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

Abstract

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 5h 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.

Protective Effect of Strawberry Extract against Inflammatory Stress Induced in Human Dermal Fibroblasts

“…The endotoxin LPS constitutes an outer membrane structure and an important virulence factor of the cell wall of Gram-negative bacteria, commonly used as an inflammatory agent [ 18 , 19 ]. HDF represent a standard cell model for different kinds of toxicity determinations [ 20 , 21 ]; it is also known that HDF recruit immune cells via soluble mediators and contribute to the progression of inflammatory processes [ 22 , 23 ]. For this reason it is very important to better understand how fibroblasts produce and secrete different inflammatory mediators, in relation to LPS-induced stress.…”

Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

“…2A). This effect was blunted or not observed in cells treated with defined media containing the DMSO vehicle, reflecting a difference from the effect of components of the CAS vehicle (Malpass et al, 2013). STE significantly decreased the IL8 expression (relative to vehicle control), while TPM significantly increased the response after 5 h in culture, and these effects were not due to nicotine.…”

“…Elevated levels of IL8 in in vitro studies negatively affect the morphology and function of fibroblasts (Iocono et al, 2000). It has been observed that α-amylase is responsible for a CAS-induced increase in genes for certain pro-inflammatory cytokines including IL8 and VCAM1 in HDFa (Malpass et al, 2013). Our data indicate that STE inhibits or reverses this stimulation of IL8 expression, suggesting that STE may reverse some of the effects of salivary pro-inflammatory mediators.…”

Efeito da fotobiomodulação sobre o estresse oxidativo e o dano no DNA induzido pelo bussulfano em doses encontradas na saliva: estudo in vitro

“…Foi constituída saliva artificial com base em Malpass et al (2013), com a seguinte composição: mucina (1.35 g/l), cloreto de potássio (475 mg/l), cloreto de sódio (700 mg/l), cloreto de cálcio (185 mg/l), fosfato de potássio (420 mg/l), cloreto de magnésio (105 mg/l), uréia (45 mg/l), D-(+)-glicose (100 mg/l), α-amilase (100,000 U/l), lisozima (750 U/l) e fosfatase ácida (4 U/l). Esta saliva artificial, por vezes chamada de saliva artificial completa, é utilizada para experimentos in vitro, a fim de criar um ambiente oral artificial válido.…”

“…Esta saliva artificial, por vezes chamada de saliva artificial completa, é utilizada para experimentos in vitro, a fim de criar um ambiente oral artificial válido. Segundo Malpass et al (2013) Bezinelli et al, 2015). A irradiação foi realizada com a fibra óptica mantida em posição perpendicular ao fundo da placa.…”