Ozonated water modulates cell proliferation and vascular density in solid ehrlich tumor of mice

Abstract

Background: The solid Ehrlich tumor (SET) is a transplantable experimental neoplasm that mimics mammary adenocarcinoma in female mice, widely used to investigate tu- mor physiology, behavior, and therapeutic interventions. Among emerging approaches, ozone therapy has gained attention in human and veterinary medicine, prompting stud- ies 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). Immunohistochem- istry with anti-CD31 and anti-Ki-67 antibodies assessed vascular and cellular proliferation, respectively, considering peri- and intratumoral regions. Results: Increased CD31 expres- sion 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.