# Investigating the Influence of Conventional vs. Ultra-High Dose Rate Proton Irradiation Under Normoxic or Hypoxic Conditions on Multiple Developmental Endpoints in Zebrafish Embryos

## TL;DR

This study examines how ultra-high dose rate proton irradiation affects zebrafish embryos under different oxygen conditions, finding that it can reduce toxicity in some cases.

## Contribution

The study demonstrates the FLASH effect in zebrafish embryos under both normoxic and hypoxic conditions, highlighting tissue-specific responses.

## Key findings

- Ultra-high dose rate proton irradiation reduces toxicity in zebrafish embryos compared to conventional dose rates.
- The protective effect of ultra-high dose rate irradiation is observed in pericardial edema under normoxic conditions and in head and eye size under hypoxic conditions.
- The FLASH effect appears to be tissue- and endpoint-dependent, with oxygenation playing a variable role.

## Abstract

FLASH radiotherapy is based on the selective sparing of normal tissue side effects, keeping the same anti-tumor effectiveness, when the irradiation is performed at an ultra-high dose rate (i.e., >40 Gy/s). In this study, we explore the response of zebrafish embryos after proton irradiation at conventional and ultra-high dose rates, in terms of multiple developmental endpoints. The role of oxygenation at the time of irradiation is also explored. Overall, our data indicate that the so-called FLASH effect can be observed either under normoxic and hypoxic conditions, being more or less pronounced depending on the specific tissue and endpoint considered. This suggests that the sparing effect can have a significant dependence on the specific tissue, and that oxygenation could play a different role depending on the tissue type.

Objectives: To investigate how the FLASH effect modulates radiation response on multiple developmental endpoints of zebrafish embryos under normoxic and hypoxic conditions, after irradiation with proton beams at a conventional and an ultra-high dose rate (UHDR). Methods: Embryos were obtained from adult zebrafish and irradiated with a 228 MeV proton beam 24 h post-fertilization (hpf) at a dose rate of 0.6 and 317 Gy/s. For the hypoxic group, samples were kept inside a hypoxic chamber prior to irradiation, while standard incubation was adopted for the normoxic group. After irradiation, images of single embryos were acquired, and radiation effects on larval length, yolk absorption, pericardial edema, head size, eye size, and spinal curvature were assessed at specific time points. Results: Data indicate a general trend of significantly reduced toxicity after exposure to a UHDR compared to conventional regimes, which is maintained under both normoxic and hypoxic conditions. Differences are significant for the levels of pericardial edema induced by a UHDR versus conventional irradiation in normoxic conditions, and for eye and head size in hypoxic conditions. The toxicity scoring analysis shows a tendency toward a protective effect of the UHDR, which appears to be associated with a lower percentage of embryos in the high score categories. Conclusions: A radioprotective effect at a UHDR is observed both for normoxic (pericardial edema) and hypoxic (head and eye size) conditions. These results suggest that while the UHDR may preserve a potential to reduce radiation-induced damage, its protective effects are endpoint-dependent; the role of oxygenation might also be dependent on the tissue involved.