# Ozonated Water Modulates Cell Proliferation and Vascular Density in Solid Ehrlich Tumor of Mice

## TL;DR

Ozonated water initially slows tumor cell growth in mice but may later increase blood vessel density, suggesting mixed effects on tumor progression.

## Contribution

This study reveals the dual effects of ozonated water on tumor cell proliferation and vascular density in mice.

## Key findings

- Ozonated water reduced tumor cell proliferation after five days of treatment.
- At 30 days, ozonated water increased blood vessel density, especially with repeated applications.
- Treatment discontinuation favored increased vascular density in the long term.

## Abstract

The solid Ehrlich tumor (SET) is an experimental model in mice used to study cancer. Ozone therapy has been suggested as a complementary treatment in medicine, but its effects on tumors are not fully understood. This study tested whether ozonated water could influence tumor blood vessels and cell growth. Mice with SET were treated with different doses of ozonated water, given either inside or around the tumor, and compared to control groups. The researchers measured blood vessel formation by CD31 and tumor cell growth by Ki-67. Results showed that ozonated water reduced tumor cell proliferation after five days, and at 30 days, it increased blood vessel density, especially with repeated applications. These findings suggest that ozone therapy may have both beneficial and harmful effects on tumors, helping to slow cell growth in the short term, but possibly favoring tumor progression over time.

Background: The solid Ehrlich tumor (SET) is a transplantable experimental neoplasm that mimics mammary adenocarcinoma in female mice, widely used to investigate tumor physiology, behavior, and therapeutic interventions. Among emerging approaches, ozone therapy has gained attention in human and veterinary medicine, prompting studies to clarify its mechanisms and potential applications. This study evaluated vascular and tumor cell proliferation in SET of mice treated with ozonated water under different protocols. Methods: A total of 99 animals were allocated into four groups: ozonated water at 104 mM/5 ppm (G1, n = 30), 208 mM/8 ppm (G2, n = 30), vehicle control with 0.9% saline (G3, n = 30), and negative control (GCN, n = 9). Subgroups were established according to administration routes (intratumoral or peritumoral), number of applications (one or two), and observation periods (24 h, five days, or 30 days). Immunohistochemistry with anti-CD31 and anti-Ki-67 antibodies assessed vascular and cellular proliferation, respectively, considering peri- and intratumoral regions. Results: Increased CD31 expression was detected at 30 days in treated groups compared to controls, particularly after two intratumoral applications and in all peritumoral (PT) protocols. Ki-67 expression was reduced after five days in the treated groups, indicating decreased cell proliferation relative to controls. A positive correlation was observed between peri- and intratumoral CD31 immunostaining. Conclusion: Ozonated water reduced tumor cell proliferation in the medium term, but treatment discontinuation favored increased vascular density in the long term. These findings suggest caution in the oncological use of ozone, as it may present both antineoplastic and tumor-promoting effects depending on treatment conditions.