List of abbreviations AA = anaplastic astrocytoma; AE = anaplastic ependymoma; CAT = cata- lase; CNS = central nervous system; DA = diffuse astrocytoma; DAB = di- aminobenzidine; DNA = deoxyribonu- cleic acid; E = ependymoma; GBM = glioblastoma multiforme; GPx = glu- tathione peroxidase; GRx = glu- tathione reductase; GST = glu- tathione-S-transferase; HNE = 4-hy- droxynonenal; HNE-dG = 4-hydrox- ynonenal deoxyguanosine; IMD = in- tratumoral microvessel density; LPO = lipid peroxidation; MDA = malondi- aldehyde; PA = pilocytic astrocytoma; PUFA = polyunsaturated fatty acid; ROS = reactive oxygen species; SOD = superoxide dismutase; VEGF = vascu- lar endothelial growth factor Key words: 4-hydroxynonenal, astro- cytomas, ependymomas, angiogenesis. Acknowledgments: This study was supported by the Croatian Ministry of Science, Education and Sports and by COST Actions B35 and CM0602. We wish to thank Mrs Nevenka Boric and Mrs Branka Frankovic (Zagreb Clinical Hospital Center, Zagreb, Croa- tia) for excellent technical assistance, and Laligam N Sekhar, MD, FACS (Uni- versity of Washington, Seattle, USA) for generous assistance in preparing this paper. Correspondence to: Neven Žarkovic, MD, PhD, Institute “Rudjer Boškovic”, Bijenika 54, 10000 Zagreb, Croatia. Tel +385-1-457 1234; fax +385-1-456 1010; e-mail zarkovic@irb.hr Received November 11, 2008; accepted May 14, 2009. Distribution of 4-hydroxynonenal-protein conjugates as a marker of lipid peroxidation and parameter of malignancy in astrocytic and ependymal tumors of the brain Gordana Juric-Sekhar 1 , Kamelija Zarkovic 1 , Georg Waeg 2 , Ana Cipak 3 , and Neven Zarkovic 3 1 Department of Neuropathology, Zagreb Clinical Hospital Center, Zagreb, Croatia; 2 Karl Franzens University Graz, Institute of Molecular Biosciences, Graz, Austria; 3 Institute “Rudjer Boskovic”, Zagreb, Croatia ABSTRACT Aims and background. Lipid peroxidation (LPO) is an autocatalytic process caused by oxidative stress. It results in the production of 4-hydroxynonenal (HNE), which plays a crucial role in hypoxic brain injury, neuronal degeneration and apoptosis. The aim of this study was to evaluate the expression of HNE in 120 astrocytic and 40 ependymal tumors in relation to tumor type, grade of malignancy, angiogenesis, and presence of necrosis and apoptosis. Methods. Immunohistochemical staining was performed using a monoclonal anti- body for the detection of HNE-modified proteins. Results. HNE-protein adducts were found in all tumors. The incidence of HNE-im- munopositive tumor cells increased with increasing grades of malignancy. Signifi- cantly higher HNE expression was found in tumor cells of glioblastomas multiforme than in cells of pilocytic astrocytomas (P <0.005), and in anaplastic ependymomas than in benign ependymomas (P <0.01). HNE-immunopositive tumor cells were dis- tributed more diffusely than in perivascular locations (P <0.05). Pronounced HNE- protein adducts were detected in mitotic, necrotic, and apoptotic cells. HNE was ex- pressed in the endothelium of almost all tumor vessels, but its expression in the walls of the vessels was significantly higher in diffuse and anaplastic astrocytomas than in pilocytic astrocytomas and glioblastomas multiforme (P <0.05). The number of mi- crovessels containing HNE in their endothelium and walls was significantly associat- ed with the grade of malignancy in both astrocytic (P <0.001) and ependymal tumors (P <0.05), although microvessels in pilocytic astrocytomas were significantly more numerous (P <0.05) than in diffuse astrocytomas. Conclusions. LPO seems to be a common pathological process in astrocytic and ependymal glial tumors, proportional to the level of malignancy and neovasculariza- tion. Therefore, HNE might be involved in the damage of brain cells and the induc- tion of malignancy. Introduction 4-Hydroxynonenal (HNE) is a major product of lipid peroxidation (LPO), which is formed by radical-initiated degradation of ω-6-polyunsuturated fatty acids (PUFA) such as linoleic acid and arachidonic acid. The HNE level becomes elevated in cells under oxidative stress, and this aldehyde interacts with deoxyribonucleic acid (DNA) to form 4-hydroxynonenal deoxyguanosine (HNE-dG) adduct. Such HNE-dG adducts have also been found in various normal tissues of humans and rats 1-3 . HNE forms rel- atively stable bioactive protein adducts and its biological effects are proportional to the intensity of its binding to the cellular proteins 4 . HNE is cytotoxic 5 , but it is also a Tumori, 95: 762-768, 2009