1 FERRIC IRON ACCUMULATION IN THE BRAIN - A NEW NANOTECHNOLOGY RESEARCH CHALLENGE Mohorovic L 1*, Jonjic N 2, ,Micovic V. 1, Pavelic K 3, Lavezzi AM 4 Labinac-Peteh L 5 1 Department of Environmental Medicine, University of Rijeka School of Medicine, Rijeka, Croatia 2 Department of Pathology, University of Rijeka School of Medicine, Rijeka, Croatia 3. Department of Biotechnology, University of Rijeka, Croatia 4 “Lino Rossi” Research Center for The Study and Prevention of Unexpected Perinatal Death and SIDS,Department of Surgical,Reconstructive and Diagnostic Sciences,University of Milan, Italy 5. Department of Pathology, General Hospital Pula ,Croatia * Mohorovic L, MD,PhD:
2 LABIN Raša Sv. Nedelja Pićan Kršan Study area Croatia Labin area
3
4 Objective As a main aim we want to make a contribution to the establishment about the role of environmental toxic factors on the endothelial small vessels of the brain, increasing the rate of endothelial cell apoptosis and making possible the transport of methemoglobin and heme derived ferric iron across the bloodbrain-barrier (BBB), and its accumulation in the brain parenchyma.
5 Introduction Our hypothesis originate from using epidemiologically the notion that very important environmental factors such as the Intensity of exposure, the Accumulation of oxidants , the Synergism of nitrogen oxide–sulfur dioxide metabolites , and the continuous inhalation of oxidants, nitrogen oxides, sulfur dioxide and their metabolites over longer periods, cause vascular endothelial dysfunction from the early pregnancy to the end stage leading to neurodegeneration.(1) (2)
6 The mechanisms responsible for redox-active iron accumulation in some regions of the brain in Alzheimer’s disease (AD) are unknown, nor if it is an initial event that causes neuronal death or a consequence of the disease process. However, little is still known about the chemical form of iron associated with neurodegenerative diseases, its role in neurodegeneration (if any) and its origin. Our goal is to understand iron-induced oxidative stress and point out the deleterious effects of redox-active ferric iron as a final product from methemoglobin and heme degradation. Ferric iron has a direct impact on capillary brain endothelial structure and function and in consequence of apoptosis brain parenchyma atrophy finally follows, so pinpointing specific clinical determinants for the onset and progression of AD. (2)
7
8 The Hemoglobin catabolismNO + Superoxide ( O2- ) = peroxinitrite (ONOO) a strong oxidant The erythrocytes are catabolised with the process where ONOO causes intravascular hemolysis NOx(NO and NO2) oxidation of hemoglobin oxyhemoglobin (Fe II) methemoglobin( Fe III) methemoglobinemia ERYTHROCYTES DESINTEGRATION IN PHYSIOLOGICAL CONDITION IN PATHOLOGICAL CONDITION Oxyhemoglobin Methemoglobin – an oxidant HEME as oxidant HEME as oxidant Both cause brain capillary endothelial cell damage
9 Figure 1. Difference between physiological hemoglobin catabolism and pathological methemoglobin catabolism
10 State of the art Most researchers believe the Alzheimer ‘s disease is caused by one of two proteins, one called tau, the other amyloid-beta. Now, a new UCLA study suggest a third possible cause: iron accumulation.(3) The cellular and intercellular iron transport mechanisms in the CNS are still poorly understood, while accumulating evidence suggests that impaired iron metabolism is an initial cause of neurodegeneration.(4)
11 Leung and Moody [6] have demonstrated that ferric heme is significantly more prooxidant than ferrous heme. MR imaging measures showed a T1 relaxivity that was 10 times higher for ferric than for ferrous forms of hemoglobin. (5) This justifies the study of the oxidant effect of methemoglobin catabolic products on vital organs and CNS, resulting in their dysfunction. Balla et al. posited that ferrimethemoglobin (FeIII), but not ferrohemoglobin (FeII), releases its hemes, which are then incorporated into endothelial cells, thus rapidly increasing the heme oxygenase levels of these cells. Ferritin production was also markedly increased. (6) (7)
12 Interaction Between Amyloid-beta And Ferric(III) IronMethemoglobinemia help to answer the bell about the relationship betwween amyloid-beta and iron that is poorly understood. Hershko et.al. originally identified NTBI (non transferin bound iron) is thought to play a major role in various pathological conditions that are dominated by iron overload. NTBI corresponds to iron which is not only unbound to transferrin but also does not correspond to heme or ferritin iron.(8) Evereet et. al. examined the interaction between amyloid-beta and synthetic iron(III), reminiscent of ferric iron stores in the brain, and report amyloid-beta to be capable of accumulating iron(III) within amyloid aggregates, with this process resulting in amyloid-beta mediated reduction of ferric iron(III) to a redox active ferrous iron(II) phase as substantial source of ROS production through the Fenton chemistry. (9)
13 Differently from their statement , we point out the role of methemoglobin catabolism as source of ferric(III) iron, with cytotoxic and paramagnetic properties, which in situ (intracellularly-without ferrous(II)-ferric(III) inversion), and addition to the blood circulation as NTBI (non-transferrin bound iron), which is not correspond to heme or ferritin iron( 10) act non only with amyloid-beta, but has serious consequences as endothelial brain capillary apoptosis and ferric(III) iron brain accumulation, responsable for the neurodegenerative process and some neurologic disorders such as Alzheimer’s disease, Parkinson disease, type I neurodegeneration, and other disorders.
14 Blood Brain Barrier Methemoglobin by itself, and heme, have prooxidant properties and induce structural and functional changes in the brain vascular endothelium. These changes include cell growth arrest, senescence, morphological alterations and cell apoptosis, under the influence of redox-active ferric iron (Fe3+), as a product of heme-oxygenase, responsible for methemoglobin-heme degradation. The substantial difference of intracellular ferric iron originates from the physiological hemoglobin degradation and Fenton reaction, and ferric iron originates from pathological methemoglobin catabolism occurring as the level of methemoglobin cellular uptake increases, and the resulting methemoglobinemia causes ferric iron-induced oxidative stress injury. An abundant source of cytotoxic and redox-active ferric (Fe3+) iron, without ferrous-ferric inversions “in situ”, as a cause of iron-induced oxidative stress has a direct and specific impact on the endothelial small vessels of the brain, and increases the rate of endothelial cell apoptosis, making it possible to cross over from methemoglobin and heme derived ferric iron and accumulate in the brain parenchyma.
15 Blood Brain Barrier Diameter 7,8µm Diameter µm
16 Exposure –”dirty” period Control – “clean” periodBlood and urine samples were tested 3 times with a 1 month interval in each period
17 Institute of Biology, Medical School University of Rijeka, 1990Results Exposed period : Mother methemoglobin >1.5 g/l Newborn, N= 36 Control period : Mother methemoglobin 0 g/l Newborn, N= 19 Presuming the adverse effects of mother methemoglobinemia, we analyzed the blood samples of newborns, whose mothers (122 women) were exposed during pregnancy, for SCE-sister chromathide exchange - N= 36 No statistical significant Sister chromatide exchange was found Institute of Biology, Medical School University of Rijeka, 1990
18 Neonatal jaundice incidence p= 0.034 Later heart murmur p= 0.011 Eighteen years later, by comparing the exposed and control newborns we found the following: Neonatal jaundice incidence p= 0.034 Later heart murmur p= 0.011 Dyslalia and learning deficiency p= 0.002 Earlier research found that the incidences of neonatal jaundice (p = 0.034), heart murmur (p = 0.011) and disorders such as dyslalia and learning/memory impairments (p = 0.002) were significantly higher in those children born from mothers with methemoglobinemia. Health data from Perinatal hospital departments, the Preschool Office and School Service at the Health Center for the period of eighteen years.
19 Figure 1 Figure 1 – Free iron positivity in the subcortical region (A) and parenchyma (B) of the cerebellum of a 6 month-old victim of SIDS (case no.9). Staining: Perl’s Prussian Blue reaction for ferric iron – Magnification 20x
20 Figure 2 Figure 2 – Signs of mitotic activity in iron-positive neurons. A) a neuron in telophase; B) amultinucleated neuron. Staining: Perl’s Prussian Blue reaction for ferric iron –Magnification 100x
21 Figure 3 Figure 3 – Iron positivity in capillary endothelial cells of the blood-brain barrier in a victim of SIUD aged 36 gestational weeks (case no.3). Staining: Perl’s Prussian Blue reaction forferric iron – Magnification A) 10x; B) and C) 20x
22 Figure 4 Figure 4. Free iron positivity in the cerebral parenchyma of a 77 year old patient with Alzheimer’s disease A) Focal distribution of positive glia and neurons; B) accumulation of strong ironpositive perivascular regions. Staining: Perl’s Prussian Blue reaction for ferric iron—Magnification: 40×.
23 Conclusion The Methemoglobin level is a useful biomarker but also has a degree of predictive validity.. To facilitate development of novel drugs and lay the groundwork for a new research projects with the pharmaceutical and biotechnological industry, we underline the usefulness of developing nanotechnologies being pursued to produce pharmacologically efficacious reducing agent where the methemoglobin reductase is overwhelmed, and a valuable contribution to neurodegenerative disease prevention and novel therapy aimed at reducing pathological methemoglobin ferric iron (Fe3+) back to hemoglobin ferrous iron (Fe2+). We suggest ferric iron as an originator of brain parenchyma accumulation, having an important role in crossing the brain microvessels to the neurons (neurovascular unit), causing neuronal death, continuous ageing process, and leading finally to hard neurodegenerative disorders such as Alzheimer’s disease, Parkinson disease, type I neurodegeneration, and other disorders.
24 Reference Mohorovic, L. The role of methemoglobinemia in early and late complicated pregnancy. Med. Hypotheses 2007, 68, L.Mohorovic1*, AM. Lavezzi2, S. Stifter3, G. Perry4, D. Malatestinic5, V. Micovic1, E. Materljan6, H. Haller7, O. Petrovic. Methemoglobinemia—A biomarker and a link to ferric iron accumulation in Alzheimer’s disease. Advances in Bioscience and BiotechnologVol, 2014, 5, UCLA Study Suggest Iron Is At Core Of Alzheimer’s Disease-2013 Mills, E., Dong, X.P., Wang, F. and Xu, H. (2010) Mechanisms of brain iron transport: Insight into neurodegeneration and CNS disorders. Future Medicinal Chemistry, 2,51. Leung, G. and Moody, A.R. (2010) MR imaging depicts oxidative stress induced by methemoglobin. Radiology, 257, Balla, J., Vercellotti, G.M., Jeney, V., Yachie, A., Varga, Z., Jacob, H.S., Eaton, J.W. and Balla, G. (2007) Heme, heme oxygenase, and ferritin: How the vascular endothelium survives (and dies) in an iron-rich environment. Antioxid Redox Signal, 9, Balla J. et al. Proc Natl Acad Sci USA 1993, 90:9285-9;
25 8. C. Hershko, G. Graham, G. W. Bates, E. A. RachmilewitzC.Hershko, G.Graham, G.W.Bates, E.A.Rachmilewitz..Non-specific serum iron in thalassaemia : an abnormal serum iron fraction of potential toxicity.Br.J.Haematol. 40(1978) Evereet J,Cespedes E, Shelford LR, Exley C,Collingwood JF, Dobson J, van der Laan G, Jenkins CA, Arenholz E, Telling ND. Ferrous formation following the co-aggregation of ferric iron and the Alzheimer’s disease peptide Beta-amyloid(1-42) JR Soc Interface Mar 26;11(95): Brissot P, Ropert M, Le Lan C, Loreal O. Non-transferrin bound iron : A key role in overload and iron toxicity. Biochim.Biophys.Acta –General Subjects, Volume 1820, Issue 3, March 2012; Pages
26