Life Sciences, Vol. 65, Nos. 1809, pp. 1901-1904, 1999 Comieht 0 1999 Elsevk ScienceInc. ELSEVIER PII s0024-3205(99)00444-0 Printed in the USA. All rights reserved 0024-3205B!X-set fkmt matter CENDROXYNONENAL AS A SECOND MESSENGER OF FREE RADICALS AND GROWTH MODIFYING FACTOR Neven Zarkovid, Kamelija Zarkovic’, Rudolf Jiirg Schaur3, Svorad Sto1c4, Gtinther S&lag’, Heinz Red?, Georg Waeg3, Suzana Borovic’, Iva Loncaric’, Gordana Juric’, Vladimir Hlavka’ ’ - “Rudjer Boikovic” Institute, Zagreb, Croatia; ’ - Jnstitute of Neuropathology, Medical Faculty, KBC “Rebro”, Zagreb, Croatia; ’ -Jnstitute of Biochemistry, Graz, Austria; 4 - Institute of Exp. Pharmacology SASc, Bratislava, Slovakia; ’ - “Ludwig Boltzmann” Institute of Traumatology, Vienna, Austria Summary Immunohistochemical analysis of the distribution of the lipid peroxidation product 4-hydroxynonenal (HNE) in the brain of baboons exposed to experimental hemorrhagic traumatic shock or sepsis showed that systemic oxidative stress and the thereby generated HNE affect the blood:brain barrier and the regulation of cerebral blood flow determing secondary brain damage. Similarly, HNE was determined during ischemia in the brain blood vessels of rats exposed to ischemia/reperfirsion injury of the brain. After reperfusion, HNE disappeared from the blood vessels but remained in neurones and in ghal cells. Since HNE modulates cell proliferation and differentiation (including proto-oncogcne expression), it is postulated that HNE might have prominent local and systemic effects that are not only harmful but beneficial, too, determing the outcome of various pathophysiologcial conditions based on oxidative stress. Key Words: oxidative stress, lipid peroxidation, 4-hydrox~nonenal, brain, c-fos, immunol&ochemistIy, cell growth, free radicals, reactive oxygen species Aldehyde 4-hydroxynonenal (HNE) is a lipid peroxidation product generated during pathophysiological conditions based on the production of reactive oxygen species (ROS) and is considered as a causative factor of secondary tissue damage (1). However, HNE is not only a cytotoxic mediator of the oxidative stress, but also a potent growth modifying factor and a normal constituent of various cells and tissues (1.2). The study presents some recent immunohistochemical findings on HNE distribution in the brain and its effects on cell growth regulation in vitro. Materials and Methods zyxwvutsrqponmlkjihgfedcbaZYXWVU In vitro effects of 4-hydroxynonenal HNE was prepared as previously described (3) and added to established human uterine carcinoma cell cultures (HeLa) at 50 pM final concentration (50% cytotoxicity dose). After addition of HNE to the cultures, the medium was not changed, and the cells were further cultured under standard conditions for different time schedules (from 15 min., in c-fos RT- PCR, until 3 days, for cell count determinations) (2,4,5). The intensity of incorporation of 3H- thymidine (Amersham,U.K.) added at 0.5 nCi per well was determined for the last 24 hours of culturing, when the cells were harvested with a flow harvester (Skatron, Norway) and the quantity of incorporated 3H-thymidine was measured by a D-counter (LS 7800, Beckman, USA). Evaluation of c-fos expression was done by RT-PCR using AMV-reverse transcriptase