model consisting of a continuous oral keratinocyte cell line, TR146 (Rupniak et al., 1985) grown in defined culture medium on 0.5 cm2 polycarbonate membranes (Rosdy and Clauss, 1990). OMM cultures were shipped on agar, transferred into 24 well culture plates containing maintenance medium (MDCB 153 containing 5 mg/ml insulin and 1.5 mM Ca2+) and incubated at 37 °C (5% CO2) in a humidified atmosphere. Cultures were incubated for 24 h before testing and all experiments were performed with 14 day cultures. 2.3. MTT epithelial viability assay Two hundred microlitres of test compound or control was applied to the epithelial surface of the OMM and incubated for 5 minutes at 37 °C in a 5%CO2 humidified atmosphere. BC, CPC, CA and DNCB were all tested at 1 and 5 mg/ml, NiSO4 was tested at 3 mg/ml and 15 mg/ml and SLS at 1 (0.1% w/v), 5 (0.5% w/v), 10 (1% w/v) and 15 mg/ ml (1.5% w/v). Percentages (w/v) in brackets represent the equivalent amounts used in oral healthcare products. PBS alone and 1% (v/v) DMSO in PBS were used as controls. After treatment the epithelial surface was washed with 200 μl PBS (three times) and cultures were placed into fresh medium and incubated for a further 22 h at 37 °C in a 5%CO2 humidified atmosphere. Cultures were then transferred into a new 24 well plate containing 500 μl of 0.5 mg/ml MTT in PBS and incubated for 60 minutes at 37 °C in a 5%CO2 humidified atmosphere. Cultures were then incubated with isopropanol for a further 2 h at 37 °C to extract the MTT dye and the optical density at 570 nm was determined using a Titertek Multiskan Plus plate reader (Dynatech MRW, Guernsey, Channel Islands) and epithelial viability expressed as a percentage of the control cultures. 2.4. Treatment procedure Exposure to test compounds was for 5 minutes since epithelial detachment is known to occur after 2 minutes of brushing twice a day with 1.5% SLS toothpaste (Veys et al., 1994). Therefore, the treatment procedure for all subsequent experiments, with the exception of the time course experiments, followed the same routine i.e. 5 minute exposure to the test ingredient followed by three PBS washes and 22 h recovery. 2.5. LDH cell membrane integrity assay Levels of the cytosolic enzyme, lactate dehydrogenase (LDH) were determined in the underlying culture medium collected after OMM cultures were exposed to test compounds and controls. LDH was measured using a commercially available assay kit, CytoTox96 Non-radioactive cytotoxicity assay (Promega, USA). Optical density at 492 nm was determined using a Titertek Multiskan Plus plate reader (Dynatech MRW) and LDH release expressed as a ratio of treated/PBS control treated values. 2.6. IL-1α and IL-8 measurements OMM cultures were exposed to test compounds or controls as for the MTT assay following the treatment procedure described above. The underlying culture medium was then collected and levels of extracellular IL-1α and IL-8 were measured using commercially available assay kits (R&D Systems, Abingdon, UK) following the manufacturer’s protocol. Levels of cytokine are expressed as pg/ml or stimulation index (SI) where test compound cytokine levels are normalized to levels from PBS controls. 2.7. RNA extraction and cDNA synthesis SLS (5 mg/ml) and CA (1 mg/ml) were made up using PBS to obtain the required concentration. An aliquot of 200 μl of test compound or PBS was applied to the epithelial surface and the tissue was incubated for 5 minutes at 37 °C in a 5%CO2
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humidified atmosphere. Following treatment, the epithelial surface was washed with 200 μl PBS (three times) and cultures were placed into fresh medium (1000 μl) and incubated for a further 0.5 h, 1 h, 2 h, 4 h, 8 h and 22 h at 37 °C in a 5%CO2 humidified atmosphere. At the end of this time the culture subnatants were collected and stored for assaying IL-1α and IL-8 cytokine levels using ELISA. The culture insert was removed from the medium and the polycarbonate membrane upon which the cells that are growing were cut out using a sterile blade and immediately placed into 1 ml of Ultraspec™ (Biotecx, Houston, USA) and stored at −70 °C until all samples from all time points were collected after which total RNA was extracted following the manufacturer’s instructions. Samples with intact 28S and 18S RNAs were converted to single stranded cDNA. Reverse transcription was performed using 1.5 μg of total RNA as template using the archive reverse transcription kit (Applied Biosystems, UK) in a 50 μl reaction. 2.8. Quantitative real-time polymerase chain reaction (qPCR) Real time PCR was performed on cDNA by the Genome Centre Facility (Queen Mary University of London, UK) using the TaqMan Gene Expression Master Mix and TaqMan® Gene expression arrays (Applied Biosystems). The assay IDs, IL-1α: HS00174092-m1, IL-8:HS00174013-m1 and house-keeping gene 18S rRNA: HS99999901-S1. Samples were run on 3 separate days and each sample was run in triplicate and the average point at which the signal from the sample crossed the threshold was taken (CT mean). Standard deviation and co-efficient of variance (CV) was calculated for each sample for each probe. The relative expression of the target genes were calculated by normalizing to the housekeeping gene using the formula
2−ΔΔCT . 2.9. Histology OMM inserts were collected after the 22 h period and fixed in neutral buffered formalin overnight at room temperature. The epithelium with supporting polycarbonate membrane was cut out of the insert, placed into fixative and tissue processed to paraffin wax, 5 μm sections were cut and stained with haematoxylin and eosin (H&E) and examined under a light microscope. SkinEthic Laboratories provided with every batch of cultures the depth of a representative OMM culture insert; therefore, the average depth for cultures used in these experiments was 121.7 ± 1.16 μm with a CV of 10.3%. 2.10. Statistical analysis The independent Student’s t-test (two-tailed) was used to compare the effect of test sensitizer and irritant compounds as compared to control. Differences were considered statistically significant at p < 0.05. The degree of spread of the data for each treatment regime is given as standard deviation (SD) in the text.
3. Results 3.1. Histology OMM cultures responded differently on exposure to SLS, CPC and CA and control irritant and sensitizer compounds. Cultures exposed to the control irritant BC (Fig. 1a, b), and oral care ingredients CPC (Fig. 1c, d) and SLS (Fig. 2) all caused changes to the OMM morphology. However, CA (Fig. 1e, f), and control sensitizers: DNCB (Fig. 1g, h) and NiSO4 (Fig. 1i, j) had no observable effect on tissue morphology. Treating cultures with 1 mg/ml CPC (Fig. 1c, d) caused disruption of the upper cell layers, cell membranes were disrupted with no discernible cell–cell contacts. At the higher concentration of CPC (Fig. 1d) and both concentrations of BC (Fig. 1a, b), damage was seen throughout all cell layers with individual cells appearing ruptured. SLS also caused cellular damage throughout the OMM, though with 1 mg/ml SLS (Fig. 2a) morphological changes were seen only in the upper few cell layers. Treatment with 5 mg/ml SLS altered cells throughout the OMM with cells showing disrupted cell membranes, cytoplasmic vacuolation and indistinct nuclei (Fig. 2d). Similar changes were also observed at the higher SLS concentrations (Fig. 2c, d). 3.2. Cytotoxicity Exposure to control irritant 1 mg/ml BC decreased viability significantly to 28.1 ± 3.2% (Fig. 3a) and increased LDH levels to
Fig. 1. Haematoxylin and eosin stained sections showing histological damage to OMM caused by treatment with irritants and sensitizers. Effects of irritants on OMM morphology are shown in a–d. Control irritant 1 mg/ml BC (a), 5 mg/ml BC (b) and oral healthcare ingredient 1 mg/ml CPC (c) and 5 mg/ml CPC (d). The effects of sensitizers on OMM morphology are shown in e–j with oral healthcare ingredient 1 mg/ ml CA (e), 5 mg/ml CA (f) and sensitizer controls 1 mg/ml DNCB (g), 5 mg/ml DNCB (h), 3 mg/ml NiSO4 (i) and 15 mg/ml NiSO4 (j). PBS (k) and PBS/DMSO (l) controls were also tested. Arrows indicate upper epithelial surface to which test compounds were applied; ×63 magnification.
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The oral care ingredient CA had no significant effect on viability at 1 mg/ml (Fig. 3b). Of the sensitizer controls NiSO4 had the least impact on cell viability at the concentrations tested (Fig. 3b). DNCB, the other control sensitizer had a more significant effect on viability whereby even at 1 mg/ml DNCB had a small but statistically significant effect in decreasing cell viability (p < 0.05) (Fig. 3b). However, a statistically significant decrease in viability was observed following treatment with the higher concentrations of all three sensitizers. At all concentrations tested there was no significant difference in LDH release with all three sensitizers (Fig. 4b). OMM treatment with increasing concentrations of SLS resulted in a dose dependent decrease in cell viability (Fig. 3a). At the lowest SLS concentration there was no differentiating effect on OMM viability [94 ± 12% compared to PBS control (100 ± 4.7%)], while 5 mg/ ml SLS (74.9 ± 6.9%), 10 mg/ml (69.6 ± 4.6%) and 15 mg/ml (60.6 ± 8.2%) significantly reduced viability (p < 0.05) (Fig. 3a). Similarly, 1 mg/ml SLS had a statistically insignificant effect on LDH release (2.1 ± 1) while 5 mg/ml (LDH 4.2 ± 1.7) (Fig. 4a) caused a significant increase (p < 0.05). Levels of LDH release were not significantly different between OMM exposure to 5 mg/ml, 10 mg/ ml and 15 mg/ml SLS (Fig. 4a). Overall, the irritant compounds had a more adverse impact on OMM cell viability and LDH release compared to the sensitizer compounds. Importantly, though SLS is classified as an irritant in the literature, at the concentrations tested the response of the OMM was more analogous to the responses measured with the sensitizer compounds. 3.3. Cytokine responses
Fig. 2. Effect on morphology following treatment of OMM with increasing SLS concentrations representative of the %SLS found in oral healthcare products. OMM cultures were treated with SLS at varying concentrations: (a) 1 mg/ml (0.1% w/v), (b) 5 mg/ ml (0.5% w/v), (c) 10 mg/ml (1% w/v) and (d) 15 mg/ml (1.5% w/v). Haematoxylin and eosin stained paraffin wax sections; ×63 magnification.
18.5 ± 12.0 (p < 0.05 compared to PBS control) (Fig. 4a). At 5 mg/ ml BC viability decreased further to 4.0 ± 0.4% (Fig. 3a) and LDH increased to 14.0 ± 7.0 (p < 0.05) (Fig. 4a). Similarly, exposure to CPC significantly decreased viability and increased LDH with the highest concentration being the most cytotoxic to cells (viability 7.3 ± 0.2%, LDH 20 ± 4) (Figs. 3a and 4a respectively).
The data for the release of IL-1α and IL-8 following exposure to varying concentrations of SLS, CPC, CA and control irritant and sensitizers are summarized in Figs. 5 and 6. Results are expressed as stimulation indices (SI), where stimulated levels are normalized with respect to unstimulated levels in PBS-treated controls. Where statistically significant the mean cytokine values in pg/ml ± S.D. are also shown in the text below. OMM cultures exposed to 1 mg/ml and 5 mg/ml BC irritant exhibited significantly increased IL-1α release to 120 ± 55.2 pg/ml (SI = 134), and 105.12 ± 20.2 pg/ml (SI = 118.6) respectively compared to PBS control 0.88 ± 0.12 pg/ml (p < 0.05) (Fig. 5a). There was a similar response to CPC (Fig. 5a), with the highest levels following exposure to 5 mg/ml CPC where IL-α levels increased to a maximum of 167 ± 42 pg/ml (SI = 196.8) as compared to the PBS control 0.88 ± 0.12 pg/ml (p < 0.05). While exposure to 1 mg/ml CPC significantly increased both IL-1α to 27.18 ± 3.04 pg/ml (SI = 32) and IL-8 to 1550.9 ± 139.5 pg/ml [(SI = 1.8), p < 0.05] (Fig. 6a), the higher concentration of CPC (Fig. 6a) decreased IL-8 levels to 533.9 ± 113.6 pg/ml (SI = 0.6). Similarly, BC at both 1 mg/ml (SI = 0.79) and 5 mg/ml (SI = 0.14) concentrations decreased IL-8 release (Fig. 6a). Application of sensitizers CA and NiSO4 at 1 mg/ml and DNCB at 1 and 5 mg/ml concentrations had no significant effect on IL-1α release (Fig. 5b), with CA eliciting 0.76 ± 0.20 pg/ml IL-1α (SI = 0.87); NiSO4 0.91 ± 0.17 pg/ml IL-1α (SI = 1.03); 1 mg/ml and 5 mg/ml DNCB 1.05 ± 0.56 pg/ml IL-1α (SI = 1.3); and 2.68 ± 2.11 pg/ml IL-1α (SI = 3.4) respectively (Fig. 5b). In contrast, CA stimulated an increase in IL-8 production (Fig. 6b) at both concentrations tested, with the highest levels observed following treatment with 1 mg/ml CA where IL-8 levels increased to 8369 ± 1672 pg/ml (SI = 8.97) as compared to PBS control 911.11 ± 197.84 pg/ml (p < 0.05). Likewise at 5 mg/ml CA increased IL-8 significantly to 5373 ± 3206 pg/ml [(SI = 6.12), p < 0.05] (Fig. 6b). OMM treatment with DNCB significantly increased IL-8 release (Fig. 6b) following treatment with the lower concentration of 1 mg/ml to 5405 ± 1990 pg/ml (SI = 9.1) (p < 0.05). However, treatment with the higher concentration resulted in a lesser IL-8 response of 1388 ± 1216 pg/ml (SI = 2.3). Unlike the oral care
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Fig. 3. (a) Effect of irritant compounds (oral care ingredients CPC, SLS and control BC) and (b) sensitizers (oral care ingredient CA, controls NiSO4, and DNCB) on OMM viability (N = 6, experiments were carried out on triplicate cultures on two separate occasions).
ingredient CA and control sensitizer DNCB, the sensitizer NiSO4 did not elicit a significant IL-8 response in the OMM following treatment with either concentration since 3 mg/ml NiSO4 produced 584.49 ± 82.72 pg/ml (SI = 0.64) IL-8 and 15 mg/ml NiSO 4 1478.03 ± 392.07 pg/ml IL-8 (SI = 1.60) (Fig. 6b). Exposing OMM cultures to SLS at concentrations similar to those used in toothpastes lead to our discovery that at the lower end of
the concentration range SLS initiated low levels of IL-1α release [3.6 ± 0.78 pg/ml (SI = 4.08)] (Fig. 5a). At higher SLS concentrations, IL-1α release increased significantly (p < 0.05) compared to PBS control (Fig. 5a) with 5 mg/ml, 10 mg/ml and 15 mg/ml SLS stimulating 32 ± 10 pg/ml (SI = 35.9), 42 ± 16 pg/ml (SI = 48.94), and 41 ± 11 pg/ml (SI = 48) IL-1α release respectively. The proportional increase in IL-1α release was less marked with SLS than with
Fig. 4. (a) Effect of irritant compounds (oral care ingredients CPC, SLS and control BC) and (b) sensitizers (oral care ingredient CA, controls NiSO4, and DNCB) on OMM lactate dehydrogenase (LDH) release (N = 6, experiments were carried out on triplicate cultures on two separate occasions).
Fig. 5. (a) Effect of irritant compounds (oral care ingredients CPC, SLS and control BC) and (b) sensitizers (oral care ingredient CA, controls NiSO4, and DNCB) on IL-1α release from OMM expressed as stimulation indices (SI) in relation to levels measured in controls (N = 6, experiments were carried out on triplicate cultures on two separate occasions).
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Fig. 6. (a) Effect of irritant compounds (oral care ingredients CPC, SLS and control BC) and (b) sensitizers (oral care ingredient CA, controls NiSO4, and DNCB) on IL-8 release from OMM expressed as stimulation indices (SI) in relation to levels measured in controls (N = 6, experiments were carried out on triplicate cultures on two separate occasions).
5 mg/ml BC and CPC, but greater than with CA, DNCB and NiSO4 (p < 0.05). Increasing the SLS concentration from 10 mg/ml to 15 mg/ ml did not result in a further statistically significant increase in IL1α release. At all SLS concentrations tested, there was a significant increase in IL-8 release with the most striking response following treatment with 5 mg/ml SLS, where IL-8 levels were stimulated to 7888 ± 2161 pg/ml (SI = 8.67) compared to PBS control (p ≤ 0.05) (Fig. 6a). The lowest SLS concentration 1 mg/ml caused a fivefold increase in IL-8 levels to 5147 ± 774 pg/ml (SI = 5.59, p ≤ 0.05). As with IL-1α levels, when SLS concentrations were increased from 10 mg/ml to 15 mg/ml, there was no further significant difference in the increase in IL-8 release between the higher SLS concentrations, 3357 ± 900 pg/ml IL-8 (SI = 3.98) and 3597 ± 556 pg/ml IL-8 (SI = 4.26) respectively (Fig. 6b). 3.4. Measurement of IL-1α and IL-8 expression over time To investigate the effect of SLS and CA on IL-1α and IL-8 gene expression OMM cultures were challenged with either 5 mg/ml (0.5% w/v) SLS or 1 mg/ml CA. Gene expression levels of IL-1α and IL-8 were measured at 0.5,1, 2, 4, 8 and 22 h after treatment using quantitative polymerase chain reaction (QPCR) (Figs. 7a and 8a respectively). IL-1α and IL-8 levels were also measured in culture supernatants up to 8 h following SLS or CA stimulation (Figs. 7b and 8a respectively). Subsequent treatment with SLS and CA significantly up-regulated the expression of IL-1α (Fig. 7a) where mRNA levels peaked at 4 h following treatment with both 5 mg/ml SLS and 1 mg/ml CA (Fig. 7a). IL-1α expression levels were 11.2 ± 2.4 fold higher with 5 mg/ml (0.5%) SLS where IL-1α gene levels at 4 h post SLS treatment being significantly higher than at all other time points (p ≤ 0.001). At 8 h and 22 h IL-1α levels were 4.2 ± 0.7 fold and 6.4 ± 0.7 fold higher than control but significantly lower than at 4 h (p ≤ 0.001). IL-1α gene levels increased 8.0 ± 2 fold higher with the application of 1 mg/ml CA with peak expression levels at 4 h being significantly higher than at all other time points (p ≤ 0.001). Comparison of the effect on IL-1α gene expression between the two different treatments certainly highlights a difference in the response of the OMM to SLS. Application of SLS to OMM resulted in a difference in the duration of IL-1α gene up regulation since IL1α gene levels did not diminish as they did following CA treatment but still remained significantly high 22 h post treatment. This effect was also paralleled when levels of IL-1α were measured in culture supernatants taken at the different time points (Fig. 7b) where after 8 h IL-1α levels were 35.2 ± 14.6 pg/ml (SI = 27 ± 11.2) with SLS
but only 1.8 ± 1 pg/ml (SI = 1.3) with CA. IL-1α levels measured in OMM cultures upon challenge with CA were altogether low in comparison to SLS where SIs at 0.5 h, 1 h, 2 h and 4 h were 4.2 ± 1.3, 1.6 ± 0.9, 1.2 ± 0.9 and 1.9 ± 1.3, respectively (Fig. 7b). Similarly, IL-8 gene expression was measured and while maximal IL-8 gene levels were reached 4 h following treatment with CA, the same was not observed with SLS (Fig. 8a). Furthermore, CA increased IL-8 gene expression by 14.2 ± 5.3 fold while SLS at the same time point increased IL-8 gene expression by 7.6 ± 4.9 fold. However, after 8 h SLS increased IL-8 expression by 100.4 ± 41 fold as compared to control and at 22 h induced IL-8 expression by 61.5 ± 20.4 fold. Levels of IL-8 expression were numerically higher but not significant at 8 h compared to 22 h post SLS treatment (p = 0.049) but significantly higher than at 4 h, 2 h, 1 h and 0.5 h (p ≤ 0.001) where IL-8 expression increased by 7.6 ± 4.9, 1.9 ± 0.3, 0.2 ± 0.4 and 1.8 ± 1.3 respectively (Fig. 8a). On the other hand, CA induced moderate IL-8 expression where levels dropped to a 2.1 ± 0.7 fold change at 8 h followed by no significant induction at 22 h. Cytokine levels were also assessed in supernatants from OMM cultures taken at the different time points and while CA significantly increased IL-8 to 2761 ± 355 pg/ml (SI = 4.7 ± 0.6) at 8 h SLS increased IL-8 to 4262 ± 372 pg/ml (SI = 7.2 ± 0.6) at the same time point (Fig. 8b). 4. Discussion Irritants and sensitizers were screened by assessing relative changes in viability, LDH, IL-1α and IL-8 release, to determine if an OMM could be used to identify whether groups of compounds exhibited a characteristic response profile that may predict mucosal irritation. Certainly, the OMM used in this study demonstrates a different response profile to irritants as compared to sensitizers using the multiple endpoint analysis of histology, cell viability and cytokine release. SLS concentrations between 1 and 15 mg/ml (0.5%–1.5%) were chosen for testing on the OMM since this is the concentration range of SLS that is typically found in commercial toothpastes. SLS can cause increased epithelial desquamation (Searls and Berg, 1986). Reports of mucosal sloughing, erosion and irritation have been attributed to surfactant content and therefore led to speculation that SLS may exacerbate certain mucosal conditions. This contention is supported by a study in which the effects of using SLS-free toothpaste were compared with standard SLS containing toothpaste in 47 patients with recurrent oral ulceration (Healy et al., 1999). We observed reduced ulcer incidence compared to using SLS-containing
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Fig. 7. Effect of SLS (5 mg/ml) and CA (1 mg/ml) on IL-1α expression in OMM. (a) Gene expression levels of IL-1α were measured by qPCR analysis and normalized against expression levels of 18SrRNA. Samples were run in triplicate on 3 separate days and the average point at which the signal from each sample crossed the threshold was taken, CT. The amount of target was calculated by taking the CT value of the test gene from the 18SrRNA CT. This value was then used to calculate the relative amount of template using the formula 2−ΔΔCT. (b) IL-1α release from OMM expressed as stimulation indices (SI) in relation to levels measured in controls at time points 0.5, 1, 2, 4, and 8 h following treatment. Experiments were carried out on triplicate cultures and cytokine responses from each OMM were measured in duplicate.
toothpaste, thus implicating SLS in oral mucosal damage within individuals with recurrent oral ulceration. Previous studies have illustrated the cytotoxic effects of SLS on organotypic cultures of human oral mucosa (Neppelberg et al., 2007) and monolayer cultures of gingival epithelial and gingival fibroblast cells (Babich and Babich, 1997); however, to our knowledge this is the first study using a reconstructed OMM to assess the effects of SLS on inflammatory cytokine release from oral epithelial cells. SLS concentrations higher than 0.5% have been shown to cause cell necrosis in the superficial cell layers of an oral mucosal model (Neppelberg et al., 2007). Surprisingly, Neppelberg et al. did not investigate whether necrosis stimulated the release of inflammatory cytokines, but they did postulate that it was possible that SLSinduced cell necrosis could activate an inflammatory response in the oral mucosa, thus helping to explain reports of the clinically
damaging effects of SLS in the mouth (Neppelberg et al., 2007). Our study adds to these observations, since we measured IL-1α and IL-8 in culture supernatants and found that both cytokines increased in comparison to PBS control with increasing SLS concentration. At the lowest concentration of 1 mg/ml SLS, cell viability remained high (93.6 ± 12.2%) with LDH and IL-1α release low, indicating that cellular damage was minimal. Furthermore, histological examination showed damage only to the upper two cell layers of the OMM. At the higher SLS concentrations, differentiating cytokine release attributable specifically to SLS from release due to cell death is difficult. However, by examining cytokine data following exposure to the other irritants and sensitizers screened, an indication of the levels of IL-1α and IL-8 that would be expected following substantial cell death may be obtained. For
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Fig. 8. Effect of SLS (5 mg/ml) and CA (1 mg/ml) on IL-8 expression in OMM. (a) Gene expression levels of IL-8 were measured by qPCR analysis and normalized against expression levels of 18SrRNA. Samples were run in triplicate on 3 separate days and the average point at which the signal from each sample crossed the threshold was taken, CT. The amount of target was calculated by taking the CT value of the test gene from the 18SrRNA CT. This value was then used to calculate the relative amount of template using the formula 2−ΔΔCT. (b) IL-8 release from OMM expressed as stimulation indices (SI) in relation to levels measured in controls at time points 0.5, 1, 2, 4, and 8 h following treatment. Experiments were carried out on triplicate cultures and cytokine responses from each OMM were measured in duplicate.
example, the irritant BC at a concentration of 1 mg/ml significantly reduced cell viability (28.1 ± 3.1%) and increased LDH denoting considerable cell death, but while IL-1α (SI = 134 ± 57) levels were high, IL-8 levels were low (SI = 0.8 ± 0.1). Similarly, 5 mg/ml CPC was also highly cytotoxic with IL-1α levels increased (SI = 212 ± 52.8) and IL-8 levels decreased (SI = 0.9 ± 0.2). On the other hand, the sensitizer compounds, CA, DNCB and NiSO4 were not as cytotoxic, and this resulted in an altogether different cytokine profile. CA and DNCB induced low levels of IL-1α but high levels of IL-8, whereas NiSO4 had no significant effect on release of either of the inflammatory cytokines measured. Therefore, by comparison the levels of IL-1α and IL-8 measured following treatment with the lower concentrations of SLS are not entirely the consequence of cell necrosis, but are in part due to de novo synthesis of IL-1α and IL-8. Evidence to support this observation was provided when gene ex-
pression of IL-1α and IL-8 was measured after application of 5 mg/ ml SLS, where IL-1α expression was maximal 4 h post exposure, with an 11-fold increase, whereas IL-8 expression increased 100fold and peaked 8 h after treatment. OMM cultures treated with 1 mg/ml CA also induced IL-1α and IL-8 gene expression, but the increase in IL-8 was modest as compared to SLS treatment. Thus the levels of IL-1α and IL-8 measured in culture supernatants from OMM cultures exposed to SLS are not due solely to stimulation by necrotic cells or leakage of constitutive IL-α from damaged epithelial cells, but are also the result of the induction of IL-1α and IL-8 gene expression. IL-1α is a key mediator of inflammation in skin, where it is constitutively expressed in keratinocytes (Welss et al., 2004). IL-1α released from keratinocytes following injury will induce expression of itself and other cytokines such as IL-6 and IL-8, by binding onto the IL-1 receptor type I found on the keratinocyte
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cell membrane (Corsini and Galli, 1998; Dinarello, 1998). Therefore, since damage to tissue morphology is not as profound when OMM cells are treated with 1 mg/ml and 5 mg/ml (0.5%) SLS, IL-1α released from damaged epithelial cells could induce IL-8 and further IL-1α expression from viable cells in the OMM. Consequently, IL-8 measured from these cultures could be a sum of IL-8 directly stimulated by SLS and IL-8 indirectly induced by the release of IL-1α from epithelial cells damaged by treatment with SLS. Furthermore, even at 10 mg/ml and 15 mg/ml SLS concentrations where histological examination shows substantial damage to the OMM, it is hypothesized that there remain sufficient numbers of viable cells that are able to produce IL-8 in response to SLS and IL-1α released from cells undergoing necrosis. Besides, if IL-1α levels measured with higher SLS concentrations were entirely due to cell necrosis and leakage of IL-1α from damaged cells, it is currently unclear why LDH levels remained relatively low in comparison to levels measured following BC and CPC treatments. We therefore propose that the IL-1α levels measured are a sum total of constitutive IL-1α from disrupted cells and induced IL-1α. The changes in IL-1α and IL-8 release observed using an OMM exhibit similar trends to those reported for a reconstructed human epithelial (RHE) skin model where irritants or sensitizers were applied (Coquette et al., 2003; Corsini and Galli, 1998). Even though application of DNCB to either model presented similar trends, the levels of IL-8 released from the OMM were much higher than for RHE. Similarly, levels of IL-8 observed when NiSO4 was applied to OMM would appear high in our study, when compared to those obtained by Coquette et al. suggesting that epithelial models derived from skin or oral mucosa may respond differently when challenged with the same compounds. Case reports indicate CA-containing dentifrice as causing inflammation of the lips, labial mucosa, and gingivae in sensitive individuals (Thyne et al., 1989) and topical application of 1% CA to the forearm has been reported to cause cutaneous flushing and to increase levels of circulating prostaglandins (PGE2) (VanderEnde and Morrow, 2001). CA has also been tested on human immature dendritic cells and, by analysis of the phenotypic alterations induced by CA, the sensitizing potential of CA could be predicted (Staquet et al., 2004). Certainly, from our data levels of IL-8 induced at both 1 mg/ml (0.1% w/v) and 5 mg/ml (0.5% w/v) CA concentrations were high and comparable with those obtained with DNCB which is a welldocumented strong sensitizer, suggesting potential value of the OMM in screening for ingredients which have sensitizing potential as well as screening potential irritants. In conclusion, this study is the first to show pro-inflammatory IL-1α and chemokine IL-8 release from oral epithelial cells treated with different concentrations of the common toothpaste surfactant SLS. Importantly, we have demonstrated through these studies that the OMM is a potentially valuable model for screening the relative oral irritation of new oral healthcare ingredients prior to clinical testing. Nevertheless, one must keep in mind that while this model is capable of predicting mucosal irritation it is still an in vitro model and as such it is far more sensitive. Hence, while the model is suitable for relative comparisons or for predicting trends, it is not appropriate for informing what level of oral care ingredient may cause irritation in the mouth. Conflict of interest The authors declare no potential conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version.
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Contents lists available at ScienceDirect
Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x
Characterizing the immunological effects of oral healthcare ingredients using an in vitro reconstructed human epithelial model E. Hagi-Pavli a,*, D.M. Williams a, J.L. Rowland b, M. Thornhill c, A.T. Cruchley a a b c
Bart’s and the London School of Medicine and Dentistry, Queen Mary University, UK GSK Oral Healthcare R & D, Weybridge, Surrey, UK School of Clinical Dentistry, University of Sheffield, UK
A R T I C L E
I N F O
Article history: Received 24 March 2014 Accepted 18 September 2014 Available online 5 October 2014 Keywords: Irritant Inflammatory cytokines Sodium lauryl sulphate (SLS) Human oral mucosal model Sensitizer Cinnamic aldehyde
A B S T R A C T
Oral healthcare products are well tolerated and while adverse occurrences are rare there is still a need to explore the interaction between these products and the oral mucosa. This study assessed the effects of oral healthcare ingredients: sodium lauryl sulphate (SLS), a detergent; cinnamic aldehyde (CA), a flavouring agent; and cetylpyridinium chloride (CPC), an antiseptic, using a reconstructed human oral mucosal model (OMM). Differential release of inflammatory cytokines IL-1α, IL-8 and cytotoxicity was compared with other known irritants and sensitizers to identify a signature response profile that could be associated with oral mucosal irritation. Response profiles differed with irritants being more cytotoxic. CA and control sensitizers nickel sulphate (NiSO4) and 1-chloro-2,4-dinitrochlorobenzene (DNCB) released lower levels of IL-1α than CPC and control irritant benzalkonium chloride (BC), whereas the opposite was observed for IL-8. Significant levels of IL-8 and IL-1α were released with 5–15 mg/ml (0.5–1.5% w/v) SLS. Quantitative PCR indicated that cytokine release at lower SLS concentrations is not entirely due to cell necrosis but in part due to de novo synthesis. These findings suggest that the OMM can be used to predict oral irritation thus making it a potentially valuable model for screening new oral healthcare ingredients prior to clinical release. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction The use and range of oral health products available to the consumer has increased dramatically over the last 50 years. On the whole, they are well-tolerated by the majority of users; however, some individuals appear to show adverse reactions to some of these products. Although these reactions occur rarely, there is a need to explore the interaction between oral health products and the oral mucosa. In vitro screening methods for assessing oral mucosal irritation and sensitization are valuable in assessing cytotoxicity and the determination of ingredient concentrations for the development of dentifrice formulations. Monolayer gingival fibroblasts have been used to assess the cytotoxic effects of alcohol containing mouth rinses (Poggi et al., 2003), TERT-1 cells to test a panel of dentifrice detergents (Moore et al., 2008) and gingival epithelial and fibroblast cells to assess the cytotoxicity of the antimicrobial agent triclosan, a component of both dentifrices and mouth-rinses (Babich and Babich, 1997). While monolayer culture experiments can offer an insight
* Corresponding author. Bart’s and the London School of Medicine and Dentistry, Queen Mary University, UK. Tel.: +44207 882 7141. E-mail address: [email protected] (E. Hagi-Pavli). http://dx.doi.org/10.1016/j.fct.2014.09.007 0278-6915/© 2014 Elsevier Ltd. All rights reserved.
into possible toxicological effects of oral care ingredients, they are not representative of the organizational complexity of the multilayered oral mucosa. Several in vitro 3D oral mucosal models have been engineered (Moharamzadeh et al., 2012). The commercial SkinEthic Oral Mucosal Model (OMM) is a 3D in vitro model that is based on the TR146 buccal carcinoma cell line grown on polycarbonate filters and has been extensively used to study the effects of Candida albicans (Schaller et al., 2006; Whiley et al., 2012) and surfactants (Hagi-Pavli et al., 2004; Lundqvist et al., 2002). The aim of this study was to investigate the in vitro effects of the oral healthcare ingredients sodium lauryl sulphate (SLS), cinnamic aldehyde (CA) and cetylpyridinium chloride (CPC), in concordance with other known irritants and sensitizers and in parallel with their known clinical tolerance profile. SLS is an anionic surfactant and detergent found in many oral healthcare products and cosmetics and is the most commonlyused detergent in dentifrice formulations in Europe and North America. The concentration of SLS in dentifrice typically ranges from 0.5% to 2% (Barkvoll and Rolla, 1994). Although widely tolerated in the general population for use in dentifrices, it is recognized that SLS can have potential adverse effects on the oral soft tissues particularly where the oral mucosa is compromised, sensitive or affected by erosive or ulcerative disease (Healy et al., 2000; Herlofson and Barkvoll, 1993; Skaare et al., 1997). In vitro work previously
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published from our laboratory has shown that SLS damages the permeability barrier of human oral mucosa (Healy et al., 2000); moreover, there are clinical reports of SLS eliciting inflammatory responses in oral soft tissues (Herlofson and Barkvoll, 1994, 1996a, 1996b, 1996c; Rantanen et al., 2002; Skaare et al., 1996). The U.S. Expert Panel of the Cosmetic, Toiletry and Fragrance Association, concluded in their Cosmetic Ingredient Review (CIR) for SLS that this ingredient is safe when used within cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing from the surface of the skin. In products designed for prolonged contact with the skin, concentrations must not exceed 1% since at higher concentrations irritation can occur (NICNAS, Scheme NICNaA, 2007). The cytotoxicity of SLS has been shown in vitro in gingival (Babich and Babich, 1997) and OKF6/TERT-1 oral mucosal epithelial cells derived from the floor of the mouth (Moore et al., 2008), as well as in a reconstructed human oral mucosal model derived from oral keratinocytes and fibroblasts (Neppelberg et al., 2007). The in vivo effects of a single exposure of SLS on CD4+and CD8+ T cell phenotypes and tissue levels of T-cell cytokines IL-2 and IFN-γ in murine oral mucosa have also been reported (Ahlfors et al., 2012). However, while there have been some clinical reports of SLS causing inflammation to date (Herlofson and Barkvoll, 1994, 1996b), there have been no detailed in vitro studies assessing whether SLS induces a cytokine response in human oral mucosa. CA is the main component of cinnamon bark oil or cassia oil that is commonly used as a flavouring agent in toothpaste. It is reported in the literature as a fragrance allergen, with case reports of delayed type sensitization from toothpastes containing cassia oil appearing as far back as the early 1950s and continuing to the present time (Gerberick et al., 2001; Johansen et al., 1996). This aside, the overall incidence of allergy associated with CA is low (Buckley et al., 2000). CPC can cause irritation when applied to the skin in ‘leave on’ applications (Watanabe et al., 2002) but it is also commonly found in oral care mouth rinses (Haps et al., 2008). Benzalkonium chloride (BC) is a skin irritant that has been extensively used as a model irritant in understanding skin irritation (Basketter et al., 2004; Willis et al., 1988). 1-chloro-2,4dinitrochlorobenzene (DNCB) is well-documented in the dermatological literature as a strong sensitizer (Dearman and Kimber, 1991; Poumay et al., 2004; Van Och et al., 2002), as is nickel sulphate (NiSO4) (Allenby and Basketter, 1994; Dou et al., 2003). The SkinEthic OMM was used to identify whether these groups of compounds exhibited a particular response profile that could be used to predict oral mucosal irritation with the purpose of using the OMM as part of the safety assessment of new oral care ingredients. Therefore, to assess the potential sensitivity of the in vitro model, we compared the cellular responses of the OMM to our panel of oral care ingredients with other known irritants and sensitizers. Irritants, BC, CPC and SLS and sensitizers, CA, 1- DNCB and NiSO4 were evaluated using cell viability, release of cytosolic lactate dehydrogenase (LDH); and protein and mRNA levels of interleukin-1α (IL1α) and interleukin-8 (IL-8) as markers of irritation. While measurement of cell viability and extracellular LDH release gives an indication of cytotoxicity, the release of IL-1α and IL-8 are associated with activation of early inflammatory responses essential to local defence at sites of tissue damage and infection (Dinarello, 2000; Gabay et al., 2010; Hirsiger et al., 2012; Lie et al., 2012). Epithelial cells constitutively express IL-1α thus IL-1α and cytosolic LDH release indicates that cells were undergoing necrosis. On the other hand, IL-8 release requires de novo synthesis and is important in recruitment of leukocytes to the damaged tissue (Hoffmann et al., 2002). Therefore, we hypothesized that we would observe distinct differences in response of the OMM between the classes of compounds and that these differences in viability, LDH, IL-1α and IL-8 release would allow us to see a response pattern that would permit predictions regarding oral irritation. Hence, the purpose of
this study was to characterize the interaction between a human reconstructed OMM and oral care ingredients in order to develop a model for predicting oral irritation. 2. Materials and methods 2.1. Chemicals DNCB was purchased from Fisher Scientific UK Limited (Loughborough, Leicester, UK). BC, CPC, NiSO 4 and MTT (3-[4, 5-Dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) were purchased from Sigma Aldrich (Poole, Dorset, UK). CPC, SLS, BC and NiSO4 were diluted in sterile phosphate buffered saline (PBS, Oxoid™). DNCB was first diluted in dimethyl sulphoxide (DMSO) and then further diluted with sterile PBS. SLS (Empicol 0303) and CA were a gift from GlaxoSmithKline (GSK). 2.2. Tissue culture The reconstituted human oral mucosal model (OMM) supplied by SkinEthic Laboratories, Lyon, France, is a three dimensional tissue culture model consisting of a continuous oral keratinocyte cell line, TR146 (Rupniak et al., 1985) grown in defined culture medium on 0.5 cm2 polycarbonate membranes (Rosdy and Clauss, 1990). OMM cultures were shipped on agar, transferred into 24 well culture plates containing maintenance medium (MDCB 153 containing 5 mg/ml insulin and 1.5 mM Ca2+) and incubated at 37 °C (5% CO2) in a humidified atmosphere. Cultures were incubated for 24 h before testing and all experiments were performed with 14 day cultures. 2.3. MTT epithelial viability assay Two hundred microlitres of test compound or control was applied to the epithelial surface of the OMM and incubated for 5 minutes at 37 °C in a 5%CO2 humidified atmosphere. BC, CPC, CA and DNCB were all tested at 1 and 5 mg/ml, NiSO4 was tested at 3 mg/ml and 15 mg/ml and SLS at 1 (0.1% w/v), 5 (0.5% w/v), 10 (1% w/v) and 15 mg/ ml (1.5% w/v). Percentages (w/v) in brackets represent the equivalent amounts used in oral healthcare products. PBS alone and 1% (v/v) DMSO in PBS were used as controls. After treatment the epithelial surface was washed with 200 μl PBS (three times) and cultures were placed into fresh medium and incubated for a further 22 h at 37 °C in a 5%CO2 humidified atmosphere. Cultures were then transferred into a new 24 well plate containing 500 μl of 0.5 mg/ml MTT in PBS and incubated for 60 minutes at 37 °C in a 5%CO2 humidified atmosphere. Cultures were then incubated with isopropanol for a further 2 h at 37 °C to extract the MTT dye and the optical density at 570 nm was determined using a Titertek Multiskan Plus plate reader (Dynatech MRW, Guernsey, Channel Islands) and epithelial viability expressed as a percentage of the control cultures. 2.4. Treatment procedure Exposure to test compounds was for 5 minutes since epithelial detachment is known to occur after 2 minutes of brushing twice a day with 1.5% SLS toothpaste (Veys et al., 1994). Therefore, the treatment procedure for all subsequent experiments, with the exception of the time course experiments, followed the same routine i.e. 5 minute exposure to the test ingredient followed by three PBS washes and 22 h recovery. 2.5. LDH cell membrane integrity assay Levels of the cytosolic enzyme, lactate dehydrogenase (LDH) were determined in the underlying culture medium collected after OMM cultures were exposed to test compounds and controls. LDH was measured using a commercially available assay kit, CytoTox96 Non-radioactive cytotoxicity assay (Promega, USA). Optical density at 492 nm was determined using a Titertek Multiskan Plus plate reader (Dynatech MRW) and LDH release expressed as a ratio of treated/PBS control treated values. 2.6. IL-1α and IL-8 measurements OMM cultures were exposed to test compounds or controls as for the MTT assay following the treatment procedure described above. The underlying culture medium was then collected and levels of extracellular IL-1α and IL-8 were measured using commercially available assay kits (R&D Systems, Abingdon, UK) following the manufacturer’s protocol. Levels of cytokine are expressed as pg/ml or stimulation index (SI) where test compound cytokine levels are normalized to levels from PBS controls. 2.7. RNA extraction and cDNA synthesis SLS (5 mg/ml) and CA (1 mg/ml) were made up using PBS to obtain the required concentration. An aliquot of 200 μl of test compound or PBS was applied to the epithelial surface and the tissue was incubated for 5 minutes at 37 °C in a 5%CO2
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humidified atmosphere. Following treatment, the epithelial surface was washed with 200 μl PBS (three times) and cultures were placed into fresh medium (1000 μl) and incubated for a further 0.5 h, 1 h, 2 h, 4 h, 8 h and 22 h at 37 °C in a 5%CO2 humidified atmosphere. At the end of this time the culture subnatants were collected and stored for assaying IL-1α and IL-8 cytokine levels using ELISA. The culture insert was removed from the medium and the polycarbonate membrane upon which the cells that are growing were cut out using a sterile blade and immediately placed into 1 ml of Ultraspec™ (Biotecx, Houston, USA) and stored at −70 °C until all samples from all time points were collected after which total RNA was extracted following the manufacturer’s instructions. Samples with intact 28S and 18S RNAs were converted to single stranded cDNA. Reverse transcription was performed using 1.5 μg of total RNA as template using the archive reverse transcription kit (Applied Biosystems, UK) in a 50 μl reaction. 2.8. Quantitative real-time polymerase chain reaction (qPCR) Real time PCR was performed on cDNA by the Genome Centre Facility (Queen Mary University of London, UK) using the TaqMan Gene Expression Master Mix and TaqMan® Gene expression arrays (Applied Biosystems). The assay IDs, IL-1α: HS00174092-m1, IL-8:HS00174013-m1 and house-keeping gene 18S rRNA: HS99999901-S1. Samples were run on 3 separate days and each sample was run in triplicate and the average point at which the signal from the sample crossed the threshold was taken (CT mean). Standard deviation and co-efficient of variance (CV) was calculated for each sample for each probe. The relative expression of the target genes were calculated by normalizing to the housekeeping gene using the formula
2−ΔΔCT . 2.9. Histology OMM inserts were collected after the 22 h period and fixed in neutral buffered formalin overnight at room temperature. The epithelium with supporting polycarbonate membrane was cut out of the insert, placed into fixative and tissue processed to paraffin wax, 5 μm sections were cut and stained with haematoxylin and eosin (H&E) and examined under a light microscope. SkinEthic Laboratories provided with every batch of cultures the depth of a representative OMM culture insert; therefore, the average depth for cultures used in these experiments was 121.7 ± 1.16 μm with a CV of 10.3%. 2.10. Statistical analysis The independent Student’s t-test (two-tailed) was used to compare the effect of test sensitizer and irritant compounds as compared to control. Differences were considered statistically significant at p < 0.05. The degree of spread of the data for each treatment regime is given as standard deviation (SD) in the text.
3. Results 3.1. Histology OMM cultures responded differently on exposure to SLS, CPC and CA and control irritant and sensitizer compounds. Cultures exposed to the control irritant BC (Fig. 1a, b), and oral care ingredients CPC (Fig. 1c, d) and SLS (Fig. 2) all caused changes to the OMM morphology. However, CA (Fig. 1e, f), and control sensitizers: DNCB (Fig. 1g, h) and NiSO4 (Fig. 1i, j) had no observable effect on tissue morphology. Treating cultures with 1 mg/ml CPC (Fig. 1c, d) caused disruption of the upper cell layers, cell membranes were disrupted with no discernible cell–cell contacts. At the higher concentration of CPC (Fig. 1d) and both concentrations of BC (Fig. 1a, b), damage was seen throughout all cell layers with individual cells appearing ruptured. SLS also caused cellular damage throughout the OMM, though with 1 mg/ml SLS (Fig. 2a) morphological changes were seen only in the upper few cell layers. Treatment with 5 mg/ml SLS altered cells throughout the OMM with cells showing disrupted cell membranes, cytoplasmic vacuolation and indistinct nuclei (Fig. 2d). Similar changes were also observed at the higher SLS concentrations (Fig. 2c, d). 3.2. Cytotoxicity Exposure to control irritant 1 mg/ml BC decreased viability significantly to 28.1 ± 3.2% (Fig. 3a) and increased LDH levels to
Fig. 1. Haematoxylin and eosin stained sections showing histological damage to OMM caused by treatment with irritants and sensitizers. Effects of irritants on OMM morphology are shown in a–d. Control irritant 1 mg/ml BC (a), 5 mg/ml BC (b) and oral healthcare ingredient 1 mg/ml CPC (c) and 5 mg/ml CPC (d). The effects of sensitizers on OMM morphology are shown in e–j with oral healthcare ingredient 1 mg/ ml CA (e), 5 mg/ml CA (f) and sensitizer controls 1 mg/ml DNCB (g), 5 mg/ml DNCB (h), 3 mg/ml NiSO4 (i) and 15 mg/ml NiSO4 (j). PBS (k) and PBS/DMSO (l) controls were also tested. Arrows indicate upper epithelial surface to which test compounds were applied; ×63 magnification.
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The oral care ingredient CA had no significant effect on viability at 1 mg/ml (Fig. 3b). Of the sensitizer controls NiSO4 had the least impact on cell viability at the concentrations tested (Fig. 3b). DNCB, the other control sensitizer had a more significant effect on viability whereby even at 1 mg/ml DNCB had a small but statistically significant effect in decreasing cell viability (p < 0.05) (Fig. 3b). However, a statistically significant decrease in viability was observed following treatment with the higher concentrations of all three sensitizers. At all concentrations tested there was no significant difference in LDH release with all three sensitizers (Fig. 4b). OMM treatment with increasing concentrations of SLS resulted in a dose dependent decrease in cell viability (Fig. 3a). At the lowest SLS concentration there was no differentiating effect on OMM viability [94 ± 12% compared to PBS control (100 ± 4.7%)], while 5 mg/ ml SLS (74.9 ± 6.9%), 10 mg/ml (69.6 ± 4.6%) and 15 mg/ml (60.6 ± 8.2%) significantly reduced viability (p < 0.05) (Fig. 3a). Similarly, 1 mg/ml SLS had a statistically insignificant effect on LDH release (2.1 ± 1) while 5 mg/ml (LDH 4.2 ± 1.7) (Fig. 4a) caused a significant increase (p < 0.05). Levels of LDH release were not significantly different between OMM exposure to 5 mg/ml, 10 mg/ ml and 15 mg/ml SLS (Fig. 4a). Overall, the irritant compounds had a more adverse impact on OMM cell viability and LDH release compared to the sensitizer compounds. Importantly, though SLS is classified as an irritant in the literature, at the concentrations tested the response of the OMM was more analogous to the responses measured with the sensitizer compounds. 3.3. Cytokine responses
Fig. 2. Effect on morphology following treatment of OMM with increasing SLS concentrations representative of the %SLS found in oral healthcare products. OMM cultures were treated with SLS at varying concentrations: (a) 1 mg/ml (0.1% w/v), (b) 5 mg/ ml (0.5% w/v), (c) 10 mg/ml (1% w/v) and (d) 15 mg/ml (1.5% w/v). Haematoxylin and eosin stained paraffin wax sections; ×63 magnification.
18.5 ± 12.0 (p < 0.05 compared to PBS control) (Fig. 4a). At 5 mg/ ml BC viability decreased further to 4.0 ± 0.4% (Fig. 3a) and LDH increased to 14.0 ± 7.0 (p < 0.05) (Fig. 4a). Similarly, exposure to CPC significantly decreased viability and increased LDH with the highest concentration being the most cytotoxic to cells (viability 7.3 ± 0.2%, LDH 20 ± 4) (Figs. 3a and 4a respectively).
The data for the release of IL-1α and IL-8 following exposure to varying concentrations of SLS, CPC, CA and control irritant and sensitizers are summarized in Figs. 5 and 6. Results are expressed as stimulation indices (SI), where stimulated levels are normalized with respect to unstimulated levels in PBS-treated controls. Where statistically significant the mean cytokine values in pg/ml ± S.D. are also shown in the text below. OMM cultures exposed to 1 mg/ml and 5 mg/ml BC irritant exhibited significantly increased IL-1α release to 120 ± 55.2 pg/ml (SI = 134), and 105.12 ± 20.2 pg/ml (SI = 118.6) respectively compared to PBS control 0.88 ± 0.12 pg/ml (p < 0.05) (Fig. 5a). There was a similar response to CPC (Fig. 5a), with the highest levels following exposure to 5 mg/ml CPC where IL-α levels increased to a maximum of 167 ± 42 pg/ml (SI = 196.8) as compared to the PBS control 0.88 ± 0.12 pg/ml (p < 0.05). While exposure to 1 mg/ml CPC significantly increased both IL-1α to 27.18 ± 3.04 pg/ml (SI = 32) and IL-8 to 1550.9 ± 139.5 pg/ml [(SI = 1.8), p < 0.05] (Fig. 6a), the higher concentration of CPC (Fig. 6a) decreased IL-8 levels to 533.9 ± 113.6 pg/ml (SI = 0.6). Similarly, BC at both 1 mg/ml (SI = 0.79) and 5 mg/ml (SI = 0.14) concentrations decreased IL-8 release (Fig. 6a). Application of sensitizers CA and NiSO4 at 1 mg/ml and DNCB at 1 and 5 mg/ml concentrations had no significant effect on IL-1α release (Fig. 5b), with CA eliciting 0.76 ± 0.20 pg/ml IL-1α (SI = 0.87); NiSO4 0.91 ± 0.17 pg/ml IL-1α (SI = 1.03); 1 mg/ml and 5 mg/ml DNCB 1.05 ± 0.56 pg/ml IL-1α (SI = 1.3); and 2.68 ± 2.11 pg/ml IL-1α (SI = 3.4) respectively (Fig. 5b). In contrast, CA stimulated an increase in IL-8 production (Fig. 6b) at both concentrations tested, with the highest levels observed following treatment with 1 mg/ml CA where IL-8 levels increased to 8369 ± 1672 pg/ml (SI = 8.97) as compared to PBS control 911.11 ± 197.84 pg/ml (p < 0.05). Likewise at 5 mg/ml CA increased IL-8 significantly to 5373 ± 3206 pg/ml [(SI = 6.12), p < 0.05] (Fig. 6b). OMM treatment with DNCB significantly increased IL-8 release (Fig. 6b) following treatment with the lower concentration of 1 mg/ml to 5405 ± 1990 pg/ml (SI = 9.1) (p < 0.05). However, treatment with the higher concentration resulted in a lesser IL-8 response of 1388 ± 1216 pg/ml (SI = 2.3). Unlike the oral care
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Fig. 3. (a) Effect of irritant compounds (oral care ingredients CPC, SLS and control BC) and (b) sensitizers (oral care ingredient CA, controls NiSO4, and DNCB) on OMM viability (N = 6, experiments were carried out on triplicate cultures on two separate occasions).
ingredient CA and control sensitizer DNCB, the sensitizer NiSO4 did not elicit a significant IL-8 response in the OMM following treatment with either concentration since 3 mg/ml NiSO4 produced 584.49 ± 82.72 pg/ml (SI = 0.64) IL-8 and 15 mg/ml NiSO 4 1478.03 ± 392.07 pg/ml IL-8 (SI = 1.60) (Fig. 6b). Exposing OMM cultures to SLS at concentrations similar to those used in toothpastes lead to our discovery that at the lower end of
the concentration range SLS initiated low levels of IL-1α release [3.6 ± 0.78 pg/ml (SI = 4.08)] (Fig. 5a). At higher SLS concentrations, IL-1α release increased significantly (p < 0.05) compared to PBS control (Fig. 5a) with 5 mg/ml, 10 mg/ml and 15 mg/ml SLS stimulating 32 ± 10 pg/ml (SI = 35.9), 42 ± 16 pg/ml (SI = 48.94), and 41 ± 11 pg/ml (SI = 48) IL-1α release respectively. The proportional increase in IL-1α release was less marked with SLS than with
Fig. 4. (a) Effect of irritant compounds (oral care ingredients CPC, SLS and control BC) and (b) sensitizers (oral care ingredient CA, controls NiSO4, and DNCB) on OMM lactate dehydrogenase (LDH) release (N = 6, experiments were carried out on triplicate cultures on two separate occasions).
Fig. 5. (a) Effect of irritant compounds (oral care ingredients CPC, SLS and control BC) and (b) sensitizers (oral care ingredient CA, controls NiSO4, and DNCB) on IL-1α release from OMM expressed as stimulation indices (SI) in relation to levels measured in controls (N = 6, experiments were carried out on triplicate cultures on two separate occasions).
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Fig. 6. (a) Effect of irritant compounds (oral care ingredients CPC, SLS and control BC) and (b) sensitizers (oral care ingredient CA, controls NiSO4, and DNCB) on IL-8 release from OMM expressed as stimulation indices (SI) in relation to levels measured in controls (N = 6, experiments were carried out on triplicate cultures on two separate occasions).
5 mg/ml BC and CPC, but greater than with CA, DNCB and NiSO4 (p < 0.05). Increasing the SLS concentration from 10 mg/ml to 15 mg/ ml did not result in a further statistically significant increase in IL1α release. At all SLS concentrations tested, there was a significant increase in IL-8 release with the most striking response following treatment with 5 mg/ml SLS, where IL-8 levels were stimulated to 7888 ± 2161 pg/ml (SI = 8.67) compared to PBS control (p ≤ 0.05) (Fig. 6a). The lowest SLS concentration 1 mg/ml caused a fivefold increase in IL-8 levels to 5147 ± 774 pg/ml (SI = 5.59, p ≤ 0.05). As with IL-1α levels, when SLS concentrations were increased from 10 mg/ml to 15 mg/ml, there was no further significant difference in the increase in IL-8 release between the higher SLS concentrations, 3357 ± 900 pg/ml IL-8 (SI = 3.98) and 3597 ± 556 pg/ml IL-8 (SI = 4.26) respectively (Fig. 6b). 3.4. Measurement of IL-1α and IL-8 expression over time To investigate the effect of SLS and CA on IL-1α and IL-8 gene expression OMM cultures were challenged with either 5 mg/ml (0.5% w/v) SLS or 1 mg/ml CA. Gene expression levels of IL-1α and IL-8 were measured at 0.5,1, 2, 4, 8 and 22 h after treatment using quantitative polymerase chain reaction (QPCR) (Figs. 7a and 8a respectively). IL-1α and IL-8 levels were also measured in culture supernatants up to 8 h following SLS or CA stimulation (Figs. 7b and 8a respectively). Subsequent treatment with SLS and CA significantly up-regulated the expression of IL-1α (Fig. 7a) where mRNA levels peaked at 4 h following treatment with both 5 mg/ml SLS and 1 mg/ml CA (Fig. 7a). IL-1α expression levels were 11.2 ± 2.4 fold higher with 5 mg/ml (0.5%) SLS where IL-1α gene levels at 4 h post SLS treatment being significantly higher than at all other time points (p ≤ 0.001). At 8 h and 22 h IL-1α levels were 4.2 ± 0.7 fold and 6.4 ± 0.7 fold higher than control but significantly lower than at 4 h (p ≤ 0.001). IL-1α gene levels increased 8.0 ± 2 fold higher with the application of 1 mg/ml CA with peak expression levels at 4 h being significantly higher than at all other time points (p ≤ 0.001). Comparison of the effect on IL-1α gene expression between the two different treatments certainly highlights a difference in the response of the OMM to SLS. Application of SLS to OMM resulted in a difference in the duration of IL-1α gene up regulation since IL1α gene levels did not diminish as they did following CA treatment but still remained significantly high 22 h post treatment. This effect was also paralleled when levels of IL-1α were measured in culture supernatants taken at the different time points (Fig. 7b) where after 8 h IL-1α levels were 35.2 ± 14.6 pg/ml (SI = 27 ± 11.2) with SLS
but only 1.8 ± 1 pg/ml (SI = 1.3) with CA. IL-1α levels measured in OMM cultures upon challenge with CA were altogether low in comparison to SLS where SIs at 0.5 h, 1 h, 2 h and 4 h were 4.2 ± 1.3, 1.6 ± 0.9, 1.2 ± 0.9 and 1.9 ± 1.3, respectively (Fig. 7b). Similarly, IL-8 gene expression was measured and while maximal IL-8 gene levels were reached 4 h following treatment with CA, the same was not observed with SLS (Fig. 8a). Furthermore, CA increased IL-8 gene expression by 14.2 ± 5.3 fold while SLS at the same time point increased IL-8 gene expression by 7.6 ± 4.9 fold. However, after 8 h SLS increased IL-8 expression by 100.4 ± 41 fold as compared to control and at 22 h induced IL-8 expression by 61.5 ± 20.4 fold. Levels of IL-8 expression were numerically higher but not significant at 8 h compared to 22 h post SLS treatment (p = 0.049) but significantly higher than at 4 h, 2 h, 1 h and 0.5 h (p ≤ 0.001) where IL-8 expression increased by 7.6 ± 4.9, 1.9 ± 0.3, 0.2 ± 0.4 and 1.8 ± 1.3 respectively (Fig. 8a). On the other hand, CA induced moderate IL-8 expression where levels dropped to a 2.1 ± 0.7 fold change at 8 h followed by no significant induction at 22 h. Cytokine levels were also assessed in supernatants from OMM cultures taken at the different time points and while CA significantly increased IL-8 to 2761 ± 355 pg/ml (SI = 4.7 ± 0.6) at 8 h SLS increased IL-8 to 4262 ± 372 pg/ml (SI = 7.2 ± 0.6) at the same time point (Fig. 8b). 4. Discussion Irritants and sensitizers were screened by assessing relative changes in viability, LDH, IL-1α and IL-8 release, to determine if an OMM could be used to identify whether groups of compounds exhibited a characteristic response profile that may predict mucosal irritation. Certainly, the OMM used in this study demonstrates a different response profile to irritants as compared to sensitizers using the multiple endpoint analysis of histology, cell viability and cytokine release. SLS concentrations between 1 and 15 mg/ml (0.5%–1.5%) were chosen for testing on the OMM since this is the concentration range of SLS that is typically found in commercial toothpastes. SLS can cause increased epithelial desquamation (Searls and Berg, 1986). Reports of mucosal sloughing, erosion and irritation have been attributed to surfactant content and therefore led to speculation that SLS may exacerbate certain mucosal conditions. This contention is supported by a study in which the effects of using SLS-free toothpaste were compared with standard SLS containing toothpaste in 47 patients with recurrent oral ulceration (Healy et al., 1999). We observed reduced ulcer incidence compared to using SLS-containing
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Fig. 7. Effect of SLS (5 mg/ml) and CA (1 mg/ml) on IL-1α expression in OMM. (a) Gene expression levels of IL-1α were measured by qPCR analysis and normalized against expression levels of 18SrRNA. Samples were run in triplicate on 3 separate days and the average point at which the signal from each sample crossed the threshold was taken, CT. The amount of target was calculated by taking the CT value of the test gene from the 18SrRNA CT. This value was then used to calculate the relative amount of template using the formula 2−ΔΔCT. (b) IL-1α release from OMM expressed as stimulation indices (SI) in relation to levels measured in controls at time points 0.5, 1, 2, 4, and 8 h following treatment. Experiments were carried out on triplicate cultures and cytokine responses from each OMM were measured in duplicate.
toothpaste, thus implicating SLS in oral mucosal damage within individuals with recurrent oral ulceration. Previous studies have illustrated the cytotoxic effects of SLS on organotypic cultures of human oral mucosa (Neppelberg et al., 2007) and monolayer cultures of gingival epithelial and gingival fibroblast cells (Babich and Babich, 1997); however, to our knowledge this is the first study using a reconstructed OMM to assess the effects of SLS on inflammatory cytokine release from oral epithelial cells. SLS concentrations higher than 0.5% have been shown to cause cell necrosis in the superficial cell layers of an oral mucosal model (Neppelberg et al., 2007). Surprisingly, Neppelberg et al. did not investigate whether necrosis stimulated the release of inflammatory cytokines, but they did postulate that it was possible that SLSinduced cell necrosis could activate an inflammatory response in the oral mucosa, thus helping to explain reports of the clinically
damaging effects of SLS in the mouth (Neppelberg et al., 2007). Our study adds to these observations, since we measured IL-1α and IL-8 in culture supernatants and found that both cytokines increased in comparison to PBS control with increasing SLS concentration. At the lowest concentration of 1 mg/ml SLS, cell viability remained high (93.6 ± 12.2%) with LDH and IL-1α release low, indicating that cellular damage was minimal. Furthermore, histological examination showed damage only to the upper two cell layers of the OMM. At the higher SLS concentrations, differentiating cytokine release attributable specifically to SLS from release due to cell death is difficult. However, by examining cytokine data following exposure to the other irritants and sensitizers screened, an indication of the levels of IL-1α and IL-8 that would be expected following substantial cell death may be obtained. For
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Fig. 8. Effect of SLS (5 mg/ml) and CA (1 mg/ml) on IL-8 expression in OMM. (a) Gene expression levels of IL-8 were measured by qPCR analysis and normalized against expression levels of 18SrRNA. Samples were run in triplicate on 3 separate days and the average point at which the signal from each sample crossed the threshold was taken, CT. The amount of target was calculated by taking the CT value of the test gene from the 18SrRNA CT. This value was then used to calculate the relative amount of template using the formula 2−ΔΔCT. (b) IL-8 release from OMM expressed as stimulation indices (SI) in relation to levels measured in controls at time points 0.5, 1, 2, 4, and 8 h following treatment. Experiments were carried out on triplicate cultures and cytokine responses from each OMM were measured in duplicate.
example, the irritant BC at a concentration of 1 mg/ml significantly reduced cell viability (28.1 ± 3.1%) and increased LDH denoting considerable cell death, but while IL-1α (SI = 134 ± 57) levels were high, IL-8 levels were low (SI = 0.8 ± 0.1). Similarly, 5 mg/ml CPC was also highly cytotoxic with IL-1α levels increased (SI = 212 ± 52.8) and IL-8 levels decreased (SI = 0.9 ± 0.2). On the other hand, the sensitizer compounds, CA, DNCB and NiSO4 were not as cytotoxic, and this resulted in an altogether different cytokine profile. CA and DNCB induced low levels of IL-1α but high levels of IL-8, whereas NiSO4 had no significant effect on release of either of the inflammatory cytokines measured. Therefore, by comparison the levels of IL-1α and IL-8 measured following treatment with the lower concentrations of SLS are not entirely the consequence of cell necrosis, but are in part due to de novo synthesis of IL-1α and IL-8. Evidence to support this observation was provided when gene ex-
pression of IL-1α and IL-8 was measured after application of 5 mg/ ml SLS, where IL-1α expression was maximal 4 h post exposure, with an 11-fold increase, whereas IL-8 expression increased 100fold and peaked 8 h after treatment. OMM cultures treated with 1 mg/ml CA also induced IL-1α and IL-8 gene expression, but the increase in IL-8 was modest as compared to SLS treatment. Thus the levels of IL-1α and IL-8 measured in culture supernatants from OMM cultures exposed to SLS are not due solely to stimulation by necrotic cells or leakage of constitutive IL-α from damaged epithelial cells, but are also the result of the induction of IL-1α and IL-8 gene expression. IL-1α is a key mediator of inflammation in skin, where it is constitutively expressed in keratinocytes (Welss et al., 2004). IL-1α released from keratinocytes following injury will induce expression of itself and other cytokines such as IL-6 and IL-8, by binding onto the IL-1 receptor type I found on the keratinocyte
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cell membrane (Corsini and Galli, 1998; Dinarello, 1998). Therefore, since damage to tissue morphology is not as profound when OMM cells are treated with 1 mg/ml and 5 mg/ml (0.5%) SLS, IL-1α released from damaged epithelial cells could induce IL-8 and further IL-1α expression from viable cells in the OMM. Consequently, IL-8 measured from these cultures could be a sum of IL-8 directly stimulated by SLS and IL-8 indirectly induced by the release of IL-1α from epithelial cells damaged by treatment with SLS. Furthermore, even at 10 mg/ml and 15 mg/ml SLS concentrations where histological examination shows substantial damage to the OMM, it is hypothesized that there remain sufficient numbers of viable cells that are able to produce IL-8 in response to SLS and IL-1α released from cells undergoing necrosis. Besides, if IL-1α levels measured with higher SLS concentrations were entirely due to cell necrosis and leakage of IL-1α from damaged cells, it is currently unclear why LDH levels remained relatively low in comparison to levels measured following BC and CPC treatments. We therefore propose that the IL-1α levels measured are a sum total of constitutive IL-1α from disrupted cells and induced IL-1α. The changes in IL-1α and IL-8 release observed using an OMM exhibit similar trends to those reported for a reconstructed human epithelial (RHE) skin model where irritants or sensitizers were applied (Coquette et al., 2003; Corsini and Galli, 1998). Even though application of DNCB to either model presented similar trends, the levels of IL-8 released from the OMM were much higher than for RHE. Similarly, levels of IL-8 observed when NiSO4 was applied to OMM would appear high in our study, when compared to those obtained by Coquette et al. suggesting that epithelial models derived from skin or oral mucosa may respond differently when challenged with the same compounds. Case reports indicate CA-containing dentifrice as causing inflammation of the lips, labial mucosa, and gingivae in sensitive individuals (Thyne et al., 1989) and topical application of 1% CA to the forearm has been reported to cause cutaneous flushing and to increase levels of circulating prostaglandins (PGE2) (VanderEnde and Morrow, 2001). CA has also been tested on human immature dendritic cells and, by analysis of the phenotypic alterations induced by CA, the sensitizing potential of CA could be predicted (Staquet et al., 2004). Certainly, from our data levels of IL-8 induced at both 1 mg/ml (0.1% w/v) and 5 mg/ml (0.5% w/v) CA concentrations were high and comparable with those obtained with DNCB which is a welldocumented strong sensitizer, suggesting potential value of the OMM in screening for ingredients which have sensitizing potential as well as screening potential irritants. In conclusion, this study is the first to show pro-inflammatory IL-1α and chemokine IL-8 release from oral epithelial cells treated with different concentrations of the common toothpaste surfactant SLS. Importantly, we have demonstrated through these studies that the OMM is a potentially valuable model for screening the relative oral irritation of new oral healthcare ingredients prior to clinical testing. Nevertheless, one must keep in mind that while this model is capable of predicting mucosal irritation it is still an in vitro model and as such it is far more sensitive. Hence, while the model is suitable for relative comparisons or for predicting trends, it is not appropriate for informing what level of oral care ingredient may cause irritation in the mouth. Conflict of interest The authors declare no potential conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version.
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