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Ozone (O3) is a major air pollutant and is well-known for its very strong oxidative actions with beneficial effects in purifying various matrixes.In particular, ozone is frequently used for chemical industrial applications replacing chorine as a purifying and sterilizing agent for matrixes such as air, water and soil.In recent years there has been an ever increasing interest in ozone for its potential use in biological systems and for medicinal applications.Ozone converts to reactive oxygen species (ROS) upon contact with biological membranes, which ultimately are responsible for its systemic oxidative effects.In addition, ozone has been proposed as a cellular metabolic activator and immunomodulator.Numerous reports have entered the literature on the medicinal application of ozone in altemative medicine.However, the majority of the data from these reports has come from uncontrolled investigations and unsubstantiated medicinal claims for a variety of therapeutic area, including disease of the brain, pain, and treatment of HIV/AIDS.The safety concerns and the medicinal claims have prompted us to conduct several in vitro and in vivo investigations on the biological activity of ozone.The experimental protocols were designed to evaluate the effects of ozone in isolated organ experiments, as well as following acute and chronic administration to intact test animals in an in vivo exposure model.Since ozone would be administered by inhalation, the possible effects of ozone on rodent respiratory tissue were studied during in vitro experiments.Subsequently, the intact animal in vivo studies were conducted.The successful experimental design allowed for the dose-and time-dependent investigations into the effects of inhaled ozone on the central nervous system, and to measure subsequent effects on the behaviour of the animal.These ozone studies allowed for effective investigation of the biological activity of ozone as well as the mechanisms involved in ozone-induced oxidative stress, particularly in the brain.The data further showed that the responses after ozone treatments differ in a concentration-dependent manner with respect to acute versus chronic treatments.In conclusion, these studiesprovided information on the on the safety and biological activity of ozone and that oxidative stress conditions in biological systems, have significant biological and behavioural effects.The data from these studies further caution against ad hoc systemic use of ozone for medicinal purposes.