Complete Artificial Saliva Alters Expression of Proinflammatory Cytokines in Human Dermal Fibroblasts

toxicological sciences 134(1), 18–25 2013 doi:10.1093/toxsci/kft103 Advance Access publication April 29, 2013 Complete Artificial Saliva Alters Expression of Proinflammatory Cytokines in Human Dermal Fibroblasts Gloria E. Malpass,*,1 Subhashini Arimilli,† Gaddamanugu L. Prasad,‡ and Allyn C. Howlett* *Department of Physiology and Pharmacology and †Department of Microbiology and Immunology, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157; and ‡R&D Department, R. J. Reynolds Tobacco Company, Winston-Salem, North Carolina 27102 To whom correspondence should be addressed at Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Medical Center Boulevard, Winston-Salem, NC 27157. Fax: (336) 713-1545. E-mail: . 1 Received January 25, 2013; accepted April 22, 2013 Artificial saliva (AS), sometimes called complete artificial saliva (CAS), is a saliva substitute prepared according to the protocol of Chou and Que Hee (1994) and is often employed as a vehicle for orally used test articles including smokeless tobacco products (Pappas et al., 2008). Alternatively, dimethyl sulfoxide (DMSO) is often used when testing smoked tobacco products, where total particulate matter from smoke generated by puffing is typically collected on filter pads and eluted off using this solvent (Johnson et al., 2009). In preliminary studies comparing the effects of different tobacco products on gene expression in cultured normal adult human dermal fibroblasts (HDFa), we observed that gene expression for proinflammatory cytokines interleukin 8 (IL8) and tumor necrosis factor-α (TNF-α) and for vascular adhesion molecule 1 (VCAM1), a gene upregulated by inflammatory cytokines TNF-α and IL1 (Carlos and Harlan, 1994), increased after 5-h exposure to CAS, but not DMSO (Supplementary fig. 1). These findings suggested that components of CAS alter the expression of proinflammatory cytokines. Because AS is essential for controlled in vitro experiments in the study of test articles used in the oral cavity, it should react with test materials in a manner similar to that of natural saliva in order to create a valid artificial oral environment (Leung and Darvell, 1997). To mimic many of the properties of human saliva, CAS is buffered at pH 7.0 (phosphate and calcium) and contains mucin, α-amylase, lysozyme, acid phosphatase, and urea (Chou and Que Hee, 1994). Calcium and phosphate help maintain tooth mineral integrity. Mucins protect the tooth surface against demineralization of enamel and promote remineralization, provide lubrication, bind to toxins, agglutinate bacteria, interact with host cells, and may protect the esophagus in gastroesophageal reflux disease (Castagnola et al., 2011; Dodds et al., 2005). α-Amylase, the most abundant protein in human saliva, catalyzes the breakdown of starch and glycogen to maltose, inhibits the adherence and growth of bacteria to epithelial surfaces (Papacosta and Nassis, 2011), and binds with high affinity to certain oral streptococci (Scannapieco et al., 1993). Lysozyme, a relatively minor component of saliva, has antimicrobial properties as a result of disrupting bacterial cell walls (Dodds et al., 2005; Humphrey and Williamson, 2001; Papacosta and Nassis, 2011). Acid phosphatase frees phosphate groups from other molecules during digestion. Saliva also contains urea that works together with bicarbonates and phosphates to modulate pH and buffering capacity of this fluid. The studies reported herein demonstrate that CAS alters the expression of certain proinflammatory cytokines, © The Author 2013. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: . Complete artificial saliva (CAS) is a saliva substitute often used as a vehicle for test articles, including smokeless tobacco products. In the course of a study employing normal adult human dermal fibroblasts (HDFa) as a model in vitro, we discovered that CAS as a vehicle introduced a significant change in the expression of proinflammatory cytokines. To determine the effects of CAS on gene expression, real-time quantitative reverse-transcriptase PCR gene array analysis was used. Results indicate that robust changes in the expression of the proinflammatory cytokine interleukin 8 (IL8) and the vascular cell adhesion molecule 1 (VCAM1) occur within 5 h of exposure to CAS. To determine whether CAS also alters cytokine release into the culture media, cytometric bead array assays for human inflammatory cytokines were performed. Analysis shows that CAS induced the release of IL8 and IL6. This study focused on determining which components in CAS were responsible for the proinflammatory response in HDFa. The following components were investigated: α-amylase, lysozyme, acid phosphatase, and urea. Results demonstrated that enzymatically active α-amylase induced gene expression for proinflammatory cytokines IL8, IL6, tumor necrosis factor-α, and IL1α and for VCAM1. Therefore, it is important to carefully evaluate the “vehicle effects” of CAS and its components in in vitro toxicology research. Key Words: vascular adhesion molecule 1; human dermal fibroblasts; tumor necrosis factor-α; interleukin 8; α-amylase. ARTIFICIAL SALIVA AND CYTOKINES  19 including IL8, and VCAM1, a gene induced by proinflammatory cytokines, in HDFa. To determine which components of CAS induce the expression and release of proinflammatory cytokines in HDFa, we investigated α-amylase, lysozyme, acid phosphatase, and urea. We report data indicating that α-amylase is responsible for this proinflammatory response. We caution that CAS may not be an inactive vehicle in studies involving immune function. Materials and Methods Cell culture for gene expression and cytokine release studies. HDFa were maintained in serum-complete media composed of FBM supplemented with 10% FBS, GlutaMAX I (2mM), 1% Pen-Strep, and phenol red (2μM) in a humidified 5% CO2 incubator at 37°C. To initiate the experiments, HDFa were plated at a density of 4 × 105 cells in 60-mm tissue culture dishes in serumcomplete media, such that cells were 50 to 80% confluent and actively proliferating at the time of each experiment. After 20–24 h, serum-complete media were removed, cells were rinsed twice with DPBS, and media were changed to serum-free defined media composed of FBM, GlutaMAX I, Pen-Strep, phenol red, and fibroblast supplemental growth factors (hydrocortisone hemisuccinate [1 µg/ml], human serum albumin [500 µg/ml], linoleic acid [0.6µM], lecithin [0.6 µg/ml], rh FGFβ [5 ng/ml], rh EGF/TGF [5 ng/ml], β-1 supplement [30 pg/ ml], rh Insulin [5 µg/ml], and ascorbic acid [50 µg/ml]). Appropriate treatment or vehicle was added, and cells were incubated at 37°C and 5% CO2. At the designated time following treatment (1) culture media were collected for determination of released cytokines, and (2) cells were collected for RNA isolation. Gene expression arrays. (...truncated)