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Fiziologia - Physiology  2008.18.4(60)CONTENTS
1. REVIEW: Correlation between HSP and Different Diseases .......................................................................................................................4
Carmen Tatu, V Ciocotisan, Gabriela Tanasie, Florina Bojin, Daniela Puscasiu, RF Tatu, Carmen Bunu
2. Moderate Alcohol Consumption and the Oxidants/Antioxidants Imbalance in Rat Pregnancy...................................................................7
Mihaela Rudeanu, Adriana Muresan, Simona Tache, Remus Moldovan, Doina Daicoviciu, Nicoleta Decea
3. Oxidant/Antioxidant Ratio: A Predictor for a Local Postoperative Wound Evolution ................................................................................11
Vrabete Maria, Parvu Alina, Parvanescu H, Novac M, Brujan Iulia, Amarascu Marina
4. Correlations between Oxidative Stress and Endothelin-1 Levels in Patients with Rheumatoid Arthritis...................................................14
Doina Baltaru, Irina Chis, Adriana Marton, Marius-Ionut Ungureanu, Ramona Simedrea, Adriana Muresan, Philippe Hoang, Nicoleta Decea, Mihai Dobre, Zubedat Mosa
5. Prevalence and Resistance to Antibiotics in Oral Viridans Streptococci....................................................................................................18
Dorina Dugaesescu, Camelia Szuhanek, Liliana Dragomirescu, Elena Hogea, Mihaela Craciunescu, Delia Berceanu-Vaduva,
Delia Muntean, Mihaela Popa, Ciprian Pilut, Roxana Rominu, Ramona Pirvan, Roxana Zugravu, Denis Serban
6. Patterns of Resistance to Antimicrobial Agents Observed in Acinetobacter Spp. Strains Isolated from Intensive Care Unit........................22
Liliana Dragomirescu,Monica Licker, Elena Hogea, Victor Dumitrascu, Marinela Holospin, Bianca Podac, Dorina Dugaesescu, Dorel Sandesc, Roxana Moldovan
7. Acinetobacter Strains Isolated from Nosocomial Infections....................................................................................................................25
Delia Muntean, Monica Licker, Delia Berceanu-Vaduva, Elena Hogea, Liliana Dragomirescu, Ciprian Pilut, Mihaela Craciunescu,
Mihaela Popa, Marcela Adamut, Dorina Dugaesescu, Denis Serban, Roxana Moldovan
8. Photodynamic therapy with TMPP and ZnTMPP in tumor bearing rats...................................................................................................28
Olteanu Elena-Diana, Adriana Filip, Simona Clichici, Adriana Muresan, Doina Daicoviciu, Rodica-Mariana Ion

CUPRINS
1. REIEW: HSP si corelatia cu diferite boli....................................................................................................................................................4
Carmen Tatu, V Ciocotisan, Gabriela Tanasie, Florina Bojin, Daniela Puscasiu, RF Tatu, Carmen Bunu
2. Consumul moderat de alcool şi balanţa oxidanţi/antioxidanţi în gestaţie la şobolani...............................................................................7
Mihaela Rudeanu, Adriana Muresan, Simona Tache, Remus Moldovan, Doina Daicoviciu, Nicoleta Decea
3. Raportul oxidanti/antioxidanti: un predictor al evolutiei spre vindecare a plagii postoperatorii..............................................................11
Vrabete Maria, Parvu Alina, Parvanescu H, Novac M, Brujan Iulia, Amarascu Marina
4. Corelatii intre stresul oxidativ si nivelul endotelinei-I la pacientii cu poliartrita reumatoida . .................................................................14
Doina Baltaru, Irina Chis, Adriana Marton, Marius-Ionut Ungureanu, Ramona Simedrea, Adriana Muresan, Philippe Hoang, Nicoleta Decea, Mihai Dobre, Zubedat Mosa
5. Prevalenta si rezistenta la antibiotice a Streptococilor Viridans orali......................................................................................................18
Dorina Dugaesescu, Camelia Szuhanek, Liliana Dragomirescu, Elena Hogea, Mihaela Craciunescu, Delia Berceanu-Vaduva, Delia Muntean,
Mihaela Popa, Ciprian Pilut, Roxana Rominu, Ramona Pirvan, Roxana Zugravu, Denis Serban
6. Modalitati de dobandire a rezistentei la agentii antimicrobieni investigate la tulpinile de Acinetobacter
Spp. izolate din unitatile de Terapie Intensiva...........................................................................................................................................22
Liliana Dragomirescu, Monica Licker, Elena Hogea, Victor Dumitrascu, Marinela Holospin, Bianca Podac, Dorina Dugaesescu, Dorel Sandesc, Roxana Moldovan
7. Tulpini de Acinetobacter izolate din infectii nosocomiale......................................................................................................................25
Delia Muntean, Monica Licker, Delia Berceanu-Vaduva, Elena Hogea, Liliana Dragomirescu, Ciprian Pilut, Mihaela Craciunescu,
Mihaela Popa, Marcela Adamut, Dorina Dugaesescu, Denis Serban, Roxana Moldovan
8. Terapia fotodinamica cu TMPP si ZnTMPP la soareci purtatori de tumori.................................................................................................28
Olteanu Elena-Diana, Adriana Filip, Simona Clichici, Adriana Muresan, Doina Daicoviciu, Rodica-Mariana Ion
2008.18.4(60)  Fiziologia - Physiology

3

REVIEW
CORRELATION BETWEEN HSP AND DIFFERENT DISEASESCARMEN TATU1, V CIOCOTISAN2, GABRIELA TANASIE1, FLORINA BOJIN1, DANIELAPUSCASIU1, RF TATU1, CARMEN BUNU1
1
Victor Babes University of Medicine and Pharmacy Timisoara, Romania
2
Ricercatore del Dipartimento di Bioingegneria, Politecnico di Milano, Italy

ABSTRACT
Circulating heat shock proteins (HSP) has been shown to correlate in diseases or disorders in which there is destruction or damage to target tissues or organs,including cardiovascular diseases, a wide range of human cancers and numerous autoimmune disorders such as autoimmune arthritis, type 1 diabetes
mellitus, atherosclerosis, multiple sclerosis, and other autoimmune reactions
Key words: heat shock proteins, cancer, neurodegenerative diseases, cardiovascular diseases

INTRODUCTION
Heat shock proteins are highly conserved proteins that, when produced
intracellularly, protect stress exposed cells. Heat shock response is nature’s device
to protect cells against environmental and physiological stresses. Cells under stresseither mount heat shock response and survive, or succumb to the stress and die(9). This phenomenon is conserved through evolution. The sequential molecular
events in heat shock response are extensively characterized. Its typical features
include drastic repression of normal transcription and translation pathways, andactivation of heat shock gene family by heat shock factor (HSF) (9). Circulating HSP
has been shown to correlate in diseases or disorders in which there is destruction
or damage to target tissues or organs, including cardiovascular diseases. Higher
circulating levels of HSP70 predict the future development of cardiovascular diseasein established hypertensives and a recent study demonstrating a decrease in HSP60
and HSP70 with advancing age (6).

Roles of HSPs in Cancer
Recently the roles of Hsps in cancer are attracting increasing attention andhave accelerated the study of heat shock response mechanism. Hsp expression
levels are high in various tumors, compared to normal cells. Overexpression of Hsps
inhibits the apoptotic machinery and promotes the scavenge of the misfolded
proteins in proteasome-mediated degradation (1, 9), possibly inducing the resistance to chemotherapy. The depletion of Hsp27, Hsp70 and Hsp90 using RNA interference or inhibitors, induces the cell growth arrest or cell death (4, 9). Thermotolerant cells induced by chronic heat shock stress showed the inhibition of JNK
activation and the rapid deactivation in response to heat shock (9). Studies onthe
up-and down-regulation of various Hsps, obviously suggest that Hsps expression
is well associated with the resistance of cell death in various tumors. Therefore, thestrategy to inhibit the expression and biological function of Hsps has great potential in cancer treatment. Hsp expression has been analyzed in relation to the histopathological characteristics of the tumor tissues (eg, tumor type, grade of differentiation), with the expression of other molecules (eg, estrogen receptors, c-myc,

mutated p53), and with patient parameters like sex and age (5). In addition, levelsof circulating Hsps and anti-HSP antibodies have been correlated with patient andtumor characteristics. Hsps are overexpressed in a wide range of malignant cells
and tissues. The presence of Hsps and antibodies to the Hsps in the serum of cancer
patients is still a new research area. Although it seems that autoantibodies tocertain Hsps are of significance as tumor markers in osteosarcomas, ovarian cancer
and others, at present, we need more studies to draw a clear conclusion on thisimportant subject. The same applies to the study of the polymorphism of the
Hsp70-2 gene. HSP expression becomes deregulated in cancer leading to elevatedexpression (3). Elevated HSP expression promotes cancer by inhibiting programmed
cell death (Hsp27, Hsp70) and by promoting autonomous growth (Hsp90) andleads to resistance to chemotherapy and hyperthermia. Tumor HSPs have anotherproperty that can be exploited in therapy. They are immunogenic and can be used
to form the basis of anticancer vaccines. Elevation in HSP levels may thus havecompeting effects in tumor growth, being required for tumor cell survival but
conferring a hazard for cancer cells due to their immunogenic properties. This dichotomy is also reflected by the approaches used to target HSP in therapy. Hsp
expression levels can help indicate the presence of abnormal changes during theprocess of carcinogenesis (in certain tissues). For example, Hsp27 is overexpressedin hyperplastic endometrium, and this protein appears as a marker of squamous
metaplasia in the uterine cervix; Hsp10 and Hsp60 are related with the process ofcarcinogenesis of the uterine cervix and colon; and Hsp70 is associated with carcinogenesis of the oral epithelium and as a marker of early hepatocellular carcinoma.
In oesophageal carcinomas, Hsp27 decreases during the carcinogenesis that ends
in adenocarcinomas but increases during the carcinogenesis that ends in squamous
carcinomas. Then, Hsps can be used as subrogate biomarkers of certain cancers.
The increased transcription of HSPs in tumor cells is due to loss of p53 function andto higher expression of the proto-oncogenes HER2 and c-Myc, and is crucial totumorigenesis (4). The HSP family members play overlapping, essential roles intumor growth both by promoting autonomous cell proliferation and by inhibiting
death pathways. Hsp expression correlates with the degree of differentiation incertain tissues. Hsps associated with higher differentiation are: Hsp27 and Hsp90

Received September 2008. Accepted October 2008. Address for correspondence: Dr. Carmen Tatu, Physiology Department, Victor Babes University of Medicine and Pharmacy, Eftimie Murgu Square No. 2A, 300041,
Timisoara, Romania; phone/fax: 0040256220479, e-mail: carmen.tatu@umft.ro4

Fiziologia - Physiology  2008.18.4(60)in endometrial carcinomas, Hsp27 in squamous carcinomas (uterine cervix, oral
epithelium), and Hsp27 as a marker of keratinocyte differentiation in the skin. In
contrast, Hsps associated with poor differentiation are Hsp70 in cancers of the breast,
ovary, and oral epithelium, Grp78 in lung carcinomas, and Hsp27 in astrocytomas.
At present, we do not have a clear explanation for these disparities and associations.
Hsp70 has been involved not only with poor tumor differentiation but also withincreased cell proliferation (breast, uterine cervix, lung), lymph node metastasis(breast, colon), increased tumor size (uterine cervix), presence of mutated p53(breast, endometrium), and higher clinical stage (oral, colon, melanoma). Several
Hsps are coexpressed in cancer tissues; in addition, certain Hsps can be significantly associated with other molecules. For example, Hsp27 has been associatedwith ERα in female breast carcinomas and endometrial carcinomas, but this protein
did not appear associated with ERα in male breast carcinomas, cervical uterine
carcinomas, hepatocellular carcinomas, and meningiomas (tissues that may express
ERα). It is interesting that Hsp27, which was first described as an estrogen-regulated protein, is significantly associated with ERα in the female breast and endometrium. These 2 organs are under strong estrogen and progesterone regulation.
Doxorubicin (DOX) is a widely used antitumor drug, but its application is limited
because of its cardiotoxic side effects (7). Hsp20 has been recently shown to protect
cardiomyocytes against apoptosis, induced by ischemia/reperfusion injury or byprolonged beta-agonist stimulation. However, it is not clear whether Hsp20 would
exert similar protective effects against DOX-induced cardiac injury. Actually, DOX
treatment was associated with downregulation of Hsp20 in the heart. To elucidate
the role of Hsp20 in DOX-triggered cardiac toxicity, Hsp20 was first overexpressedex vivo by adenovirus-mediated gene delivery. Increased Hsp20 levels conferredhigher resistance to DOX-induced cell death, compared to green fluorescent protein
control. Furthermore, cardiac-specific overexpression of Hsp20 in vivo significantly ameliorated acute DOX-triggered cardiomyocyte apoptosis and animal
mortality. Hsp20 transgenic mice also showed improved cardiac function andprolonged survival after chronic administration of DOX. The mechanisms underlying these beneficial effects were associated with preserved Akt phosphorylation/
activity and attenuation of DOX-induced oxidative stress (7). Coimmunoprecipitation studies revealed an interaction between Hsp20 and phosphorylated Akt. Accordingly, BAD phosphorylation was preserved, and cleaved caspase-3 was decreased in DOX-treated Hsp20 transgenic hearts, consistent with the antiapoptotic effects of Hsp20. Parallel ex vivo experiments showed that either infection
with a dominant-negative Akt adenovirus or preincubation of cardiomyocytes withthe phosphatidylinositol 3-kinase inhibitors significantly attenuated the protective
effects of Hsp20. Taken together, our findings indicate that overexpression of Hsp20
inhibits DOX-triggered cardiac injury, and these beneficial effects appear to be
dependent on Akt activation. Thus, Hsp20 may constitute a new therapeutic target
in ameliorating the cardiotoxic effects of DOX treatment in cancer patients. Hsp70
has been described as an important molecule in the assembly and trafficking ofsteroid receptors, and in breast cancer, Hsp70 has been found associated with ERα(5). It is of interest to mention that Hsp70 can increase ERα transcriptional activity
and growth in MCF-7 breast cancer cells (11), which in turn may explain the increased cell proliferation found in breast tumor biopsy samples that express Hsp70(5). In addition, Hsp70 has been associated and complexed with mutant p53 incancer cell lines (5, 11). This association has been studied in several cancer tissues,and the results have shown this association in certain cases only.The expression ofcertain Hsps can be correlated with the carcinogenic process as well as with thedegree of differentiation and cell proliferation, and moreover, they have been implicated in the regulation of apoptosis (5). The prognosis of a particular cancer
patient is very important in the clinic to individualize cancer treatments, to plan
the patient's follow-up, and to answer questions from the patient or relatives.
2008.18.4(60)  Fiziologia - Physiology

Overtherapy with cytotoxic drugs can be avoided in cancer patients if they arecorrectly identified as having good prognosis and vice versa. Hsp27 expression has
been associated with poor prognosis in ovarian, gastric, liver and prostate cancer,
and osteosarcomas (5). In contrast, Hsp27 expression has been associated withgood prognosis in endometrial adenocarcinomas, oesophageal cancer, and in
malignant fibrous histiocytomas. Although there are fewer studies in other cancers,
the data suggest that Hsp27 has no prognostic value in head and neck squamouscancer, bladder and renal cancer, and leukemia (except when associated withother markers). Hsp70 expression is correlated with poor prognosis in breast cancer,
endometrial cancer, uterine cervical cancer, and transitional cell carcinoma of the
bladder. This is consistent with the Hsp70 associations with poor differentiation,
lymph node metastasis, increased cell proliferation, block of apoptosis, and higher
clinical stage, which are markers of poor clinical outcome. In contrast, high Hsp70
expression was correlated with good prognosis in oesophageal cancer, pancreaticcancer, renal cancer, and melanoma. Hsp70 expression showed no correlation withprognosis in ovarian cancer, oral cancer, head and neck squamous cancer, gastricand prostate cancer, and leukemia (5). Hsp90 expression in cancer tissues andpresence of autoantibodies to Hsp90 have been correlated with poor prognosis inbreast cancer. In contrast, Hsp90 expression is associated with good prognosis inendometrial cancer. Loss of Hsp90 (and Hsp60) expression has been associated inbladder carcinoma with invasive recurrence risk. Hsp90 expression was of no
prognostic value in ovarian and oral cancer (5).

Roles of HSPs in neurodegenerative diseases
One of the characteristics of neurodegenerative diseases such as Alzheimer’s
disease (AD), Parkinson’s disease (PD), familial amylotrophic lateral sclerosis (FALS)
and poly Q disease is the formation of plaques/inclusion bodies which co-localize
with various chaperones and components of the ubiquitinproteasome degradation
system (5, 11). In AD, Hsp70 is found in the extracellular senile plaques and isbelieved to play a role in phagocytic digestion of amyloid plaques by microglia (5).
The unusual extracellular accumulation of chaperones is suggested to be facilitatedby calcium-induced interaction with lipid rafts (2). In AD intracellular myloid-β(Aβ) starts cellular dysfunction before it accumulates in extracellular plaques.
Grp78 interacts with amyloid precursor protein (APP) and inhibits the secretionof Aβ40 and Aβ42, suggesting that Grp78 might retain APP in the endoplasmic
reticulum and protect APP from β/γ-secretase cleavage into Aβ. Tau, a neuronal
microtubule binding protein that contributes to microtubule stability in normal
condition, is hyperphosphorylated in pathologic conditions and accumulates in
the neurofibrillary tangles, which are regarded as a hallmark of AD. At the sametime, Hsp27 is reported to bind to a hyperphosphorylated tau variant in humanbrain samples (13). Down regulation of Hsp70 and Hsp90 using RNA-mediatedinterference and Hsp90 inhibitor, geldanamycin, induced tau aggregation, andoverexpression of these chaperones showed the opposite effect. This suggests thatHsp70 and Hsp90 maintain tau in a soluble, functional conformation and prevent
tau aggregation (9). Expression in the brain of Hsp70 and Hsp27, is notable because
both proteins are highly inducible in glial cells and neurons following a wide range
of noxious stimuli including ischemia, epileptic seizure and hyperthermia (8). In
the central nervous system, constitutive expression of Hsp27 is limited to many(but not all) sensory and motor neurons of the brain stem and spinal cord, while
there is little or no constitutive expression of Hsp70. However, inducible expressionof both Hsp70 and Hsp27 is present in many areas of the brain and retina and isassociated with cellular resistance to a variety of insults. Molecular chaperones are
involved in most neurodegenerative diseases, providing protection at several levels.
5This shows that modulators of chaperone expression can have therapeutic benefits
for neurodegenerative diseases.
Heat Shock Protein and Its Role in Cardiovascular Disease
The expression of HSP in the early stages of cardiovascular disease might result
from one or a combination of factors. For example, risk factors for atherosclerosis
such as hyperlipidemia, diabetes, smoking, and hypertension cause oxidative stress.
Oxidative stress, in turn, may lead to the induction of HSP expression in vascularsmooth muscle cells (6, 10). In addition, prior research indicates that circulating
HSP70 levels predict the development of cardiovascular disease in subjects withestablished hypertension (6, 12) These authors suggest that HSP70 protects against
or modifies the progression of atherosclerosis in this subject group. The evidence issuggesting a protective role of HSP70 for cardiovascular diseases.
REFERENCES

1. Aghdassi, A, Phillips, P, Dudeja, V, Dhaulakhandi, D, Sharif, R Heat shock protein
70 increases tumorigenicity and inhibits apoptosis in pancreatic adenocarcinoma,
Cancer Res. 2007; 67:616−625.
2. Broquet AH, Thomas G, Masliah J, Trugnan G, Bachelet M, Expression of themolecular chaperone Hsp70 in detergent-resistant microdomains correlates with its
membrane delivery and release, J. Biol. Chem, 2003; 278:21601−21606.
3. Calderwood SK, Ciocca DR, Heat shock proteins: stress proteins with Janus-like
properties in cancer, Int J Hyperthermia. 2008;24(1):31-9.
4. Calderwood SK, Khaleque MA, Sawyer DB, Cicca DR, Heat shock proteins in cancer:
chaperones of tumorigenesis, Trends Bichem, 2006; 31:164-172.

5. Ciocca DR, Calderwood SK, Heat shock proteins in cancer: diagnostic, prognostic, predictive, and treatment implications, Cell Stress Chaperones, 2005;
10(2):86–103.
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Bausero M, Ogawa K, Perls T, Asea A, Cardiovascular Disease Delay in Centenarian
Offspring: Role of Heat Shock Proteins, Ann NY Acad Sci 2004; 1019: 502–505.
7. Fan GC, Zhou X, Wang X, Song G, Qian J, Nicolaou P, Chen G, Ren X, Kranias EG, Heatshock protein 20 interacting with phosphorylated Akt reduces doxorubicin-triggered
oxidative stress and cardiotoxicity, Circ Res. 2008, 21;103(11):1270-9.
8. Franklin TB, Krueger-Naug AM, Clarke DB, Arrigo AP, Currie RW, The role of heatshock proteins Hsp70 and Hsp27 in cellular protection of the central nervous system,
Int J Hyperthermia. 2005;21(5):379-92.
9. Hee-Jung K, Hwang N, Lee K, Heat Shock Responses for Understanding Diseasesof Protein Denaturation, Mol Cells, 2007; 23(2):123-131.
10. Liao DF, et al. Purification and identification of secreted oxidative stress- induced
factors from vascular smooth muscle cells, J Biol Chem, 2000;275:189–196.
11. Muchowski P, Wacker LJ, Modulation of neurodegeneration by molecular chaperones, Nat. Rev. Neurosci, 2005;6:11−22.
12. Pockley AG, et al. Serum heat shock protein 70 levels predict the development of atherosclerosis in subjects with established hypertension, Hypertension,
2003;42:235–238.
13. Shimura H, Miura-Shimura Y, Kosik KS, Binding of tau to heat shock protein 27
leads to decreased concentration of hyperphosphorylated tau and enhanced cell
survival, J. Biol. Chem, 2004;279:17957−17962.
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activity, Proc 1st Int Cong Stress Responses Biol Med, 2003,10–14:72-73.

HSP SI CORELATIA CU DIFERITE BOLIREZUMATS-a demonstrat ca exista o corelatie intre proteinele de stres din circulatie (HSP) si anumite maladii in care se observa distrugerea tesutului sau organului
tinta. In aceste categorii de boli sunt incluse maladiile cardiovasculare, o larga varietate de cancere, numeroase boli autoimune cum ar fi artrita, diabetul
zaharat de tip 1, ateroscleroza, scleroza multipla si altele.
Cuvinte cheie: proteine de stres, cancer, maladii neurodegenerative, boli cardiovasculare

6

Fiziologia - Physiology  2008.18.4(60)MODERATE ALCOHOL CONSUMPTION AND THE OXIDANTS/ANTIOXIDANTS IMBALANCE IN RAT PREGNANCYMIHAELA RUDEANU, ADRIANA MURESAN, SIMONA TACHE, REMUS MOLDOVAN, DOINADAICOVICIU, NICOLETA DECEA
Physiology Department “Iuliu-Hatieganu” University of Medicine and Pharmacy Cluj-Napoca

ABSTRACT

Alcohol induced oxidative stress (AIOS) and alcohol induced nitrosative stress (AINS) are linked to metabolism of ethanol, involving both mitochondrialand microsomal systems. Low ethanol intake is known to have a beneficial effect on cardiovascular disease; it attenuates hypertension and atherosclerosis.
Chronic low ethanol intake confers its beneficial effect mainly through its quality to increase antioxidant capacity and lower advanced glycation end products.
Moderate alcohol consumption determined significant decrease of MDA, PC and SH and did not significant increase DH in non pregnant rats. Moderate alcohol
consumption determined significant increase of MDA, and PC and significant decrease of DH in prim pregnant rats. Moderate alcohol consumption determinedsignificant increase of MDA, and PC and significant decrease of DH and SH in multi pregnant rats.
Key words: alcohol, oxidative stress, pregnancy, rat

INTRODUCTION
The alcohol (ethanol) is the most widely consumed drug worldwide. The
immoderate alcohol consumption is associated with high levels of morbidity,
mortality and antisocial actions. A high-dose chronic alcohol consumption induces:iron overhead, alcoholic skeletal and heart myopathy, chronic alcohol mediatedliver disease, neurological disorders (14,4,7,12,8,6), alcohol-induced congenital
malformations (17), neuro-endocrine alterations in adult offspring (5) and others
liver and lung diseases (11,13).
The pathogenic mechanisms that lie between alcohol intake and morbidity
involve multiple pathways. Recent studies indicate that reactive oxygen species(ROS) and reactive nitrogen species (RNS) play an important role. ROS and RNS
are able to cause various cellular injuries, such as DNA damage, lipid peroxidationand protein modifications (1,18).
Alcohol induced oxidative stress (AIOS) and alcohol induced nitrosative stress(AINS) are linked to metabolism of ethanol, involving both mitochondrial andmicrosomal systems. AIOS and AINS are the results of a combined impairment ofantioxidant defenses (SOD, CAT, GSH/GSSG, vitamin E and A, Se) and the productionof ROS and RNS (3).
Moderate or low chronic alcohol consumption is associated with a lower risk of
all-cause mortality and morbidity (8). Low ethanol intake is known to have a beneficial effect on cardiovascular disease; it attenuates hypertension and atherosclerosis.
Chronic low ethanol intake confers its beneficial effect mainly through its quality toincrease antioxidant capacity and lower advanced glycation end products.

OBJECTIVES
The aims of the present study are to follow the influence of moderate ethanol
intake on the balance oxidants/antioxidants in non pregnant female rats and to study
the influence of moderate ethanol intake on the balance oxidants/antioxidants inpregnant primipara and multipara female rats.

METHODS
Experimental groups were formed of Wistar female rats, whichwere used for analysis and divided into six groups (n = 10 rats/group), with aweight of 200-220 grams, as follows:
Group I – control non pregnant female rats
Group II – primipare females
Group III – multipare females
Group IV – non pregnant female rats exposed to alcoholGroup V – primipare females exposed to alcoholGroup VI – multipare females exposed to alcohol
The experiment was made in the Laboratory for experimental exploration at
the Department of Physiology of “Iuliu Hatieganu” University in Cluj-Napoca in the
period May-June 2008.
Animals were prepared for gestation using Ladosi method (10) adapted forrodents. The alcohol administration consisted in quantities of 0.5 ml 20% alcohol
per animal/day by gavages.
Biochemical methods
Several parameters were identified in the serum, including: a) indicators of
the oxidative stress (malonaldehyde, fluorimetric Conti method, 1991; carbonylated
proteins, Reznick method, 1994), and b) indicators of the oxidative defense (hydrogen donor capacity, Janazewska method, 2002; content of sulphydril groups,
Hu method, 1994).
The samples were analyzed in the Laboratory for experimental oxidative
stress exploration at the department of Physiology of “Iuliu Hatieganu” University
in Cluj-Napoca.
The blood to analyze was taken from the retro orbital sinus of the rats for thecontrol group and the pregnant group (day 21-23), respectively.
Statistical analysis
Results were given as measures of central tendency and dispersion. The t-test

Received November 2008. Accepted December 2008. Address for correspondence: Dr. Mihaela Rudeanu, Physiology Department, “Iuliu Hatieganu” University of Medicine
and Pharmacy Cluj-Napoca, Clinicilor Street 3-5, 400006, Cluj Napoca, Romania
2008.18.4(60)  Fiziologia - Physiology

7

for equal or unequal variances (the variances were tested with Levene test) was used
for comparisons between two means of independent groups. The variables were normal distributed, the normal distribution were tested with Kolmogorov-Smirnov test.
Statistical significance was assessed with p less than 0.05 and two-tailed tests,
computed using SPSS 13.0 and Statistica 7.0.

RESULTS
Alcohol exposure in non pregnant females (Group IV) (Table I
and II) resulted in significant decrease of MDA, PC and SH (in comparison with non
pregnant unexposed to alcohol females - Group I); DH values that didn’t significantly
differ from non pregnant unexposed to alcohol females (Group I).
In primipare females (Group V) (Table I and II) alcohol exposure
induced significant increase of MDA (in comparison with primipara gestation females
unexposed to alcohol - Group II), while the level of MDA was not significantly different from the level of MDA of non pregnant unexposed to alcohol females (GroupI); significant increase of PC (in comparison with primipare females unexposed toalcohol - Group II), also increase PC in comparison with non pregnant unexposed
to alcohol females (Group I); the values of SH were not significantly different fromprimipare females unexposed to alcohol (Group II); significant decrease of DH (incomparison with primipare females unexposed to alcohol - Group II), the level of
MDA was not significantly different from the level of MDA of non pregnant females
exposed to alcohol (Group IV).
In multipare females (Group VI) (Table I and II), the following
were noticed: significant increase of MDA (compared with multipare females
unexposed to alcohol - Group III); significant increase of PC (compared multipare
females unexposed to alcohol - Group III), the level of PC was not significantly different from the level of PC of non pregnant females unexposed to alcohol (GroupI); significant decrease of SH (compared with multipare females unexposed toalcohol - Group III), the level of SH was not significantly different from the level of
SH of non pregnant exposed to alcohol females (Group I); significant decrease of
DH (compared with multipare females unexposed to alcohol - Group III), the levelof DH was not significantly different from the level of DH of non pregnant females
exposed to alcohol (Group IV).
The groups exposed to alcohol (I, II, III) and the groups unexposed to alcohol(IV, V, VI) are summarized in Table I.

The p probabilities (Table II) show significant differences between the groups
exposed and unexposed to alcohol.
Table II. The probability p between study groups for the indicators ofantioxidants / oxidants scales
Statistical significance p between groups
GroupsMDA
PC
SHDH
Group IGroup IIGroup III
Group IGroup IGroup IIGroup IV
Group IV
Group VGroup IGroup IGroup IIGroup IIGroup III
Group III-

Group IV
Group VGroup VI
Group IIGroup III
Group III
Group VGroup VI
Group VI
Group VGroup VI
Group IV
Group VI
Group VGroup IV

0.001
0.004
0.04
0.007
0.00001
0.00010.001
0.42
0.00040.36
0.00010.21
0.007
0.0001
0.051

0.003<0.0001
0.003<0.0001
0.003
0.0004<0.0001
0.002
0.27
0.04
0.53
0.004
<0.0001
0.78<0.0001

0.03
0.14
0.001
0.61
0.002
0.0001
0.01
0.01
0.95
0.62
0.62
0.04
0.20
0.001
0.0003

0.06
0.002
0.02
0.001
0.004
0.67
0.16
0.29
0.88
0.62
0.55
0.01
0.01
0.049
0.01

Correlation between the indicators of antioxidants / oxidants scales
Our study set off acceptable correlation between MDA and PC in all studiedgroups (Table III). But, the correlation was in some groups positive, in others was
negative. We can say that the correlation is between size (they grow/shrink in thesame way), but there wasn’t correlation between direction. The same affirmation
we put in evidence between SH and DH (Table III).
Between PC and DH there was an acceptable correlation in 4 from 6 groups(Table III). We can say that PC and DH are significantly correlated (Table IV).
In Table III there is presented the coefficient of correlation Pearson r between
the indicators of antioxidants / oxidants scales.

Table I. Descriptive measures of indicators for the antioxidants / oxidants scales in study groups
Conf. interval of
Mean
St. dev. St. error
95%
MDA
1.87
0.36
0.11
1.61
2.13
MDA
PC
1.58
0.38
0.12
1.31
1.85
PC
Group IGroup IV
SH
0.21
0.06
0.02
0.17
0.26
SH
DH
32.23
5.03
1.59
28.64 35.83
DH
MDA
1.45
0.24
0.08
1.27
1.62
MDA
PC
0.66
0.22
0.07
0.50
0.82
PC
Group IIGroup V
SH
0.20
0.04
0.01
0.17
0.23
SH
DH
42.06
6.12
1.93
37.68 46.44
DH
MDA
1.01
0.16
0.05
0.89
1.12
MDA
PC
1.09
0.22
0.07
0.93
1.25
PC
Group III
Group VI
SH
0.30
0.05
0.01
0.27
0.33
SH
DH
40.85
6.43
2.03
36.25 45.45
DH
8

-

Mean

St. dev.

St. error

Conf. interval of 95%

1.27
1.06
0.08
36.01
2.06
1.91
0.22
33.34
1.17
1.71
0.23
33.71

0.35
0.31
0.16
3.32
0.54
0.29
0.03
4.68
0.16
0.49
0.04
5.78

0.11
0.10
0.05
1.05
0.17
0.09
0.01
1.48
0.05
0.16
0.01
1.83

1.02
0.84
-0.03
33.64
1.68
1.70
0.20
29.99
1.05
1.35
0.20
29.58

1.52
1.28
0.20
38.38
2.45
2.12
0.24
36.68
1.28
2.06
0.25
37.84

Fiziologia - Physiology  2008.18.4(60)Table III. The coefficients of correlation Pearson r between MDA, PC, SH and DH in the study groups
The coefficient of
MDA – PC
MDA - SHMDA - DHPC -SH
correlation Pearson r
Group I
0.41**-0.01*
0.03*-0.58***
Group II
-0.50**-0.21*
0.52***
0.35**
Group III
0.35**
0.15*
0.33**
0.02*
Group IV
-0.75****-0.06*
0.56***
0.17*
Group V
0.43**
0.49**
0.13*
0.05*
Group VI
-0.25**-0.35**
0.32**
0.47*** inexistent or weak correlation , ** acceptable correlation,*** good correlation, **** very good correlation (Colton classification)

PC - DHSH - DH-0.17*-0.33**
0.05*-0.61***-0.32**
0.11*

0.48**-0.42**-0.44**-0.49**
0.65***
0.51***

The coefficients of correlation Pearson r between MDA, PC, SH and DH in all 6 studied groups are reproduced in Table IV.
Table IV. The coefficients of correlation Pearson r between MDA, PC, SH and DH in all 6 groups
Groups I-VI (n=60)

MDA - PC

MDA - SHMDA - DHPC -SHPC - DHSH - DHThe coefficients of correlation Pearson r

0.33

-0.04

-0.14

0.13

-0.49

0.05

p

0.01

0.79

0.29

0.31

0.00010.72

DISCUSSIONS
During the normal pregnancy an oxidative and nitrosative stress is produced
affecting the balance O/AO, O is raised and AO is decreased (15).
The mechanism of SON production in the normal pregnancy are: maternal
metabolism and rate of oxygen utilization at the mitochondrial level increase withconsecutive production of oxygen radicals, the ischemia-reperfusion caused byincreased O2 consumption, and the physiological hypoxia which produces variationof the pO2 at the placental level.
The endothelial mechanical stress (2) can be explained as increased estrogenand choriogonadotropic hormone levels. Stimulation of the hormonal secretion has a
pro-oxidant effect. The sources of SRO and SRN are: maternal, placental and vascular.
The ROS and RNS have benefic effects during gestation (16) due tothe fact that they control cellular growing, differentiation and transformation into
embryo and fetus (O2·¯, H2O2 (low quantities) → the regulation molecules → thetranscription factors and gene expression modifications), as well as regulation ofvascular tone in fetoplacental blood vessel (O2·¯, NO·¯, ONOO¯). ROS and RNS are
involved in metabolism of arachidonic acid with endoperoxides and PGI2 formation,
resulting in implications of GC and GMPc system.
The negative effects of SRO and SRN are could be differentiated into:a) cellular destruction and inflammation due to activation of neutrophiles withincrease activity of myeloperoxidasis and NADPH oxidation, resulting in increased
SRO and increased neutrophils aggregation to the endothelium, ischemia andinjuries, and b) teratogen and toxic effects.
The chronic maternal alcohol intake in large quantities and the crossing overto placenta produces the fetal alcoholic syndrome and becomes a risk factor for theembryonic development, fetal development (teratogenic effects) and for the adultdescendents (neuroendocrine, pulmonary, hepatic diseases and mental retardation).
The teratogen, neurotoxic and hepatotoxic effects of the maternal alcohol
intake induced to the descendents, experimentally proven, can be due to direct
peroxidative mechanism of the alcohol or indirect peroxidative mechanism ofacetaldehyde ( the alcohol metabolite) (11, 13).
Adverse oxidative effects of ethanol-exposed female rats can be ameliorated
by antioxidative treatment vitamin E (α-tocopherol) (17).
2008.18.4(60)  Fiziologia - Physiology

The data in the literature have shown that antioxidant pretreatment withmelatonin and U83836E does not ameliorate alcohol-induced Purkinje cell loss inthe developing rat cerebellum (9).
Our experiments show that moderate intake of alcohol on witness non pregnant
animals induces a reduction of SO.
Pregnancy in females induced a series of changes, depending on study group(Table II). For primipare females (Group II): significant decrease of MDA
and PC compared with non pregnant females (Group I); the values of SH were notsignificantly different from the values of SH of non pregnant females (Group I);
significant increase of DH compared with non pregnant females (Group I).
For multipare females (Group III): significant decrease of MDA and
SH in comparison with non pregnant females (Group I) and, also for primipare
females (Group II), significant decrease of PC in comparison with non pregnant
females (Group I), but significant increase for the primipare females (Group II),significant increase of DH in comparison with non pregnant females (Group I), but
no significant difference compared with primipare females (Group II).

CONCLUSIONS
Moderate alcohol consumption determined significant decrease of MDA, PC
and SH and did not significant increase DH in non pregnant rats.
Moderate alcohol consumption determined significant increase of MDA, andPC and significant decrease of DH in primipare rats.
Moderate alcohol consumption determined significant increase of MDA, andPC and significant decrease of DH and SH in multipare rats.
REFERENCES
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65(3):278-290.
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4. Dejica D (sub red.). Statusul oxidativ în bolile interne. Ed. Casa Cărţii de Ştiinţă, ClujNapoca, 2000: 130, 413-431

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5. Dembele K, Yao XH, Chen L, Nyomba BL. Intrauterine ethanol exposure results in hypothalamic oxidative stress and neuroendocrine alterations in adult rat offspring. Am J Physiol
Regul Integr Comp Physiol. 2006, 291(3):R796-802
6. Enache M, Van Waes V, Vinner E, et al. Impact of an acute exposure to ethanol on theoxidative stress status in the hippocampus of prenatal restraint stress adolescent male rats.
Brain Res. 2008, 29;1191:55-62
7. Fernandez-Solà J, Preedy VR, Lang CH et al. Molecular and cellular events in alcohol-induced
muscle disease. Alcohol Clin Exp Res. 2007, 31(12):1953-1962
8. Ferreira MP, Willoughby D. Alcohol consumption: the good, the bad, and the indifferent.
Appl Physiol Nutr Metab. 2008, 33(1):12-20
9. Grisel JJ, Chen WJ. Antioxidant pretreatment does not ameliorate alcohol-induced Purkinje
cell loss in the developing rat cerebellum. Alcohol Clin Exp Res. 2005, 29(7):1223-1229
10. Ladoşi I. (sub red.). Embriotehnologie animală. Ed. Victor Melenti, Cluj-Napoca, 1999:
49-51
11. Lee RD, An SM, Kim SS et al. Neurotoxic effects of alcohol and acetaldehyde during
embryonic development. J Toxicol Environ Health A. 2005, 68(23-24):2147-2162

12. Mantena Mantena SK, King AL, Andringa KK et al. Novel interactions of mitochondria andreactive oxygen/nitrogen species in alcohol mediated liver disease. World J Gastroenterol.
2007, 13(37):4967-4973
13. Perez MJ, Velasco E, Monte MJ, Gonzalez-Buitrago JM, Marin JJ. Maternal ethanol
consumption during pregnancy enhances bile acid-induced oxidative stress and apoptosis
in fetal rat liver. Toxicology. 2006 Aug 15;225(2-3):183-194
14. Sies SH. Oxidative stress: Oxidants and antioxidants. Experim Physiol, 1997, 82: 291295
15. Todea C. Statusul oxidativ în patologia ginecologică şi obstetricală. Ed. Casa Cărţii deŞtiinţă, Cluj-Napoca, 2008, 49-53
16. Vasdev S, Gill V, Singal PK. Beneficial effect of low ethanol intake on the cardiovascularsystem: possible biochemical mechanisms. Vasc Health Risk Manag. 2006, 2(3):263-276
17. Wentzel P, Rydberg U, Eriksson UJ. Antioxidative treatment diminishes ethanol-induced
congenital malformations in the rat. Alcohol Clin Exp Res. 2006, 30(10):1752-1760
18. Wu D, Zhai Q, Shi X. Alcohol-induced oxidative stress and cell responses. J Gastroenterol
Hepatol. 2006, 21 Suppl 3:S26-29

CONSUMUL MODERAT DE ALCOOL ŞI BALANŢA OXIDANŢI/ANTIOXIDANŢI ÎN GESTAŢIE LA ŞOBOLANIREZUMATStresul oxidative (AIOS) şi nitrozativ (AINS) indus de alcool sunt corelate cu metabolismul etanolului, implicând atât sistemul mitocondrial şi microzomal.
Aportul redus de etanol este cunoscut a avea un effect benefic în patologia cardiovasculară. Consumul cronic redus de etanol este benefic în principal princalitatea lui de a creşte capacitatea antioxidantă şi reducerea produşilor finali de glicare. Consumul moderat de alcool a determinat scăderi semnificative de
MDA, PC şi SH şi nu a crescut semnificativ DH la şobolanii negestanţi. Consumul moderat de alcool a determinat creşteri semnificative de MDA şi PC şi scăderi
semnificative ale DH la şobolani în prima sarcină. Consumul moderat de alcool a determinat creşteri semnificative de MDA şi PC şi scăderi semnificative ale
DH şi SH la şobolanii cu sarcini multiple.
Cuvinte cheie: alcool, stress oxidative, sarcină, şobolan

10

Fiziologia - Physiology  2008.18.4(60)OXIDANT/ANTIOXIDANT RATIO: A PREDICTOR FOR A LOCALPOSTOPERATIVE WOUND EVOLUTION
VRABETE MARIA1, PARVU ALINA2,
AMARASCU MARINA1
1
Deptartment of Pathophysiology - Plastic Surgery -UMF Craiova
2
Deptment of Pathophysiology, UMF Cluj-Napoca

PARVANESCU H1,

NOVAC M1,BRUJAN IULIA1,

ABSTRACT
Surgical procedures often unbalance the organic systems, leading to a tendency to one or many others disequilibrium. Besides the operative intervention itself
and many well-known clinical risk factors, including immobility, infections, a.s.o. there are various other perioperative factors that are being demonstrated tointerfere with all the systems, of organism such as hypothermia, metabolic acidosis, fluid-coagulant imbalance, volume expanders, decreasing of antioxidant
capacity of plasma and the receptivity to therapy. The first several hours after surgery there are marked increases to a hypercoagulable and hypofibrinolytic
state and increasing of reactive O2 species (ROS). The levels of mediators released in this period, fluctuates rapidly and their degree of perturbation is dependent not only on the type, degree and duration of surgery, but also on the timing of blood collection. We have tried to introduce a new method of laboratory,
research, proposed by Oczan E (12), to reveal the role of antioxidant capacity of plasma, in a dynamically evolution.
Key words: oxidants, wound healing, antioxidant capacity of plasma

INTRODUCTION
Early phase of reactivity, in post-anesthetic-surgical stress, to surgical patients,
contributes significantly, to create the wound healing and the typology of morphoclinical evolution. The interindividual variability response to surgical stimuli is
profound, because of genomic variations within most of genes, in each patients,
every gene encoding proteins, involved in signal transduction, in inflammatory
process (Toll receptors), implicated in surgical wound extent and in individual
cellular response to injury. Clinical significance of genomic markers remain to betested, and may be used to participate to elect the strategy of therapeutic management into the future. Many authors (14,15) consider that individual genotype
is implying and is strongly influenced by the balance between the two systems[oxidants (O)/ antioxidants (Ao)], which has a crucial role upon the cellular cycle andcellular homeosthasis, both, interacting and inter-influencing the wound healing.
Because of these effects (9,14,15) it is appreciated that to modulate the oxidative
stress, or the antioxidant capacity of serum, become one of the best therapeuticmanner, for obtaining an efficient wound healing. It is known that the oxidants/
antioxidants systems
are interactive, so the potent free radical reactions, starting with OH_, do not
end in only a one-step reaction; generally, they continue, even forming a free radical
chain reaction, and antioxidants prevent the prolongation of this kind of reactions.
To show the antioxidant responses of the antioxidant components of serum, against
the ascendant potent free radical reactions, is one of the major pathophysiologicalmechanism of organism defense, which could be included through the therapeutic
objectives of surgical patients’ management.
Aims of the study were the following: 1.To evaluate thekind of wound healing (per primam, per secundam, a.s.o.) in postoperative period, tosurgical patients, who received antioxidants, associated to conventional therapeutic
schema; 2. To observe if the reactive oxigen species (ROS) increase production in
operated region and could induce, at a distance, into the healthy (“naïve”) vascularendothelium, the same alterations; 3. To evaluate the quality of a novel parameter
offered, by using an indirect performant and automated bioquantitative assay,
proposed by Oczan Erel (12); 4. To estimate the quality of tissular response, during
the period of wound healing process, correlated to O/Ao equillibrium, before theappearance of any perturbation, at a distance, and may be to block this one, bymeans of a locally therapy.

MATERIALS AND METHODS
Our clinical study was made upon: healthy subjects, volunteers who gave
us, their acceptance and surgical patients from the Plastic and Reconstructive
Surgery, from the Hospital nr.1 Craiova, UMF Craiova. The study was approved by
Ethics Commitee of UMF Craiova. The study included: 60 patients, age: 40-60 years,
submitted to a mild anesthetic surgical stress, divided into four groups: group A:10
cases(17%), control, healthy volunteers, group B: 28 cases (48%): with localised
focus of bacterial infection, or infectiouse complications, at risk, group C: 12 cases(20%): several with multi-organic-dysfunctions (MODS) and failure (MOF), group
D:10 cases (17%), with SIRS: 6 cases: septic shock (2 cases) and death (2 cases).
For critically ill patients the scores: SOFA and APACHE, were calculated. Fromeach group we have determined, from blood (serum) the values of circulating
proinflamatory cytokines (IL1, TNF alfa), C reactive protein (CRP), procalcitonin(PCT), coagulabillity status of plasma, leukocytes, thrombocytes number, total
antioxidant response (TAR), or total antioxidant capacity of plasma (TAOP). The
study was approved by Ethical Committee of University of Medicine and Pharmacyand Hospital no.1 of Craiova.
Technique
Chemicals: Ferrous ammonium sulfate, ortho-dianisidine dihydrochloride(3-3V-dimethoxybenzidine), vitamin C (L(+) ascorbic acid), bilirubin, uric acid,
reduced glutathione (GSH), (F)-catechin, 5,5V-dithiobis-(2-nitrobenzoic acid)

Received August 2008, Accepted October 2008. Address for correspondence: Dr. Maria Vrabete, Pathophysiology Department – Plastic Surgery, Craiova, Romania
2008.18.4(60)  Fiziologia - Physiology

11Fig.1. Plasma total peroxide µmol. H2O2.(p < 0.001)

Fig.2. Plasma TAOP: µmol.Trolox equiv/l (p<0.001)

Fig.3. TAR: mmol.Trolox equiv/l

Fig.4. OSI (p<0.001)(DTNB), ethylenediaminetetraacetic acid (EDTA), 2,2V-azino-bis (3- ethylbenzthiazoline-6-sulfonic acid) (ABTS), potassium persulfate, glucose, ribose, saccharoseand sodium citrate were purchased from Sigma Co. and Merck Co. The watersoluble
analogue of vitamin E (Trolox; 6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylicacid) was from Sigma- Aldrich Chemical Co. All chemicals were ultra pure grade andtype I reagent grade deionized water was used. Venous blood samples obtained
were collected into tubes (and heparinized tubes) and serum (and plasma) was
separated from cells by centrifugation at 1500 _ g for 10 min. Serum (and plasma)
samples were run immediately or stored at _80jC. Antioxidants: stock solutions(1.0 mM) of ascorbic acid, glutathione and (F)-catechin were separately preparedin saline solution (0.9% NaCl). Uric acid and solid bilirubin were dissolved in 10
mM NaOH solution. Trolox was dissolved in phosphate buffer (10 mM, pH 7.4).
Apparatus: A Cecil 3000 spectrophotometer with a temperature controlled cuvetteholder (Cecil), and an Aeroset automated analyzer (Abbott). Assay principle of themethod: a standardized solution of Fe2+–o-dianisidine complex reacts with astandardized solution of hydrogen peroxide by a Fenton-type reaction, producing
OH_. These potent ROS oxidize the reduced colorless o-dianisidine molecules
to yellow-brown colored dianisidyl radicals at low pH. The oxidation reactions
progress among dianisidyl radicals and further oxidation reactions develop. The
color formation is increased with further oxidation reactions. Antioxidants in thesample suppress the oxidation reactions and color formation. This reaction can bemonitored by spectrophotometry. Assay calibration: the suppression of the color
formation is calibrated with Trolox, which is widely used as a traditional standard
for TAR measurement assays, so the results in this assay are expressed as in termsof millimolar Trolox equivalent per liter. Oczan Erel (12).

non- receiving to antioxidant postoperative exogenous therapy, didn’t receive
antioxidants, before intervention.
TAR and TAOP values were different in control, face to group B, receiving “peros”fruits, vegetables, rich in antioxidant polyphenolic compounds, in the first 4- 12hours, postoperative. The total antioxidant capacity of group B was little increased,
face to group A. For C-D groups, a great potent free radical reactions were dominant,an implication of plasma antioxidant capacity to assure the balance between oxidants/antioxidants, was associated to a low TAR serum level. (Figures 1, 2, 3, 4)
PCT values rise earlier, before CRP and CK, and have a high specificity, being atrue marker for patients with multiorgan dysfunctions and failure. We appreciatedthat the increasing of plasma concentrations of CRP is associated to minor infections,but do not adequately reflected the severity of infections.
Hipercoagulability was common, in postoperative status in 70% of cases, from
B, C and D groups. We observed also, great fluctuations of thrombocytes count, ingroup D, and considered the decreasing of their value as a dilutional thrombocytopenia, not as a direct effect of oxidants upon the leukocytes, thrombocytes and/or erhytrocytes.

RESULTS
Morphological and clinical aspects of surgical wounds showed the threekinds of healing: per primam, per secundam and local complications associatedwith systemic bad evolution.
Cellular edema was the most important clinical sign, which was correlated
to a lower TAR level. We observed the appearance of the resistance to antibio-or/
and corticoid therapy, in T2 period to cases from C, D groups; the last group,
12

DISCUSSION
After tissue injury, produced by programmed trauma (surgical act) or activation of inflammatory defence reaction, the endothelial cell properties may bereversed. The endothelium properties of endothelial cells are due to three types ofactivities: a) control of vaso-regulation, by the release of vasomotor components
as endothelin ET-1 prostacyclin (PGI2) and nitrite oxide (NO); b) anti-thrombotic,
anticoagulant properties, such as the capture and degradation of thrombogenous
substances (ADP, 5-HTP) and through the effects of active products on platelets; c)anti-adhesive properties, exerted by endothelial cells, express adhesion molecules,
that can be modulate by mechanical or biochemical stimulations. Variations in local
shear and oxidative stress may also modify the secretion of vasomotor substances
and the postoperative locally evolution could take any way. Reactive oxygen species (ROS) are produced in metabolic and physiological processes, and oxidative
reactions occurred in organisms, remove them via enzymatic and non-enzymatic
antioxidative mechanisms. In intra-and postoperative periods, the tissues’incisions
Fiziologia - Physiology  2008.18.4(60)create the conditions for apoptosis and also, to shift the oxidative/ antioxidativebalance, toward the oxidative status. ROS are responsible for the oxidative injury
of biomolecules, indirectly, by producing OH_ via Fenton reaction and/or ironcatalyzed Haber– Weiss reaction. Oxidized molecules have the capacity to form new
radicals, leading to radical chain reactions that consume the antioxidant capacity ofplasma. Decreasing of antioxidant molecules generates the disequilibrium oxidants/
antioxidants and creates the conditions to amplify the intracellular force of contraction that could dislocate the cytoplasm from the cell membrane. The intercellular
gaps and cross bridging disappear, so the hydrostatic and osmotic gradient direct
the circulation of water, through extracellular to intracellular space and in opposite
sense, making possible a massive cellular death and blocking the wound efficienthealing. This status of irreversibility and dezorganisation could be explained bymeans of the activation of an autocrin and paracrin mechanism from the altered
endothelial cells (EC), which is transferred and extended at a distance.
Our results sustain that the mechanism generating the local and at a distancecellular destruction, is represented by the alteration of antioxidant capacity ofcirculatory components, differentiated, from one region to another. Because themeasurement of different antioxidant molecule separately, is not practical andantioxidant effects of them are additive, actually, total antioxidant response (TAR) ofa sample is measured and the result obtained is defined and recognized by the following names: total antioxidant capacity, total antioxidant activity, total antioxidant
power, total antioxidant status. By means of the new parameter proposed (12), it
could be evaluated the capacity of tissues to resist to injury or to be sensible to theapplied therapy. Understanding the basic mechanisms underlying the locally woundhealing failure, suggests that efficient therapies have to be developed, and applied
early, after pathophysiological principles, before the appearance of the irreversible
changes, and to reduce the death rates. Rau B and Steinbach G (13) sustain thepredictive power of TAOP and PCT, and consider these tests to be almost equal, tothat of fine needle biopsy-as the gold standard.

CONCLUSIONSThe novel parameter obtained by using an indirect performant and automatedbioquantitative assay, proposed by Oczan Erel, is sensible, and may reflect the realtissular response, during the period of wound healing process.
Correlated to a local O/Ao disequilibrium, before the development of edema, orin the first 4 hours postoperative, TAOP could be used to select an adequate therapy,
to block a bad evolution of wound.
The two periods of endogenous antioxidants rising level (at 4 and 24 hours
postoperative), showed the favorable role of the adaptive mechanism of the organism sustaining the favorable effect of surgical intervention.
Early prognosis about the wound evolution, elaborated in immediate postoperative period, associating the exogenous antioxidants to conventional therapeutic
schema, could prevent the wound evolution toward non-healing.

The final conclusion is those that to use the combinative therapies, accordingwith the pathophysiological mechanisms, and especially with their pivotal expression: the lowering of TAR.
REFERENCES
1. Benzie IF, Strain JJ. Ferric reducing/antioxidant power assay: direct measure of
total antioxidant capacity of biological fluids and modified version for simultaneous
measurement of total antioxidant power and ascorbic acid concentration. Methods
Enzymol 1999;299:15-27
2. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of‘‘antioxidant power’’: the FRAP Assay. Anal Biochem 1996;239:70-6
3. Ching S, Ingram D, Hahnel R, Beilby J, Rossi E. Serum levels of micronutrients,
antioxidants and total antioxidant status predict risk of breast cancer in a case control
study. J Nutr 2002;132(2):303-6
4. Ghiselli A, Serafini M, Natella F, Scaccini C. Total antioxidant capacity as a tool
to assess redox status: critical view and experimental data. Free Radic Biol Med
2000;29(11):1106-14
5. Halliwell B, Gutteridge JMC, editors. Free radicals in biology and medicine. Third
ed. Oxford: Oxford Science Publications; 2000; p. 617-24
6. Janaszewska A, Bartosz G. Assay of total antioxidant capacity: comparison of four
methods as applied to human blood plasma. Scand J Clin Lab Invest, 2002;62(3):231-6
7. Kaplan IV, Attaelmannan M, Levinson SS. Fibrinogen is an antioxidant that protects
lipoproteins at physiological concentrations in a cell free system. Atherosclerosis
2001;158:455-63
8. Kampa M, Nistikaki A, Tsaousis V, Maliaraki N, Notas G, Castanas E. A new automatedmethod for the determination of the total antioxidant capacity (TAC) of humanplasma, based on the crocin bleaching assay. BMC Clin Pathol 2002;28(2):3
9. Koracevic D, Koracevic G, Djordjevic V, Andrejevic S, Cosic V. Method for the measurement of antioxidant activity in human fluids. J Clin Pathol 2001;54(5):356-61
10. Leenen R, Roodenburg AJ, Tijburg LBM, Wiseman SA. A single dose of tea withor without milk increases plasma antioxidant activity in humans. Eur. J Clin Nutr
2000;54(1):87-92
11. Mayer M. Association of serum bilirubin concentration with risk of coronary
artery disease. Clin Chem 2000;46:1723-7
12. Oczan. E. Clinical Biochemistry 37 (2004) 112-119
13. Rau B., Steinbach G. The role of procalcitonin and IL8 in the prediction of infected
necrosis in acute pancreatitis. Gut 1997;41:823-40
14. Reinhardt K, Meisner M, Hartog C. Diagnosis in sepsis: Novel and Conventional
Parameters. Adv. in Sepsis 2001; 1(2): 42
15. Stuber F, Book M. The role of genomic polymorphism in sepsis. Adv. in Sepsis,
2001; 1(2):56
16. Vallet B. Vascular nitric oxide during sepsis: from deficiency to overproduction.
Adv in Sepsis, 2001; 1(2)17. Young IS, Woodside JV. Antioxidants in health and disease. J Clin Pathol
2001;54:176–86

RAPORTUL OXIDANŢI/ANTIOXIDANŢI: UN PREDICTOR AL EVOLUŢIEISPRE VINDECARE A PLĂGII POSTOPERATORIIREZUMAT
Actul chirurgical este definit ca un stres sau o trauma programata, efectuata in favoarea pacientului, cu acordul acestuia. Postoperator, reactia de aparare aorganismului fata de trauma se insoteste de tendinta la dezechilibrare a homeostaziei celulare, care se reflecta in alterarea procesului de vindecare a plagii
chirurgicale. In afara interventiei chirurgicale, care stimuleaza direct apoptoza, sunt activati o serie de factori, care interfereaza cu subsistemele organismului, pe unele dintre aceste, putandu-le deregla: termoreglarea, echilibrul fluido-coagulant, oxidanti/antioxidanti, acido-bazic, etc. Se stie ca in primele ore
postoperator, se produce o stare de hipercoagulabilitate si cresterea producerii speciilor reactive de O2. Nivelul mediatorilor eliberati in aceasta perioada, este
oscilant iar gradul perturbarii este dependent nu doar de durata si intensitatea actului operator dar si de momentul si de tehnicile de recoltare a probelorde sange. Studiul nostru a incercat sa introduca o metoda performanta de laborator, cu ajutorul careia sa se evidentieze nivelul seric al capactitatii oxidantea serului apreciindu-se si rolul central al acestei valori, in cadrul reactiei de aparare tisulara, postinjurie.
Cuvinte cheie: oxidanti, vindecarea plagii, capacitate antioxidanta a plasmei
2008.18.4(60)  Fiziologia - Physiology

13

CORRELATIONS BETWEEN OXIDATIVE STRESS AND ENDOTHELIN-1 LEVELS IN PATIENTS WITH RHEUMATOID ARTHRITISDOINA BALTARU1, IRINA CHIS2, ADRIANA MARTON3, MARIUS-IONUT UNGUREANU3, RAMONA SIMEDREA2, ADRIANA MURESAN2, PHILIPPE HOANG4, NICOLETA DECEA2, MIHAIDOBRE3, ZUBEDAT MOSA3
1”Constantin Papilian” Military Emergency Hospital, Cluj- Napoca, Romania
2
Physiology Department, „Iuliu Hatieganu”University of Medicine and Pharmacy,Cluj Napoca, Romania
3
Student, „Iuliu Hatieganu”University of Medicine and Pharmacy, Cluj Napoca, Romania
4
Centre Tomberg, Bruxelles, Belgium

ABSTRACTBACKGROUND. Rheumatoid arthritis (RA), a systemic chronic autoimmune inflammatory disease, is associated with excessive cardiovascular mortality, whichcannot be explained by the traditional risk factors alone. There is growing evidence that inflammation plays an important role in the initiation and progressionof atherosclerosis. One of the hypotheses incriminates systemic inflammation in inducing early endothelial dysfunction. Rheumatoid arthritis is also associated with an overproduction of oxygen reactive species and there is data about their involvement in the decrease of endothelial nitric oxide synthase (eNOS)
activity. PURPOSE. We decided to investigate the endothelial function and the levels of vascular oxidative stress in patients with RA, in order to highlightthe fact that the overproduction of reactive oxygen species (ROS) is involved in the development and progression of atherosclerosis in these patients as wellas in the excessive cardiovascular mortality. METHODS. Twenty RA patients with a mean age of 50.7, with ACR 50 response to DMARD’S by the ACR criteria,
were enrolled. They had a mean disease duration of 6.5 years and had no traditional cardiovascular risk factors except the age. Twenty healthy age matchedvolunteers were enrolled to form the control group. Pulse wave velocity (PWV) was measured in all subjects. Serum levels of endothelin-I, lipid peroxides (LP),carbonylated proteins (CP), ceruloplasmin (C) and donor hidrogen ability (DHA) were determined in all subjects. RESULTS. Results (mean±SD) in patientswith RA and healthy patients, respectively, were as follows: PWV was significantly increased (P<0,001), correlated with a significantly increased production of endothelin-I (P<0,001), LP (P<0,01) and CP (P<0,001) and a significant decrease of serum antioxidant capacity: C (P<0,01) and DHA (P<0,001).
CONCLUSIONS. Patients with RA have an altered endothelial activity and increased oxidative stress levels which aggravate the endothelial dysfunction, andthis should be a powerful enough reason to initiate a long-term, efficient therapy, in order to reduce cardiovascular mortality.
Key words: Rheumatoid arthritis, endothelin-I, oxidative stress, endothelial dysfunction, pulse wave velocity.

.

INTRODUCTION
Rheumatoid arthritis (RA) is a common chronic disease characterized by persistent inflammation of multiple joints. RA is associated with an increased mortality
from cardiovascular causes, which exceeds that of the general population (1, 2, 3).
Recent reports demonstrate that endothelial function is reduced in RA patientswith high inflammatory activity (4, 5, 6). Although the underlying mechanisms forendothelial dysfunction in RA are poorly understood, it is postulated that systemic
inflammation may be involved in the early vascular damage (7, 8, 9). In supportof this hypothesis, anti–tumor necrosis factor (anti-TNF) therapy has been shown
to improve endothelial dysfunction in patients with active RA (6, 10).
The endothelium plays a pivotal role in the regulation of vascular tone andstructure through the release of various vasoactive agents, such as vasodilators andvasoconstrictors. It has been recognized that alterations of endothelial function

are involved in the development and progression of atherosclerosis and its clinicalcomplications (11).
Endothelial dysfunction is actually considered to be a reduced bioavailability of
nitric oxide (NO), which is a major endothelium-dependent vasodilator. Endothelium
dependent NO is also known to have other anti-atherosclerotic properties, including the inhibition of cell growth, leukocyte adhesion, and platelet adherence andaggregation. Regulatory mechanisms that control vascular NO bioavailability inpathophysiologic states are complex. It has been reported that vascular productionof reactive oxygen species (ROS), such as superoxide (O-2), is increased in hypertension, atherosclerosis, or diabetes (12). O-2 reacts rapidly with NO, resulting in theformation of peroxynitrite (ONOO-), which could lead to a loss of bioactivity of NO.
The increased oxidative stress may affect the synthesis of NO. Vascular NO is mainly
synthesized by endothelial NO synthase (eNOS) from the precursor L-arginine.

Received November 2008. Accepted December 2008. Address for correspondence: Dr. Doina Baltaru, Physiology Department, “Iuliu Hatieganu” University of medicine and Pharmacy, Cluj-Napoca, Romania

14

Fiziologia - Physiology  2008.18.4(60)However, previous data demonstrate that increased oxidative stress promotes adysfunctional eNOS, which generates O-2 instead of NO (13). This dysfunctional
enzyme is termed uncoupled eNOS.
Endothelins are potent 21 amino acid vasoconstrictor isopeptides produced indifferent vascular tissues, including vascular endothelium. Endothelin-I mediatedvasoconstrictor tone increases with age and contributes to the pathogenesis of
hypertension (14).
Our aim in this study was to bring new data concerning the correlation between
ROS production and endothelin-I levels in the progression of endothelial dysfunction
in patients with RA.

Figure 2 shows a significant increase of endothelin-I serum levels in patientswith RA (group number 2) comparatively to the control group.
Serum levels of endothelin-1

1086fmol/ml

4
2

MATERIALS AND METHODS
Twenty RA patients with a mean age of 50.7, with ACR 50 response to DMARDs
by the ACR criteria, were enrolled. They had mean disease duration of 6.5 years
and had no traditional cardiovascular risk factors, except the age. Twenty healthy
age-matched volunteers were enrolled to form the control group.
The subjects included in the study were divided into 2 groups:-1st Group- Control group, 20 age-matched healthy patients;-2nd Group- 20 patients with RA.
For all the subjects included in the study, endothelial dysfunction
was assessed by measuring the pulse wave velocity (PWP) between common
carotid artery and radial artery, by means of a COMPLIOR device. Serum levels ofendhothelin-I (by ELISA method) were determined in all subjects. The oxidative
stress was assessed using the following markers: lipid peroxides (LP, by Satohmethod) (15) and carbonylated proteins serum levels (CP, by Reznick method)(16)- for the free radicals; ceruloplasmin (C, by Ravin method) (17) and hydrogen
donor ability (HDA, by Hatano method) (18)- for the antioxidant capacity.
Statistical validation of our results was made by standard deviation method
and student test. All the studies were conducted in accordance with the National
Institute of Health and the experiments were carried out as per the Institutional
Ethics Committee.

RESULTS
Figure 1 shows the PWP in patients with RA comparatively with the control
group. A significant increase of PWP can be observed in patients with RA (thesecond group).

0

1st Group

Fig.2. Serum levels of endothotelin-I were significantly increased (P<0.001)
in the 2nd Group (9.01±0.98 fmol/ml), as compared to the 1st Group (1.81±0.83
fmol/ml)
Figures 3 and 4 show the levels of free radicals: lipid peroxides and carbonylated
proteins levels in the serum of control and experimental group. A significant increasedoxidative stress was observed la pacientii cu RA.
P<0.001

Serum levels of lipid peroxides

2
1.5
nmol/ml

0.5
0

m/s 4

2nd Group

Serum levels of carbonylated proteins3

P<0.0012
10

6

1st Group

2nd Group

Fig.4. CP were significantly increased (P<0.001) in the 2nd Group (2.58±0.34
nmol/mg), as compared to the 1st Group (1±0.2 nmol/mg)

20

1st Group

Fig.3. LP were significantly increased (P<0.01) in the 2nd Group (1.73±0.34
nmol/ml), as compared to the 1st Group (1.35±0.2 nmol/ml)

nmol/mg

P<0.001

P<0.01

1

Pulse wave velocity

8

2nd Group

1st Group

2nd Group

Fig.1. PWV was significantly increased (P<0.001) in the 2nd Group
(7.89±0.98 m/s), as compared to the 1st Group (4.04±0.16 m/s).
2008.18.4(60)  Fiziologia - Physiology

Figures 5 and 6 show the activities of ceruloplasmin and hydrogen donor abilityof control and experimental groups. A significantly decreased oxidative stress wasobserved in RA patients.

15

Serum levels of ceruloplasmin

34

P<0,01

32
mg% 3028
26

1st Group

2nd Group

Fig.5. Ceruloplasmin was significantly decreased (P<0.01) in the 2nd Group(28.5±8.74 mg%), as compared to the 1st Group (32.73±4.9 mg%)
Hydrogen donor ability

50

P<0,001

40
%

30
20
10
0

1st Group

2nd Group

Fig.6. HDA was significantly decreased (P<0,001) in the 2nd Group(41.4±10.5 %), as compared to the 1st Group (50±1.5%)

DISCUSSIONS
Endothelial dysfunction has been associated with a number of pathophysiological processes. Oxidative stress appears to be a common denominator underlying
endothelial dysfunction in cardiovascular diseases. Superoxide anion, the hydroxyl
radical (·OH), H2O2, and peroxynitrite (ONOO¯·) are produced in the vessels underboth normal and stress conditions such as inflammation, injury and aging. Reactive
oxygen species promote the contraction of vascular smooth muscle cells by facilitating the mobilization of calcium and increasing the sensibility of the contractile
proteins to calcium ions (19).
Aging, hypertension, endothelial injury, as well as dyslipidemia (in particular
hypercholesterolemia), smoking and increased body mass index are major risk factors for the development of atherosclerosis. They are all associated with increasedoxidative stress levels and endhothelial dysfunction (20, 21, 22).
Endothelin-1 is the main endothelin generated by the endothelium and probably the most important in the cardiovascular system. Endothelin-I acts throughspecific receptors termed ET(A), represented only on smooth muscle cells and having
the functions of growth promotion and contractions mediator, and ET(B), locatedboth on smooth muscle cells, where they evoke contractions, and on endothelial
cells, inducing relaxation through the production of the endothelium-derived
relaxing factor nitric oxide. In physiological conditions endothelin-1 administration causes vasodilation and vasoconstriction at low and high concentrations,
respectively (14).
There is substantial evidence supporting the fact that ROS and RNS (nitrogen
reactive species) contribute to the initiation of RA lesions. These active toxic products
can be produced through different mechanisms by the neutrophils, chondrocytes,
and panos macrophages as well as by the xantin-oxidase in the synovial membrane
through the hypoxia-reperfusion process (23).
Oxidative stress in the articulation becomes apparent both because of anincreased production of ROS and RNS, and because of a decrease in the antioxidant
16

activity, which cannot fight the oxidative aggression on the synovial liquid, synovial
membrane and other articular components, including chondral and bone structures.
There are mechanisms which can limit these processes, but they are perturbed anddiminished by hypoxia and intrasynovial acidosis, which increase the formationof ROS and RNS.
Our study confirmed the fact that ROS and the concomitant decrease of theantioxidant capacity, correlated with an increased endothelin-I production may
play an important role in the RA endothelial dysfunction.

CONCLUSIONS
Patients with RA have a significantly increased pulse wave velocity, as compared to the control group, reflecting the presence of endothelial dysfunction.
The serum level of endothelin -I is significantly higher in patients with RA
comparatively to the control group, therefore contributing to the increase ofendothelial dysfunction.
We have noticed a significantly increased generation of LP and CP and a decrease in the HDA and plasma level of ceruloplasmin, as markers of overproductionof reactive oxygen species in patients with RA.
Antioxidants will be an important option for a better management of endothelial dysfunction in patients with RA.
REFERENCES
1. Del Rincon I, Williams K, Stern MP, Freeman GL, Escalante A. High incidence of cardiovascular events in a rheumatoid arthritis cohort not explained by traditional cardiac risk factors.
Arthritis Rheum., 2001; 44: 2737–45.
2. Symmons DP, Jones MA, Scott DL, Prior P. Long term mortality outcome in patients withrheumatoid arthritis: early presenters continue to do well. J Rheumatol., 1998; : 1072–7.
3. Wolfe F, Mitchell DM, Sibley JT, Fries JF, Bloch DA, Williams CA, et al. The mortality ofrheumatoid arthritis. Arthritis Rheum., 1994; 37:481–94.
4. Bergholm R, Leirisalo-Repo M, Vehkavaara S, Makimattila S, Taskinen MR, Yki-Jarvinen
H. Impaired responsiveness to NO in newly diagnosed patients with rheumatoid arthritis.
Arterioscler Thromb. Vasc. Biol., 2002; 22: 1637–41.
5. Gonzalez-Juanatey C, Testa A, Garcia-Castelo A, Garcia-Porrua C, Llorca J, Vidan J, et al.
HLA-DRB1 status affects endothelial function in treated patients with rheumatoid arthritis.
Am. J. Med., 2003; 114: 647–52.
6. Hurlimann D, Forster A, Noll G, Enseleit F, Chenevard R, Distler O, et al. Anti-tumor necrosis
factor-α treatment improves endothelial function in patients with rheumatoid arthritis
Circulation, 2002; 106: 2184–7.
7. Kaplan MJ, McCune WJ. New evidence for vascular disease in patients with early rheumatoid arthritis. Lancet, 2003; 361: 1068–9.
8. Sattar N, McCarey DW, Capell H, Mclnnes IB. Explaining how high-grade systemic inflammation accelerates vascular risk in rheumatoid arthritis. Circulation, 2003; 108: 2957-63.
9. Van Doornum S, McColl G, Wicks IP. Accelerated atherosclerosis: an extraarticular featureof rheumatoid arthritis? Arthritis Rheum., 2002; 46: 862–73.
10. Gonzalez-Juanatey C, Testa A, Garcia-Castelo A, Garcia-Porrua C, Llorca J, Gonzalez-Gay
MA. Active but transient improvement of endothelial function in rheumatoid arthritis patients
undergoing long-term treatment with anti-tumor necrosis factor α antibody. Arthritis
Rheum., 2004; 51: 447–50.
11. Higashi Y, Yoshizumi M. New methods to evaluate endothelial function: method forassessing endothelial function in humans using a strain-gauge plethysmography: nitricoxide-dependent and -independent vasodilation. J. Pharmacol. Sci., 2003; 93: 399-404.
12. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant
stress. Circ. Res., 2000; 87: 840–4.
13. Zou MH, Shi C, Cohen RA. Oxidation of the zinc-thiolatecomplex and uncoupling ofendothelial nitric oxide synthase by peroxynitrite. J. Clin. Invest., 2002; 109: 817–26.

Fiziologia - Physiology  2008.18.4(60)14. Stauffer BL, Westby CM, DeSouza CA. Endothelin-1, aging and hypertension. Curr Opin
Cardiol., 2008; 23(4): 350-5.
15. Satoh K. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin. Chim. Acta 1978; 90: 37- 43.
16. Reznik AZ, Packer L. Oxidative damage to proteins: spectrofotometric method for carbonyl
assay. Meth. Enzymol. 1994; 233: 357-363.
17. Manta I, Cucuianu M, Benga G, Hodârnău A. Metode biochimice în laboratorul clinic.
Ed. Dacia, 1976: 346-347.
18. Amstrong D. Free radical and antioxidant protocols. Humana Press Inc., Totowa, New
Jersey, 1998: 15- 27.

19. Van der Loo B, Labugger R, Skepper JN, Bachschmid M, Kilo J, Powell JM, et al. Enhanced peroxynitrite formation is associated with vascular aging. J Exp Med., 2000; 192:
1731-44.
20. Brandes RP, Fleming I, Busse R. Endothelial aging. Cardiovascular research. 2005; 66:
286-294.
21. Csiszar A, Ungvari Z, Edwards JG, Kaminski P, Wolin MS, Koller A, et al. Aging-induced
phenotypic changes and oxidative stress impair coronary arteriolar function. Circ res.,2002; 90:1159-66.
22. Félétou M and Vanhoutte PM. Endothelial dysfunction: a multifaceted disorder. Am J
Physiol Heart Circ Physiol. 2006; 291: H985-H1002.
23. Dejica D. Stressul oxidativ în bolile interne. Casa Cărţii de Ştiinţă, Cluj-Napoca, 2000.

CORELAţII îNTRE STRESUL OXIDATIV şI NIVELUL ENDOTELINEI- I LAPACIENţII CU POLIARTRITă REUMATOIDăREZUMATINTRODUCERE. Poliartrita reumatoida (PAR), o boala inflamatorie cronica sistemica autoimuna, este asociata cu o mortalitate cardiovasculara excesiva, care nu
poate fi explicata doar prin factorii de risc traditionali. Exista tot mai multe dovezi care sustin faptul ca inflamatia joaca un rol esential in initierea si progresia
aterosclerozei. Una din ipoteze incrimineaza inflamatia sistemica in inducerea disfunctiei endoteliale. Poliartrita reumatoida este de asemenea asociata cu osupraproductie de specii reactive ale oxigenului (ROS, si exista informatii privind implicarea acestora in scaderea activitatii NO sintetazei endoteliale (eNOS).
OBIECTIVE. Am decis sa investigam functia endoteliala si nivelurile stresului oxidativ vascular la pacientii cu PAR, pentru a evidentia faptul ca supraproductia
de ROS este implicata in dezvoltarea si progresia aterosclerozei la acesti pacienti si, prin urmare, determina mortalitatea cardiovasculara excesiva intalnita inaceste cazuri. METODE. Au fost inclusi in studiu 20 de pacienti cu PAR avand o medie a varstei de 50,7 ani, cu raspuns ACR 50 la DMARD conform criteriilor ACR.
Ei aveau o medie a duratei bolii de 6,5 ani si nu au avut alti factori de risc traditionali, cu exceptia varstei. Al doilea grup, grupul martor, a fost format din 20
de voluntari sanatosi. A fost masurata capacitatea undei de puls (PWV) la toti subiectii inclusi in studiu. Au fost determinate nivelurile serice ale endotelinei-I,
ale peroxizilor lipidici (LP), proteinelor carbonilate (CP), ceruloplasminei(C) si capacitatea donarii de hidrogen (DHA) la toti subiectii. REZULTATE. Rezultatele(media±S) la pacientii cu PAR si, respectiv, la grupul martor, au fost dupa cum urmeaza: PWV a fost semnificativ crescuta (P<0,001), corelata cu o cresteresemnificativa a productiei de endotelina-I (P<0,001), LP (P<0,01) si CP (P<0,001) si cu o scadere semnificativa a capacitatii antioxidante serice C (P<0,01)
si DHA (P<0,001). CONCLUZII. Pacientii cu PAR au o activitate endoteliala alterata asociata cu o crestere a stresului oxidativ care agraveaza disfunctia endoteliala, iar acest lucru ar trebui sa fie un motiv suficient pentru initierea unei terapii eficiente pe termen lung, cu scopul de a reduce mortalitatea de cauzacardiovasculara.
Cuvinte cheie: Poliartrita reumatoida, endotelina-I, stres oxidativ, disfunctie endoteliala, velocitatea undei de puls.

2008.18.4(60)  Fiziologia - Physiology

17

PREVALENCE AND RESISTANCE TO ANTIBIOTICS IN ORAL
VIRIDANS STREPTOCOCCIDORINA DUGAESESCU1, CAMELIA SZUHANEK2, LILIANA DRAGOMIRESCU1, ELENA HOGEA1, MIHAELA CRACIUNESCU1, DELIA BERCEANU-VADUVA1, DELIA MUNTEAN1, MIHAELAPOPA1, CIPRIAN PILUT1, ROXANA ROMINU3, RAMONA PIRVAN1, ROXANA ZUGRAVU1, DENISSERBAN1
1
Department of Microbiology – University of Medicine and Pharmacy „Victor Babeş”Timisoara
2
Department of Pedodontics and Orthodontics, Faculty of Dental Medicine - University of Medicine and Pharmacy „Victor Babeş”Timisoara
3
Department of Prostheses Technology and Dental Materials, Faculty of Dental Medicine -University of Medicine and Pharmacy „Victor Babeş”Timisoara

ABSTRACT
Aims: This purpose of this study was to determine the prevalence of species of oral viridans streptococci and their resistance to antibiotics.
Method: Isolation was carried out on blood agar 5%. The identification of the viridans streptococci species was made using API system (20 biochemical tests).
Susceptibility to antibiotics was tested by disk-diffusion method Kirby-Bauer.
Results: Of the 54 samples collected from dental plaque, carious lesions and throat swab were isolated in 47 strains of viridans streptococci. Their distribution was: 29.78% S. mitis, 27.65% S. mutans, 14.89% S. sobrinus, 10.63% S. oralis, 8.51% S. salivarius, 4.25% S. anginosus and 4.25% S. sanguis. In termsof resistance to antibiotics, the order was: Ciprofloxacin (46.8%), Tetracycline (40,4%), Gentamicin (27.6%), Erythromycin (25,5%), Clindamycin (21,2%),
Penicillin (17%), Doxycycline (15%) and Ampicillin (10,6%). It was not reported any resistant strain to Vancomycin.
Conclusions: Although viridans streptococi are part of the normal flora of the oral cavity which provides colonization resistance at this level, preventing
colonization of other pathogenic species, they nevertheless represent a reservoir of the determinants of acquired resistance that can transmit these species(S. pyogenes, S. pneumoniae and S. agalactiae).
Keywords: dental plaque, cavities lesions, antibiotics resistance, viridans streptococci.

INTRODUCTION
Oral viridans group streptococci are part of the normal flora of the mouthwhich adheres to different surfaces providing oral mucosal colonization resistanceand inhibiting bacterial colonization of other pathogens species. At this level, thesemicroorganisms occur at the rate of 28% in dental plaque, 29% crevicular fluid,
45% on the tongue and 46% in saliva. Oral species viridans streptococci have avery different distribution in the mounth. Thus, Streptococcus salivarius prevails on
the surface of tongue, while Streptococcus mitis is more prevalent at the mucosalof mouth. Streptococcus mutans and Streptococcus anginosus meet particularly inarea teeth surfaces (8,9).
Dental plaque is initially colonized with Streptococcus sanguis and Streptococccus mitis, like supragingival dental plaque with Streptococccus gordonii andthe subgingival dental plaque with Streptococcus anginosus and Strepococcus
salivarius. Streptococcus mutans and Streptococcus sobrinus are the species mostcommonly isolated in carious lesions and dental plaque. The species often involved
Table I. Distribution of samples collected by age group
Collected samples
Age
4-8 years
Throat swab
6
Dental plaque6
Dental caries
6
Total
18

8-12 years
6
6
6
18

in infections in patients with neutropenia are Streptococcus oralis, Streptococcus
mitis, Streptococcus salivarius and Streptococcus anginosus. Anyway their presencein the blood can sometimes be associated with severe infections like bacterial endocarditis especially in pacients with valvular prostheses where Streptococcus sanguis,
Streptococcus mitis, Streptococcus mitis, Streptococcus oralis and Streptococcus
gordonii are often isolated (2,6,11).

MATERIAL AND METHOD
54 samples have been collected from healthy children and young people aged
between 4 and 18 years. The samples were collected in a private Orthodontics
Pedodontics dental office in the period November 2007 - March 2008. Sampling
was collected with sterile swabs, which subsequently were suspended in Stuart
carriage medium. The samples were collected on the throat swab, supragingival
dental plaque and dental caries.

18 years
6
6
6
18

Total
18
18
18
54

Received July 2008. Accepted October 2008. Address for correspondence: Dr. Dorina Duaesescu, Microbiology Department, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Eftimie Murgu SquareNo. 2A, 300041 Timisoara, Romania

18

Fiziologia - Physiology  2008.18.4(60)The samples were processed in the Laboratory of Bacteriology Departmentof UMF “Victor Babes”Timisoara, and in the private laboratory for medical analysis
of Timisoara.
In the first day, the swabs shown areas with sheep blood agar 5%, which werethen incubated at 37° C for 24h in 5% CO2. The next day colonies suspected viridans
streptococci were prick out in the Columbia agar +5% sheep blood sectors and thenafter 24h at 37° C in 5% CO2 were identified the species taken in the study.
Identifying the oral viridans group streptococci has been made on the morphotinctorial character (gram positive in chain coccal bacteria), cultural character (colony
with small dusty area haemolysis alpha-green) and metabolic character, using API
20 strep galleries (bio Merieux): 20 biochemical tests – making the reading at 4h
after incubated at 37°C (1, 5).
Subsequently, the viridans streptococci species isolated were tested for antibacterial susceptibilities. For interpretation of the antibiogram testing of viridans
streptococci strain disk diffusion by the method Kirby-Bauer and breakpoints
procedures were used according to NCCLS (National Committee for Clinical nLaboratory Standard) standard and criteria. The viridans streptococci strains were tested
against 9 antibiotics: Penicillin (P), Ampicillin (AM), Erythromycin (E), Clindamycin(CM), Tetracycline (TE), Doxycycline (DO), Gentamicin (GM), Ciprofloxacin (CIP)and Vancomycin (VA) (3).

S. sanguis
12,5%
S. mitis37%

S. salivarius
25%
S. anginosus

S. oralis
12,5%

12,5%

Fig. 1. Distribution of viridans streptococci ssp. isolated from throat swabs

In terms of age group, most strains of viridans streptococci isolated from throatswabs were from the children aged 4-8 years: 3 strains represented by S. mitis,
2 strains S. salivarius and which 1 strains by S. oralis, S. anginosus
and S.sanguis.
In dental plaque were isolated 23 strains of viridans streptococci representing
48.93% of all strains (47 strains).
S. sobrinus

S. mitis

17%

22%

S. sanguis

RESULTS AND DISCUSSION
From the 54 samples collected were isolated a number of 47 strains of viridans
streptococci belonging to 7 species: S. mitis, S. mutans, S.anginosus,
S. oralis, S. salivarius, S. sanguis and S. sobrinus.

4%

S. salivarius
9%
S. oralis

S. anginosus

13%

4%

S. mutans
31%

Fig. 2. Distribution of viridans streptococci ssp. isolated from dental plaqueMost strains of viridans streptococci were isolated as expected, in dental plaque:23 strains (48.93%) and then throat swabs 16 strains (34%) and a lower numberof carious lesions, only 8 strains (17.02%).
From the pharyngeal swabs, it were isolated 8 strains of viridans streptococci
representing 17.02% of total strains isolated from the 35 children taken into thestudy.

Most viridans streptococci were isolated from children aged between 8-12
years and 18 years younger (8 strains). Large numbers of viridans streptococci
isolated from these age group is explained by a greater amount of dental plaquepresent at the oral cavity and possibly impercipient hygiene from this young
compared with other children where the parents probably education in the processof bringing their say.

Table II. Prevalence viridans streptococci in samples collected
Viridans streptoEvidence collected
Throat swab
Dental plaquecocci ssp..
N oFrequency
N oFrequency
strains(%)
strains(%)
S. mitis
3
37.1
5
21.73
S. mutans
7
30.43
S. anginosus
1
12.5
1
4.34
S. oralis
1
12.5
3
13.04
S. salivarius
2
25
2
8.69
S. sanguis
1
12,5
1
4,34
S. sobrinus
4
17.39
Total viridans
8
100
23
100
streptococi ssp.

2008.18.4(60)  Fiziologia - Physiology

Dental caries
N oFrequency
strains
(%)
6
37.25
6
37.25
1
6.25
3
18,75
16
100

TOTAL
strains
N oFrequenstrainscy (%)
14
29.78
13
27.65
3
4.25
5
10.63
4
8.51
2
4.25
7
14.89
47
100

19

In the carious lesions were isolated but only 16 strains of viridans streptococci,
representing 34.04% of all strains isolated (47 strains).
A number of studies appreciates that have identified 10 areas that are ingood correlation with viridans streptococci mainly S. mutans, of wich 5 are in thehindquarters interproximale face and 5 in the vestibular.
Also other tests have shown that approximal and interproximale regions
and fissures areas is a rich reservoir of S. mutans, because in these areas are lessaffected by the methods of oral hygiene.

S. mitis

S. sanguis

S. sobrinus

S. oralis

S. mutans

S. anginosus

S. salivarius

%
16

14.9

14
12.7

12
10.6

10.6

10
8.5

8.5

8.5

8
6.4

6.4

6.4

6.4

6.4

6.4

6.4

6
4
2

4.25

4.25
.

2.12

2.12

4.2

4.2

4.2

4.2

2.12

2.12

2.1.

AM

E

CM

2.1

4.25

2.1

4.2

2.1

2.1

4.2

2.12

0

S. sobrinus

P

19%

S. mitis

S. oralis

37,5%

6%

S. mutans
37,5%Fig. 3. Distribution of viridans streptococci ssp. isolated from dental caries

After antibiograms for the 47 strains of viridans streptococci isolated andidentified in samples collected in the past to determine sensitivity to antibiotics
in the 9 to do the testing.
Viridans streptococci resistance to antibiotics is a phenomenon acquired andis transmitted by determinants of resistance (plasmids), mostly as a consequenceof the administration of antibiotics.
Resistance of 47 strains of viridans streptococci isolated to antibiotics has
been tested in the following order: Ciprofloxacin (46.8%), Tetracycline (40.4%),
Gentamicin (27.6%), Erytromycin (25.5%), Clindamycin (21.2%), Penicillin (17%),
Doxycycline (15%) and Ampicillin (10.6%). To Vancomycin was not reported any
resistant strain.
Among viridans streptococci isolated S. mitis presented the greatest resistance.
In order of frecquency were followed S. oralis and S. mutans.
Viridans streptococci are considered bacteria with low virulence. They don’t
posses endotoxins and not secret any exotoxins. They are very sensitive to the litic
actions of serum and lysosomal enzymes. Although some species secret proteolytic
enzymes, these are not responsible for the production of infection.
Ciprofloxacin
46.8%

Penicillin
17%

Ampicillin
10,6%
Erytromycin
25,5%

Gentamicin
27,6%

Doxycycline
14,9%

Clindamycin
21,2%
Tetracycline
40,4%Fig. 4. Viridans streptococci resistance to antibiotics

20

TE

DOX

GM

CIP

Fig. 5. Viridans streptococci species resistance to antibiotics

Infections caused by viridans streptococci usually are the result of penetration and their accidental spread in the body, especially to people susceptible or
immunocompromised. Only their virulence factor is represented by their ability
to adhere to heart valve offense leading to the installation endocarditis presence
dextran extracellular playing a particular role in this case.
Clinical observations have shown that bacteremia consecutive dextran production by viridans streptococci increased incidence of endocarditis compared withstreptococci non-production dextran. Studies have sown that dextran produced
by strains of viridans streptococci increases their ability to adhere to heart valve in
vitro and bring on endocarditis at the experience animals (2).
On the other hand, the production of dextran modifies the response to medical
therapy. Thus endocarditis caused by strains of streptococci producing dextran ismore resistant to treatment with Penicillin, compared with the non-production of
dextran. Studies have shown that the experimental treatment endocarditis with
Penicillin associated with dextranase increase the rate of sterilization in the valve
than treatment only with Penicillin (4,12).
Also very effective to treat severe infection with strains has proved a
combination of beta-lactams (Penicillin) with aminoglycosides (Gentamicin or
Streptomycin). A number of other beta-lactams such as cephalosporins in vitro
have showe a similar activity on Penicillin to the viridans streptococci. Ceftriaxone
in particular (IIIrd generation cephalosporins) has proved extremely effective on
viridans streptococci involved in the etiology of serious infections: endocarditis,
meningitis or septicemias (7,10).
Other antibiotics that have proven effective in vitro on viridans streptococci:
Vancomycin, Teicoplanin and Imipenem, while: Tetracycline, Chloramphenicol,
Erytromycin, Clindamycin and fluoroquinolones have veritable activity which is
exemplified in our study.

CONCLUSIONS
Even if viridans streptococci are part of normal flora of the oral cavity, great
importance should be given to this group of bacteria because of their involvementin the etiology of dental cavity, gingivitis and other periodontal diseases, but
especially in the endocarditis.
Monitoring of resistance to antibiotics is important to differentiate the therapeutical failure from a reinfection. For example in the case of endocarditis:

Fiziologia - Physiology  2008.18.4(60)Therapeutical failure: in patients with recurrent endocarditis who were nottreated properly during the acute episode, the same strain is isolated,
reinfection: another strain is isolated.
Viridans streptococci resistance to antibiotics can be transferred through thedeterminants of resistance acquired a plasmid in may pathogenic species suchas Streptococcus pyogenes, Streptococcus pneumoniae and Streptococcus
agalactiae.
Special attention should be given to oral hygiene, which influences the incidence and severity of the disease at this level (dental caries, periodontal diseases,
gingivitis and stomatitis), and the distance complications, especially in patientswith risk.
REFERENCES
1. Collins CH, Lyne PM, Grange JM, Falkinham Jo, editors. Collins &Lynes Microbiological Methods. *th ed, Arnold, London, 2004.
2. Husain E, Whitehead S, Castell A, Thomas EE, Speert DP. Viridans Streptococcci
bacteremia in children with malignancy: relevance of species identification andpenicillin susceptibility. Pediatr Infect Dis J, 2005, 24: 563-6.
3. National Committee for Clinical Laboratory Standards. Performance Standards
for Antimicrobial Disk Susceptibility Tests. Approved Standard M2-A*. Wayne, PA:
NCCLS, 2003.
4. Malhotra-Kumar S, Lammens C, Martel A, Mallentjer C, Chapelle S, Verhoeven J. Oropharyngeal carriage of macrolide-resistant viridans group streptococci: aprevalence study among healthy adults in Belgium. J Antimicrob Chemother, 2004,
53:271-6.
5. Moldovan R, Licker M, Berceanu Vaduva D, Craciunescu M, Dan L, Branea D, Hogea E,
Popa M, Muntean D. Microbiologie – Indreptar de lucrari practice, Lito UMFT, 2002.
6. Mrazova M, Docze A, Buckova E, Bucko l, Kacmarikova M, Grey E. Prospective national
survey of viridans streptococcal bacteraemia risk factors, antibacterial susceptibility
and outcome of 120 episodes. J Infect Dis. 2005, vol 37, issue 9 pg: 637-41.
7. Rotimi VO, Salako NO, Mokaddas E, Philip L, Rajan P. High frequency of isolation of
antibiotic-resistant oral Viridans streptococci from children in Kuwait. J Chemother,
2005, 17:493-501.
8. Rozkiewicz D, Daniluk T, Sciepuk M, Zaremba L, Cylwik-Rokicka D, Stokowska W.
The prevalence rate and antibiotic susceptibility of oral viridans streptococci in healty
children population, Adv Med Sci 2006, vol. 51 suppl 1, pg 191-5
9. Ruoff KL, Whiley RA, Beighton D, Streptococcus In: Manual of clinical microbiology.
Murray PR, Baron EJ, Jorgensen JH, Pfaller Ma, Yolken RH, editors 8th ed., Washinton,
DC American Society for Microbiology Press, 2003, 1: 405-21.
10. Stapteton P, Adams V, Pike R, Lucas V, Roberts G Mullany P, Rowbury R. Characterisation of viridans group streptococci with different levels of Tet(M)- mediatedTetracycline resistance. Int Antimicrob Agents, nov 2004 (vol 24, issue 5, pg 493-43.
11. Westling K, Ljungman P, Thalme A, Julander I. Streptococcus viridans septicaemia:
a comparison study in patients admitted to the departments of infectious diseases
and haematology in a university hospital. Scand J Infect Dis, 2002, 34: 316-9.
12. Westling K, Julander I, Ljungman P, Heimdahl A, Thalme A, Nord CE. Reduced
susceptibility to penicillin of viridans group streptococci in the oral cavity of patientswith haematological disease. Clin Microbial Infect, 2004, 10: 899-903.

PREVALENŢA ŞI REZISTENŢA LA ANTIBIOTICE A STREPTOCOCILORVIRIDANS ORALIREZUMAT
Scop. Acest studiu a urmărit determinarea prevalenţei speciilor de streptococi viridans orali şi rezistenţa lor la antibiotice..
Metodă. Izolarea s-a efectuat pe geloză sânge 5% la 37°C în atmosferă deCO2, iar identificarea speciilor de streptococi viridans s-a făcut cu ajutorul sistemului
API (20 teste biochimice). Sensibilitatea la antibiotice s-a testat prin metoda difuzimetrică Kirby-Bauer.
Rezultate. Din cele 54 probe recoltate din placa dentară, carii dentare şi exudate faringiene, s-au izolat 47 tulpini de streptococi viridans: 29,78% S. mitis,
27,65% S. mutans, 14,89% S. sobrinus, 10,63% S. oralis, 8,51% S. salivarius, 4,25% S, anginosus şi 4,25% S. sanguis. Sub aspectul rezistenţei la antibioticeordinea a fost: Ciprofloxacin (46,8%), Tetraciclină (40,4%), Gentamicină (27,6%), Eritromicină (25,5%), Clindamicină (21,2%), Penicilină (17%), Doxiciclină
(15%) şi Ampicilină (10,6%). La Vancomicină toate tulpinile de streptococi viridans au fost sensibile.
Concluzii. Deşi streptococii viridans fac parte din flora normală a cavităţii orale unde asigură colonizarea de rezistenţă la acest nivel, împiedicând colonizarea
altor specii mai patogene, ei reprezintă totuşi un rezervor de determinanţi ai rezistenţei dobândite ce se pot transmite la aceste specii (S. pyogenes, S. pneumoniae, S. agalactiae).
Cuvinte cheie: placa dentara, carii dentare, rezistenta la antibiotice, streptococi viridans

2008.18.4(60)  Fiziologia - Physiology

21

PATTERNS OF RESISTANCE TO ANTIMICROBIAL AGENTSOBSERVED IN ACINETOBACTER SPP. STRAINS ISOLATEDFROM INTENSIVE CARE UNIT
LILIANA DRAGOMIRESCU1, MONICA LICKER1, ELENA HOGEA1, VICTOR DUMITRASCU2,
MARINELA HOLOSPIN3, BIANCA PODAC3, DORINA DUGAESESCU1, DOREL SANDESC4,ROXANA MOLDOVAN1
1
Department of Microbiology, University of Medicine and Pharmacology “Victor Babes”Timisoara
2
Department of Pharmacology, University of Medicine and Pharmacology “Victor Babes”Timisoara
3
Clinical County Hospital Timisoara, Central Laboratory
4
Clinical County Hospital Timisoara, Intensive Care Unit

ABSTRACT
Background: The present study was conducted in order to study the resistance to antimicrobial agents of Acinetobacter spp. strains among clinical isolates frompatients hospitalized in the Intensive Care Unit (ICU) wards of Clinical County Hospital Timisoara. Methods: A total of 60 Acinetobacter spp. strains were isolated
from patients hospitalized in the ICU wards. Strains were isolated from specimens such as: bronchial aspirate, wound secretions, blood, cerebrospinal fluid, central
venous catheter, urine, sputum. All samples were inoculated on blood agar supplemented with 5% sheep blood and lactose agar (AABTL, MacConkey). The plates
were incubated aerobically for 24 hours. Smears were made from samples and stained by the Gram’s stain. Acinetobacter spp. strains were identified using Vitek
32 automated system, with GNI (Gram Negative Identification) cards, and susceptibility testing was performed using GNS (Gram Negative Susceptibility) cards.
Results: Between June 2006 and July 2007, a number of 60 Acinetobacter spp. strains were isolated, mainly from bronchial aspirate (48.33%). The mostfrequent encountered β-lactam resistance phenotype was addition of mechanisms (43.33%); resistance to gentamicin-amikacin was expressed in 31.67%
of all Acinetobacter spp. strains; 73.33% strains were resistant to quinolones. Meanwhile, 51.37% of all strains presented carbapenem resistance, aztreonam
resistance reached 76.67%, and trimethoprim/sulfamethoxazole was resistant in 75% cases.
Conclusions: Acinetobacter spp. strains have predominated in bronchial aspirates, mainly because the specific of ICU procedures. Multdrug-resistance(MDR)
was expressed for β-lactam antimicrobials, carbapenems, aminoglicosides, quinolones and others. The number of MDR Acinetobacter spp. strains is growing
worldwide, making prevention, surveillance and control programs very necessary.
Key words: Acinetobacter spp, Intensive Care Unit, resistance phenotypes, antimicrobials.

INTRODUCTION
Species of the genus Acinetobacter are strictly aerobic nonfermentativegram-negative bacilli, catalase-positive and oxidase-negative (7). Acinetobacter
spp are widely distributed in nature. They are able to survive on various surfaces(both moist and dry) in the hospital environment, thereby being an importantsource of infection in debilitated patients; it is an oportunistic germ that causes
nosocomial infections, especially in the Intensive Care Units, where the numberof immunocompromised patients is high (5). The role of the Acinetobacter spp
strains in the etiology of nosocomial infections has increased in the last three
decades and outbreaks of Acinetobacter infections were raported globally (3,9).
Acinetobacter spp. strains are frequently encountered in the Intensive Care Units,
mainly because of the increasing number of invasive diagnosis and treatment
procedures used in the ICU in the latest years and because of this microorganism’s
capacity to survive long periods in the environment and on the hands of medical
staff (6). The risk factors for nosocomial Acinetobacter spp. infection are represented
by previous hospitalisation, the immunocompromised patient, mechanicalventilation, cardiac or respiratory insufficiency, previous infections and antibiotic
treatment and the presence of central or vesical catheters (4). There are differences
between susceptibility to antimicrobials of Acinetobacter baumannii strains

and other species of the genus, A.baumanii being the most resistant.
Acinetobacter spp. strains have became resistant to the vaste majority ofantibacterials available, including aminoglycosides, quinolones, and extended
spectrum β-lactam antimicrobials (9). The majority of strains is resistant tocephalosporines, while resistance to carbapenems is often encountered. These
multidrug-resistant (MDR) strains are in some cases susceptible only to polymyxines (colistin and polymyxin B), which were not used for a few decades and havemore toxic effects than commonly used antimicrobials (8).

MATERIAL AND METHODS
The study was conducted at the Department of Microbiology of Universityof Medicine and Pharmacy “Victor Babes”, Timisoara. Between June 2006 andJuly 2007, a total of 60 Acinetobacter spp. strains were isolated from the patients
admitted to the ICU wards of Clinical County Hospital Timisoara. Patients included
were from both sexes and were all over 15 years old. Prior to inclusion in thisstudy, they have all been hospitalised for at least 48 hours, a requirement to define
nosocomial infection. The only exclusion criterion was those patients who were on
antibiotic therapy.

Received August 2008. Accepted November 2008. Address for correspondence: Dr. Liliana Dragomirescu, Microbiology Department, “Victor Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu
Square No. 2A, 300041, Timisoara

22

Fiziologia - Physiology  2008.18.4(60)Specimens such as bronchial aspirate, sputum, wound secretion, blood, urine,
cerebrospinal fluid were collected. All samples were inoculated on blood agar
supplemented with 5% sheep blood and lactose agar (AABTL and MacConkey agar)(6). The plates were incubated aerobically for 24 hours. Smears were made fromsamples and stained by the Gram’s stain. Isolation and identification of strains were
performed in the Microbiology Laboratory of the Hospital. Strains were identified
using Vitek 32 automated system (bioMerieux, France) using GNI (Gram Negative
Identification) cards, and the susceptibility tests were performed using GNS cards(Gram Negative Susceptibility) with the determination of Minimum Inhibitory Concentration (MIC) for the following antimicrobials: amikacin, aztreonam,
cefepime, ceftazidime, ciprofloxacin, gentamicin, imipenem, netilmicin, pefloxacin,
piperacillin, piperacillin/tazobactam, ticarcillin, ticarcillin/clavulanate, tobramicin,
trimethoprim/ sulfamethoxazol.
The age of the patients ranged from 15 to 91 years (Fig. 1). There were 38 males(63.33%) and 22 females (36.67%). There were 29 specimens of bronchial aspirate,
11 specimens of wouns swabs and six blood cultures (Table I).

3 strains (5%) as Acinetobacter lwoffii. The total number of polymicrobial samples
was 22, A. baumanii being associated with other Gram-negative germs (ex. Klebsiella pneumoniae, Providencia stuartii), but mostly with Staphylococcus aureus
strains (in 19 cases). Staphylococcus aureus strains associated with A.baumanii was
methicillin sensible (MSSA – 7 strains, 36.84%) or methicillin-resistant (MRSA-12 strains, 63.16%) (Fig.3). These A.baumanii - MRSA associations were 100%
isolated from bronchial aspirates. Acinetobacter spp. strains showed resistance todifferent classes of antimicrobials: extended spectrum β-lactams, carbapenems,
quinolones, aminoglycosides.
A.baumannii +
MRSA
37%

A.baumannii +
MSSA
63%

ov er 80 y ears
70-79 y ears
60-69 y ears
50-59 y ears

Distibution in age groups

40-49 y ears
30-39 y ears

The resistance to β-lactams (2) was revealed by the presence of Acinetobacter
spp. penicillinase-producing strains (6.67%) or cephalosporinase-producing strains(10%); 18 Acinetobacter spp. strains were penicillinase- and cephalosporinaseproducing strains, and the vaste majority (26 strains, representing 43.33%) wereβ-lactam resistant through addition of mechanisms; only 5% of the strains wereβ-lactam susceptible (three strains out of the 60 isolated) (Table II).

15-29 y ears

0

2

4

6

8

10

12

14

16

Fig. 1. Distribution of patients in various age groups

Table I. Samples isolated from patients
Samples
Bronchial aspirate
Wound secretion/swab
Blood
Cerebrospinal fluid
Central venous catheter
Sputum
Urine
Total

No.
29
11
6
4
4
3
3
60

Percentage
48.33%
18.33%
10.00%
6.67%
6.67%
5.00%
5.00%
100%

RESULTS AND DISCUSSIONSIn the same period, a total of 23 strains of Acinetobacter spp. were isolated
from other Surgical Units of the Clinical County Hospital Timisoara (Plastic Surgery –11 strains, Surgical Clinics I, II and III – 7 strains, Orthopedy – 2 strains, Urology – 2strains, Neurosurgery – 1 strain), compared to those 60 strains of Acinetobacter
spp. isolated from the Intensive Care Unit (Fig. 2).
8%2% 2% 1%ICU
Plastic Surgery

14%

General Surgery
Ortopedy
Urology73%

Neurosurgery

Fig. 2. Acinetobacter spp. isolates from different Surgical Units of ClinicalCounty Hospital Timisoara (June 2006 – July 2007)

Out of the 60 Acinetobacter spp stains isolated from ICU patients, 57 strains(95%) were identified as Acinetobacter calcoaceticus baumannii complex, and2008.18.4(60)  Fiziologia - Physiology

Fig. 3. Association of Acinetobacter baumannii with Staphylococcus aureus
MSSA – methicillin-susceptible Staphylococcus aureus; MRSA – methicillinresistant Staphylococcus aureus

Table II. β-lactam resistance phenotypes in Acinetobacter spp.
Resistance phenotypes
Susceptible
Penicillinase
Cephalosporinase
Penicillinase + cephalosporinase
Addition of mechanisms
Total

Number of strains
3
4
6
18
26
60

Percentage
5.00%
6.67%
1.00%
30.00%
43.33%
100%

Strains of Acinetobacter spp. presented susceptibility to aminoglycosides(2) in only 6.67% of all strains, resistance phenotypes most encountered were GA(resistance for gentamicin and amikacin – 19 strains, 31.67%), followed by GTA(resistance for gentamicin, tobramicin and amikacin – 15 strains, 25%). Resistanceto all 4 aminoglycosides tested (GNTA phenotype – resistance to gentamicin,
netilmicin, tobramicin and amikacin) was described in 7 Acinetobacter spp.
strains (11.67%) (Table III).
Table III. Aminoglycoside resistance phenotypes in Acinetobacter spp.
Resistance phenotypes
Susceptible
G
A
N
GA
GT
GN
GTA
GNA
GTN
GNTA
Total

Number of strains
4
4
5
1
19
1
1
15
1
2
7
60

Percentage
6.67%
6.67%
8.33%
1.67%
31.67%
1.67%
1.67%
25.00%
1.67%
3.33%
11.67%
100%
23

Quinolone resistance (2) is frequently encountered in Acinetobacter spp
strains. 44 strains (73.33%) showed ciprofloxacin and pefloxacin resistance, while
only 4 strains (6.67%) remained susceptible to quinolones (Table IV).
Table IV. Quinolone resistance phenotypes in Acinetobacter spp
Resistance phenotypes
Susceptible
Ciprofloxacin resistance
Pefloxacin resistance
Ciprofloxacin + Pefloxacin resistance
Total

Number of strains
4
7
5
44
60

Percentage
6,67%
11,67%
8,33%
73,33%
100%

31 strains of Acinetobacter spp. (51.67%) were resistant to carbapenems,
and 46 strains (76.67%) were resistant to aztreonam. Resistance to trimethoprim/sulfamethoxazol was encountered in 45 Acinetobacter spp. strains (75%).
Literature data show an increased and alarming number of multidrug-resistantAcinetobacter spp. strains (1,3). In Europe, studies such as SENTRY, MYSTIC and
ESAR (European Surveillance of Antibiotic Resistance) confirmed the increasing
number (9). Although antimicrobial resistance of Acinetobacter spp. appears to
be increasing across Europe, it is difficult to estimate accurately the extent of thisemerging problem, partially because the published susceptibility data are basedon different methods. A reference method for susceptibility testing and MIC breakpoints should be established to better monitor trends of resistance. Surveillance ofantimicrobial resistance, the study of resistance mechanisms, the use in medical
practice of antimicrobials for which Acinetobacter spp. is still susceptible (colistin,azithromycin, doxycycline, rifampin) (1,8), the development of new drugs, andthe prevention of the spread of multiresistant strains, are all important measuresrequired to control the impact of these multiresistant bacteria (4).

CONCLUSIONS
A total of 60 Acinetobacter spp. strains were isolated from patients
hospitalised in the Intensive Care Unit of Clinical County Hospital Timisoara between
June 2006 – July 2007. The most frequent encountered species was Acinetobacterbaumannii (95%), both from monomicrobial and polymicrobial samples (mainly

bronchial aspirates). The most frequent association of germs was represented byA.baumannii + MRSA (63.16%).
The number of Acinetobacter spp. strains isolated in the same period fromthe other Surgical Units of this hospital was 23, compared to the 60 strains isolated
from ICU; this situation is due to the specific procedures of this Unit.
Acinetobacter spp. strains presented multidrug-resistance (including
β-lactams, carbapenems, aminoglycosides, quinolones) in high percentage.
One alternative in current treatment of nosocomial Acinetobacter spp.
infections is represented by other antimicrobials for which Acinetobacter spp.
strains are still susceptible (colistin, azithromycin, doxycycline, rifampin).
Multidrug-resistant Acinetobacter spp. strains are isolated with increasing
frequence in healthcare facilities worldwide, and the treatment implies high economic costs. The development of innovative control strategies is needed in order
to limit the spread of these pathogens.
REFERENCES
1. Falagas ME, Karveli EA. The changing global epidemiology of Acinetobacterbaumannii infections: a development with major public health implications. Clin
Microbiol Infect. 2007 Feb;13(2):117-9.
2. Jehl F, Chromarat M. De la antibiograma la prescriptie
3. Jones RN. Resistance patterns among nosocomial pathogens: trends over the
past few years. Chest 2001;119:397-404S
4. Jones RN, MastertonR. Determining the value of antimicrobial surveillance
programs. Diagn Microbiol Infect Dis 2001;41:171-175
5. Licker M, Moldovan R. Rezistenta la antibiotice – istorie si actualitate: studii
efectuate in spitalele clinice universitare. Timisoara: Eurostampa; 2002;105-108.
6. Lorian V. Antibiotics in laboratory medicine. Philadelphia, Lippincott
Wiliams&Wilkins 2005;815-847
7. Moldovan R, Licker M, Doroiu M et al. Microbiologie – Indreptar de lucrari
practice. Timisoara; Lito UMFT; 2002;93-97
8. Motaouakkil S, Charra B. Colistin and rifampicin in the treatment of nosocomial infections from multiresistant Acinetobacter baumannii. B J Infect. 2006
Oct;53(4):274-8.
9. Van Looveren M, Goosens H, AARPAC Steering Group. Antimicrobial resistance of Acinetobacter spp. in Europe. Clin Microbiol Infect. 2004;10:684–704. doi:
10.1111/j.1469-0691.2004.00942

MODALITATI DE DOBANDIRE A REZISTENTEI LA AGENTII ANTIMICROBIENIINVESTIGATE LA TULPINILE DE ACINETOBACTER SPP. IZOLATE DIN UNITATILE DE TERAPIE INTENSIVAREZUMAT
Scopul acestei lucrari este studierea sensibilitatii tulpinilor de Acinetobacter spp la chimioterapicele antiifectioase, tulpini izolate de la pacienţi internati în Clinica de Anestezie si Terapie
Intensiva a Spitalului Clinic Judetean de Urgenta Timisoara in perioada iunie 2006 – iulie 2007.
Metoda: S-au izolat 60 tulpini de Acinetobacter spp. de la pacienti internati in Clinica de Terapie Intensiva. Tulpinile au fost izolate din diverse produse patologice reprezentate de: aspirate bronsice, secretii din plaga, sange pentru hemoculturi, urină, sputa, lichid cefalorahidian. Toate produsele patologice au fost insamantate pe agar cu 5% sange de berbec si pe medii
lactozate (AABTL, MacConkey). Placile au fost incubate in aerobioza cu 10%CO2 timp de 24 ore. S-au efectuat frotiuri colorate Gram din produsele patologice. Tulpinile au fost identificate
cu ajutorul analizorului automat Vitek 32 folosind carduri GNI (Gram Negative Identification), iar testarea sensibilitatii la chimioterapice antiinfectioase s-a realizat cu ajutorul aceluiasi
analizor automat, folosind carduri GNS (Gram Negative Susceptibility).
Rezultate: In perioada iunie 2006 – iulie 2007, s-a izolat un numar de 60 tulpini de Acinetobacter spp. in special din aspirate bronsice (48,33%). Fenotipul de rezistenţă la β-lactamine
cel mai frecvent intalnit a fost prin acumulare de mecanisme (43,33%); rezistenta la aminoglicozide s-a exprimat cel mai frecvent sub forma fenotipului GA (rezistenta la gentamicinaamikacina, 31,67%), iar la quinolone rezistenta s-a instalat la 73,33% din tulpinile de Acinetobacter spp. Rezistenta la carbapeneme a fost de 51,37%, la aztreonam 76,67%, iar latrimetoprim/sulfametoxazol de 75%.
Concluzii:Tulpinile de Acinetobacter spp. au predominat în aspirate bronsice, in special datorita specificului Clinicii deTerapie Intensiva. Multirezistenta la chimioterapice antiinfectioases-a exprimat in cazul β-lactaminelor, carbapenemelor, aminoglicozidelor, quinolonelor si a altor clase de chimioterapice antiinfectioase. Numarul tulpinilor de Acinetobacter multirezistente este in continua crestere, facand astfel foarte necesare programele de control, prevenire si supraveghere a tulpinilor multidrug-rezistente (MDR).
Cuvinte cheie: Acinetobacter spp, Terapie Intensiva, chimioterapice antiinfectioase, fenotipuri de rezistenta.

24

Fiziologia - Physiology  2008.18.4(60)ACINETOBACTER STRAINS ISOLATED FROM NOSOCOMIALINFECTIONSDELIA MUNTEAN, MONICA LICKER, DELIA BERCEANU-VADUVA, ELENA HOGEA, LILIANADRAGOMIRESCU, CIPRIAN PILUT, MIHAELA CRACIUNESCU, MIHAELA POPA, MARCELAADAMUT, DORINA DUGAESESCU, DENIS SERBAN, ROXANA MOLDOVAN
Microbiology Department-University of Medicine and Pharmacy “Victor Babes”Timisoara

ABSTRACT
Aims: The aim of our study was to determine the prevalence of Acinetobacter strains, isolated from patients hospitalized in Intensive Care Unit (ICU), andtheir resistance phenotypes.
Methods: Identification of germs was performed by the API system (ID 32 GN) and susceptibility tests by disk-diffusion tests (CLSI standards). We categorizedthese strains according to their phenotypic patterns.
Results: In our study undertaken over a period of six months (Jan-June 2008) from 482 samples (bronchoalveolar fluids, wound secretions, urines, blood,etc.) we isolated 466 microbial strains with nosocomial potential, from which, 28 strains (6%) were Acinetobacter spp. The percentage of multidrug resistantisolates showing resistance to two or more antibiotics was 82.14% (23 strains).
Conclusions: Acinetobacter baumannii is responsible for nosocomial infections, especially pulmonary and urinary tract infections. These infections are difficult
to eradicate in part because of the multiple resistance of this organism to antibiotics.
Keywords: nosocomial infections, resistance phenotype, Acinetobacter

INTRODUCTION
Members of the genus Acinetobacter are ubiquitous, free living, small
aerobic Gram negative coccobacilli that prefer moist environment (4). They areusually considered to be opportunistic pathogens, and recently have been reported
to cause a number of outbreaks of nosocomial infections in hospitalized patientslike septicaemia, pneumonia, wound sepsis, endocarditis, meningitis and urinary
tract infection (UTI) (8,12). Interpreting the significance of isolates from clinicalspecimens is often difficult, because of the wide distribution of Acinetobacterin nature and its ability to colonise healthy or damaged tissue (6).
The genus Acinetobacter comprises 17 validly named and 14 unnamed(genomic) species. Some unrelated (genomic) species have common designations,while some other species seem to be congruent but have different names. The
knowledge of the biology or ecology of acinetobacters at species level is limited.
This is due to the fact that identification of acinetobacters at species level is difficult. A phenotypic species identification system has been described and a varietyof genotypic methods has been explored and applied to investigate the diversity
or phylogeny in the genus. These methods include high resolution fingerprinting
with AFLP, PCR-RFLP with digestion of PCR amplified sequences, and analysis ofvarious DNA sequences. Of these, AFLP analysis and amplified 16SrRNA ribosomal
DNA restriction analysis have been validated with large numbers of strains of all
described species. Nucleotide sequence based methods are expected to be thestandard for identification in the near future (2).

However, because routine identification in the clinical microbiology laboratory
is not yet possible, they are divided and grouped into three main complexes:
Acinetobacter calcoaceticus-baumannii complex: glucose-oxidisingnonhemolytic, (A.baumannii can be identified by OXA-51 typing)
Acinetobacter lowffii: glucose-negative nonhemolytic
Acinetobacter haemolyticus: hemolytic

MATERIAL AND METHODS
In a period of six months (Jan-June 2008) we collected 482 samples from 444
patients hospitalized for at least 48 hours in the Intensive Care Unit`s of Clinical
Emergency County Hospital Timisoara.
From 457 positive samples (bronchoalveolar fluids, wound secretions, urines,
blood, peritoneal fluids, catheter tips) we isolated 466 microbial strains with nosocomial potential, from wich 28 were Acinetobacter spp. strains.
All the samples were inoculated on 5% sheep blood agar and MacConkey
agar at 37°C for 24 hours.
The identification of germs was based on colonial appearance and biochemical
characteristics. Identification of Gram negative germs was performed by the API
system (ID 32GN/BioMerieux). Acinetobacter species differentiation was doneon basis of glucose oxidation, haemolysis, growth at 37 and 44°C, susceptibility
to penicillin and chloramphenicol discs.

Received July 2008. Accepted October 2008. Address for correspondence: Dr. Delia Muntean, Microbiology Department, “Victor Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu Square No.
2A, 300041, Timisoara

2008.18.4(60)  Fiziologia - Physiology

25

The sensitivity of isolated germs to antimicrobials was tested using standardised Kirby-Bauer technique (CLSI standards) with manual and automatic reading
methods (Osiris-BioRad Laboratories). We categorized these germs according totheir phenotypic patterns.

RESULTS
From a total of 466 isolates of various bacteria obtained from different specimens, 28 (6%) isolates belonged to the Acinetobacter spp. Species differentiation of the isolates showed a predominance of A. baumannii –complex at
92.85%, while A. lowffii showed an isolation rate of 7.14%.
The distribution of these strains from various specimens (bronchoalveolar
fluids, wound secretions, urines, blood, peritoneal fluids, catheter tips) are asshown in Table I.
Table I. Clinical specimens showing isolation rates and antibiotic
resistanceSpecimen

Isolates

Bronchoalveolar fluids

A. baumannii
A. lowffii

Wound secretions
A. baumannii
UrinesA. baumannii
Blood
A. baumannii
Peritoneal fluids
A. baumannii
Catheter tips
A. baumannii*MDR: multidrug resistantNo./%of isolates
12/ 46.15%
2/ 100%
5/ 19.23%
6/ 23.07%
1/ 3.84%
1/ 3.84%
1/ 3.84%

No./%
MDR*
1 2 /
52.17%
2/ 8.69%
4/ 17.39%
5/ 21.73%
0/ 0%
0/ 0%
0/ 0%

The percentage of multidrug resistant isolates showing resistance to two or
more antibiotics was 82.14% (23 strains). The highest was seen in bronchoalveolar
fluid and urine specimens at 60.86% and 21.73% respectively. Only 3 (10.71%) of
isolates were sensitive to all antibiotics exception trimethoprim-sulphamethoxazole.
Resistance phenotypes in Acinetobacter spp. are presented in Table II.
Table II. Resistance phenotypes in Acinetobacter spp. isolates

Antibiotics

Resistance pheno- No.
%
types
wild phenotype3
10.71%
b-lactam antibiotics
PASE
2
7.14%
CASE
1
3.57%
PASE+CASE
16
57.14%
Mechanisms accumu- 6
21.42%
lation17.85%
wild phenotype
535.71%
G
10
35.71%
Aminoglycosides
GT
10
3.57%
GN
1
2
7.14%
GTN
Fluoroquinolones
wild phenotype
6
21.42%
resistant phenotype
2278.58%
Trimethoprim-sulphame- wild phenotype
00%thoxazole
resistant phenotype
28
100%
Doxiciclyne
wild phenotype
2278.58%
resistant phenotype
6
21.42%
Chloramphenicol
wild phenotype
517.85%
resistant phenotype23
82.14%
Legend: PASE-penicillinase, CASE-cephalosporinase, G-gentamycine, Ttobramycine, Nt-netilmicin, A-amikacin
26

DISCUSSIONS
Studies on Acinetobacter in various countries (13) have shown a predominanceof isolation from urine (21-27%) and tracheo-bronchial secretions (24.8-48.8%).
In this study, respiratory secretions samples were predominantly received forbacterial diagnosis (50%).
A. baumannii is naturally resistant to first and second generation cephalosporins and at low levels to trimethoprim. Acquired resistance in this species
is secondary to the presence of transposable elements or of plasmids (7,10). The
percentage of b-lactam antibiotics resistance, in our study, was 89.29%, higher
than in other European countries. In this study Acinetobacter strains were resistantin all cases to trimethoprim-sulphamethoxazole.
Aminoglycoside resistance in Acinetobacter spp. is common and due to
enzymes which modify the antibiotics. 3’-Phosphotransferase types I and II,
2’-acetyltransferase, 3-acetyltransferase type I, 6’-acetyltransferase, 9-adenylyltransferase, 3”,9-adenylyltransferase, and 2”-adenylyltransferase have been
detected in this bacterial genus (1,3,5,9,10,11). Since it is inactivated only by
the 6’-acetylating enzyme, amikacin remained until recently the most active
aminoglycoside for infections due to Acinetobacter spp. We did not isolate any
strains with amikacin resistance.

CONCLUSIONS
Acinetobacter baumannii showed predominance amongst the isolated species.
Another identified species was Acinetobacter lowffii.
Acinetobacter baumannii is responsible for nosocomial infections, especiallypulmonary and urinary tract infections. These infections are difficult to eradicate inpart because of the multiple resistance of this organism to antibiotics.
Multi-drug resistant Acinetobacter nosocomial infection has emerged as anincreasing problem in intensive care units. These infections are difficult and costly
to treat. The analysis of risk factors and susceptibility pattern will be useful inunderstanding epidemiology of this organism in a hospital setup.
REFERENCES
1. Bergogne-Berezin, E., M. L. Joly, N. Moreau, and F. Le Goffic. Aminoglycosidemodifying enzymes in clinical isolates of Acinetobacter calcoaceticus. Curr. Microbiol.
1980; 4:361-364
2. Dijkshoorn L. The Diversity of the Genus Acinetobacter. Acinetobacter Molecular
Biology (Gerischer U, ed.). Caister Academic Press, 2008
3. Dowding, J. E. Novel aminoglycoside modifying enzyme from a clinical isolate ofAcinetobacter. J. Gen. Microbiol. 1979; 110:239-241
4. Gerner-Smidt P. Taxonomy and epidemiology of Acinetobacter infections. Rev
Med Microbiol 1995;6:186-97
5. Gomez-Lus R, Larrad L, Rubio-Calvo MC, Navarro M, Asierra MP. AAC(3) and AAC(6’)
enzymes produced by R plasmids isolated in general hospital, p. 295-303. In S.
Mitsuhashi, L. Rosival, and V. Krcmery (ed.), Antibiotic resistance. Springer-Verlag
KG, Berlin, 1980
6. Henricksen SD. Moraxella, Acinetobacter and Mimae. Bacterial Rev 1973;37:522-61
7. Hinchliffe E, Vivian A. Naturally occurring plasmids in Acinetobacter calcoaceticus:a P class R factor of restricted host range. J. Gen. Microbiol. 1980; 116:75-80
8. Levi I, Rubinstein E. Acinetobacter infections-overview of clinical features. In :Bergogne-Berezin I, Joly-Guilloo MI,Towner KJ,editors. Acinetobacter: microbiology,
epidemiology, infections, management. Boca Raton, CRC Press. 1996;101-15
9. Murray BE, Moellering RC Jr. Aminoglycosidemodifying enzymes among clinicalisolates of Acinetobacter calcoaceticus subsp. anitratus (Herellea vaginicola): explanation for high-level aminoglycoside resistance. Antimicrob. Agents Chemother.
1979; 15:190-199

Fiziologia - Physiology  2008.18.4(60)10. Murray BE, Moellering RC Jr. Evidence of plasmid-mediated production ofaminoglycoside-modifying enzymes not previously described in Acinetobacter.
Antimicrob. Agents Chemother. 1980; 17:30-36
11. Shimizu TJ, Inoue M, Mitsuhashi S, Naganawa H, Koudo S. Enzymatic adenylylation of spectinomycin by Acinetobacter calcoaceticus, subsp. anitratus. J. Antibiot.
1981; 34:869-875

12. Towner KJ. Clinical importance and antibiotic resistance of Acinetobacter spp. J
Med Microbiol 1997;46:721-46
13. Villers D, Espase E, Coste-Burel M, Pharm D et al. Nosocomial Acinetobacterbaumannii infections: Microbiological and clinical epidemiology. Ann Intern Med
1998;129:182-9

TULPINI DE ACINETOBACTER IZOLATE DIN INFECŢII NOSOCOMIALEREZUMAT
Scop: Determinarea prevalenţei tulpinilor de Acinetobacter spp., izolate de la pacienţi spitalizaţi într-o Secţie de Terapie Intensivă, şi fenotipurile lor derezistenţă.
Metode: Germenii au fost identificaţi utilizând galerii API (ID 32 GN), iar pentru testarea sensibilităţii am folosit metoda difuzimetrică (conform standardului CLSI). Ulterior, tulpinile au fost încadrate în fenotipuri de rezistenţă.
Rezultate: Din 482 produse patologice (aspirate bronşice, secreţii de plagă, urini, sânge,etc.), recoltate timp de 6 luni (Ianuarie-Iunie 2008), am izolat
466 germeni cu potenţial nosocomial, dintre aceştia, 28 de tulpini (6%) au fost reprezentate de Acinetobacter spp. Un număr de 23 de tulpini (82,14%)
au fost catalogate drept tulpini multirezistente deoarece au prezentat rezistenţă la două sau mai multe antibiotice.
Concluzii: Acinetobacter baumannii este frecvent implicat în infecţiile nosocomiale, în special ale tractului respirator şi urinar. Aceste infecţii sunt dificil detratat în parte şi datorită multirezistenţei acestor germeni.
Cuvinte cheie: infecţii nosocomiale, fenotipuri de rezistenţă, Acinetobacter2008.18.4(60)  Fiziologia - Physiology

27

PHOTODYNAMIC THERAPY WITH TMPP AND ZNTMPP IN
TUMOR BEARING RATSOLTEANU ELENA-DIANA1, ADRIANA FILIP1SIMONA CLICHICI1ADRIANA MURESAN1, DOINADAICOVICIU1RODICA-MARIANA ION2
1
University of Medicine and Pharmacy “Iuliu Hatieganu”, Department of Physiology, Cluj-Napoca,
2
Valahia University/ ICECHIM, Targoviste/ Bucuresti, Romania

ABSTRACT

Photodynamic therapy (PDT) is a relatively new and highly promising treatment for cancer. It uses porphyrin derivates (photosensitisers), which are being
activated in the presence of light and generate reactive oxygen species. To improve this treatment new photosensitisers are being synthesized and tested.
The aim of this study is to assess the effect of PDT with 5,10,15,20-tetrakis (methoxyphenyl) porphyrins (TMPP) and zinc-5, 10,15,20-tetrakis (methoxyphenyl)
porphyrins (Zn-TMPP) on serum levels of reactive oxygen species (ROS) in male rats with experimentally Walker carcinoma sarcoma 256.
The rats were randomly divided into groups as follows: group 1 - no treatment, group 2 - the tumors were only irradiated, group 3 - were given TMPP 10mg/
kg peritumoral and irradiated, group 4 - were given Zn-TMPP 10mg/kg peritumoral and irradiated, group 5 - were given 5-aminolevulinic acid (5-ALA) andirradiated. Laser light (at fluence rate 100 J/cm2 and pulse frequency rate 10 kHz) at 685 nm was delivered to tumors for 15 minutes following 24 h after drug
administration.
Serum was analyzed for ROS levels: malondialdehyde (MDA), carbonylated protein (CP), the hydrogen donating ability and total SH groupings at 24 h after
treatment.
The groups treated with TMPP and Zn-TMPP was compared with those given 5-ALA regarding the ROS levels in plasma.
The MDA levels were lower than those obtained with 5-ALA (TMPP: 1.37±0.13; ZnTMPP: 1.44±0.21 nmoles/mg protein vs. 3.37± 0.70 nmoles/mg protein,p<0.01).
Regarding the levels of carbonylated proteins PDT with TMPP and ZnTMPP showed significantly increased levels compared to the control group (TMPP:
1.08±0.39; ZnTMPP: 1.03±0.22 vs. control: 0.61±0.07 nmoles/mg protein; p<0.01).
The levels of SH groupings were increased after the 5-ALA treatment and also after TMPP and Zn-TMPP but they didn’t reach statistical significance. Our results
suggest that PDT with TMPP and ZnTMPP perturbs the normal redox balance and induces citotoxic effects.
Key words: Photodynamic therapy, ROS, TMPP, ZnTMPPINTRODUCTION
Photodynamic therapy (PDT) is a relatively new and highly promising treatment
for cancer. It consists of the administration of a photosensitizer, which is selectively
retained by tumor cells and the subsequent irradiation with visible light in the presence of oxygen, which specifically inactivates neoplastic cells (10). Basically, twotypes of reactions can occur after photoactivation of the photosensitizer. One involves
the generation of free radicals (type - I photochemical reaction) and the other the
production of singlet molecular oxygen, (type II) as the main species responsible
for cell inactivation. Evidence favors the role of the type - II process in cells, although
the photodynamic process of the sensitizers on neoplastic tissues is still not wellunderstood (11). Besides the direct effect on the tumor cells, PDT also destroys themalignant cell by indirect paths such as affecting the endothelial cells of the tumor
vascularisation determining vascular damage leading to the blockage of the blood
supply to the tumor (10). In addition to this direct killing process, tumor eradication
also arises from an acute inflammatory response featured by an increased level ofvarious mediators in the PDT-treated tumor area (IL-1 β, G-CSF, IL- 8, MIP–2) (2).
To improve this treatment new photosensitizers are being synthesized and tested.
The tetrapyrrolic macro cycles have been used in PDT the last few years withpromising results both in the treatment and detection of malignant tumors. The
5,10,15,20-tetrakis (4-methoxyphenyl) porphyrin is a tetrapyrrolic macrocycle witha high coefficient of absorption in visible light and with a long life of the triplet

state. The methoxy substitutes offer important photophysics and photosensitizing
properties, and also they increase the polarity of the compound but in the meantime they preserve its lipophilic character, which confers a good interaction withthe tissue. The formation of metalloporphyrin complexes with Zn or Cd causeschanges in the free-base photophysical properties by increasing the duration of thetriple and of the quantity of singlet oxygen leading to an increased photodynamic
effect (8). In previous studies, the photodynamic activity of 5,10,15,20-tetrakis(4methoxyphenyl) porphyrin was studied in different biomimetic and biologicalmedia. This synthetic porphyrin and its complexes with metals are effective photosensitizers, and can be used as model compounds to investigate the theoreticaland instrumental aspects of PDT (5).
Taking into account the promising results of the in vitro testing of theseporphyirins, the aim of this study is to evaluate the presence of oxidative stress inthe plasma by indirect methods caused by PDT with TMPP and ZnTMPP in male
Wistar rats with experimentally Walker carcinoma sarcoma 256.

MATERIALS AND METHODS
Animal model
In this study we used male Wistar rats, 10 weeks old, weighing 180-200g.
The animals were fed a standard diet and had water ad libitum. The environmental
conditions were standard, with a temperature of 19-23°C and 12:12-hr light:darkReceived September 2008. Accepted October 2008. Address for correspondence: Olteanu Elena-Diana, 7 Popa Sapca Street. Bl. 44, Sc. D, Ap. 3, phone: 0040726240602, e-mail: ariana_di@yahoo.com

28

Fiziologia - Physiology  2008.18.4(60)cycle through the entire experiment. The PDT experiments were in agreement with
The National Ethical Committee’s Guidelines on Animal Welfare, Romania.
The Walker carcinoma-sarcoma 256 was obtained from tumor bearing rats offered by The University of Agriculture Sciences and Veterinary Medicine, Cluj-Napoca.
The tumor was maintained in The Biobase of the Physiology Department of UMP”Iuliu Hatieganu”, Cluj Napoca by successive inoculations at a 30 days period. The
tumoral material was harvested in sterile conditions (Betadine was used to clean
the tegument above the tumor) from the tumor-bearing rat; the rat was anesthetized prior to this procedure using xylazine and ketamine (2:1). The harvesting
was done after the tegument and conjunctive fibers that form the tumor capsulewere dissected. The harvested tumor was put in a Petri platelet and mixed with asterile NaCl 0,9% solution and Penicillin G and was cut in small pieces ready for
inoculation using a scalpel. Small bits of the tumor were inoculated subcutaneousin the right flank using a trochar. Prior to this procedure the rats were anesthetizedand the teguments were disinfected using Betadine. At the site of inoculation asolution of Penicillin G was applied.
Light experiment
The photodynamic therapy experiments were carried out when the tumor
reached a volume of 1cm3 at a variable interval of 21- 30 days from inoculation. The
animals were first anesthetized with a mixture of ketamine 90mg/kg and xylazine
10mg/kg and shaved. After 24h from the administration of the porphyrins, thetumors were irradiated using red light applied directly on the tegument above thetumor (λ=685 nm), at a light dose of 100 J/cm2 and a medium power of 25 W
with a frequency of 10Hz for 15 minutes. The light source was Therapeutic LASERModel D-68.
The animals were randomly divided in 5 groups each containing 10 animals:
group 1 (CONTROL) - no treatment;group 2 (LIGHT) - the tumors were only irradiated;group 3 (TMPP) - were given TMPP 10mg/kg b. w. peritumoral and irradiated;group 4 (ZnTMPP) - were given Zn-TMPP 10mg/kg b.w. peritumoral andirradiated;group 5 (5-ALA)- were given 5-aminolevulinic acid (5-ALA) 250mg/kg b.w.,
peritumoral and irradiated.
The animals were sacrificed after 24h from the irradiation and blood was
harvested on EDTA.
Drug administration
For administration TMPP and ZnTMPP were dissolved just prior to use in PBS
and the pH adjusted to 7 with NaOH 1N. The animals were injected with TMPP and
ZnTMPP 10mg/kg b.w. in 0.75 ml solution, PBS was neutralized prior to use with asolution of NaOH 1N. The TMPP and ZnTMPP were synthesized by Prof. PhD Rodica
Mariana Ion (ICECHIM Bucharest). All the solutions were protected from light withaluminum sheets. Five-aminolevulinic acid (purity 98%) was obtained from SigmaAldrich Inc. (Germany). 50 mg of 5-ALA chlorhydrate were dissolved in 0.75ml PBS.
PBS was neutralized prior to use with a solution of NaOH 1N. Each animal received adose of 250mg/kg b.w. of 5-ALA. Absolute ethanol, HCl, ethilacetate and n-butanol
were purchased from Chimopar (Bucharest). Thiobarbituric acid and KH2PO4,
Streptomycin, DNPH, guanidine chloral hydrate, 1,1-diphenil-pycrilhydrazile,
dithiobisnitrobenzoic acid, Tris were purchased from Sigma-Aldrich Chemicals GmbH
(Germany), EDTA-Na2, trichloracetic acid from Merck KgaA Damstadt (Germany).
All the reagents were of analytical grade.
To evaluate the presence of oxidative stress in the plasma we used indirect
methods. These quantify the lesions produced by the reactive oxygen species on
2008.18.4(60)  Fiziologia - Physiology

the organism’s biomolecules, the so-called effect biomarkers. In our study weevaluated the products resulted from the oxidation of lipids by determining themalondialdehyde (1) with the fluorescence method and also the effects on proteinsdetermining the carbonylated proteins (9). We also evaluated the antioxidant capacity by determining the hydrogen donating ability (4) and total SH groupings (3).
Statistical Analysis
The data were analyzed using the SPSS 16 program. They were tested forstatistical significance using the Mann-Whitney test and the Kruskal-Wallis test,
the criteria for statistical significance was p<0.05.

RESULTSThe groups that received PDT treatment with TMPP and ZnTMPP were comparedto the groups that received 5-ALA and also with two control groups: one thatreceived no treatment and the other one was only exposed to light without being
given the photosensitisers.
Malondialdehyde (MDA)
Malondialdehyde is a marker that shows the effect of the reactive oxygen
species on lipids. The values that were registered for TMPP and ZnTMPP are lower
then those obtained by PDT with 5-ALA (TMPP: 1.37±0.13: ZnTMPP: 1.44±0.21
nmoles/mg protein vs. 3.37± 0.70 nmoles/mg protein, p<0.01). And they werealso significantly lower then those obtained in the control group 2.41±0.18 nmoles/
mg protein and only with light 2.35±0.17 nmoles/mg protein, p<0.01 (Fig.1).To
appreciate if there is an overall difference between the value of MDA in this groups
we used the Kruskal-Wallis test and we obtained a p=0.001, so the difference isstatistically significant. When comparing the MDA values between the group that
underwent PDT with TMPP and that with ZnTMPP we found that there was nodifference between the two groups

Fig.1. The mean level of malondialdehyde in the plasma of tumor bearing rats,= p<0.05 compared to the control group

Carbonylated proteins (CP)The plasma levels of carbonylated proteins in PDT with TMPP and especiallywith ZnTMPP is lower than that of PDT with 5-ALA, but not statistically significant,but when compared to the control group the values are significantly higher (TMPP:
1.08±0.39; ZnTMPP: 1.03±0.22 vs. control: 0.61±0.07 nmoles/mg protein;p<0.01) (Fig.2). Also, the values of CP after PDT with 5-ALA are higher than thoseof the control group (p=0.014). The Kruskal-Wallis test showed a statisticallysignificant difference between the 5 groups (p=0.011).

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DISCUSSION

Fig.2. The mean level of carbonylated proteins in the plasma of tumor bearing,= p<0,05 compared to the control group

The total hydrogen donating ability (DH)The antioxidant capacity evaluated using the test with DPPH shows an increase
in all the groups compared to the control group. The values of DH are significantly
higher in the groups treated with 5-ALA, TMPP and ZnTMPP compared to the control
group (p<0.05) (Fig.3). And also the group that received PDT with ZnTMPP has asignificantly higher value of DH compared to that treated with 5-ALA (p=0.009).
The Kruskal-Wallis test showed also statistical significance with a p=0.004.

Fig.3. The mean level of the total hydrogen donating ability in the plasmaof tumor bearing rats, = p<0.05 compared to the control group

4. The SH groupings
The levels of SH groupings were increased after the 5-ALA treatment and also
after TMPP and ZnTMPP but they didn’t reach statistical significance (Fig.4).

One of the challenges of PDT in the treatment of cancer is to find an ideal
photosensitizer that should be a chemically pure drug, with a preferential uptake
in tumors, rapid clearance, and a strong absorption peak at light wavelengths>630 nm (2). The PS that are now in use fail to reach the criteria for the ideal PS,
so new drugs are being developed and some of them such as ZnTMPP and TMPP
showed promising results when tested in vitro. In vitro, the survival curves of Hep-2
cells, treated with TMPP, were markedly dependent on the light wavelength ranges
used for irradiation; also singlet oxygen (1O2) was found to be the main species
responsible for cell inactivation (6).
We found that PDT with ZnTMPP and TMPP influence the oxidative stressparameters in plasma increasing significantly the level of carbonylated proteins(fig.2), which leads us to the conclusion that their antitumor effect is mainly due
to the effect of these porphyrins on proteins. Surprisingly, when we look at theeffect that these porphyrins have on the lipids by measuring the oxidative stresseffects using MDA in fluorescence we observed that they are not affected byTMPPand ZnTMPP (fig.1).
We also evaluated the antioxidant capacity, we used the test with DPPH and
we found higher levels than those obtained in the control group (fig.3).
Also in vitro studies showed that the cytotoxic effect in a human carcinoma
cell line increases in the order: CuTMPP << TMPP < ZnTMPP approximately CdTMP(2). Our studies didn’t reveal any statistical significant difference between TMPP
and ZnTMPP regarding the effects of the oxidative stress on the serum parameters
we evaluated.
To prove the efficiency of these porphyrins in vivo we compared the effects
they have on the oxidative stress parameters in plasma with those obtained with
5-ALA. The second-generation photosensitizer 5-ALA received approval for thetreatment of cancerous lesions in 1999. The use of 5-ALA PDT has several advantages over PDT with porfimer sodium: there is a more rapid clearance (limiting skinphotosensitivity to 1–2 days), it can be applied topically for the treatment of skincancer and orally for cancer in the oral cavity or digestive tract, and greater tumor
selectivity is achieved. The disadvantage of 5-ALA is that it is strongly hydrophylic
and therefore not able to enter cells easily. This led to the development of several
alkyl esters of 5-ALA that do penetrate the cell easier (3).
Our results showed that 5-ALA is more effective than TMPP and ZnTMPP
regarding the parameters that we studied, but although the methods we used offera good overall image of the effects of the oxidative stress parameters they don’t
quantify the singlet oxygen generated in the tumor, they don’t offer informationon the intimate mechanism of cell destruction, and also they can be influenced byother compounds with similar structures. So before we decide on the efficiency ofthese porphyrins further in vivo studies should be conducted first to see if the dose
and the incubation period are proper; by histological means we should evaluate thetumor necrosis and the effect of these porphyrins on tumor vasculature, knowingthat an important role in PDT is played by the destruction of the vessels that offer
blood supply to the tumor. Other tests that should be performed: specific tests todetermine the cellular organites (mitochondria, lysosomes) affected by PDT with
TSPP, to quantify the genes’ expression that are induced by PDT cyclooxigenase 2,
hemoxigenase 1, aldehiddehydrogenase, citocrome p450, RhoB, HSP27, HSP70,
to quantify the level of singlet oxygen generated in the cells, the TUNEL test to
determine the apoptosis in the tissues.

CONCLUSION
Fig.4. The mean level of total SH groupings in the plasma of tumor bearing rats

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PDT with TMPP and ZnTMPP determines increased levels of protein carbonyls
in plasma in Walker bearing rats. We found no difference between the rats treatedwith TMMP and those treated with ZnTMPP. The results obtained using 5-ALA
Fiziologia - Physiology  2008.18.4(60)were better than those obtained with the two new porphyrins. We also observed
an important increase in the levels of compounds that measure the antioxidant
capacity (total antioxidant capacity and SH groupings); this may lead to a decreasein the damage done by the reactive oxygen species, namely in the values obtained
for malondialdehyde.
Additional studies are necessary in order to establish the mechanisms, whichlead to tumor destruction by PDT with these porphyrins.
Acknowledgements
The research was financed by the Ministry of Education, Research and Youthby CEEX Program (102/2006). I would like to thank the University of Medicine andPharmacology “IULIU HATIEGANU” Cluj-Napoca for the opportunity to performresearch, to the Department of Physiology for the support throughout the project.
Also I would like to give special thanks to Mrs. Daicoviciu for doing the oxidative
stress parameters determination and to the project director: Prof. PhD MD Simina
Dreve for all her support.
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5,10,15,20-tetrakis(methoxyphenyl)porphyrin. Journal of Molecular Structure,
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Oncologist, 2006, 11(9): 1034-1044

TERAPIA FOTODINAMICĂ CU TMPP ŞI ZNTMPP LA ŞOARECIPURTĂTORI DE TUMORIREZUMAT
Terapia fotodinamică (PDT) este un tratament relativ nou şi foarte promiţător în cancer ce implică utilizarea derivaţilor de porfirine ca substanţe fotosensibilizante care, activate în prezenţa luminii, generează specii reactive ale oxigenului, citotoxice. Pentru a îmbunătăţi efectele acestui tratament s-a încercat
testarea de noi substanţe fotosensibilizante.
Scopul studiului a fost să evalueze efectele PDT cu 5,10,15,20-tetra(metoxifenil) porfirină (TMPP) şi zinc-5,10,15,20-tetra(metoxifenil) porfirină (ZnTMPP)
asupra nivelelor serice ale speciilor reactive ale oxigenului (ROS) la şobolani masculi purtători de carcino-sarcom Walker 256.
Şobolanii au fost împărţiţi aleator în 5 grupuri după cum urmează: grupul 1- control, grupul 2- tumori iradiate, grupul 3–animale tratate peritumoral cu TMPP
10 mg/kgc şi apoi iradiate, grupul 4 – animale tratate peritumoral cu Zn MPP 10mg/kgc şi iradiate, grupul 5 – animale tratate cu acid 5- aminolevulinic (250mg/kgc) şi iradiate cu lumină roşie aplicată direct pe tegumentul suprajacent tumorii (λ=685 nm), în doză de 100 J/cm2, la o putere medie de 25 W şi frecvenţă
de 10 Hz, timp de 15 minute la 24 de ore de la injectarea porfirinelor. La 24 ore după tratament s-au determinat din ser malondialdehida (MDA), proteinele
carbonilate (CP), capacitatea de donor de hidrogen şi grupările SH totale iar rezultatele au fost comparate cu cele obţinute după PDT cu 5-ALA.
În PDT cu TMPP şi ZnTMPP nivelul MDA în ţesutul tumoral a fost mai scăzut comparativ cu 5-ALA (TMPP: 1,37±0,13; ZnTMPP: 1,44±0,21 nmoli/mg proteină
faţă de 3,37±0,7 nmoli/mg proteină, p<0,01). În ceea ce priveşte nivelul proteinelor carbonilate concentraţia acestora creşte semnificativ comparativ cu lotul
martor (TMPP: 1,08±0,39; ZnTMPP: 1,03±0,22 vs. control: 0,61±0,07 nmoli/mg proteină; p<0,01). Apărarea antioxidantă apreciată prin conţinutul în grupăril
sulfhidril (SH) se modifică după tratamentul cu cele trei porfirine dar nesemnificativ statistic.
Rezultatele obţinute sugerează că PDT cu TMPP şi ZnTMPP modifică echilibrul redox şi determină efecte toxice asupra macromoleculelor din celule.
Cuvinte cheie: terapie fotodinamică, ROS, TMPP, ZnTMPP

2008.18.4(60)  Fiziologia - Physiology

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Fiziologia - Physiology  2008.18.4(60)