*3.1. DNA Damage, Insu*ffi*cient DNA Repair and Cell Cycle Arrest*

Administration of IR to salivary glands activates an array of signaling pathways that influence the development of acute hyposalivation. Within minutes of IR exposure, DNA double-strand breaks were detected in mouse parotid glands using a neutral comet assay and were associated with increased phosphorylation of the H2A histone family member X (referred to as γH2AX) [59]. Furthermore, DNA strand breaks were insufficiently repaired in parotid glands due to reduced activity of the stress-induced deacetylase, sirtuin-1, that results in reduced phosphorylation of the DNA repair protein, NBS1, likely due to inadequate deacetylation that is necessary for optimum kinase function and initiation of the DNA damage response [59]. Insulin-like growth factor (IGF)-1 pretreatment in mice preserved salivary gland function following IR exposure [5]. Mice pretreated with IGF-1 had reduced γH2AX levels, increased NBS1 phosphorylation and improved DNA repair capabilities. Blocking sirtuin-1 activity using a pharmacological inhibitor in combination with IGF-1 therapy decreased DNA repair efficiency, confirming the importance of sirtuin-1-mediated DNA repair in conserving parotid gland function post-IR, especially in the context of IGF-1-mediated preservation of glandular function [59].

Following IR-induced damage, salivary glands insufficiently undergo cell cycle arrest that would allow for complete DNA repair. Following 5 Gy IR, parotid glands exhibit reduced cell cycle arrest, with a low percentage of cells in the G2/M phase at 8 h post-IR, as well as reduced levels of the cell cycle arrest gene, *p21*, at 24 h post-IR [47]. At 8 and 24 h post-IR, there were elevated levels of total and phosphorylated p53 tumor suppressor protein and increases in the truncated, inhibitory isoform of the p53 homolog, p63, (ΔNp63) which is known to block transcription of genes, including *p21*. In the IR-induced salivary gland damage model, IGF-1 pretreatment reduced salivary dysfunction in mice through induction of cell cycle arrest by increasing *p21* transcription due to reduced ΔNp63 binding and increased p53 binding to the *p21* promoter 8 h post-IR [47]. Interestingly, pretreatment of mice with roscovitine, a cell cycle inhibitor, 2 h prior to IR, increased G2/M phase cell cycle arrest and p21 protein content within 6 h post-IR [72]. Compared to vehicle treatment, roscovitine increased phosphorylation of protein kinase B (Akt), a master regulator of cell survival, and mouse double minute 2 homolog (MDM2), an E3 ubiquitin ligase that negatively regulates p53, at 6 h post-IR, which correlates with reduced apoptosis at 24 h post-IR and improved salivary output at days 3 and 30 post-IR [72]. These results confirm the importance of cell cycle inhibition immediately following IR-induced damage to enhance DNA repair and reduce apoptosis in salivary glands.

#### *3.2. Reactive Oxygen Species Generation*

Reactive oxygen species (ROS) production is a known consequence of IR treatment and typically induces cellular damage immediately following IR exposure. In rats receiving 5 Gy IR, there was a significant reduction in the activity of the free radical scavenging enzymes superoxide dismutase, glutathione peroxidase and glutathione S-transferase that correlates with elevated levels of the oxidative stress markers, malondialdehyde and xanthine oxidase, as well as increased levels of peroxynitrite, nitric oxide synthase and nitric oxide in salivary glands at day 10 post-IR [61]. In mouse primary submandibular gland (SMG) cells, mitochondrial ROS levels were increased by days 1–3 post-IR with a reduction in ROS levels observed in cells deficient in transient receptor potential melastatin-related 2 (TRPM2), a calcium-permeable cation channel that is activated by oxidative stress and the DNA damage responsive protein, poly (ADP-ribose) polymerase 1 (PARP1), which correlates with improved salivary secretory function post-IR [45]. Furthermore, pharmacologically quenching ROS levels with Tempol improved salivary gland function in mice post-IR [46]. Another group showed that ROS and malondialdehyde levels remained elevated at day 7 post-5 Gy IR in SMGs, but were reduced by adenoviral induction of Sonic Hedgehog signaling at day 3 post-IR, which promoted DNA damage repair [60]. In rats receiving 18 Gy IR, there were elevated levels of the ROS-generating enzyme, NADPH oxidase at days 4–7 post-IR and increased DNA oxidation, measured as enhanced oxidized deoxyguanosine production by 4 days post-IR [58]. This phenotype was reversed following treatment with the antioxidant, α-lipoic acid, that correlated with increased amylase content and salivary function in SMGs [58]. Taken together, these results indicate that IR-induced ROS generation is detrimental to salivary gland function.
