Dietary nitrate supplementation prevents radiotherapy-induced xerostomia

Thank you for submitting your article "Dietary nitrate supplementation prevents radiotherapy-induced xerostomia" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Wafik El-Deiry as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Fred Bunz (Reviewer #1); Changyu Zheng (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

The reviewers were enthusiastic about the findings and their potential for practical application in the clinic to address xerostomia. Revisions are suggested by each of the reviewers, including more discussion of limitations of the evidence for the role of EGFR signaling, nitrate signaling and free radicals.

Reviewer #1:

Xerostomia, a frequent side-effect of radiotherapy to the head and neck, can compromise dental health, nutrition and speech. As a result, affected patients often experience a significant decline in their quality of life. Current measures used to prevent or manage radiation-induced xerostomia are often ineffective. In the present study, the authors set out to determine whether dietary supplementation with inorganic nitrate might be a useful approach to the prevention and/or management of this important manifestation of radiation toxicity.

The in vivo studies were based on the miniature pig, a previously established model of salivary gland dysfunction that recapitulates many of the radiation responses observed in human tissues. Dietary nitrate was administered either prophylactically, prior to irradiation, or therapeutically to irradiated animals. Pre-treatment with nitrate resulted in the preservation of salivary gland function, longitudinally assessed by salivary flow rate and local blood flow, in animals that were subsequently treated with clinically-relevant doses of ionizing radiation. Histologically, these pre-treated animals also exhibited elevated levels of acinar glad proliferation and lower levels of local fibrosis, a late effect of radiotherapy that is observed in human patients. In striking contrast, nitrate supplementation administered after irradiation did not appear to ameliorate these long-term toxicities.

Molecular analyses of parotid tissues supported these impressive clinical observations, as did in vitro studies of human parotid gland cells. An important finding was the dose-dependent relationship between nitrate administration and the expression of the transmembrane protein sialin. Sialin acts as a local transporter of nitrate and was shown here to enhance proliferation and suppress apoptosis in this cell type, in what the authors describe as a positive feedback loop. This novel hypothesis provides a plausible explanation for the observed clinical and histological effects of nitrate pre-treatment.

The mechanistic studies of how sialin might trigger cell proliferation were exclusively focused on the activity of the EGFR-AKT-MAPK pathway. The activation of this pathway by sialin and nitrate seemed real, but modest in magnitude. Compared to the other sections, this part was somewhat underdeveloped. From the data presented, it is difficult to conclude that EGFR activation would account for the dramatic clinical and histologic effects of pre-treatment. This is a relatively minor weakness, which could be addressed by adding further context to the discussion. Overall, this was a rigorous study that presents a new, highly translatable approach to radiation-induced xerostomia.

The question of whether the EGFR-AKT-MAPK is responsible for the observed effects could simply be addressed in a revised discussion. It would suffice to mention the caveat that additional pro-proliferative or anti-apoptotic mechanisms, not addressed in this study, could conceivably be involved.

Second, it might also be helpful to contextualize the doses of sodium nitrate used in vivo. Would similar dosage in humans be tolerable? What was the level of nitrates in the non-supplemented feed? In the average human diet?

Reviewer #2:

Manuscript of "Dietary nitrate supplementation prevents radiotherapy-induced xerostomia" by Xiaoyu Feng, et al., is an interesting article. The presented data suggest that nitrate, dietary nitrate supplementation, effectively prevented radiotherapy-induced-induced xerostomia via the EGFR-AKT-MAPK signaling pathway, suggesting that dietary nitrate supplementation may provide a novel, simple, safe and effective way to prevent IR-induce xerostomia. Their experimental design is straightforward. Most results are solid. This manuscript will be a useful reference for the field.

Following comments are needed to clarify.

1. Please check if "Mechanically" in line 26 is right word.

2. Would like to check if nitrate affects free radicals, which are produced during irradiation, and mitochondrial functions. If authors don't know yet, hope authors can try to understand these in their future study.