**4. Bystander E**ff**ect: Potential Role for Purinergic Signaling**

Ionizing radiation affects cells within the radiation field and indirectly on adjacent, non-irradiated cells and tissue. This phenomenon, called the bystander effect, has been described for multiple cancer models [110–114] and has been investigated in relation to the protection of non-irradiated tissue [115], therapeutic approaches to cancer progression [112,116] and secondary radiation-induced neoplasms [112,117]. The bystander effect can be initiated by the release of several signaling molecules from irradiated cells, including ROS, nitric oxide (NO) or cytokines such as TGF-β1 [111,118] and through direct intercellular interactions via gap junctions or membrane channels [119–121] (Figure 2). While sparing techniques in cancer therapies for RT have attempted to remove salivary glands from the radiation field and/or reduce the overall radiation dose delivered to the glands, bystander effects still persist, suggesting that other radioprotective and regenerative approaches are needed [122].

**Figure 2.** Radiation-Induced Bystander Effects. The bystander effect can be propagated by intercellular communication between adjacent cells via gap junctions or by autocrine or paracrine signaling processes whereby NTPs, nitric oxide (NO), reactive oxygen species (ROS), cytokines (e.g., TGF-β), or other second messengers elicit a response in non-irradiated cells. Created with Biorender.com.

In the past ten years, mounting evidence demonstrates that purinergic signaling can mediate the IR-induced bystander effect in adjacent, non-irradiated cells [110,122–128]. Extracellular nucleotides such as ATP (eATP), which are released into the extracellular space in response to cellular damage including ionizing radiation [124,128], act as autocrine or paracrine signaling molecules via activation of P2 receptors (P2Rs) on nearby cells [122–124,128–134]. In response to γ-irradiation, both human and murine cell lines have been shown to release ATP, thereby activating G protein-coupled P2Y [110,129,135] and ATP-gated ionotropic P2X receptors [126,136], which initiate purinergic signaling. Although no studies have yet investigated this bystander effect in IR-induced salivary gland dysfunction, we and others have reported on the expression of P2Y2, P2X7 and P2X4 receptors in salivary gland epithelia of mice [137–139] and humans [139], suggesting that irradiated salivary glands in vivo should be highly sensitive to elevated levels of IR-induced eATP release. Mechanisms of P2 receptor signaling relevant to the bystander effect have been investigated in multiple tissues [110,122–128] and their potential roles in IR-induced salivary gland damage are summarized in Figure 3.

**Figure 3.** Purinergic Signaling in Bystander Effects. In response to elevated extracellular ATP (eATP) levels following irradiation, P2Y2R and P2X7R, which are expressed in murine and human salivary glands, may mediate a number of bystander effects. Through activation of the NLRP3 inflammasome, P2X7Rs promote ROS production, growth factor maturation (e.g., IL-1β) and subsequent release and apoptosis. Activation of either P2Y2R or P2X7R causes increased [Ca2<sup>+</sup>]i and subsequent downstream signaling processes (e.g., ERK1/2 signaling, gene expression changes, inflammatory processes). P2Y2R activates phospholipase C (PLC) resulting in the production of inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG), in turn mobilizing intracellular Ca<sup>2</sup><sup>+</sup> and activating protein kinase C (PKC), respectively, and subsequent downstream signaling. Through its C-terminal Src-homology 3 (SH3) domain, the P2Y2R is involved in Src-dependent activation of growth factor receptors (e.g., EGFR, VEGFR-2) and downstream MAPK signaling as well as transactivation of EGFRs through activation of metalloproteases, ADAM10 and ADAM17. P2Y2Rs may also contribute to the bystander effect by promoting latent TGF-β signaling. DAG: diacylglycerol; PLC: phospholipase C; PKC: protein kinase C; ERK: extracellular signal-regulated protein kinase; JNK: c-Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; COX: cyclooxygenase; PGE2: prostaglandin E2; ADAM: A Disintegrin And Metalloprotease. Created with Biorender.com.
