Proteome and Transcriptome in Irradiated Glands Figure 3. Fold change in gene expression following irradiation. Relative quantification (RQ) values of Muc19, Cdh22, Psp, Prb1, CysS, Mgmt, cyclin d1, p21, p57 and Ywhaq gene expression during the 12 weeks post-irradiation. Validation of microarray values for salivary gland functional gene expression following irradiation by TaqMan real-time PCR. The data are expressed as means of n = 3. *P,0.05, **P,0.005, ***P,0.001 compared to control levels, unless otherwise stated. doi:10.1371/journal.pone.0040636.g003 therapy used on human patients is a fractionated dose [29]. However, in experimental animal models, it has been shown that a single high dose or its equivalent dose as fractionated IR lead to essentially the same end result, i.e. reduced saliva secretion [6,7]. We therefore used a single dose in our experiments. In an initial confirmation of previous experiments, we found that a single 15- Gy dose of IR to the head and neck in a rat model reduces saliva output to 50% of normal [12,30] as well as inducing minimal salivary tissue damage with no signs of fibrosis [2,11,12]. To better understand the mechanisms underlying salivary gland IR damage, we performed both a proteomic analysis of the saliva and gene array of the SSG tissue before and after IR. As IR affects protein production, we examined its global changing effect on the saliva proteome by comparing it to non- irradiated animals and to previous reported proteomic maps [31,32,33,34]. Proteomic analysis of oral fluid (whole saliva) protein composition represents the summed effect of the IR treatment on all glands, not only on the SSGs. It reflects at least two different consequences of the IR: (i) reduced salivary- originated proteins and (ii) increased serum-derived proteins. The reduction in salivary proteins in the oral fluids occurred in two steps and became larger with time. Four weeks after IR, expression of most proteins decreased, followed by partial recovery at week 8 and a further decrease at week 12 post-IR. Comparison of these results to the gene-array assay showed partial similarity at the 4-week time point (pattern 1, Fig. 2B); however at the later time points, while gene expression recovered, protein levels remained low. This decrease in protein synthesis might be explained by the transcriptomic data showing damage to protein synthesis, processing or trafficking pathways (pattern 1). The increase in serum proteins, mainly albumin, in the saliva indicates damage to the tight junctions in the salivary cell membrane causing loss of barrier function between serum and secretory ductal glandular compartment components. Tight junctions are also needed for directional fluid movement from the acini to the ducts and therefore, their damage may contribute to a reduction in the efficiency of fluid secretion by the glands. These findings are also supported by our real-time PCR results showing down- regulation of cell-adhesion molecules such as Cdh22, and by our microarray results showing down-regulation of genes encoding epithelial cell-adhesion molecule (Epcam) and gap junction beta 2 (Gjb2). Gene-array analyses showed that following IR, the expression of 95 genes was modified following two general patterns. The most prominent pattern, shown by 81 of the genes, was a damage-like response. The genes exhibiting this pattern were grouped into categories associated mainly with protein synthesis and secretion (endoplasmic reticulum, protein transport, golgi apparatus, protein folding and secretory granule), indicating a reduction in the cell’s ability to produce and secrete protein products not only as a direct result of possibly lower transcription levels but also as a result of damage to protein synthesis, processing or trafficking pathways. PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e40636