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Fiziologia - Physiology  2009.19.3(63)CONTENTS
1. Review - New Approaches to Adaptation.........................................................................................................................................................4
Petru Derevenco
2. Mechanisms Involved in Mesenchymal Stem Cells Differentiation towards the Epithelial Lineage. Effects of Chemical Inductors........................7
Gabriela Tanasie, Florina Bojin, C.A. Tatu, Oana Gavriliuc, Carmen Tatu, Daciana Nistor, Hortensia Ionita, Carmen Bunu, V. Paunescu
3. Ontogenetic Changes of Bone Mineral Composition.......................................................................................................................................13
Olga Tagadiuc, V. Gudumac, V. Reva
4. Glutathione Peroxidase Activity Affects Prognosis in non-ST Segment Elevation Acute Coronary Syndrome.....................................................16
Violeta Sapira, Elvira Craiu, Cecilia Adumitresi
5. The Involvement of Oxidative Stress in the Ulcerous Pathology Induced in Rats by Aspirin...............................................................................20
Simona Martura, Doina Daicoviciu, Soimita Suciu, Adriana Muresan, Doinita Crisan , Petru A. Mircea, Simona Valean
6. Variations of some Saliva Markers of the Oxidative Stress in Patients with Orthodontic Appliances..................................................................26
Olteanu C, Muresan A, Daicoviciu D, Tarmure V, Olteanu I
7. Consumption of Calorigenic Foods in Adolescence..........................................................................................................................................30
Oana Suciu, Brigitha Vlaicu
8. Molecular Changes in Precancerous Lesions of the Larynx..............................................................................................................................33
Raluca Horhat, Delia Horhat, Marioara Poenaru, Stan Cotulbea, Nicolae Balica, Valeria Mocanu
9. The Hypolipemiant Effect of High Doses Atorvastatin Therapy in Diabetic Patients with Acute Myocardial Infarction.......................................37
Adriana S Potra, Virgil M Luca, Constantin O Luca
10. Urban Noise Assessment in Timisoara..........................................................................................................................................................40
Ernest Putnoky

CUPRINS
1. Review - Noi abordari in adaptologie..............................................................................................................................................................4
Petru Derevenco
2. Mecanisme implicate in diferentierea celulelor stem mezenchimale spre linia epiteliala. Efectul unor inductori chimici....................................7
Gabriela Tanasie, Florina Bojin, C.A. Tatu, Oana Gavriliuc, Carmen Tatu, Daciana Nistor, Hortensia Ionita, Carmen Bunu, V. Paunescu
3. Modificari ontogenetice in compozitia osoasa minerala.................................................................................................................................13
Olga Tagadiuc, V. Gudumac, V. Reva
4. Activitatea glutation peroxidazei afecteaza prognosticul in sindromul coronarian acut fara supradenivelare de segment ST...........................16
Violeta Sapira, Elvira Craiu, Cecilia Adumitresi
5. Implicarea stresului oxidativ in patologia ulceroasa indusa de aspirina la sobolani.........................................................................................20
Simona Martura, Doina Daicoviciu, Soimita Suciu, Adriana Muresan, Doinita Crisan , Petru A. Mircea, Simona Valean
6. Variaţii ale unor markeri salivari ai stresului oxidativ la pacienţii purtători de aparate ortodontice.................................................................26
Olteanu C., Muresan A., Daicoviciu D., Tarmure V., Olteanu I.
7. Consumul de alimente calorigene în adolescenţă..........................................................................................................................................30
Oana Suciu , Brigitha Vlaicu
8. Alterari Moleculare In Leziunile Precanceroase Laringiene............................................................................................................................33
Raluca Horhat, Delia Horhat, Marioara Poenaru, Stan Cotulbea, Nicolae Balica, Valeria Mocanu
9. Efectul hipolipemiant al dozelor crescute de Atorvastatin la pacientii diabetici cu infarct miocardic acut.........................................................37
Adriana S. Potra, Virgil M. Luca, Constantin O. Luca
10. Evaluarea zgomotului urban in municipiul Timisoara...................................................................................................................................40
Ernest Putnoky
2009.19.3(63)  Fiziologia - Physiology

3review
NEW APPROACHES TO ADAPTATIONPETRU DEREVENCO
Academy of Medical Sciences, Branch of Cluj, Romania

ABSTRACTThis topic has been widely explored from the adaptation by natural selection (Darwin) to the general adaptation syndrome (Selye). This paper is focused on
nowadays issues of the research on adaptation linked to stress. Allostasis is an oscillatory stabilization which allows the adjustment to various states. “Allostaticload” is the cumulative cost ongoing to repeated cycles of adaptation by physiological responses to stress and to possible adverse effects. “Psychological resilience”, a component of positive adaptation during stress, represents the capacity to take on a traumatic event leading to disturbances and the subject’s ability
to offer protection against future challenges. Has been proved the importance of “posttraumatic growth and of thriving” – after psycho traumas; it explains
way a large proportion of subjects submitted to adverse events recover fast. “Maladaptation” leads to PTSD and to other stress-syndromes which comprise
psychoneurological, immunological, occupational and endocrine mechanisms. In contrast to the chronic or severe stress, a light or moderate stress named
eustress has positive stimulating issues, related especially to the secretion of endorphins. Is in progress the foundation of adaptology which integrates the
structural, functional and biopsychosocial sides of adaptation. In Romania, adaptology is promoted by several publications. We mention two books: “Human
adaptation” (Badiu & Papari, 1999) and “Stressology, adaptology and mental health” (S. Riga & D. Riga, 2008).
Key words: allostasis, resilience, posttraumatic growth, stress-syndromes, eustress, adaptology

INTRODUCTION
The topic of adaptology is very large.
The first scientific account belongs to the Lamarckian concept of the inheritanceof acquired traits. The main crucial advance has been performed by Darwin with histheory of evolution by natural selection, developed later by neodarwinism.
Selye’s general adaptation syndrome is a seminal contribution to the links
between stress and adaptation.
The above mentioned historical steps covered to explore adaptive processes
can not be detailed in this paper; its aim is to discuss only the present-day directions
dealing with the relationships between adaptation and various aspects of stress.
Further on will be underlined six main dimensions of this area of interest.

ALLOSTASISThe term “allostasis”has been introduced by Sterling & Eyer (28). Its definitionand peculiarities have been outlined by Mc Ewen (19-21).
Mc Ewen & Seeman (22) and Sterling (27) published comprehensive papers
dealing with principles of allostasis, pathophysiology, therapeutic aspects andthe links to stress.
Complementary information is given by Mc Ewen & Wingfield (23) and in
Wikipedia (35). Are proposed two forms of allostatic load resulting in different
responses: type 1 allostatic overload found especially among animals when energy
demand exceeds supply, and type 2 when allostatic overload occurs when there issufficient or even excess energy consumption accompanied by social conflicts or
dysfunctions; this type present particularly in human society.
Allostasis means maintaining stability through change.This notion has been applied
to the adjustment of the cardiovascular system, but can be used to explain other physiological processes of the body, such as secretion of cortisol or catecholamine (22).

Allostasis has a wider implication in the regulator mechanisms of theorganism, such as homeostasis (3) and can be even used instead of the term
“stress”. Allostatic adaptive systems have much broader boundaries thanhomeostasis. They enable to respond to many physiological states: awake,asleep, standing, exercising, isolation, hunger, extreme temperatures, danger,
microbial infections (19).
“Allostatic load”is the cumulative cost to cover repeated adaptive cycles of allostasis,experienced by the body as well as the turning on as shutting off of the neuroendocrine,
systemic, and behavioral or by positive or sometimes negative events (22).
Examples of allostatic loads are the adverse effects of job strain on the
cardiovascular system and the inhibition of cellular immunity resulting fromchronic stress.
“Allostatic support” refers to mechanisms that confer resistance to individuals
making them more robust to various kinds of stress and chronic illness.
The above described theoretical information is illustrated in Figure 1.

Fig. 1. The stress response and development of allostatic load (19)Received August 2009. Accepted September 2009. Address for correspondence: Prof. Petru Derevenco, Academy of Medical Sciences, Branch of Cluj, Romania

4

Fiziologia - Physiology  2009.19.3(63)Mc Ewen (13) described four types of physiological response to an allostaticload: a. normal oscillatory responses over time; b. lack of adaptation with disturbed oscillations; c. prolonged response without recovery; d. inadequate low
responses.
Physiological and pathophysiological effects of allostasis involve the action of
glucocorticoids (GC) through intracellular, protoplasmatic steroid receptors and of
catecholamines (CA) via membrane receptors and the second-messenger system.
As well GC and CA act finally at the cell nucleus.
PSYCHOLOGICAL RESILIENCEThis psychological dimension represents the capacity to take on a traumatic
event which could lead to functional disturbances and even to psychiatric conditions
and to recover successfully.
Resilience implies good outcomes regardless of high-risks status, constantcompetition under stress, and efficient coping mechanisms. The negative effects
of adverse life situations depend on the vulnerability of subjects which in stressful
situations can be directed as well to pessimistic as to optimistic behaviors.
This dynamic process has been firstly described by Emmy Werner (34) on
Taiwanese children, which grew up in very bad conditions. The results showedthat one third of these youngsters did not exhibit destructive behaviors; this fact is
explained by Werner by their resilience.
In 1980’s this research topic emerged from observations on children of
schizophrenic mothers.
Later, the resilience concept has been developed by Bonnano (2) in the USA
and Cyrulnik (9) in France. Further details can be found in Wikipedia (36).

c. Life-philosophy with shifts in an existential perspective.
Tedescky et al. (31) have designed a PTG inventory measuring the positive
reactions of traumas. In Romania, Kallay (16, 17) has studied systematically this
topic.
Posttraumatic growth explains why large proportion of subjects submitted
to adverse events recovers successfully and fast returning to their pre-traumafunctioning. Most important is the fact that sometimes the initial functional levelis even raised. The psychological background of these positive reactions implies theintervention of meaning making. For details see Kallay & Miclea (18).

MALADAPTATION AND STRESS-SYNDROMES
Maladaptation leads to various stress-syndromes.
This topic has been developed in the last decades in several directions.
Horowitz (13, 15) has explored systematically the clinical significance of
traumatic reactions, designed an event scale and elaborated a theory of traumatic
reactions based on a social-cognitive approach.
A comprehensive book is devoted to the treatment of stress response syndromes (14). Other Horowitz’s contributions are reported by Kallay (16).
The neuro-endocrine mechanisms of the stress-syndrome have been studied
extensively by Chrousos (6, 7), the occupational outcomes by Theorell (33), and theendocrine syndromes by Coculescu (3).
The other directions of this topic deal with the cardiovascular stress-syndrome(for instance Tako-Tsubo syndrome) and with stress-syndromes described in exercise(overtraining, female athlete triad). The literature on the theories, models andpeculiarities of posttraumatic stress reactions is very wide. For details see Kallay(16) and Derevenco (10-12).

POSTTRAUMATIC GROWTH
The negative reactions to psycho traumas and extreme events (grief, torture,
natural catastrophes, etc.) have been widely explored and categorized in DSM and
ICD (see also the following section).
The possible/potential responses to trauma are outlined in Figure 2.

EUSTRESSIn contrast to the familiar terms distress and strain, eustress is less used in
the stress topic.
Eustress, aiming a light or moderate stress with positive stimulating issues, hasbeen introduced by Selye and mentioned in his later writings (26). His expression“stress is the salt of life” belongs probably to eustress.
Eustress implies the secretion of endorphins and of other neuromediators
including likely serotonin. The exercise-dependence of some athletes is probably
related to the euphoric state produced by moderate exercise and social environmental factors.
Eustress represents a basis of the stress-inoculation method with preventive
and curative effects.

ADAPTOLOGY

Fig. 2. Potential responses to trauma (O’Leary & Ickovics) (5)An adverse event can induce four main consequences shown in Fig. 2.
The thriving reaction has been studied especially in USA by Carver (5), Calhoun& Tedescky (4), Tedescky & Calhoun (29, 30), who proposed a model of posttraumatic growth (PTG). Tedescky et al. (31) identified three main dimensions of PTG:
a. Friendly and altruistic interpersonal relationships;
b. Self-perception with better acceptance of limitations of the self;
2009.19.3(63)  Fiziologia - Physiology

At present is in progress the foundation and promotion of adaptology, a new,
distinct discipline which integrates the structural, functional and biopsychosocial
components of adaptology.
This area of interest deserves to be explored in details elsewhere.
For the time being, we mention some contributions of the Romanian scientists.
A pioneer of studies on adaptation is Schneider (25). He organized also a firstmeeting devoted to adaptology – topic of the 15th national Conference of Physiology, being author/coauthor of 16 works aiming adaptation. Recently, he has been
published 2 comprehensive volumes.
The authors of the first book (“Human adaptation”) are Badiu & Papari (1). This
book comprises four parts including 21 chapters. Part I, general adaptology, Part
5

II, ontogenetic adaptology, Part III, environmental adaptology, Part IV, adaptology
and physical exercise.
The second valuable volume signed by S. Riga & D. Riga is entitled“Stressology, adptology and mental health”(24). The section adaptology comprises
five chapters: 1. adaptation, coping maladaptation, troubles of adaptation; 2.
homeostasis, allostasis, vitality and vulnerability; 3. the stress binom: distress andeustress; 4. the general syndrome of adaptation; 5. capacity to cope with stress,
antistress. S. Riga and D. Riga have also published other four papers on adaptation
between 1988 and 2002.

CONCLUSION
Allostasis, resilience, posttraumatic growth, stress-syndrome, and eustressoffer at present and in perspective a roadmap for the development of adaptology
as a new integrative science and for surpassing over simplified and narrow-minded
views on stress.
REFERENCES
1. Badiu G, Papari A. Human adaptology. “Andrei Saguna” Foundation Ed., Constanta, 1998.
2. Bonanno GA. Loss, trauma, and human resilience. How weunderstand the human capacity to thrive after extremely adverse
events. Amer. Psychologist 2004; 39:20-28.
3. Cannon WB. The wisdom of the body. New York, Norton, 1932.
4. Calhoun LG, Tedescky RG. Beyond recovery from trauma: implications for clinical practice and research. J. of Social Issues, 1998;
54:summer.
5. Carver CS. Resilience and thriving. Issues, models and linkages.
J. of Social Issues 1998; 54:245-266.
6. Chrousos GP. Stress, chronic inflammation, and emotional andphysical well-being: concurrent effects and chronic squeal. J. Allery
Clin. Immunol. 2008; 106:S275-S291.
7. Chrousos G, Gould AD. The concept of stress and stress syndromes. JAMA 1992; 269:1242-52.
8. Coculescu M. Psycoendocrine stress induced syndromes. Rev.
Roum. Physiol. 1989 ; 26 :233-253.
9. Cyrulnik B, Seron C. La resilience ou comme sensation de sessouffrances. Paris Ed. Fabert 2004.
10. Derevenco P. Aspects of posttraumatic stress in Romania.
Cognition, Brain, Behavior V (1);2001;29-34.
11. Derevenco P. Psychological and medical consequences of Holocaust on its survivors. Cognition, Brain, Behavior VIII (1);2004:79-85.
12. Derevenco P, Baban A, Dumitrascu D et al. PTSD – psychophysiological and medical aspects. Rom. J. Physiol. 1993; 30:194-206.
13. Horowitz M. Stress response syndrome 1st ed. Northvale, Jason

Aronson, 1976. (4th ed.) 2001.
14. Horowitz M. Treatment of stress response syndromes. Washington, London American Psychiatric Pub. 2003.
15. Horowitz M, Field N, Classen C. Stress response syndrome andtheir treatment in L. Goldberger, S. Breznitz (eds.). Handbook ofstress (2nd Ed.) New York, The Free Press 1993:753-773.
16. Kallay E. Trauma, trauma theories, and possible posttraumatic
reactions. Cognition, Brain, Behavior 2004; VIII (4):55-74.
17. Kallay E. Posttraumatic growth and meaning making (Abstract)
PhD dissertation. Cluj-Napoca, Babes-Bolyai University 2006.
18. Kallay E, Miclea M. The role of meaning in life in adaptation
to life threatening illness. Cognition, Brain, Behavior 2007; XI (1):153-174.
19. McEwen B. Protective and damaging effects of stress mediators.
NEJM 1998; 398:171-179.
20. McEwen BS. Allostasis and allostatic load in G. Fink (ed.).
Encyclopedia of stress vol. 1, San Diego, Academic Press 2000;135150.
21. McEwen B. Physiological neurobiology of stress and adaptation.
Central role of the brain. Physiol. Rev. 2007; 37:873-904.
22. Mc Ewen B, Seeman T in John & Caterine Mac Arthur Research
Network on Socioeconomic Status and Health.
23. Mc Ewen BS, Wingfield JK. The concept of allostasis in biology
and biomedicine. Horm. Behave. 2003; 43:2-15.
24. Riga S, Riga D. Stressology, adaptology and mental health.
Bucharest, Cartea Universitara, 2008.
25. Schneider F. Introduction in clinical Physiology. Timisoara, Facla
Ed., 1977: 147-149, 152.
26. Selye H. Stress without distress, Philadelphia, Lippincott
1974.
27. Sterling P. Principles of allostasis: optimal design, predictive
regulation, pathophysiology and rational therapeutics in J. Schulckin (ed.) Allostasis, homeostasis and the costs of adaptation.
Cambridge, Cambridge Univ. Press 2004; 1-36.
28. Sterling P, Eyer J. Allostasis. A new paradigm to explain arousal
pathology in S. Fisher and J. Stearon (Ed.) Handbook of stress,
cognition and health. New York, J. Wiley 1998.
29. Tedescky RG, Calhoun LG. Trauma and transformation. Growing
the aftermath of suffering. Thousand Oaks, Sage 2995.
30. Tedescky RG, Calhoun LG. Posttraumatic growth. Conceptual
foundation and empirical evidence. Psychological Enquiry 2004;
15:1-18.
31. Tedescky RG, Park C, Calhoun LG. The posttraumatic growthinventory. Measuring the positive enquiry of trauma. J. TraumaticStress 1996; 9:455-471.
32. The 17th National Conference of Physiology. Adaptology. Rom.
Soc. Physiol. Sc. (Abstracts). Arad, Sept. 25-26, 1998 FiziologiaPhysiology, 8(3): 1-121.
33. Theorell T. Stress syndromes. Annals Clinical Research 1987;
19:53-61.
34. Werner E, Smith RS. Vulnerable but invincible. A study on
resilient children. New York, Mc Graw-Hill, 1982.

NOI ABORDARI IN ADAPTOLOGIEREZUMAT

Problematica adaptarii, de la teoria evolutionista (Darwin) la sindromul general de adaptare (Selye), a fost larg explorata. Lucrarea puncteaza unele directii
actuale vizand studiul adaptarii si relatiile acestuia cu stresul. Allostaza este stabilirea oscilatorie ce asigura adaptarea la variate stari ale organismului.
“Incarcarea alostatica” este costul cumulativ de a parcurge cicluri repetate de adaptare prin reactii la stres, cu posibile efecte adverse. Rezilienta psihologica,componenta a adaptarii pozitive la stres, consta in a lua act de un eveniment traumatizant care provoaca disfunctii fiziologice si abilitatea de a oferi protectie
provocarilor viitoare. Intre raspunsurile la evenimente adverse, s-a dovedit importanta cresterii posttraumatice si a reusitei (thriving) care explica de ce oproportie insemnata de subiecti supusi psihotraumelor se reechilibreaza rapid. Maladaptarea se traduce prin sindromul dereglarilor posttraumatice de stressi prin alte sindroame care comporta mecanisme pe plan psihoneurologic, im unologic, ocupational, endocrin. Spre deosebre de stresul cronic sau prelungit(distres), eustresul, deci stresul usor/moderat, are valori pozitive, stimulatoare, legate in special de secretia endorfinelor. In prezent se contureaza adaptologia,
disciplina de integrare a aspectelor structurale, functionale, biopsihosociale ale adaptarii. Adaptologia este promovata in Romania prin mai multe publicatii.
Semnalam cartile “Adaptologia umana” (Badiu, Papari, 1999) si “Stresologie, adaptologie si sanatate mintala” (S. Riga, D. Riga, 2008).
Cuvinte cheie: adaptare, alostaza, rezilienta, crestere posttraumatica, sindroame de stres, eustres, adaptologie

6

Fiziologia - Physiology  2009.19.3(63)MECHANISMS INVOLVED IN MESENCHYMAL STEM CELLSDIFFERENTIATION TOWARD EPITHELIAL LINEAGE. THE
EFFECT OF SOME CHEMICAL INDUCTORSGABRIELA TĂNASIE1,2, FLORINA BOJIN1, C. A. TATU1,2, OANA GAVRILIUC1, CARMENTATU1,2, DACIANA NISTOR1,2, VICTOR CIOCOTISAN4, HORTENSIA IONIŢĂ3, CARMEN
BUNU1, V. PĂUNESCU1,2
1
Department of Physiology and Immunology, University of Medicine and Pharmacy Victor Babes, Timisoara
2
Laboratory of Immunology, Clinical County Hospital Timisoara
3
Clinic of hematology, University of Medicine and Pharmacy Victor Babes, Timisoara
4
Department of Physiology, Heidelberg Medical University, Germany

ABSTRACT
Adult mesenchymal stem cells (MSCs) are extremely attractive in order to study the regeneration and reparation features in various type of tissues. For maintaining the differentiation potential of MSCs there are used several strategies: the in vitro culture ofmesenchymal cells in media supplemented with specific growth factors, the transfection of MSCs with the genes which are specificto the differentiated cells, co-culture of MSCs with differentiated cells from the target tissue. In this study we verify the effect ofsome chemical inductors to differentiate adult MSCs into epithelial-like cells. We used various cytokines and growth factors, addedin the culture media alone or in combination. The results were analyzed using immunocytokemistry and molecular biology (PCR)
methods. The experiments revealed that MSCs differentiation toward the cells expressing epithelial markers is relatively easily toobtain using a certain combination of inductors, without genetic manipulation of the cells
Key words: mesenchymal stem cells, epithelial differentiation, epithelial-like cells, chemical induction
INTRODUCTION
The mesenchymal stem cells (MSCs) role in the adult human body is thegeneration of mesenchymal cell lines and afterwards, through mesenchymal
progenitors is involved in development, maintaining and restoration of connective
and muscular tissue. Having these outstanding properties MSCs represents thebest alternative for cell therapy applications; they have self renewal, great plasticity
with differentiation potential in functional cell lines. Their efficiency was proven innumerous therapeutic protocols: bone marrow recovery, osteogenesis imperfecta,bone regeneration. Another advantage seems to be the fact that MSCs could beobtained from autologous sources (bone marrow) eliminated thus the complicationof allogeneic transplantation. In past years were developed many researches in
the field of MSC and now we have lot of information regarding plasticity, homing
and differentiation potential in vivo, but less known are the intrinsic mechanisms
which allow the self-renewal and differentiation. The understanding of thesemechanisms at the molecular level is still a challenge for the medical community
and the elucidation of mechanisms could offer a supplementary safety to celltherapies and make possible a large scale application of these methods (1,2). The
MSCs capacity to differentiate in various cell lines was extensively studied beginningwith the 1960. The results confirm the possibility to obtain specialized cells in vitro
and in vivo. MSCs pluripotency is explained based on two theoretical models. The
first one considering all of MSCs as pluripotent cells and differentiation could be
induced by various environment factors. In the differentiation process MSCs loose

their self-renewal capacity, passing through intermediary states (mesenchymal
progenitors) and finally acquiring the characteristic phenotype of specialized cells.
The alternative model, which is more confirmed by experimental studies sustainedthat the major determinant of pluripotency is the MSCs heterogeneity a property
identified even in a single cell derived colonies. From the entire population of adherent cells only 30% are having a tri-differentiation potential (adipocytes/osteoblasts/chondrocytes) and the others having only a double osteo/chondrogenic or a unique
osteogenic potential (3). A possible explanation is that in bone marrow resideprimitive pluripotent MSC with limited capacity of self renewal which generates
mesenchymal progenitors in various differentiation studies.
The concept of plasticity means the property of stem cells to differentiate
in a distinct cell line apart from the originated tissue. The in vitro differentiation
techniques are based on using a differentiation agent, co culture with specific cells
or structures and modification in some gene expression. In vivo the differentiation
process can be followed by marked SC transplantation in experimental animals or
CS transplantation in genetic modified animals (with deficiency on some cellularlines). The differentiation methods used for the MSCs plasticity studies included
MSCs transplant, chemical stimulation in culture and culture in special conditions.
Till now there are cited successful differentiation processes through the cell lines
belonging to all the embrionary layers. By MSC transplantation was obtained
differentiation in: astrocytes (4); neurons; epithelial cells from skin, digestive andrespiratory tract (5), cardiomyocites, (6), chondrocytes, adipocytes and marrow

Received July 2009. Accepted September 2009. Address for correspondence: Dr. Gabriela Tanasie, MD, PhD, Physiology Department, University of Medicine and Pharmacy “Victor Babes”
Timisoara, 2A Eftimie Murgu Square, 300041, Romania, e-mail: gtanasie@umft.ro2009.19.3(63)  Fiziologia - Physiology

7stromal cells (7). After chemical stimulation MSC was differentiate in: adipocytes
The induction medium was supplemented with 200 μ M indomethacin(1-metil 3izobutilxantine, dexametasone, insulin and indhometacin), chondrocytes(Sigma), 1 μg/mL rosiglitasone, 1 mM dexamethasone, 0.5 mM isobutylmeth(without bovine serum and stimulation with TGF β), osteoblasts (dexametasone,ylxanthine (IBMX). (8)β-glicerophosphate and ascorbate), neurons and astrocytes (EGF and BDNF or
2. Osteogenic differentiation
media for21 days, with mediaThechangesof 3 wastimes
a week.
β-mercaptoetanol), skeletal muscle fibers and cardiomyocites(5-azacitidine).
medium culture
replacedwith osteoinductor medium, containing
1.
Adipocytic
differentiation
After prolonged cultures and co culture with another cell lines was obtained the
50µg/ml ascorbic acid 2-phosphate (Sigma), 10nM dexamethasone (Sigma) and
The induction medium was
supplementedwith200 (9)
ȝ M indomethacin (Sigma), 1 ȝg/mL rosi
MSC differentiation into neurons.
10mM
ß-glycerol phosphate(Sigma).
mM
dexamethasone,
0.5
mM
isobutylmethylxanthine(IBMX). (8)In our study we proposed to evaluate the differentiation of human adult bone
3. Chondrogenic differentiation2.
Osteogenic
differentiation
marrow derived mesenchymal stem cells in to epithelial like cells by stimulation
The chondrogenic media consisted of 30 µg/ml ascorbate-2-phophate, ITS
waswith
osteoinductor
medium,with some chemical inductors: growth factors and cytokines. The medium culture
premix(BD replaced
Biosciences) and10ng/ml
TGF-β1 (R&D Systems)(10). containing 50µg/ml as
2-phosphate (Sigma), 10nM dexamethasone(Sigma)and10mM
ß-glycerol
phosphate(Sigma)To evaluate the occurrence of differentiation, at the end of this period thecells
3. Chondrogenic differentiation
were fixed with 10% formalin for 10 minutes and stained with specific antibodies. To
The chondrogenicmonitor
media
consisted
ofan antiFAB430 µg/ml
ITS pr
MATERIAL AND METHODS
adipogenic
differentiation,
antibody ascorbate-2-phophate,
was used (R&D Systems),
Biosciences) and 10ng/mlasTGF-ȕ1(R&D
Systems)(10).
osteogenic marker was used antiosteocalcin antibody (DAKO) and for evidentiaThe experiment was developed after obtaining the agreement of Ethics ComTo evaluate the occurrenceof differentiation,end of thisperiodtion of chondrogenic
induction the choice atwasthethe antiaggrecan
antibody(R&Dthe cells were
mittee of the University of Medicine and Pharmacy Victor Babes Timisoara. The
10% formalin for 10 minutes
andstainedwithspecific
antibodies.
Tomonitor
Systems). For further confirmation of differentiation, RNA extraction and RT-PCRadipogenic diffbone marrow samples (2-4 ml) was harvest from 5 patients (4 females, 1 male;an antiFAB4 antibody wasanalysis
usedof(R&D
Systems), as osteogenic marker was used antiosteocalc
gene expression have also been performed. For RNA extraction the Gen
medium age 45+/-2) suffering by different forms of anemia. In accordance with
(DAKO) and for evidentiationofchondrogenic
induction the choice was the antiaggrecan antib
Elute Mammalian Total RNA Miniprep Kit (Sigma) was used in accordance withthe working protocol previously established, the written consent was obtained
Systems). For further confirmation of differentiation, RNA extraction and RT-PCR analy
the manufacturer instructions. The RNA quality and concentration was determinedfrom patients. The sternal punction maneuvers were performed using specificexpression have also been performed. For RNA extraction the Gen Elute Mammalian T
using a spectrophotometer (Nanodrop 2000C, ThermoScientific) and measuring the
instruments, in conditions of perfect safety and sterility, with minimum invasive
Miniprep Kit (Sigma) was used in accordance with the manufacturer instructions. The RNA
absorption at 260 and 280 nm. For reverse-transcription the One Step RT-PCR kit
procedure for the patients.
concentration was determined using a spectrophotometer (Nanodrop 2000C, ThermoScie(Quiagene) was used and the thermal conditions were specific for each primer pair.
measuring the absorption at 260 and 280 nm. For reverse-transcription the One Step R
The primer sequences used were found in literature (11) or were custom designed
Isolation and culture of mesenchymal stem cells(Quiagene) was used and the thermal conditions were specific for each primer pair. The prime
according to Gene Bank data (Table I).
The procedure used for mesenchymal stem cells isolation was based on
used were found in literature (11) or were custom designed according to Gene Bank data (Tabl
plastic adherence, following a protocol described elsewhere. Briefly, the procedure
Table I. Primers used for RT-PCR
steps are:Table I. Primers used for RT-PCR- The mononuclear cells (MNCs) were separated from the whole bone marPrimer
sequencePPARȖ2
S: 5'GCTGTTATGGGTGAAACTCTG 3'
row cells by density gradient centrifugation using Ficoll-Paque plus (Amersham
AS: 5'ATAAGGTGGAGATGCAGGCTC 3'
Biosciences Inc.)
Lipoprotein
S: 5'GAGATTTCTCTGTATGGCACC 3'- the MNCs were seeded on a cell concentration of 50,000 cells/cm2 in 25 cm2
lipaseAS: 5'CTGCAAATGAGACACTTTCTC 3'
plastic T flasks in on IMDM media (Invitrogene) supplemented with 10% fetal bovine
FAB 4 (AP2) S: 5'-GTACCTGGAAACTTGTCTCC 3’
AS: 5'-GTTCAATGCGAACTTCAGTCC 3’
serum (Sigma), 2 ng/ml Fibroblast Growth Factor (R&D Systems).
GAPDH
S: 5’GGGCTGCTTTTAACTCTGGT 3’- Complete medium change was done at day 1 to 3 after the initiation of
AS: 5’TGGCAGGTTTTTCTAGACGG 3’
culture.
Alkaline
S: 5'TGGAGCTTCAGAAGCTCAACACCA 3'
phosphatase
AS: 5'ATCTCGTTGTCTGAGTACCAGTCC 3'- The cultures were maintained at 37℃in a humidified atmosphere with 5%
Osteocalcin
5'ATGAGAGCCCTCACACTCCTC 3'
CO2.
5' GCCGTAGAAGCGCCGATAGGC 3'- The medium was replaced one to two times every week, every third to
CBFA-1
S : 5’CCACAGAACCACAAGTGCGG 3’(Runx2)
AS : 5’ACGGAGCACAGGAAGTTGGG 3’
fourth day.
Osterix
S : 5'-CAGCTGCCATCTTAGATGTGC 3’- When layers were subconfluent (70-75% confluence), cells were treated with
AS : 5'-CCATTCCACAATGTTCTCTCC 3’
0.25% (v/v) trypsin/1 mM EDTA (Sigma) and replated at 1000 cells/cm2.
Agreccan
5'GCCTTGAGCAGTTCACCTTC 3'
5'CTCTTCTACGGGGACAGCAG 3'- for determination of cell concentration the standard procedure of cell contCollagen type 5'CCCTTTTTGCTGCTAGTATCC 3'
in Neubauer counting chamber (hemocytometer) was followedX
5'CTGTTGTCCAGGTTTTCCTGGCAC 3'
The cells were examined daily using an inverted microscope (Olympus) in
Collagen type 5'GAACATCACCTACCACTGCAAG 3'II
5'GCAGAGTCCTAGAGTGACTGAG 3'
order to detect morphological changes and the initiation of confluence. The cell
viability was determined both in the moment of isolation and placement in culture
Inductionof epithelial-likecells
Induction of epithelial-like cells
medium (day 0 of each passage), as well as at different
time intervals,
during
For this 200
study MSCsForatthispassage
were3 were
used.
of experiments,
thestudy MSCs at3passage
used.InIn aafirstfirst
set of set
experiments,
thetheir evolution, using small amounts of cellular suspension (approximately
with one ofmedia
thewascytokines
factors:
growth factor (EGF
supplementedandwith growth
one of the cytokines
and epidermal
growth factors: epidermalµl). We used Trypan Blue microscopic method: deadsupplemented
cells, having lysis of cellular
keratinocyte
growth factorgrowth(KGF)
ng/ml,
bone
morphogenic
protein(BMP4)
factor10(EGF)
20 ng/ml,
keratinocyte
growth factor(KGF) 104ng/ml,
bone 10 ng/ml, tu
membrane, would be stained in blue, while viable cells,
having intact membrane,
factor beta 1 (TGF beta1) morphogenic
20 ng/ml,protein
tumor
growthfactor3 factor(TGF
25 ng/ml, hepatoc
4 (BMP4)
10 ng/ml,
tumorbeta
growthbetabeta3)
1 (TGF beta1)
would not be colored.
factor (HGF)
10 ng/ml, beta
cellulin
100 ng/ml,
25 ng/ml,
fibroblast
20 ng/ml,
tumor growth
factor betanicotinamide
3 (TGF beta3) 25 ng/ml,
hepatocyte
growth growth fac
Confirmation of differentiation potential characteristic
for mesenchymal
10 ng/ml, insulin like growth(IGF2)
50 ng/ml.
All
reagents
purchased from R@
factorfactor(HGF) 10II
ng/ml,
beta cellulin
100 ng/ml,
nicotinamide
25 wereng/ml, fibroblaststem cells
experiment
a combination
fibroblast
factor(FGF)
10Allng/ml, EGF 20
factor (FGF1) 10ofng/ml,
insulin likegrowth
growth factor
II (IGF2)
50 ng/ml.
For this study MSCs at 3rd passage were used. TheAlso,
cultures ansubsequently
were with growth
KGFof 310
ng/ml
out. were purchased.
from R@D Systems. Also, an experiment with a combinamaintained in this media for 21 days, with media changes
times
a week.was carriedreagents
In a second set ofthe (FGF)
effectof different
combination
tion ofexperiments,
fibroblast growth factor
10 ng/ml,
EGF 20 ng/ml and
KGF 10 ng/ml of this indu
1. Adipocytic differentiation
evaluated. Four groups of experiments were used and the exact composition of the media i
figure 1.
Fiziologia - Physiology  2009.19.3(63)8Fig.2. MSCs, day 5, ob.20x

Confirmation of differentiation potential characteristic for mesenchymal stem cells
The differentiation studies occurred in MSCs at passage 2. The cells were able to differentia
all three lines (adipocytic, osteogenic and chondrogenic) which are characteristic
stromal/mesenchymal progenitors. The characteristic markers for those three lineages were express
the gene level and were evidenced by RT-PCR reaction (Figure 3). The presence of some markers aprotein expression level could be evidenced also, using immunocytochemistry staining (Figures 4, 5,

ROUP 1DMEM +

10% FCS

was carried out. .
GROUP 4GROUP
GROUP
In a second
set of 2experiments, the effectof different3 combination of thisinductors were
evaluated.
•DMEM +•DMEMwere+ used and the exact•DMEM+ Four groups of experimentscomposition of the media is shown in figure 1.
•10% FCS•10% FCS•10% FCSGROUP 2
ng/ml
FGF 10 ng/ml GROUP 1•IGF2 50

GROUP 3ng/ml
GF2 50 ng/ml •10% FCS•KGF 10•10%
FCS•10% FCS•DMEM +•DMEM +

•FGF 10 ng/ml•IGF2 50 ng/ml

GROUP 4
•IGF2 60 ng/ml•HGF 10 ng/ml•KGF 10 ng/ml•10% FCS•BMP4 10 ng/ml•DMEM +

•EGF 30•KGF
ng/ml
KGF 10 ng/ml •IGF2 50 ng/ml
10 ng/ml•IGF2 60 ng/ml•EGFng/ml
30 ng/ml•BMP4 10
EGF 20 ng/ml •KGF 10 ng/ml•EGF 20 ng/ml•EGF 20 ng/ml•BMP4 10 ng/ml•DMEM +•HGF 10 ng/ml•KGF 10 ng/ml•EGF 20 ng/ml•BMP4 10 ng/ml•KGF 10 ng/ml

Fig.3. RT-PCR for MSCs
afterRT-PCR
2 weeks
in adipocytic
osteogenic(O) andFig.3.
for MSCs
after 2 weeks (A),in adipocytic(A), osteogenic(O)chondrogenic (C) media•KGF 10 ng/ml •EGF 10 ng/ml•HGF 10 ng/ml•HGF 10 ng/ml•EGF 10 ng/ml

Fig.1. The composition of media for epithelial induction on each groupand chondrogenic (C) media

Fig.1. The composition of media for epithelial induction on each group

Induction of epithelial-like cells
The results of RT-PCR expression of cytokeratin 19 is shown in figure 7. The
the presence Toofevaluate
somecharacteristic
markers
undifferentiatedMSCs respectively
the presenceof some characteristic
markers forfor
undifferentiatedcytokeratin expression was enhanced by media supplementation with EGF, KGF,
e at the dayMSCs
14 respectively
the cells
werelineage
fixed
10%
formalinminutesfor epithelial
at thewith
day 14 thecells were
fixed with 10%for 10 BMP4,
IGF2 and and
HGF. Thestained
addition of a with
mixture containing EGF, KGF and FGF seems
formalin for
10 minutes and stainedwith specific antibodies:(Becktonanti-vimentin(Beckton
Dickinson)
and anti-vimentin
anti-pancytokeratin(R&D
Systems).
For
to decrease
the cytokeratin
expression. The
effect is possible to be generated by FGF
Dickinson) and anti-pancytokeratin (R&D Systems). For further confirmation of
addition
becauseinexperiments
using
only
n of differentiation, RNA extraction and RT-PCR analysis of gene expression haveFGF as supplement the cytokeratindifferentiation, RNA extraction and RT-PCR analysis of gene expression have alsod. The primers
used were
forusedcytokeratin(R&D
and for E-cadherin (R&D
been performed.
The primers
were for cytokeratin19
19 (R&D
Systems)Systems)
and forE-cadherin (R&D Systems)

RESULTS
Isolation and culture of mesenchymal stem cells

re of mesenchymal
cells
From all thestembone marrow
samples were obtained primary cultures of adherbone marrow
samplescellswere
culturesent, spindle-shape(Figure obtained2). The cells had aprimary
good proliferation
capacity andof adherent, spindle-shape cells
are able proliferation
to survive in our lab for 5capacity
to 6 passages. Theandcell viability
each passage
lls had a good
are onable
to survive in our lab for 5 to 6
wasaround90-95%.
iability on each passage was around 90-95%.
Fig.4.
adipocytic
media.
FAB4.
ob. 10x
10x
Fig.4.MSCs
MSCsininFig.4.
adipocytic
forob.FAB4.
ob.
MSCs in adipocytic
media.IHC
IHC for FAB4.
10x

Fig.2. MSCs,
dayday5,5, ob.
ob.20xFig. 2. MSCs,
20x

Confirmation characteristicof differentiation potential
characteristic for mes-stem cells
fferentiation potential
for mesenchymal
Fig.5. MSCsin
in osteogenic
media.media.
IHC
forosteocalcin
osteocalcin
ob. 20x
Fig.5. MSCs in media.
osteogenic
IHC forob. 20x
enchymalstem cells in MSCs at passage 2. The cellsFig.5.
ntiation studies
occurred
wereMSCs
able toosteogenic
differentiate
inIHC for osteocalcin ob. 20x
differentiation studies occurred in MSCs at passage 2. The cells were able to(adipocytic, Theosteogenic
and chondrogenic) which are characteristic fordifferentiate in all three lines (adipocytic, osteogenic and chondrogenic) which are
expression was also very weak (Figure 7).
mal progenitors.
Thefor characteristic
markers
for those
lineageswere expressed atcharacteristic
stromal/mesenchymal progenitors.
The characteristic
markersthreeforRegarding the second set of experiments, in all studied groups some modificawere evidencedby
RT-PCR
reaction(Figure3).
The
presenceofsomemarkers
those three lineages were expressed at the gene level and were evidenced by RT-PCR
tions of cell morphology
were noticed.at
Thetheproliferation rate seems to be slower, cells
reaction(Figure 3). The presencesome markers
at the protein expression level
evel could be
evidenced
also, ofusing
immunocytochemistry
staining(Figures4,for5,the6).
became polygonal
and, mainly
cells from group 3 was noticed a tendency of
could be evidenced also, using immunocytochemistry staining (Figures 4, 5, 6).

2009.19.3(63)  Fiziologia - Physiology

multilayered culture (figure 8). A specific marker for MSCs, vimentin, had a weaker
expression in the epithelial inducing media in comparison with undifferentiated9

Fig.6. MSCs in chondrogenic media. IHC for aggrecan ob. 20x

Fig.5. MSCs in osteogenic media. IHC for osteocalcin ob. 20x

Fig.5. MSCs in osteogenic media. IHC for osteocalcin ob. 20x

Fig.10. Pancitokeratin expression in MSCs placed in undifferentiated media (left) and in epithelial inducing media (right)

Fig.6. MSCs in chondrogenic media. IHC for aggrecan ob. 20x

Fig.11.
RT-PCR
for Citokeratin
experimentalgroupsFig.11.
RT-PCR
for Citokeratin19and
19and E-cadherin
E-cadherin ininexperimentalgroups1 the expression of E-cadherin and cytokeratin was not detectable, suggesting a
possible role of FGF in inhibition of epithelial lineage.

of epithelial-like cells
e results of RT-PCR expression of cytokeratin 19 is shown in figure 7. The cytokeratin
Fig.6. MSCs in chondrogenic
media.media.
IHC forforaggrecanob. 20x IGF2 and HGF. The
MSCs in chondrogenic
aggrecanob. 20x BMP4,
n was enhanced by media Fig.6.
supplementation
withIHC EGF,
KGF,
DISSCUSIONf a mixture containing EGF, KGF and FGF seems to decrease the cytokeratin
expression. The
MSCs (figure 9). In parallel, the expression of pancytokeratin is stronger in theof MSCs differentiation in the cells belonging to all the emossible to be generated by FGF addition because in experiments using only FGFTheasprocesses
supplement
culturessupplemented
withepithelial-induction
cocktail(figure10).Thebest
resultsbryonic
layersarepoorlyunderstood. Although exist a theoretical model which
ratin
was
alsonoticed
very weak (Figure
7).
elial-likecells
or the expression
cells from
group
3 was
of multilayered
culture (figure 8). A specific marker for
were obtainedin group 3.a tendency
Regarding thedetection of expressionof cytokeratin 19
try
toexplaintheprocess
sat the molecular and genetic level (12). According tovimentin,
expression
epithelialculture
inducingmedia
in comparison
for thecells fromhad
groupa 3 weaker
was noticed
a tendencyinofthe
multilayered
8). A specificfor withsMSCs,
ofexpressionof cytokeratin
19
is(figureshown
in marker
figure7. The cytokeratinforthe RT-PCR
cells
from
group
32awasnoticeda 5tendency
culture(figure8).
Acomparison
specificfor
MSCs,
vimentin,
weaker
in ofthemultilayered
epithelial
inducing
media
withundifferentiatedMSCs
parallel,expression
isinstronger
in marker
theculturesthismodel
the differentiation process have to distinct phases. In the first step, Go/
1had(figure39). In
4expression
6the7
8
9of pancytokeratin
10
11 12
13
MSCs,
vimentin,
had a(figure
weakerthe epithelial
inducing
media
inobtained
comparison
with 3. and HGF. The
undifferentiated9). Inexpression
parallel,thein(figure
expressionof pancytokeratin
is werestrongerin the culturesnhancedbymedia
supplementation
with
EGF,
KGF,
BMP4,
IGF2
upplemented
withMSCs
epithelial-induction
cocktail
10).Thebest resultsinG1group
MSC
suffer transcriptional modifications and generate mesenchymal progeniundifferentiatedMSCs(figure9). In parallel,the(figure
expression
pancytokeratin
is strongerin
culturessupplemented
withepithelial-induction
10).Thebest resultsby
wereobtained(figurein thegroup
3.theseRegardingthe detectionof expression
ofcocktail
cytokeratin
19
andof E-cadherin
RT-PCR
11);
ure
containing
EGF,
KGF
and
FGF
seems
to
decreasethecytokeratin
expression.
The or self-renewal. The result is a cell
Regardingthedetectionofexpressionofcytokeratin
19
andE-cadherin
by
RT-PCR(figure
11);
these
tors
but
without
any alteration of phenotype
supplemented
withepithelial-induction
cocktail(figure10).Thebest
results
wereobtained
in
group
3.
markers
very well 19
expressed
incultured
group 2,in3 media
and 4 without
notablewith
differences
betweenTGF
groups.
In cells
PCR
forarecytokeratin
inofMSCs
supplemented
TGF
beta1(1),beta3(2),beta-cellulin
markersarevery
wellbyexpressed
inaddition
group
3 and
4 withoutnotabledifferences
betweengroups.
In
cells
Regardingthedetection
expressionofand
E-cadherin(figure11);
theseFig.7.
RT-PCR
forE-cadherin
cytokeratin
19 2,
incytokeratin
MSCs
cultured
in19media
supplemented
withbyTGF RT-PCR
beta1(1),
TGFusing
identicalwiththemothercell
anda mesenchymal pluripotent progenitor witho(4),begenerated
FGF
becauseinexperiments
only
FGF
as
supplement
rom
group
1
the
expressionofandcytokeratinwasnotdetectable,suggestingapossiblerole
EGF(5),
FGF(6),
nicotinamide(7), HGF(8),KGF(9)
IGF2(10),
EGF+KGF+FGF(11),undifferentiated
MSCs
from
groupvery
1 thewell
expressionof E-cadherin
cytokeratinwasnot(7),
detectable,suggesting
possiblerolein (3),
group
2,and
3EGF(5),and
4 withoutnotabledifferences
betweena groups.
In
cells
beta3(2),beta-cellulin
BMP4(4),
FGF(6),
nicotinamide
HGF(8),
KGF(9)
IGF2(10),
ofmarkers
FGF in are
inhibitionof expressed
epithelial
lineage.
restricteddifferentiation
potential
butphonotypical similar. After that the pluripotent(12),
negative
control(13)
of FGF
inwas
inhibitionof epithelial
lineage. (Figure
ression
also
very
weak
7).(12),fromgroup
1 the expressionof E-cadherin
and cytokeratinwasnegativenot detectable,
EGF+KGF+FGF(11),undifferentiated
MSCs
control (13) suggesting a possible role
progenitor cells generate by division tri and bipotent progenitors which differ onlyof FGF in inhibition of epithelial lineage.
garding the second set of experiments, in all studied groups some modificationsof level.
cellThe differentiation potential is restricted to osteogenic
at the transcriptional
1
2
3
4
5
6 7
8
10 11 12 13
gy were noticed.
The
proliferation
rate seems
to 9be slower,cells became polygonal
and,/ mainly/ chondrogenic
adypogenic for tripotent progenitors and to muscle/tendon forthe bipotent progenitors. In the second step bi and tripotent progenitors generate
unipotent specific progenitors for each line, with the phenotype modification andytokeratin 19 in MSCs cultured in media supplemented with TGF beta1(1), TGF beta3(2), beta-cellulin
becoming finally adult cells with the specific specialized structures. The microar5), FGF(6), nicotinamide (7), HGF(8), KGF(9) IGF2(10), EGF+KGF+FGF(11), undifferentiated MSCs
ray analysis of global gene expression identified the genes which are involved in(12), negative control (13)the MSCs differentiation process. The genes which trigger the progression toward
Fig.8. MSCsMSCs
in epithelial
inducing
3), day
day20,
20,obob20x
20x
in epithelial
inducingmedia
media(group(group3),
Fig.8.Fig.8.
MSCs in epithelial
inducing
media(group3), day
20, ob 20x
a specialized cell line were evidenced by analyzing the transcriptional profiles
in epithelial inducingin
media(group3), day 20, ob groups
20x
the second set Fig.8.
of MSCs
experiments,
allstudiedsomeof threemodifications
cell undifferentiated MSC. For analyses
cell lines: osteoblasts,ofchondrocytes,
noticed. The proliferation rate seems to be slower, cells becamewaspolygonal
and,genome
mainlyused Affymetrix humanU133 array set (13). The genes with strong
expression in the differentiation process was identified and divided into three
categories depending on the cell type. There was identified eight genes expressedin the differentiation process toward all three cell lines, which suggest that they
are major determinants of the differentiation process: period homolog1 (PER1),
nebulette (NEBL), neuronal cell adhesion molecule (NRCAM), FK506 binding
Fig.9. Vimentin expression in MSCs placed in undifferentiated media (left) and in epithelial inducing media (right)protein 5 (FKBP5), interleukin 1 type II receptor (IL1R2), zinc finger protein 145
Fig.9. Vimentin expressionin MSCs
placedininMSCs
undifferentiatedmedia (left)in andepithelial
inducing media (right)(ZNF145), tissue inhibitor of metalloproteinase 4 (TIMP4), serum amyloid A2. The
Fig.9. Vimentin
expression
placed in undifferentiatedmediaand(left)
in epithelial
Fig.9. Vimentin expression in MSCs placed in undifferentiated media (left) and in epithelial inducing media (right)
inducing media (right)
functions of these gene address to the large spectrum of biological processes: celladhesion, the tertiary structural protein organization, the citoskeletal organizationand inflammatory response. The genes involved in final steps of differentiation
process were not identified.
The cell plasticity is a hallmark of embrionary development. Although wasdemonstrated that MSCs can differentiate in vitro in a raw of cell types with thepossibility to use them for therapeutic purposes in organ and tissues reconstruction.
Fig.10. Pancitokeratin expression in MSCs placed in undifferentiated media (left) and in epithelial inducing media (right)
The molecular mechanisms involved in MSCs plasticity and in MSCs transdifferentiation
are still unclear. The cells have a heterogeneous genetic pattern suggestingFig.10.
Pancitokeratin expression in MSCs placed in undifferentiated media (left) and in epithelial inducing media(right)Fig.10. Pancitokeratin expression in MSCs placed in undifferentiated media (left) and in epithelial inducing media (right)
Fig.10. Pancitokeratin expression in MSCs placed in undifferentiated media (left) and in epithelial
that they are in a permanent „standby stat” in which lot of genes is expressed at
inducing media (right)
the minimal level making possible the changing of the cell fate. Some researchers
showed that this remarkable plasticity appear mainly in the cells isolated fromand E-cadherin by RT-PCR (figure 11); these markers are very well expressed inbone marrow which could under adequate circumstances differentiate into anygroup 2, 3 and 4 without notable differences between groups. In cells from groupcells belonging to the embryonic layers. (14) Is still controversially if in some cases
10

Fig.11. RT-PCR for Citokeratin 19and E-cadherin in experimental groups

Fig.11.RT-PCR
RT-PCR for
for Citokeratin 19andFig.11.
19and E-cadherin
E-cadherinininexperimental
experimentalgroups
groupsFiziologia - Physiology  2009.19.3(63)is involved a fusion process with the differentiated cells likes epithelial, Purkinje ormuscular cells. The bone marrow derived stromal cells can differentiate in renal
epithelial and non epithelial cells. The evidence of this fact appears when thefemale kidney is transplanted into the male and in the transplanted kidney could
be identified the epithelial cells positive for Y chromosome (15). There are many
experiments in animals which demonstrate that the cells from bone marrow canform bronchiolar epithelium and type 2 pneumocytes. Kotton et al. demonstrate
in an experiment in which the MSCs were injected in mice with bleomycin induced
pulmonary lesions that the injected cells differentiate in type 1 pneumocytes (16)
MSCs can differentiate in vivo in gastrointestinal epithelial cells like esophageal andgut epithelium (17); it was noticed that the administration of the bone marrowderived mesenchymal progenitors leads to the differentiation in the crypt cells
which represent the proliferative compartment from the tissue. Regarding the
MSCs differentiation in epidermal cells, Herzog et al. showed that MSC can reachat the cutaneous lesion site and differentiate in proliferative keratinocytes which
later are integrated in the scar tissue. (18) For maintaining of MSCs differentiation
potential lot strategies are mentioned, like cell culture in the presence of specificgrowth factors, enrichment of the number of undifferentiated cells prior the additionof differentiation factors or even co culture of MSCs with the cells belonging to thespecific tissue. On the other hand the analysis of the in vitro MSCs differentiationin the conditions mimicking the in vivo cell microenvironment could omit someessential factors for MSCs commitment toward a certain cell line. These factors aresoluble molecules and their receptors (TGFbeta), molecules of the extracellularmatrices (collagen, proteoglycans), cytoskeletal actine, transcription factors (Cbfa1/
Runx2, PPARgamma, Sox9, MEF 2). Multiple protein interactions (focal adhesion,
cytoskeleton) allow the cells to generate complex signals which lead to various in
vitro cells behaviors. The initiation of cell adhesion integrin mediated has a great
impact on cell proliferation. The integrins can regulate in a cooperative manner themembers of cycline family and interfere in the cell cycle progression (19). Establishment of specific stimuli integrin-extracellular matrices can induce an increased
expression of the genes involved in the differentiation processes.

CONCLUSIONS
From the anterior presentation we can conclude that adult MSC are extremely
attractive for the researchers involved in the study of the various tissues regenerationrestoration processes. In the scientific literature can found references to the plastic
potential of MSC to the mesodermal cell lines: bone, cartilage, adipocytes. There
are data about the differentiation inducing agents and about the molecular events
which can induce the cell switching from stem cell state able of self renewal in acommitment state toward a specific cell lineage. Concerning the capacity of MSC
to differentiate in the cells belonging to the other embrionary layers there are lotof studies regarding in vivo and in vitro generation of the cell with neuronal characteristics; also a lot of interests was noticed regarding the possibility of obtaining
the insulin secretory cells by manipulating the MSC. Concerning the differentiationin the epithelial lineage there are very few studies and the existing ones are about
the murine MSC or referring to the in vivo experiments. Our study demonstrated
that MSCs differentiation toward the cells expressing epithelial markers is relatively
easily to obtain using a certain combination of inductors, without genic manipulation of the cells.
AKNOWLEDGEMENTS: The work within this paper was financial
supported by a national PNCD-II grant, IDEI 198/2007 (financing agency CNCSIS)

2009.19.3(63)  Fiziologia - Physiology

REFERENCES
1. Zipori D. The Stem State: Plasticity Is Essential, Whereas Self-Renewal and Hierarchy Are Optional. Stem Cells 2005, 23:719-726.
2. Bianco P, Gehron Robey P. Marrow stromal stem cells. JCI 2000,
105: 1663-1668.
3. Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according toa hierarchical model. J. Cell Sci 2000, 113:1161-1166.
4. Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate
throughout forebrain and cerebellum, and they differentiate into
astrocytes after injection into neonatal mouse brains. Proc. Natl.
Acad. Sci. U. S. A. 1999; 96:10711-16.
5. Jiang Y, Jahagirdar BN, Reinhard RL, Schwartz RE, Keenek CD,
Ortiz-Gonzalezk XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du
J, Aldrich S, Lisberg A, Lowk WC, Largaespada DA, Verfaillie CM.
Pluripotency of mesenchymal stem cells derived from adult marrow, Nature 2002;418:41-49.
6. Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human Mesenchymal Stem Cells Differentiate to a Cardiomyocyte Phenotype
in the Adult Murine Heart. Circulation. 2000;105:93-98.
7. Liechty KW, MacKenzie TC, Shaaban AF, Radu A, Moseley AB,
Deans R, Marshak DR, Flake AW. Human mesenchymal stem cells
engraft and demonstrate site-specific differentiation after in uterotransplantation in sheep. Nat. Med. 2000;6:1282-86.
8. Hwang JH, Shim SS, Seok OS, Lee HY, Woo SK, Kim BH, Song HR, Lee
JK, Park YK. Comparison of cytokine expression in mesenchymal
stem cells from human placenta, cord blood, and bone marrow, J
Korean Med Sci. 2009 Aug;24(4):547-54.
9. Eslaminejad MB, Yazdi PE. Mesenchymal Stem Cells: In Vitro
Differentiation among Bone and Cartilage Cell lineages. Yakhteh
Medical Journal 2007; 9(3): 158-169.
10. Malladi P, Xu Y, Chiou M, Giaccia AJ, Longaker MT. The effectof reduced oxygen tension on hondrogenesis and osteogenesis
in adipose-derived mesenchymal cells. Am J Physiol Cell Physiol,
2005 Apr;290(4):C1139-46.
11. Schutze N, Noth U, Schneidereit J, Hendrich C, Jakob F. Differential
expression of CCN-family members in primary human bonemarrow-derived mesenchymal stem cells during osteogenic,chondrogenic and adipogenic differentiation. Cell Commun Signal.
2005, 17;3(1):5.
12. Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells:
characterization, differentiation, and application in cell and genetherapy. J. Cell. Mol. Med. 2004;8(3):301-316.
13. Song L, Tuan RS. Transdifferentiation potential of humanmesenchymal stem cells derived from bone marrow, FASEB J.
2004;18:980-982.
14. Alison MR, Poulsom R, Otto WR, Vig P, Brittan M, Direkze
NC, Lovell M, Fang TC, Preston SL, Wright NA. Recipes for adultstem cell plasticity: fusion cuisine or ready made? JClinPathol
2004;57:113-120.
15. Poulsom R, Forbes SJ, Hodivla, Dilke K et al. Bone marrowcontributes to renal parenchymal turnover and regeneration.
JPathol. 2001;195:93.
16. Kotton DN, Ma BY, Cardoso WV, et al. Bone marrow-derived
cells as progenitors of lung alveolar epithelium. Development
2001;128:5181-88.
17. Păunescu V, Deak E, Herman D, Siska IR, Tănasie G, Bunu
C, Anghel S, Tatu CA, Oprea TI, Henschler R, Rüster B, Bistrian R,
Seifried E. In Vitro Differentiation of Human Mesenchymal StemCells to Epithelial Lineage. J Cell Mol Med 2007;11:1-8.
18. Herzog E, Chai L, Krause D. Plasticity of bone-marrow derived
stem cells. Blood 2003; 102:3483-93.
19. Docheva D, Popov C, Mutschler W, Schieker M. Human
mesenchymal stem cells in contact with their environment:
surface characteristics and the integrin system. J.Cell.Mol.Med.
2007;11(1):21-38.

11

MECANISME IMPLICATE ÎN DIFERENŢIEREA CELULELOR STEMMEZENCHIMALE SPRE LINIA EPITELIALĂ. EFECTUL UNORINDUCTORI CHIMICIREZUMAT

Celulele stem mezenchimale adulte (CSM) sunt extreme de attractive pentru studiul proceselor de reparatie si regenerare in diferite tipuri de tesuturi. Pentru
mentinerea potentialului de diferentiere al CSM au fost utilizate o serie de strategii: cultivarea celulelor in vitro in mediu suplimentat cu factori de cresterespecifici, transfectia MSC cu genele specifice celuelor differentiate, cu-cultivarea MSC cu cellule differentiate din tesutul tinta. In experimental nostrum am
utilizat diferiti factori de crestere si cytokine adaugati in mediul de cultura singuri sau in combinatii. Rezultatele au fost evaluate prin imunocitochimie si
metode de biologie moleculara (PCR). Studiul a demonstrat ca diferentierea CSM la celule ce exprima markeri de tip epitelial este relativ usor de realizat
utilizand anumite combinatii de agenti inductori, fara a fi necesara manipularea genelor celulare.
Cuvinte cheie: celule stem mezenchimale, diferentiere epiteliala, celule epitelial-like, inductie chimica

12

Fiziologia - Physiology  2009.19.3(63)ONTOGENETIC changes OF bone MINERAL Composition
OLGA TAGADIUC1, V. GUDUMAC1, V. REVA2
1
State University of Medicine and Pharmacy "N. Testemitanu"
2State University, Republic of Moldova

ABSTRACTThe goal of the study was to assess the dynamic of the bone mineral content at different ontogenetic stages. In the study were involved 80 rats that were divided
in 4 groups by age – young, adult, old and senile. The dynamics of the calcium and phosphate quantities in the bone were similar – the maximal amounts
were revealed in adult rats and their concentrations decreased gradually in old and senile animals. The highest mean concentrations of magnesium, chloride,potassium and copper were revealed in young animals (1.46 ± 0.21, 3.53 ± 0.28, 0.02 and 0129 ± 2.97 ± 0.18 mM/g tissue). The amounts of copper andsodium changed from one age group to another – moderate decreased in the adult compared with young (by 33% and 51%, p < 0.0001), increased in theold compared with those adults (by 13% and 62%, p < 0.05) and subsequently decreased again in the bone of senile rats (by 18% and 73%, p < 0.005). The
amount of zinc was virtually identical in the bone of young, adult and old rats and varied between 0.017 and 0.0183 mM/g bone tissue while the sulphates
concentration increased significantly in adult animals versus the young (14 times) and decreased gradually with age in the subsequent groups.
Our results revealed divers and significant quantitative changes of the mineral compound's content in the bone at different ontogenetic stages, their knowledge
being important in the evaluation of bone status in different physiological and pathological conditions.
Keywords: bone, mineral composition, ontogenetic changes

INTRODUCTION
Along with increasing life expectancy and changing age structure of population, diseases of the osteo-articular system have grown in importance. Thus, investigations were directed to a greater extent to bone metabolism and the most commonbone disease – osteoporosis. Particularly intense were studied bone composition
and metabolism in persons during risk periods –postmenopause and senile.
Strategies for prevention and treatment methods developed and implemented,
also were targeted at older people, aimed at halting the decrease bone mineraldensity and recovery of lost bone.
However, it was acumulated a significant amount of information on theparentage in childhood and adolescence of many factors that determine the quality of bone tissue and predispose to osteoporosis (1) .Osteopenia and osteoporosis
are not an exclusively adults and elderly issue, there are a lot of evidence of direct
correlation between mineral density in elderly bone and bone mass accumulated
in the first two decades of life and increased risk of osteoporosis if the accumulated
peak bone mass was less than optimal (5).
Most studies have focused on the evaluation of specific markers of bone
remodeling that can be assessed in blood or urine (2) in various primary andsecondary osteo-articular diseases (6), on establishing correlations between bonemineral density and the dynamics of bone tissue markers in normal and pathologicalconditions (4), the analisys of drugs and osteotrope remedies influence on bone
remodeling (3), etc.
However, there is no sufficient scientific data about the detailed bone composition and the dynamic of individual compounds in various diseases at different ages,
necessary to build a scientific based diagnosis and differential, coherent prevention
and therapy strategies.
It is obviously necessary to study the particularities of composition andmetabolism of bone at different ontogenetic stages of development, to determine
the parameters and characteristics of each age, to create database that would
differentiate the age-related physiological changes from the pathological one
determinated by various osteo-articular conditions or bone diseases.
Research goal was to determine the peculiarities of the bone mineral composition at different ontogenetic stages of development in experiment.

MATERIAL AND METHODSThe experiences were performed on 80 rats of different age:• group I - young rats until sexual maturation (2 months);• group II - adult rats in reproducible period (6 months);• group III - old rats in postmenopause period (18 months);• gloup IV - senile rats (24 months).
Rats were sacrificed under light narcosis with diethyl ether. Femoral bones
were extracted, stripped of adjacent soft tissues and released of the bone marrowby repeated washing with glacial solution of 0.9% NaCl. Subsequently femoral
bones were triturate in liquid nitrogen to the state of powder.
In the femoral bone powder was determined the amount of some bone mineralcompounds. The calcium, phosphorus, magnesium, copper and chlorine contents
were assessed with standard kits Elitech Diagnostic (France), according to the
instructions attached. The amount of sodium, potassium and zinc was assessed withstandard kits Centronic GmbH (Germany), according to the instructions attached. The
amount of sulfates was determined turbidimetric with barium chloride.
The results were evaluated statistically according to Student t-criterion,
neparametric Mann-Whitney criterion and the correlation coefficient r (Statistica
6.0, Stat Soft Inc.., 2002).
The research was approved by the Ethics of biomedical research Board of

Received August 2009. Accepted September 2009. Address for correspondence: Dr. Tagadiuc Olga, B.P.Hasdeu Street 14/2, Mun. Chişinău, Republica Moldova, Postal code 2005, e-mail:tagadiuc_olga@yahoo.co.uk
2009.19.3(63)  Fiziologia - Physiology

13the State University of Medicine and Pharmacy "N. Testemitanu" from Republicof Moldova.

old compared with those adults (13%, p<0.05) and subsequently decreased again
in the bone of senile rats (18%, p<0.005). Similar changes but less pronounced
in magnitude of sodium content had been revealed in bone tissue of experimental
rats (Figure 1).

RESULTS AND DISCUSSION
It was established that the contents of calcium and phosphate in bone tissueof rats are subjected to similar changes, but of different magnitudes (Table I). The
results show the highest average concentrations of calcium and phosphate in adultanimals. The content of calcium increased significantly in adult rats compared withyoung (12%, p <0005) and decreases gradually thereafter, the differences being
statistical conclusive between the concentrations detected in the adult animals andin the senile one (8%, p <0.05). The content of phosphates in the bone of adultrats is higher than in the young by 6% (p <0.05), in the old – by 8% (p <0.01)
and the senile rats – by 5%. Thus, the total amount of phosphates in bone tissue ofrats is changing less pronounced than that of calcium, the contents of these mineralelements are correlated between them. Strong positive correlation were revealed inyoung (r = 0.79, p <0.0001) and old rats (r = 0.91, p <0.0001).

3,5
3

mM/g bone

2,5

1

PhosphateI4.86 ± 0.10

3,44 ± 0,09

II

5.45 ±
0.18***I

3.64 ± 0.08*I

3

**II

1

Group I

3,51+0,2

3,35+0,4

3,44+0,1

2

3,64+0,1

5,02+0,1

5,45+0,2

4

5,41+0,4

5

4,86+0,1

6

I
II
III
IV

0,02 ± 0.21
1.460

0.115 ± 0.02

3.53 ± 0.28

0.655 ± 0.03****I

0.068 ± 0.002****I

2.24 ± 0.09****I****I

0.073 ± 0.007

0.162 ± 0.02****I,II

The amount of zinc was virtually identical in the bone of young, adult and oldrats and varied between 0.017 and 0.0183 mM/g bone tissue (Figure 2). Only insenile animals was detected a significantly higher amount of zinc in bone tissue –0.0287 mM/g bone tissue (p < 0.0001). The average content of sulphates in thebone tissue of young rats was 21 times less (p <0.0001) compared to adult andold animals. In senile rats were detected in comparisone with adult and old animals
moderate decreased concentrations of sulfates (24%, p < 0.05 in both cases).

b)
0,0287±0,001
2,5

0,03

mM/g bone

0,03

0,0183±0,001

0,0181±0,001

0,017±0,003

2

0,02

1,5

0,02

1

0,01

0,5

0,01
0,00

Group IGroup IIGroup III

Group IV0Grob)Fig.2. Age depending dynamics of the zinc (a) and sulphates (b) con

ontogenetic stages
Sulfates (mM/g bone)Note. a) the concentrationsof zinc and sulphates are expressed in mMb) figure includes M ± m.
2,5Table II. The contents of magnesiumand chlorine in the bone tissue of rats of different ages
0,0183±0,001
0,0181±0,001
0,03
0,017±0,003 Chloride
Group
Magnezium
PotasiummM/g bone

CopperZink

0,03 of magnesium and chlorine in the bone tissue of rats of different ages
Table II. The contents

0.660 ± 0.06

Group IVa)

The highest average concentrations of magnesium, chlorine, potassium andcopper in the bone tissue were attested in young animals, respectively, – 1.46 ±0.21, 3.53 ± 0.28, 0.02 and 0129 ± 2.97 ± 0.18 mM/g tissue (Table II). In adultrats decreases the amount of magnesium compared with young animals (69%, p< 0001) and no significant changes were established in the following age groups.
Same dynamic was characteristic for the potassium level – the highest concentration
was detected in young animals, and then it decreased by about 50% (p < 0005)
and remains virtually at the same level in the bone of adult, old and decrepit rats
(0.068 – 0.080 mM/g tissue). The level of chlorine in bone was moderate decreasedin the adult rats compartiv with young (27%, p < 0.0005) and more marked inold animals compared with adult (with 93%, p < 0.0001) and young one (95%,a) In group IV (senile rats) the chlorine content significantly increased
p < 0.0001).
Zink 255%, p <0.0001), but not tocomparative to the levels found in old animals (by0,0287±0,001
the specific quantities of mature or young one were detected.

III

Group III(mM/g bone tissue)
Note: a) the content of sodium and cooper is expressed as mM/g bone tissue;b) figure includes M±m.

Note a) table includes M±m; b) the concentrations of calcium and phosphate are expressed as
mM/g bone tissue; c) the reliability of differences caused by age: *-p<0.05, **-p<0.01, ***-p<0.005,****-p<0.001; the number indicates the group that the comparisont was carried out with

0,02

Group II
SodiumCa
Pi
5.02 ±
IV
3.51 ± 0.17
0.09*II,IV
Note a) table includes M ± m; b) the concentrations of calcium and phosphate are expressed as mM/g bone tissue; c) the
reliability of differences caused by age: * - p < 0.05, ** - p < 0.01, *** - p < 0.005, **** - p < 0.001; the number indicates thegroup that the comparisont was carried out with.

I

0,803±0,1

Note.
theAgecontentof sodiumcopperexpressedbonetissue;Fig.a)1.
dependingdynamicsandof sodium
andiscooper
contentsasin mM/gbone tissueof ratsb) figure includes M ± m.

0II

1,15±0,1

mM/g

Calcium

3.35 ± 0.42

2,27±0,1

0

Table I. The concentrations of calcium and phosphate in bone tissue of rats of different ages
Group

5.41 ± 0.40

1,51,86±0,1

2,00±0,1
2,34±0,5

0,5Table I. The concentrations of calcium and phosphate in bone tissue of rats of different agesIII2

Fig.1.Age depend
and copper conte(mM/g bone tissu

3,04±0,2

2,97±0,22

2,353±0,182,302±0,28

1,5
MODIFICARILEONTOGENETICE1,754±0,08
ALE COMPOZITIEI

0,01 ± 0.03
IV
0.645
0.080 ± 0.004****I; ***II
0.58 ± 0.04****I,II,III
Note. a) the table includes the M ± m; b) the contents of magnesium, chloride and potassium are expressed as mM/g bone
0,01
tissue; c) the reliability of differences caused by age: * - p < 0.05, ** - p < 0.01, *** - p < 0.005; **** - p < 0.001; the number
indicates the group that the comparison was carried out with

1

0,115±0,02
REZUMAT0,5
Note. a) the table includes the M ± m; b) the contents of magnesium, chloride and potassium are Obiectivul cercetării a fost evaluarea compoziĠiei minerale osoase
0,000
expressed as mM/g bone tissue; c)Groupthe Ireliability ofGroupdifferences
causedby IIIage: * - p <Group
0.05,IV** - p <IIGroupGroup I efectuateGroup II pe 80
Group
III
GroupdivizaĠi
IV
ExperienĠele
au fostúobolaniîn 4 loturi – tiner
0.01, *** - p < 0.005; **** - p < 0.001; the number indicates the group that the comparison was
S-au depistatt modificări de acelaúi sens ale cantităĠilor de calciu úi fcarried out with
Fig.2. Age depending dynamics of the zinc (a) and sulphates(b)contents
in thebonerats atin the
differentAge depending
dynamicsof the zinc(a) andtissuesulphatesof(b) contentsbone tissue ofadulte úiFig.în2.următoarelegrupedevîrstă
diminuează
treptat. ConcentraĠ
ontogenetic stages
rats at different ontogenetic stages
The amount of copper changed from one age group to another-moderate úi cupruînĠesutulosos
seatestă
laanimaleletinere(respectiv, 1.46
Note. a) the inconcentrations
zinc and tissue;
sulphates are expressed in mM/g bone tissue;
Note. a) the concentrations of zinc and sulphates are expressedmM/gofbone
decreased in the adult compared with young (33%, p<0.0001), increased in the 0.18 mM/g         b)figureincludesM±m.
Ġesut).
ConcentraĠiile
de
cupru
úi
sodiu
alternează
de lab) figure includes M ± m.
comparativ cu cei tineri (cu 33% úi 51%, p < 0,0001), creúte la cei băt- Physiology
 2009.19.3(63)14
p < 0,05) úi ulterior Fiziologiaiarăúi descreútela úobolanii
senili comparativConĠinutul de zinc este practic identic la úobolanii tineri, adulĠi úi bătrî
MODIFICARILE ONTOGENETICE ALE COMPOZITIEIMINERALE OSOASE
osos, pe cînd concentraĠia sulfaĠilor în os creúte semnificativ la animori), odată cu înaintarea în vîrstă înregistrîndu-se descreúterea treptatăREZUMAT
Studiul efectuat denotă modificări semnificative cantitative úi variat
Obiectivul cercetării a fost evaluarea compoziĠiei minerale osoase la diferite etape ontogenetice de dezvoltare.

Table III. Correlations between the contents of the mineral compoundsof the bone tissue of rats of different ages

Table III. Correlations between the contents of the mineral compounds of the bone tissue of rats of different ages.
Young
Adult
Old
SenileCa Mg NaPi
Cl
Ca Mg NaPi
Cl
Ca Mg Na
Pi
Cl
Ca
0.79 0.57 0.8 0.54
0.61
Mg 0,79
0.75 0.68
0.52
1.0 0.8 0.68
Na 0,57 0.75
0.72 0.65-051 053
1.0
0.8 0.68
Pi 0,8 0.68 0.72
0.6
0.52 -0.51-0.58 0.88 0.8 0.8
0.77
Cl 0,54 0.61 0.65 0.6
0.6
0.53 -0.58
0.68 0.68 0.77
Note a) table includes the values of correlation coefficient r;b) in all cases p < 0.05.

Note a) table includes the values of correlation coefficient r,b) in all cases p<0.05. 1.

Many correlations between the quantities of mineral compounds in bone tissue
were found. These correlations vary in number, power and related substances inanimalsof different age (Table III).
The greatest number of singifcant positive correlations of medium and strong
intensity with r between 0.54 – 0.80 (p < 0.05) were attested in young and senile
rats. In adult rats a moderate number of medium intensity correlations both positive
and negative were found. The strongest positive (r between 0.71 and 0.89, p <0.05)
correlations were established in old animals.

REFERENCES
1. Bailey DA, McKay HA, Mirwald RL et. al. A six-year longitudinal
study of the relationship of physical activity to bone mineral accrual in growing children: the university of Saskatchewan bonemineral accrual study. J Bone Miner Res 1999; 14: 1672-1679.
2. Cremers S, Bilezikian JP, Garnero P, Bone markers – new aspects.
Clin Lab 2008; 54 (11–12): 461-471.
3. Garnero P, Biomarkers for osteoporosis management: utility indiagnosis, fracture risk prediction and therapy monitoring. Mol
Diagn Ther 2008; 12(3): 157-170.
4. Honig St, Treatment Strategies for Patients with Low Bone Mass
(The Younger Postmenopausal Female). Bulletin of the NYU Hospital for Joint Diseases 2008; 66(3): 240-243.
5. Saggese G, Barancelli GI, Bertelloni S, Osteoporosis in children
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CONCLUSIONS
Research data attested statistically significant changes, but at moderate scaleof the calcium and phosphate content – cardinal constituent elements of bonemineral phase, in animals of different age.
2. Quantities of magnesium, chlorine, copper, sodium, potassium, zinc andsulphates undergo significant age-dependent changes in the bone tissue of rats.
3. Correlations between the quantities of the mineral elements of the bone
tissue of rats vary in number, intensity and type of the related substances in animals
of different age.

MODIFICARILE ONTOGENETICE ALE COMPOZITIEI MINERALEOSOASEREZUMAT

Obiectivul cercetării a fost evaluarea compoziţiei minerale osoase la diferite etape ontogenetice de dezvoltare. Experienţele au fost efectuate pe 80 şobolani
divizaţi în 4 loturi – tineri, adulţi, bătrîni şi senili.
S-au depistatt modificări de acelaşi sens ale cantităţilor de calciu şi fosfaţi, care ating valori maxime la animalele adulte şi în următoarele grupe de vîrstă
diminuează treptat. Concentraţiile medii maxime de magneziu, clor, potasiu şi cupru în ţesutul osos se atestă la animalele tinere (respectiv, 1.46 ± 0.21, 3.53
± 0.28, 0.02 and 0129 ± 2.97 ± 0.18 mM/g ţesut). Concentraţiile de cupru şi sodiu alternează de la o grupă de vîrstă la alta – scade la cei adulţi comparativ
cu cei tineri (cu 33% şi 51%, p < 0,0001), creşte la cei bătrîni comparativ cu cei adulţi (cu 13% şi 62%, p < 0,05) şi ulterior iarăşi descreşte la şobolanii senili
comparativ cu cei bătrîni (cu 18% şi 73%, p < 0,005). Conţinutul de zinc este practic identic la şobolanii tineri, adulţi şi bătrîni şi variază între 0,017 şi 0,0183
mM/g ţesut osos, pe cînd concentraţia sulfaţilor în os creşte semnificativ la animalele adulte comparativ cu cele tinere (de 14 ori), odată cu înaintarea în vîrstă
înregistrîndu-se descreşterea treptată a cantităţii lor.
Studiul efectuat denotă modificări semnificative cantitative şi variate ca direcţie ale concentraţiilor substanţelor minerale în ţesutul osos al şobolanilor ladiferite etape ontogenetice de dezvoltare, cunoaşterea căreea este necesară pentru evaluarea stării ţesutului osos în diverse condiţii fiziologice şi patologii
Cuvinte cheie: ţesut osos, compoziţie minerală, modificări ontogenetice2009.19.3(63)  Fiziologia - Physiology

15

GLUTATHIONE PEROXIDASE ACTIVITY AFFECTSPROGNOSIS IN NON-ST SEGMENT ELEVATION ACUTE
CORONARY SINDROM
VIOLETA SAPIRA1, ELVIRA CRAIU2, CECILIA ADUMITRESI3
University of Medicine, Constanta, Romania
1
Department of Neurology
2
Department of Cardiology
3
Department of Physiology and Pathophysiology

ABSTRACTIntroduction: Cellular antioxidant enzymes such as glutathione peroxidase play a central role in the control of reactive oxygen species. In vitro, data andstudies on animal models suggest that these enzymes may protect against atherosclerosis, but few are known for their relevance to human disease.
Aim: We have examined the association between redox status and the prognosis of non ST elevated acute coronary syndrome.
Methods: We have evaluated 55 patients, age under 75, consequently hospitalized in the Cardiology Clinic of Emergency Clinic Hospital of Constanta,
during May 2008 – May 2009, diagnosed with non ST elevated acute coronary syndrome and 19 healthy volunteers (without cardiovascular affections,
hypercholesterolemia, diabetes and non-smoking). The patients were divided in two groups: the first group - patients with unstable angina (37 patients, outof which 10 were readmission in the following 6 months) and the second group – patients with non-Q wave myocardial infarction (18 patients, out of which
6 were readmission in the first 6 months after the heart attack). Glutathione peroxidase (GPx) activity was measured over a fixed time: T1 – the first 24 hours
after hospital admission, T2 – at 48 hours and T3 – at discharge. After discharge, the patients were monitored and the following data was recorded: monthsof follow-up, death due cardiovascular cause and onset of major cardiovascular events.
Results: This prospective study of patients admitted with non-ST-segment elevation acute coronary syndrome (unstable angina and non-Q wave myocardial
infection) showed a direct association between baseline GPx and the onset of major acute coronary events in the group with unstable angina and inversely
associated with future fatal and non-fatal cardiovascular events in the group with non-Q wave myocardial infarction.
Conclusion: In the case of the patients with non-ST-segment elevation acute coronary syndrome, the antioxidative enzyme GPx seems to protect against
adverse oxidative effects. The analysis of GPx activity provides superior information on cardiovascular risk assessment compared with the measurement oftraditional risk factors alone.
Key words: acute coronary syndrome, biological markers/blood, glutathione peroxidase, oxidation-reduction, oxidative stress

INTRODUCTION
Controversial data existing concerning the relation between the activities ofscavenger antioxidant enzymes and coronary heart disease (CHD) risk.
Oxidative stress may be defined as an imbalance between the production anddegradation of reactive oxygen species such as super oxide anion, hydrogen peroxide,
lipid peroxides and peroxynitrite. Aerobic organisms possess antioxidant defense
systems that deal with reactive oxygen species (ROS) produced as consequence of
aerobic respiration and substrate oxidation. Low levels of ROS are indispensable
in many biochemical processes, including intracellular signaling, defense against
microorganisms and cell function. In contrast, high dose and/or inadequate removalof ROS, results in “oxidative stress”, which has been implicated in the pathogenesisof many cardiovascular diseases, including hypercholesterolemia, atherosclerosis,
hypertension, diabetes, and heart failure. Enzymatic inactivation of reactive oxygen
species is achieved mainly by glutathione peroxidase, superoxide dismutase, andcatalase (7, 8). In mammalian cells, glutathione and the glutathione peroxidases
are the principal antioxidant defense system (13, 20).
There are at least four different glutathione peroxidases, all of them containing
selenocysteine at their active sites (2).

Glutathione peroxidase 1, the ubiquitous intracellular form and key antioxidant
enzyme within most of the cells, including those of endothelium, uses glutathione
to reduce hydrogen peroxide to water and lipid peroxides to their respective alcohols(6,9), and it also acts as a peroxynitrite reductase (17).
On the basis of the experimental evidence, we addressed the hypothesis thatenhanced activity of cellular glutathione peroxidase 1 would be protective against
cardiovascular events in patients with coronary artery disease.

METHODS
We have evaluated 55 patients, age under 75, consequently hospitalized in the
Cardiology Clinic of Emergency Clinic Hospital of Constanta, during May 2008 – May
2009, diagnosed with non ST elevated acute coronary syndrome and 19 healthy
volunteers (without cardiovascular affections, hypercholesterolemia, diabetes andnon-smoking) – control group.
The patients were informed about the study protocol and a written consent
was obtained from each patient. All the patients were evaluated within the same
clinical and paraclinical protocol: medical history, physical examination, 12-leadelectrocardiography, biochemical analysis.

Received June 2009. Accepted July 2009. Address for correspondence: Dr. Violeta Sapira, Neurology Department, University of Medicine Constanta, Romania, e-mail:violetasapira@yahoo.com
16

Fiziologia - Physiology  2009.19.3(63)The patients were divided in two groups: group I - patients with unstableangina (37 patients, out of which 10 were readmission in the following 6 months– group IA and 27 without being re-hospitalized – group IB) and group II – patients with non-Q wave myocardial infarction (18 patients, out of which 6 were
readmission in the first 6 months after the heart attack – group IIA and 12 withgood evolution, without being re-hospitalized – group IIB).
Glutathione peroxidase activity was measured over a fixed time: T1 – in thefirst 24 hours after hospital admission, T2 – at 48 hours and T3 – at discharge.
After discharge, the patients were monitored by means of a telephone survey, apersonal interview, a chart consultation or any other combination of these methods.
The following data was recorded: months of follow-up, death due cardiovascularcause and onset of major cardiovascular events (MACEs defined as cardiovasculardeath, readmission with acute coronary syndrome or the need for coronary
revascularization).
Glutathione peroxidase (GPx) in whole blood quantitative determination isbased on Paglia and Valentine method. GPx catalyse the oxidation of gluthatione(GSH) by Cumene Hydroperoxide. In the presence of gluthatione reductase (GR)and NADPH the oxidized glutathione (GSSG) is immediately converted to the
reduced form with a simultaneous oxidation of NADPH to NADP+. The decreased
in absorbance at 340nm is measured.
The main reaction is:
GPX

2GSH + ROOH 
→ ROH + GSSG + H2OGR

GSSG + NADPH + H+ → NADP+ + 2GSH
Glutathione peroxidase concentration was calculated using the following
formula: U/l whole blood = 8412 x ∆A 340 nm/min and expressed in U/l.
Through routine methods we have determined: the complete blood count(CBC) and the lipidic profile of the patients (high cholesterol, LDL - cholesterol,
HDL – cholesterol and triglyceride)
The presence of diabetes at baseline was defined as fasting plasma glucose>110 mg/dl or use of oral hypoglycemia agents or insulin (19). A surrogate marker
for obesity content is the body mass index (BMI), which is determined by weigh(kilos), divided by the square of the height in meters. In clinical terms, a BMI of
25-29 kg/m2 is called overweight; higher BMI (30 kg/m2) are called obesity. The
waist circumference was measured on admission, midway between the last rib andiliac crest and the average of 2 measured was recorded (12).
All data is presented as the mean and standard deviation. Continuous data
analyzed using Student test for independent samples. The hazard ration and their
95 percent confidence intervals are reported. The p-value is two-sides; a p-valueof less was considered to indicate statistical significances.

RESULTS AND DISCUSSION
The study’s results are presented as tables and graphs.
Table I offers the base-line characteristics of the 55 study participants. It canbe noticed that there are no important differences between the two study groups
as far as it concerns the clinic characteristics of the patients. Also, there are no significant variations of the incidence of cardiovascular risk factors (diabetes, obesity,
dyslipidemia). The age of the patients hospitalized with non-Q wave infarction issignificantly bigger than the age of the patients with unstable angina (65+/-9.57
vs 60.86+/-10.49, pT=0.0036). In table I it is also shown a significant grow of
the fibrinogen (535±163.22 vs 646±193.23, pT=0.043), total creatine kinase(128±76.21 vs 639±680.34, pT=0.0055), troponin I (0.409±0.39 vs 35.2±40.84,
pT=0.0219), and of the leucocytes number (7.96±2.16 vs 9.71±2.31, pT=0.0119)
at the patients with non-Q wave myocardial infarction.

35.2±40.84, pT=0.0219), and of the leucocytes number (7.96±2.16 vs 9.71±2.31, pT=0.0119) at thepatients with non-Q wave myocardial infarction.
.
Table I. Base-line characteristics of the study patients
Table I. Base-line characteristics of the study patients

Characteristics
mean age
sex

women
male

body-mass index
diabetes (%)
history of
miocardial
infarction (%)
hypertension (%)
lipid variables

inflammatory
variablesCBCpatients with nonQ infarction
32.73 % (N = 18)

60.86±10.49191826.8±3.4
18.92 (7)65±9.579926.4±4.1
27.78 (5)p valueNS
0.0036NS
NS
NS
NS

16.21 (6)33.33 (6)NS

35.14 (13)66.67 (12)NScholesterol (mg/dl)
LDL- cholesterol (mg/dl)
HDL- cholesterol (mg/dl)
triglycerides (mg/dl)

230±61.48130±33.98
67±16.97
156±64.55

226± 74.28
129 ± 31.46
62±16.46
180±60.71

NS
NS
NS
NS

ESR (mm/h)
fibrinogen (mg/dl)
Total CKCK – MBTnI
RBC (10^6/mm3)
HGB (g%)
HCT (%)WBC (10^3/mm3)
PLT (10^3/mm3)22±17.64535±163.22
128±76.21
12±16.65
0.409±0.39
4.4±0.5
13.29±1.65
40.39±4.62
7.96±2.16
215.49±62.24

30±25.73646±193.23
639±680.34
27.25±24.78
35.2±40.84
4.51±0.63
13.51±1.8541.18±5.37
9.71±2.31
263.22±102.57

NS
0.043
0.0055NS
0.0219NSNS
NS
0.0119NS

Continuous numerical
datadata
werewereexpressed
as mean as± std.dev.;
not significant;Continuous
numerical
expressedmean ±NS,std.dev.;
NS, ESR,not erythrocyte
significant; ESR,
erythrocyte
sedimentation
rate; total-CK,
total
creatinekinase;I; TnI,
troponin
I; RBC,
red blood cell;
sedimentation
rate; total-CK,
total creatine
kinase;TnI, troponin
RBC, redblood cell;
HGB, hemogloHGB, hemoglobin; HCT, hematocrit; WBC, white blood cell; PLT, platelets
bin; HCT, hematocrit; WBC, white blood cell; PLT, platelets

At the patients with unstable angina (UA) and good evolution (group IB) we can see a significant
decrease GPx at the time of hospitalization toward the witness lot, but also toward the group withunstable angina and unfavorable evolution (group IA), followed by a significant grow within 48 hours,
and at the discharge the GPx values returning to normal (Fig. 1). Unlike the patients with unstable angina
and good evolution, at the patients with unstable angina who have been re-hospitalized in the following 6
months, it is noticeable a precocious grow of GPx, grow that is still available after 48 hours, the returning
to normal values being slower that in the cases of patients with favorable evolution within 6 months ofsurveillance (Table II). No significant difference is noticeable of the GPx activity within 48 hours and at
externalization between the two lots (IA and IB) of patients with unstable angina.

At the patients with unstable angina (UA) and good evolution (group IB)
we can see a significant decrease GPx at the time of hospitalization toward thewitness lot, but also toward the group with unstable angina and unfavorable
evolution (group IA), followed by a significant grow within 48 hours, and at thedischarge the GPx values returning to normal (Fig. 1). Unlike the patients with
Table II. The variations of glutathione peroxidase at the patients with unstable angina (UA)unstable angina and good evolution, at the patients with unstable angina who
6 months, it is48
noticeable a precocious
Pacientshavewith been re-hospitalized in the following
Admissionhours
Discharge
unstable angina
(T1)
(T2)
(T3)
grow of GPx, grow that is still available
after
48
hours,
thereturningtonormal
age
u/l
u/gHb
u/l
u/gHb
u/l
u/gHb
values being slower that in the cases of patients with favorable evolution within 6months of surveillance (Table II). No significant difference is noticeable of the GPx
activity within 48 hours and at externalization between the two lots (IA and IB) of
patients with unstable angina.
Table II. The variations of glutathione peroxidase at the patients with unstable angina (UA)
Table II. The variations of glutathione peroxidase at the patients with unstable angina (UA)Pacients withunstable angina
Control group
UA withcardiovascular
event
Group I A

UA without
cardiovascular
event
Group I B

age

Admission(T1)
u/l
198.444
16.964

u/gHb
1.491
0.303

averagestd.dev.
pT (group IA vs control
group)
pT (T1 vs T2 and T3)
pT (T2 vs T3)

63.70
9.44

256.900
55.551

1.956
0.412

averagestd.dev.
pT (grup IB vs control
group)
pT (T1 vs T2 and T3)
pT (T2 vs T3)
pT (IA vs IB)

59.81
10.83

averagestd.dev.

48
hours
(T2)
u/l

u/gHb

Discharge
(T3)
u/l

u/gHb

272.500
48.415

1.969
0.508

207.100
33.351

1.550
0.367

0.009

178.74145.191

0.001
0.512

1.354
0.399

274.29680.5130.047< 0.001< 0.001

0.001

0.935

0.458
0.026
0.002
1.980
0.653

197.22252.491

1.465
0.368

0.911
0.172
0.000
0.505

At the patients with myocardial infarction (MI) non-Q that were re-hospitalized
in the first 6 months after the discharge it is noticeable o quick, significant growof GPx toward the witness lot but also toward the patients with IM non-Q withgood evolution after 6 months (317.667 vs 198.44 vs 142.917, pT<0.05) (Table
III). Afterwards, the GPx values are significantly decreasing and remain decreased
until the discharge, unlike the patients with good evolution, the significance of theGPx (U/L)

300

250

200

150

100

50

0

2009.19.3(63)  Fiziologia - Physiology

patients withunstable angina
67.25 % (N = 37)

UA with cardiovascular event

UA without cardiovascular event

control group

198.44

198.44

T1

256.90178.74T2

272.50274.29T3

207.10197.22

Fig. 1 The average variations of GPx at the patients with unstable angina (UA)17

variations being statistical (T<0.05) (Fig.2)

The enzymes in the GPx family scavenge ROS in the vascular and protect
the bioavailability of NO, thereby maintaining normal endothelial function andantithrombotic vascular milieu. The cellular isoform of GPx (GPx-1) has impaired
endothelium-dependent vasodilatator function (7), and decreased levels of GPx-1
in humans have been recently associated with coronary heart disease in a dosedependent manner (11).
This prospective study of patients admitted with non-ST-segment elevation
acute coronary syndrome (unstable angina and non-Q wave myocardial infarction)
showed a direct association between baseline GPx and the onset of MACEs in thegroup with unstable angina and inversely associated with future fatal and non-fatal
cardiovascular events in the group with non-Q wave myocardial infarction.
Although the association between oxidative stress and the development ofboth endothelial dysfunction and coronary arteriosclerosis has been studied before(18,21,22,23), the prognostic role of markers of oxidative damage had comparatively
received less attention and the results so far are not clear. Indeed, in the particularcase of GPx, only 3 reports (4,10,15) have examined its association with the onset
cardiovascular events and the results (an inverse association between higher GPx
and the rate of adverse event during follow-up) were partly in disagreement withthose of our study. Admittedly, those study populations comprised mostly persons
with stable ischemic heart disease, whereas in our study all patients were admittedwith on-ST-segment elevation acute coronary syndrome. Another factor that must
be taken into consideration is that the method by which we have determined the
GPx was different from the one used in the earlier studies, which might also yield
different results.
Most prognostic studies have analyzed the onset of cardiovascular events inrelation to systemic oxidative status (1,3,5,13,15,23) whereas the role of antioxidant
status has received less attention, perhaps because its action was assumed to beprotective against cardiovascular events during follow-up. Our study show that,in the event of unstable angina, higher GPx probably reflects greater antioxidant
response due to greater oxidative status, and that this response, at least during theacute phase, is not associated with a better prognosis but with a worse one. In
opposition, at the patients with non-Q wave myocardial infarction, the decreased
level of GPx at externalization is associated with the growth of the risk of futurecardio-vascular complications (cardiovascular death, readmission with acutecoronary syndrome or the need for coronary revascularization).

GPx (U/L)
300

250

200

150

100

50

0

UA with cardiovascular event

UA without cardiovascular event

control group

198.44

198.44

T1

256.90178.74T2

272.50274.29T3

207.10197.22

Fig. 1 The average variations of GPx at the patients with unstable angina (UA)GPx (U/L)
350

300

250

200

GPx (U/L)
350

150

300

250

100

200

150

50

100

50

0

MI 0nonQ with cardiovascular event
control group

T1

T2

T2
T3

MI nonQ without cardiovascular event

MI nonQ with cardiovascular event

control group

198.44

T1
T3

MI nonQ without cardiovascular event

198.44

198.44317.67317.67142.92179.67207.17179.67180.17210.58198.44142.92207.17

Fig. 2 The average variations
180.17 of GPx at the patients with non-Q wave
210.58myocardial infarction

At the patients with myocardial infarction (MI) non-Q that were re-hospitalized in the first 6
months after the discharge it is noticeable o quick, significant grow of GPx toward the witness lot but also
2 The withaverageof GPxthe patientswith6 months
non-Q wave
myocardial
infarction
toward Fig.
the patients
IM variations
non-Q withgoodatevolution
after(317.667
vs 198.44
vs 142.917,
pT<0.05) (Table III). Afterwards, the GPx values are significantly decreasing and remain decreased until
the discharge, unlike the patients with good evolution, the significance of the variations being statistical(T<0.05) (Fig.2) Table III. The variations of glutathione peroxidase at the patientswith non-Q wave myocardial infarction

Table III. The variations of glutathione peroxidase at the patients with non-Q wave myocardial infarction
Pacients with
MI nonQ
Control group
MI nonQ withcardiovascular
event
Group II A

MI nonQ
without
cardiovascular
event
Group II B

48
hours
(T2)
u/l

u/gHb

Discharge
(T3)
u/l

u/gHb

179.667
11.776

1.539
0.438

180.167
35.114

1.430
0.449

age

Admission(T1)
u/l
198.444
16.964

u/gHb
1.491
0.303

averagestd.dev.
pT (grup IIA vscontrol group)pT (T1 vs T2 andT3)
pT (T2 vs T3)

67.00
7.18

317.667
89.415

2.398
0.595

0.023

0.062

0.322

0.009

0.006
0.937

averagestd.dev.
pT (grup IIB vscontrol group)pT (T1 vs T2 andT3)
pT (T2 vs T3)
pT (IIA vs IIB)

64.00
10.73

averagestd.dev.

142.91737.049
0.000

0.004

1.066
0.296

207.167
33.701

1.484
0.233

210.58326.294

0.736

0.666

0.003

0.030
0.824
0.049

0.023

1.571
0.347

The enzymes in the GPx family scavenge ROS in the vascular and protect the bioavailability ofNO, thereby maintaining normal endothelial function and antithrombotic vascular milieu. The cellularisoform of GPx (GPx-1) has impaired endothelium-dependent vasodilatator function (7), and decreased

18

CONCLUSIONS• The coronary acute syndromes are associated with the alternation of thebalance between the oxidant and anti-oxidant systems.
• GPx is an essential component of the enzymatic system of anti-oxidant
defense of the body.
• At the patients with non-Q wave myocardial infarction who have beenreadmission, the GPx values have precocious increased (at 24 hours from hospitalization). Afterwards, the values have decreased, remaining under the values of thecontrol group also at externalization.
• At the patients with unstable angina with unfavorable evolution, after 24hours from hospitalization, the GPx values were bigger that at the patients withunstable angina with good evolution.
• Taking into consideration the anti-oxidant mechanism’s dynamic related
to the evolution of the patients with non-ST-segment elevation acute coronary
syndrome, the anti-oxidant therapy could be a good method to improve theprognostic (25)

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status be considered in interventional trials with antioxidants?
Heart, 2004;90(6):598–602.

ACTIVITATEA GLUTATION PEROXIDAZEI AFECTEAZAPROGNOSTICUL IN SINDROMUL CORONARIAN ACUT FARASUPRADENIVELARE DE SEGMENT ST
REZUMAT

Enzimele celulare antioxidante, ca glutation peroxidaza detin un rol central in controlul speciilor reactive de oxigen. In vitro, datele si studiile efectuate pe animale
sugereaza ca aceste enzime au rol protector impotriva aterosclerozei, dar sunt putine date relevante la om. Scopul studiului a fost determinarea asocierii dintre
statusul redox si prognosticul pacientilor cu sindrom coronarian acut fara supradenivelare de segment ST (angina instabila si infarct miocardic fara unda Q). Metoda:
Au fost evaluati 55 de pacienti, cu varsta sub 75 ani internati consecutiv in Clinica de Cardiologie a Spitalului Clinic de Urgenta Constanta in perioada mai 2008 – mai2009 cu diagnosticul de sindrom coronarian acut fara supradenivelare de segment ST si 19 persoane voluntare (fara afectiuni cardiovasculare, hipercolesterolemie,
diabet zaharat si nefumatoare). Pacientii au fost impartiti in doua grupuri: grup I – pacientii cu angina instabila (37 pacienti, din care 10 au necesitat reinternare inurmatoarele 6 luni) si grup II – pacientii cu infarct miocardic acut fara unda Q (18 pacienti, din care 6 pacienti au suferit reinternare in primele 6 luni post-infract).
Activitatea glutation peroxidazei (GPx) a fost determinata la perioade fixe: T1 – primele 24 de ore de la internare, T2 – la 48 ore de la internare si T3 – la externare.
Dupa externare pacientii au fost monitorizati si s-au notat urmatoarele date: perioada de urmarire, decesul de cauza cardiovasculara si incidenta evenimentelor
cardiovasculare majore. Rezultate: Studiul demonstreaza o asociere directa intre nivelul GPx si incidenta evenimentelor coronariene majore in grupul cu angina
instabila si o asociere inversa cu evenimentele cardiovasculare fatale sau non-fatale in grupul pacientilor diagnosticati cu infarct miocardic fara unda Q. Concluzii:
La pacientii cu sindrom coronarian acut fara supradenivelare de segment ST enzima antioxidanta GPx pare a avea un rol protector impotriva efectelor oxidative
adverse. Analiza activitatii GPx poate oferi informatii superioare in evaluarea riscului cardiovascular comparativ cu masurarea doar a factorilor de risc traditionali.
Cuvinte cheie: sindrom coronarian acut, markeri biologici/sange, glutation peroxidaza, oxidare-reducere, stress oxidativ
2009.19.3(63)  Fiziologia - Physiology

19THE INVOLVEMENT OF OXIDATIVE STRESS IN THE
ULCEROUS PATHOLOGY INDUCED IN THE RATS BY ASPIRINSIMONA MARTURA1, DOINA DAICOVICIU2, SOIMITA SUCIU2, ADRIANA MURESAN2, DOINITACRISAN3, PETRU A. MIRCEA1, SIMONA VALEAN1, OANA DAMIAN4
1
Medical Clinic I
2
Physiology Department
3
Pathological Anatomy Department
University of Medicine and Pharmacy "Iuliu Hatieganu" Cluj-Napoca, Romania
4"Heinrich Braun Krankenhaus" Zwickau, Germany

ABSTRACT
Various studies show that reactive oxygen species (ROS) are involved in ulcerous disease. This has multiple etiopathogenetic factors, among which the administration of NSAIDs plays an important role. The digestive lesions may be prevented by the association of proton pump inhibitors (PPI), and there are also
data that indicate the advantages of the therapeutic supplementation of potentially antioxidant potions.The aim of the study was to evidence the growthof the oxidative stress level due to the administration of aspirin, and its decrease following the association of the antiulcerous protection, the antioxidant.
There are considerable variations of the oxidant-antioxidant balance among the study groups. The aspirin treatment alters considerably the serum oxidative
stress parameters and the gastric tissue in favor of prooxidants. The preventive administration of PPI considerably improves the status of serum antioxidants.
PPI therapy decreases the level of tissue antioxidants (probably due to excessive consumption), but pathomorphologically improves the gastric mucous
membrane. The association with antioxidants decreases the level of reactive oxygen species (ROS) and improves the anatomo-pathological aspect of thegastric mucous membrane.
Key words: oxidative stress, NSAIDs, peptic ulcer

INTRODUCTION
Peptic ulcer and gastritis have multiple etiopathogenetic factors, and one of
the major factors of aggression is the formation of a reactive oxygen species (ROS).
The involvement of free radicals derived from oxygen, such as the superoxide anion,hydrogen peroxide and hydroxyl radical, is demonstrated in the pathogenesis of theischemic lesions of the gastro-intestinal mucous membrane, as well as in othertypes of its lesions that are induced by NSAIDs, ethanol, food diet and Helicobacter
pylori (23). The reactive oxygen species generated by the metabolism of arachidonic
acid, blood platelets, macrophages and cells of smooth brawn may contributeto the formation of gastric mucous membrane lesions. Therefore, through thescavenger action against free radicals, the reactive oxygen metabolites may be
useful by protecting the gastric mucous membrane from oxidative lesions or by
the acceleration of the healing of gastric ulcers (19).Gastric ulcerations associatedwith NSAIDs appear in 30% of the patients which require to be hospitalized and areassociated with a high mortality rate (13). NSAIDs are frequently prescribed due totheir efficiency in fighting pain, inflammation and fever. Their use is associated withthe appearance of some digestive side effects, such as erosions of the gastric mucous
membrane, ulcerations, bleeding, perforation, as well as the high risk of hemorrhage
from the preexisting peptic ulcers. The pathogenesis of gastrointestinal lesions
induced by NSAIDs may depend on the independent processes of prostaglandins,such as oxidative phosphorylation, the decrease of the proliferation of the mucous
membrane cells and the activation of the neutrophils, followed by the increase ofendothelial adhesion. Finally, they produce the decrease of microvascularizationand the hyperproduction of reactive oxygen metabolites, able to induce tissue

oxidative lesions which probably play an important role in the pathophysiology ofdigestive ulcerations induced by NSAIDs (5,10). NSAIDs inhibit the cyclooxigenase
isoforms and decrease the level of prostaglandin E2 in the gastric mucous membrane,
causing ulceration, and it delays the cure of ulcer by the reduction of the level of
prostaglandins and the prevention of angiogenesis mediated by these. In addition to this, NSAIDs decrease the gastric production of mucus, which may lead tohemorrhagic ulcer. So, the drugs that prevent the progression of ulcer by antioxidant
action and also promote the secretion of PGE2, gastric mucus and increase factors,
may accelerate the cure of gastric ulcer (4). The proton pump inhibitors (PPI) arecurrently used in the treatment and prevention of gastroduodenal lesions induced
by NSAIDs, their efficiency being related to the inhibition of gastric acid secretion.
The gastric protection provided by pantoprazol is also given by its ability to interferewith NSAIDs secondary oxidative and inflammatory lesions. Pantoprazol acts withthe help of direct and indirect antioxidant mechanisms, protects the gastric mucous
membrane against the oxidative injuries caused by focal ischemia and the activationof neutrophils induced by NSAIDs (10). Vitamin C is a hydrosoluble antioxidant thateliminates the reactive oxygen species (ROS), among which: superoxide radical,hydroxyl radical, hydrogen peroxide, singlet oxygen and hypochlorous acid; it plays
the role of antioxidant of vitamin E, by reducing the radical of vitamin E at the level ofliquid / water. It prevents the burn of neutrophils in the endothelium by its scavenger
action on the derived ROS at the level of the activated neutrophils. Vitamin E is aliposoluble antioxidant and plays the role of destroying the bonds in the peroxidation
process of the cell membrane lipids. At the same time, it is a scavanger of ROS, suchas superoxide radical, hydroxyl radical and singlet oxygen. Vitamin E exerts an anti-

Received March 2009. Accepted June 2009. Address for correspondence: Dr. Simona Martura, Medical Clinic I, University of Medicine and Pharmacy "Iuliu Hatieganu" Cluj-Napoca, Romania

20

Fiziologia - Physiology  2009.19.3(63)inflammatory action by inhibiting the O2 production - in the activated neutrophils,
the adhesion of neutrophils to endothelial cells and the transendothelial migrationof neutrophils (17). The action of vitamin E is enhanced by selenium and protects
other liposoluble antioxidants; it protects especially the lipids of the membranes(11). Selenium is part of the composition of a primordial enzyme that fights ROS(glutathione peroxidase- through selenomethionine and selenocysteine). Vitamin
A presents precursors of vegetal origin (carotenoids) which may be used as verystrong antiradicals; the most common are beta-carotene, lycopene and lutein (9).
It is well known that in the case of chronic peptic ulcers and gastroduodenitis, theroutine of applying the neutralizing or protective therapy, or agents that decrease
gastric secretion, not only produces the reduction of the symptoms and the remissionof the inflammatory changes, but also has an inhibitory effect on the engenderingof free oxygen radicals. The results of the researches of other authors emphasize
the favorable effect of vitamin C supplementation in patients with chronic recurrent
gastroduodenitis, and a decrease in the risk of development of gastric cancer isalso suggested for these patients. Even though the benefits of antioxidant vitaminsupplementation are demonstrated in the treatment of some gastric diseases (forexample for vitamin C), there are still not sufficient studies available to formulate
specific therapeutic recommendations (3).

MATERIAL AND METHODSThe experimental study that we performed was one of prospective longitudinaltype, and the collection of the data was the exposed – non-exposed type.
The major objective of this study was to monitor the variation of oxidative stressparameters as a result of aspirin administration; in parallel, the secondary objectives
were to monitor the changes in the general state and the pathomorphological stateat the level of gastric tissue.
The purpose of this study was to demonstrate the increase of oxidative stress(OS) after the administration of aspirin and the decrease of OS as a result of theanti-ulcer protection, antioxidant therapy, respectively.

Conditions
The researches were conducted on white male Wistar rats, aged approximatively 10 weeks old, with a weight between 170 – 250 g. The animals were kept in
the biobase of the Physiology Department of ”Iuliu Haţieganu”UMPh Cluj-Napoca,
under standard laboratory conditions, at a temperature of 23 +/- 0C, with a durationof 12 hours of exposure to light and 12 hours exposure to darkness, with water andfood ad libitum. The animals had a period of 1 week to adapt themselves before
the experiment started.

Groups4 groups of 10 male Wistar rats, aged approximatively 10 weeks old, with aweight between 170 – 250 g, were included in the study.
- group I (control) –received distilled water by gavage for 7 days;- group II –received by daily gavage aspirin suspended in carboxymethylcellulose, in a dose of 200 mg/Kg body weight for 7 days;- group III –received by daily gavage aspirin suspended in carboxymethylcellulose, in a body dose associated with PPI (pantoprazol in a dose of 60 μmol/Kg
body weight) for the 7 days (4)- group IV –received by daily gavage aspirin suspended in carboxymethylcellulose, in a dose of 200 mg/Kg body weight associated with PPI (pantoprazol in adose of 60 μmol/Kg bodyweight) and a prepared antioxidant, each capsule of which2009.19.3(63)  Fiziologia - Physiology

contained: selenium 50 μgrams, provitamin A (beta-carotene) 10 mg, vitamin C(ascorbic acid) 100 mg and vitamin E (DL-tocopherol acetate) 40 mg (11).
After the last dose received by gavage, the animals were not given food for 24hours before they were sacrificed. The animals included in the study were individuallyweighed both at the start and at the end of the experiment.

Biological samplingBlood samples were taken from the internal angle level of the eye – the venous
orbital sinus – with the help of a capillary tube (approximately 1 ml/experimental
animal). The blood was immediately centrifuged for 5 minutes at 3500 rotations/min. After the plasma was obtained, it was immediately frozen and kept at atemperature of -800 C until it was processed. After blood sampling, the animals
were euthanized by cervical dislocation, the necropsy was performed. The abdomen
was cut open along the white line, the peritoneal cavity was examined and thestomach was exposed. The stomach was harvested in isotonic solution of KCI, on
ice, and subsequently it was cut along the great curvature so that the macroscopic
examination could be done. During the evaluation, the samples were kept on ice.
After the completion of the process of counting the lesions, parts of the gastricperiulcerous zone were excised and frozen for the histopathological examinationand in order to determine the oxidative stress markers. The samples were kept at atemperature of -800 C until they were processed.
The macroscopic examination of the gastric lesions
The lesions were categorized as follows:- score 0 – no ulcerations or up to 3 punctiform ulcerations- score 1 – more than 3 punctiform ulcerations- score 2 – between 1 and 5 small-size ulcers (<2 mm)- score 3 – more than 5 small ulcers with a diameter smaller than 2 mm
- score 4 – one or more gigantic ulcers, with a diameter of more than 2 mm (12,13).
The indicators which were determined from the blood and from the gastrictissue were:- prooxidants: malondialdehyde (MDA) and the carbonilated proteins (CP)- antioxidants: the hydrogen donor capacity (HD) and the thiol group(sulfhydryl) (SH)

Methods to determine the oxidative
stress indicators
Malondialdehyde (MDA) was determined through the method of fluorescein
dosage, according to Conti. The concentration values were expressed in nmol/ml,
for the serum and in nmol/mg protein, for the gastric tissue (14).The carbonylated
proteins (CP) were dosed using Rezsnick’s method. The results were expressed in
nmol/ml, for the serum and in nmol/mg protein, for the gastric tissue (15).The
determination of the hydrogen donor capacity (HD) was made using a dosage
method elaborated by Janaszewska. The results were expressed both in the serum
and in the gastric tissue, as the inhibition percentage of the free radical (i%) (16).
The determination of the content of the total thiol group (sulfhydryl) (SH) was madeaccording to the dosage method of Hu. The values were expressed in μmol/ml in
the serum and in nmol/mg protein, for the gastric tissue (17).
The statistical study of results
It was performed using the SPSS 13 program. For the detection of the significance of the statistical difference from the average, the „t” Student test (for two
21

groups) and ANOVA (for three or more groups) were used. Two aspects were studied
in the „t” Student test: the descriptive analysis of the data and the analysis of thesignificance of the difference between the average values; a 95% confidence interval
was used; the results in which p < 0.05 were considered to be eloquent.

RESULTS
Evolution of the oxidative stress markers
Serum MDA had the following variations: an insignificant increase of the values
in group II compared to the control group; an insignificant decrease in group II
compared to the first two groups; a significant decrease of the values of group IV
compared to those of the control group and group II, with an insignificant variationcompared to group III (Fig. 1).No significant variations in serum CP values weredetected (Fig. 2).Serum HD presented the following alterations: they decreased
significantly in group II compared to the control group; they increased significantly
in group III compared to group II; otherwise, the variations of the values between
the groups were insignificant (Fig. 3). For the serum thiol groups, the following were
noticed: they decreased significantly in group IV compared to group II; they increased
significantly compared to group III; they did not vary significantly compared to thecontrol group (Fig. 4). Malondialdehyde at gastric tissue level presented: a significant
decrease in group II compared to the control group; a significant decrease in group
IV compared to the first two groups, with insignificant variations compared to thecontrol group (Fig.5). The levels of carbonylated proteins (CP) in the gastric tissue
did not undergo significant alterations between the rat groups under observation(Fig. 6). The level of hydrogen donors (HD) in the gastric tissue presented differences
between the groups: they decreased significantly in group II compared to the control
and in group IV compared to group II, the rest of the variations between the groups
were insignificant (Fig. 7).Except one insignificant difference between groups II and
III, the thiol groups (SH) varied as follows: they decreased significantly in the last
three groups compared to the control group, meaning that they were situated at alower level in group IV compared to the rest of the groups (Fig.8).

Fig. 3. Variation of serum hydrogen donor capacity

Fig. 4. Variation of serum sulfhydryl groups

Fig. 5. Variation of malondialdehyde in the gastric tissue

Fig. 1. Variation of serum malondialdehyde

Fig. 6. Variation of carbonylated proteins in the gastric tissue

Fig. 2.Variation of serum carbonylated proteins

22

Evolution
No significant changes were identified in the general state or in the evolutionof individual weight. Mortality was less than 5%, and was most probably caused
by individual sensitivity.

Fiziologia - Physiology  2009.19.3(63)abundant in the areas of discontinuity of the mucous membrane, deep chorion and submuco
the muscularis of the mucous membrane). Group III presented an undamaged surface epit
chorion contained a mixed inflammatory lymphoplasmacytic and neutrophilic infi
inflammatory cells were more abundant in the deep region of the mucous membrane and at
the muscularis of the mucous membrane.Group IV also presented an undamaged surface epith
no erosions or ulcerations, but it had a reduced inflammatory lymphoplasmacytic infiltrate, lo
level of the deep chorion. Some lymphocytes in the muscularis of the mucous membran
observed, but with absent polymorphonuclear neutrophils (Fig.9).

Fig. 7. Variation of the hydrogen donor capacity in the gastric tissue

Fig. 9. Histopathological appearance of the gastric mucosa in the studied groups

Fig. 9. Histopathologicalappearance of CP
the gastric mucosa
in the studied
MDAHD
SH groups
serum of thetissuetissueserumbalancetissueserumgroups tissue
Table I. The alterations
indicatorsserumof the oxidant/antioxidantin the studied
Average

1.350

MDA

0.167

serum
tissueStandard
MDA
Average0.2361.350 0.071
0.167
deviation
serum
tissueGroup I
AverageStandard1.3800.236 0.101
0.071
deviation
Average
1.350
0.167Group II Group
Standard
I Average
0.101Standard0.3521.380 0.037
0.236
0.071
deviationGroup
II Standard
deviation
0.352
0.037
Average1.0351.380 0.062
0.101
Averagedeviation
Group Group II Average
1.035
0.062
Standard
0.352
0.037
Group
III
Standarddeviation
0.023
III
Standard0.658
deviation
0.658
0.023
Average
1.035
0.062
deviation
Group
Average
III
Standard0.0200.020 0.180
Average
0.180
0.658
0.023
Group Groupdeviation
IV
IVStandard
Standard
0.059
Average0.4420.442
0.020 0.059
0.180
deviation
deviation
Group

Group I

Fig. 8. Variation of sulfhydryl groups in the gastric tissue

0.921

serum

CP

0.970

tissueHD

26.090SH

23.1300.175

CP 0.450
0.445
0.921
0.970
serum
tissue
0.926
0.850
0.445
0.450
0.921
0.970

serum
tissueserum
tissueHDSH
9.182
26.09023.1306.264
0.175 0.227
0.242
serum
tissueserum
tissue16.4106.26424.830
9.182
0.227 0.221
0.081
26.090 23.130
0.175
0.242

0.445
0.372

9.182
7.114

0.242
0.081
0.099

Table I. The alterations of the indicators of0.372
thebalancein the0.218
studied groups
0.926 oxidant/antioxidant
0.850
0.221 0.172
0.099
0.392 16.410
7.11424.8304.523
0.450
0.392

6.264
4.523

0.227
0.172

0.081
0.218

0.926
0.850
24.8309.790
0.221 0.156
0.099
0.860
1.037 16.410
24.3700.105
The macroscopic examination of the gastric lesions
0.860
1.037
24.370
9.790
0.156
0.105
0.372
0.392
7.114
4.523
0.172
0.218
The macroscopic examination of the gastric mucous membrane, after the stomach
0.157
0.420
6.468
3.593
0.033
0.051
0.157
0.420
6.468
3.593
0.033
0.051
0.860
1.037
24.370
9.790
0.156
0.105
was sectioned along the great curvature, showed that the animals in the control group
0.910
1.005 16.220
16.22018.530
18.530
0.065
0.910
1.005
0.196 0.196
0.065
0.157
0.420
6.468
3.593
0.033
0.051
did not present any pathological alterations.The second group showed lesions of the
0.321
0.305
6.623
4.334
0.025
0.262
0.910
1.005
16.22018.530
0.196
0.065
0.321
0.305
6.623
4.334
0.025
0.262
gastric mucous membrane categorized according to the score presented before: 40%
IV
Standard
0.442
0.059
0.321
0.305
6.623
4.334
0.025
0.262
Table
II.
The
statisticalsignificance
(p)
of
the
differenceintheindicatorsoftheoxidant/antioxidantbalancebetweendifferent
deviation
Table
II.
The
statisticalsignificance
(p)
of
the
differenceintheindicatorsoftheoxidant/antioxiscore 1, 60% score 4.The third group presented lesions with a smaller degree of sever-Table II. The statistical significance (p) of the difference in theindicators of the oxidant/antioxidant balance between different
groupsdant balance between
differentgroupsTable II. The statistical significance (p) of the difference ingroups
theOxidative
indicatorsstressof theindicatorsoxidant/antioxidant balance between different
ity compared to group II, 40% presented score 1 lesions, and 60% presented score 0groupsOxidative stress indicators
Comparison between
lesions.The forth group presented score 0 lesions of the mucous membrane , meaning
Oxidative stress indicators
MDA
CP
DHD
SH
the groups:
betweenthat the mucous membrane was affected comparably to the control group and less Comparison
Comparison between
MDAMDA
CP
DHD
SH
serum
tissueserum CP tissue
serumDHD tissue
serumSH tissue
the groups:
the groups:
severely compared to the animals of group II, group III, respectively (Fig. 2).
0.832
serum
tissueserum
tissueserum
tissueserum
tissueHistopathological examination
No pathological changes were found at the level of the gastric mucous membraneof the control group. The gastric mucous membrane of the rats of group II presented:
focal erosions and profound areas of ulcerations, superficial chorion with small hemorrhagic foci, an inflammatory neutrophilic infiltrate more abundant in the areas of
discontinuity of the mucous membrane, deep chorion and submucosa (close to themuscularis of the mucous membrane). Group III presented an undamaged surface
epithelium; the chorion contained a mixed inflammatory lymphoplasmacytic and neutrophilic infiltrate. The inflammatory cells were more abundant in the deep region of themucous membrane and at the level of the muscularis of the mucous membrane.GroupIV also presented an undamaged surface epithelium, with no erosions or ulcerations,but it had a reduced inflammatory lymphoplasmacytic infiltrate, located at the levelof the deep chorion. Some lymphocytes in the muscularis of the mucous membrane
could be observed, but with absent polymorphonuclear neutrophils (Fig.9).

I-II
I-III
I-IV
II-III

I-II

serum

I-II
I-III

0.8320.080

I-III
I-IV

0.080
0.020

I-IV
II-IIIII-III
II-IV
II-IV
III-IV

0.832

tissue
0.010

serum
0.974

tissue0.510

serum 0.570 tissue 3.10
0.002

0.010
0.0003

0.974
0.680

0.510
0.720

0.002
0.630

0.570
0.00002

3.10
0.120

0.680
0.950

0.720
0.830

0.630
0.320

0.00002
0.07

0.120
0.065

0.950
0.572

0.830
0.350

0.320
0.009

0.07
3.140

0.065
4.35

0.010

0.0003
0.660

0.974

0.080

0.0003

0.680

0.020

0.660

0.950

0.020
0.088

0.088
0.030

0.660
0.040

0.040
0.002

0.0880.960
0.030 0.040
0.002
0.0004

0.510
0.720

0.830

0.002
0.630

0.320

0.570

serum
0.00070.00070.00023.10

0.00020.000

tissue0.00070.000020.120

0.0002

0.07

0.065

0.000

4.35
0.040
0.057

0.777

0.572
0.920

0.350
0.340

0.009
0.950

3.140
0.004

4.35
0.010

0.572
0.920
0.724

0.350
0.340
0.870

0.009 0.004
3.1400.010
0.950
0.004
0.001
0.040

0.000
0.777
0.777
0.040

III-IV
0.0004
0.724
0.870
0.057
0.030 0.960 0.002
0.920
0.340 0.004
0.950 0.001 0.0040.040 0.010
0.040
Table III. Distribution and categorization of the gastric lesions observed by macroscopic examination
Group IGroup IIGroup III
Group IV
Table
III.
Distribution
andcategorizationofthegastric
lesions
observedbymacroscopic
examination
Score
100% 0.724 60%0.004
100%
III-IV
0.96000.00040.870 Group
0.001
0.040
0.057
Group
Group
Group- IV
Score 1- I
40% II
40%III
Score 20
100%
-60%
100%
Score
Score 3140%
40%
-Score-Score 42- the gastric lesions-Table III. Distribution
and categorizationofobserved by- -macroscopic examination
Score
-60%
Score 3- II
- Group IV
Group-I
Group
Group
III
Score 4
60%
-

II-IV

Table III. Distribution and categorization of the gastric lesions observed
by macroscopic examination

Score 0
100%
60%
100%
Score 140%
40%
DISCUSSIONS
ThereScore
werein the- oxidant-antioxidantbalance between
the study
2 significant differences
DISCUSSIONS
groups.The
results
the researches
years suggest -that freeoxygenradicals
coScore3 ofsignificant- over the- the are
There
were
differencesin pastthe- oxidant-antioxidantbalancebetween
study
responsible,
inadditiontotheetiologic
factor,forthelesionsofthemucousmembraneofthedigestive- over the past
60%years suggest thatgroups.The Score
results4 of the researches
free oxygen radicals
are cotract
and
for
the
initiationoftheinflammatory
process.
The
inflammation,
which
is
a
defensereaction
responsible, in addition to the etiologic factor, for the lesions of the mucous membrane of the digestivefromat the sametime
a cause of process.
tissue damage.
During the which
respiratory
explosionof thetract theand body,
for theisinitiationof theinflammatory
The inflammation,
is a defense
reaction
phagocytesof thealmost
3.2 million
radicals,
and also
3.6
from the body,is atinflammatory
the same timecells,
a causeof tissue
damage.superoxide
During theanion
respiratory
explosionof theDISCUSSIONS
phagocytes of the inflammatory cells, almost 3.2 million superoxide anion radicals, and also 3.6
There were significant differences in the oxidant-antioxidant balance between the study
groups.The results of the researches over the past years suggest that free oxygen radicals are coresponsible, in addition to the etiologic factor, for the lesions of the mucous membrane of the digestive
tract and for the initiation of the inflammatory process. The inflammation, which is a defense reaction
from the body, is at the same time a cause of tissue damage. During the respiratory explosion of thephagocytes of the inflammatory cells, almost 3.2 million superoxide anion radicals, and also 3.6

2009.19.3(63)  Fiziologia - Physiology

23

DISCUSSIONSREFERENCES

There were significant differences in the oxidant-antioxidant balance between
the study groups.The results of the researches over the past years suggest that freeoxygen radicals are co-responsible, in addition to the etiologic factor, for the lesionsof the mucous membrane of the digestive tract and for the initiation of the inflammatory process. The inflammation, which is a defense reaction from the body, isat the same time a cause of tissue damage. During the respiratory explosion of thephagocytes of the inflammatory cells, almost 3.2 million superoxide anion radicals,
and also 3.6 millionperoxide hydrogen molecules are generated in one second. The
stimulated phagocytes represent one of the main sources of free oxygen radicals
in the body (3).Our study has demonstrated that the administration of aspirin
significantly alters serum and tissue oxidative stress parameters (meaning that it
alters the oxidant-antioxidant balance in favor of the first). The rats that were given
aspirin alone presented the appearance of necrotic areas of the mucous membrane,
associated with extensive polymorphonuclear cell infiltrate. The results of our research are correlated with evidence showing that NSAIDs, acting through local andsystemic mechanisms, lead to ischemic and inflammatory changes which result ingastric neutrophil infiltration, the release of oxygen metabolites and the peroxidation of the cellular mucous membranes. In particular, focal ischemia produced by
the adhesion of neutrophils to the vascular endothelium, the capillary connection
and the intravascular deposit of fibrin were recognized as an early event in thepathogenesis of gastric oxidative injuries produced by NSAIDs. These alterations
are further amplified by subsequent tissue infiltrations of polymorphonuclear cells,
activation due to which a massive release of oxygen radicals and other inflammatory mediators responsible for epithelial lesions is produced (10). The preventive
administration of PPI has significantly improved the serum antioxidant status. There
are studies that prove that they alone have antioxidant properties (2,7,10,21). Locally, the administration of PPI partially prevents gastric lesions or, at least, reduces
their severity. In parallel, a reduction of tissue antioxidants was noticed, probably
due to excessive consumption which contributes to local defense. The addition ofantioxidants further decreases the oxidative excess and improves the macroscopic
and anatomo-pathological aspect of the gastric mucous membrane. These aspects
are to be found in other studies supporting that the addition of vitamin E to dietis able to protect the cellular mucous membrane against the alterations producedin the case of ischemia-reperfusion lesions induced in rats, by the inhibition oflipid peroxidation and the interference with neutrophilic infiltrates (16). Anotherexperimental study of gastric lesions produced by ethanol or HCl showed thatvitamin A and beta-carotene are able to significantly prevent the number and theseverity of the lesions of the gastric mucous membrane, but these agents fail tocompensate the decrease of the SOD activity of the mucous membrane diminishedby the administration of HCl (15). We obtained the same findings regarding the
local antioxidant capacity in the study above. Another experimental study demonstrated that selenium and vitamin E treatment in rats with stress induced ulcers
significantly reduced the acid base gastric secretion, their combination producing abetter inhibition of gastric acid secretion compared to their individual effects (1). In
conclusion, we may state that the parallel administration of drugs that decrease acidgastric secretion (PPI) significantly reduces the risk and size of lesions produced byaspirin administration. At the same time, the association in treatment of productswith an antioxidant potential, further improves the defense of the gastric mucous
membrane through the growth of the antioxidant status, and results in an obvious
decrease of tissue lesions.

1. Al-Moutairy AR, Tariq M. Effect of vitamin E and selenium on
hypothermic restraint stress and chemically-induced ulcers. Dig
Dis Sci. 1996 Jun; 41(6):1165-71.
2. Augusto AC, Miguel F, Mendonca S, et al. Oxidative stress expression status associated to Helicobacter pylori virulence in gastricdiseases. Clinical Biochemistry 2007 (40);615-622.
3. Bala G, Czerwionka-Szaflarska M, Drewa G, et al. An evaluationof the impact of supplementation with antioxidants vitamins onoxidation stress parameters in children with chronic recurrent
gastroduidenitis. Med Sci Monit 2002; 8(1):14-18.
4. Banerjee D, Maity B, Nag SK, et al. Healing Potential of Picrorhiza
kurroa (Scrofulariaceae) rhizomes against indomethacin-induced
gastric ulceration: a mechanistic exploration. BMC Complementaryand Alternative Medicine 2008; 8(3):1-14.
5. Blandizzi C, Fornai M, Colucci R, et al. Lansoprazole prevents
experimental gastric injury induced by non-steroidal anti-inflammatory drugs through a reduction of mucosal oxidative damage.
World J. Gastroenterol 2005; 11(26): 4052-60.
6. Cioli V, Silvestrini B, Dordoni F. Evaluation of the potential of gastric ulceration after administration of certain drogs. Experimental
and molecular pathology 1967;6:68-83.
7. Conti M, Morand PC, Levillain P et al. Improved Fluorometric
Determination of Malonaldehyde. Clin. Chem. 1991;37(7):12731275.
8. Demir S, Yilmaz M, Koseoglu M, et al: Role of free radicals in peptic
ulcer and gastritis. Turk J. Gastroenterol. 2003;14(1):39-43.
9. Favier A. Le stress oxidant- Interet conceptual et experimental
dans la comprehension des mecanismes des maladies et potential therapeutique. http://www.maomusique.com/uploaded/ Joylulu/
Favier.pdf
10. Fornai M, Natale G, Colluci R et al. Mechanisms of protection
by pantoprazole against NSAID- induced gastric mucosal damage.
Naunyn-Schimiedeberg”s Arch Pharmacol 2005;372(1):79-87.
11. Hauret MC. Les antioxydants http://www.chateaudavanton.
com/blog/index.php?2007/02/16/17-les-antioxydants
12. Hu ML. Methods in Enzymology, 1994;233:380-384.
13. Jainu M, Vijai Mohan K, Shyamala Devi CS: Gastroprotective
effect of Cissus quadrangularis extract in rats with experimentallyinduced ulcer. Indian J. Med. Res. 2006;(123):799-806.
14. Janaszewska A, Bartosz G: Assay of total antioxidant capacity:
comparison of four methods as applied to human blood plasma.
Scand. J. Clin. Invest.2002;62:231-236.
15. Mozsik G, Javor T, Toth G, et al. Interrelationships between
the gastric cytoprotective effects of vitamin a and beta-carotene
and the gastric mucosal superoxid dismutase activity in rats. Acta
Physiol Hung.1984;64 (3-4):315-8.
16. Naito Y, Yoshikawa T, Matsuyama K et al. Effect of vitamin E ingastric mucosal injury induced by ischaemia-reperfusion in nitricoxide-depleted rats. Aliment Pharmacol Ther 1999;13:553-559.
17. Ohta Y, Kobayashi T, Imai Y, et al. Effect of Oral Vitamin E Administration on Acute Gastric Mucosal Lesion Progression in Rats
Treated with Compound 48/80, a Mast Cell Degranulator. Biol.
Pharm. Bull.2006;29(4):675-683.
18. Passoni CR, Coelho CA. Ascorbic acid supplementation has acytoprotective effect on secondary billiary cirrhosis: experimental
study in young rats. J Pediatr (Rio J). 2008;84(6):522-8.
19. Repetto MG, Llesuy SF. Antioxidant properties of naturalcompounds used in popular medicine for gastric ulcers. Brazilian
J Medical & Biological Research 2002;35:523-534.
20. Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol. 1994;
233:347-357.
21. Suzuki M, Suzuki H and Hibi T. Proton Pump Inhibitors andGastritis. J. Clin. Biochem. Nutr.2008;42:71-75.
22. Tajuddin, Ahmad S, Latif A, et al. Effect of 50% ethanolic extractof Syzygium aromaticum (L.) Merr. &Perry. (clove) on sexual behaviour of normal male rats. BMC Complement Altern Med. 2003;3:6

24

Fiziologia - Physiology  2009.19.3(63)IMPLICAREA STRESULUI OXIDATIV IN PATOLOGIA ULCEROASA
INDUSA DE ASPIRINA LA SOBOLANIREZUMAT

Numeroase studii arata faptul ca speciile reactive ale oxigenului (SRO) sunt implicate in boala ulceroasa. Aceasta recunoaste factori etiopatogenetici multipli,
intre care un loc important il ocupa administrarea de AINS. Leziunile digestive pot fi prevenite prin asocierea inhibitorilor pompei de protoni (IPP), existand sidate care indica avantajele suplimentarii terapeutice aditionale cu preparate cu potential antioxidant. Scopul studiului a fost de a demonstra cresterea niveluluistresului oxidativ consecutiv administrarii de aspirina si, respectiv, scaderea acestuia secundar asocierii terapiei de protectie antiulceroasa, respectiv antioxidanta. Am constatat ca intre loturile din studiu exista variatii semnificative ale balantei oxidanti-antioxidanti. Tratamentul cu aspirina modifica semnificativ
parametrii de stres oxidativ serici si din tesutul gastric in favoarea prooxidantilor. Administrarea preventiva de IPP imbunatateste semnificativ statusul seric
antioxidant. Terapia cu IPP scade nivelul de antioxidanti tisulari (probabil datorita consumului in exces), insa imbunatateste morfopatologic tesutul gastric.
Asocierea de antioxidanti scade nivelul speciilor reactive ale oxigenului (ROS) si amelioreaza aspectul anatomo-patologic al mucoasei gastrice.
Cuvinte cheie: stres oxidativ, AINS, ulcer peptic.

2009.19.3(63)  Fiziologia - Physiology

25

VARIATIONS OF SOME SALIVA MARKERS OF THE OXIDATIVESTRESS IN PATIENTS WITH ORTHODONTIC APPLIANCESOLTEANU C1, MURESAN A2, DAICOVICIU D2, TARMURE V1, OLTEANU I3,
KEULARTS MLW IRENE4
1
Department of Orthodontics, „Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca
2
Department of Physiology, „Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca
3
Department of Biochemistry, „Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca
4
Department of Clinical Genetics, Maastricht University, Holland

ABSTRACTThe use of orthodontic appliances in the treatment of the various dento-maxillar anomalies most frequently presume the application of high intensity forces,
non-physiological, which always will produce an inflammatory response localised around the tooth or the teeth subjected to displacement. The presence ofan inflammatory process at this level will produce an increased synthesis of free radicals, secondary followed by the oxidative stress.
In order to asses the level of the oxidative stress, a series of its markers are used, including the reactive species of oxygen and nitrogen, and the oxidation
products in particular, such as lipid peroxides and oxidized proteins respectively.
Thus, the objective of the present study is to determine and compare the levels of some oxidative stress markers (malondialdehyde, ceruloplasmine, hydrogen
donors) in saliva of patients with orthodontic appliances, before and after the initiation of the treatment.
The patients included in this study were 7 girls and 4 boys, with the average age of 9.9 years, in which orthodontic biomechanical appliances were applied,in the purpose of treatment of some dento-maxillar anomalies. Saliva has been collected before the initiation of the orthodontic treatment, at 1 hour, at 24hours, and at 7 days from from the orthodontic appliance application.
The variations in the concentrations of the saliva markers of the oxidative stress reached a maximum at 24 hours from the debut of the treatment for ceruloplasmine and malondialdehyde, and at one hour for the hydrogen donors respectively, while at 7 days from the device application the concentrations wereclose to the initial values.
The health condition at the level of the oral cavity in the patients taken into study did not change during the research. These results demonstrate that theutilization of an orthodontic appliance changes the levels of the saliva markers of oxidative stress, but these variations, even statistically significant, do not
determine the appearance of certain pathological processes at the level of the oral cavity.
Key words: ceruloplasmine, malondialdehyde, hydrogen donors, saliva, orthodontic appliances

INTRODUCTION
Free radicals, such as the reactive species of oxygen, result in a high diversityof biochemical reactions produced during the progress of the cellular functions(e.g. the mitochondrial metabolism). The formation of these free radicals (prooxidants) in physiological conditions is balanced through their consumption by
the antioxidants.
The oxidative stress is produced as a consequence of an unbalance between thesynthesis and the neutralization of the pro-oxidants. A high number of pathologicalprocesses in the human organism can affect this balance by the increasing of thefree radicals production related to the existing antioxidants.
Formation of pro-oxidants and their effect upon the cellular functions (which
can finally lead to apoptosis) have the name of oxidative stress. Sies H., in his paper:„Oxidative stress: from basic research to clinical application”, published in 1991 (1),stated the first definition of the oxidative stress – „an unbalance in the balance
pro-oxidants / antioxidants behalf pro-oxidants, with possible repercussions on
the organism”.
The oxidative stress involves the adverse effects of oxygen and of other free
radicals on the living tissues (2). The free radicals are unstable molecules, withincreased reactivity, because they have a free electron, which combines with various

cellular components, such as DNA, proteins, lipids and fatty acids. These reactions
are finally followed by damages in the DNA molecules, mitochondrial malfunctions,alteration of plasma membrane and eventually the cell death.
A high variety of biological processes, such as the anti-microbial defence,
inflammation, carcinogenesis, and the aging as well imply the involvement of thefree radicals (3, 4), leading thus to oxidative stress. The DNA alteration, the cellularmalfunctions, and other pathological processes mediated by these free radicals
represented in time sources of interest for biochemists and physicians.
The main free radicals are represented by the reactive species of oxygen (ROS)
and nitrogen (RNS).
The settlement of a certain causative relation between the oxidative stress andthe physiological or pathological processes in which is involved, is achieved by theassessment of the oxidative stress markers in different fluids of the human body.
The biomarkers are defined as characteristics which can be objective measured,and evaluated as indicators of the normal biological processes, of the pathologicalprocesses, or of the pharmacological responses after a therapeutic intervention(5).
A series of markers of the oxidative stress are used, including the reactive speciesof oxygen and nitrogen, but most of them have a limited value because of a reduced

Received July 2009. Accepted August 2009. Address for correspondence: Dr. Cristian Olteanu, Department of Orthodontics, „Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca,
e-mail: cristidolteanu@yahoo.com

26

Fiziologia - Physiology  2009.19.3(63)method with 2-tiobarbituric acid, and reading the extinction at 530 nm.
The assessment of ability of hydrogen donor is based on the reduction of the stable radical 1, 1diphenil-2-picrilhydrazil by a series of antioxidant compounds, pursued by the colour change from violet
to wan yellow, monitorised by the modification of absorbance at 520 nm.
Ceruloplasmine is an alpha-2 globulin, which in vitro displays properties of phenoloxidase, andcatalyzes the oxidation of p-phenylendiamine to a compound with a violet colour having a maximum
absorbance at 530 nm.
sensibility or specificity, or because they need invasive methods of collection.
phenoloxidase,and catalyzes
the inoxidation
to a compound
Concerning
the statisticalanalysis,order toof p-phenylendiaminecompare the mean values
Student t-test was
As ROS/RNS are unstable products, these reactive species were quantified bywith a violet colour having a maximum absorbance at 530 nm.
the values
have displaiednormalto compare
distribution.
The values
statisticalanalysis was
the evaluation of their stable metabolites, such as nitrates or nitrites, as well asperformed, because
Concerning
the statisticalanalysis, ina orderthe meanStudent
withwastheperformed,
Statistical Package(SPSS),a normal
and withdistribution.
the statisticalThesoftware ofby the measurement of the oxidized products’ concentration: lipid peroxides andaccomplishedt-testbecause for
the Social
valuesSciences
have displaied
oxidized proteins respectively.
statistical analysis was accomplished with the Statistical Package for Social Sciences
Microsoft Office.

Malondialdehyde (MDA) is an aldehyde produced by the peroxidative(SPSS), and with the statistical software of Microsoft Office.
degradation of the unsaturated lipids. The MDA excess secondary arisen after the
RESULTSlipid peroxidation is combined with the free amino groups in the protein structure,
TheRESULTS
average level of the oxidative stress markers in the saliva from the patients taken into study,
leading to the appearance of proteins with modified structure.
beforepresentedstressin Table
I. in the saliva from the patients
Ceruloplasmine, which is the major copper transporter within the organism,at the time intervals
The mentioned
average levelof theis oxidative
markers
displays an increased level in the acute and chronic inflammations, thus representing
taken into study, at the time intervals mentioned before is presented in Table I.
an acute phase reactant.
Table I. The mean concentration of the oxidative stress saliva markers in the patients taken into study
Table I. The mean concentration of the oxidative stress saliva markers in the patients taken into study
The using of the orthodontic appliances in order to treat various dento-maxillar
anomalies presumes most frequently the application of some forces of increased
MOMENT
intensity, non-physiological, which always will generate an inflammatory answer
zero
1 hour
24 hours
7 dayslocalized around the tooth or teeth which need to be displaced. And, as was alreadyCeruloplasmine
2.277
3.627
4.881
2.768
mentioned, any inflammatory process stimulates a higher synthesis of free radicals,( mg%)
resulting in oxidative stress.
Malondialdehyde
0.263
0.309
0.536
0.309
In these conditions, the objective of this study is represented by the quanti(nm/ml)
H+ donors
20.127
14.581
16.10919.472
fication of the levels of some markers of the oxidative stress (malondialdehyde,(inhib. %)ceruloplasmine, hydrogen donors) in the saliva of patients carrying orthodontic
appliances, before, and after 1 hour, 24 hours, and 7 days from the beginning of
Changes in the concentrations of ceruloplasmine (mg%), malondialdehyde (nm/the orthodontic treatment.
Changesin the concentrations of ceruloplasmine (mg%), malondialdehyde (nm/ml), andml), and hydrogen donors (inhib. %) in saliva are presented in Figures 1, 2 andhydrogen donors
3. (inhib. %) in saliva are presented in Figures 1, 2 and 3.
SUBJECTS AND METHOD
The written consent of the parents of the child patients taken into this study5
was obtained after a previous description of the study’s protocol. In order to asses
54,5
the saliva levels of the oxidative stress markers, 11 patients were taken into study,
4 4,5
in which 142 measurements were made. The 11 patients, 7 girls and 4 boys
3,5
4
(between 8 and 12 years, average age 9.9 years), displayed the following criteria
3
for including in study:
2,5 3,5• Well general status, without known affections;
2
Ceruloplasmine3• Lack of antibiotic therapy within the last 6 months;
1,5
2,5• Absence of anti-inflammatory drugs administration in the month before
1
2
Ceruloplasmine
the study;
0,5
0 1,5
• Healthy periodontal tissues and appropriate oral hygiene.
zero 1 hour 24 7 days
In each patient a brief instruction for teeth brushing and oral hygiene was made1
hours
before the beginning of the orthodontic treatment. The orthodontic treatment for all
0,5
11 patients was performed using biomechanical orthodontic appliances.
Fig.1. Changes in the ceruloplasmine concentration in saliva during the study (mg%)
The samples of saliva were collected as follows:0• Moment 0 – before the initiation of the orthodontic treatment;
zero 1hour 24 7days• Moment 1 – at 1 hour from the device application;
0,6
hours
• Moment 2 – at 24 hours from the device application;
0,5• Moment 3 – at 7 days from the device application.
In order to perform the study, unstimulated saliva was collected, by theFig.1.
0,4 Changes in the ceruloplasmine concentration in saliva during the study (mg%)
expectoration method, for 2 minutes. The test tubes with saliva were then kept at
0,3-80°C until the measurements were performed.
Malondialdehyde
0,2Malondialdehyde was dosed, after the previous protein precipitation, by thespectrophotometric method with 2-tiobarbituric acid, and reading the extinction
0,1
at 530 nm.
0The assessment of ability of hydrogen donor is based on the reduction of thezero 1 hour 24 7 days
stable radical 1, 1-diphenil-2-picrilhydrazil by a series of antioxidant compounds,
hours
pursued by the colour change from violet to wan yellow, monitorised by themodification of absorbance at 520 nm.
Fig. 2. Changes in the malondialdehyde concentration in saliva during the study (nm/ml)
Ceruloplasmine is an alpha-2 globulin, which in vitro displays properties of2009.19.3(63)  Fiziologia - Physiology

27

25
20
15
10

H+ donors

5
0

zero 1 hour

24 7 days
hours

Fig. 3. Changes of the hydrogen donor ability in saliva during the study (inh.%)DISCUSSIONSAt the moment 0, before the application of the orthodontic appliance, thesaliva levels of ceruloplasmine and malondialdehyde were ranging in normal limits(average: 2.27 mg% for ceruloplasmine, and 0.26 nmols/ml for malondialdehyde
respectively).
At 1 hour from the beginning of the force action, the saliva concentrationsof ceruloplasmine and malondialdehyde display a slight increasing, statisticallysignificant (p<0.01), but without implications on the pathology of the oral cavity(3.62 mg%, and 0.30 nmols/ml respectively).
At 24 hour from the device application, the levels of ceruloplasmine and malondialdehyde in saliva have a maximum of 4.88 mg%, and 0.54 nmols/ml respectively,
values which are statistically significant increased (p<0.01), but again without theappearance of certain pathological changes at the level of the oral cavity.
At 7 days from the beginning of force action, the saliva levels of the twomarkers decrease, coming close to the values recorded before the application of
the orthodontic appliance.
Before the beginning of the orthodontic treatment, the ability of hydrogen
donor in saliva has the mean value of 20.13 inhibition %. At 1 hour from the force
application, the minimum of level is reached, respectively a mean value of 14.58
inhib.% decrease which is statistically significant (p<0.01). At 24 hours from theforce application, the value increases at an average of 16.11 inhib. %, while at 7days from the debut of the orthodontic treatment, the level returns to values close
to those evaluated at the moment 0, respectively an average of 19.48 inhib.%. All
these variations in the ability of hydrogen donor are statistically significant, but
without implications on the oral pathology.
A series of studies have proven increases of the plasma concentrations ofmalondialdehyde and ceruloplasmine in patients with rheumatoid arthritis (6,7), as
well as of the saliva level of these markers of the oxidative stress in the same disease(8), in which one of the articulations of interest is the temporo-mandibular joint.
Because the use of the orthodontic appliances for the treatment of the various
dental anomalies will produce an inflammatory response, localized around the toothor the teeth to be displaced, the results of this study are in correspondence with thedata reported in the literature.
Taking into account there is not known literature data to mention the variationof the hydrogen donors in the context of our study, a possible explanation for thereach of their minimal level at 1 hour from the beginning of the treatment could
be the direct mechanism of annihilation of the oxygen free radicals by hydrogen,with water formation.
28

Therefore, the oxidative stress markers in saliva display a series of variations
along the study, but which do not lead to the appearance of some pathologicalprocesses in the oral cavity of the patient with orthodontic appliance. The study
proved that the highest variation in the levels of oxidative stress markers in thesaliva of the patient with an orthodontic appliance is measured at 24 hours fromits application for ceruloplasmine and malondialdehyde, and at 1 hour in the caseof the hydrogen donors respectively, and at 7 days the values being close to theinitial ones. These variations will be repeated after each activation of the device.
Variations of the oxidative stress markers in saliva are statistically significant, butthey do not change the health status of the oral cavity, which denotes that eventhese markers undergo important variations, these do not prove the appearanceof a pathologic process in the oral cavity in patients with orthodontic appliances.

CONCLUSIONS• The application of an orthodontic appliance modifies the saliva markers of
the oxidative stress, but these variations, even statistically significant, do not change
the health status at the level of the oral cavity.
• Even if the application of an orthodontic appliance is followed by the appearance of an inflammatory process localized around the tooth which needs tobe displaced (otherwise strictly necessary to achieve the dental displacements),
and consecutively the appearance of the oxidative stress at this level, this does not
manifest generally at the level of whole oral cavity.
• This is due to the fact that the inflammatory process is a localized one, andthe markers of the oxidative stress are diluting in the oral cavity, thus their variations,even statistically significant in the saliva of the patient carrying an orthodontic appliance, do not produce the appearance of pathological changes at this level.
• The application of an orthodontic appliance does not produce pathologicalchanges in the oral cavity of the patient to be treated, in the conditions in which theforces which act on the tooth to be displaced are close to the physiological forces.

REFERENCES
1. Sies H. Oxidative stress: from basic research to clinical application. Am J Med. 1991 Sep 30; 91(3C): 31S-38S
2. Pugliese PT. The skin, free radicals, and oxidative stress.
Dermatol Nurs 1995 Dec; 7(6): 361-9
3. Sies H. Biochemistry of oxidative stress. Angew Chem Int Ed
Engl 1986; 25: 1058-71
4. Halliwell B, Gutteridge JMC. Free radicals in biology andmedicine. 2nd ed Oxford UK: Clarendon Press, 1989
5. Dalle-Donne I, Rossi R, Colombo R, Giustarini D, Milzani a.
Biomarkers of oxidative damage in human disease. Clin Chem
2006 Feb 16; 52: 601-23
6. Kiziltunc A, Cogalgil S, Cerrahoglu L. Carnitine and antioxidant levels in patients with rheumatoid arthritis. Scand J
Rheumatol 1998; 27: 441–5.
7. Ozturk HS, Cimen MY, Cimen OB, Kacmaz M, Durak I. Oxidant/
antioxidant status of plasma samples from patients with rheumatoid arthritis. Rheumatol Int 1999; 19: 35–7.
8. Nagler RM, Salameh F, Reznick AZ, Livshits V, Nahir AM.
Salivary gland involvement in rheumatoid arthritis and its relationship to induced oxidative stress. Rheumatology (Oxford).
2003 Oct; 42(10): 1234-41.

Fiziologia - Physiology  2009.19.3(63)VARIAŢII ALE UNOR MARKERI SALIVARI AI STRESULUI OXIDATIVLA PACIENŢII PURTĂTORI DE APARATE ORTODONTICEREZUMAT

Utilizarea aparatelor ortodontice pentru tratarea diverselor anomalii dento-maxilare presupune cel mai frecvent aplicarea unor forţe de intensitate crescută,nefiziologice care întotdeauna vor determina apariţia unui răspuns inflamator localizat în jurul dintelui sau dinţilor care urmează a fi deplasaţi. Prezenţa unuiproces inflamator la acest nivel va produce o sinteză crescută de radicali liberi, cu apariţia secundară a stresului oxidativ.
Pentru determinarea nivelului stresului oxidativ, sunt utilizaţi o serie de markeri ai acestuia, incluzând aici speciile reactive ale oxigenului şi azotului, dar maiales produşii care sunt oxidaţi, respectiv peroxizii lipidici şi proteinele oxidate.
Astfel, obiectivul lucrării îl constituie determinarea nivelului unor markeri ai stresului oxidativ (malondialdehida, ceruloplasmina, donorii de hidrogen) însaliva pacienţilor purtători de aparate ortodontice, înainte şi după debutul tratamentului.
Pacienţii cuprinşi în studiu au fost 7 fete şi 4 băieţi, cu vârsta medie 9,9 ani, la care s-au aplicat aparate ortodontice mobilizabile, în scopul tratării unor anomalii
dento-maxilare. Saliva a fost recoltată înainte de debutul tratamentului, la 1 oră, 24 ore şi 7 zile de la aplicarea aparatului ortodontic.
Variaţiile concentraţiilor markerilor salivari ai stresului oxidativ prezintă un maxim la 24 ore de la debutul tratamentului pentru ceruloplasmină şi
malondialdehidă, respectiv la o oră pentru donorii de hidrogen, la 7 zile de la aplicarea aparatului, concentraţiile apropiindu-se de valorile iniţiale.
Pe tot parcursul desfăşurării cercetării, starea de sănătate de la nivelul cavităţii orale a pacienţilor luaţi în studiu nu s-a modificat. Aceste rezultate demonstrează
că aplicarea unui aparat ortodontic modifică markerii salivari ai stresului oxidativ, dar aceste variaţii deşi sunt semnificative statistic, nu determină apariţia
unor procese patologice la nivelul cavităţii orale.
Cuvinte cheie: ceruloplasmina, malondialdehida, donori de hidrogen, saliva, aparate ortodontice

2009.19.3(63)  Fiziologia - Physiology

29

CONSUMPTION OF CALORIGENIC FOODS IN ADOLESCENCEOANA SUCIU 1, BRIGITHA VLAICU 1,2
1“Victor Babes” University of Medicine and Pharmacy Timisoara, Romania
2

Institut of Public Health “Prof.Dr. L. Georgescu”, Timisoara, Romania
ABSTRACT
Daily consumption of cereals and derivates contributes to covering ¾ of the glucide requirements, ½ of the energetic needs and up to ½ of the protein
necessities of the organism. Fast-food products are rich in fats, sugar and salt, and they are included into the lifestyle of adolescents and they often replace
traditional nutrient sources. The working method was a transversal populational study, by use of the CORT 2004 questionnaire for the investigation of somehealth risk behaviours in young subjects, on a representative population of adolescents in Timiș County, including 2908 pupils. We found that 96.4% of theadolescents consume bread daily, ¾ of the girls eat 2-7 slices of bread a day, while ½ of the boys consume at least 8 slices daily. The consumption of pasta,
rice, cereals is present daily in 10.5% of the young subjects, with a 1.3/1 ratio between boys and girls. Chips and snacks were consumed by 20.5% of theadolescents, hamburgers and pizza by 10.2% and fried potatoes by 15.5%. The results may corelate mainly with an energetic intake replacing basic foods
and with a deficit of high biological value proteins.
Key words: adolescents, alimentation, fast-food

INTRODUCTION

mETHODS

Cereals represent an important source of vegetal proteins, with a content
of 7-16 g%, represented in proportion of 430-40% by gliadin and gluteins, andby low quantities of albumins, nucleoalbumins and globulins (1). Regarding thebiological value, they do not signify a qualitative fulfilment of the food portion
because proteins in cereals and in dry vegetables contain all essential aminoacids
but not in optimal proportions for the organism (2).
Among glucids, starch has the highest proportion, at an average of 80-90% in
cereals and 50-55% in dry vegetables, and this cathegory of foods is an importantsource of B group vitamins, vitamin E and minerals, especially phosphorus (200400 mg%), potasium (100-350 mg%) and magnezium (50-150 mg%) and theylack vitamin C (3).
The requirement of cereals and cereal derivates in adolescence: 300-450 g bread/day (6-11 slices of 40 g); 60 g cereal derivates/zi (1 porion of 60 g). Reported to thecaloric value of the portion, these foods will cover, on average, 50% of the needs,
meaning 70-80% of the necessary glucides and 45-50% of the protein needs (4).
In the great fast-food era, good home-learned practices are replaced by some
not very advisable feeding trends. There is a tendency to rapidly eating spicy foods,
sandwiches, conserved food, steaks, pasta, avoiding vegetables, fruits, bread (5).
Consumption of fast-food products causes the dramatical increase in thenumber of obese individuals. Obesity is no longer an American problem and it isspreading throughout the world at an increasing rate (6).
Many snacks have a very high level of fats, sugar or salt, ingredients whichshould be restricted to a small part of foods (7). Eating a healthy diet does not mean
complete elimination of favourite foods, but young people must be selective andlimit the total of fats, saturated fats, cholesterol or sodium. Consumer organizations worldwide released the code which bans promotion of junk food amongchildren. The code is meant as a strategy against obesity and diseases occuring asa consequence of uncontrolled diets, because statistics show that, worldwide, over
177 million children are threatened by obesity. The code addresses the marketingsystem for foods with high caloric value, rich in fats, sugar and salt.

The study was performed on a representative population of adolescents in highschools, postgraduate and apprentice schools in Timiș county, in urban areas, and it
included a total of 2908 pupils aged between 14 and 25 years (99% for the group ofadolescents aged between 15-19 years), 51.5% girls and 48.5% boys (9).
The work method was a transversal populational study, by use of the group andanonimous CORT 2004 questionnaire for investigating some health risk behaviours
in young subjects, designed by a CNCSIS accredited research team, by adaptation ofsome international questionnaires (ESPAD, YRBSS) to the reality of Romanian life,in the period 2003-2005 (10).

RESULTS AND DISCUSSIONS
1. Bread consumption (Figure 1)
The cereal intake in the daily portion of most young people, 96.4%, was coveredby consumption of bread, and only 3.6% of the adolescents did not consume this aliment. Among the young bread consumers, 36.0% reported a 2-4 slices a day intake(around 80-160 g), 27.8% of them consumed 5-7 slices daily (around 200-280 g),
and 32.6% of them ate more than 8 slices of bread a day (over 320 g).
% 60

50.5

50
40

28.4

27.3

30

20.7

Boys
Girls

28.2
21.7

20
6.4

More than 10
slices daily

8-10 slices
daily

5-7 slices
daily

2.1

2-4 slices
daily

0

9.6
5.1

Did not
consume10

Fig.1. Percentual distribution of boys and girls according to the frequencyof bread consumption during one week

Fig.1. Percentual distribution of boys and girls according to the frequency of bread consumption during one week

Received March 2009. Accepted June 2009. Address for correspondence: Dr. Oana Suciu, „Victor Babes” Universityof Medicine and Pharmacy Timisoara, 2A Eftimie Murgu Square, 300041,
70
63.7
69.8
%
Timisoara, Romania, phone/fax: 0256220479
60
50

Fiziologia - Physiology  2009.19.3(63)
Boys

40

Girls

30
20

13.5

10.9

10.5

10.4

ot
e

es
ek

1.9

es
ek

0

8.0 7.4
4.0

ily

10

re
er

30

Between the frequencies of bread consumption depending on gender, there arestatistically significant differences, with a χ2 value of 459.7 and a probability cutoff% 60 differences in favour of boys who recorded a daily bread consumption
p<0.001,
50.5
of over5010 slices (around 400g or more). Half of the girls, 50.5%, and 20.7% of theboys consumed between 2-4 slices of bread a day, and the percent of those who do
not eat40bread is double for girls, 5.1% as compared to only 2.1% in boys.
28.4

27.3

30

Boys
Girls

28.2

differences between genders are not statistically significant. Thus, 48.0% of boys
50

48.0

46.2

and 4546.2% of girls, consumed 1-3 times a week, and 19.3% of boys and 21.7% of%girls,40had a daily intake of chips and snaks. Only 16.9% of boys and 16.6% of girls
35 consume these foods.
did not
30
Boys
25
15.7
16.9(Figure 4) Girls
3.2. Consumption of hamburgers
and pizza
20
16.6
15.5
12.2
An important percentof the sample,
57.5%, reported hamburger consump15
11.6
7.7 9.5
tion10with a frequency of 1-3 times a week, which represents a higher proportion
as compared
to chips and snaks consumers, 10.2% consuming at least once a day,50
and 24.8%of the young subjects never consuming these foods.

20

Boys
Girls

40
30
10.9

10.5

0

10.4

Did not
consume1-3 times
per week

4-6 times
per week

1.9

Once daily0

8.0 7.4
4.0

2 or moretimes per
day

10

13.5

10

Did not
consume1-3 times
per week

20.2
8.3 6.7

7.7
4.6

Boys
Girls

5.6

2.6

Did not
consume50

29.2

1-3 times
per week

30

4-6 times
per week

60

20

Once daily

40

69.8

Once daily

63.7

2 or moretimes perday

70

2 or moretimes perday

More than 10
slices daily

8-10 slices
daily

5-7 slices
daily

2-4 slices
daily

Did not
consume%4-6 times
per week

21.7 cereals (Figure 2)
2. Consumption of pasta, rice,
20sample, most young subjects, 66.9%, consumed cereal derivates, others
Per
9.6
6.4
than bread, with a frequency of 1-3 times a week. The 4-6
times a week
consumption
10
5.1
was recorded2.1in 12.0% of the adolescents, 10.5% of them reporting an intake of at
As for hamburgers and pizza consumption, there is a statistically significant
least once
difference between consumption frequencies in boys and girls, with χ2 of 39.6 and
0 a day, and 10.6% did not eat food from this cathegory.
Differences between percentual distribution in girls and boys, depending on
a probability cutoff p<0.001. An increased proportion persists in boys, 59.2% andthe frequency of cereal derivates consumption are statistically significant, with agirls, 55.8%, who consumed hamburgers and pizza 1-3 times a week, and 12.3%
Fig.3.ofPercentual
distributionof boys
girlsconsumed
accordingtheseto thefoods
frequency
chips
and snaks consumption during one
χ2 value of 19.5 and a probability cutoff p=0.001, and they are in favour
boys.
of boys and8.2%andof girls
at leastofonce
a day.
Thus, 12.0% of boys and 9.3% of girls consumed this type of food at least once a
60
day. The percent of boys, 13.5%, who had an intake of cereal derivates 4-6 times a
59.2
55.8
%
is higher ofthanin girls,
10.5%.
Fig.1. Percentualweek
distribution
boys
and girls
according to the frequency of bread consumption during one
week
50
20.7

Fig.4. Percentual distribution of boys and girls according to the frequencyof hamburgers
and pizzatoconsumption
during one
week
Fig.4. Percentual distribution of boys andgirls according
the frequencyof hamburgers
and pizza consumption durin
week3.3. Fried potatoes consumption (Figure 5)

Fig.2. Percentual distribution of boys and girls according to the frequency

pasta,according
rice, cereals consumption
during oneofweek.2. Percentual distribution of boys andofgirls
to the frequency
pasta, rice, cereals consumption during
In theonecaseweekof fried potatoes, 56.4% of the adolescents consumed them at least

3. Consumption of fast-food
3.1. Consumption of chips and snacks (Figure 3)
Regarding the consumption of chips and snacks, most young subjects, 47.0%,
reported consumption 1-3 times a week, and the proportion of adolescents who
consumed these ”unhealthy” foods, at least once a day, is rather important, 20.5%.
Among the two genders, the hierarchy of consumption frequency of chips andsnaks is identical and similar to consumption frequencies for the entire sample, and
50

48.0

% 45

%

60

55.1

57.6

50

46.2

40

20

Did not
consume1-3 times
per week

4-6 times
per week

Once daily

0

6.8

10.2

9.2

8.7

5.4

4.1

Did not
consume

10

Boys
Girls

19.1

1-3 times per
week

11.6

16.6

23.2

4-6 times per
week

9.5

16.9

15.5

30

Once daily

7.7

12.2

Boys
Girls

2 or moretimes per day

15.7

2 or moretimes per
day

40
35
30
25
20
15
10
5
0

1-3 times a week, and to these subjects a 21.1% is added represented by young
subjects who consumed this food 4-6 times a week, and only 7.0% of them did
not eat this aliment.
Like in the case of chips, snaks, hamburgers and pizza, fried potatoes werealso more frequently eaten by boys. Gender differences, regarding fried potatoes

Fig.5. Percentual distribution of boys and girls according to the frequencyof fried potatoes consumption during one week

Fig.3. Percentual distribution of boys and girls according to the frequencyof chips and snaks consumption during one week

distributionof weekboys and girls according to the frequency of fried potatoes consumption during one w
g.3. Percentual distribution of boys and girls according to the frequency of chips and Fig.5.
snaks Percentual
consumption
during one

2009.19.3(63)  Fiziologia - Physiology
60
59.2
55.8
%

31

50
40
30

29.2

Boys
Girls
CONSUMUL
DE ALIMENTE CALORIGENE ÎN ADOLESCENğĂ

consumption, is statistically significant, with χ2 of 31.20 and a probability cutoff
p<0.001 and it is in favour of boys. Thus, 78.3% of the boys and 76.7% of the girls
consumed this type of food 1-6 times a week, and 17.0% of the boys and 14.1%of the girls consumed fried potatoes at least once a day. Only 4.7% of the boys anda double percent, 9.2% of the girls did not consume it.

CONCLUSIONS
Daily consumption of cereals and derivates contributes to covering ¾ of theglucidic needs, ½ of the energetical needs provided by glucides, and especially bystarch, and up to ½ of the proteic needs.
Daily intake of bread is present in 96.4% of the teen-agers, 4/3 of the girls
consume 2-7 slices of bread daily, whereas ½ of the boys eat at least 8 slices/day
and 1/3 of the boys consume more than 10 slices/day. By age groups, consumption
differences are not significant.
The consumption of cereal derivates, other than bread, (pasta, rice, cereals)
is present daily in 10.5% of the adolescents, with a boys/girls consumer ratio of
1.3/1. The most frequent consumption is 1-3 times/week throughout the entire
sample and in all age groups.
The obtained results may corelate with an energetic intake replacing deficiencies found in meat, milk and eggs intake, but also with a deficit in high biologicalvalue proteins obtained from animal foods.
Fast food aliments are rich in fats, sugar and salt and are included into thelifestyle of adolescents. They often replace traditional nutritional sources, such ascereal derivates, dry and fresh vegetables, fruits (11).
Daily consumption of chips and snaks has been reported by 20.5% of theadolescents, and the boys/girls consumer ratio was 1/1.2. Reported to age groups,
the daily consumption was 25.3% in 15 year olds and 12.7% in 19 year olds.
Daily consumption of hamburgers and pizza is declared by 10.2% of theadolescents, and with a boys/girls consumer ratio of 1.5/1. In 15 year old subjects
the consumer percent was 11.7% and in 19 year olds it was 9.3%.
Daily consumption of fried potatoes was reported by 15.5% of the adolescents,
with a boys/girls consumer ratio of 1.1/1 and with 15.8% consumption in 15 year

olds and 10.7% in 19 year old subjects.
High/excessive consumption of fast-food type aliments increases the risk ofobesity, diabetes, depressive moods (7).
REFERENCES
1. Boutelle K, Fulkerson J, Neumark-Sztainer D, et al., Fast food forfamily meals: relationships with parent and adolescent food intake,home food availability and weight status. Public Health Nutrition,
2007, 10(1):16-23
2. De Garine I, Socio-cultural aspects of alimentary behavior. Attempted classification of food prohibitions. Aspects socio-culturels
des comportements alimentaires. Essai de classification des interdits
alimentaires. Journal Article Maroc Medical, 1989, 47(508):764-73
3. Gosnell BA, Mitchell JE, Lancaster KL, et al., Food presentationand energy intake in a feeding laboratory study of subjects withbinge eating disorder. International Journal of Eating Disorders,
2001, 30(4):441-6
4. Isnard-Mugnier P, Vila G, Nollet-Clemencon C, et al., Etude
controlee des conduites alimentaires et des manifestations emotionnelles dans une population d'adolescentes obeses. Archives
Francaises de Pediatrie, 1993, 50(6):479-84
5. Jeffery RW, French SA, Epidemic obesity in the United States: arefast foods and television viewing contributing? American Journalof Public Health, 1998, 88:277—280
6. Negrişanu G, Treaty nutrition, Brumar Publishing, Timişoara,
2005, 259-277
7. Sjoberg A, Hallberg L, Hoglund D, et al., Meal pattern, foodchoice, nutrient intake and lifestyle factors in The Goteborg Adolescence Study. Eur J Clin Nutr., 2003, 57(12):1569-78
8. Strain GW, Nutrition, brain function and behavior. Journal Article,
Research Support, U.S. Gov't, P.H.S., Psychiatric Clinics of North
America, 2001, 4(2):253-68
9. Yamamoto J.A., Yamamoto J.B., Yamamoto B.E., et al., Adolescent
fast food and restaurant ordering behavior with and without
calorie and fat content menu information. Journal of Adolescent
Health, 2005, 37(5):397-402
10. ***, United States Department of Agriculture, Dietary Guidelines for Americans. U.S. Department of Health and Human Services, 2005, www.healthierus.gov/dietaryguidelines
11. ***, Grant A CNCSIS code 1167, 2003-2005, Assessing the sizeof risk behaviors in high school and young people in secondary
education, post, professional and academic Timis County, 2004.

CONSUMUL DE ALIMENTE CALORIGENE ÎN ADOLESCENŢĂ
Rezumat

Consumul zilnic de cereale şi derivate de cereale contribuie la acoperirea a 3/4 din necesarul de glucide, 1/2 din necesarul energetic şi până la 1/2 din necesarulde proteine. Alimentele de tip fast-food sunt bogate în grăsimi, zahăr şi sare, sunt incluse în stilul de viaţă al adolescenţilor şi înlocuiesc frecvent sursele
tradiţionale de nutrienţi. Metoda de lucru a fost studiul populaţional transversal, prin aplicarea chestionarului CORT 2004 de investigare a unor comportamente cu risc pentru sănătate la tineri, pe o populaţie reprezentativă de adolescenţi din judeţul Timiş, totalizând 2908 elevi. S-a constatat că, 96,4% dintre
adolescenţi consumă zilnic pâine, respectiv, 3/4 dintre fete consumă 2-7 felii de pâine/zi, în timp ce 1/2 dintre băieţi consumă cel puţin 8 felii/zi. Consumul
de paste făinoase, orez, cereale, este prezent zilnic la 10,5% dintre tineri, cu un raport al consumatorilor băieţi/fete de 1,3/1. Chips-urile şi snacks-urile au fostconsumate de 20,5% dintre adolescenţi, hamburgerii şi pizza de 10,2% dintre ei, iar cartofii prăjiţi de 15,5% dintre tineri. Rezultatele obţinute se pot corela,în principal, cu un aport energetic care înlocuieşte alimentele de bază şi cu un deficit de proteine de valoare biologică superioară.
Cuvinte cheie: adolescenţi, alimentaţie, fast-food32

Fiziologia - Physiology  2009.19.3(63)MOLECULAR CHANGES IN PRECANCEROUS LESIONS OF
THE LARYNX
RALUCA HORHAT, DELIA HORHAT, MARIOARA POENARU, STAN COTULBEA, NICOLAE
BALICA, VALERIA MOCANUENT Clinic Timisoara
UMFT “Victor Babes”Timisoara

Abstract
Precancerous lesions of the larynx are imtermediary states between the hpysiological mucosa and cancer.
Recent molecular pathology studies in head and neck cancer support a carcinogenesis model in which the development of a field with genetically alteredcells plays a central role. Information from the genetic analysis of laryngeal cancer has grown enormously in the last 20 years.
There are two classes of genes implicated in the progress of malignancy: the oncogenes and the tumor supressor genes.
The molecular biology techniques have identified mutations at the level of chromosomes 9p, 3p, 17p, 11q and also the accumulation of nuclear proteins as
Ki-67 and PCNA. These genetic alterations are represented by the inactivation of tumor suppressor genes- p16, p53, FHIT- and by the activation of oncogenescyclin D1.
Advances in the understanding of the molecular basis of the laryngeal carcinogenesis will help in the identification of new molecular markers that could be
used for a more accurate diagnosis and assessment of prognosis and may open the way for novel approaches to treatment and prevention.
Keywords: benign scuamous hyperplasia, dysplasia, carcinoma in situ, oncogenes, tumor supressor genes, nuclear proteins

INTRODUCTION
The same year Watson and Crick published their article on the tridimensional
structure of the DNA 50 years ago, Slaughter et all, described for the first time thephenomenon of “field cancerization”, hypothesizing that there are alterations at thelevel of the entire mucosa determined by carcinogens in patients with cancer of theupper aero-digestive tract. The authors examined pathology slides from 783 patientswith head and neck cancers in an effort to identify alterations of the epitheliumthat surrounds tumors and to explain its clinical behaviour. 11% of the patients
presented histologic alterations independent of the tumor area. At the time there
was no molecular basis for their observations, but the term has been used to describe
three phenomena: (1) wide field of aero-digestive mucosa tends to be affected by
the premalignant disease; (2) frequent occurrence of multiple primary tumors in
the epithelial areas affected by widespread premalignant disease and (3) possible
distant related primary tumors in the upper aero-digestive tract.
In 1990, Fearon and Vogelstein proposed the first model for the molecular
progression of the colorectal cancer stipulating that tumors progress by the activationof the oncogenes and the inactivation of the tumor suppressor genes, each producing
growth advantage for a clonal population of cell. Generally there are specific genetic
events in a distinct order, but the order in which they are produced isn’t necessarily
the same for a certain type of tumor. Genetic events summation determines the
progression of the tumor (Ha and Califano, 2003)(13).
Laryngeal carcinogenesis is a multistage process, initiated by various carcinogens (tobacco, alcohol, gastro-esophageal reflux, the human papillomavirus, etc.)
which induce genetic alterations and if they remain undetected by the DNA repairing
mechanisms, they confer growing advantage with increased cellular proliferation(Monier R et al., 2008)(20).

The aim of this article is to review and highlight some of the recent advancesin the molecular biology of the laryngeal carcinogenesis with particular clinicalrelevance for the diagnosis, management and prognosis of the precancerous lesionsof the larynx and laryngeal carcinoma.

A GENETIC MODEL FOR THE LARYNGEALCARCINOGENESISThe transition from the normal epithelium to the scuamocellular carcinoma of
the larynx is a long, complex and multistage process, with the accumulation ofgenetic alterations- between 6 and 10- that translate initially by the presence of
precancerous conditions (benign squamous hyperplasia, dysplasia, carcinoma insitu) and which can suffer malignant transformation (Braakhuis B et al., 2004)(2).
The number of genetic alterations grows with the malignity level established by
the histologic examination (see Figure 1)(Califano et al., 1996)(5).

Fig. 1. Genetic model for the laryngeal carcinogenesis (Califano et al., 1996)(5)

There are two classes of genes involved in the progress of malignancy: the oncogenes and the tumor supressor genes. The activation of the oncogenes induces theinitiation and progression of the tumor while the inactvation of the tumor suppressor
genes contributes to the progression of cancer (Crissman J et al., 2004)(7).

Received March 2009. Accepted June 2009. Address for correspondence: Dr. Raluca Horhat, ENT Clinic, ”Victor Babes” University of Medicine and Pharmacy Timisoara, 2A Eftimie Murgu Square,
300041, Timisoara, Romania, phone/fax: 0256220479
2009.19.3(63)  Fiziologia - Physiology

33Normal cells present protooncogenes that turn into oncogenes when activatedby different carcinogens, radiations or viruses. This activation is produced by chromosomal translocation, gene amplification, point mutations or deletions. Oncogenes
activity is expressed by the level of the growing factors and of their receptors, thesignal transducer systems and the nuclear proteins.
The tumor suppressor genes, active in normal cells, suffer mutations ordeletions under the action of the same agents, which leads to their inactivationand consecutive disregulation of the pathways that control cellular growth anddifferentiation.
Studies have demonstrated that patterns of genetic alterations can be used to
predict the behavior and tumorigenetic potential of pre-malignant laringeal lesions.
In a retrospective study, it has been demonstrated that loss of chromosomes 9p21
and 3p14 were more frequent in patients whose premalignant disease eventuallydeveloped into scuamous cell carcinoma (Chang S et al., 2008)(6).
Cromosome 9p
The 9p21-22 deletion is the most frequent genetic alteration in laryngeal
carcinogenesis, occuring in about 70% cases (Kim et al., 2004)(18). At this levelp16/p14ARF gene, responsible of tumor supressor activity is located. This alteration
is found in 20% of the benign squamous hyperplasias and in 57% of the dysplasiasof the larynx (Califano et al., 1996)(5).
Gene p16 encodes a 16kDa protein, part of the cyclin dependent kinase inhibitors (CDKIs), among p15WAF/CIP1 and p27KIP1. The product of the p16 (CDKN2A/
MTS1) gene binds to CDK4 and CDK6, inhibiting their association with the cyclinD1.
The inhibition of cyclin D1/CDK4/6 complex activity prevents the Rb phosphorylation and the release of E2F, thus preventing the G1-S transition in the cell cycle.
The genetic alterations that inactivate p16 gene offer growth advantage to the cell,
contributing in the carcinogenesis process (Hardisson D, 2003)(15).
Gene p14ARF inhibits the association between p53 and its inhibitor, MDM2,
resulting an antiproliferative effect (Ha P et al., 2008)(14).
The p16/p14ARF genes can be inactivated by an alternative mechanism by
the promoters hypermethylation (Fan, 2004)(8). This is an epigenetic phenomenon
consisting in the abolishment of the gene expression without mutations in thesequence of the genetic code. The methylation of the cytosine residues at thelevel of the CpG regions of the promoters is responsible of the reduced expression(Baylin and Herman, 2000)(1). Thus, in a significant number of cases the inactivation of the p16 gene can occur in the absence of homozygous deletions. Loss ofgene function due to promoter hypermethylation has several characteristics thatbear striking similarity to loss of the tumor suppressor gene function by somatic
mutation. First, promoter methylation in one allele is frequently accompanied bydeletion of the opposite allele, resulting in loss of heterozygosity of the gene. Second,
gene inactivation in association with promoter hypermathylation is fully heritable.
Finally, loss of gene function due to epigenetic alterations leads to selective growthadvantage in a manner identical to loss of tumor suppressor gene function due tosomatic mutation (Herman and Baylin, 2000)(16).
Chromosome 3p
Another genetic alteration that occurs early in the laryngeal carcinogenesis
process is the deletion at 3p14.2, present in 16% of the laryngeal squamous benign
hyperplasias, 52% of the dysplasias and 60% of the carcinoma in situ (Califano etal., 1996)(5).
At this level is located the FHIT (fragile histidine triad), member of the histidine
triad superfamily. It seems to work as a preapoptotic tumor supressor gene, that,when affected by deletions, can induce multiple epithelial tumors. The low levelof protein expression of the FHIT associates low rates of apoptosis and increased
34

tumoral cell proliferation (Kim M. and Califano J, 2004)(18).
Chromosome 17p
At the level of the 17p13.1 chromosome there is located the p53 tumor supressor gene, one of the most involved genes in carcinogenesis. It was initially thought
to be an oncogene, as there was aberrant overexpression of mutated forms of TP53,
but it was subsequently shown that these forms were not functional (Oliver et al.,2002)(21). It is constituted of 11 exons that encode a nuclear phosphoprotein withfunctions in the regulation of gene transcription, DNA synthesis and reparation, cell
cycle coordination and reparation, cell cycle coordination and apoptosis. Thus, theloss of function of the p53 gene determines genomic instability and accumulationof genetic alterations.
Most frequently, mutations involve guanine, including G:C to T:A transversions and G:C to A:T translocations at the level of non GpC sites. These changes are
frequently associated with benzo(α)pyren, nitrosamines and oxygen reactive species
from tobacco smoke (Fan CY, 2001)(8). These selective mutations seem to be causedby the formation of specific compounds between DNA and the carcinogens mentioned above and usually affect the codons 238-248 (Hardisson D, 2003)(15).
The product of the mutant p53 gene, characterized by conformational changes,
has a prolonged half-life and stability, and can be detected at nuclear level by IHC
staining. Molecular assessment of surgical margins for the TP53 mutations observedin primary laryngeal carcinoma have shown that if the margins are positive for theclonal genetic alteration, there is an increased risk of local recurrence (Ueno T. etal., 2003)(25).
Califano et al. Found p53 mutations in 11% of the benign squamous cell
hyperplasia of the larynx, in 33% of the dysplastic lesions and 47% of the carcinoma in situ.
Chromosome 11q
On the 11q13 chromosome there are located the bcl-1, int-2, hst-1 and cyclin
D1/ PRAD1 genes. The function of the cyclin D1 protooncogene, also known as
PRAD1 and CCND1, is the Rb activation by phosphorilation, forming complexes withthe cyclin dependent kinase cdk 4 and cdk 6, facilitating the progression from theG1 phase to the S phase of the cell cycle (Kim m. et al., 2004)(18).
The cyclin D1 supraexpression shortens the G1 interval and reduces the mitogen
dependence for the cellular proliferation.
Alterations in the cyclin D1 expression are found according to Califano et al. in
6% of the benign squamous hyperplasia, 29% of the dysplasia lesions and 40%of the carcinoma in situ.
Amplification of the cyclin D1 gene may also have prognostic significance. In ahomogenous cohort of 51 patients with laryngeal carcinoma treated surgically withor without postoperative radiotherapy, Belacosa et al. demonstrated independent
prognostic values for both tumor stage and cyclin D1 gene amplification in predicting
poor overall survival (Jeannon J.P. et al., 2004)(17).
Beside the activation of the oncogenes and the inactivation of the tumor suppressor genes, laryngeal carcinogenesis is also characterized by the supraexpressionof proteins as result of gene amplification and the increased DNA transcription andtranslation.
The epidermal growth factor receptor (EGFR)The EGFR gene is located on the chromosome 7p12 and it has 110kb. It is atransmembranar receptor, regulator of growth. Its structure consists of a glycoprotein of 170 kDa, that influences cell division, migration, adhesion, differentiation
and apoptosis, acting on the tyrosine kinase pathway, activating STATs through
Fiziologia - Physiology  2009.19.3(63)phosphorilation (Pomerantz R.G. and Grandis G.R., 2004)(23).
The EGFR overexpression is an early event in laryngeal carcinogenesis. Its level
increases with the degree of the epithelial lesions (Gale N.et al., 2008)(11). The
retinoic acid, used in the treatment of the laryngeal precancerous lesions, decreases
the level of the EGFR mRNA by low gene transcription.
Ki-67
Ki-67 is a nuclear protein expressed only in cells actively going through cell
cycle and it can be used to appreciate the proportion of proliferating cells in a
population. It can be determined by IHC techniques and quantitated by determining
the percent of nuclei that stain (Brown J et al., 2003)(4).
As a marker of cell activity, Ki-67 can be useful in the prognosis of laryngeal
precancerous lesions. The researchers at Fox Chase Center in Philadelphia observedthat Ki-67 is nine fold more increased in the basal layer of the dysplastic lesions
compared to normal mucosa and more increased in the superficial layers in highgrade dysplasia compared to low grade ones (Koch W., 1999)(19).
In another report, 9 of 10 low grade preneoplastic laryngeal lesions thateventually progressed to invasive scuamous cell carcinomas, had detectable Ki-67
staining. In contrast only 2 of 22 lesions that did not progress expressed Ki-67. Thus,
as a marker of biologic agressiveness in premalignant lesions, the sensitivity andspecificity of Ki-67 approached 90% (Pignataro L. et al., 1998)(22).
The proliferating cell nuclear antigen (PCNA)
PCNA is a nuclear protein as Ki-67 that appears late in the G1 phase, increases
in S phase and decreases through G2 and M phases of the cell cycle. It is involvedin the regulation of DNA synthesis and cell proliferation. It can be detected by IHC
techniques with PC10 antibody (Sarac S. et al.)24.

CONCLUSIONSThe term “field cancerization” reunites today more concepts that its initial use
50 years ago. What at the beginning was only the description of histologic changes
includes today the molecular bases of the transformation of the normal epitheliumin premalignancy and cancer.
Precancerous lesions of the larynx are intermediary states between the physiological mucosa and cancer. The development of molecular techniques promises
to facilitate early diagnosis, the creation of screening protocols for population atrisk and a better surveilance for cancer treated patients. The creation of a molecularprofile of the lesions will facilitate the analysis of surgical margins, the predictionof the behavior of precancerous lesions and laryngeal malignancy and the therapy
response.
The use of adenoviral vectors to restore p53 expression, demethylating agentsto re-express p16, anti-EGFR immunotherapy and small molecule kinase inhibitors
are in various stages of testing and clinical trials.
REFERENCES
1. Baylin SB, Herman JG. DNA hypermetilation in tumorigenesis:
epigenetics joins genetics. Trends Genet 2000; 16:168-174.
2. Braakhuis B, Leemans R, Brakenhoff R. A genetic progression
model of oral cancer: current evidence and clinical implications. J
Oral Pathol Med 2004;33:317-322.
3. Braakhuis B, Leemans R, Brakenhoff R. Expanding fields of genetically altered cells in head and neck squamous carcinogenesis.
Seminars in cancer biology 2005;15:113-120.

2009.19.3(63)  Fiziologia - Physiology

4. Brown J, Xu H, Nishitani J, Mohammed H, Osborne R, Teklehaimanot S, Gill G, Liu X. potential biomarkers for head and necksquamous cell carcinoma. Laringoscope 2003;113:393-400.
5. Califano J, van der Riet P, Westra W, Nawroz H, Clayman G, Piantadosi S, Corio R, Lee D, Greenberg B, Koch W, Sidransky D. Genetic
progression model for head and neck cancer: implications for fieldcancerization. Cancer Research 1996;56:2488-92.
6. Chang S, Califano J. Current status of biomarkers in head andneck cancer. J Surg Oncol 2008;97:640-643.
7. Crissman J, Visscher D, Sarcar F. Premalignant lesions of the upper
aero-digestive tract: biomarkers of genetic alterations, proliferationand differentiation. J Cell Bioch 2004;53:192-198.
8. Fan CY. Epigenetic alterations in head and neck cancer: prevalence, clinical significance and implications. Current Oncology
Reports 2004;6:152-161.
9. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759-767.
10. Gale N, Zidar N, Kambic V, Poljak M, Cor A. Epidermal growth factor receptor, c-erb-2 and p53 overexpressions in epithelial hyperplastic lesions of the larynx. Acta Otolaryngol 1997;527:105-110.
11. Gale N, Michaels L, Luzar B, Poljak M, Zidar N, Fischinger
J, Cardesa A. current review on squamous intraepithelial lesions of the larynx. Histopathology 2008 DOI: 10.1111/j.13652559.2008.03111.x
12. Gallo O, Vincentis M, Rocca C, Moi R, Simonelli L, Minni A,
Shaha A. Evolution of precancerous laryngeal lesions. Head andNeck 2000;23:42-47.
13. Ha P, Califano J. The molecular biology of mucosal field cancerisation of the head and neck. Crit Rev Oral Biol Med 2003;14:363369.
14. Ha P, Chang S, Glazer C, Califano J, Sidransky D. moleculartechniques and genetic alterations in head and neck cancer. Oral
Oncology 2008.
15. Hardisson D. molecular pathogenesis of head and necksquamous cell carcinoma. Eur Arch Otorhinolaryngol 2003;260:502508.
16. Herman JG, Baylin SB. Promoter-region hypermethylation andgene silencing in human cancer. DNA Methylation and cancer. Edited by Jones PA, Vogt PK. New York: Springer Verlag, 2000:35-50.
17. Jeannon JP, Soames JV, Aston V, Stafford FW, Wilson JA. Molecular markers in dysplasia of the larynx: expression of cyclindependent kinase inhibitors p21, p27 and p53 tumour suppressor
gene in predicting cancer risk. Clin Otol 2004;29:698-704.
18. Kim M, Califano J. Molecular pathology of head and neck cancer.
Int J Cancer 2004;112:545-553.
19. Koch W. Clinical implications of biomarkers in head and neckcancer. Current Oncology Reports 1999; 1:129-137.
20. Monier R, Tubiana M. cancerogenese. Accroissement desconnaissances et evolution des concepts. Oncologie 2008;10:319347.
21. Oliver M, Eeles R, Hollstein M, Khan MA, Harris CC, Hainaut P.
The IARC TP53 database: new online mutation analysis and recommendations to users. Hum Mutat 2002;19:607-614.
22. Pignataro L, Capaccio P, Pruneri G. the predictive valueof p53, MDM2, cyclin D1 and Ki-67 in the progression of low
grade dysplasia towards carcinoma of the larynx. J Laringol Otol
1998;112:455-459.
23. Pomerantz RG, Grandis GR. The epidermal growth factor receptor signaling network in head and neck carcinogenesis andimplications for targeted therapy. Semin Oncol 2004;31:734-443.
24. Sarac S, Ayhan A, Hosal AS. Prognostic significance of PCNA
expression in laryngeal cancer. Arch Otolaryngol Head Neck Surg
1998;124:1321-1324.
25. Ueno T, Hoshii Y, Cui D, Kawano, Gondo T, Takahashi M, Ishihara
T. Immunohistochemical study of cytokeratins in amyloid deposits
associated with squamous cell carcinoma and dysplasia in the oralcavity, pharynx and larynx. Pathol Int 2003;53:265-269.

35

ALTERARI MOLECULARE IN LEZIUNILE PRECANCEROASELARINGIENE
RezumatLeziunile precanceroase ale laringelui sunt stari intermediare intre mucoasa fiziologica si cancer.
Studiile recente de biologie moleculara in cancerele capului si gatului au scos in evidenta un model al carcinogenezei in care rolul central este jucat de campul
de celule alterate genetic. In ultimii 20 de ani s-a obtinut o cantitate enorma de informatii din analiza gentica a cancerului laringian.
In progresia malignitatilor sunt implicate doua clase de gene: oncogenele si genele supresoare tumorale.
Tehnicile de biologie moleculara au identificat mutatii la nivelul cromozomilor 9p, 3p, 17p, 11q si acumularea de proteine nucleare precum Ki-67 si PCNA.
Aceste alterari genetice sunt reprezentate de inactivarea unor gene supresoare- p16, p53, FHIT-si de activarea oncogenelor-ciclina D1.
Progresele in intelegerea bazei moleculare a carcinogenezei laringiene vor ajuta la identificarea de noi markeri moleculari care ar putea fi folositi pentru undiagnostic mai exact si pentru stabilirea prognosticului si ar putea deschide calea unor noi protocoale terapeutice si de preventie.
Keywords: hiperplazie scuamoasa benigna, displazie, carcinoma in situ, oncogene, gene supresoare tumorale, proteine nucleare

36

Fiziologia - Physiology  2009.19.3(63)THE HYPOLIPEMIANT EFFECT OF HIGH DOSESATORVASTATIN THERAPY IN DIABETIC PATIENTSWITH ACUTE MYOCARDIAL INFARCTIONADRIANA S. POTRA, VIRGIL M. LUCA, CONSTANTIN O. LUCA
Institute of Cardiovascular Disease Timisoara“Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania

ABSTRACTIn cardiovascular patients, the presence of diabetes mellitus is a major cardiovascular risk, according to all the clinical evidences in the present. However,there is a lack of information on the acute coronary patients with diabetes mellitus witch arrived late at specific medical cardiovascular assistance, becauseof the distance.
This trial evaluates the long term hypolipemiant effect of the early therapy with high doses Atorvastatin 80 mg in diabetic coronary patients hospitalized in
The Cardiology Department of The Cardiology Institute in Timisoara.
We studied 352 acute coronary patients, only 81 being with diabetes mellitus type 2. The period from the first symptomatic evidence to the hospitalisation
was late, 8-12 hours. The present observational study is undertaken in accordance with all valid ethical principles, all the patients included benefited frompharmaceutical therapy conformable with the current therapeutic guides. Written informed conscience was obtained from all patients. We had two groupsof patients: the control group (A) and the group of patients who received the early therapy with high doses of Atorvastatin 80 mg (B).
At the baseline, the level of seric lipids was similar for both lots of patients with an average level of the LDL cholesterol of 124 mg/dl, triglycerides 184 mg/dl
and HDL 46 mg/dl. At 6 months the reduction of the level of total cholesterol, of the LDL and of the triglycerides was complete in the lot of patients with highdose of Atorvastatin; this fact maintained at 12 months and at 24 months. By the end of the study, after 24 months treatment, the diabetic coronary patientstreated with 80 mg Atorvastatin had mean reduction of LDL cholesterol of 61%, approximately 90% of the maximum reduction in plasma LDL cholesterollevels was achieved by the end of the first 2 weeks.
Patients treated with 80 mg Atorvastatin had reductions in total cholesterol of 46% and reductions of Apo B of 50%, respectively. Atorvastatin 80 mg reduced
plasma triglycerides concentrations in 24 months with 25%. There was no consistent pattern in the percent changes from baseline for HDL cholesterol, apo
A-I and LP (a).
The present observational study is undertaken in accordance with all valid ethical principles, all the patients included benefited from pharmaceutical therapy
conformable with the current therapeutic guides. Written informed consent was obtained from all patients.
The results of our trial confirm and sustain all the present medical evidences, that the early therapy with high doses of statin has a lot of benefits in short andlong term evolution of coronary patients.
Key words: acute myocardial infarction, Atorvastatin, hypolipemiant, triglycerides, cardiovascular risk

INTRODUCTION
Diabetes has been shown to increase the cardiovascular risk in all populations
studied. However, there is a lack of information on the acute coronary patients withdiabetes mellitus witch arrived late at specific medical cardiovascular assistance,
because of the distance. The objective of this analysis was to evaluate the longterm evolution of these type of patients with high doses of Atorvastatin therapy
80 mg.

MATERIAL AND METHOD
The present observational study is undertaken on acute coronary patients withdiabetes mellitus non insulin-dependents, adults (above 18 years), that hospitalized
relatively tardy, 8-12 hours after the onset of the myocardial infarct, regardless ofthe presence or absence of dyslipidemia.
We excluded the coronary patients that were scheduled for revascularizationinterventions, the myocardial infarct patients with Q wave in the last 4 weeks,coronary by pass undertaken in the last 3 months, PTCA in the last 6 months, severe
anaemia (HGB < 8mg%), dialysis patients (IRC stadium IV), insulin-dependent
diabetes, pregnancy and nursing.
The present observational study is undertaken in accordance with all valid
ethical principles, all the patients included benefited from pharmaceutical therapy
conformable with the current therapeutic guides. Written informed consent was
obtained from all patients. The hospitalized patients, with acute myocardial infarct(with or without pre-hospital thrombolysis) and severe unstable angina, weretreated and monitored adequately during the hospitalization period and received
high doses of Atorvastatin (80mg/day) after which they were attentively monitored
both clinically and biologically at 6 months, 12 months and 24 months. All thepatients received recommendations for hygiene-dietetic regime, conformable withthe current therapeutic guides.
After 24-96 hours of hospitalization, the acute coronary patients received
Atorvastatin in maximum dose of 80mg/day; the clinical and paraclincal monitoringof the patients has been duly undertaken, both during the hospitalization period

Received March 2009. Accepted June 2009. Address for correspondence: Dr. Adriana Potra, Institute of Cardiovascular Disease, ”Victor Babes” University of Medicine and Pharmacy Timisoara, 2A
Eftimie Murgu Square, 300041, Timisoara, Romania, phone/fax: 0256220479
2009.19.3(63)  Fiziologia - Physiology37

en informed consent was obtained from all patients. The hospitalized patients, with acutenfarct (with or without pre-hospital thrombolysis) and severe unstable angina, were treatedd adequately during the hospitalization period and received high doses of Atorvastatin
and after the hospitalization at 6, 12 and 24 months. The clinical and paraclinicalAt the baseline, the level of seric lipids was similar for both lots of patientsfter which they were attentively monitored both clinically and biologically at 6 months, 12evolution of these patients was compared with hospitalized patients with similar
with an average level of the LDL cholesterol of 124 mg/dl, triglycerides 184 mg/dl
24 months. All the patients received recommendations for hygiene-dietetic regime,
characteristics who received usual pathology specific treatment, except the maxiand HDL 46 mg/dl. At 6 months the reduction of the level of total cholesterol, ofwith the current
therapeutic guides.
mum Atorvastatin dose administered just after admission in the study. Preliminary
the LDL and of the triglycerides was complete in the lot of patients with high dose
24-96 hoursanalysesof hospitalization,
acutecoronary
Atorvastatin
in at 12 months and at 24 months (Table I).
of the results at 2 weeks, 3themonths,6 months,
12 months andpatients
24 months receivedof Atorvastatin;
this fact maintained
se of 80mg/day;theclinicalandparaclincal
monitoringofthepatients
has
been
duly
from the hospitalization of acute myocardial infarct patients were effectuated.
By the end of the study, after 24 months treatment, the diabetic coronary
oth during the hospitalization
periodafter thehospitalization
12 andmonths.
Upon entering the baseline
phaseandand continuing
throughout
the therapy at 6,patientstreated24with
80 mg atorvastatin had mean reduction of LDL cholesterol
and paraclinicalevolutionofthesepatients
wascomparedwith
hospitalized
patients
withphase of the study, patients were counselled on to use of the National Institutes ofof 61%, approximately 90%of the maximum reduction in plasma LDL cholesterol
cteristics who Healthreceivedusualpathology
specifictreatment,
exceptthemaximum
Atorvastatin(NIH) NCEP Diet. This diet limits dietary cholesterol to <300 mg/d and total
levels was achieved by the end of the first 2 weeks.
tered just afterin thewithstudy.
analysesof the results
at 2 treatedweeks,
3 had dose-related reductions from baseline in totalfats toadmission<30% of total calories,<10% ofPreliminary
total calories from saturated
fats, 10%
Atorvastatinpatients
nths, 12 months
and 24 months
fromfrom
themonosaturated
hospitalization
infarct andpatients
from polysaturated
fats and 10-15%
fats. of acute myocardial
plasma cholesterol
Apo B (Table I). Patients treated with 80 mg Atorvastatin had
ted.
reductions in total cholesterol of 46% and reductions of Apo B of 50%, respectively.
entering the baseline
phase ANALYSIS
and continuing throughout the therapy phase
thereduced
study,STATISTICAL
Atorvastatinof80 mg
plasma triglycerides concentrations in 24 months withcounselled on toTheuseof theof aHealthThislimitssamplesize ofNational
the study wasInstitutes
chosen to detectsignificant(NIH)
linear doseNCEP Diet.
25%. There
was dietno consistentpattern in the percent changes from baseline for HDL
sterol to <300effectmg/dtotal fats
to <30%
total
calories,
total calories
for aand25% difference
betweenthe meanof
percentchangesof controlwith
and the<10% ofcholesterol,
apo A-I and fromLP (a).
highest dose of Atorvastatin.
Statisticalanalysesfrom
were performed
with SAS statistical
10% from polysaturated
fats and
10-15%
monosaturated
fats.
package. Analyses included data from all patients with at least one baseline and one

SAFETY

double blind measurement of the parameter of interest regardless of the patient
No serious adverse effects appeared in more than 1% of the patients. Increased
AL ANALYSIS
complianceprotocol.
ANOVA
was
used
to
evaluatetheeffectof
Atorvastastinonthehepatictransaminases
times the normal value) appeared in 8 patients (4.5%),
ample size of the study was chosen to detect a significant linear dose effectfor a(three
25%
percentchangefrom
thebaselinein
LDL
cholesterol,theprimary
efficacy
parameter.
for
whom
the
treatment
withtween the mean percent changes of control and the highest dose of Atorvastatin. StatisticalAtorvastatin was stopped and who where excluded
BaselineSAS
was define
as the meanpackage.
of each patient’s
LDL cholesterol
values at start,
study. No caseof documented
myositis was registered.
e performed withstatistical
Analyses
includeddata fromfrom
allthepatients
with
at
with
the
analysisofpercentchangefrom
thebaseline
being
performed
at
the
last
eline and one double blind measurement of the parameter of interest regardless of the patient
visit of thestudy.used
On thetobasisevaluateof this model,thea sequential,
trend test
rotocol. ANOVA
waseffect“stepof down”
Atorvastastinon theDISCUSSIONS
percent change
wasperformed
todeterminethesignificance
of
the
duff
effect.
Dunnett’s
test
was
Atorvastatina rapid onset
line in LDL cholesterol, the primary efficacy parameter. Baseline was define
as thehasmeanof of action; approximately 90% of LDL-cholesterolusedtocompare
each
group
when
the
percentchangewasnotmonotonic
acrossreductionfrombaselineoccurred
LDL cholesterol values at start, with the analysis of percent change from the baseline being within the first 2 weeks of treatment. Reduction
All analyses
usingofa two-sided
significancelevel of 5%.
of LDLdown”
cholesteroltrend
was 61%the last visit theof dosethelevels.
study.
On werethe donebasis
this model,
a sequential,“step
testin Atorvastatin 80 mg group of patients, after 24The
same
analysiswasperformedforsecondaryefficacy
parameters
exceptthatmonths.
Inadditionto
LDLcholesterol, apo B, the major protein component ofd to determine the significance of the duff effect. Dunnett’s test was used to compare eachthe baseline value for Apo-I, apo-B and Lp (a) was the mean of the measurements
Ldl cholesterol, was reduced from baseline with 51% after 24 months. There were
he percent change was not monotonic across the dose levels. All analyses were done using aduring the study.
no clinically important changes in HDL cholesterol, Apo A-I or LP (a). Triglycerides
nificance level of 5%. The same analysis was performed for secondary efficacy parameters
were reduced from baseline with 32% after 24 months of treatment with 80 mg
e baseline value for Apo-I, apo-B and Lp (a) was the mean of the measurements during the

RESULTS
Atorvastatin.
From a total of 584 acute coronary patients, we included 352 patients with
Atorvastatin was well tolerated in this study. No serious adverse effects apacute myocardial infarction and severe unstable angina in the study, and only 81
peared in more than 1% of the patients. Increased hepatic transaminases (threepatients with diabetes mellitus, in accordance with the preset inclusion criteria, thetimes the normal value) appeared in 8 patients (4,5%), for whom the treatmenta total of 584
acutecoronary
patients,
included
3522005.
patients
withmyocardial
periodof the inclusionof the patients
being weFebruary
2004-DecemberThe
withacute
Atorvastatin
was stopped and who where excluded from the study. No cased severe unstableanginainthestudy,
andonly
81
patients
withdiabetes
mellitus,
moment of the admission of the patients to the study overlapped with the momentof documented myositis wasin
registered.
with the presetofinclusion
criteria,
the period
the inclusion
ofwasthe patientsThisbeing
February
their hospitalization.
The maximum
daily doseof
of Atorvastatin(80mg/day)study suggeststhat 80 mg Atorvastatin is very effective in precociousber 2005. Theadministered
momentto aoflot thethepatientspatients
withthewith acute myocardial infarction.
of 179admission
patients, the restofof thereceivedtothethe
usualstudy overlapped
treatment of diabetic
patients
eir hospitalization.
maximum
daily dose
Atorvastatin(80mg/day) was administeredtreatmentThe
accordingto the valid therapeutic
guides of(otherstatins in usual doses),
79 patients, therestof thepatients
usualtreatment according
to the valid
meaning
the control
lot wascomposed ofreceived
173 patients.theThe clinicaland paraclinical
CONCLUSIONS
uides (other statinsdoses),
meaningat 6,thecontrol
lotfrom
wasofpresent
173study
patients.
monitoringinofusual
all the patientswas undertaken
12 and24 months
thecomposedTheproves that the early administration of 80mg/day Atorvasand paraclinicalmonitoring
ofclinical
all thepatients wasmonthsinclusionto the study. The
and demographicaldata ofundertaken
the patients fromat
the 6, 12 andtatin in24
diabetic
patientsfrom
with acute myocardial infarction late hospitalised, withto the study. two
Thegroups
clinicalandatdemographicaldatato ofthe (Table
patients
two STgroups
were similar
the moment of the inclusion
the studyI). from theor without
segment weredepression, is necessary immediately after the ischemic
event and in the long run, because it reduces the recurrence of the ischemic events
moment of the inclusion to the study (Table I).
Table I. Adjusted mean percent changes from baseline in lipid and apolipoprotein
in long term evolution and especially the ischemic cardiovascular recurrences thatat 24 months
therapyand apolipoprotein values at 24require
Adjusted mean percent changes from values
baseline
in lipidmonthstherapy
hospitalization.
Percent (%) change
Control
Atorvastatin 80 mg
The aggressive decrease of the values of the serum lipids at the administraLDL Cholesterol
7.6-61.0*
tion of Atorvastatin in maximum dose of 80mg/day is very effective in short time,
Total cholesterol
4.8-45.7*
approximately 90% of LDL-cholesterol reduction from baseline occurred within
HDL cholesterol
-2.5
3.5*
the first 2 weeks of treatment.
Triglycerides
-0.7
-27.2
Atorvastatin was generally well tolerated in this patient population. There
Apo A-I
-3.5
0.8
were
no documented cases of myositis, witch is the most serious adverse effect of
Apo B
5.8
-50.3*
statins.
Levels of serum transaminases exceeding 3 times the ULN were detected
Lp (a)
7.1-14.2*
in 8 patients, but these patients were excluded.
* Significant different than control by sequential, step-down, trend test, p<0.05
In conclusion, the results of this trial indicate that the treatment with high doses
38

Fiziologia - Physiology  2009.19.3(63)Atorvastatin 80 mg may provide adequate therapy for diabetic patients with acute
myocardial infarction late hospitalised.

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Kevin A. Primary evaluation and management of statin therapy
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treatment for preventing deaths from coronary heart disease:modelling study. Douglas G Manuel, Kelvin Knowing, Peter Danuseputro, Jenny Lim, Cameron A Mustard, Geofrey M Anderson,
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4. Miller KL, Pollack CV Jr, Petterson Ed. Moving from evidence topractice in the care of patients who have acute coronary syndrome.
Cardiol Clin. 2006; 24(1): 87-102.
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Am J Cardiol.2007; 99(6C):1E-35E.
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7. Bavry AA, Kumbhani DJ, Helcon TJ, Borek PP, Mood GR, Bhatt
DL. Late thrombosis of drug-eluting stents: a meta-analysis ofrandomized clinical trials. Am J Med. 2006; 119(12): 1056-1061.
8. The Expert Panel. Report of The National Cholesterol Education
Program Expert Panel on detection , evaluation and treatment ofhigh blood cholesterol in adults. Arch Intern Med. 1988:148:3669.
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Metab Cardiovasc Dis. 1992;2:113-156.
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11. The long term intervention with pravastatin in Ischemic Disease (LIPID) Study Group. Prevention of cardiovascular eventsand death with pravastatin in patients with coronary heart disease and broad range of initial cholesterol levels. N. Engl. J Med.
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EFECTUL HIPOLIPEMIANT AL DOZELOR CRESCUTE DEATORVASTATIN IN TERAPIA PACIENTILOR DIABETICI CU
INFARCT MIOCARDIC ACUT
REZUMAT
Diabetul zaharat este un factor de risc major suplimentar la patientii cardiaci, cu atat mai mult la pacientii coronarieni acuti, conform tuturos evidentelor cliniceactuale. Totusi, exista o lipsa de evidente clinice effectuate la pacienti diabetici coronarieni acuti care sunt spitalizati tardiv pentru asistenta medicala de specialitate, datorita distantei. Prezentul studiu evalueaza efectul hipolipemiant al terapiei precoce cu atorvastatin in doza maxima, la pacientii diabetici coronarieni
acuti, spitalizati tardiv pentru acordarea asistentei medicale de specialitate in Clinica de Cardiologie a Institutului de Boli Cardiovasculare Timisoara.
Din totalul de 352 pacienti coronarieni acuti monitorizati, 81 pacienti au fost diabetici. La 8-12 ore de la debutul bolii. Terapia administrata a fost conform
ghidurilor terapeutice in vigoare, in lotul A (lot control), iar in lotul B (lotul cu atorvastatin) s-a administrat tuturor pacientilor, imediat de la internare (precoce) doza maxima de atorvastatin 80 mg/zi, cu monitorizare la 6, 1 si 24 luni. La debut, valorile serice lipidice au fost similare in cele doua loturi de pacienti:
LDL_colesterol 124 mg/dl, triglyceride 184 mg/dl, HDL 46 mg/dl. Dupa 24 luni de tratament, s-au inregistrat diferente semnificative din punct de vedere
statistic intre cele doua loturi: LDL-colesterol a scazut cu 61%, colesterolul total a inregistrat o scadere cu 45,7%, cresterea HDL-colesterolului cu 3,5%, iar
Apo B a inregistrat o scadere cu 50,3%, comparative cu lotul control, in care s-a inregistrat o crestere cu 7,6% a LDL-colestrol, cu 4,8% a valorilor colesterolului
total, scaderea HDL-colesterol cu 2,5% si cresterea Apo B cu 5,8%. De mentionat ca complianta pacientilor la trtament a fost similara in cele doua loturi,
inclusive aparitia efectelor adverse secundare.
Rezultatele acestui studiu, confirma si sutin evidentele clinice effectuate pana in present, terapia precoce cu doze maxime de statine aducand numeroase
beneficii pe termen mediu si lung in evolutia pacientilor coronarieni.
Cuvinte cheie: infarct miocardic acut, Atorvastatin, hipolipemiant, triglyceride, risc cardiovascular2009.19.3(63)  Fiziologia - Physiology

39

URBAN NOISE ASSESSMENT IN TIMISOARA
ERNEST PUTNOKY
Ministry of Environment IB Timisoara

ABSTRACT
Noise is a modern plague of the urban society. In an effort made to asses the noise level of the urban area of Timisoara, a number of 103 noise level measurements, each 8 hours long were performed in the daytime. The measurements show that the urban noise in generated mainly by road traffic, affecting
significantly residential, recreational and medical treatment areas.
Keywords: urban noise, daytime noise measurements, road traffic

INTRODUCTION
Noise consists of all unwanted sound - sound that is loud, unpleasant or
unexpected. It has been increasing in urban areas to the point where it has becomea matter of public concern.
The effects of noise can vary from one individual to another. However, a WHO
report entitled "Community Noise - Environmental Health Criteria", published in
1996, and highlights such effects as disturbance of sleep, auditory or physiologicaleffects (basically cardio-vascular) and interference with communication.
Initially, action to reduce noise was not considered an environmental priority- unlike action to reduce air pollution, for example. The effects of noise are unspectacular, and the decline in quality of life was accepted by the general public as being
an inevitable consequence of technological progress and urbanization (1).
However it has been estimated that around 20 percent of the European Union's
population or close to 80 million people suffer from noise levels that scientists andhealth experts consider to be unacceptable, where most people become annoyed,
where sleep is disturbed and where adverse health effects are to be feared. An
additional 170 million citizens are living in so-called 'grey areas' where the noiselevels are such to cause serious annoyance during the daytime (2).
MATERIALS AND METHODSIn 2008, a number of 103 8h-daytime measurements were conducted in
randomly selected measurement points in the urban area of Timisoara. The measurement points were later classified by the function of the selected urban area:• Traffic area (roads, public transportation paths & railways)• Industrial area
• Residential area
• Recreational area (playgrounds & parks)• Medical treatment & care area

Traffic area measurements were made at the edge of the pathway (25m fromthe axis in the railways case), industrial and medical treatment area measurements
were made along the specific perimeter, recreational area measurements were madeinside the area, as central as possible and in the residential area measurement wereconducted at a distance of 3m form the façade of the buildings, facing the noise
sources, according to Romanian measurement standards (6).
The measuring instrument chosen was a Bruel&Kjaer 2238 Mediator (Class
1 Integrating Sound Level Meter with logging software) fitted with an outdoor
kit (free-field microphone with windscreen, cable preamplifier extension andtripod)The microphone was set at a height of 1.3 m form the ground level, accordingto Romanian noise measurement standards (6).
A 100 ms sampling rate was selected in order to maximize the amount ofrecorded data (288,000 samples/measurement) for statistical purposes and forfurther analysis.
Before each measurement the SLM was recalibrated using a class 1 acoustic
calibrator.
Measurement data included LAeq (A-weighted equivalent noise level) L90(background noise) and L10 (maximum noise). Lowest sound pressure levels andpeak levels were also recorded. The measurement unit was the dBA (A-weighted
decibel) (5).

RESULTS
Road traffic measurement conducted on the main roads at the city limits
area indicated higher noise levels due to higher vehicle speed and increased traffic
density. It is known that at speeds above 50 km/h tire noise becomes significant in
the overall noise emission of a vehicle. The equivalent noise level was in all cases
above 60 dBA, although the background noise was in some 3rd category roads under
45 dBA indicating that the flux of vehicles was not constant (7). However, in 2nd
category roads, the background noise was permanently over 55 dBA (Table I).

Received March 2009. Accepted April 2009. Address for correspondence: Ernest Putnoky, Ministry of Environment – IB Timisoara, Romania

40

Fiziologia - Physiology  2009.19.3(63)c measurement conducted Road
on theat LA
theeqmotors,
city limits
area indicatedhigherchillers, sawing mills, dryers anthemainuse roadsof electriccompressors,ventilators,
Area
(dBA)
higher vehicle speed and increased
traffic density.
It is anknown
thatconstantat speeds
aboveThese
50 areas were characterized by aequipment
generating
almostnoise.
1 in
Giroc
Centru noise
comes significant
the–overall
emission of 62.6
a vehicle. The equivalent noise level
noise, above 50 dBA,
and occasionally with high peak levels during loading/unloading
Halta Semenic
bove 60 dBA, 2although
the background noise was 60.2
in some 3rd category roads under 45
Calea Girocului 113
(UM)
70.8
operations.
Ininall2ndcases
measurements
category
roads, theresults were influenced by the n
hat the flux of3 vehicles
was notconstruction
constant(7).
However,
4 Calea Girocului – generated
Maldini noise (Table
70.9 IV).
was permanently over 55 dBA (Table I).
5
6
7
8
91
10
2
11
3
12
4
13
5
6
14
7
15

Calea Girocului – Vile Braytim
64.5
Calea Girocului-Albac
67.6
Table I. Road
Area Measurements
Table I. Road Area Measurements
DN6 - Vama Timisoara
71.4
LAeq
Versului Road Area
66.0
(dBA)
Uranus68.4
Giroc – Centru
62.6
Padurice
67.5
Halta Semenic
60.2
Sirius65.9
Calea Girocului 113 (UM)70.8
Brancoveanu- Snagov
69.5
Calea
Girocului– Maldini
70.9
Aradului – Vile Braytim
72.1
Calea Girocului
64.5
Calea
Girocului-Albac67.6
Amurgului
73.5
DN6 -Sagului
Vama Timisoara
71.4
Calea
72.4

IV. Industrial Area Measurements
Table IV.Table
Industrial
Area Measurements
Nr.
LAeq
Industrial Area
crt.
(dBA)
1
Prelucrare lemn, Dragalina
54.8
2
Spalatorie auto, Eroilor
55.7
3
Centrala, Piata Victoriei
66.9
4
Prelucrari metalice, Dragalina
59.7
5
Tinichigerie, Titulescu
50.0
6
Depozit,Take Ionescu
67.1
8 Versului
66.0
7
Fabrica mobilier, Brediceanu
65.0transportation,9 in Uranus
the case of trams, generates a 68.4
high level of noise.
Atanaverage58Chiller, Aries
58.8
10 Padurice
67.5
passage, with 11a peak
of
85-97
dBA,
on
canyon-likestreets(GheorgheDoja),
tram
9
Service auto, Eroii de la Paulis
59.9
Sirius65.9
uld be a serious
especiallyat high speeds andpoorly maintainedrails andCaprioareicars. A
12 issue,Brancoveanu- Snagov
69.5
10 Brutarie,
51.8
13
Calea
Aradului72.1
s at the Drubeta – Eneas junction where the improper maintenancethe curved
rails
11 ofBenzinarie,
Lidia
65.0
14
Amurgului
73.5
5.9 dBA, the distance between the rails and the façade of the residentialbuildings
in Buziasuluithe
12 Fabrica
boltari,
52.4
15 Calea Sagului
72.4
n 3 meters (Table II).
13 Fabrica electrice, Orion
50.8

Table II. Public Transportation – Trams MeasurementsintervalAtof timethe passages
transportation,
caseof trams, generates
a high level oflongnoise.
anbetween
average
5 permits the 8 hours value ofTableinII.thePublicTransportation– Trams Measurements
LAeq
tosettlebetween
58
dBA
and
68
dBA. In some sectors railway noisepassage, with a peak Publicof 85-97
dBA, onResidential
streets
tramlevels
had isa(Gheorghewide rangeDoja),
of noiserecorded, noise generated aTransportation- canyon-like
LAareaseq
combined with road traffic noise leading to increased noise levels. Studies
uld be a serious issue, especiallyathighspeedsandpoorly
rails
and
cars.
road
traffic,
mainlybecausemaintained
ofhavetheshown
sizethat,andpavingofAtheand railway
becauseTrams(dBA)
psychologically,
peopletendstreets
to support easiernoise of the conts at the Drubeta–
Eneas
junctionwheretheimpropermaintenanceof
thecurvedrails
than roadtrafficwith
noise evenrailway noise levelsnoise
are higher
thanaroad
1 Rebreanu
of vehicle traffic62.6(4). There areareas
lowwhenbackgroundandpassage of fe
traffic noise levels(3) (Table III).in the
5.9 dBA, the distance
betweenbuildings
2 Drubeta- Eneasthe rails and the façade
85.9 of the residential
Codrului Street, 75.0
LAeq 47.1 dBA,Noiseandalmostcontinuous
in theareas
industrialwith
area, LAeq
ranging between
50 dBA andnoise
67 dBA, levels of 72
n 3 meters (Table
3 II).
Gheorghe Doja

Aradului. Romanianrecommend a maximum accepted outdoor level of
4 Piata Resita
59.8 standards
chillers,
sawing mills, dryers and other stationary equipment generating an
Table
PublicTransportation– Tramsnoise
Measurements
equivalentlevel for residential
areas.
limit
surpassedin themajority of case
5 II.Iosif
Nemoianu
68.2
almost constantnoise.This
These areas
wereischaracterized
by a highbackgroundLA
6 PiataPublic
MariaTransportation 67.5eq
noise, above 50 dBA, and occasionally with high peak levels during loading/(dBA)
unloading, maintenance and construction operations. In all cases measure7 Gara Nord Trams
69.8
was mainly generated by the use of electric motors, compressors, ventilators,

1
Rebreanu
62.6
ments results were influenced by the nearby road traffic generated noisetransportation,in the case of trams, generates a high2The public
Drubeta- Eneas85.9level of(Table IV).
anGheorgheaverage
5 minutes
car passage,
peak of 75.0
85-97
dBA,
ys crossing noise.
the3 Attown
have
an perimpactonwitha asmallpercentageof thepopulation.
Doja
Residential
areas had a wideTrainrange of noise levels recorded, noise generon
canyon-likestreets(GheorgheDoja),
tram
generated
noise
could
be
a
4 but
PiatatheResita59.8
ated almost
entirely by road permits
traffic, mainly because of the size and paving ofhigh peak levels,
relatively long interval oftime between
the passages
seriousespeciallyat high speeds and poorly maintained rails
and cars.
the streets and because of the continuity and amount of vehicle traffic (4).
5 issue,Iosif
Nemoianu
68.2
of LAeq toA particular
settlebetween58 dBAand
68wheredBA.
In some sectors
railway noise is
case
was
at
the
Drubeta–EneasjunctiontheimproperThere are areas with low background noise and a passage of few cars/hour, like
6
Piata Maria
67.5
maintenance of the curved rails led to a LAeq of 85.9 dBA, the distance beCodrului Street, LAeq 47.1 dBA, and areas with almost continuous noise levels
7
Gara Nord
69.8
tween the rails and the façade of the residential buildings in the area beingof 72 dBA, like Calea Aradului. Romanian standards recommend a maximum
road trafficlessnoise
leading
have shown that,than 3 meters(Table II). to increased noise levels. Studies
accepted outdoor level of 50 dBA daytime equivalent noise level for residential
railways crossing
town
have an noise
impactasmallsmall percentageofpeopletend tosupporteasiertheroadtrafficnoise
even
ys crossing
theThetown
haveanrailway
impacton aonthan
percentageofpopulation.
Trainareas.the
This
limit
iswhensurpassedrailway
in themajority of cases (Table V).
thepopulation.
Trainpassages
generatehighpeaklevels,buttherelatively
in the recreational
areas (parks and playgrounds) are relatively
high thanpeakroadlevels,butnoise
the relatively
interval
thelevels
passages
permits
gher
traffic
levels (3)long(TableIII). of time betweenNoisehigh due to thevicinity of noise
the city’s mainof LAeq to settle between
58 dBA and 68 dBA. In some sectors
railway
is roads. Especially for playgrounds
Table III. Railway Measurements

Table III. Railway Measurements
Railway Area

1
2
3
4
5

Spitalul Nou
DAB staĠia Meteo
Gheorghe Lazăr
Halta Semenic
Gara Sud

LAeq
(dBA)
67.7
63.7
67.0
58.2
68.4

that were built beside busy roads, for easy access, this positioning leads to an
exposure of children to high levels of noise and air pollutants. Neverthelessthere are still parks relatively calm during daytime like the Roses Park with a
LAeq of only 49.7 dBA and a background noise of 43.3 dBA (Table VI).
Unfortunately a large number of medical treatment units and care facilities
are built near major roads, traffic noise having a significant impact upon thiskind of area. From 14 hospitals and other units investigated none was measured
with a daytime outdoor LAeq smaller than 55 dBA which is with 10 dBA morethan the maximum limit set for medical areas (Table VII and VIII).

Fiziologia- Physiology
e industrial 2009.19.3(63)area, LAeq ranging
between
50 dBA and 67 dBA, was mainly generated byc motors, compressors, ventilators, chillers, sawing mills, dryers and other stationary
ting an almost constant noise. These areas were characterized by a high backgroundBA, and occasionally with high peak levels during loading/unloading, maintenance and

41

LAeq
(dB
A)
Barbu Iscovescu
55.9
Aleea Ripensia 26
60.3
Piata Mărăúti
71.0
Calea Martirilor – Lidiainvestigated66.5
investigated nonenone
was measured
measured with
with aa daytime
daytime outdoor
outdoor LAeq
LAeq smaller
smaller than
than 55
55 dBAdBA ww
wasdBA moremore than
than thethemaximum limit
limit set
set forfor medical
medical areas
areas (Table(Table VII
VII andand VIII).
VIII).
Calea Martirilor 37 dBA
67.7maximum
Table V. Residential Area Measurements
Table VII. Recreational Area – Playgrounds Measurements
Codrului
47.1
Table
V.
Residential
Area
Measurements
Table VII.
VII. Recreational
Recreational Area
Area –– Playgrounds
PlaygroundsMeasurements
Measurements
Aurelianus
56.6
Table
LA
LAeqeqLA
Mihai Viteazul
68.5eqRecrational Area
Area -- Playgrounds
Playgrounds
Recrational
Residential Area
(dB
(dBA)
(dBA)
Calea Martirilor – Siemens
66.6
A)PiaĠa Eforie
Eforie53.5
11 PiaĠa
53.5
Brediceanu
14
70.7
2
Calea
Aradului
–
MoĠul
67.1
Barbu Iscovescu
55.9
2
Calea Aradului – MoĠul
67.1
EroilorRipensia 26
60.4
3
Calea
Aradului
–
Sever
Bocu
65.6
Aleea
60.3
3
Calea Aradului – Sever Bocu
65.6
NicolaeTitulescu - Dragalina
68.6
Bucovinei
49.4
Piata
Mărăúti
71.0
44 Bucovinei
49.4
5
PiaĠa
Dacia
1
53.6
PiaĠa
Traian55.2
Calea Martirilor – Lidia
66.5
5PiaĠa Dacia 1
53.6
6
PiaĠa
Dacia
2
57.6
6
PiaĠa Dacia 2
57.6
Calea
Martirilor 37
67.7PiaĠa Romanilor
58.2
7
Eroilor–ParculConstructorilor
58.8
7
Eroilor–ParculConstructorilor
58.8
Codrului47.1
Calea Aradului 18
72.8
Aurelianus
56.6
Table VIII. Medical Treatment and Care Area Measurements
Take Ionescu
69.0
Table VIII.
VIII. Medical
Medical Treatment
Treatment andand Care
CareArea
AreaMeasurements
Measurements
Table
Mihai
Viteazul
68.5
Mures 33
67.2
LAeqeqLA
Calea
Martirilor – Siemens
66.6
Medical Treatment
Treatment && CareCareArea
Area
Piata Unirii
58.6
Medical(dBA)(dBA)
Brediceanu 14
70.7
Piata Concordia
70.5
Maternitatea Bega
Bega -- Victor
Victor Babeú
Babeú12
12
67.9
11
Maternitatea
67.9
Eroilor
60.4
Piata Dr Russel 5
72.4Mărăúti
71.8
2
Clinicile Noi
Noi -- PiaĠa
PiaĠa Mărăúti
2
Clinicile
71.8
Nicolae Titulescu - Dragalina
68.6
Spitalul Militar
Militar -- Gheorghe
Gheorghe Dima
Dima
69.1
Tudor
Vladimirescu–
Mangalia
65.6
33
Spitalul
69.1PiaĠa Traian
55.2
4
Clinica
Oftalmologie
1
Mai
3
70.4
4
Clinica
Oftalmologie
1
Mai
3
70.4
CaleaRomanilor
Sagului 37
69.7
PiaĠa
58.2
5
Spitalul
Copii
3
Hematologie
5
Spitalul
Copii
3
Hematologie
VictorAradului
Babes - Cluj
77.0
Calea
18
72.8
Republicii
68.0
Republicii
68.0
Iepurelui–
Piata
Varfulcu
Dor
71.1
Take Ionescu
69.0
Spitalul Copii
Copii 33 Ortopedie
Ortopedie -- Iosif
Iosif
66
Spitalul
Simion33
Barnutiu 4
63.0
Mures
67.2
Nemoianu
65.8
Nemoianu
65.8
Piata
58.6
MuresUnirii
33
69.6
7
Spitalul
5
Maternitate
16
Decembrie
7
Spitalul 5 Maternitate - 16 Decembrie
24
71.5
Piata
70.5
24
71.5
IancuConcordia
Vacarescu 8
59.5
8SpitalulPsihiatrie
IancuVăcărescu
PiataDr
Russel5
72.4
8SpitalulPsihiatrie
IancuVăcărescu
Lidia 100
64.2
23
55.9
23
55.9
Tudor
Vladimirescu
65.6
Brancoveanu
52A – Mangalia
69.2
Spitalul CFR
CFR -- Tudor
Tudor Vladimirescu
Vladimirescu
62.7
99
Spitalul
62.7
Calea Sagului 37
69.7
Piata Aurel Vlaicu – str. Anton Pann
63.7
10 Spitalul
Spitalul Clinic
Clinic 11 JudeĠean
JudeĠean –– Dr.
Dr.
10
Victor Babes - Cluj
77.0
Liviu Rebreanu
70.5
Stanca59.7
Stanca59.7
Iepurelui – Piata Varful cu Dor
71.1
11 Spitalul
Spitalul 11 Ortopedie
Ortopedie -- Aleea
Aleea SănătăĠii
SănătăĠii
55.2
11
55.2
SplaiulGalati
6
67.8
Simion Barnutiu 4
63.0
12 Clinica
Clinica 11 Pediatrie
Pediatrie -- PăltiniúPăltiniú
61.1
12
61.1
Roma 33
54.5
Mures
69.6
Residential Area

1
2
3
4
5
6
7
8
9
10
1
11
2
12
3
13
4
5
14
6
15
7
16
8
17
9
18
10
19
11
20
12
21
13
22
14
23
15
24
16
25
17
18
26
19
27
20
28
21
29
22
30
23
31
24
32
25
33
26

13
13

Clinica dede Cardiologie
Cardiologie -- Spitalul
Spitalul Nou
Nou
Clinica

27 Iancu Vacarescu 8
59.5
13
70.1
13
70.1
28
Lidia
100
64.2
els in the recreational areas (parks and playgrounds) are relatively
highAustria
due ––toIosif
theBulbuca
1469.2
14 Casa
Casa
Austria
Iosif
Bulbuca
69.2
29 Brancoveanu 52A
69.2
y’s main roads. Especially for playgrounds that were built beside busy roads, for easy
30 Piata Aurel Vlaicu – str. Anton Pann
63.7
ioning leads 31
to anLiviu
exposure
and air pollutants.
Rebreanuof children to high levels
70.5 of noiseCONCLUSIONSCONCLUSIONS
Observer’sof
pointonlyof view, the crushing majority of the urban noisee are still parks32relatively
calm6during daytime like theSplaiul Galati
67.8Roses Park withByatheLAeq
By
the
Observer’s
point
of
view,
the
crushing
majority
of
the
of
view,
the
crushing
majority
oftraffic.
the Inurban
urban
noise source
sourc
sourcesin
Timisoara
duringdaytimeare represented
by the road
96% of noise
33 ofRoma
54.5
ackground noise
43.3 dBA (Table VI).
thecases,
noise
wasgeneratedbythetraffic
of
cars,
vans,
trams,
motorcycles
andduring daytime are represented byby thethe roadroad traffic.
traffic. In
In 96%
96% ofof thethe cases,
cases, noise
noise waswas generate
generatlorries. Thisandtype oflorries.
noise affectsThis
all studied
areasofin anoisesignificant
degree, leading
vans, trams, motorcycles
type
all
Table VI. Recreational Area –of
Parkcars,
Measurements
motorcycles
andlorries.
This
type
of
noise affects
affects
alltostudiedstudied areas
areas
els in the recreational
areas (parks
playgrounds)
areproposal
relativelyhightothe should
Table VI. Recreational
Areaand– ParkMeasurements
the proposal
a newdue
sortof noisebe introducedin the regulations,degree,leading to thethataa new
sort
of
noise
shouldbethat thatnewsortof source
noise source
sourceshouldbe introduced
introduced inin thethy’s main roads. Especially for playgrounds
that were
built
besidebusy
roads,
foreasy
to categorybeconsideredasaseparate
category
from
fixed
and
mobilesources:thetrafficLAeq
beconsideredasaseparate
from
fixed
and
mobilesources:thetrafficnoise
category from fixed and mobile sources: the traffic noise
Recreational Area - Parks
noise as anda collective
ioning leads to an exposure of children
to high (dBA)levels of noise
airphenomenon.
pollutants.
phenomenon.
Road
noise
increases
proportionallywith speed for all categories of vehicles.
Parc Pârvan
60.6
e are still parks1 relatively
calm during daytime
like
the
Roses
Park
with
a
LAeq
of
only
Road noise increasesRoadproportionally
with
speed
all
of vehicles.
proportionallywith
speed forfor
all categories
categories
material,
pavement
design,road
maintenanceand timingof the traffic lights of vehicles.
2 of Parc
CoposuVI).
56.6road maintenanceackground noise
43.3CorneliudBA (Table
pavement design,andtimingofthetraffic
lightscontribute
maintenanceandthe traffic
as wellwell totocontribute as wellto thetiming
noise levelof
generatedby the roadlights
traffic. contribute as3
Parcul UniversităĠii generated by the54.7
road traffic. Although high values of noise were measured, railway noise in Timisoara is
Table
VI. Recreational
Area – Park Measurements
4
Parc
Doina
Although62.4
high valueslimitedof tonoise
railway
noise
in
noise
were
measured,
railwaynoise
in Timisoara
Timisoara isis limi
lima narrowwere
path andmeasured,affects a small percentageof thepopulation.
LA
5
ParculRozelor
49.7
eq
path
and
affectsasmall
percentageofthepopulation.
Stationarynoise
sourcesintheindustrial
area
represent
only
4%
of
the
total
percentage of the population.
Recreational Area - Parks(dBA)
6
Parc Padurice
48.6noise sources
cases. With
enlargementof the cityarea
limits,areas tendonly
to move4%
outsideof
Stationaryinindustrial
represent
sources
inthethetheindustrial
areaindustrial
represent
only
4%of thethe total
total c17
Parc
Pârvan60.6
ParculCopiilor - Beethoven
53.2
the
town,
therefore
thepressureontheresidential
andprotected
area
diminishes
enlargement of the city limits, industrial
industrial areas
areas tend
tend toto movemove outside
outside thethe town,
town, therefore
therefore
28
Parc
Corneliu
Coposu – Eroilor
56.6
with the passageof time.
ParculConstructorilor
59.0protected significantly
the residential andarea
diminishes
significantly
withthepassage
of
time.
area
diminishes
significantly
withthepassageoftime.
3
Parcul UniversităĠii
54.7
Noise
levels in(recreational
protected areas (recreationaland medicaltreatment)
treatment) is high9Parcul Zona Soarelui
58.6 in protected
Noise levels
areas
and
isis highprotected
areas(recreationaland medical
medicaltreatment)high inin thethe4Parc Doina
62.4
in
the
daytime,inthemajority
of
cases
being
generated
by
the
road
traffic
due
tomajority
of
cases
being
generated
by
the
road
traffic
due
to
the
fact
that
Timisoara’smgenerated
by
the
road
traffic
due
to
the
fact
that
Timisoara’sm5Parcul Rozelor
49.7
areas
and
medicalfacilities
arelocated
near
very
busy
roads.
located
near
very
busy
roads.
tely a large numberofmedicaltreatment
units
and
care
facilities
are
built
near
major
6
Parc Padurice
48.6
Fiziologia - Physiology  2009.19.3(63)42
7ParculCopiilor- Beethovene having a significant
impactupon
this kind of area.53.2
From 14 hospitals and other units
8
Parcul Constructorilor – Eroilor
59.0
9Parcul Zona Soarelui
58.6

tely a large number of medical treatment units and care facilities are built near major

the fact that Timisoara’s main recreational areas and medical facilities are locatednear very busy roads.

REFERENCES

1.The European Commission: The Green Paper on Future Noise
Policy (COM(96) 540), Bruxelles 1996
2. The European Parliament and Council: Directive 2002/49/EC Assessment and Management of Environmental Noise, Bruxelles
2002
3. World Health Organization, Regional Office For Europe Euro-pean Centre For Environment And Health: Noise Guidelines For
Europe, 1999
4. Drăgănescu G: Vibraţii şi zgomote, Ed. Politehnica, Timişoara,
2000
5. STAS 10009-88: Acustica urbană. Limite admisibile ale niveluluide zgomot urban.
6. STAS 6161/3-82: Acustica urbană. Metode de determinare anivelului de zgomot în localităţile urbane.
7. STAS 10144/3-91: Elemente geometrice ale străzilor. Prescripţii
de proiectare.

EVALUAREA ZGOMOTULUI URBAN IN MUNICIPIUL TIMISOARA
REZUMAT
Zgomotul este o problema a societatii urbane moderne. Intr-o incercare de a evalua nivelul de zgomot urban in municipiul Timisoara, au fost efectuate 103
masuratori ale nivelului de zgomot, pe timp de zi, cu o durata de 8 ore pentru fiecare masuratoare. Masuratorile au indicat faptul ca zgomotul urban este
generat in mare masura de catre traficul rutier si afecteaza semnificativ zonele rezidentiale, recreationale si de tratament medical din oras.
Cuvinte cheie: zgomot urban, masuratori pe timp de zi ale nivelului de zgomot, trafic rutier

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Fiziologia - Physiology  2009.19.3(63)