ACTA MEDICA MARTINIANA - uniba.sk · Chemoprevention is the use of pharmacological agents that...

ACTA MEDICAMARTINIANA

Journal for Biomedical Sciences,Clinical Medicine and Nursing

Contents3

Chemoprevention of breast cancerKubatka P., Pec M., Zihlavnikova K., Ahlers I.

11Chromosomal Aberrations in Relation to Polymorphisms of GST Genes

in Slovak Footwear-WorkersMatakova T., Halasova E., Musak L., Statelova D., Janickova M., Krajcovic A., Dobrota D.

17Micromorphological changes of peripheral nerve induced by gamma radiation

Boselova L., Fusekova E., Balentova S., Adamkov M., Ochodnicka E.

22Nosocomial pneumonia at selected clinics of Martin Faculty Hospital

Busikova J., Murajda L.

26Risks of ultraviolet radiation – health protection of university students

Jakusova V., Murajda L., Poliacek I., Jakus J.

Published by the Jessenius Faculty of Medicine in Martin,Comenius University in Bratislava, Slovakia

ISSN 1335-8421 Acta Med Mart 2010, 10(1)

maketa 10/1:MAKETA 7/1 6/16/10 7:59 AM Stránka 1

E d i t o r – i n – C h i e f :Javorka Kamil, Martin, Slovakia

I n t e r n a t i o n a l E d i t o r i a l B o a r d :Belej Kamil, Martin, SlovakiaBelova Nina, Sofia, Bulgaria

Bohlin Kajsa, Stockholm, SwedenDanko Jan, Martin, Slovakia

Honzikova Natasa, Brno, Czech RepublicJakus Jan, Martin, Slovakia

Javorka Kamil, Martin, SlovakiaKliment Jan, Martin, SlovakiaLehotsky Jan, Martin, Slovakia

Mares Jan, Praha, Czech RepublicMechirova Eva, Kosice, SlovakiaMistuna Dusan, Martin, SlovakiaMokan Marian, Martin, SlovakiaMokry Juraj, Martin, SlovakiaMusial Jan, Krakow, Poland

Plank Lukas, Martin, SlovakiaStasko Jan, Martin, Slovakia

Stransky Albert, Martin, SlovakiaTatar Milos, Martin, Slovakia

Zibolen Mirko, Martin, Slovakia

E d i t o r i a l O f f i c e :

Acta Medica MartinianaJessenius faculty of Medicine, Comenius University

(Dept. of Physiology)Mala Hora 4

036 01 MartinSlovakia

Instructions for authors: http://www.jfmed.uniba.sk (Acta Medica Martiniana)

© Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia, 2010

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CHEMOPREVENTION OF BREAST CANCER

KUBATKA P.1, PEC M. 1, ZIHLAVNIKOVA K. 1, AHLERS I.2

1Department of Medical Biology, Jessenius Faculty of Medicine, Comenius University, Martin and 2 Departmentof Biological and Ecological Sciences, Faculty of Science, P. J. Safarik University, Kosice, Slovak Republic

A b s t r a c tBreast cancer represents one of the most important problems of clinical and experimental oncology. Excluding can-

cers of the skin, breast cancer is the most frequent neoplasm in women, forming approximately 30 % of all tumours. Theknowledge on the possibility and suitability of the prevention of breast cancer rose sharply in last years. The strategy ofchemoprevention seems to be promising for reducing cancer incidence both in well-defined high-risk individuals and alsoin the general population. Several clinical studies have shown that in high-risk women for breast cancer, the drugs suchas non-steroidal inflammatory drugs, selective oestrogen receptor modulators and retinoids, may reduce this risk.Chemopreventive studies carried out on model laboratory animals have an important role in new drugs efficacy evalua-tion in pre-clinical tests. Our group tested several groups of chemopreventive substances in female rat mammary car-cinogenesis.

Key words: chemoprevention, breast cancer, non-steroidal inflammatory drugs, selective oestrogen receptor modula-tors, retinoids

INTRODUCTION

Mammary gland cancer is a serious worldwide medical problem; this disease exhausts patientsboth physically and emotionally. Recently (2007) 178,480 of new cases of invasive breast cancerwere expected in USA, as well as an estimated 62,030 additional cases of in situ breast cancers.Approximately 40,460 women were expected to die in consequence of the disease (1).Chemoprevention is a rapidly growing area of oncology. In the beginning of the 21st centurya vision of chemoprevention as an effective tool to fight with breast cancer exists.Chemoprevention is the use of pharmacological agents that inhibit or reverse the process of car-cinogenesis. Whilst chemotherapy is targeted at people with manifest disease, primary chemo-prevention is directed at individuals who are healthy, although those within high risk of the dis-ease (e.g. family history, mutated genes carriers). As treatment (surgical, chemotherapy, radio-therapy) of breast cancer in many cases brings little or no success (depending on the stage of thedisease), it is necessary to be concentrated on the prevention not only from ethical but also fromeconomical reasons (2).

The aim of the chemopreventive trials is to find an efficient substance that can be administeredfor a long period with minimum adverse effects. The drugs with pleiotropic effects are very suit-able for the chemoprevention. In the clinical oncology, some classes of new agents, namely non-steroidal anti-inflammatory drugs, selective oestrogen receptor modulators, and retinoids, arediscussed (3). Moreover, the drugs primary used in the treatment of any disease with anticar-cinogenic side effect are also statins, antidiabetics or pineal hormone melatonin – they may bepotentially profitable in the breast cancer chemoprevention. Our working group tested all theabove mentioned classes of drugs.

Chemopreventive animal studies play an important role in the evaluation of the effects of newdrugs in oncochemopreventive projects. Rodent models have been important in both breast can-cer etiology and treatment strategies of this malignancy. Experimental mammary carcinogenesisin female rats induced by various chemocarcinogens, especially by N-methyl-N-nitrosourea(NMU) and 7, 12-dimethylbenz(a)anthracene (DMBA) is often used model for the study of chemo-preventive drug’s role in the process of breast cancer initiation. Our experiments showed, that

A C T A M E D I C A M A R T I N I A N A 2 0 1 0 10 / 1 3

A d d r e s s f o r c o r r e s p o n d e n c e:Assoc. Prof. Peter Kubatka, RNDr., PhD., Department of Medical Biology, Jessenius Faculty of Medicine, ComeniusUniversity, Mala Hora 4, 037 54 Martin, Slovakia, Phone: 043/4131425, Fax: 043/413 63 32, e-mail: [email protected]


the application of carcinogen between 40th – 55th postnatal days (early adolescence when pro-liferation of mammary gland structures reaches its maximum) significantly improved the induc-tion of mammary gland tumours in outbred Sprague-Dawley rats (4). The chemoprevention isinitiated one week prior to carcinogen application and then continues 15-25 weeks till the ter-mination of our experiments. The basic parameters of carcinogenesis in experimental oncologyare evaluated: incidence, frequency, latency and volume of tumours.

The main aim of this review is to summarize our results of the chemoprevention of rat mam-mary carcinogenesis using different classes of drugs. In this paper, the current clinicalapproaches in the breast cancer chemoprevention will be resumed.

NON-STEROIDAL ANTI-INFLAMMATORY DRUGS

Non-steroidal anti-inflammatory drugs (NSAIDs) are the important group of substancesinhibiting the manifestation of inflammation (pain, oedema etc.). NSAIDs belong to the most fre-quently used drugs worldwide; with as many as 20 to 30 % of the population in the developedcountries taking prescribed forms of these agents (5). The mechanism of their action is based onthe decrease of prostanoid synthesis through cyclooxygenase-1 (COX-1), COX-2 and lipoxygenaseinhibition. COX-2 is supposed to have more important role in carcinogenesis than COX-1. Theincreased activity of COX-2 isoform was found in colorectal, oesophageal, gastric and mammarycarcinomas. Expression of COX-2 may influence the process of carcinogenesis via several mech-anisms: peroxidase activity of COX-2 supports the conversion of procarcinogens to carcinogens(6), inhibitory effect of prostaglandins in immune reactions was found (7), as well as angiogenet-ic (8) and antiapoptotic effects (6). Enhanced invasion of tumour cells with increased expressionof COX-2 was observed. Brueggemeier et al. (9) pointed to linear dependence between aromatasegene (CYP 19) expression and expression of COX-2 in mammary carcinoma. The above mentionedfacts predetermine the use of NSAIDs in mammary carcinogenesis prevention.

At present, medical practice prefers NSAIDs with predominant inhibition of COX-1 (mostlyibuprofen and diclofenac), with predominant COX-2 inhibition (nimesulide) and selective COX-2inhibition: coxibs (celecoxib, rofecoxib, parecoxib, etoricoxib, lumiracoxib, valdecoxib). From thepharmaco-dynamic point of view, inhibitors COX-1 (acetylsalicylic acid, indomethacin andibuprofen) display serious side effects on the gastrointestinal tract associated with their long-term application. These undesirable effects are markedly reduced in administration of COX-2inhibitors (nimesulide, meloxicam, coxibs), supporting their preference in long-term use. On theother hand, several recent studies pointed to an increased incidence of thromboembolic eventsassociated with the use of coxibs (10,11).

The results from epidemiological studies indicate the obvious chemopreventive effect of NSAIDsin female breast cancer. The regular ibuprofen and aspirin use decreased the mammary car-cinogenesis incidence by 50 and 40%, respectively (12,13). Moreover several retrospective clinicalstudies aimed at a long-term application of various NSAIDs reported significant decrease in theincidence of colon cancer (14), prostate (15) and lung cancer (16). The experimental studies car-ried out in laboratory animals confirmed the chemopreventive effects of NSAIDs in various neo-plasias including breast cancer (17,18).

The experiments with indomethacin administered in drinking water conducted by our group inDMBA-induced rat mammary carcinogenesis reported apparently decreased tumour incidenceand frequency and prolonged latency by 59 days as opposed to controls (19). Chemopreventiveeffect of diclofenac administered in drinking water to female rats significantly decreased mam-mary tumour incidence, frequency and volume (20). Tumour suppressive effect of nimesulide(applied subcutaneously) was found out by our group in NMU-induced rat mammary carcino-genesis in (21). No oncostatic effect of nimesulide in above-mentioned experiment was found inDMBA-induced mammary carcinogenesis.

Our further studies were focussed on preventive effects of coxibs in experimental mammarycarcinogenesis. Dietary administered rofecoxib in NMU-induced rat mammary carcinogenesis isthe first report on significant tumour - suppressive effects of this drug in experimental mamma-



ry carcinogenesis. Antitumor effect of rofecoxib, with a clear dose- dependent manner, wasrecorded in all evaluated parameters of mammary carcinogenesis (22). The study focussed onprevention of NMU-induced mammary carcinogenesis by etoricoxib administered with diet tofemale rats reported a slight tumour - suppressive effect documented by decrease in tumour inci-dence, frequency and prolonged latency by 7 days (23). In our next study, dietary administeredcelecoxib markedly decreased tumour frequency (24).

The results from our above-mentioned experiments and also the data from clinical and epi-demiological studies point out to preventive effects of NSAIDs in mammary carcinogenesis thatcannot be overlooked. Removal of side effects of coxibs may offer a new promise for their use incancer prevention.

TAMOXIFEN AND RALOXIFENE

Oestrogens have an important role in the process of breast cancer initiation. Binding to oestro-gen receptors (ER) is the first active step in target tissue. Selective oestrogen receptor modulators(SERMs) competitively inhibit the binding of oestrogens to ER and through this action have animportant role in the treatment of oestrogen-dependent breast tumours. Tamoxifen acts asantioestrogen in breast tissue whereas in other tissues acts as partial agonist. Its oestrogen-likeeffects were observed in the increase of bone density, in the decrease of serum cholesterol andof low-density lipoproteins. On the other hand tamoxifen has an oestrogenic effect on uterus (25).The results of our study showed the potent inhibitory effect of tamoxifen: chemically-inducedmammary carcinogenesis in female rats was totally suppressed in both NMU and DMBA exper-iment (26). The efficacy of tamoxifen was confirmed also in other experimental studies where themammary gland carcinomas were induced by NMU (27) or DMBA (28).

In 1998, a randomized trial of more than 13.000 women demonstrated, than tamoxifen couldreduce the risk of invasive and in situ ER-positive breast cancer in high risk women (25). Womenwho received tamoxifen had decreased cancer risk by 42% in comparison with control group.A preventive effect of tamoxifen was also observed in international randomized prevention trialinvolving more than 7000 women. The breast cancer risk was reduced by 26% in the women whoreceived tamoxifen (29). Administration of tamoxifen resulted in some risks in both trials, par-ticularly an increased risk of endometrial cancer.

Raloxifene - a derivative of benzothiophene represents the second generation of SERMs.Originally it was developed for treatment and prevention of postmenopausal osteoporosis.However, its oestrogen-antagonistic properties in other tissues of the organism were alsoobserved. Clinical studies have shown oestrogen-like effects in the bone density increase andtotal serum cholesterol and low density lipoproteins decrease in postmenopausal women (30).Raloxifene appeared to be effective in the prevention of experimental mammary carcinogenesis infemale rats induced by NMU (31) or DMBA (32). In our experiments, a markedly decreasedtumour incidence and frequency and lengthened latency period after raloxifene administrationwere found compared to group without chemoprevention (33,34).

The study initiated in 1994 and aimed at osteoporosis prevention in postmenopausal women(n=7.705), investigate also the possibilities of raloxifene to decrease breast carcinoma incidence(The Multiple Outcomes of Raloxifene Evaluation). Researches noticed significant lowered risk ofbreast cancer (35). A study comparing the effectiveness of the 2 drugs – The Study of Tamoxifenand Raloxifene found that raloxifene decreased the risk of invasive breast cancer to the samedegree as tamoxifen, although it did not have the same protective effects against in situ cancer(36). However raloxifene had a lower risk of some adverse effects such as uterine cancer andblood clots in the legs or lungs, compared to tamoxifen.

RETINOIDS

Retinoids have been studied as chemopreventive agents in clinical trials due to their estab-lished role in carcinogenesis. Retinoids effect through their interaction in retinoid receptors. They



can act both as anti-initiators and anti-promoters; alter carcinogen metabolism, cell division anddifferentiation, as well as expression of oncogenes and biosynthesis of polyamines andprostaglandins (37). Retinoids can induce apoptosis, carcinogenesis could be influenced by theirimmunostimulative activities – they activate cell-mediated cytotoxicity and increase of naturalkillers (38). The positive effects of retinoids on chemically - induced mammary carcinogenesiswere reported by Moon et al. already in 1976 (39). Two experiments investigating the effects ofretinyl acetate were performed at our laboratory. In both of them, female Sprague-Dawley ratsbred in the same conditions were used, but in the first experiment mammary carcinogenesis wasinduced by DMBA (40) and in the second by NMU (41). In the first experiment retinyl acetate sig-nificantly reduced the incidence and frequency of tumours and lengthened the latency comparedto control group. In the second experiment retinoid lengthened latency by 16 days; an additiveeffects of combined administration of retinyl acetate and melatonin in this experiment wereobserved in tumour incidence and latency period of tumours, this combination of drugs demon-strably inhibited tumour burden in rats.

The most widely studied retinoid in clinical trials on breast cancer chemoprevention due to itsselective accumulation in breast tissue and to its favourable toxicological profile is fenretinide. Ina phase III breast cancer breast cancer prevention trial, fenretinide showed a durable trend toa reduction of second breast malignances in premenopausal women (42). This result was asso-ciated with a favourable modulation of circulating insulin-growth factor-1 and its main bindingprotein IGFBP-3, which have been associated with breast cancer risk in premenopausal women.Novel retinoid X receptor-selective retinoids (or rexinoids) have been shown to suppress the devel-opment of breast cancer in several animal models with minimal toxicity (as the consequence totheir selective binding to rexinoid receptors) (43,44) and are intensively studied either alone or incombination with SERMs, both in vivo and in vitro. The trial with rexinoid bexarotene in womenwith high breast cancer risk is currently underway.

MELATONIN

Melatonin (5-methoxy-N-acetyltryptamine) is a pineal gland hormone, produced also in theretina and gastrointestinal tract. Antitumour effect of melatonin (MEL) was observed mainly inoestrogen-sensitive tumours (mammary gland and prostate tumours), but also in cervicaltumours, Lewis lung carcinoma, hepatocarcinoma, melanoma and ovarian carcinoma (45,46).Numerous experimental studies pointed to tumour - suppressive effects of MEL via several mech-anisms. Blask et al. (46) emphasised possible antioestrogenic effect of MEL in NMU-induced ratmammary carcinogenesis. Blask and Hill (47) points to a possible tumour-suppressive effect ofMEL by inhibition of linoleic acid and its metabolite 13-hydroxyoctadecadienoic acid utilisation.Besides the antioxidative properties with free radical scavenging, MEL displays someimmunomodulatory and especially antiinflammatory activities. MEL stimulates the productionof IL-2, IL-6, IL-12 and depresses the effectiveness of IL-8 and tumour necrosis factor-α (48).

Anticarcinogenic effect of MEL administered in drinking water alone or in combination withanother chemoprevention was investigated in our studies. Continuous administration of MEL ata concentration of 100 μg/ml during 25 weeks (MEL was administered after DMBA application)reduced mammary tumour frequency per animal (40). More pronounced anticarcinogenic effectwas observed when MEL was administered before carcinogen application. Prolonged latency peri-od was found out by Bojková et al. (41) in NMU-induced carcinogenesis with MEL applied indrinking water (20 μg/ml). In the experiment carried out by Kubatka et al. (33) MEL applied indrinking water at a concentration of 4 g/ml markedly reduced tumour frequency in NMU-induced carcinogenesis. In our next experiment, MEL applied at the same dose significantlydecreased the incidence and frequency of mammary tumours (34). On the other hand, in thatexperiment a paradoxical increase in the tumour volume occurred. Obvious anticarcinogeniceffect of indomethacin and MEL in DMBA-induced carcinogenesis was recorded in the experi-ment by Môciková-Kalická et al. (19); however, the addition of MEL did not substantially influ-enced the anticarcinogenic effect. No additive oncostatic effect of MEL in combination with nime-



sulide on female rat mammary carcinogenesis was observed in further experiment (21).Administration of MEL influenced the mammary carcinogenesis parameters in female ratsinduced by NMU and psychoemotional stress (immobilization) (49). One of the possible causesexplaining weaker antineoplastic effect of MEL in any of our experiments is the use of the doseshigher than “physiological” concentrations. Those could have influenced the modulation of den-sity and/or affinity of expected MEL receptors through which MEL can manifest antioestrogeniceffects. More research should be done to find the proper dose, frequency and application methodof MEL combined with other chemopreventive substances.

AROMATASE INHIBITORS AND OTHER CHEMOPREVENTIVE SUBSTANCES

The enzyme aromatase catalyzes the last step of oestrogen synthesis. Recently, potent third-generation aromatase inhibitors have been introduced for breast cancer treatment in post-menopausal women: the steroidal exemestane and the non-steroidals letrozole and anastrozole.The concept of the key role of in situ oestradiol synthesis rather than uptake of plasma oestra-diol by breast tissue in carcinogenesis exists (50). For these reasons, intratumoural aromatasecould be an important therapeutic target against breast cancer.

Using the female rats with functional ovaries, we have mimicked a chemoprevention of breastcancer in premenopausal high-risk woman. In our experiments, the significant chemopreventiveeffects of dietary administered letrozole (51) and anastrozole (52) were found. Based on the sup-pression of mammary carcinogenesis without changes in plasma oestradiol levels in rats afteranastrozole treatment, our results distinctly confirm the hypothesis of the crucial role of in situoestradiol biosynthesis in breast tissue. On the other hand, a neoplastic effect of exemestane inour study was observed (53). It is postulated that the effects of exemestane and its metabolite 17-hydroexemestane, with the chemical structure related to the androstenedione, may be a result oftheir androgenic action through the androgen receptor (54).

Data from experimental studies in vivo suggested anticarcinogenic effects of statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors). Pravastatin administered in water and sim-vastatin administered in food were shown to suppress the development of colon cancer in mice(55). Pravastatin administered in rats has been shown to reduce the incidence and volume ofhepatic neoplastic nodules and colon carcinogenesis (56). Some epidemiologic studies suggestedthat statins may prevent certain malignancies, including melanoma (57), and breast (58), colon(59), uterine (60) and prostate (61) cancers. Antitumour properties of statins in breast cancerhave not been tested so far, therefore original experimental studies are necessary, which mayconfirm expected tumour - suppressive effects of statins in mammary carcinogenesis. Our pilotstudy demonstrated an apparent antineoplastic effect of dietary administered atorvastatin inmammary gland carcinogenesis in female rats (sent for publication).

Antidiabetic drugs, mainly biguanids and thiazolidinedines, have been found to have variouseffects on cancer in experimental systems and in epidemiologic studies (62,63,64). The chemo-preventive effect of peroral antidiabetic metformin in mammary carcinogenesis in female rats wasevaluated by our group (65). Metformin administered in drinking water, did not significantly alterthe tumour growth although a delay in tumour onset was recorded after higher metformin dose.In our recent study, dietary administered pioglitazone had a significant antineoplastic effect inrat mammary carcinogenesis (sent for publication).

CONCLUSIONS

Chemoprevention offers promise as a strategy for reducing morbidity and mortality from breastcancer in women. Our working group has shown that NSAIDs – indomethacin, diclofenac, nime-sulide, robecoxib, etoricoxib and celecoxib demonstrated antineoplastic effects in experimentalmammary carcinogenesis. The optimal type, dose and treatment duration of NSAIDs is still not



defined together with the feasibility of such an intervention in risk population. There may bea role for NSAIDs in combination with endocrine therapies as either an adjuvant or palliativetreatment for women with established breast cancer. As to long term NSAIDs application, thereis an effort to find highly selective COX-2 inhibitors with prominent oncostatic properties andminimal adverse effects. The SERMs are a logical choice for the chemoprevention because of theirantiestrogenic effects in the breast. Despite the large potential breast cancer risk reduction oftamoxifen, its risk management is still not routine. Raloxifene was proposed as an alternative totamoxifen based on its oestrogen antagonist effects in the mammary gland and its relative safe-ty. Retinoids are being extensively evaluated in clinical trials in cancer prevention. Great interesthas been recently focussed on rexinoids, which have strong antineoplastic potential with limitedtoxicity. With exception of above mentioned three classes of drugs, there are several groups ofother substances with an antineoplastic potential in cancer disease yet.

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Received: September 25, 2009Accepted: November 26, 2009



CHROMOSOMAL ABERRATIONS IN RELATION TO POLYMORPHISMS OF GST GENES IN SLOVAK FOOTWEAR-WORKERS

MATAKOVA T. 1, HALASOVA E.2*, MUSAK L.2, STATELOVA D.3, JANICKOVAM.3, KRAJCOVIC A.4 , DOBROTA D.1

1 Department of Medical Biochemistry, Comenius University, the Jessenius Faculty of Medicine, 2 Department ofMedical Biology, Jessenius Faculty of Medicine, Comenius University, 3 Clinic of Stomatology and Maxillofacial Surgery,

Jessenius Faculty of Medicine, Comenius University, 4 Department of Public Health, Jessenius Faculty of Medicine,Comenius University, Martin, Slovak Republic

A b s t r a c tEmployees in the footwear manufacturing industry are routinely exposed to complex mixtures of solvents used in

cleaning and as diluents in glues, primers, and degreasers. The aim of this study was to determine the genotoxic effectsin a group of footwear-workers occupationally exposed to solvent-based adhesives and solutions containing organic sol-vents, mainly toluene.

Cytogenetic analysis in peripheral blood lymphocytes was used to compare 40 shoe workers (14 men and 26 women)and 31 control subjects selected from general population not exposed to any mutagenic or carcinogenic agents.Frequencies of damaged cells, chromosomal aberrations, including breaks and rearrangements were scored for bothgroups. Polymorphisms in genes GSTT1, GSTM1, and GSTP1, determined by PCR-based methods, were used as bio-markers of susceptibility.

The exposed group showed a significant increase of total chromosomal aberrations (CAs) in comparison with control(2.65±0.24%; 1.54±0.12 respectively). The frequency of chromosome-type aberrations (CSA) was 3.9 time higher (P<0.001)in the exposed than in the control group (2.07±0.19%; 1.52±0.12%, respectively). The frequency of chromatide-type abe-rations (CTA) was significantly lower (P<0.05) in the exposed group than in the control group (0.58±0.2%; 1.02±0.11% res-pectively). GSTT1 null subjects exhibited significantly higher (P<0.05) frequency of CSAs than GSTT1 positive subjects(2.00±0.43%; 1.40±0.25%). Different genotype of GSTM1 and GSTP1 did not show any influence on frequency of evalu-ated parameters.

Key words: occupational exposure, chromosomal aberration, glutathione S-transferase, polymorphisms

INTRODUCTION

Footwear manufacture workers are routinely exposed to complex mixtures of solvents, such asbenzene, acetone, n-hexane, methyl ethyl ketone, and large amounts of toluene used in cleaningand diluents in glues, primers, and degreasers (1). Employment in the shoe production is asso-ciated with an enhanced risk of cancer (primarily nose and nasal sinuses as well as leukemia)(2,3). Benzene is well-known clastogen that requires metabolic activation to be mutagenic (4).The genotoxic metabolites as phenol, catechol, hydroquinone and p- or o-bezoquinone are alsothought to play an important role in benzene myelotoxicity and leukemogenesis (5,6). The abovementioned metabolites of benzene can cause DNA damage via DNA alkylation or induction ofoxidative stress but they may also act as aneuploidogens, causing microtubule disruption (7).

People employed in footwear industry are at an increased risk of leukemia and nasal cancers(8). Some studies indicated an excessive risk of other cancers as well, for example cancers of tra-chea, lung, stomach, bladder and kidney (9,10,11,12)

Cytogenetic analysis in peripheral lymphocytes is a routine method used to evaluate an expo-sure to clastogens (13,14) and cytogenetic parameters may provide information on structuralchromosomal damage often predictive for increased cancer risk (15).

Polymorphic genes of low penetrance but with high allele frequency involved in the metabolismof xenobiotics may modulate the levels of biomarkers arising from environmental and/or occu-


A d d r e s s f o r c o r r e s p o n d e n c e: Assoc.Prof. Erika Halasova, RNDr. PhD., Department of Medical Biology, Comenius University, Jessenius Faculty ofMedicine, Malá Hora 4, 03601 Martin, Slovak RepublicPhone: +421-43-4131425; Fax: +421-43-4136770; E-mail: [email protected]


pational exposure to genotoxicants (16,17). Many studies have shown elevated cancer pronenessfor individuals carrying the potential at-risk alleles of metabolic genes but a number of contro-versial results has also been obtained. Reasons for controversial data and recommendations todesign studies aimed to measure the effect of polymorphic metabolic genes on cancer suscepti-bility have been suggested (18,19,20).

The purpose of the present study was to assess CA frequencies in peripheral blood lympho-cytes of workers engaged in the production of shoes from a factory in Partizanske, Slovakia, andunexposed controls as markers of genotoxic biological effect. Subsequently, the possible modu-latory effect of gene polymorphisms (GSTM1 and GSTT1 deletion polymorphisms and GSTP) ofbenzene metabolizing enzymes on CA frequencies was also analyzed.

SUBJECTS AND METHODS

Study population and sample collection:The study involved 40 footwear industry workers (14 males and 26 females), employed at the

factory with occupational exposure to adhesives and solutions containing organic solvents. Thecontrol group was recruited from occupationally unexposed individuals (11 males and 20females). The characteristics of studied populations are presented in Table 1. The workers andthe controls were asked to read and sign an informed consent in agreement with requirementsof Ethical Commission for Research.


Table 1 Characteristics of study subject

Total no. of individualsAge ± S.D. (years)RangeGenderMaleFemaleTime of exposure ± S.D.(years)range

Exposed group

4049.05±11.68

34-66

14 (35%) 26 (65%)

19.04±10.12 2-30

Control group

31 47.22±7.81

30-67

11 (35.5%) 20 (64.5%)

- -

S.D. – standard deviation

Chromosomal aberrations (CAs) analysis:Chromosome aberration analysis was performed on peripheral blood lymphocytes. Two paral-

lel cultures were incubated for 48h at 38°C. Colcemid (Gibco) Invitrogen, Paisley (UK), in finalconcentration 0.02μg.ml-1 was added to 2h prior to processing. The cultures were centrifugedand subjected and hypotonic shock for 2min in 0.075 M KCl at 37°C. The collected cells werefixed in acetic acid: methanol (1:3) twice and air-dried preparations were then prepared. Air-driedslides were stained with 10% Giemsa. For each person 100 metaphases were analyzed. CAs inperipheral blood lymphocytes include chromosomal brakes and exchanges visible in attestedmetaphase-stage cells. They are usually divided in the chromosome-type aberrations (CSAs) andchromatide-type aberrations (CTAs). Identification of the type of aberrations was performedaccording to the standard classification (21).

Genotype analysis:Genomic DNA was isolated using standard techniques (proteinase K digestion, the phenol

/chloroform extraction and ethanol precipitation, dissolved in TE buffer (pH = 7.5) from blooddrawn into 4.5 ml EDTA tubes and stored at -20oC until use. The concentration of DNA wasadjusted to 100 μg/ml and the DNA was stored at -20oC. All genotyping analyses were PCR-


based, with a total volume of 25 μl for each reaction. In all reactions a positive and negative con-trol is included. As a quality control, 10-20% of all samples are repeated as blind duplicates.

The GSTM1 and GSTT1 genetic polymorphisms are determined simultaneously by multiplexPCR (22). The absence of the GSTM1- and/or GSTT1-specific PCR-product indicated the corre-sponding null genotype. β-globin was amplified in the same reaction as an internal positive con-trol. PCR-reaction products are analyzed by gel electrophoresis (3% agarose) and ethidium bro-mide staining for the presence of 273 bp GSTM1 product, 480 bp GSTT1 product, and 320 bpβ-globin product.

GSTP1 A to G polymorphism at codon 105 is determined by PCR of 176 bp fragment andrestriction fragment length polymorphism (RLFP). An amount of 10μl of the amplicon was digest-ed for 4 hours with 5 units of the restriction enzyme Alw 26I at 37°C. Three variants were iden-tified: Ile/Ile, Ile/Val, and Val/Val (23).

Statistical analysis:Statistical analysis was carried out with the use of Statgraphics programme, version 7 (LEAD

technologies, Manugistics, Cambridge, MA). Mann-Whitney U-test was used to test the differ-ences between the groups. Spearman correlation analysis was employed to calculate the corre-lation between individual parameters. Kruskal-Wallis test was performed to evaluate associationsbetween biomarkers and particular genotypes. Heterozygous (Ile/Val) and homozygous variant(Val/Val) genotypes were considered as one group due to the low number of individuals bearinghomozygous variant (Val/Val) genotype.

RESULTS

A significant difference (P<0.001) in the frequency of total CAs between the exposed and con-trol groups was recorded (2.65±0.24%; 1.54±0.12% respectively). The frequencies of chromosome-type aberrations were 3.9 time higher (P<0.001) in the exposed than in the control group(2.07±0.19%; 0.52±0.12%, respectively). On the other hand, frequencies of CTAs were signifi-cantly lower (P<0.05) in exposed group in comparison to control (0.58±0.2%; 1.02±0.11% respec-tively). The data are shown in the Table 2.


Table 2 Frequency of chromosomal aberration in exposed workers and control individuals

Exposed group (n = 40)Control group (n = 31)

CAs ± SEM (%)

2.65±0.24***

1.54±0.12

CTA ± SEM (%)

0.58±0.2*

1.02±0.11

CSA ± SEM (%)

2.07±0.19***

0.52±0.12

CAs – chromosomal aberrations (total), CTA – chromatide-type aberrations, CSA – chromosome-type aberrations,*(P<0.05), ***(P<0.001), SEM – standard error of mean

Table 3 Frequencies of total chromosomal aberrations, chromatide type, and chromosome-type aberrations stratifiedaccording to the polymorphism in GSTM1, GSTT1, and GSTP1 genes.

GSTT1 positiveGSTT1 nullGSTM1 positiveGSTM1 nullGSTP1Ile/IleIle/Val or Val/Val

CAs±SEM (%)

2.47±0.26 2.83±0.302.66±0.33 2.50±0.35

2.66±0.37 2.50±0.33

CTA±SEM (%)

1.06±0.17 0.83±0.47 1.00±0.17 1.00±0.31

0.66±0.16 1.25±0.27

CSA±SEM (%)

1.40±0.25 2.00±0.43*

1.67±0.44 1.50±0.36

2.00±0.41 1.25±0.35

CAs – chromosomal aberrations (total), CTA – chromatide-type aberrations, CSA- chromosome-type aberrations, SEM – standard error of mean, *(P<0.05)


GSTT1 null subjects demonstrated significantly higher (P<0.05) frequency of CSAs than GSTT1positive subjects (2.00±0.43%; 1.40±0.25% respectively). Similarly, frequencies of CTAs werehigher in individuals with variant-type Ile/Val (1.25%) genotype in GSTP1* Ile105Val than in indi-viduals with wild-type Ile/Ile (0.66%) genotype but the difference was not significant (Table 3).We did not find any differences in the frequency of chromosomal aberrations regarding polymor-phisms of GSTM1 and GSTP1 genes.

DISCUSSION

Our study showed significant increase in frequency of structural chromosomal aberrationsamong exposed individuals over that found in the control population. This finding is in accor-dance with previous studies on workers that have been exposed to benzene (10,24,25). Ourobservation of 3.9-times higher frequency of CSAs, which is considered as genetically more seri-ous damage, suggests the formation of DNA double strand breaks caused by chemicals used inshoes factory, especially by benzene and toluene.

The controversial result of significantly lower frequency of CTAs in workers exposed to differ-ent mutagens was commented in many previous works as well (26,27,28,29). The reason of thisphenomenon could be probable lay in the DNA repair system (30).

We did not find any significant relation between the frequency of chromosomal aberrations andworking period, gender and age. Similarly, no female-male, age and working period differences inthe response to benzene exposures were detected in the study performed by Kasuba et al. (31).

Several studies have reported that polymorphisms of genes coding for detoxification enzymesprobably play a role in final genotoxic effect of chemicals and their intermediates (1,32,33).Moreover, epidemiological studies on Nordic and Italian cohorts have previously shown a clearassociation between increased CA frequency, gene polymorphisms and cancer risk in individualsexposed to environmental and occupational pollutants (34,35,36).

GSTs are known to catalyze the detoxification of various reactive toxic and mutagenic compoundsincluding epoxides resulting from cytochrome P450 mediated metabolism of polycyclic aromatic hydro-carbons as well as lipid and DNA products of oxidative stress (37,38). Reduced detoxification capac-ity of GSTM1 null individuals have been reported by CA assay. However, our results do not confirmthese findings. We did not observe any differences between GSTM1 positive and null genotype.

The next important isoenzyme of GST family is GSTT1. GSTT1 catalyzesthe conjugation of rel-atively small molecules, such as methylene chloride, ethylene dibromide, and the epoxidesderived from isoprene (39). Given the importance of GSTs in the detoxification of electrophilic car-cinogens, GSTT1 null genotypes has become the object of many studies because homozygousdeletions of GSTT1 are expected to result in an impaired ability to detoxify carcinogenic com-pounds and may place GSTT1 null individuals at increased cancer risk (40). In our study we alsoshowed that GSTT1 null genotype had been associated with an increased frequency of chromo-somal aberrations in the exposed workers.

GSTP is an isoform which is the most abundant in the lungs; thus; it has particular impor-tance in the detoxification of inhaled toxicants (41). Polymorphism in codon 105 of GSTP1 pro-duces an enzyme with a different thermal stability and substrate affinity (42). In our work we didnot find any influence of GSTP1 genotypes on the level of chromosomal aberrations. The resultsmust be cautiously interpreted, owing to the relatively low number of individuals with varianttype of genotype included in this study.

Polymorphisms of xenobiotic-metabolizing enzymes may markedly influence individual abilityto activate or detoxify genotoxins which affect the extent of genetic damage formed. Therefore,they could modulate susceptibility to exposure-associated diseases. However, the polymorphismsof DNA repair genes can also influence the effect of genotoxic agents and the final result dependson the cooperation of many systems involved in the defense lines of the cell. A search for the opti-mal and effective monitoring tools to identify the susceptible individuals seems to be the mostimportant role for current and future research. We hope that our findings will help to establishprinciple of mentioned role.



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A c k n o w l e d g e m e n t s:This work was supported by the grant AV 4/0013/05.

Received: February 2,2010Accepted: April,20,2010



MICROMORPHOLOGICAL CHANGES OF PERIPHERAL NERVE INDUCED BYGAMMA RADIATION

BOSELOVA L., FUSEKOVA E., BALENTOVA S., ADAMKOV M., OCHODNICKA E. Department of Histology and Embryology, Jessenius Faculty of Medicine, Comenius University, Martin, Slovak

Republic

A b s t r a c tIonizing radiation, currently used in the radiotherapy, influences not only tumor cells, but can cause also significant

changes of nerves, blood vessels and surrounding tissues.We studied the effect of the low dose of gamma radiation on the development of alterations in myelinated nerve fibres

of n. ischiadicus of rat after whole-body irradiation with single dose 3 Gy of gamma rays. The alterations of nerve fibreswere examined in various intervals after exposure. Fluoro-Jade B histochemistry method was used to label of changesin the axons. Changes in the axons and myelin sheaths were studied also with other staining and impregnating meth-ods (Toluidin blue, Holmes, OsO4). In our experiment male adult rats survived one, 5 and 10 days after exposure.

The response of nerve fibres to ionizing radiation is induction of alterations in axon, involved the axoplasmic vacuola-tion and accumulation of neurofibrils. This determined as acute post-irradiation changes, which were observed 1-dayafter exposure. Alterations of myelin sheath, that independent of axonal changes, were observed 5th day after radiation,and involved elongation of Ranvier node, dilatation of Schmidt-Lanterman incisures and intramyelinic splitting. With pro-longation of survival period after exposure these changes led to segmental demyelinization of some nerve fibres.

Key words: Ionizing radiation; Myelinated axon; Myelin sheath; Fluoro-Jade B method

INTRODUCTION

Ionizing radiation, currently used in the treatment of various tumour diseases, influences notonly tumour cells, but can cause significant damage on nerves, blood vessels and surroundingtissues. On the other hand, some authors (5) stated that low doses of ionizing radiation havea beneficial influence on epineural scar after surgical or local trauma of peripheral nerve.

Many authors pointed out the negative influence of ionizing radiation to nerve tissues andcalled attention to the identical sensibility of nerve and glial cells. Kamarád (6) studied the mor-phological changes after gamma radiation in cortex cerebri of the experimental animals and hedescribed a moderate alteration or even full death of cells, depending on irradiation dose.Tumanov and Malamud (12) described the influence of radiation on neuroglial cells of the CNSand found out that astrocytes responded by their enlargement. They described the dilated endo-plasmatic reticulum and mitochondria on ultrastructural level. They observed two types of mito-chondria - dark ones with an osmiophilic matrix, and light ones with a pale matrix and reducedcristae. Some authors (3) observed influence of radiation on Purkyně cells of cerebellum anddetermined their sensitivity, which was manifested by various densities, where dark cells wereprevailing. Other authors (1) described cytogenetic effect of ionising radiation on cells subven-tricular zone of the cerebral lateral ventricles. Some authors, who are observing influence of radi-ation on peripheral nerves, either in experiments or at tumour therapies, suggest a lower sensi-bility of peripheral nerves to radiation, compared with CNS, and they put it down to their lowermetabolic activity (4).

The aim of the present study was to demonstrate alteration in myelinated nerve fibres ofn. ischiadicus of rat after low dose of ionizing radiation. We have concentrated on the changes inaxon and myelinated sheath. Our intention was to compare the Fluoro-Jade B method, as a verysensitive and reliable method for histochemical localization of neuronal degeneration includingaxon, with other staining and impregnating methods.


A d d r e s s f o r c o r r e s p o n d ence:RNDr. Ludmila Boselova, CSc., Department of Histology and Embryology, Jessenius Faculty of Medicine, ComeniusUniversity, Malá Hora 4, 036 01 Martin, Slovak RepublicPhone: +421 43 4131531; E- mail: [email protected]


MATERIAL AND METHODS

In the experiment we used 2 - 4 month old males white rat of Wistar strain. Weight of the usedanimals moved between 190 to 300 g. The animals were exposed to the whole-body irradiationwith single dose 3 Gy of gamma rays using a 60Co source (apparatus TERAGAM 02 UJP Praque).The animals survived one, 5 and 10 days after exposure. The animals were divided into 4 groups,5 rats in each. First group included non-irradiated animals, whose n. ischiadicus was sampledat the same time as n. ischiadicus of experimental animals. In the second group, the animalssurvived 1 day after exposure. In the third group, there were the animals with 5-day survivalafter irradiation. The fourth group comprised the animals with 10-day survival after exposure.

After survival period the animals were overdosed with ether anaesthesia and after sacrifice sci-atic nerves were fixed 10 min. in situ with 4% formalin solution and immersed in same solutionfor 3 days and paraffin embedded according to standard procedures. For light microscopy we pre-pared 6 m thick sections and staind by HE, toluidine blue, and the impregnation methods byHolms and OsO4. For observation with the aid of fluorescence microscope, we employed Fluoro-Jade B. This method is used for the proof of degenerative changes in neuron including axon atvarious types of damages, but Fluoro-Jade B does not stain myelin sheaths. We observed thechanges of myelinated sheaths with the aid of the above-mentioned methods. The slides were cutin transversal and longitudinal directions.

In the course of the experiment, all animals were getting their food and water ad libitum andthe animals were kept under standard conditions.

RESULTS

In our experiments, we have evaluated micromorphological changes of the myelinated axons ofn. ischiadicus of rat after their whole-body irradiation using single dose 3 Gy of gamma rays. Theanimals exposed to the whole-body irradiation suffered neither from behaviour nor from mobilitydisorders. The morphological image of the examinated animals was compared with a control group.

1 day after irradiationDuring examination of the longitudinal and transversal sections from experimental animals

with 1-day survival after exposure stained by Holmes method, were observed only moderatechanges of some axons. These changes were manifested by accumulation of neurofibrils and axo-plasmic vacuolation caused by dilatation of mitochondria and endoplasmic reticulum. Thisreminded varicosities of axons observed on longitudinal sections (Fig. 1). We have found ontransversal sections that neurofibrils are more concentrated on central zone of the axon (Fig. 2).The described changes have been mostly found on large calibre fibres. In this experimentalgroup, some undamaged fibres were also found. 1-day after radiation no alterations of myelinsheath including paranodal areas were observed.


Fig. 1 Varicosity of axons and accu-mulation of neurofibrils is seen at lon-gitudinal sections of nerve fibres.1-day after radiation, Holmes, 240xmagnification.


The Fluoro-Jade B histochemistry method has shown a moderate positivity in axon (Fig. 3).


Fig. 2 At transversal section is seenconcentration of neurofibrils in centralzone of axon. 1-day after radiation,Holmes, 240x magnification.

Fig. 3 Degenerating axons exhibita green fluorescence (asterisk), whilehealthy axon are unstained. 1-dayafter radiation, Fluoro-Jade B method,240x magnification.

5 days after irradiationIn the experimental group with 5-day survival on longitudinal sections stained by OsO4 method

we observed retraction of the myelin sheath from the paranodium (Fig. 4), that was due to dis-ruption of axoglial junction. Elongation of the nodes of Ranvier is a characteristic feature for thisgroup, disregarding the type of myelin alterations at internodal level. Changes myelin sheath atinternodal level were more expressive than in the previous group of animals. The alterations wererelated to disturbed integrity and compactness of myelin sheath, dilatation of Schmidt-Lantermann incisures (Fig. 5). Some myelin sheaths were split in the place Schmidt-Lantermannincisure and their sheath was separated into two parts. In most fibers myelin sheaths were split-ting along the whole internodium. A tubular shape of myelin sheath is generally preserved,though the myelin sheath at internodal level is split, swollen or destroyed. This type of alterationsof myelin sheath in most of the nerve fibres is characterised by an intact axon. The Fluoro-JadeB method has not shown any positivity.


10 days after irradiationIn the experimental group of animals with 10-day survival, we observed myelinated nerve fibres

with compact myelin sheath and unchanged axons. Sporadically, a segmental demyelinizationwas observed. In endoneurium an increased number of cell nuclei was found, which we put intoconnection with increased activity of Schwann cells. The Fluoro-Jade B method has not shownany positivity.

DISCUSSION

Our experiments demonstrate response of nerve fibres of n. ischiadicus of rat to low dose (3 Gy)of ionizing radiation. Radiation induced the moderate changes on myelinated nerve fibres, whichwe observed by means of the methods selected by ourselves. The first changes were observed 1-day after exposure, involved axoplasmic vacuolation and accumulation of neurofibrils in axon.The similar changes in terms of accumulation of microtubules were observed by Vujaskovic et al.(13) in canine sciatic nerve after radiation, these alterations are suggestive of radiation-inducedhypoxia that damages microcirculation. Accumulation of microtubules in myelinated axons ofn. ischiadicus of quinea pig after ischemia was described also by Bošelová et al. (2001).Axoplasmic vacuolation in sciatic nerve was described also by Coban et al. (2). 1-day after radi-ation no alterations of myelin sheaths including paranodal areas were observed. First changeson myelin sheaths were observed 5-days after exposure and involved elongation of Ranvier node,which we ascribe to the damage of paranodal region, manifested by retraction of myelin sheath.Elongation of Ranvier node is a frequent finding also at toxic damage of peripheral nerve (8).Pursuant to Masurovsky et al. (7), we have found out that the Ranvier node is a very sensitivearea to radiation effects. The mentioned authors pointed out the large sensitivity of Schwanncells, which are the first to be hit by radiation, and this leads to alteration of myelin sheath.Changes on the intermodal level were dilatation of Schmidt-Lantermann incisures andintramyelinic split. With prolongation of the survival period after radiation these changes led tosegmental demyelinization. The segmental demyelinization was also described by Penatti et al.(10) in n. opticus after UV-radiation. The unrepeated 3 Gy dose used in our work caused mod-erate changes in axon and myelinated sheath of nerve fibres, which are reversible because 10thday after irradiation, the reproduced cores of Schwann cells were observed, what we regard as


Fig. 5 Alterations of myelin sheath is seen at transver-sal section. 5-day after irradiation, OsO4, 200x magnifi-cation.

Fig. 4 Alterations of myelin sheath at internodal level andretraction of myelin sheath from paranodium (arrow). 5-day after radiation, OsO4, 240x magnification.


a manifestation of reparation. It is known from literature that the Schwann cells play a key rolenot only in a degeneration process, where they help to remove the remains of damaged parts ofmyelinated nerve fibres, but also in regeneration process.

The Fluoro-Jade B histochemistry method, declared in literature (9,11) as a very reliable forillustration of degenerating neurons including axon. In our experiment the Fluoro-Jade B isdemonstrated as a useful histochemical method only for degeneration of axons. All damages ofmyelin sheaths were demonstrated with other above mentioned staining methods. Fluoro-Jade Bdoes not stain myelin sheath. In our experiment axonal changes were seen only in group 1-dayafter radiation, where Fluoro-Jade B histochemical method showed moderate positivity. Withtime of survival after radiation more intensive were changes on the myelin sheath.

We can conclude that response of nerve fibres to low dose of ionizing radiation at whole-bodyapplied is induction of alterations in axon, involved the axoplasmic vacuolation and accumula-tion of neurofibrils. The first sign of myelin damage is an increase in nodal length. Changes ofmyelin sheath on the intermodal level are dilatation of Schmidt-Lantermann incisures andintramyelinic splitting. With prolongation of the survival period after exposure these changes ledto segmental demyelinization in some nerve fibres.

REFERENCES

1. Bálentová S, Račeková E, Mišúrová E. Effect of paternal exposure to gamma rays juvenile rat forebrain.Neurotoxicology and Teratology 2007; 29: 521 – 526.

2. Coban YK, Ciralik H and Kurutas EB. Ischemic preconditioning the reduces severity of ischemia-reperfusion injuryof peripheral nerve in rats. Journa of Brachial Plexus and Peripheral Nerve Injury. 2006; 1:2: 1-5.

3. Domoráková I, Zachariaš L, Mechírová E, Eliášová. Radiation - induced damage of the grey matter in central nerv-ous system in rats. Sborník Lékařsky. 2001; 102, No.2: 137-143.

4. Gillette EL, Mahler PA, Powers BE, Gillette SM, Vujaskovic Z. Late radiation injury to muscle and peripheral nerves.Int J. Radiat Oncol Biol Phys 1995; 31: 1309 – 1318.

5. Görgülu A, Uzal C, Doganay L, Imer M, Eliuz K, Cobanoglu S, et al. The effect of low- dose external beam radiationon extraneural scarring after peripheral nerve surgery in rats. Neurosurgery 2003; 53, No. 6: 1389-1396.

6. Kamarád V. Acute radiation sickness-morphology of CNS syndrome. Acta Universitatis Palackianae Olomoucensis,1989; 121: 11-144.

7. Masurovský EB, Bunge MB and Bunge R. Cytological studies of organotypic cultures of rat dorsal root ganglia fol-lowing X irradiation in vitro. The Journal of Cell Biology 1967; 32: 197-318.

8. Mello M, Mellová Y, Výbohová D, Kunertová L, Hešková G, Marčeková M. Sledovanie priameho účinku lokálnychanestetík na periférny nerv. Morfológia v súčasnosti 2006. p. 230 – 233.

9. Mitrušková B, Orendáčová J and Račeková E. Flouro Jade-B Detection of Dying Cells in the SVZ and RMS of AdultRats After Bilateral Olfactory Bulbectomy. Cellular and Molecular neurobiology, 2005; Vol. 25, No 8: 1255 – 124.

10. Penatti A, Monteiro ML, Manchiori PE and Scaff M. Superior orbital petrositis and late ischaemic monocular blind-ness induced by intense UV radiation exposure .Occup. Med. 2002; Vol.52, No.4: 219-221.

11. Schmued LC, Albertson Ch, Slikker W Jr.: Flouro-Jade. A novel flourochrome for the sensitive and reliable histo-chemical localization of neuronal degeneration. Brain Research 1997; 751: 37 – 46.

12. Tumanov VP, Malamud MD. Izmenenija centralnoj nervnoj sistemy pri termičeskoj lučevoj i ombinirovanoj travme.Štiinca, Kišinev 1977. p. 117.

13. Vujaskovic Z, Gillette SM, Powers BE, Thurmond N, Gillette EL, Colacchio TA. Ultrastructural morphometric analy-sis of peripheral nerves after intraoperative irradiation. International journal of radiation biology. 1995: 68: 71-76.

A c k n o w l e d g e m e n t sThis work was supported by the grant AV 4/2026/08. This work was supported by project „Center of TranslationalMedicine“ co-financed from EC sources and European Regional Development Fund.

Received: March,24,2010Accepted: April,29,2010



NOSOCOMIAL PNEUMONIA AT SELECTED CLINICS OF MARTIN FACULTY HOSPITAL

BUSIKOVÁ J., MURAJDA L.�Clinic of Infectology and Travel Medicine, Jessenius Faculty of Medicine, Comenius University and Martin Faculty

Hospital, Department of Public Health, Jessenius Faculty of Medicine, Comenius University, Martin, Slovak Republic

A b s t r a c tIntroduction: Nosocomial pneumonia (NP) is the second most frequent nosocomial infection markedly participating in

increasing hospitalized patient mortality and morbidity. NP increases financial expenses due to a prolonged period ofhospitalisation and application of an expensive anti-infectious therapy (1).

Methodology: We were monitoring the representation of aetiological agents and length of hospitalisation of patientswith NP at the First Surgical Clinic, Neurosurgery and the Clinic of Anaesthesiology and Intensive Medicine in MartinFaculty Hospital for the period from 2002 to 2008.

Results: The most frequent NP agent, found by means of bronchoalveolar lavage (BAL), was Klebsiella pneumoniaethat caused 34 of all 127 detected NP cases. The polymicrobial character of NP has been recorded in 43 cases. The aver-age length of hospitalization was 16.61 days (SD ± 11.88) in all monitored patients.

Discussion: Gram-negative bacteria are as agents in 55-85% and gram-positive microorganisms in 20-30% NP cases(7). Gram-negative bacteria have participated in NP genesis in our research (65% NP cases). We have detected gram-pos-itive bacteria in 22% NP cases. The data on the most frequently detected bacterial microorganisms found by us are inconformity with the data in world literature. NP genesis prolongs the stay in health care institution for 7 to 9 days (8).The prolongation of hospitalization due to NP at individual clinics in Martin Faculty Hospital correspond to informationin the literature.

Key words: nosocomial pneumonia, aetiological agent, length of hospitalization

INTRODUCTION

Nosocomial infections belong to serious infections occurring within 48-72 hours after patientadmittance to health care institution, unless the incubation period of the infection is known.Pneumonias manifesting themselves after patient´s release or transfer to other health care insti-tution also belong here in case their occurrence is connected with previous stay in heath careinstitution (2). Nosocomial pneumonia (NP) is the second most frequent nosocomial infection incritically sick patients, and it participates markedly in increasing morbidity and mortality as wellas in increasing financial expenses connected with the application of an expensive anti-infectioustherapy and prolonged period of hospitalization – from 4 to 13 days (1).

The representation of aetiological agents, responsible for NP occurrence, differs depending onhealth care institution and particular ward. NP of bacterial aetiology asserts itself in 80-90% ofcases. Pseudomonas aeruginosa and Enterobacteriaceae participate most frequently in NP gen-esis among gram-negative bacteria. Gram-positive bacteria (in particular, Staphylococcusaureus, Streptococcus species) occur in 35-39% of NP cases. 10-20% of NP cases are caused byviruses and less than 1% NP cases are caused by fungi (3,4,5,6).

The representation of individual pathogens responsible for NP genesis is changed gradually.Acinetobacter species, Enterobacter species, methicilin-resistant Staphylococcus aureus come tothe fore among NP agents. The occurrence of Klebsiella pneumoniae and Pseudomonas aerugi-nosa in NP cases remains stable or descending. About a half of NP cases is of polymicrobial aeti-ology. A probable NP agent is determined in a large extent by the seriousness of basic illness,length of hospitalization and previous ATB therapy (7).

The goal of our research was to find the most frequent NP agents and to record an averagelength of hospitalization in NP patients at the First Surgical Clinic, Neurosurgery and The Clinic


A d d r e s s f o r c o r r e s p o n d e n c e:Juliana Busiková, MD, Clinic of Infectology and Travel Medicine, JLF UK and MFN, Kollarova 2, 036 59 MartinPhone: 043 4203 539,e-mail: [email protected]


of Anaesthesiology and Intensive Medicine in Martin Faculty Hospital for the period from 2002to 2008.

MATERIAL AND METHODS

We were monitoring patients with NP, hospitalized at the First Surgical Clinic, Neurosurgeryand the Clinic of Anaesthesiology and Intensive Medicine in Martin Faculty Hospital for the peri-od from 2002 to 2008. Source of information about patients with NP was anti-infectives indica-tions database of RAT programme (rational anti-infective therapy). NP was specified according tocriteria of Centers for Disease Prevention and Control (CDC). We have concentrated on the detec-tion of NP agents and on the monitoring of the length of hospitalization in patients with this diag-nosis.

We were investigating the number of cases with the detected NP pathogen by means of bron-choalveolar lavage (BAL), the occurrence of polymicrobial and monomicrobial NP, and capture ofindividual aetiological agents responsible for NP genesis. At the same time we were monitoringthe length of hospitalization in NP patients at individual clinics of Martin Faculty Hospital.Subsequently, we have calculated the average length of hospitalization of the patients with NP atindividual clinics and for all the monitored clinics together. We have compared the data with theinformation on average length of hospitalization of patients without NP at selected clinics ofMartin Faculty Hospital for the period from 2002 to 2008, obtained from Martin Faculty HospitalInformatics Department.

RESULTS

We have detected 62 NP cases from among 11 680 hospitalized patients at the First SurgicalClinic, 34 NP cases from among 4835 hospitalized patients at Neurosurgery, and 31 NP casesfrom among 1471 hospitalized patients at the Clinic of Anaesthesiology and Intensive Medicineby means of monitoring NP at the selected clinics of Martin Faculty Hospital.

Aetiological agent has not been identified in 65 cases (51%) from among all detected NP cases(127 cases) at the monitored clinics of Martin Faculty Hospital in 2002-2008. Aetiological agenthas been identified by means of BAL in remaining 62 cases (49%). The polymicrobial characterof NP was recorded in 43 NP cases (69%) (Fig.1).


Fig.1: Percentage representation of detected aetiological agents responsible for NP gene-sis at the selected clinics of Martin Faculty Hospital in 2002-2008


The most frequent NP agent detected by means of BAL was Klebsiella pneumoniae (43 cases),followed by Enterococcus species (18 cases) and Pseudomonas aeruginosa (17 cases) (Table 1).


Table 1 Aetiological agents responsible for NP genesis at the selected clinics of Martin Faculty Hospital

Aetiological agents

Klebsiella pneumoniae

Enterococcus species

Pseudomonas aeruginosa

Candida species

Acinetobacter species

Escherichia coli

Enterobacter species

MRSA*

Other staphylococci

Other bacteria

Proteus mirabilis

Citrobacter freundii

Stenotrofomonas maltophilia

Acinetobacter baumanii

Number NP

34

18

17

14

13

9

9

8

5

5

4

3

3

2

%

23.6

12.5

11.8

9.7

9.0

6.3

6.3

5.6

3.5

3.5

2.8

2.1

2.1

1.4

* methicilin resistant Staphylococcus aureus

The average length of hospitalization of all monitored patients with NP was 16.61 days (SD ±11.88). The longest average length of hospitalization was found at the First Surgical Clinic (17.98days, SD ± 13.32). The average length of hospitalization was 15.97 days (SD ± 12.16) atNeurosurgery, the shortest average length of hospitalization from among all monitored clinicswas recorded at the Clinic of Anaesthesiology and Intensive Medicine (14.58 days, SD ± 8.68)(Fig.2).

We have found significant difference between groups of patients with NP (X = 16.61 days, SD ±11.88) and without NP (X = 6.72 days*) at the selected clinics of Martin Faculty Hospital by com-parison of the average length of hospitalization for the period from 2002-2008.

*Average value calculated on the basis of data obtained from Martin Faculty HospitalInformatics Department.

DISCUSSION

We have detected aetiological agent in 49% NP cases and polymicrobial character has beendetected in 69% cases out of this number by monitoring the identification of NP agent.


Gram-negative bacteria as agents were found in 55-85% and gram-positive microorganisms in20-30% NP cases (7). Gram-negative bacteria have participated in NP genesis in our research(65% NP cases). Klesiella pneumoniae (24%) and Pseudomonas aeruginosa (12%) have been themost abundant ones. We have detected gram-positive bacteria in 22% NP cases. The data on themost frequently detected bacterial microorganisms found by us are in conformity with data inworld literature.

Candida species has been found in 10% of NP cases. Candida species does not belong to fre-quent NP agents, and a positive cultivation may indicate rather the colonisation of respiratorypassages (7%).

We have not detected any NP of viral origin in our research which could be caused by diagnosticdifficulties. The clinical picture of viral pneumonia has few special attributes to be distinguishedfrom bacterial NP, and suitable culture and serological examinations are seldom prescribed (7%).

The finding of NP prolongs the stay in health care institution for 7 to 9 days (8). The averagelength of hospitalization in patients with NP at the selected clinics of Martin Faculty Hospitalhas been by 10 days longer than in other hospitalized patients in our research, which accountsfor not negligible increase in financial expenses for treatment of patients with NP.

CONCLUSION

In Martin Faculty Hospital we suggest a strict hygienic-epidemiological programme and barri-er nursing technique, especially hand hygiene, and to improve testing for aetiologic agents of NP.

A thorough knowledge of the issues of the most frequent aetiological agents, responsible for NP,is crucial in starting a suitable initial antibiotic therapy leading definitely to the reduction ofmorbidity and length of hospitalization of patients with NP.

REFERENCES

1. Hoenig SM, Truwit JD. Ventilator-associated pneumonia: diagnosis, treatment, and prevention. Clin Microbiol Rev2006; 19(4):637-57.

2. Krkoška D. Nozokomiálne nákazy. In: Bálint O a kol. Infektológia a antiinfekčná terapia. Martin: Osveta; 2000. p.342-8. 3. Torres A, Ewig S, Lode H, Carlet J. Defining, treating and preventing hospital acquired pneumoniae: European per-

spective. Intensive Care Med 2009; 35:9-29.4. Celis R, Torres A, Gatell JM, Almela M, Rodríguez-Roisin R, Agustí-Vidal A. Nosocomial pneumonia. A multivariate

analysis of risk and prognosis. Chest 1988; 93(2):318-24. 5. Fein A, Grossman R, Ost D, Farber B, Cassiere H. Diagnosis and management of pneumonia and other respiratory

infections. 5th ed. United States of America: Professional Communications, Inc.; 1999. p. 133-49. 6. Máderová E, Šrámová H. Respirační nákazy. In: Šrámová H. et al. Nozokomiální nákazy. Praha: Maxdorf; 1995.

p. 210-16. 7. Lynch JP. Hospital-acquired pneumonia. Chest 2001; 119(2): 373-84.8. Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated

pneumonia. Am J Respir Crit Care Med 2005; 171(4):388-416.

Received: February 2,2010Accepted: May,21,2010



RISKS OF ULTRAVIOLET RADIATION – HEALTH PROTECTION OF UNIVERSITY STUDENTS

1JAKUSOVA V., 1MURAJDA L., 2POLIACEK I., 2JAKUS J.1Department of Public Health Jessenius Faculty of Medicine, Comenius University,

2 Department of Medical Biophysics, Jessenius Faculty of Medicine, Comenius University, Martin, Slovak Republic

A b s t r a c tIntroduction: It is generally acknowledged that there is a correlation between the incidence of ultraviolet burden dis-

eases and an overexposure of the body to ultraviolet radiation (UVR). The main goal of our study was to determine thepersonal protection of students against the harmful effects of UVR.

Methods: We performed a questionnaire study of UVR in years 2005 and 2008 at 2 Slovak universities - ComeniusUniversity in Bratislava, the Jessenius Faculty of Medicine in Martin (JFMED) and the University of Žilina (UŽ).

Results: The study includes 478 university students, 403 from JFMED and 75 from UŽ; 315 respondents in 2005 and163 in 2008. The number of students avoiding solaria increased in 2008 (p<0.001) compared to 2005 because of thereduction of those attending solaria occasionally (p<0.001). A low number of regular users of solaria in 2005 did notdecrease in 2008 significantly. The proportion of students not familiar with the term “skin photo-type“ was not signifi-cantly different in 2005 and 2008. However, the number of those who knew their skin type rose in 2008 (p<0.05) com-pared to the number in 2005. No statistically significant improvement in the protection of students against UVR (theratios of respondents using sunscreens, wearing sunglasses, and wearing the hat and clothes) was found in 2008 com-pared to 2005.

Conclusions: This study showed the particular types of personal protection of university students, future doctors andbioengineers. Higher level of students’ awareness against the harmful effects of UVR and improved safety behaviour relat-ed to UVR exposure was proved. The study brings new insights on the surveillance and protection against the harmfuleffects of UVR in university students.

Key words: Ultraviolet Radiation, Health Protection, University Students, Questionnaire.

INTRODUCTION

The damage of human body by non-ionizing radiation becomes a significant medical problemof today. Successful prevention of serious diseases resulting from non-ionizing radiation expo-sure would not be possible without study and understanding the effects of ultraviolet radiation(UVR) on human body (1). Higher intensities of UVR coming to the Earth surface due to thereduced concentration of the atmospheric ozone (”ozone hole”) represent an important danger tohuman health. Spectral characteristics as well as the intensity of solar radiation, particularly itsUV component, is determined by the position of Sun, by properties of the atmosphere and theEarth surface, by geographical, time, meteorological, and some environmental factors (2).

Computer simulations resulted in disconcerting predictions that e.g. 10% reduction in ozonelayer can cause 300,000 new cases of skin tumour (of non-melanoma type) and 4,500 new malig-nant melanomas (3). It has been also predicted that present reduction in the ozone concentra-tion will fully manifest (changes in ecosystem, the prevalence of skin cancer etc.) within next 20-50 years.

METHODS

We present a questionnaire study performed in years 2005 and 2008 at 2 Slovak universities- Comenius University in Bratislava, the Jessenius Faculty of Medicine in Martin (JFMED) andthe University of Žilina (UŽ). We queried a total number of 478 university students about the pro-


A d d r e s s f o r c o r r e s p o n d e n c e :Assoc. Prof. Viera Jakušová, Ing. PhD., MPH., Department of Public Health, Jessenius Faculty of Medicine, Sklabinska26, 036 01 Martin, Slovak RepublicPhone: 421-43-4132507, e-mail: [email protected]


tection of health against UVR. The distribution of respondents between the years and the uni-versities is presented in Table 1.

Table 1 The numbers of university students involved in the study.

University Students in the year Total

2005 2008# % # % # %

JFMED 287 60.0 116 24.3 403 84.3UŽ 28 5.9 47 9.8 75 15.7Total 315 65.9 163 34.1 478 100.0

In 2005 137 students of JFMED involved in our survey studied 1st year of medical programme, 37students the 2nd year of Public Health programme, and 113 students the 3rd and the 5th years ofthe Nursing programme. Twenty-eight students of UŽ were future biomedical engineers. In 2008eighty respondents represented future medical doctors (1st year of medical study), 36 future nurses (3rdyear of nursing study) and 47 future bio-engineers of UŽ. There were 367 (76.8 %) females and 111(23.2 %) males participating in the survey. The average age of our respondents was 23 years.

Students freely answered anonymous questionnaire that queried their tanning in solaria, theskin type of respondents, and the protection against the UVR used by themselves. Respondentsreturned 89.7 % of questionnaires, 92.7 % in 2005 (95.1 % at JFMED, 90.3 % at UŽ) and 86.7 %in 2008 (73.3 % at JFMED, 100 % at UŽ).

The data were processed by a PC programme GraphPad InStat by the analysis of contingencytables using chi-square and Fisher’s tests. The level of statistical significance was set up at p<0.05.

The above mentioned procedures were carried out in accordance with the ethical standardsand with the Helsinki Declaration from 1975 as revised on 1983.

RESULTS

Fourteen of our respondents (3 %) regularly used solaria. Low percentage of them was detect-ed already in 2005 (Fig. 1). However, the number of respondents avoiding the artificial exposureby UVR in solaria (together 364 students; 76 %) increased in 2008 (142; 87 %; p<0.001) com-pared to 2005 (222; 70 %), mainly because marked reduction of students tanning in solaria occa-sionally in 2008 compared to 2005 (p<0.001; Fig. 1). The number of sporadic solaria users wasaltogether 100 (21 %).


Fig. 1 Percentages of university students using solaria in years 2005 and 2008. xxx, p<0.001 comparing with 2005


The query about the skin “photo-type“ was answered as follows: 317 (66.3 %) respondents knewit, 121 (25.3 %) respondents did not, and 40 (8.4 %) of them were not familiar with the term. Theproportion of students not familiar with the term skin “photo-type“ was not significantly differ-ent in 2005 and 2008. However, the number of those who know their skin type rose in 2008(p<0.05) compared to such number in 2005 (Fig. 2).


Fig. 2 Percentages of university students who know, do not know, or are not familiar with the term“skin photo-type“ in years 2005 and 2008. x, p<0.05 compared to 2005

A high percentage of respondents (452; 94.6 %) protected themselves against extensive exposeto UVR by sunscreens, in addition, 348 (72.8 %) of them wore sunglasses, and 244 (51 %) worehat and clothes. No protection was reported by 26 (5.4 %) students.

No statistically significant differences in the ratios of respondents protecting themselvesagainst UVR by sunscreens, by wearing sunglasses and by wearing the hat and clothes in 2005and 2008 were found.

We also have not found any significant differences in analyzed characteristics in students ofJFMED and UŽ.

DISCUSSION

UVR is a natural component of the environment. Everyone is exposed to UVR to both positive(vitamin A production, an increase of immunity, behavioural aspects) and negative impacts (dis-orders of skin, eye, a decrease of immunity, etc.). World Health Organization monitors effects ofionizing as well as non-ionizing radiation on population every year. Higher risks of health dam-age due to climatic changes (global warming, ozone hole etc.) and artificial UVR produced byindustry or solaria were identified.

High ratio of returned questionnaires during our survey confirms deep interest of students -future medical doctors, nurses, and biomedical engineers - in the topic.

It was gratifying to find that only few of our respondents regularly used solaria (Fig. 1) and alsothat the number of students using the solaria seldom has been decreasing. This is a very prom-ising trend because in some European countries (particularly Northern Europe) there is stilla high prevalence of those taking artificial sunbath in solaria (4,5). Recent studies proved therelation between the tanning in solaria and the incidence of malignant melanomas (6). The num-ber of new cases of malignant melanoma has sharply risen during last 30 years. For example, inthe U.S.A. there were diagnosed 55,100 new patients including 7,910 fatal events among 1.5 mil-lion participants in 2004 (7). Tanning per se always implies damage to cellular DNA in the skinwith unpredictable longterm consequences. As a precaution, it would be reasonable to reduceyearly doses as much as possible. Some epidemiological studies indicate an increased risk ofmalignant melanoma if a person has more than 10 sessions in a solarium per year (8).


Nonetheless, there is still an increase in the number of solaria per year. For example, in 2008and 2009, the total number of licensed solaria in Slovakia was 761 and 823, respectively (per-sonal information from Slovak Ministry of Health). It is recommended that solaria should be reg-istered and solaria staff provided with professional education, that a systematic control of solar-ia operation be introduced, and the users be given instruction that must include informationabout protection (particularly wearing sunglasses). Similar survey as the present one was per-formed by us on population in the region of Liptovský Mikuláš and Ružomberok. It was foundthat the solaria were used mostly by 26-45 year females with a secondary education (9).

It is very positive that more than half of the respondents knew their skin type and that thisnumber has been rising (Fig. 2). Knowledge of the skin type is necessary in order to make cor-rect calculations regarding reasonable durations of UVR exposure. To reduce and potentially pre-vent damage due to UVR exposure, it is essential that students of primary and secondaryschools, if not the whole population, should be educated on this point (9, 10).

Our analysis disclosed that university students protect themselves against UVR mostly by sun-screens (typically with the factor No. 15 to 25) combining it with sunglasses and hat and lightsummer clothing. There are also very few respondents (and the number was decreasing; Fig. 3)not protecting themselves against UVR at all in our study. Our aforementioned survey in theregions of Liptovský Mikuláš and Ružomberok showed similar results to the actual ones in termsof the type of health protection against UVR (the only difference was the usual factor of sun-screens used - No. 5 to 15). However, among 168 inhabitants of Ružomberok and LiptovskýMikuláš areas who did not receive any specific UVR education, more than 16% did not use anykind of UVR protection (that was more than 6 fold higher than in our students in 2008). It seemsthat the level of general (i.e. primary, secondary, or university) education may greatly influencethe quality of UVR protection used. In this respect our former study (11) proved that medical staffin hospitals protected themselves in 95% of cases, and the medical doctors in 100% of the cases.Present study confirmed the relation: higher the knowledge and education the higher is the self-responsibility.


Fig. 3 The protection against UVR used by students in years 2005 and 2008.

Our study emphasizes the need of education and formation of new habits in protecting chil-dren, youngsters, and adults against harmful effects of non-ionizing radiation. For example, typ-ical example of “melanoma” patient is an indoor worker (exposed very little to the UVR during theyear) having 1-2 extreme UVR exposures per year during the holidays. Appropriate habits needto be adopted, encouraged and strengthened also in the parents, teachers, medical staff, publichealth professionals and other representatives. Only a systematic and long term educational pro-gramme, along with comprehensive protection policies, will reduce the burdens of diseasesresulting from excessive exposure to UVR.

sunscreens sunglasses hat and clothes no protection


REFERENCES

1. Jakušová V, Jakuš J. Ultraviolet radiation – A Need for Education of Further Medical Personell. Rev Nurs Lab Tech2005; 11(4):237-9. (in Slovak)

2. Brűhl C, Crutzen PJ. On the disproportionate role of tropospheric ozone as a filter against solar UV radiation.Geophys Res Lett 1989; 16:703-6.

3. Intersun. The Global UV Project: a guide and compendium. Radiation and Environmental Health Unit, Protection ofthe Human Environment. WHO, Geneva, 2003, 19p.

4. Jalarvo V. Use of sunbeds by Finnish population. Helsinki: STUK-A181, Radiation and Nuclear Safety Authority;2000.

5. Boldeman C, Bränström R, Dal H, Kristjansson S, Rodvall Y, Jansson B, Ullén H. Tanning habits and sunburn ina Swedish population age 13-50 years. Eur J Cancer 2001; 37:2441-8.

6. Lajčíková A, Pekárek L. Ultraviolet Radiation and its effect on the health. Hygiena 2009; 54(2):57-61. (in Czech)7. Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, Feuer EJ, Thun MJ. Cancer statistics, 2004. CA

Cancer J Clin 2004; 54:8-29.8. Westerdahl J, Olsson H, Masbäck A, Ingvar Ch, Jonsson N, Brandt L, Jönsson PE, Möller T. Use of Sunbeds or

Sunlamps and Malignant Melanoma in Southern Sweden. Am J Epidemiol 1994; 140(8):691-9.9. Jakušová V, Švandová M, Valach M. Postoje obyvateľstva k možnostiam ochrany zdravia pri nadmernej expozícií

ultrafialovému žiareniu. XVII. vedecko-odborná konferencia s medzinárodnou účasťou Životné podmienky a zdravie.Štrbské Pleso, 2009.

10. Jakušová V. Risks of Ultraviolet Radiation and Mobile Communication -Analysis of Knowledge, Protection of Healthand the Efficiency of University Student Education – the future medical staff. Bratislava. (“PhD. Thesis”). 2006: 117p.

11. Tisoňová V, Jakušová V. What Does the Medical Staff Know about Ultraviolet Radiation? Florence plus 2007; 3(9):1-4. ISSN 1801-464X. http://www.florence.cz/plus.php?stat=614 (in Slovak)

A c k n o w l e d g m e n t s:This work was supported by the Center of Experimental and Clinical Respirology. The project is co-financed from „ECsources” – ERDF – European Regional Development Fund.

Received: May,6,2010Accepted: June,7,2010



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