UDK: 613 - hrana-ishrana.orghrana-ishrana.org/wp-content/uploads/2019/01/Hrana-i-ishrana-59-br-1.pdf · analize rizika u nacionalno zakonodavstvo je u funkciji unapređenja međutrgovinske

1BEOGRAD 2018

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UDK: 613.2

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1BEOGRAD 2018

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UDK: 613.2

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Vlasnik i izdavač / Owner and Publisher:Društvo za ishranu Srbije 11000 Beograd, Savska 9/III

Izdavački savet / Editorial Committee:Ljiljana Trajković Pavlović, Dragojlo Obradović, Bato Korać, Ida Leskošek-Čukalović, Petrica Ružić , Anita Klaus, Ivan Stanković, Nađa Vasiljević, Nedeljko Radlović, Slavica Šiler-Marinković, Suzana Dimitrijević, Vera Katić, Jasna Mastilović, Vesna Vučić

Glavni i odgovorni urednik / Editor in Chief:Prof. dr Bato Korać

Zamenik glavnog i odgovornog urednika:

Prof. dr Petrica Ružić

Uređivački odbor / Editorial Board:Evangelos Polychronopoulos (Gračka), Heiner Boeing (Nemačka), Philip Calder (Velika Britanija), Stefaan de Henauw (Belgija), Marie Kunesova (Češka), Budimka Novaković (Srbija), Ana Stančić (Srbija), Aleksandra Korać (Srbija), Zorica Nikolić (Srbija), Sladjana Šobajić (Srbija), Bojana Vidović (Srbija), Miomir Nikšić (Srbija), Dušica Stojanović (Srbija), Milka Popović (Srbija), Sladjana Žilić (Srbija)

Lektor za engleski jezik: Danica Pavlović

Grafička obrada i lektura: Dušan Ćasić

Uredništvo i administracija:11000 Beograd, Savska 9/II; p. fah: 333 Tel.: ++381(0)11 420-2998

ČASOPIS IZLAZI DVA PUTA GODIŠNJE

U troškovima štampanja časopisa učestvuje Ministarstvo prosvete, nauke i tehnološkog razvoja Republike Srbije

Štampa:Ton Plus, Danila Lekića Španca 31 11077 Beograd, Tel.: 011/3016624

Tiraž: 100 primeraka

SADRŽAJ / CONTENTS

Reč urednika / Word from the editor ........................................................................ 2

PREGLEDNI RADOVI / REVIEWS

Dragan Milićević, Saša Janković, Srđan Stefanović Mikotoksini: analiza rizika, principi i procedure ............................................... 3 Mycotoxins: risk analysis, general principles and procedures

Sandra Jakšić, Sunčica Kocić-Tanackov, Milica Živkov Baloš Kontrola mikotoksina u Republici Srbiji i Evropskoj uniji sa aspekta

zakonske regulative ................................................................................................. 12 Mycotoxin control in Republic of Serbia and European union

from the aspect of the legislation

ORIGINALNI RADOVI / ORIGINAL PAPERSAna Stančić, Aleksandra Korać, Vesna Otašević, Aleksandra Janković, Bato Korać The effect of simvastatin in pancreas of diabetic rats .................................. 19 Efekat simvastatina u pankreasu dijabetičnih pacova

Boban Stanojević, Mirjana Đukic, Ivana Stevanović, Milica Ninkovic, Ana Djuric, Borko Gobeljic, Milan Apostolovic, Ana Pantelic, Goran Zebic, Lidija Todorovic, Tijana Bojic, Kiril Savovski Zeolite pretreatment accomplishes partial brain radioprotective

role by reducing iron and oxidative /nitrosative stress in rats .................. 26 Predtretman zeolitom ostvaruje delimično radioprotektivni efekat na

mozak smanjujući gvožđe i oksidativni/nitrozativni stres kod pacova

Teodora Obradović, Biljana Radišić, Anita Agić, Davor Korčok, Duško Mirković, Vesna Spasojević-Kalimanovska, Jelena Kotur-Stevuljević Comparison of the antioxidative action of the two N-acetylcystein

containing products: Propomucil® and Fluimucil® in a group of smokers who have seasonal coughing problems ......................................... 33 Poređenje antioksidativnog delovanja dva proizvoda koji sadrže

N-acetilcistein: Propomucila® i Fluimucila® u grupi pušača sa sezonskim kašljem

24. SUSRETI NUTRICIONISTALjiljana Trajković-Pavlović Javnozdravstveni značaj kontrole ostataka mikotoksina u hrani ............. 42

PRIKAZI KNJIGA

Instrumentalne metode, autori Mirjana Medenica, Nataša Pejić ............... 44 Mikotoksini u lancu hrane: hemijski, biološki i zdravstveni aspekt,

autor Dragan Milićević ............................................................................................ 45 Ohratoksin A u hrani za životinje, štetni efekti, detekcija i mogućnost

zaštite, autori Jelena Nedeljković-Trailović, Srđan Stefanović .................. 47

Uputstvo autorima / Instruction to Authors ................................................... 48

VOL. 59 BEOGRAD, 2018. BROJ 1

ISSN 0018-68727UDK: 613.2

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018.2

Društvo za ishranu Srbije je jedna od najstarijih zajed-nica naučnih radnika i stručnjaka u Srbiji koja ima za cilj da bude nacionalni servis stručne i šire javnosti. Od svog osnivanja 1956. godine objedinjuje istraživače iz oblasti agronomije, biohemije, hemije, farmacije, hu-mane i veterinarske medicine, dijetetike i drugih srod-nih naučnih disciplina koji brinu o proizvodnji i bez-bednosti hrane i zdravlju nacije, posebno dece.HRANA I ISHRANA je pregledni časopis Društva za ishranu Srbije koji od 1960. godine prati osnovne po-stulate Društva u cilju poboljšanja i unapređenja jav-nog zdravlja. Časopis objavljuje originalne istraživačke i pregledne članke, teme za diskusiju, vesti i informaci-je, kao i ekspertske izveštaje o svim aspektima hrane i ishrane.Časopis je mesto koje obezbeđuje da se glas naučne zajednice čuje i blagovremeno prenose informacije o hrani i ishrani stanovništvu, mesto za sve napredne ide-je i kritičko razmišljanje.Osnovni cilj časopisa HRANA I ISHRANA je integraci-ja znanja fundamentalnih istraživanja nauke o hrani i ishrani u zdravlju i bolesti. Paralelno sa istraživanjem molekularnih mehanizama delovanja hrane, časopis je usmeren na praćenje zakonodavstva i etičkih stan-darda, što je danas veliki izazov u svetu kada se radi o hrani.Urednički tim očekuje interaktivnu saradnju i pomoć svih naučnika kojima je hrana i ishrana više od znače-nja tih reči, etička kategorija koja ima za cilj poboljšanje zdravlja nacije.

Prof. dr Bato Korać Glavni i odgovorni urednik

Serbian Nutrition Society is one of the oldest commu-nities of scientists in Serbia, which aims to be a national service and a service to all people. Since its establish-ment in1956, it unites researchers, doctors, educators and all followers who care about the health of the na-tion, especially children.The Food and Nutrition is a peer reviewed journal of the Serbian Nutrition Society which since 1960 follows the basic postulates of the Society, with the aim to improve and promote public health. The Journal pub-lishes original research and review articles, topics for discussion, news and information, and expert reports on all aspects of food and nutrition.The Journal is a place that ensures that the voice of the scientific community is heard and timely transmits information related to food and nutrition to the popu-lation, the place for all the advanced ideas and critical thinking.The primary aim of the Food and Nutrition journal is to integrate knowledge of fundamental research of food science and nutrition in health and diseases. In parallel with the research of molecular mechanisms of action of food, the scope of the Journal is to monitor legislatures and ethical standards, which today is a great challenge in the world when food is concerned.The editorial board expects interactive cooperation and assistance from all scientists to whom food and nutrition are more than the meaning of those words, an ethical category aimed at improving the health of the nation.

Prof. dr Bato Korać Editor in Chief

DRUŠTVO ZA ISHRANU SRBIJE, ČASOPIS „HRANA I ISHRANA˝SERBIAN NUTRITION S0CIETY, JOURNAL ˝FOOD AND NUTRITION˝

Beograd, Savska 9/II, tel: 011/420 2998; p. fah: 333Žiro račun: 355-1032408-17

Reč urednika / Word from the editor

Ukoliko želite da naručite određeni broj časopisa, možete se obratiti na sledeći mail: [email protected].

Ukliko želite da se učlanite u Društo za ishranu Srbije, neophodno je da popunite zahtev koji se nalazi na internet stranici Društva za ishranu Srbije www.hrana-ishrana.org i uplatite godšnju

članarinu u iznosu od 1000 dinara na sledeći žiro račun 355-1032408-17.

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018. 3Milićević i sar., Mikotoksini: analiza rizika, principi i procedure

UVOD

Bezbednost hrane predstavlja ključni faktor u zaštiti zdravlja ljudi i ekonomskom razvoju zemalja širom sve-ta. Podaci Svetske zdravstvene organizacije (SZO) uka-zuju da su kontaminirana hrana i voda uzročnik preko 200 oboljenja kod ljudi, da godišnje do 30% svetske populacije oboli od određenog vida oboljenja izazva-nih hranom i vodom, a da 2,2 miliona ljudi, od toga 1,9 miliona dece, umre od posledica bolesti [1]. Zbog stalnog rasta međutrgovinske razmene i migracije stanovništva, može se očekivati trend porasta obolje-nja izazvanih hranom. U tom segmentu odgovornost državnih organa usmerena je ka uspostavljanju efika-snog sistema bezbednosti hrane, koji uključuje integri-san i dobro koordinisan longitudinalan sistem od njive do trpeze.

Savremeni sistem bezbednosti hrane koji ima za cilj visok nivo zaštite zdravlja i interesa potrošača bazira se na analizi rizika. Ovakav holistički-sveobuhvatni pristup koji u sebi integriše naučne, stručne i socioekonomske faktore, a sve u cilju donošenja pravovremenih mera kontrole i prevencije je opšte prihvaćen kao najpo-godniji način identifikacije opasnosti i procene mogu-će veze između opasnosti i pojave oboljenja kod ljudi izazvanih hranom [2]. Takođe, implementacija principa analize rizika u nacionalno zakonodavstvo je u funkciji unapređenja međutrgovinske razmene [3].

Analiza rizika je naučno zasnovan proces koji uklju-čuje tri odvojena, ali sistemski povezana elementa [4]:

– procenu rizika,– upravljanje rizikom i– međusobnu komunikaciju o riziku između svih učes-

nika zainteresovanih za bezbednost hrane.Da bi se očuvala naučna objektivnost, procena rizi-

ka i upravljanje rizikom su funkcionalno odvojeni pro-cesi. U svetlu bezbednosti hrane opasnost (hazard) se definiše kao biološki ili hemijski agens/činilac u hrani ili stanje hrane, koji može potencijalno da izazove štetno dejstvo na zdravlje ljudi, dok je rizik faktor verovatnoće štetnog uticaja na zdravlje i ozbiljnost tog uticaja, kao posledica postojanja opasnosti.

Bazirano na podacima Globalnog monitoringa za-štite životne sredine – monitoring kontaminacije hrane i programu procene rizika od mikotoksina (engl. Global Environment Monitoring System – Food Contamination Monitoring and Assessment Programme – GEMS/Food, Joint Food and Agriculture Organization of the United Nations, World Health Organization – FAO/WHO), kao i na podacima drugih nacionalnih agencija, mikotoksini po učestalosti pojavljivanja, nutritivno-zdravstvenim poremećajima i ekonomskim štetama predstavljaju ve-oma ozbiljan problem u sistemu snabdevanja stanov-ništva hranom, naročito u zemljama u razvoju [5].

Mikotoksini: analiza rizika, principi i procedure*

Kratak sadržaj

Bezbednost hrane se definiše kao skup preventivnih mera u cilju sprečava-nja oboljenja ljudi izazvanih hranom. Mikotoksini su toksični sekundarni metaboliti, proizvodi sekundarnog metabolizma toksigenih plesni koji u organizam ljudi i životinja dospevaju najčešće preko kontaminirane hra-ne. Oboljenja koja u tom slučaju nastaju predstavljaju tipične alimentar-ne intoksikacije-mikotoksikoze, najčešće kao rezultat hronične izloženosti. Radi se o vrlo stabilnim hemijskim jedinjenjima koje uobičajeni tehnološki postupci prerade ne uništavaju, stoga je njihovo prisustvo u hrani prak-tično nemoguće izbeći. Smatra se da su ljudi i životinje najčešće izloženi simultanom dejstvu više mikotoksina, najčešće u niskim koncentracijama. Negativan efekat prisustva mikotoksina u hrani ne odražava se samo na bezbednost hrane, već i na nacionalnu ekonomiju i međutrgovinsku razme-nu. Savremeni pristup bezbednosti hrane zasniva se na analizi rizika. Ana-liza rizika, naročito od mikotoksina u našoj zemlji je nedovoljno razvijena. Stoga ovaj rad ima za cilj da informiše naučnu i stručnu javnost ne samo o značaju mikotoksina, već o mehanizmu analize rizika od mikotoksina.

Ključne reči: mikotoksini, analiza rizika, bezbednost hrane, javno zdravlje

Dragan Milićević, Saša Janković, Srđan StefanovićInstitut za higijenu i tehnologiju mesa, Kaćanskog 13, 11040 Beograd

Autor za korespondenciju: Dragan Milićević, Institut za higijenu i tehnologiju mesa, Kaćanskog 13, 11040 Beograd, Srbija, E-mail: [email protected]

UDK: 663/664:[615.918:528.28COBISS.SR-ID 271829772

* Rad je saopšten 19. juna 2018. godine na stručnom skupu „Javnozdravstveni značaj kontrole ostataka mikotoksina u hrani” čiju organizaciju je sufinansiralo Ministarstvo prosvete, nauke i tehnološkog razvoja Republike Srbije.

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018.4Milićević i sar., Mikotoksini: analiza rizika, principi i procedure

Podaci o zastupljenosti mikotoksina na našim pro-storima su nedovoljni i može se reći nesistematični. Po-java aflatoksinske krize od 2012, za koju se može reći da još uvek traje, ukazala je na značaj istraživanja u ovoj oblasti i podigla svest kod svih učesnika u sistemu bez-bednosti hrane. Iako postoji obilje literature o analizi rizika od hemijskih hazarda i mikotoksina, smatrali smo za shodno da naučnu i stručnu javnost informišemo o savremenim saznanjima i stavovima o analizi rizika od mikotoksina sa posebnim osvrtom na aktuelno stanje sistema bezbednosti hrane u Srbiji.

Procena rizika

Procena rizika je kvalitativni i/ili kvantitativni proces saznanja o potencijalnoj opasnosti od pojedinih age-nasa (mikrobiološki, hemijski, fizički) u hrani na zdravlje ljudi, kako bi se evaluirao sistem bezbednosti hrane i shodno tome donele strategije za unapređenje javnog zdravlja. Glavne osobine koje karakterišu procenu rizika su naučno utemeljen pristup, nezavisnost, transparen-tnost, ponovljivost i multidisciplinarni pristup u reša-vanju određenog problema. Naučno utemeljen pristup se zasniva na svim naučnim dokazima kojima raspolažu relevantne međunarodne organizacije u oblasti bez-bednosti hrane kao što su Zajednički ekspertski komi-tet Svetske zdravstvene organizacije za aditive hrane (engl. Joint FAO/WHO Expert Committee on Food Addi-tives-JECFA), telo osnovano od strane SZO, Agencija za zaštitu životne sredine Sjedinjenih Američkih Država (engl. United States Environmental Protection Agency-US EPA), Agencija za hranu i lekove (engl. Food and Drug Administration-US FDA), a u Evropskoj uniji (EU) za tu vrstu aktivnosti zadužena je Evropska agencija za bezbednost hrane (EFSA) (engl. European Food Safety Authority-EFSA). Naučni odbor sa svojih 10 naučnih panela koje čine vodeći naučnici u svojim oblastima. Naučni odbor daje savete o međusektorskim pitanjima, dok se paneli bave procenom rizika pružajući stručna uputstva u konkretnim oblastima. Za kontaminente u lancu hrane zadužen je CONTAM Panel. U zavisnosti od raspoložive infrastrukture, analiza rizika spada u delokrug rada nacionalnih agencija. Detaljan princip i metod za procenu rizika od hemijskih kontaminenata u hrani opisan je u monografiji SZO “Principles and Met-hods for the Risk Assessment of Chemicals in Food” [6].

Identifikacija i karakterizacija hazarda

Toksikološki, procena rizika od mikotoksina koji mogu imati štetan efekat na zdravlje ljudi pod određenim uslovima, počinje opisom njihove hemijske struktu-re i fizičko-hemijskih karakteristika. U narednoj fazi proučavaju se faktori od značaja za formiranje ili sinte-zu mikotoksina, koji će pored ostalog omogućiti razvoj preventivnih mera, kontrole i tretmana.

Nakon definisanja fizičko-hemijskih karakteristika mikotoksina, pristupa se prikupljanju toksikološki re-levantnih podataka o biološkim efektima jedinjenja

na žive sisteme (in vivo i in vitro). Studije na in vivo modelima sprovode se u cilju prikupljanja podataka o toksikokinetici i toksikodinamici mikotoksina, kao i dozno-zavisnom odgovoru organizma na jedinjenje. Toksikokinetika proučava podatke o načinu apsorpcije, distribuciji, biotransformaciji, eliminaciji i pojavi rezi-dua mikotoksina i/ili njegovih metabolita. U narednoj fazi analize rizika ispituje se toksikodinamika, odnosno štetni efekti mikotoksina na određena (ciljna) tkiva. Ova faza ispitivanja sprovodi se na eksperimentalnim životinjama najčešće na miševima i pacovima, kao i na primarnim ćelijskim kulturama izolovanim iz tkiva lju-di i životinja, subcelularnim organelama (mitohondrije ili mikrozomi) ili prečišćenim ciljnim molekulima (npr. enzimi, receptori) [7]. U pojedinim slučajevima kao što je aflatoksin (AF), ohratoksin A (OTA), fumonizin (FUM), trihoteceni (TCT), koriste se i epidemiološki podaci zabeleženi u pojedinim regionima. In vitro istraživa-nja imaju važnu ulogu u definisanju načina delovanja jedinjenja (engl. mode of action-MoA), nakon nalaza na eksperimentalnim životinjama. Ukoliko jedinjenje ispoljava kancerogeni efekat podaci o MoA su daleko kompleksniji jer ne uključuju samo pojavu kancera, već i pojavu sistemskih degenerativnih oboljenja, kao što je ubrzano starenje, imune disfunkcije, kardiovaskular-na i neurodegenerativna oboljenja, kao i uticaj na re-produktivne organe. U svakom slučaju podaci o geno-toksičnosti su obavezan segment istraživanja štetnog efekta jedinjenja [8].

Internacionalna agencija za izučavanje raka (IARC) (engl. International Agency for Research on Cancer) je 1993. evaluirala kancerogena svojstva AFT, OTA, TCT, ZEA i FB. Aflatoksini su svrstani u grupu kancerogenih jedinjenja (grupa 1), dok su nefrotoksični OTA i kancer promoter FB1 i FB2 svrstani u grupu 2B (mogući kan-cerogeni). ZEA koji ispoljava estrogena svojstva i TCT sa izraženim imunosupresivnim efektom, svrstani su u grupu 3 kao nekancerogena jedinjenja, ali sa štetnim efektom po zdravlje ljudi. Pored navedenih mikotoksi-na, patulin, sterigmatocistin, ergotalkaloidi i Alernaria toksini predmet su evaluacije od strane međunarodnih organizacija. Sveobuhvatna studija o štetnim efektima mikotoksina na zdravlje ljudi objavljena je u publikaci-jama IARC [9], JECFA [10] i Milićević i sar. [11]. Klasifika-cija kancerogenih jedinjenja prema IARC prikazana je u Tabeli 1.

U procesu karakterizacije hazarda neophodno je odrediti kvantitativni odnos između doze i odgovora, odnosno njenog štetnog uticaja na zdravlje ljudi, kako bi se identifikovala polazna tačka (engl. Point of De-parture-POD) za dalju analizu rizika. Za genotoksične kancerogene u hrani, EFSA je primenila princip grani-ce izloženosti (engl. Margin of exposure-MoE). Granica izloženosti predstavlja odnos između referentne doze (engl. Benchmark dose-BMD) za štetni efekat utvrđen na eksperimentalnim životinjama i procenjenog unosa hazarda. Referentna doza (BMDL10) za AFB1 i OTA iznosi 170 ng/kg tm/dnevno i 21.0 µg/kg tm/dnevno, ponao-sob. Uopšteni stav je da ukoliko je MoE>10 000 može


se smatrati da je rizik nizak, dok u slučaju ukoliko je MoE <10 000 smatra se da rizik postoji [12,13].

S obzirom na činjenicu da za genotoksične kance-rogene ne postoji prihvatljiv ili tolerantni dnevni unos, kao i nemogućnost postizanja nultog rizika, u tom slu-čaju primenjuje se koncept razumno ostvarive niske koncentracije (engl. As low as reasonably achievable-ALARA). U takvim uslovima svi učesnici u lancu hrane u obavezi su da tragaju za rešenjima koja će omogućiti smanjenje izloženosti onoliko koliko je to ekonomski i tehnički moguće.

U slučaju ne-genotoksičnih jedinjenja kao što su aditivi hrane i rezidue primenjuje se koncept prihvat-ljivog dnevnog unosa (engl. Acceptable Daily Intake-ADI) ili referentna doza (engl. Reference Dose RfD), koja se definiše kao procenjena količina supstance u hrani koja se može uneti u organizam tokom kraćeg perioda vremena izražena na telesnu masu (mg/kg tm/dnevno), tokom jednog dana, a da ne predstavlja rizik po zdravlje. Za kontaminente hrane izračunava se to-lerantni dnevni unos (engl. Tolerable Daily Intake-TDI), dok se za jedinjenja sa kumulativnim svojstvima pri-menjuje koncept maksimalno tolerantni nedeljni unos (engl. maximum tolerable weekly intake – MTWI). TDI ili ADI predstavlja odnos između maksimalno netoksične doze (engl. noobservable adverse effect level – NOAEL) i faktora sigurnosti (engl. uncertainty factor-UF). Kori-šćenje sigurnosnog faktora uzima u obzir mogućnost postojanja razlika između čoveka i eksperimentalnih životinja i pri tome se smatra da je čovek osetljiviji (do 10 puta), iako to nije uvek slučaj. Takođe se u ob-

zir uzimaju varijacije koje postoje u osetljivosti prema delovanju ispitivanih supstanci u okviru same humane populacije (trudnice, deca, stare, bolesne osobe). Uku-pna vrednost faktora sigurnosti obično iznosi 100 (10 puta zbog ekstrapolacije rezultata animalnih testova na čoveka i 10 puta zbog razlika unutar ljudske popu-lacije). Tolerantni dnevni unos za pojedine mikotoksine prikazan je u Tabeli 2.

U integrisanom sistemu bezbednosti hrane, inkor-poriranom u savremenom konceptu SZO Jedno zdrav-lje (engl. One-Health), hrana za životinje predstavlja prvu kariku u lancu bezbednosti hrane. Serije afera koje su potresle evropski sistem bezbednosti hrane, kao što je pojava BSE, dioksina, melamina i razni vidovi falsifikovanja hrane, a u našem regionu aflatoksinska kriza, ukazuju na nedostatak sveobuhvatnog i celovi-tog pristupa u proceni i upravljanju rizikom. Procena rizika kod životinja konceptualno je ista kao i u slučaju zdravlja ljudi, međutim brojne fiziološke i metaboličke različitosti između životinjskih vrsta (monogastrične životinje i preživari), kao i unutar samih vrsta (tovna i mlečna grla), dosta otežavaju procenu rizika u delu koji se odnosi na toksikokinetiu i toksikodinamiku jedinje-nja. Takođe, razlike u načinu ishrane otežavaju procenu izloženosti [14]. Direktivom EU [15] propisan je obave-zan monitoring kontaminenata u hrani za životinje u cilju zaštite zdravlja životinja i moguće pojave rezidua u primarnim proizvodima animalnog porekla.

Tabela 1. Klasifikacija mikotoksina prema IARC

Mikotoksin Grupa Biološki efekat Referenca

Aflatoksini 1 Kancerogeno, teratogeno, mutageno, hepatotoksično [16, 17]

Ohratoksin A

2B

kancerogeno, teratogeno, nefrotoksično [18]

Fumonizin B1 i B2 kancerogeno, neurotoksin [18, 19]

Fusarin C[16, 18]

Sterigmatocistin Kancerogeno, teratogeno, mutageno, hepatotoksično

Citrinin

3

[16]Ciklohlorotin

Deoksinivalenol Imunotoksično, Hematotoksično[18]

Fusarenon H

Kojić kiselina [20]

Luteoskurin [16]

Nivalenol Imunotoksično, Hematotoksično [18]

Patulin Neurotoksično, imunotoksično i antibakterijsko [16]

Penicilinska kiselina[16]

Rugulosin

HT-2 i T-2 toksin Imunotoksično, Hematotoksično[18]

Zearalenon Hiperestrogenizam

Napomena: Grupa 1 – dokazano kancerogeni agens, Grupa 2A – verovatno kancerogeni agens, Grupa 2B – mogući kancerogeni agens, Grupa 3 – nije klasifikovan kao kancerogen.


Procena izloženosti

Procena izloženosti je kvantitativno determinisana i definiše se količinom nekog jedinjenja kojoj je izložena određena populacija. Radi se o veoma složenom pro-cesu koji zahteva multidisciplinarni pristup kako bi se utvrdilo koji mikotoksini predstavljaju značaj za javno zdravlje, kako na nacionalnom, tako i regionalnom ni-vou [29]. Stoga su u rešenje ovog problema uključene naučne discipline kao što je: toksikologija, analitička hemija, nutricionizam i matematičko-statističke disci-pline. U osnovi postoje dva koncepta procene izlože-nosti stanovništva mikotoksinima. To su deterministički i probabilistički model. Klasičan deterministički pristup procene izloženosti stanovništva mikotoksinima se zasniva na količini mikotoksina u pojedinim vrstama hrane i proceni količine konzumirane hrane. Međutim, na konzumiranje hrane utiču brojni faktori od kojih su najznačajniji: individualne varijacije, starost, sezonske i geografske varijacije, kulturološke, verske i ekonomske specifičnosti. Stoga je deterministički model predmet osporavanja i kao alternativa ovom modelu nameće se probabilistički metod. Kod probabilističkog načina, prilikom procene izloženosti umesto fiksnih vrednosti uzimaju se u obzir kvalitativne i kvantitativne varijacije koje utiču na konzumiranje hrane, u cilju što precizni-je procene izloženosti. Procenjeni nivo izloženosti mi-kotoksinima se izražava u ng mikotoksina/kg telesne mase/dnevno.

Podaci o prosečnoj potrošnji hrane mogu se preu-zeti iz Sistema globalnog monitoringa životne sredine/baza podataka o potrošnji hrane (http://www.who.int/nutrition/databases/en/). Statistički podaci o kon-zumiranju hrane za određene kategorije stanovništva na nivou Evropske unije (EU) mogu se preuzeti iz baze podataka Evropske agencije za bezbednost hrane [30] i mogu se koristiti za procenu izloženosti.

Za pravilnu procenu izloženosti ljudi mikotoksini-ma, kao i za kasniju fazu koja se odnosi na upravljanja rizikom, neophodni su podaci o stepenu kontaminacije (koncentracija) mikotoksina u pojedinim vrstama hrane

[5]. Ovi podaci dobijaju se iz više izvora i to: sistemskim praćenjem (monitoring), kontrolom, podacima Sistema globalnog monitoringa životne sredine/baza podataka o potrošnji hrane (GEMS/Food data base), podacima iz naučne literature, rezultatima istraživanja. Podaci o proceni izloženosti treba da pruže podatke relevantne za karakterizaciju rizika, odnosno za poređenje dobi-jenih podataka sa tolerantnim dnevnim unosom (TDI) mikotoksina propisanim od strane JECFA i EFSA i vezani su prevashodno za zastupljenost mikotoksina u hrani, s jedne strane i socioloških, ekonomskih i kulturoloških navika u ishrani stanovništva, sa druge strane.

Karakterizacija rizika

Karakterizacija rizika predstavlja završnu fazu u proce-ni rizika i definiše se kao kvalitativna i/ili kvantitativ-na procena verovatnoće pojave hazarda i posledica koje mogu nastati po zdravlje određene populacije na osnovu podataka dobijenih u prethodnim aktivno-stima. Za genotoksične kancerogene u ovoj fazi pre-računava se MoE, kao što je to opisano u potpoglav-lju identifikacija i karakterizacija hazarda i na osnovu dobijene vrednosti procenjuje se rizik. Vrednost 10000 kao granica izloženosti predstavlja grubu klasifikaciju različitog stepena zabrinutosti i dobijena je na osnovu ekstrapolacije podataka između vrsta, različitosti unu-tar vrsta i dodatnim specifičnostima karakterističnim za toksikološka ispitivanja. Za ne-genotoksične hazarde sa pragom toksičnosti karakterizacija rizika predstavlja poređenje preporučenih podataka o PDU, TDU ili PNU (Tabela 2) sa podacima o izloženosti datom hazardu dobijenim probabilističkim ili determinističkim meto-dom istraživanja. U odnosu na dobijene podatke tj. u kom odnosu stoje dobijeni podaci sa preporučenim vrednostima procenjuje se rizik [14].

Upravljanje rizikom

Nakon što je utvrđen štetan efekat određenog agensa, neophodno je doneti odluku koje mere treba predu-

Tabela 2. Preporuke za tolerantni unos mikotoksina (TDU)

Mikotoksin TDU Referenca

AF ALARA – as low as reasonably achievable [21]

OTA TNU: 120 ng/kgTM, bazirano na LOAEL od 8 µg/kgTM kod svinja. [22]

T-2 i HT-2 toksin TDU zbirno: 0,1 µg/kgTM/dnevno, bazirano na BMDL05 od 10 µg/kgTM/dnevno kod svinja (UF 100).

[23]

ZEA TDU: 0,25μg/kgTM/dnevno, bazirano na NOEL od 10 μg/kgTM/dnevno kod svinja (UF 40). [24]

Patulin TDU: 0,4 µg/kgTM/dnevno, bazirano na NOEL od 43μg/kgTM/dnevno kod pacova (UF100). [25]

DON TDU: 1 μg/kgTM/dnevno, bazirano na BMDL05 od 110 μg/kgTM/dnevno kod miševa (UF 100).

[26]

Nivalenol TDU: 1,2μg/kgTM/dnevno, bazirano na BMDL05 od 350 μg/kgTM/dnevno kod pacova (UF 300).

[27]

FB MTDU zbirno: 2 μg/kgTM/dnevno, bazirano na BMDL10 od 165 μg/kgTM/dnevno kod miševa (UF 100).

[28]


zeti kako bi se eliminisao i/ili smanjio štetan efekat na zdravlje ljudi. Ovaj postupak predstavlja upravljanje ri-zikom. Iako su procesi analize rizika funkcionalno odvo-jeni, oni su sistemski povezani i za pravilno donošenje zakonske regulative koja vodi ka uspostavljanju maksi-malno dozvoljenih količina (MDK) neophodna je dobra komunikacija između institucija koje obavljaju analizu rizika i upravljanje rizikom. Pored naučnih činjenica treba uzeti u obzir i socioekonomske faktore društva u celini, kulturološke i verske specifičnosti, tehničko--tehnološku opremljenost institucija nadležnih za bezbednost hrane, kao i druge faktore [31]. Suštinski upravljanje rizikom se sastoji iz četiri faze:– preliminarne aktivnosti vezane za upravljanje

rizikom,– procene mogućih opcija upravljanja rizikom,– implementacije donetih odluka,– monitoringa i kontrole sprovođenja donetih odluka.

Menadžment kontrole hrane definisan je kao „oba-vezna regulatorna aktivnost sprovođenja zakona i pro-pisa o hrani od strane državnih organa, u cilju pružanja zaštite potrošača i osiguranja bezbednosti i kvaliteta hrane tokom celokupnog lanca hrane, u skladu sa stan-dardima i zahtevima bezbednosti hrane” [32]. Može se definisati i kao integrisan kontinuiran proces planiranja, organizovanja, praćenja, koordiniranja i komuniciranja, sa odlukama i aktivnostima koje se baziraju na analizi rizika, kako bi se obezbedila bezbednost i kvalitet, kako domaće tako i uvezene hrane [33].

Harmonizacija u primeni principa i metodologije analize rizika, naročito u delu koji se odnosi na uspo-stavljanje zakonske regulative ima za cilj da omogući slobodan protok robe i kapitala, odnosno međutrgovin-sku razmenu [33]. Za donošenje standarda o bezbed-nosti hrane – koji predstavljaju osnovu zakonodavstva zemalja članica Svetske trgovinske organizacije (engl. World Trade Organization – WTO), kao i zemalja koje pretenduju da budu članice – zaduženo je telo SZO Ko-misija kodeksa alimentarijusa (engl. Codex Alimenta-rius Commission – CAC). U okviru CAC, telo odgovorno za upravljanje rizikom kontaminenata hrane je Kodek-sov komitet za aditive hrane (engl. Codex Committee on Food Additives – CCFAC), čija uloga je da:− zaštiti zdravlje potrošača, s jedne strane, a da

osigura slobodan protok robe i kapitala, sa druge strane,

− promoviše koordinaciju rada na svim standardima između vladinih i nevladinih organizacija,

− određuje prioritete i inicira pripremu vodiča i standarda. U postupku donošenja bezbednih nivoa konta-

minenata u hrani, telo SZO odgovorno za procenu rizika JECFA obezbeđuje podatke o PDU, TDU ili TNU mikotoksina, na osnovu kojih CAC donosi standarde o maksimalno dozvoljenim količinama mikotoksina u hrani. S obzirom na to da standardi nisu obavezujući i da su u formi preporuke, važan činilac u svetskoj eko-nomiji za liberalizaciju režima međunarodne trgovine

predstavlja Svetska trgovinska organizacija (STO) (engl. World Trade Organization–WTO). Članice STO su vezane Sporazumom o primeni sanitarnih i fitosanitarnih mera (engl. Agreement on the Application of Sanitary and Phytosanitary Measures–SPS Agreement) koje, direktno ili indirektno, mogu da utiču na međunarodnu trgovi-nu. Članice su dužne da osiguraju da se svaka sanitarna ili fitosanitarna mera primenjuje samo u obimu koji je neophodan da se zaštiti život i zdravlje ljudi, životinja i biljaka, da je zasnovana na naučnim principima pro-cene rizika i da se ne održava na snazi bez dovoljno naučnih dokaza – ALOP koncept (Approppiate Level of Protection) [33]. Iako je koncept Ciljevi bezbednosti hra-ne (engl. Food Safety Objective-FSO) prvenstveno na-menjen mikrobiološkim hazardima u hrani, on se može uspešno primeniti i na hazarde hemijskog porekla, kao što su mikotoksini [34].

Unutar EU za procenu rizika odgovorna je organi-zacija EFSA, dok je upravljanje rizikom institucionalizo-vano unutar Generalnog direktorata za zdravlje i zašti-tu potrošača [35]. Na osnovu toksikoloških podataka i podataka o zastupljenosti mikotoksina, Regulativama EU [36, 37, 38] definisano je prisustvo 10 mikotoksina u hrani i to: aflatoksina, deoksinivalenola (DON), fumoni-zina, ohratoksina A (OTA), zearalenona, T-2 i HT-2 triho-tecena i patulina, dajući više od 50 mikotoksin-matriks kombinacija.

Komunikacije o rizikuKomunikacija o riziku se definiše kao razmena infor-macija i mišljenja o aktuelnim rizicima i faktorima koji dovode do pojave rizika, između svih učesnika u lancu bezbednosti hrane [39]. Glavni cilj komunikacije o rizi-ku jeste da se poveća svest i saznanje kod svih učesnika u lancu hrane (državne institucije, potrošači, industrija, naučne institucije, mediji i dr.), kako bi se donele racio-nalne odluke za procenu i upravljanje rizikom, kao i da se stanovništvo pravovremeno informiše radi sticanja poverenja u sistem bezbednosti hrane [40].

Komunikacija o riziku se mora zasnivati na principi-ma koji su neophodni za razvoj i održavanje poverenja svih učesnika. Ključni principi komunikacije o riziku su:− otvorenost, − transparentnost, − pravovremenost i− odgovornost.

Otvorenost u radu podrazumeva mogućnost da svi učesnici u analizi rizika imaju mogućnost da učestvu-ju u dijalogu. Transparentnost implicira uspostavljanje seta odluka, praksi i procedura kojima se omogućava svim učesnicima da razumeju mehanizam donošenja odluka. Pravovremenost i odgovornost u komunikaciji rizika su neophodne kako bi se izbegle afere i druge negativne asocijacije kojima su skloni mediji. Metodi kojima se može obavljati komunikacija rizika su:− mediji (elektronski),− veb stranice,− elektronska pošta (e-mail),


− publikacije, štampa, posteri, bilbordi, − sastanci, konferencije, radionice (workshops),

vebinari, konsultacije, − mreža interesnih grupa,− socijalne mreže (Facebook, Twitter), − blogovi itd.

Principi, ključni faktori za razvoj i značaj komuni-kacije o riziku detaljno su opisani u izdanju SZO Risk communication applied to food safety: handbook [41].

Sistem bezbednosti hrane u Srbiji

Imajući u vidu činjenicu da je bezbednost hrane jedan od strateških ciljeva Republike Srbije u postupku pri-druživanju EU, novi sistem bezbednosti hrane, čiji je cilj obezbeđenje visokog nivoa zaštite života i zdravlja ljudi, kao i zaštite interesa potrošača, utemeljen na načelima analize rizika, predostrožnosti i transparentnosti, uspo-stavljen je u R. Srbiji donošenjem Zakona o bezbedno-sti hrane [42]. Iako postoje delimične neusaglašenosti postojećeg zakona sa Direktivom EU [43], novi sistem bezbednosti hrane bazira se na integrisanom i sveobu-hvatnom principu od njive do trpeze, uz odgovornost svih učesnika u lancu hrane. Jedna od novina ovog zakona je da definiše odgovornost subjekta koji uče-stvuju u poslovanju hranom za bezbednost i kvalitet hrane i hrane za životinje u svim fazama proizvodnje, prerade i prometa proizvoda, dok su za sprovođenje nacionalnog monitoring plana odgovorni inspekcijski organi. Službene kontrole (inspekcijski nadzor) utvrđe-ne su Zakonom o inspekcijskom nadzoru [44] i drugim zakonima.

Institucije uključene u sistem bezbednosti hrane

U skladu sa zakonom o bezbednosti hrane nadležnosti u oblasti bezbednosti hrane podeljene su u zavisnosti od vrste hrane i faze proizvodnje i prometa, između Ministarstva poljoprivrede, šumarstva i vodoprivrede i Ministarstva zdravlja. Unutar Ministarstva poljoprivre-de, nadležnosti su podeljene između Uprave za vete-rinu, Uprave za zaštitu bilja i Sektora poljoprivredne inspekcije, dok je u Ministarstvu zdravlja za poslove bezbednosti hrane nadležna sanitarna inspekcija. U ostale subjekte u bezbednosti hrane spadaju Direkcija za nacionalne referentne laboratorije, laboratorije za is-pitivanja u oblasti bezbednosti hrane i Stručni savet za procenu rizika u oblasti bezbednosti hrane.

Nacionalni program za monitoring rezidua

U pogledu hemijskih hazarda efikasan sistem bezbed-nosti hrane mora se bazirati na naučno zasnovanim činjenicama kako bi se pravovremeno utvrdila poten-cijalna opasnost [45]. Priroda opasnosti, značaj i faza u lancu hrane u kojoj se opasnost može pojaviti, su od suštinskog značaja za donošenje strategije upravljanja rizikom. U tom kontekstu Nacionalni program za moni-toring rezidua (NPMR) u živim životinjama i primarnim

proizvodima životinjskog porekla je od suštinskog zna-čaja za bezbednost hrane jer predstavlja mehanizam za utvrđivanje stepena usaglašenosti sa nacionalnim zakonodavstvom i često služi kao rani indikator poten-cijalnog rizika.

U Srbiji se monitoring rezidua sprovodi od 1972. go-dine, kada je započela kontrola prisustva teških metala, organohlornih pesticida i antibiotika u tkivima zaklanih životinja. Nacionalni program za monitoring rezidua se zasniva na principima EU zakonodavstva, prvenstve-no direktiva 96/23/EC [46], kojom se definišu principi i zadaci monitoringa, grupe farmakološki aktivnih sup-stanci i kontaminenata koje treba ispitivati, strategija uzorkovanja, kao i mere koje treba preduzeti ukoliko se utvrdi prisustvo nedozvoljene količine rezidua. Di-rektivu 96/23/EC [46] u delu koji se bavi minimalnim brojem uzoraka koji se moraju ispitivati u odnosu na proizvodnju iz prethodne godine, dopunjuje odluka 97/747/EC [47] iz 1997. godine, koja definiše uzorkova-nje mleka, jaja, divljači i meda. Za uspešnu realizaciju NPMR neophodno je ostvariti saradnju svih relevantnih činilaca (agencija za lekove, inspekcijski organi, labo-ratorije), kako bi se planom obuhvatile one supstance koje se najčešće primenjuju, a koje mogu predstavljati problem sa aspekta javnog zdravlja [48].

Planom monitoringa predviđena je analiza prisustva OTA u jetri i bubrezima zaklanih svinja i živine i AFM1 u sirovom mleku. Od samog uspostavljanja NPMR rea-lizacija poverena je Institutu za higijenu i tehnologiju mesa iz Beograda. U skladu sa Zakonom o bezbedno-sti hrane donet je set propisa kojima je regulisano si-stemsko praćenje mikotoksina u hrani biljnog porekla, potpunim i dopunskim smešama [49], odgovornost su-bjekta u poslovanju hranom za životinje da u bilo kojoj fazi proizvodnje, prerade i prometa uspostavi, sprovodi i održava postupke koji su zasnovani na analizi rizika i kritične kontrolne tačke (engl. Hazard Analysis and Critical Control Point- HACCP) [50]. Uz male neusagla-šenosti sa evropskim propisima, uspostavljene su mak-simalno dozvoljene količine mikotoksina u hrani i hrani za životinje [51,52].

Perspektive i preporuke

Iako je Zakon o bezbednosti hrane proaktivan, zasno-van na standardima CAC i principima evropskog zako-nodavstva, postoje određeni nedostaci u njegovom razvoju, implementaciji, kontroli sprovođenja i evalua-ciji efikasnosti u cilju daljeg unapređenja sistema bez-bednosti hrane. Dokumenti FAO/WHO [32] zasnovani na obimnom iskustvu u različitim sistemima, pružaju korisne sugestije kako da se sistem bezbednosti hra-ne efikasno uspostavi i održava. Primenom ključnih strukturalnih komponenti iz vodiča FAO/WHO [32] omogućena je identifikacija značajnih indikatora i pa-rametara sistema bezbednosti hrane, kao što je jačina, slabost, potencijalne mogućnosti i opasnosti (engl. SWOT, strength-S, weakness-W, potential opportuniti-es-O, threats-T). Upotrebom SWOT analize kao osnove,


moguće je izvesti preporuke za unapređenje nacional-nog sistema bezbednosti hrane.

Kao jačina postojećeg sistema može se istaći harmo-nizacija domaćeg zakonodavstva sa zakonodavstvom i principima EU, koja sa druge strane može predstavljati i opasnost. Formiran je Stručni savet za procenu rizika u oblasti bezbednosti hrane. Laboratorije za ispitivanja u oblasti bezbednosti hrane se opremaju visokosofisti-ciranom opremom i moraju biti akreditovane u skladu sa zahtevima standarda ISO 17025. Kao slabost siste-ma treba istaći dvojna ovlašćenja u kreiranju politike, zakonodavstva i kontrole u oblasti bezbednosti hrane, opterećenost inspekcijskih organa Uprave za veterinu obimom posla u odnosu na ljudske i materijalne resur-se. Iz tog razloga nedostaju adekvatni podaci na lokal-nom nivou. Kontrola u objektima se ne obavlja u skla-du sa procenom rizika, te se i zakonodavstvo ne može bazirati na analizi rizika. U određenom broju slučajeva inspekcijski nadzor se obavlja tek nakon što se problem pojavi. Neophodno je formiranje tela/organizacije koja će se baviti prikupljanjem i analizom rezultata službe-nih kontrola i ostalih podataka u svrhe naučne procene rizika na području bezbednosti hrane i hrane za živo-tinje. Svakako, ozbiljnu slabost predstavlja nedostatak nacionalne baze podataka o populacionim karakteristi-kama ishrane i sastavu hrane. I pored dugotrajnih naja-va, nacionalna referentna laboratorija nije osposoblje-na za rad, a Sistem brzog obaveštavanja i uzbunjivanja (RASFF) nije razvijen i implementiran.

Mogućnosti

Klimatske promene i posledično neuobičajena konta-minacija kukuruza i mleka AF u zemljama regiona, uka-zuje da nacionalni programi za monitoring, prevenciju i kontrolu mikotoksina separatno na nacionalnom ni-vou ne mogu dati sistemsku analizu i rešenje proble-ma kontaminacije hrane mikotoksinima. Formiranje regionalnog centra za procenu rizika od mikotoksina kao i primena informacionih tehnologija u distribuciji podataka i informacija bi bila u funkciji unapređenja sistema bezbednosti hrane. Klimatske promene nemi-novno zahtevaju razvoj prediktivnih modela za spreča-vanje kontaminacije mikotoksinima.

Opasnosti

Uvoz prehrambenih proizvoda konstantno raste, sa dominantnim učešćem hrane iz rizičnih regiona. Broj laboratorija koje obavljaju analize hrane je u stalnom porastu, a nisu na adekvatnom tehničko-tehnološkom nivou opremljenosti. Inspekcijski sistem nije zasnovan na analizi rizika.

ZAHVALNICA

Istraživanje prezentovano u okviru ovog rada je podr-žano od strane Ministarstva prosvete, nauke i tehnološ-kog razvoja Republike Srbije u okviru projekta TR31006.

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MYCOTOXINS: RISK ANALYSIS, GENERAL PRINCIPLES AND PROCEDURES

Abstract

Food safety is considered as a set of preventive measures aimed to preventing the illness resulting from the consumption of contaminated food. Mycotoxins are toxic secondary metabolites of toxigenic molds that enter humans and animals most commonly via contaminated foods. Mycotoxins ingestion can produce both acute and chronic diseases-myotoxicosis, most commonly as a result of chronic exposure. In general, mycotoxins are known as very stable chemical compounds that are not destroyed by conventional technological processing, which makes it practicaly im-possible to avoid in food. Thus, it is assumed that humans are constantly exposed to mycotoxins, most often in low concentrations. The negative effect of the presen-ce of mycotoxins in foods reflects not only on the food safety but on the national economy and trade. Modern approach to food safety is based on risk analysis. Risk analysis, especially mycotoxins in our country, is underdeveloped. Therefore, this paper aims to inform the scientific and professional community not only about the significance of mycotoxins, but also about the mechanism of risk analysis of mycotoxins.

Key words: mycotoxins, risk analysis, food safety, public health

Dragan Milićević, Saša Janković, Srđan Stefanović

Institut za higijenu i tehnologiju mesa, Kaćanskog 13, 11040 Beograd

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018.12Jakšić i sar., Kontrola mikotoksina u Republici Srbiji i Evropskoj uniji sa aspekta zakonske regulative

UVODDanas je poznato nekoliko stotina mikotoksina, među-tim samo za neke je dokazano i potvrđeno toksično, teratogeno, mutageno i karcinogeno delovanje. Većinu mikotoksina od značaja po zdravlje ljudi i životinja pro-dukuju plesni iz rodova Aspergillus, Penicillium i Fusari-um i to su mikotoksini iz grupe aflatoksina, ohratoksina, trihotecena, fumonizina, zearalenon, citrinin i patulin [1]. Internacionalna agencija za istraživanje kancera, International Agency for Research in Cancer (IARC) je klasifikovala aflatoksine kao karcinogene za ljude, dok su ohratoksin A i fumonizini klasifikovani kao mogu-ći karcinogeni [2]. Iako trihoteceni i zearalenon nisu karcinogeni, oni mogu izazvati niz neželjenih efekata po zdravlje kako ljudi, tako i životinja [3]. Mikotoksini najčešće kontaminiraju žitarice [4,5], ali se putem lanca ishrane prenose i do ljudi [6,7]. S obzirom na akutne i hronične toksične i karcinogene efekte, u cilju zaštite zdravlja ljudi i životinja, veoma je važna kontrola miko-toksina u hrani i hrani za životinje [8].

Ovaj rad daje pregled zakonske regulative koja se odnosi na maksimalno dozvoljene količine mikotoksina u hrani i hrani za životinje, ali i ostale zakonske okvire u kojima funkcioniše kontrola mikotoksina u Republici Srbiji. Paralelno sa prikazanim stanjem zakonskih akata u Srbiji, dat je prikaz evropskih regulativa koje se od-nose na kontrolu mikotoksina, a sa kojima se usklađuje kontrola bezbednosti hrane u Srbiji.

MAKSIMALNO DOZVOLJENE KOLIČINE MIKOTOKSINAU cilju zaštite zdravlja ljudi i životinja, mnoge države su donele specifične regulative za maksimalno dozvolje-ne nivoe mikotoksina u hrani i hrani za životinje. Glavni faktor za naučnu zasnovanost propisa je analiza rizika, ali su i drugi faktori, kao što su mogućnost adekvatnog uzorkovanja i analitičke procedure važni kod donošen-ja pravilnika o mikotoksinima. Takođe su u procesu do-nošenja propisa važni socioekonomski faktori kao što su troškovi, trgovina i dovoljna snabdevenost hranom. Propisi o mikotoksinima su najstroži u Evropskoj uniji (EU), gde različite organizacije i institucije doprinose organizovanoj borbi protiv mikotoksina [9].

Specifični propisi o mikotoksinima pojavili su se sedamdesetih godina XX veka, 10 godina od otkri-ća aflatoksina. U novom milenijumu, nakon brojnih istraživanja o mikotoksinima, došlo se do harmoni-zacije regulative o mikotoksinima u okviru EU i to na osnovu analize i karakterizacije rizika, kao i procene izloženosti sprovedenih od strane međunarodnih or-ganizacija kao što su JECFA (FAO/WHO Joint Expert Committee on Food Additives and Contaminants) i EFSA (European Food Safety Authority) [9]. Danas su u EU na snazi harmonizovane maksimalno dozvoljene granice, kao i maksimalno preporučene granice (tzv. indicative levels) za određene mikotoksine u različitoj hrani [10–19] i u hrani za različite kategorije i vrste ži-votinja [17, 20, 21].

Kontrola mikotoksina u Republici Srbiji i Evropskoj uniji sa aspekta zakonske regulative*

Kratak sadržaj

Prisustvo mikotoksina u hrani je važan faktor koji određuje njenu bez-bednost. Bezbednost hrane u Srbiji je definisana i obuhvaćena Zakonom o bezbednosti hrane. Na osnovu ovog zakona, donet je niz pravilnika koji definišu maksimalno dozvoljene količine mikotoksina u različitim namirnicama i hrani za životinje. Takođe se pravilnicima definiše pro-gram monitoringa koji obuhvata kontrolu mikotoksina u mleku i hra-ni za životinje. Zakon određuje nadležnosti u kontroli različite hrane u različitim fazama proizvodnje i prometa. Službene laboratorije za kontrolu mikotoksina moraju biti akreditovane prema standardu SRPS ISO 17025, a u toku je izrada pravilnika o ovlašćivanju laboratorija. Sistem brzog upozoravanja za hranu i hranu za životinje obuhvaćen je pomenutim Zakonom kao i odgovarajućim pravilnikom, ali podaci nisu dostupni. Dalje usavršavanje i usaglašavanje zakonodavstva o kontroli mikotoksina i bezbednosti hrane sa evropskim zahtevima je neophodno zbog zaštite zdravlja ljudi i životinja, ali i radi neometanog i bržeg eko-nomskog razvoja poljoprivrede i proizvodnje hrane u Srbiji.

Ključne reči: mikotoksini, kontrola, bezbednost hrane, zakoni

Sandra Jakšić1, Sunčica Kocić-Tanackov2, Milica Živkov-Baloš1

1 Naučni institut za veterinarstvo „Novi Sad“, Rumenački put 20, 21000 Novi Sad2 Univerzitet u Novom Sadu, Tehnološki fakultet, Bulevar cara Lazara 1, 21000 Novi Sad

Autor za korespondenciju: Sandra Jakšić, Naučni institut za veterinarstvo, „Novi Sad“, Rumenački put 20, 21000 Novi Sad, Srbija E-mail: [email protected]

UDK: 614.31(497.11) 614.31(4-672EU)

COBISS.SR-ID 271831052

* Rad je saopšten 19. juna 2018. godine na stručnom skupu „Javnozdravstveni značaj kontrole ostataka mikotoksina u hrani” čiju organizaciju je sufinansiralo Ministarstvo prosvete, nauke i tehnološkog razvoja Republike Srbije.

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018. 13Jakšić i sar., Kontrola mikotoksina u Republici Srbiji i Evropskoj uniji sa aspekta zakonske regulative

Hrana

Maksimalno dozvoljene količine mikotoksina u hrani u Srbiji propisane su Pravilnikom o maksimalno dozvo-ljenim količinama ostataka sredstava za zaštitu bilja u hrani i hrani za životinje i o hrani i hrani za životi-nje za koju se utvrđuju maksimalno dozvoljene koli-čine ostataka sredstava za zaštitu bilja, Sl. glasnik RS 22/2018 [22]. Ove maksimalno dozvoljene količine se usaglašavaju sa EU regulativama još od 2011 godine. Izuzetak su propisi koji se odnose na aflatoksin M1, T2 i HT2 toksin kao i za citrinin. U EU su 2013 [17] donete preporuke za T2 i HT2 toksine u vidu indikativnih nivoa. Prisustvo citrinina u hrani propisano je 2014. godine [18]. T2 i HT2 toksini, kao ni citrinin u važećem Pravilni-ku za hranu u Srbiji [22], nisu propisani, dok se propisi za aflatoksin M1 razlikuju u odnosu na evropske norme po maksimalno dozvoljenoj količini. Naime, zbog kon-taminiranosti kukuruza u Srbiji aflatoksinima 2013. go-dine [23] i nemogućnosti postizanja stroge i niske mak-simalno dozvoljene količine aflatoksina M1 u mleku u Srbiji, granica se preispituje na svakih 6 meseci (Tabela 1) [22,24] i određuje na osnovu dostupnih podataka službenih laboratorija o kontaminiranosti mleka, ali i hrane za životinje. Zbog već pomenutih socioekonom-

skih faktora procena je da se trenutno (do 30. 11. 2018) može tolerisati kao bezbedno za zdravlje ljudi 0,25 µg/kg aflatoksina M1 u mleku, a da se granica nakon toga spušta na 0,05 µg/kg. Prisustvo mikotoksina u hrani za odojčad i malu decu, u Srbiji je propisano Pravilnikom o dijetetskim proizvodima [25] koji je usklađen sa EU direktivama iz te oblasti.

Hrana za životinje

Maksimalno dozvoljene količine mikotoksina u hrani za životinje propisane su Pravilnikom o izmenama pravil-nika o kvalitetu hrane za životinje [26]. Ovaj Pravilnik je samo delimično u skladu sa EU regulativom koja se odnosi na hranu za životinje [17, 20, 21]. U Srbiji sve maksimalno dozvoljene količine imaju isti rang, dok u EU jedino za aflatoksine u hrani za životinje postoje direktive (maximum content) [20], a za ostale mikotok-sine maksimalni limiti označeni su kao preporuke, od-nosno vodiči [21] ili indikativni nivoi [17]. Osim toga, maksimalno dozvoljena koncentracija aflatoksina B1 u hranivima u Srbiji je viša (30 µg/kg) u odnosu na EU (20 µg/kg, EC 100/2003). U Srbiji nije propisano, niti po-stoje preporuke ni indikativni nivoi za prisustvo fumo-nizina, T2 i HT2 toksina u hrani životinje, iako postoje podaci o njihovom prisustvu kako u kukuruzu, tako i u smešama za ishranu životinja [27, 28].

ZAHTEVI ZA METODE ODREĐIVANJA MIKOTOKSINA

Mikotoksini su u uzorcima hrane i hrane za životinje prisutni u veoma niskim koncentracijama. Sa druge strane, uzorci su veoma različiti, te je različite nivoe koncentracije mikotoksina potrebno dokazati i izme-riti u veoma kompleksnim matriksima. U cilju tačnog, preciznog i pouzdanog obezbeđenja usklađenosti is-pitivanog uzorka sa važećim zakonima neophodno je imati pouzdane i precizne analitičke metode. Danas se za određivanje mikotoksina primenjuju različite meto-de i analitičke tehnike [29]. Najčešće se za određivanje mikotoksina u velikom broju uzoraka primenjuje ELI-SA (Enzyme-Linked Immunosorbent Assay), ali tačni-je, potvrdne metode određivanja mikotoksina podra-zumevaju primenu tečne ili gasne hromatografije sa UV, fluorescentnom ili masenom detekcijom [27]. Ove metode su uglavnom i standardizovane kako u EU (Eu-ropean Committee for Standardization, CEN) [30], tako i u Srbiji (Institut za standardizaciju Srbije) [31]. Me-tode za određivanje mikotoksina standardizovane od strane CEN nisu obavezne za službenu kontrolu hrane u EU. Međutim, ove metode se mogu lako koristiti za službene potrebe za kontrolu namirnica, jer njihove karakteristike ili performanse ispunjavaju kriterijume utvrđene direktivama EU za uzorkovanje i analizu [32]. Prilikom akreditacije u Srbiji (prema SRPS ISO 17025) laboratorija mora da zadovolji zahteve date u standar-

Tabela 1. Izmene propisa o maksimalno dozvoljenim vrednos-tima aflatoksina M1 u mleku u Srbiji [22,24]

Maksimalno dozvoljena koncentraci-ja (µg/kg)

Referenca Period važenja

0,05„Službeni glasnik RS” 28/2011

04.05.2011. – 01.03.2013.


01.03.2013. – 20.03.2014.


20.03.2014. – 01.07.2014.


01.07.2014. – 14.07.2014.


15.07.2014. – 31.12.2014.


01.01.2015. – 06.10.2015.


07.10.2015. – 05.04.2016.


06.04.2016. – 05.10.2016.


06.10.2016. – 05.03.2017.


05.03.2017. – 06.09.2017.


07.09.2017.– 28.03.2018.


28.03.2018. – 30.11.2018.


dnoj metodi ili validaciji proizvođača ELISA kita. EU di-rektiva 38/2005 određuje metode uzorkovanja u cilju službenih kontrola nivoa fuzarijumskih toksina u hrani i hrani za životinje [33]. Uredba Evropske komisije broj 401/2006 [32] detaljnije i preciznije definiše parametre kontrole kvaliteta kako bi se osigurala pouzdanost me-toda uzorkovanja i analize za zvaničnu kontrolu nivoa mikotoksina u prehrambenim proizvodima. Ova ured-ba je izmenjena u pogledu metoda uzorkovanja velikih šarži kao i skrining metoda regulativom 519/2014 [34]. U Srbiji postoji Pravilnik o metodama uzorkovanja i is-pitivanja hrane radi utvrđivanja ostataka sredstava za zaštitu bilja u hrani [35], i postoje uputstva o uzorkova-nju u okviru Pravilnika o utvrđivanju Programa moni-toringa hrane životinjskog porekla [36], odnosno hrane za životinje [37], ali ne postoji poseban dokument koji bi detaljno definisao uzorkovanje u cilju određivanja mikotoksina. Uzorkovanje hrane za životinje je propisa-no Pravilnikom iz 1987. godine [38], koji takođe nije do-voljno prilagođen uzorkovanju u cilju što pouzdanijeg određivanja kontaminanata kao što su mikotoksini. U monitoringu hrane za životinje, laboratorija primenjuje akreditovanu metodu za određivanje mikotoksina koju navodi u izveštaju ispitivanja, dok je metoda o analizi aflatoksina M1 definisana pomenutim Pravilnikom o monitoringu [36].

NADLEŽNOSTI INSTITUCIJA I ORGANIZACIJA U KONTROLI MIKOTOKSINA

Zakon o bezbednosti hrane

U Srbiji se Zakonom o bezbednosti hrane uređuju opšti uslovi za bezbednost hrane i hrane za životinje, oba-veze i odgovornosti subjekata u poslovanju hranom i hranom za životinje, sistem brzog obaveštavanja i uz-bunjivanja, hitne mere i upravljanje kriznim situacija-ma, higijena i kvalitet hrane i hrane za životinje [39]. Cilj ovog Zakona, između ostalog, je da obezbedi visok nivo zaštite života i zdravlja ljudi i zaštitu interesa po-trošača i u pogledu kontaminacije hrane mikotoksini-ma i drugim nepoželjnim materijama. Prema Zakonu, bezbednost hrane i hrane za životinje na teritoriji Re-publike Srbije, u okviru svojih ovlašćenja obezbeđuju subjekti koji se bave proizvodnjom i prometom hra-ne iz Centralnog registra i drugi subjekti: Ministarstvo poljoprivrede, šumarstva i vodoprivrede kao centralni organ; Ministarstvo zdravlja i laboratorije. Poslove or-gana državne uprave u smislu ovog zakona, u oblasti bezbednosti hrane obavljaju: veterinarska inspekcija, fitosanitarna inspekcija, poljoprivredna inspekcija, kao i granična veterinarska i poljoprivredna inspekcija i to u zavisnosti od vrste uzorka i faze u kojoj se vrši kontrola.

Osnovna pravila EU u pogledu zakona o hrani i hra-ni za životinje propisana su Uredbom (EZ) br. 178/2002 Evropskog parlamenta [40]. U regulatornom razvo-ju kontrole mikotoksina u EU značajnu ulogu igraju evropske organizacije. One uključuju Sistem brzog

upozoravanja za hranu i hranu za životinje (RASFF) [41], Evropsku agenciju za sigurnost hrane (EFSA European Food Safety Authority) [42], Evropski odbor za standar-dizaciju (CEN) [30] i EU i nacionalne referentne labora-torije za mikotoksine (EU-RL i NRL) [43].

Savet/Agencija za bezbednost hrane

Evropska agencija za sigurnost hrane (EFSA) je neza-visno telo Evropske komisije, osnovano 2002. godine. EFSA objavljuje savete u vidu naučnih mišljenja, koji predstavljaju glavnu naučnu osnovu za pripremu pro-pisa. Radi razmatranja pitanja vezanih za procenu rizika u oblasti bezbednosti hrane u Srbiji, a u skladu sa Zako-nom o bezbednosti hrane, 11. 05. 2017. godine je osno-vana posebna radna grupa – Stručni savet za procenu rizika u oblasti bezbednosti hrane [44]. Stručni savet je dužan da u radu primenjuje i koristi preporuke, smerni-ce i informacije dostupne od strane Evropske agencije za bezbednost hrane (EFSA). Stručni savet u skladu sa Zakonom obavlja sledeće poslove: priprema stručna i naučna mišljenja; unapređuje i koordiniše i primenjuje metode za procenu rizika; tumači i razmatra mišljenja o proceni rizika; pruža naučnu i stručnu pomoć u kri-znim situacijama u oblasti bezbednosti hrane; daje mi-šljenja o merama za unapređenje sistema bezbednosti hrane; obaveštava javnost; priprema vodiče za dobru poljoprivrednu, proizvođačku i higijensku praksu, daje preporuke za stručno usavršavanje i edukaciju u oblasti bezbednosti hrane i hrane za životinje.

Sistem brzog obaveštavanja

RASFF (Rapid Alert System for Food and Feed) je sistem brzog uzbunjivanja i obaveštavanja za hranu i hranu za životinje u EU, koji je uspostavljen kao mreža za izveštavanje o direktnoj ili indirektnoj opasnosti koja potiče od hrane i hrane za životinje za zdravlje ljudi, a odnosi se i na kontaminaciju hrane mikotoksinima. Sistem brzog obaveštavanja i uzbunjivanja obezbeđuje brzi protok informacija između nadležnih organa drža-va članica (uključujući zemlje EFTA/EEA), same Evrop-ske komisije i EFSA i dostavljanje podataka o službenim kontrolama i o preduzimanju odgovarajućih kontrolnih aktivnosti i/ili mera. U slučaju problema u lancu ishrane sa direktnim rizikom za zdravlje ljudi, RASFF olakšava preduzimanje neophodnih mera za obezbeđenje si-gurnosti potrošača. Obaveštenja RASFF-a koriste se ta-kođe za razvoj novih propisa EU i zaštitnih mera i time doprinose unapređenju i održavanju evropske bezbed-nosti hrane. Na portalu RASFF od 1981. godine do 28. 04. 2018. zabeleženo je 11153 prijavljena slučaja hrane kontaminirane mikotoksinima, od toga je 27 slučajeva bilo poreklom iz Srbije (https://webgate.ec.europa.eu/rasff-window/portal/).

U Srbiji je Zakonom o bezbednosti hrane [39] obu-hvaćen i Sistem brzog obaveštavanja i upozoravanja. Njime upravlja Ministarstvo poljoprivrede, šumarstva i vodoprivrede, a nadležni Ministar bliže propisuje način


uspostavljanja i organizaciju sistema. U sistemu brzog obaveštavanja i uzbunjivanja učestvuje i Ministarstvo nadležno za poslove zdravlja, Stručni savet, Nacionalne referentne laboratorije i laboratorije iz člana 20. ovog Zakona. Pravilnik o načinu uspostavljanja i organizaci-je sistema brzog obaveštavanja i uzbunjivanja za bez-bednost hrane i hrane za životinje [45] ima za cilj da obezbedi uspostavljanje Sistema brzog obaveštavanja i uzbunjivanja na teritoriji Republike Srbije, kao i uklju-čivanje tog sistema u sistem brzog obaveštavanja za bezbednost hrane ili hrane za životinje Evropske unije, a donet je 16. jula 2013.

Laboratorije

Zajednički istraživački centar Evropske komisije/In-stitut za referentne materijale i merenja (European Commission’s Joint Research Centre/Institute for Re-ference Materials and Measurements Geel, Belgija) predstavlja referentnu laboratoriju EU za mikotoksine (EU-RL) [45]. EU-RL za mikotoksine je kreiran kako bi, iz-među ostalog koordinirao aktivnosti vezane za razvoj, poboljšanje i primenu pripreme uzoraka i analitičkih metoda za službenu kontrolu kontaminacije hrane i hrane za životinje mikotoksinima [9]. U zemljama EU tenderom se vrši odabir referentnih laboratorija za 22 vrste ispitivanja hrane i hrane za životinje, a one ispu-njavaju uslove i obaveze prema Uredbama 882/2004 [46] i 625/2017 [47]; podležu nadzoru EU komisije i finansiraju se iz budžeta EU i komercijalnim ispitiva-njima. Nacionalne referentne laboratorije u državama članicama ovlašćuju se prema Uredbama 882/2004 i 2017/625 i nacionalnom zakonodavstvu; broj labo-ratorija određuju prema mogućnostima i potrebama; one su i službene laboratorije, vrše ispitivanja i komer-cijalnih uzoraka, a finansiraju se prema mogućnostima budžeta države [48].

U Srbiji je za poslove laboratorijskog ispitivanja i nji-ma povezane stručne poslove u lancu hrane osnovana Direkcija za nacionalne referentne laboratorije [49], kao organ uprave u sastavu Ministarstva poljoprivrede, šu-marstva i vodoprivrede, međutim ona za sada ne obu-hvata kontrolu mikotoksina. Laboratorijska ispitivanja u oblasti bezbednosti hrane i hrane za životinje, kao i sprovođenje programa monitoringa mogu da se ustu-pe laboratorijama putem konkursa koji raspisuju nad-ležna Ministarstva, a koji se objavljuje u “Službenom glasniku Republike Srbije”. Monitoring jeste sprovođe-nje niza planiranih aktivnosti koje se odnose na praće-nje ili merenje, kako bi se dobio uvid u pravilnu prime-nu propisa o hrani i hrani za životinje, kao i propisa iz oblasti zdravlja i dobrobiti životinja i zdravlja bilja [39]. Na osnovu Zakona o bezbednosti hrane, na godišnjem nivou se donose Pravilnici o utvrđivanju programa mo-nitoringa bezbednosti hrane životinjskog porekla [36] i hrane za životinje [37], koji obuhvataju monitoring aflatoksina M1 u mleku i aflatoksina B1, ohratoksina A, zearalenona, fumonizina B1 i B1, i T2 toksina u hranivi-ma biljnog porekla, potpunim i dopunskim smešama

za ishranu životinja. Na osnovu podatka monitoringa, kao i ostalih službenih kontrola mikotoksina u hrani, nadležna Ministarstva prate stanje kontaminiranosti žitarica i hrane mikotoksinima i u skladu sa tim daju se određene preporuke o preventivnim merama i službe-nim kontrolama, ali se i donose odgovarajuće promene zakonske regulative.

U toku je priprema Pravilnika o ovlašćivanju služ-benih i nacionalnih referentnih laboratorija za hranu, hranu za životinje i zdravlje životinja [48], koji bi treba-lo da usaglasi službene kontrole hrane sa zakonodav-stvom Evropske Unije i Regulativama 882/2004 [46] i 625/2017 [47]. Prema ovim regulativama se definišu nadležna tela, službene laboratorije za odgovarajuće oblasti, kao i uslovi koje laboratorije moraju da obez-bede. Izrada nacionalnog zakonodavstva o ovlašćenji-ma službenih i nacionalnih referentnih laboratorija za kontrolu hrane, hrane za životinje i zdravlje životinja mora biti u skladu sa: Zakonom o bezbednosti hrane Republike Srbije [39]; Zakonom o veterinarstvu Repu-blike Srbije [50]; Međunarodnim standardom ISO/IEC 17025 i Uredbama EU: 882/2004 [46] i 625/2017 [47] (primena april, decembar 2018) U zemljama članicama EU službene laboratorije se ovlašćuju prema Uredba-ma 882/2004 i 625/2017 i nacionalnom zakonodavstvu; broj laboratorija se određuje prema potrebama i vrše ispitivanja službenih, kao i komercijalnih uzoraka [48].

Akreditacija

Akreditaciono telo Srbije (ATS) [51] je nacionalno telo za akreditaciju, koje utvrđuje kompetentnosti tela za ocenjivanje usaglašenosti za obavlјanje poslova ispi-tivanja mikotoksina u hrani i hrani za životinje.

U Evropi su države članice EU i EFTA-e uspostavi-le mrežu nacionalnih akreditacionih tela pod nazivom Evropska organizacija za akreditaciju (EA) [52], koja omogućava da se kompetentnost svih laboratorija, kontrolnih i sertifikacionih tela ocenjuje po istim prin-cipima u skladu sa međunarodnim standardima i pro-cedurama. Zadatak akreditacionih tela u skladu sa spo-razumima, koje potpisuju sa EA, je da vrše promociju akreditacije i ekvivalentnost sistema akreditacije čime se smanjuje broj nepotrebnih provera proizvoda u me-đunarodnoj trgovini i stvaraju se uslovi za slobodan protok lјudi, roba, usluga i kapitala. ATS je 2012. godine postao punopravni član EA potpisivanjem sporazuma za laboratorije za ispitivanje, čime se priznaju ekviva-lentnost i pouzdanost izveštaja i sertifikata, izdatih u Srbiji od strane akreditovanih laboratorija širom Evro-pe. To znači da se izveštaji o ispitivanju mikotoksina izdati u akreditovanim laboratorijama u Srbiji priznaju u svim članicama potpisnicama sporazuma sa EA. Tre-nutno je u Srbiji za analizu aflatoksina akreditovano 30 laboratorija, za ohratoksin 21, za zearalenon 19, deoksi-nivalenol 18, fumonizin 10, T2 toksin 7, patulin 7, dok za citrinin nema akreditovanih laboratorija [51]. Ovi po-daci obuhvataju akreditaciju različitih metoda i tehnika u različitim vrstama uzoraka.


ZAKLJUČAK

Zakon o bezbednosti hrane u Srbiji na celovit i sveo-buhvatan način definiše nadležnosti, kontrolu i obez-beđenje bezbednosti hrane. U okviru ovog zakona do-neti su pravilnici koji delimično usaglašavaju kontrolu hrane u pogledu mikotoksikološke ispravnosti sa EU. Monitoring hrane, kao i preventivne mere koje se spro-vode u cilju sprečavanja kontaminacije hrane mikotok-sinima trebalo bi da doprinesu približavanju strogoj regulativi EU. S obzirom na specifičnost kontaminacije mikotoksinima, odnosno njihovu nehomogenu raspro-stranjenost u uzorcima, trebalo bi raditi na izradi novog pravilnika o uzorkovanju za potrebe analize mikotoksi-na, kao na pravilniku o detaljnijim zahtevima za skri-ning metode, koje se veoma često koriste. U pogledu sistema kontrole, donošenjem Pravilnika o ovlašćivanju laboratorija, očekuje se unapređenje rada laboratorija. Sistem brzog obaveštavanja i uzbunjivanja bi trebalo da bude transparentniji, te da na taj način doprinese većem poverenju u bezbednost hrane.

ZAHVALNICA

Istraživanje prezentovano u okviru ovog rada je podr-žano od strane Ministarstva prosvete, nauke i tehnološ-kog razvoja Republike Srbije u okviru projekta TR31071.

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MYCOTOXIN CONTROL IN REPUBLIC OF SERBIA AND EUROPEAN UNION FROM THE ASPECT OF THE LEGISLATION

Abstract

The presence of mycotoxins in food is an important factor that determines its safety. Food safety in Serbia is defined and encompassed by the Food Safety Law. On the basis of this law, a set of rules were adopted defining the maximum permitted quantities of mycotoxins in various foodstuffs and animal feeds. Also, these rules define monitoring programs that include the control of mycotoxins in milk and animal feed. The law de-termines competencies in controlling different foods at different stages of production and trade. Official laboratories for the control of mycotoxins have to be accredited ac-cording to SRPS ISO 17025 standard, while development of the rules on laboratory au-thorization is in progress. The Rapid Alert System for Food and Feedingstuffs is covered by the said Law as well as the relevant Regulations, but the data are not available. Fur-ther improvement and harmonization of legislation on the control of mycotoxins and food safety with European requirements is necessary for the protection of human and animal health, but also for the smooth and faster economic development of agriculture and food production in Serbia.

Key words: mycotoxins, control, food safety, regulatios

Sandra Jakšić1, Sunčica Kocić-Tanackov2, Milica Živkov-Baloš1

1 Scientific Veterinary Institute „Novi Sad”, 2 University of Novi Sad, Faculty of Technology

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018. 19Stančić et al., The effect of simvastatin in pancreas of diabetic rats

INTRODUCTION

Statins represent potent inhibitors of cholesterol bio-synthesis, as they inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate limiting enzyme in this biosynthetic pathway. Consequently, statins are well established as first-line therapy for decreasing cardiovascular morbidity and mortality in patients with and without coronary disease. Due to its lipid-lower-ing effects they are also used in the treatment of di-abetes-associated hyperlipidemia. However, there are some concerns, coming from the growing experimen-tal and clinical research of statins in diabetes, about the effect of statins on glucose metabolism and glycemic control. Some reports indicate an adverse [1, 2] or no effect of statins on insulin sensitivity [3, 4] while others reported a beneficial effect [5, 6]. Similar discrepancies exist in findings related to the effect of statins on β-cell mass/function, the second pathological hallmark of the diabetic state. For example, Urbano et al. [7] has found that atorvastatin impairs mitochondrial function in hu-man pancreatic islet and rat β-cells in vitro. In contrast, recently published in vivo studies showed that statins improved islet function in streptozotocin-induced di-abetes and obesity acting through anti-inflammatory and proliferative pathways in β-cells [8, 9]. The wide insight into literature data suggests that the effect of statins on glucose metabolism and insulin sensitivity

depends on the type, dose and duration of statin treat-ment as well as the model system (in vitro or in vivo) [10–12].

The special focus in diabetes treatment nowadays is to restore β-cell population (mass) and consequently their function. The neogenesis of β-cells from endo-crine and exocrine non-insulin producing progenitors gave some promising results in the treatment of ex-perimentally induced diabetes. Several stimuli/condi-tions have been shown to induce pancreatic potency to regenerate β-cells, including gastrin and epidermal growth factor, extendin-4, duct ligation etc. [13–16]. Data from our previous study extended this list with L-arginine [17]. Namely, we observed that L-argi-nine-NO producing pathway induces β-cell neogenesis in alloxan-induced diabetes acting through the com-plex transcriptional regulation of β-cell mass/function.

In the light of such conflicting data regarding sta-tin effects in diabetes, we aimed to examine whether statins affect β-cell regeneration in diabetic pancre-as. Accordingly, the localization/expression of insulin, along with specific insulin-related and proliferative transcription factors including pancreas duodenum homeobox-1 (PDX-1) and proliferating cell nuclear anti-gen (PCNA), was examined in the pancreas of rats with alloxan-induced diabetes after 12–day treatment with simvastatin.

The effect of simvastatin in pancreas of diabetic rats

Abstract

Growing evidences suggest that statins exert several cholesterol-indepen-dent pleiotropic effects in diabetes, but there is no consensus whether they are positive or negative. To shed more light on this issue, we examined the effect of simvastatin on β-cell regeneration in a diabetic state. Diabetes was induced in male Mill Hill rats with a single alloxan dose (120 mg/kg). Both non-diabetic and diabetic groups were additionally separated into two sub-groups: treated with simvastatin (5 mg/kg/day, i.g., 12 days) and control. Treatment of diabetic animals started after diabetes induction (glucose level ≥ 12 mmol/L). Our findings revealed that there is no increase in the area of insulin-immunopositive cells neither normalization of serum insulin level after simvastatin treatment of diabetic animals, although simvastatin increased nuclear immunopositivity for pancreas duodenum homeobox-1 (PDX-1) and proliferating cell nuclear antigen (PCNA). The data from this study suggest that 12-day treatment with simvastatin did not improve dia-betes-induced disturbances in β-cell mass/function.

Key words: Statins, diabetes, insulin, β-cell regeneration

Ana Stančić1, Aleksandra Korać2, Vesna Otašević1, Aleksandra Janković1, Bato Korać1,2

1 University of Belgrade, Institute for Biological Research “Siniša Stanković”, Bulevar despota Stefana 142, Belgrade, Serbia;

2 University of Belgrade, Faculty of Biology, Belgrade, Serbia.

Address of correspondence:

Professor Bato Korać, PhDUniversity of Belgrade, Institute for Biological Research “Siniša Stanković”, Department of Physiology Bulevar despota Stefana 142, 11060 Belgrade, SerbiaTel.: (381-11) 2078-307, FAX: (+381 11) 2761-433 e-mail: [email protected]

UDK: 615.224.036:616.379-008.64-056.24COBISS.SR-ID 271833868

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018.20Stančić et al., The effect of simvastatin in pancreas of diabetic rats

MATERIALS AND METHODS

Animals and diabetes induction

The experiments were approved by the Ethical Com-mittee for the Treatment of Experimental Animals of the Institute for Biological Research ‘Siniša Stankov-ić’, Belgrade. Male Mill Hill hybrid hooded, 3-month-old rats (Rattus norvegicus, Berkenhout, 1769) were used. They were divided into two groups: diabetic and non-diabetic rats. Alloxan (Sigma, Germany) was ap-plied to induce diabetes. After a 12 h fasting period, animals received a single alloxan dose of 120 mg/(kg body weight) i.p. Rats with blood glucose level ≥ 12 mmol/L, measured by glucose oxidase reagent strips (GlucoSure test, “Prizma”, Kragujevac, Serbia) were considered diabetic. Both diabetic and non-diabetic groups were additionally separated in two subgroups. One subgroup was treated with simvastatin (5 mg/kg/day) through gastric gavage, for 12 days. The second subgroup received intragastric infusion of saline and served as a control. Treatment of diabetic rats started after diabetes induction. The rats were maintained in individual cages with food and water ad libitum. Each experimental group consisted of six animals. After 12 days of treatment, blood glucose level was determined and the rats killed by decapitation. The pancreas was dissected out within 3 min and thoroughly rinsed with physiological saline to remove traces of blood.

Biochemical tests

Blood was collected, allowed to clot and centrifuged (3500 g) to prepare serum. Serum insulin levels were es-timated by radioimmunoassay (INEP, Belgrade, Serbia). Serum total and HDL (high-density lipoprotein)-cho-lesterol as well as triglyceride levels were determined by an autoanalyzer (Spectrum CCx, Abbott, Chicago,IL, USA). LDL (low-density lipoprotein)-cholesterol was calculated from measured values of total cholesterol, triglyceride and HDL-cholesterol according to the Frie-dewald calculation [18].

Student’s t test was used to evaluate significance of the differences. Statistical significance was accepted at P <0.05.

Immunohistochemistry

Immediately after dissection and washing, samples of pancreatic tissue were fixed in 10% formaldehyde at 4 °C overnight and processed routinely for embed-ding in paraffin. 5 μm-thick serial pancreatic sections were deparaffinized and rehydrated. Immunoreactivity was assessed by the avidin-biotin-peroxidase method (following the manufacturer’s instructions; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sections were incubated with 3% H2O2 in methanol for 10 min at am-bient temperature to block endogenous peroxidase, followed by three 5 min washes in 0.015 mol/L phos-phate-buffered saline (PBS; pH 7.4) and incubated with

1.5% normal goat serum (ABC Staining System, Santa Cruz Biotechnology) in PBS for 60 min at ambient tem-perature to block non-specific sites. Primary antibod-ies used in this study were anti-PDX-1 (1:1000; Abcam, Cambridge, UK) and anti-PCNA (1:200; Santa Cruz Bio-technology, Santa Cruz, CA, USA). The sections were incubated with the primary antibody in PBS overnight at 4 °C, followed by two 5 min PBS washes, incubated with 1:200 IgG biotinylated serum goat anti-rabbit (ABC Staining System, Santa Cruz Biotechnology) in PBS for 60 min at ambient temperature, followed by two 5 min PBS washes. After that, AB reagent (ABC Staining Sys-tem, Santa Cruz Biotechnology) was added for 30 min at ambient temperature, followed by three 5 min PBS washes and incubation with 0.02% H2O2 and 0.075% diaminobenzidine (Sigma) in 0.05 mol/L Tris buffer, pH 7.6, for 10 min in a dark room. Rinsing in distilled water and counterstaining with hematoxylin completed the experimental schedule.

Insulin and PDX-1 were detected by confocal mi-croscopy. Insulin was detected with anti-insulin anti-body (1:1000; Abcam) in combination with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:200; Abcam). For PDX-1 staining, the slides were incubated with anti-PDX-1 antibody (1:600; Abcam) followed by incubation with Alexa Fluor 568-labelled secondary antibodies (1:2000; Invitrogen, Paisley, UK). Confocal images were acquired with a Carl Zeiss confo-cal laser scanning microscope LSM510 and a Windows NT operating system. The examination of fluorescent immunolabelled samples was performed under Ar la-ser lamp (FITC+Alexa Fluor 568 double-stained sam-ples were excited with 488 nm and 543 nm light, re-spectively).

The specificity of the immune reaction, for both immunofluorescence and routine immunohistochem-istry, was tested by replacing the primary antibody with a non-immune rabbit serum or by incubating the sections with the secondary antibody alone.

RESULTS

Metabolic data

As it is obvious from Figure 1A, there are no significant differences in blood glucose level in non-diabetic an-imals, control and simvastatin-treated, between two time points: 1) before treatment (beginning of the ex-periment) and 2) after 12–days of treatment (end of the experiment). Three time points were represented for glucose level in untreated and simvastatin-treated diabetic animals: 1) before diabetes induction; 2) at the beginning of diabetic state and/or treatment; 3) 12–days after diabetes induction and/or treatment with simvastatin (Figure 1B). Level of blood glucose was significantly decreased after 12–days treatment of di-abetic animals with simvastatin (point 3) compared to the value detected in those animals before treatment (point 2).


Figure 2 summarizes the changes of serum insulin, total, HDL-, LDL-cholesterol and triglyceride levels in non-diabetic and diabetic animals. In contrast to the trend for normalization of glucose after simvastatin treatment of diabetic animals, serum insulin level was significantly decreased in those animals compared to control, showing similar value as in untreated diabe-tes (Figure 2A). Interestingly, total and LDL cholesterol were significantly increased in both simvastatin-treated groups, non-diabetic and diabetic, compared to con-trol (Figure 2B). Furthermore, LDL cholesterol was high-er in simvastatin-treated diabetic animals in relation to non-treated diabetic ones. In contrast, diabetic animals treated with simvastatin showed lower triglyceride lev-els compared to both control and untreated diabetic animals.

Localization/expression of insulin

As expected, insulin immunopositive areas were signifi-cantly decreased in pancreas of diabetic animals (Figure 3B) compared to controls (Figure 3A). Simvastatin tre-atment of both non-diabetic and diabetic animals did

Figure 1. Changes of blood glucose in non-diabetic and diabetic rats. Blood glucose levels in non-diabetic animals, control and simvastatin-treated, are present-ed for two time points: 1) before treatment (beginning of experiment) and 2) after 12–days of treatment (end of experiment) (A). Glucose levels in untreated and simvastatin-treated diabetic animals are presented for three time points: 1) before diabetes induction; 2) at the beginning of diabetic state and/or treatment; 3) 12–days after diabetes induction and/or treatment with simvastatin (B). The values represent the mean ± S.E.M of six animals per group. *Compared with value at time point 1, ***p < 0.001; # compared with value at time point 2, # #p < 0.01.

Figure 2. Serum insulin and cholesterols/triglyceride levels in non-diabetic and diabetic rats at the end of experiment (A and B, respectively). Bars represent the mean ± S.E.M of six animals per group. *Compared to control, *p < 0.05, **p < 0.01, ***p < 0.001; # compared to diabetes, # # #p < 0.001.

Figure 3. Localization of insulin immunofluorescence in pancreas of control (A), diabetes (B), simvastatin (C) and diabetes + simvastatin (D) animals. Pancreas sec-tions were double-labelled with anti-insulin antibody, and fluorescence analyzed by confocal microscope. In-sulin staining appears as green fluorescence. Scale bar, 20 μm.


not significantly affect the pattern of insulin immuno-positivity observed in the appropriate untreated group (control and diabetic, respectively) but significantly re-duced insulin-positive signal in both groups, indicating suppression of insulin synthesis or additional secretion.

Localization/expression of PDX-1 and PCNA

The results of immunohistochemical staining of PDX-1 are shown on Figure 4. Simvastatin significantly incre-ased the number of immunopositive nuclei as well as cytoplasmic immunopositivity in pancreas of diabetic animals in relation to non-treated diabetic animals. Cytoplasmic immunopositivity is especially strong in tissue of non-diabetic animals treated with simvastatin compared to control.

Figure 5 shows immunopositivity of PCNA in exam-ined conditions. The number of immunopositive nu-clei was increased after simvastatin treatment of both non-diabetic and diabetic animals compared to control and non-treated diabetic animals, respectively. Besides, there was strong immunopositivity in pancreatic cyto-plasm of simvastatin-treated diabetic animals.

DISCUSSION

Growing experimental and clinical research of statins in diabetes clearly suggests cholesterol-independent effects of this drug in this widespread metabolic dis-ease. The results of the present study extended the knowledge on the effect of statins in diabetic pancre-atic tissue. We have shown in this study that 12–day treatment with simvastatin did not restore insulin level in the serum and population of insulin-positive β-cells in the pancreas of diabetic animals. Furthermore, sim-vastatin treatment of both non-diabetic and diabetic animals, did not significantly affect the pattern of in-sulin immunopositivity observed in the appropriate untreated group (control and diabetic, respectively), but significantly reduced insulin-positive signal in both groups. The observed results suggest that short-term treatment with statins has no potential to induce β-cell regeneration.

Wide usage of statins for the treatment of lipid me-tabolism disturbances of various etiologies opens an important issue about the safety of their use related to glycemic control and glucose metabolism. The main concerns come from clinical studies mainly showing increased risk of development of diabetes type 2 in the subject using statins [19, 20]. However, data from basic molecular studies are inconsistent. Some of them speak in favor beneficial effects of statins on insulin re-sistance and β-cells, other reported negative impact of statin therapy while some showed no effects. In the study presented herein simvastatin reduced insu-lin-positive signal of both non-diabetic and diabetic animals.

PDX-1 has been described as an important tran-scription factor that determines endocrine and be-ta-cell destiny [21–24]. Manipulation of this transcrip-tion factor and its signaling pathways has become the dominant approach for β-cell regeneration and non-β to β-cells reprograming in states where the function of the endocrine pancreas is compromised, including diabetes. We have shown that the L-arginine-NO pro-ducing pathway induces activation (nuclear localiza-tion) of PDX-1 in the diabetic pancreas [17]. Besides, the increased colocalization of this transcription factor with insulin in endocrine and especially exocrine pan-creatic tissue, clearly suggests that L-arginine induces β-cell regeneration in diabetic pancreas through trans-differentiation of exocrine into endocrine cells.

Comparison of these results with the results of oth-er in vivo studies examining effects of statins on β-cell

Figure 4. Immunohistochemical staining for PDX-1 in pancreas of control (A), diabetes (B), simvastatin (C), diabetes + simvastatin (D) animals. Numerous nuclei with strong PDX-1 staining can be seen in endocrine and exocrine pancreas of simvastatin-treated diabetic group (D). Scale bars, 20 μm.

Figure 5. Immunohistochemical staining for PCNA in pancreas of control (A), diabetes (B), simvastatin (C), diabetes + simvastatin (D) animals. Simvastatin treat-ment induced strong cytoplasmic and nuclear immu-nopositivity in diabetic pancreas (D). Scale bars, 20 μm.


function establishes the notion that the effect of these drugs will depend on the dose and duration of their application. In the present study simvastatin was ad-ministrated in a dose of 5 mg/kg body weight for 12 days. Li et al. [9] have found increases in islet function and proliferation in streptozotocin-induced diabetes after simvastatin treatment in a dose of 3 mg/kg/day but the duration of the treatment was much longer, lasting for 8 weeks. Similar results were found in obese mice treated with atorvastatin with higher dose (30 mg/kg/day) and longer treatment (58 days) [8].

Interestingly, although there is no increase in β-cell population after simvastatin treatment, nuclear local-ization of PDX-1 and PCNA was significantly increased in pancreas of treated animals. The activation of PDX-1 and PCNA in both endocrine and exocrine tissues, has been previously correlated by others [25, 26] and our-selves [10] with proliferation and regeneration of pan-creatic islet. So, it seems likely that simvastatin adminis-trated in the present schedule (time, dose) recruits the transcriptional program for β-cell regeneration without achieving the complete process.

Considering the absence of normalization of serum insulin level by simvastatin, we were surprised that this drug counteracted hyperglycemia in diabetic animals. We suggest the possibility that restitution of hypergly-cemia by simvastatin results from the peripheral mech-anisms operating in regulation of plasma glucose level but not from the direct effect of the drug on the pan-creatic tissue. There are growing data from in vitro and in vivo studies speaking in favor our hypothesis. The beneficial effect of statins, including simvastatin [27], on insulin sensitivity have been reported in diabetic patients and animal models of obesity and diabetes. For example, Wong et al. [28] reported that atorvasta-tin increased glucose uptake in skeletal muscle leading to improved insulin sensitivity in Zucker lean and fatty rats, while Lalli et al. [29] described molecular mech-anisms of improved insulin signaling in the liver and skeletal muscle in rats fed a high-fat diet.

However, simvastatin did not normalize total cho-lesterol level in diabetic rats and even increased LDL cholesterol. Such an unexpected outcome of statin treatment over cholesterol level and inhibition of its synthesis is not an isolated case reported for statin ad-ministration in rats. For example, Ness et al. [30] have found that lovastatin increased HMG-CoA reductase activity in rat liver. Furthermore, Isusi et al. [31] have shown that there are differences in the capacity of var-ious types of statins on cholesterol metabolism in liver, while simvastatin has been described as less effective in reduction of cholesterol ester synthesis. Besides, rats have low cholesterol levels that are generally unaffect-ed by statins [32].

According the data from this study we conclude that short-term simvastatin treatment does not im-prove diabetes-induced decrease in β-cell mass/func-tion and/or induces β-cell regeneration. These data are

in accordance with growing evidence that question ex-clusively beneficial effects of statin administration and could be important for defining appropriate clinical use and schedule of statin treatment in non-diabetic and diabetic associated metabolic disturbances.

Acknowledgments: This work was supported by the Ministry of Education, Science and Technological De-velopment of the Republic of Serbia, Grant No. 173055.

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Efekat simvastatina u pankreasu dijabetičnih pacova

Kratak sadržaj

Sve je više dokaza da statini ostvaruju holesterol-nezavisne efekte u dijabe-tesu, ali ne postoji saglasnost da li su oni pozitivni ili negativni. U pokušaju da pojasnimo ovaj problem, ispitivali smo efekat simvastatina na regene-raciju β-ćelija u dijabetesu. Dijabetes je indukovan kod mužjaka Mill Hill pacova jednom dozom aloksana (120 mg/kg). I nedijabetične i dijabetične životinje su podeljene u dve podgrupe: tretirane simvastatinom (5 mg/kg/dan, i.g., 12 dana) i kontrolne. Tretman dijabetičnih životinja započelo je nakon indukcije dijabetesa (nivo glukoze ≥ 12 mmol/L). Naši rezultati po-kazuju da nema povećanja površine insulin-imunopositivnih ćelija niti nor-malizacije nivoa insulina u serumu nakon tretmana dijabetičnih životinja simvastatinom, iako je simvastatin povećao nukleusnu imunopozitivnost pankreasnog PDX-1 (engl. pancreas duodenum homeobox-1) i PCNA (engl. proliferating cell nuclear antigen). Rezultati studije pokazuju da 12-dnevni tretman simvastatinom nije poboljšao dijabetesom indukovane promene u masi i funkciji β-ćelija.

Ključne reči: statini, insulin, dijabetes, regeneracija β-ćelija

Ana Stančić1, Aleksandra Korać2, Vesna Otašević1, Aleksandra Janković1, Bato Korać1,2

1 Univerzitet u Beogradu, Institut za biološka istraživanja “Siniša Stanković”, Bulevar despota Stefana 142, Beograd, Srbija;2 Univerzitet u Beogradu, Biološki fakultet, Belgrad, Srbija.

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018.26Stanojević et al., Zeolite pretreatment accomplishes partial brain radioprotective role by reducing iron and oxidative ...

INTRODUCTION

Zeolite, a natural clinoptilolite, is a strong non-selec-tive adsorbent and/or ion exchange agent for differ-ent compounds, including metals, mycotoxins, some strains of bacteria, gases, essential nutrients, etc. [1–3]. Adsorptive potential of zeolite grows with the decrease of particle size, which is why we used micronized clinoptilolite (MZC).

Taken orally, zeolite remains intact within the al-imentary tract. It is used worldwide as a supplement for animal feed and in human medicine [4] . However, there is plenty of evidence (pro and contra) regarding daily intake of zeolite in humans [5].

In respect to its binding properties, zeolite reduces bioavailability of essential transition metals (iron (Fe), copper (Cu), manganese (Mn), etc., which contribute

to free radical production through Fenton like reac-tions). Also, zeolite absorbs gases released during food fermentation within the alimentary tract (oxygen (O2) and nitric oxide (NO), which altogether indicates the certainty of its antioxidative role [6]. Homeostasis of essential metals is particularly important for regular development and physiological functioning of living organisms [7]. Iron is the most abundant transition ele-ment in humans with ascertained physiological impor-tance. Nevertheless, Fe contributes to oxidative stress (OS) development, via Fenton reaction, where it reacts with hydrogen peroxide (H2O2) to form the potent hy-droxyl radical (HO•) [8]. Reactive oxygen and nitrogen species (ROS, RNS) affect all classes of biomolecules (proteins, lipids and DNA), also, they alter cell signaling pathways, gene expression deplete energy and even-tually, lead cells into death by apoptosis [9].

Zeolite pretreatment accomplishes partial brain radioprotective role by reducing iron and oxidative /nitrosative stress in rats

Abstract

The aim of our study was to test the effect of subacutely applied micronized zeolite [micronized clinoptilolite (MZC)] on brain sta-tus of iron (Fe), reactive oxygen and nitrogen species (ROS, RNS), and radioprotective role against brain oxidative/nitrosative stress (OS/NS) initiated by single ionizing radiation of 2 or 10Gray (Gy).

Wistar rats on normal (n=18) and 5% MZC supplemented diet (n=18), during 4 weeks, were internally subdivided into 3 subgro-ups (6 rats in each subgroup), with one of subgroup remaining as a control, and the other two subjected to single ionizing ra-diation of 2Gy or 10Gy. Thus, we had groups on normal diet: C – controls, 2Gy and 10Gy; and on 5% MZC supplemented diet: MZC, MZC+2Gy and MZC+10Gy. Concentrations of nitrates (a final RNS metabolite) and superoxide anion radical (O2

•-) (an ini-tial ROS) were measured in homogenates of selective vulnerable brain regions (cerebellum, hippocampus and forebrain cortex), while Fe was determined in whole brain of rats. Results documen-ted a significant drop of Fe in MZC and MZC+2Gy/10Gy groups; decrease of O2

•- and nitrate in MZC group; almost equal drop of O2

•-, in 2Gy and MZC+2Gy groups; and nitrate increase in 10Gy and MZC+10Gy groups. We confirmed that subacute MZC pretre-atment contributes to partially accomplished brain radioprotecti-ve effect in rats exposed to single radiation dose of 2Gy and 10Gy, probably due to reduced OS/NS and Fe.

Key words: brain, iron, ionizing radiation, nitrosative stress, oxi-dative stress, zeolite (MZC)

Boban Stanojević1, Mirjana Đukić2*, Ivana Stevanović3, Milica Ninković3, Ana Đurić2, Borko Gobeljić2, Milan Apostolović4, Ana Pantelić5, Goran Zebić6, Lidija Todorović1, Tijana Bojić1, Kiril Savovski1

1 Department for Radiobiology and Molecular Genetics 080, Institute of Nuclear Sciences “Vinča”, University of Belgrade, Belgrade, Serbia2 Department of Toxicology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia 3 Institute for Medical Research, Military Medical Academy, Medical Faculty MMA, University of Defense, Belgrade, Serbia 4 University of Belgrade – School of Medicine, Institute for Orthopedic-Surgical diseases “Banjica”5 Department of Chemistry, Scientific Institution, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia6 Department for Food Technology, Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080 Belgrade-Zemun, Serbia

* Corresponding author:

Mirjana Đukić, PhD, ProfessorDepartment of Toxicology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia, e-mail: [email protected]; [email protected] Phone (office): +381 11 3951 308 Fax (office): +381 11 3972840

UDK: 615.038:549.67 COBISS.SR-ID 271835916

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018. 27Stanojević et al., Zeolite pretreatment accomplishes partial brain radioprotective role by reducing iron and oxidative ...

It is well known that ionizing radiation induces OS in living organisms [10]. We supposed that nitrosative stress (NS) will develop in irradiated rats as well, based on the fact that blood vessel endothelium produces NO which spontaneously reacts with superoxide anion radical (O2

•-) and produces harmful RNS, peroxynitrite anion (ONOO–). The final metabolic products of entire-ly generated RNS are nitrates (NO3

–) [9]. Additionally, we assumed that reduced bioavailability of Fe, NO and O2 from gastrointestinal tract (GIT) during prolonged MZC intake will result in systemic OS and NS decrease, including brain. Since previous studies confirmed that ionizing radiation causes increase of OS and NS [11, 12], we were tempted to check if the prolonged MZC pretreatment will have a radioprotective role in the brain , by analyzing certain OS/NS parameters in rats.

The use of zeolite as a food supplement is based on its detoxification capabilities due to ability to bind various toxins within the GIT [13–15]. Furthermore, ze-olite improves some physiological occurrences in treat-ed animals, such as growth improvement, reproductive performance, boosts immune system, etc. [16–18].

Accordingly, the aim of our study was to test if oral pretreatment with 5% MZC, for 4 weeks, will make any influence on brain status of Fe, ROS and RNS and addi-tionally, will radioprotective role against brain OS/NS initiated by single ionizing radiation doses, 2Gray (Gy) or 10Gy, will be attained.

MATERIALS AND METHODS

Experimental animals

Adult male Wistar rats (weighing 220–250g) were kept under standardized housing conditions (temperature 23±2°C, lighting 12:12 light:dark, light on from 8:00 to 20:00h) with free access to tap water and a custom pellet rat diet. Suspension of MZC was administered daily by gavage. The experimental protocol followed the “Guide for the Care and Use of Laboratory Animals” [19] and was approved by Ethical Committee for Exper-imental Animals, ,,Vinča” Institute (No. 6/12).

Experimental design

Wistar rats on normal diet were randomly subdivid-ed into three groups (n=6): C – control (not treated) and 2Gy and 10Gy groups – rats subjected to a single dose of radiation of 2Gy or 10Gy, respectively; and ac-cordingly, rats on 5% MZC supplemented diet covered three groups (n=6): MZC, MZC+2Gy and MZC+10Gy groups. The MZC amount was calculated in respect to the quantity of ingested food and rat body mass. The suspension of 0.85–1g of MZC/day (corresponds to 5% of 17–20 g of custom pellet/day) was administered orally, by gavage, during four weeks [5] . The animals gained 133.85 ± 24.7g body weight after 4 weeks. No statistically differences were observed between the C and MZC groups.

Gamma irradiation of rats was performed in the Laboratory of Radiation Chemistry and Physics, Vinča Institute of Nuclear Sciences, using 60Co gamma source designed for radiobiological and radiation chemistry experiments. The animals were confined in custom made individual cages, made of wire, sideways posi-tioned to the irradiation source and then subjected to the whole body irradiation at a dose rate of 0.167Gy/min for 12 minutes (corresponds to a total sublethal dose of 2Gy) or 60 minutes (corresponds to a total low-ered lethal dose of 10Gy) [20]. Five days after the irra-diation, the animals were anesthetized with a 50 mg of sodium pentobarbital/kg injection before being sacri-ficed (by decapitation), when brains were removed immediately and stored at -80 °C, until analyzing. The impact of the administered anesthesia on OS and NS parameters measured in brain of rats was not achieved (the results of intact and anesthetized animals were al-most identical, therefore, were not presented).

Reagents

All reagents and chemicals were of analytical grade or higher purity. In this study we used ethylenedi-aminetetraacetic acid – EDTA, nitrobluetetrazolium – NBT, nitric acid – HNO3 (65%) (Fisher Chemical, UK), perchloric acid – HClO4 (65%), hydrochloric acid – HCl, sodium hydroxide – NaOH, sulfanilic acid, N – (1–naph-thyl) amine (Sigma–Aldrich, USA), sodium nitrate – NaNO3, sodium phosphate – Na2HPO4, glycerol (Merck, Germany), Fe standard solution (AccuStandard, USA), sodium pentobarbital Vetanarcol (0.162 g/mL) (Werfft – Chemie, Austria), saline solution (0.9% w/v) and de-ionized water (Hospital Pharmacy Military Medical Academy, Belgrade, Serbia).

Analysis of iron

Brain tissue samples (around 1g) were mineralized in a mixture of concentrated HNO3 and HClO4 (4:1, v/v), heated at temperature between 250 °C and 300 °C, till dryness and diluted up to 10mL with 0.1M HNO3. The concentrations of Fe were analyzed by inductive cou-pled atomic absorption spectrometry (IC-AAS, Analyst 200, PerkinElmer), using air-acetylene flame. Standard solutions of Fe were prepared according to the Perkin-Elmer Pure Atomic Spectroscopy Standards guidelines (NIST traceable CRM, Perkin–Elmer Corporation, USA and Merck – Germany). The absorption wavelength was 305.91 nm.

Superoxide anion radical measurement

Quantification of O2•- was based on nitrobluetetrazoli-

um (NBT) reduction by O2•- to yellow colored monofor-

mazan, which absorbance was measured at 550 nm [21].

Nitrate measurement

After deproteinization, concentration of nitrates (NO3–,

the final metabolic product of all RNS) was calculated


as the difference between two sets of measurements with Griess reagent (1.5 % sulfanilamide in 1 mol HCl plus 0.15 % N-(1–naphthyl) ethylendiamine dihy-drochloride in distilled water), before (only NO2

– was measured) and after the addition of cadmium, which reduces NO3

– into NO2–

(NO2– + NO3

– were measured) [22] . The measurements were performed spectropho-tometrically at 492 nm. The results were expressed as nmol NO3

–/mg protein.

Protein determination

The Lowry method was used for protein measurement in rat vulnerable brain regions (VBR) homogenates [23].

Statistical analysis

One-way ANOVA and post-hoc Dunnett`s C tests were used (software GraphPad Prism, version 5.01) for statis-tical data analysis. Values were presented as average ± STDEV. Differences were considered statistically signif-icant for p<0.05.

RESULTS

Decrease of brain Fe concentrations was observed in all experimental groups: MZC, 2Gy and MZC+10Gy (p<0.01); 10Gy (p<0.05) and MZC+2Gy (p<0.001), compared to the controls. Also, lower Fe was found in MZC+2Gy and MZC+10Gy (p<0.05) groups than in cor-responding irradiated groups, 2Gy and 10Gy (Figure 1).

Iron

Control MZC MZC+10Gy 10Gy MZC+2Gy 2Gy0

10

20

30

** ****

***

*##

µg/g

wet

tiss

ue

Figure 1. Brain iron in the experimental groups. Brain Fe (μg Fe-/mg wet tissue) concentrations are presented as means ± SD, for 10 animals/group. Differences were considered statisti-cally significant at: *.#p<0.05, **,##p<0.01 and ***p<0.001(*-com-pared to control, #- compared to MZC group). One-way ANO-VA, Dunnett`s C test were used for statistical analysis.

Decrease of O2•- was accomplished in MZC group

(in: cerebellum (p<0.001), hippocampus and cortex (p<0.01)). Almost identical increase was accomplished in 2Gy (in: cerebellum (p<0.05) and cortex (p<0.001)), MZC+2Gy (in: hippocampus and cortex (p<0.01)) and MZC+10Gy (in: cortex (p<0.01)) groups (Figure 2).

Superoxid anion radical


100

200

300 Cer Hipp Cx

***

*

**

*****

****

**

nmol

red.

NBT

/min

/mg

prot

eins

Figure 2. Superoxide anion radical in selective vulnerable brain regiones of the treated rats. Superoxide anion radical, expressed as nmol red. NBT/min/ mg protein, was tested in VBRs: cerebellum (Cer), hippocampus (Hipp) and cortex (Cx). Values are presented as means ± SD, for 10 animals/gro-up. Differences were considered statistically significant at: *.#p<0.05, **,##p<0.01 and ***p<0.001(*-compared to control, #-compared to MZC group). One-way ANOVA, Dunnett`s C test were used for statistical analysis.

Significant increase of NO3– was reached in the

experimental groups, as follows: 10Gy group (in: cer-ebellum (p<0.05), hippocampus (p<0.001) and cortex (p<0.001)); MZC+10Gy (in: hippocampus (p<0.05) and cortex (p<0.001)); and 2Gy (in: hippocampus (p<0.01)). Contrary, NO3

– decline was documented in: MZC group

(in: cerebellum and hippocampus (p<0.01) and cortex (p<0.001)); 2Gy (in: cerebellum (p<0.001) and cortex (p<0.01)); and MZC+2Gy group (in: cerebellum and cor-tex (p<0.001)). In MZC+2Gy group, NO3

– was lower in

hippocampus (p<0.01) and cortex (p<0.05) compared to 2Gy group (Figure 3).

Nitrates


5

10

15 Cer Hipp Cx

*****

***

*

**

**

*

***

***

******

*****# #

#

nmol

NO

3- /mg

prot

eins

Figure 3. Nitrates in selective vulnerable brain regiones of rats exposed to zeolite. Nitrates (NO3

-: nmol NO3-/mg proteins),

the final metabolic product of RNS, were measured in VBRs: cerebellum (Cer), hippocampus (Hipp) and cortex (Cx). Valu-es are presented as means ± SD, for 10 animals/group. Diffe-rences were considered statistically significant at: *.#p<0.05, **,##p<0.01 and ***p<0.001(*-compared to control, #-compared to MZC group). One-way ANOVA, Dunnett`s C test were used for statistical analysis.

Superoxide anion radical


DISCUSSION

Zeolite reversibly binds gases, such as O2 and NO, as well as other nutriments, including metals (i.e. Fe) with-in GIT [24]. Reduced bioavailability of Fe, ROS and RNS in rats on 5% MZC supplemented diet during four-week diet regimen imposed a systemic effect, documented by the decreased brain levels of the aforementioned substances. Moreover, according to our results, MZC imposed a partial radioprotective role against brain OS/NS, induced by lower doses of ionizing irradiation (2Gy) in rats (Figures 1–3).

We confirmed positive association between reduced brain ROS and RNS and lower brain Fe concentrations in MZC treated rats. These occurrences confirmed that reduced bioavailability of Fe, O2 and NO is followed by a systemic drop of Fe, ROS and RNS.

The brain has high levels of O2, transitional metals and polyunsaturated fatty acids and therefore is partic-ularly susceptible to OS [25]. Excessive amounts of ROS and RNS cause OS in all neurons, yet, the vulnerability of neurons to OS varies from one brain region to anoth-er, as well as within the same brain region. Although the hippocampal regions CA1 and CA3 are next to each other and are composed of morphologically similar py-ramidal neurons, there are differences in their sensitiv-ity to OS. When exposed to pro-oxidative substances, the neurons in the CA1 region suffer massive cell death while those in CA3 mostly survive [26, 27]. Also, signifi-cant differences in vulnerability to OS of cerebellar and cortical neurons are documented. Exposed to some OS-inducing agents, such as paraquat, or conditions involving OS, such as ischemia and re-oxygenation, ex-tensive death of neurons in the cerebellum, but not in the cerebral cortex was observed [25].

Overproduction of ROS, including O2•-, H2O2, HO• and

others, can be triggered by different stimuli, including metabolism of endo- and exogenous compounds in the presence of O2, Fenton-like reactions, ionizing radi-ation, etc. Oxidative stress development assumes time and spatial spreading of free radical chain reactions in living organisms. In the Fenton reaction, a low-valent transition metal (such as Fe2+) reacts with H2O2 and produces HO•, which instantly oxidatively damages various classes of essential biomolecules such as lipids, proteins and nucleic acids [28, 29]. According to our results, reduced brain Fe level is associated with lower concentrations of O2

•- (Figures 1, 2).Keeping the homeostasis of NO sustained through-

out all body organ systems is immensely important, considering its regulatory role in many physiological processes. However, healthy blood vessel endothelium is defined by the ability to produce NO [30]. Sponta-neous reactions between NO radical (NO•) and O2

•- re-sults in ONOO– production. Peroxynitrite is an extreme-ly harmful molecule, blamed for atherosclerotic and other NS associated diseases [31].

In relation to this, MZC capability to reduce OS, as well NS in the body, including brain, was anticipated

(Figures 2, 3). Decreased concentrations of O2•- and

NO3– in all examined brain regions of rats on MZC sup-

plemented diet can be explained by the ability of zeo-lites to bind transition metals (including metals partici-pating in Fenton reaction, such as Fe) in the alimentary tract and reduce their availability to other organs. Very few studies on zeolite pointed at its indirect, antioxi-dant effect, confirmed by suppressed lipid peroxida-tion [5, 32].

However, ionizing radiation can alter molecules within the cells, both directly and indirectly, affecting cell viability. Radiation energy absorbed by tissues and fluids leads to radiolysis of water and biomolecules, resulting in consequent OS and NS development [28, 33, 34].

Human population can be exposed to the dose range of 1–10Gy during radiation therapy treatment or as the result of radiation accidents or nuclear/radiolog-ical terrorism [35].

According to our results, a higher irradiation dose caused large elevation of RNS in all examined brain regions of treated rats, while at lower doses elevation of RNS was noticed only in hippocampus. This is con-sistent with the results of previous studies that con-firm that hippocampus is one of the most sensitive brain regions to OS/NS. Radioprotective effect of MZC against brain NS induced by ionizing radiation was ac-complished only at lower dose of 2Gy in hippocampus and cortex, but not in cerebellum (Figure 3). This is in line with the results from a recent study indicating that lower doses of irradiation increased blood-brain barrier (BBB) permeability, decreased blood flow and content of antioxidants in the cerebellum, more than in other brain regions, causing OS [36].

Significantly enlarged brain NS was obtained after irradiation with 10Gy regardless of the previous dietary regimen, normal or MZC supplemented (Figure 3). This suggests that the antioxidant potential of MZC is not sufficient to reduce brain damage caused by high con-tent of ONOO– produced by the higher irradiation dose.

Herein, we demonstrated that the most resistant VBR structure to OS, initiated by 2Gy irradiation was hippocampus (no change of O2

•- level was noticed), while the most sensitive was cortex (the highest O2

•- concentration was observed). This can be explained by the different concentrations of Cu, zinc (Zn) and Mn within these brain regions. The highest concentrations of these metals were documented in hippocampus [37, 38]. These metals are cofactors of most important enzymatic antioxidants superoxide dismutases (SODs – CuZnSOD and MnSOD) which catalyze dismutation of O2

•- to H2O2. Therefore we can conclude that after exposure to 2Gy irradiation concentration of O2

•- in hip-pocampus is lower than in other brain regions due to higher total SOD activity in this brain section.

After radiation of 10Gy, no change in the O2•- con-

centration was observed, contrary to the increased NO3

– concentration in all tested brain regions of the ex-


posed rats. We assumed that O2•- spontaneously reacts

with NO• producing more potent ONOO–.Regarding NS, cerebellum was proven to be the

most resistant VBR region against NS, initiated by 2Gy irradiation (an intense NO3

– drop was documented), while hippocampus was the most sensitive. The NS respond of all VBRs was similar at 10Gy (Figures 2, 3). Nitric oxide in the brain is synthetized primarily by neuronal nitric oxide synthase (nNOS). However, in the conditions of exposure to various OS-inducing agents activation of inducible nitric oxide synthase (iNOS) can lead to overproduction of NO. Supraphysiological levels of NO cause apoptosis with resultant decreased regional neuronal function. The sensitivity of iNOS to radiation is probably the highest in the hippocampus compared to other brain structures [39].

CONCLUSION

Herein, we confirmed that decreased bioavailability of Fe, NO and O2 by subacute intake of MZC resulted in systemic drop of Fe, ROS and RNS, judging by brain Fe, ROS and RNS levels in the exposed rats. Zeolite achieved a partial antioxidative effect initiated by a lower dose of ionizing radiation and limited antioxida-tive effect initiated by a higher irradiation dose in the exposed rats.

Also, the obtained results suggest that there are differences in the sensitivity to radiation between the individual brain structures evaluated by OS/NS devel-opment that can be explained by variations in cerebro-vascular permeability, content of antioxidant enzymes and transition metals.

CONFLICT OF INTEREST STATEMENT

The authors disclosed any financial or personal rela-tionships that could inappropriately influence or bias the content of the paper.

ACKNOWLEDGEMENT

The authors greatly appreciated the courtesy of M. Sa-diković, Viridsfarm Ltd for donating MZC, as well as the reviewers’ criticisms and helpful suggestions. The study was supported by grants from the Ministry of Education and Science, Republic of Serbia (Project: No. III41018) and by the Ministry of Defense of the Republic of Ser-bia (Projects No.: MFVМА/6/15–17, MFVМА/04/16–18).

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Predtretman zeolitom ostvaruje delimično radioprotektivni efekat na mozak smanjujući gvožđe i oksidativni/nitrozativni stres kod pacova

Kratak sadržaj

Cilj naše studije je bio da ustanovimo efekat subakutno primenjenog mikro-nizovanog zeolita [mikronizovanog klinoptilolita (MZC)] na moždani status gvožđa (Fe) i reaktivnih vrsta kiseonika i azota (eng. reactive oxygen and ni-trogen species, ROS, RNS), kao i radioprotektivnu ulogu od moždanog ok-sidativnog/nitrozativnog stresa (OS/NS) iniciranog pojednačnim dozama jonizujućeg zračenja od 2 ili 10Gray (Gy) kod pacova. Wistar pacovi na če-tvoronedeljnom režimu normalne ishrane (n=18) i ishrane dopunjene 5% MZC (n=18) su interno podeljeni u 3 podgrupe sa po 6 pacova (n=6), a od toga po jedna grupa je bila kontrolna, a druge dve su podvrgnute pojedinačnim dozama zračenja od 2Gy i 10Gy. Tako, imali smo na normalnoj ishrani grupe: C-kontrolna, 2Gy i 10Gy; a na ishrani dopunjenoj 5% MZC: MZC, MZC+2Gy i MZC+10Gy grupe. Koncentracije nitrata (krajnji RNS metabolit) i superoksid anjon radikala (O2

•-) (inicijalni ROS) su merene u homogenatima selektivno osetljivih moždanih struktura (mali mozak, hipokampus i korteks), dok je Fe mereno u celom mozgu pacova. Rezultati pokazuju značajno smanjenje Fe u MZC i MZC+2Gy/10Gy grupama; smanjenje O2

•- i nitrata u MZC grupi; gotovo identičan pad O2

•- u 2Gy i MZC+2Gy grupama; i povećanje nitrata u 10Gy i MZC+10Gy grupama. Potvrdili smo da subakutni pretretman pacova MZC doprinosi delimičnom moždanom radioprotektivnom učinku kod pacova jed-nokratno izloženih zračenju od 2Gy ili 10Gy, verovatno usled smanjenjenog OS/NS i Fe.

Ključne reči: mozak, gvožđe, jonizujuće zračenje, nitrozativni stres, oksida-tivni stres, zeolit (MZC)

Boban Stanojević1, Mirjana Đukić2*, Ivana Stevanović3, Milica Ninković3, Ana Đurić2, Borko Gobeljić2, Milan Apostolović4, Ana Pantelić5, Goran Zebić6, Lidija Todorović1, Tijana Bojić1, Kiril Savovski1

1 Univerzitet u Beogradu – Institut za nuklearne nauke „Vinča”, Laboratorija za radiobiologiju i molekularnu genetiku 080, Beograd, Srbija2 Univerzitet u Beogradu – Farmaceutski fakultet, Katedra za toksikologiju, Beograd, Srbija 3 Univerzitet odbrane – Medicinski fakultet, Vojnomedicinska akademija, Institut za medicinska istraživanja, Beograd, Srbija4 Univerzitet u Beogradu – Medicinski fakultet, Institut za ortopedsko-hirurške bolesti „Banjica”, Beograd, Srbija5 Univerzitet u Beogradu – Naučna ustanova, Institut za hemiju, tehnologiju i metalurgiju, Centar za hemiju, Beograd, Srbija6 Univerzitet u Beogradu – Poljoprivredni fakultet, Katedra za tehnologiju hrane, Beograd-Zemun, Srbija

* Corresponding author:

Mirjana Đukić, ProfessorFaculty of Pharmacy, Belgrade

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018. 33Obradović et al., Comparison of the antioxidative action of the two N-acetylcystein containing products: Propomucil® and Fluimucil® ...

INTRODUCTIONOxidative stress reflects a state of disturbed balance between pro-oxidants and endogenous systems of an-tioxidative protection which can potentially damage the cell and its subtle organelles. Pathophysiology of different acute respiratory diseases also includes seg-ment of the oxidative status disorder, which contrib-utes to general inflammation. Also, the oxidative sta-tus disbalance occurs in smokers due to exposure to the free radicals from cigarettes, which are abundantly generated in tobacco smoke [1].

Acute respiratory infections are very common world-wide and also in our country. The frequency of these diseases is high and it makes a significant per-centage of total morbidity in the countries all over the world, especially among smokers [2]. High percentage of respiratory diseases in general population can be ex-plained by the direct contact between large area of re-spiratory system with external environment, with large number of different pollutants [3]. The most common manifestations of acute respiratory infections are cold, acute otitis, acute bronchitis, pneumonia, exacerbation of chronic obstructive pulmonary disease (COPD) and

Comparison of the antioxidative action of the two N-acetylcystein containing products: Propomucil® and Fluimucil® in a group of smokers who have seasonal coughing problems

Abstract

Oxidative stress reflects a state of disturbed balance between pro-oxidants and endogenous systems of antioxidative protection. N–acetyl cysteine (NAC) is well-known antioxidant for its reduction properties which originate from sulfhydryl group of cysteine. It is used as a mucolytic because it reduc-es viscosity of the bronchial secretion. It alleviates expectoration and hard breathing by disrupting disulfide bonds of mucopolysaccharide. Propolis is used for various purposes due to its antibacterial and anti-inflammatory ef-fects. Complex mixture of various compounds synergistically contributes its overall effect, and bioflavonoids, manifest antioxidant effects. The goal was to examine antioxidative effect of propolis by comparing the oxidative stress status of the respondents before and after ten-day supplementation of NAC or combined supplement product of NAC and propolis. The study includes 20 healthy respondents (18 smokers and 2 nonsmokers), divided into two groups. The first group consists of 10 respondents defined on the combined supplement product of NAC and propolis (200 mg NAC/80 mg propolis, 3 times a day) and the second group consists of 10 respondents defined on one-component preparation of NAC (200 mg NAC 3 times a day). The blood samples were taken before and after a ten-day supplementation of prepara-tions. The following parameters of the prooxidative effect in the serum were determined: products of advanced protein oxidation (AOPP), malondialde-hyde (MDA), total oxidative status (TOS), prooxidative-antioxidant balance (PAB). Also, the parameters of antioxidant protection were determined: total antioxidant status (TAS), total sulfhydryl groups (SHG), activity of superox-ide-dismutase enzyme (SOD) and paraoxonase 1 (PON1). After supplementa-tion, the combined preparation has significantly increased the parameters of antioxidant protection: SOD, PON and TAS for all subjects in the group. Moreover, one-component preparation of NAC has significantly influenced on the increase of SOD, SHG and on decrease of AOPP. Both preparations improve antioxidant protection and it is showed significant contribution of effect of propolis in combination with NAC.

Key words: oxidative stress, antioxidant protection, N-acetylcysteine, pro-polis

Teodora Obradović1, Biljana Radišić1, Anita Agić2, Davor Korčok2, Duško Mirković1,3, Vesna Spasojević-Kalimanovska1, Jelena Kotur-Stevuljević1

1 Department for Medical Biochemistry, Faculty of Pharmacy, Belgrade, Serbia2 Abela Pharm Belgrade3 Center for Medical Biochemistry, Clinical Center of Serbia, Belgrade, Serbia

Corresponding author: Jelena Kotur-Stevuljević

Department for Medical Biochemistry, Faculty of Pharmacy, Belgrade, Serbia, Vojvode Stepe 450, 011 3951272, Belgrade, Serbia

UDK: 615.233 COBISS.SR-ID 271836428

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018.34Obradović et al., Comparison of the antioxidative action of the two N-acetylcystein containing products: Propomucil® and Fluimucil® ...

bronchial asthma [4]. These infections are treated with standard antibiotics therapy, antipyretics and broncho-dilators, and especially with mucolytic agents, because one of the most common symptoms is cough.

N–acetylcysteine (NAC) can be found in clinical practice for few decades [5]. NAC is a cysteine derivate with linked acetyl group with nitrogen and as majority of thiols, it can be oxidized with different free radicals. It can serve as nucleophile and it is a good antioxidant because of its reduction properties [5]. NAC is a glu-tathione precursor and after its oral use, it becomes deacetylated in the intestines. Also, NAC reduces cys-tine from cysteine, which is an important mechanism of intracellular metabolism of glutathione in the lungs [6]. It is used as mucolytic because it reduces viscosity of bronchial secretion; it alleviates expectoration and heavy breathing by disrupting disulfide bonds of mu-copolysaccharide chains [7].

Propolis is a generic name for resins collected by bees from various plants and it is being used through centuries for various purposes due to its antibacterial and anti-inflammatory effects. Propolis is a complex mixture of various compounds which synergistical-ly contribute to its overall effect, and bioflavonoids, which manifest antioxidant effects [6]. Different studies have shown the efficiency of N–acetylcysteine as an-ti-inflammatory and antioxidative factor. Recently con-ducted studies in which subjects were both adults and children, showed the efficiency of preparation which beside the N–acetylcysteine also contains propolis [3,4,6,8–11].

It is well-known fact that tobacco smoke may con-tribute to the development of different chronic lung and cardiovascular diseases [1]. It causes lipid peroxi-dation, oxidation of proteins and thus damage to the lungs and other tissues [1]. One package of cigarettes (20 pieces) contains 1015 of oxidative radicals [1]. This current study was conducted in a group of healthy subjects who are smokers, in order to test their redox status before and after supplementation with two dif-ferent antioxidative preparations. Cough caused by smoke, followed by increased production of bronchial secretion are common symptoms that are seen with regular smokers [12]. In relation to that, the study in-cluded smokers who complained about productive cough during the winter months. The main goal of this study was to compare antioxidative effect of propo-lis combined with NAC (PropoMucil®) and mono-NAC preparation (Fluimucil®).

MATERIAL AND METHODS

The study included 20 healthy subjects (18 smokers and 2 nonsmokers), divided into two groups. The first group consisted of 10 subjects who used the com-bined preparation of NAC and propolis (PropoMucil®, AbelaPharm, Serbia) and the second group consisted of 10 subjects who used one-component preparation of NAC (Fluimucil®, Zambon, Switzerland).

The both study groups consisted of 5 younger sub-jects (22.8 ± 1.63 years) and 5 older subjects (39.6 ± 10.0 years). Age differences were separately analyzed regarding the preparations that the subjects used.

Blood was drawn after an overnight fast in vacutain-ers with serum separator gel (Becton, Dickinson and Company, Franklin Lakes, New Jersey) in two time points: firstly, at the beginning of the study and then after a ten-day supplementation with a suitable preparation with an active NAC component in a predefined dosage form and dosage regimen, as follows:

1. PropoMucil® group: 200 mg NAC/80 mg propolis, 3 times a day in the form of granules for oral solution and

2. Fluimucil® group: 200 mg NAC, 3 times a day in the form of granules for oral solution.

Comparison of redox status parameters before and after supplementation, was done by using paired T test. Comparison of parameters’ change was performed de-pending on the type of the products, as well as in two age groups by Student t test. Determination of redox status involved the analysis of several selected repre-sentative parameters, precisely prooxidants or prod-ucts of their action on biomolecules: malondialdehyde (MDA), advanced oxidation protein products (AOPP), total oxidative status (TOS) and prooxidative-antioxi-dant balance (PAB). Also, the following parameters of antioxidant protection were determined: total antioxi-dant status (TAS), total sulfhydryl groups (SHG), activity of enzymes: superoxide-dismutase (SOD) and paraoxo-nase 1 (PON1).

Principle of method for the determination of AOPP

After the addition of glacial acetic acid (40 μL) to a di-luted sample with phosphate buffer (pH 7.4) and a po-tassium iodide solution of 1.16 M (1: 5), the absorbance was measured at 340 nm, on the Ilab 300+ Instrumen-tation Laboratory, (Milan, Italy). The concentration of AOPP is expressed through chloramine T equivalents which are used to produce a standard curve at con-centrations of 10–100 μmol/L, whereby its absorption linearly increases with increasing concentration.

Principle of the method for determining TOS

The main components of the TOS system in the serum are H2O2 and lipid hydroperoxides. The oxidants pre-sented in the sample oxidize the ferro-ortho-dianiside complex in the ferric ion. The oxidation reaction is facil-itated by the glycerol molecule present in the reaction medium. The resulting ferric ion then builds a colored complex with xylenol-orange in an acidic environment. The intensity of the color is measured by spectropho-tometric method, at the Ilab 300+ Instrumentation Lab-oratory, (Milan, Italy), and it is proportional to the total content of the oxidation molecules in the sample. As standard, an aqueous hydrogen peroxide solution of


a concentration range of 10–200 μmol/L was used, which corresponds to the linearity of the method and the expected concentrations in the biological material.

Principle of the method for the MDA determination

The concentration of MDA is determined as a thiobar-bituric acid-reactive substance (TBARS) by a spectro-photometric assay based on the absorption maximum of the malondialdehyde complex and other TBARS with thiobarbituric acid. Absorbance was measured on an ELISA reader (LBK, Wien, Austria).

Determination of PAB

The PAB test determines the concentration of H2O2 in the antioxidant environment. Chromogen 3,3’,5,5’-te-tramethylbenzidine (TMB) reacts with H2O2 and with antioxidants (uric acid) at the same time, since they are in the same environment. The reaction of H2O2 and chromogen is enzymatically catalyzed by peroxidase, whereby the oxidation of TMB gives intensely blue-col-ored product. In contrast to that, a reaction between uric acid and chromogen is a non-catalyzed chemical reaction in which TMB cation is reduced to a colorless product. These two components have been selected for prooxidant and antioxidant agents because they do not react with each other and do not interfere with each other’s activity against chromogen. Based on the absorbances which are obtained in the standard solu-tions, the calibration curve is formed and used for the results calculation.

Determination of PON1

The determination of PON1 activity is based on the operation of PON1 enzymes from the sample to the substrate of paraoxone, whereby the conversion of the paraoxone to p-nitrophenol occurs, and the rate of change is monitored kinetically. The enzyme activity is determined at 25°C and at pH 8.5 using 50 mmol/L TRIS-HCl buffer in the presence of NaCl and it is ex-pressed as μmol obtained from p-nitrophenol/min/L or as IU/L. In order to determine PON1 activities, the Ilab 300+ Instrumentation Laboratory, (Milan, Italy) was used.

Determination of SOD

The method is based on the ability of SOD to inhibit the spontaneous autooxidation of adrenaline in the alkali environment (pH 10.2) because the adrenaline is quite stable in the acidic solution. Autooxidation of adren-aline was initiated by traces of heavy metals, present as impurities in reagents. The activity of this enzyme is expressed in relative units obtained by measuring the absorbance of the resulting red oxidation product of adrenaline on the Ilab 300+ Instrumentation Laboratory analyzer, (Milan, Italy). One unit of SOD activity was de-

fined as the amount of protein causing 50% inhibition of the autooxidation of adrenaline to adrenochrome.

Determination of SHG

The total content of SHG is determined by the Ellmann method, which is based on the reaction of 2,2’-dini-tro-5,5’-dithio-benzoic acid with aliphatic thiol com-pounds in the alkali environment (pH 9.0) thereby creating 1 mol of p-nitrophenol anion per mol of thiol. Since this anion is yellow, its absorbance is measured at 412 nm at the Ilab 300+ Instrumentation Laboratory, (Milan, Italy).

Determination of TAS

The total antioxidant status was determined by a col-orimetric test using stable 2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulphonic acid) (ABTS) cation as a chro-mogen. The ABTS solution itself is colorless. Oxidation to ABTS+ cations, using H2O2 in an acidic medium (pH 3.6), the solution gives a characteristic emerald color. When colored ABTS+ is mixed with an antioxidant, it is reduced to a colorless ABTS, which is manifested by color change. The color intensity decrease is propor-tional to the total concentration of the all antioxidants existing in the sample. The reaction was performed at the Ilab 300+ Instrumentation Laboratory, (Milan, Italy).

RESULTS

Table 1 shows concentrations of prooxidants and prod-ucts of their action before and after supplementation, in subjects who used the combined preparation Pro-poMucil®. Also, the concentrations/activities of antiox-idant parameters are shown in the same Table 1. After ten days of supplementation, the trend of reducing all parameters from the group of prooxidants is noticed, but this difference was not statistically significant. The data in all tables are shown as mean values and stan-dard deviations.

Table 1. Prooxidants, products of their activity and anti-oxidants before and after a ten-day supplementation with the combined preparation of NAC and propolis (PropoMucil®).

Parameter BaselineAfter the

supplementa-tion

P

MDA (μmol/L) 1.9±0.6 1.8±0.4 0.919

AOPP (μmol/L) 21.8±10.1 17.8±3.9 0.333

PAB (U/L) 134±34 130±27 0.508

TOS (μmol/L) 19.3±14.0 18.7±11.9 0.878

TAS (μmol/L) 1387±354 1546±531 0.047

SHG (mmol/L) 0.365±0.076 0.388±0.091 0.484

SOD (U/L) 118±12 137±8 0.005

PON1 (U/L) 321±279 376±331 0.012


We also noticed significant increase in the antioxi-dant protection parameters (SOD, PON1 and TAS). The obtained results are presented graphically (Figure 1), where the effect of the preparations can be noticed in each subjects individually.

The concentrations of prooxidants, the products of their activity and antioxidative parameters of the sub-jects who used the one-component preparation – Flui-mucil® before and after a ten-day supplementation, are shown at the Table 2.

Table 2. Concentrations of prooxidants, products of their action before and after a ten-day supplementation of the subjects who were defined on a one-component preparation.

Parameter BaselineAfter the

supplemen-tation

P

MDA (μmol/L) 1.60±0.60 1.50±0.50 0.594

AOPP (μmol/L) 29.4±9.4 14.7±2.8 0.047

PAB (U/L) 128±16 131±25 0.515

TOS (μmol/L) 19.5±12.2 17.1±9.5 0.507

TAS (µmol/L) 1351±345 1411±431 0.721

SHG (mol/L) 0.290±0.080 0.362±0.121 0.028

SOD (U/L) 117±17 138±8 0.005

PON1 (U/L) 325±209 340±205 0.139

Figure 1. Change in the oxidative status parameters of the subjects individually, after the combined preparation usage (PropoMucil®).

Marks: 1 – before, 2 – after, *-P<0.05 after the supplementation


Fluimucil® preparation caused significant increase of SOD enzymatic activity and also total SHG both of which are a part of general antioxidant protection. Flu-imucil® influenced significantly the decrease of AOPP. The obtained results are also graphically presented (Chart 2), where the change of parameters is presented in each subjects individually.

Comparison of the oxidative status parameters between the combined and one-component preparation, PropoMucil® vs. Fluimucil® supplementation

After examining the results for each individual prepara-tion, a statistical analysis was done by using Student’s

t test in order to estimate the differences between these two supplements. In addition to the basic pa-rameters whose concentration was directly measured, the differences in the concentration of the parameters (before – after) were calculated and these values were marked with the addition of the lower case d (differ-ence) in front of the parameter of interest.

Although the noticed differences were of the mar-ginal statistical significance, it should not be neglected even this small shift in the PON1 activity before and after supplementation (dPON, Table 3). Difference in PON1 was in favor of the combined preparation, i.e. after the Propomucil® supplementation, values were significantly higher than after the Fluimucil® supple-mentation. Similarly, as a consequence of supplemen-

Figure 2. Change in the parameters of the oxidative status of the subjects individually before and after the one-component preparation usage (Fluimucil®).

Marks: 1 – before, 2 – after, *,**,***-P<0.05, 0.01, 0.001 after the supplementation


tation, there is also a reduction in AOPP (AOPP2: Table 3). Both preparations decreased the concentration of AOPP, but there is a greater reduction in the one-com-ponent preparation – Fluimucil®. Also, the SHG concen-trations before supplementation differed slightly, but this is probably not a consequence of supplementa-tion, but because of an initial difference in the values of the subjects (SHG1, Table 3). The calculated values of the difference in the concentration/activity of parame-

ters before and after supplementation (d) in the parameter of interest, did not show a statistically significant difference (Table 3), which means that generally speaking, the both preparations equal-ly exhibit an antioxidant effect.

Comparison of antioxidative index between older and younger subjects before and after supplementation

As respondents differ by their age, the results are analyzed between the two age groups. Any vari-ation of concentration of TAS or TOS directly re-flects the antioxidant index, which is the ratio of these two parameters. Chart 3 shows antioxidant indexes with all subjects’ sub-groups (both elder-ly and younger, depending on the preparation they used). Within younger respondents group, TAS values were significantly lower than within the older respondents, also TOS values are lower, so this compensated for weaker antioxidant pro-tection. The antioxidative index was statistically higher within younger subjects group, after sup-plementation with both preparations. The Pro-pomucil® shows a higher trend in the antioxidant index increase, compared to the Fluimucil®, and we could suppose that propolis is responsible for this fortification of supplement’s antioxidative potential.

It is noted that NAC increases the activity of SOD enzymes in both age groups. Since the initial activ-ities in the younger ones were higher in relation to the elderly (SOD1; P = 0.007, Table 4), the activity after supplementation was also higher in younger subjects (SOD2; P = 0.009, Table 4). Differences in SOD activity before and after supplementation are not statistical-ly significant in these two groups (dSOD; P = 0.123,

Table 3. Comparison of oxidative status parameters before and after ten days’ supplementation with different preparations

Parameter (1-before, 2-after)

PropoMucil® Fluimucil® P

SOD1(U/L) 118±12 117±17 0.684

SOD2(U/L) 137±8 138±8 0.631

dSOD 19±9 21±6 0.684

PON1(U/L) 321±279 325±209 0.579

PON2(U/L) 376±331 340±205 0.789

dPON 55±58 16±29 0.089*

TAS1(mol/L) 1387±354 1351±345 0.796

TAS2(mol/L) 1546±531 1411±431 0.393

dTAS 159.3±200.5 60.1±161.2 0.315

SHG1(mol/L) 0.365±0.076 0.290±0.080 0.063*

SHG2(mol/L) 0.388±0.091 0.362±0.121 0.436

dSHG 0.023±0.071 0.072±0.090 0.165

AOPP1(mol/L) 21.8±10.1 29.4±9.4 0.353

AOPP2(mol/L) 17.8±3.9 14.7±2.8 0.075*

dAOPP -4.1±11.1 -4.7±9.4 0.796

* – P values indicate marginal statistical significance (0.050 < P <0.100)

Figure 3. Antioxidant index (TAS/TOS ratio) among older and younger subjects on two different kinds of therapies

P- PropoMucil®, F- Fluimucil®aa-P<0.01 vs. P group-older subjects, bb-P<0.01 vs. F group – younger subjects


Table 4). This means that the effect of NAC in both age groups is the same on the antioxidant SOD enzyme. The initial MDA values were approximately the same in both groups, however, after the NAC supplementation, a statistically significant decrease in the concentration of MDA (MDA2; P = 0.029, Table 4) was observed only in the younger group. In the overall redox balance parameter in the elderly, an unexpected increase was observed after supplementation (PAB2; P = 0.011, Ta-ble 4), while in the younger ones, these values were reduced but not significantly. The parameter showing the difference in concentration before and after, statis-tically and significantly varies (dPAB, P = 0.019, Table 4), which is reasonable with the increase in the older ones and decrease in the younger ones.

Table 4: Comparison of parameters of oxidative stress betwe-en older and younger subjects, before and after NAC supple-mentation

Parameter (1–before. 2–after)

PropoMucil® Fluimucil® P

SOD1(U/L) 109±10 126±14 0.007

SOD2(U/L) 134±7 141±7 0.009

dSOD 22.9±10.9 15.8±8.7 0.123

PON1(U/L) 343±246 303±245 0.853

PON2(U/L) 377±260 340±290 0.796

dPON 33.6±43.8 37.1±56.2 0.971

SHG1(mmol/L) 0.308±0.089 0.346±0.082 0.315

SHG2(mmol/L) 0.323±0.073 0.426±0.110 0.029

dSHG 0.015±0.068 0.080±0.082 0.105

TAS1(mmol/L) 1692±108 1046±52 <0.001

TAS2 (mmol/L) 1914±224 1043±78 <0.001

dTAS 222±199 -3.1±56.84 0.011

MDA1(umol/L) 1.88±0.45 1.60±0.73 0.143

MDA2 (umol/L) 1.87±1.88 1.42±0.59 0.029

dMDA -0.007±0.463 -0.18±0.87 1.000

PAB1(U/L) 129±29 133±24 0.912

PAB2 (U/L) 138±28 124±22 0.123

dPAB 8.95±8.87 -9.4±16.1 0.019

TOS1(mmol/L) 30.4±8.8 8.43±0.71 <0.001

TOS2 (mmol/L) 27.6±4.9 8.20±0.75 <0.001

dTOS -2.73±10.95 -0.23±1.09 0.912

AOPP1(mmol/L) 21.5±13.1 19.7±4.4 0.631

AOPP2 (mmol/L)

14.5±3.9 18.0±2.6 0.029

dAOPP -7.06±13.73 -1.7±2.7 0.971

The initial values of SHG in both groups were ap-proximately the same (SHG1; P = 0.143, Table 4). How-ever, after the NAC supplementation, a statistically sig-nificant increase in SHG (SHG2; P = 0.029, Table 4) was

observed only in the younger group of respondents. This result can be explained by the fact that the val-ues of SHG before taking supplements by the subjects defined on the one-component preparation, were significantly lower than by the subjects who used the combined preparation. The initial and final values of to-tal antioxidant status with the elderly were statistically higher in comparison to the younger ones (Table 4). In the elderly, a significant increase in TAS concentra-tion was observed compared to the younger ones in which there was no increase (TAS1; P <0.001, TAS2; P = 0.001, Table 4). The concentration of TOS is statisti-cally different before and after supplements between these two groups; the younger group has a significant-ly lower value (TOS1; P <0.001, TOS2; P <0.001, Table 4). Differences in TOS concentrations before and after supplementation in both groups were not statistically significant (dTOS; P = 0.912, Table 4). The AOPP values differ, at the end of the supplementation, the younger are more concentrated compared to the older group (AOPP2, P = 0.029, Table 4).

DISCUSSION

After a ten-day supplementation, the results showed that both preparations have a significant antioxidant effect. In order to estimate the effect of propolis in the combined preparation, these two preparations were compared. The PropoMucil® preparation did not cause a significant decrease in prooxidants, but there is obvi-ous trend in its reduction. This can be explained by the fact that, in total, respondents are still young people who, although smokers, did not have the high initial values of prooxidants, so there was not much “space” for reduction. Similar thing can be found with athletes, who are constantly challenged by increased physical activity from generating free radicals. Athletes devel-op physiological adaptation mechanisms of antioxi-dant protection that keep free radicals at a low level [13]. For now, young and healthy smokers well tolerate permanent oxidative stress caused by tobacco smoke, but it’s just a matter when the protective mechanisms became ineffective.

The main component of the both supplements is NAC, and the protective ability of NAC against oxida-tive damage can be explained by its ability to maintain intracellular reduced glutathione concentration and the ability to remove free radicals by various mecha-nisms [14]. This effect of NAC is confirmed also in this study by healthy smokers, of two different age-groups.

Various studies have shown the effectiveness of PropoMucil® as a combined preparation, which is often used in the treatment of acute respiratory infections due to its mucolytic, anti-inflammatory, antibacterial and antioxidant effect [3, 4, 6, 11]. These studies have shown that the product is suitable and safe for children and adults with acute respiratory infections and for pa-tients who are suffering from COPD. Propolis contrib-utes to the anti-inflammatory effect, precisely because


it represents a complex mixture of various compounds [15]. The anti-inflammatory effect is reflected by the presence of flavonoids, especially galangin [16]. It has been shown that flavonoid inhibits the activity of cy-clooxygenase (COX) and lipoxygenase enzymes, reduc-es the release of prostaglandin E2 and the expression of inducible isoforms of COX2 [16]. Propolis also con-tains phenylethyl ester of caffeic acid that inhibits the release of arachidonic acid from the cell membrane, suppressing the COX1 and COX2 enzymes and the COX2 gene expression [16].

One study covered only patients with chronic ob-structive disease, where it was proven that NAC com-pared to placebo has an extraordinary effect in reduc-ing oxidative stress [17]. Three studies which included healthy subjects who were smokers, have shown that supplementation of 600 mg NAC per day, significant-ly reduced prooxidants and increased antioxidant pa-rameters concentration [18 – 20]. Our current study showed similar results, which speaks in favor of the strong antioxidative effect of NAC.

CONCLUSION

After the supplementation, PropoMucil® significantly increased the parameters of antioxidant protection of SOD, PON1 and TAS. Fluimucil significantly increased SOD and SHG, from the parameters of the prooxidative status, and significantly decreased AOPP. Both prepa-rations have an effect on older and younger subjects, but younger subjects have better improvement in their overall redox status. However, in order to estimate real propolis’ influence at the antioxidant capacity in bio-logical systems, especially in the combination with NAC, it is necessary to increase the number of subjects, including healthy ones, but also patients with respira-tory diseases, which could obtain the most important benefit from this supplementation.

Acknowledgment Financial support provided by Ministry of Education, Science and Technological development Republic of Serbia (OI 175035) is greatly appreciated.

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POREĐENJE ANTIOKSIDATIVNOG DELOVANJA DVA PROIZVODA KOJI SADRŽE N-ACETILCISTEIN: PROPOMUCILA® I FLUIMUCILA® U GRUPI PUŠAČA SA SEZONSKIM KAŠLJEM

Kratak sadržaj

Oksidativni stres predstavlja stanje narušene ravnoteže između prooksidanasa i endogenih sistema zaštite. N–acetilcistein (NAC) je dobro poznat antioksidans zbog svojih redukcionih osobina, koje potiču od sulfhidrilnih grupa cisteina. Mukolitičko dejstvo ostvaruje raskidajući disulfidne mostove mukopolisaharida. Olakšava iskašl-javanje, ublažava kašalj i time popravlja kvalitet disanja, sman-juje viskoznost bronhijalnog sekreta. Propolis je smeša smola koje sakupljaju pčele sa biljaka, koristi se za različite svrhe zbog svog antibakterijskog i antiinflamatornog efekta. Kompleksna smeša različitih jedinjenja iz propolisa sinergistički doprinosi njegovom ukupnom efektu, a bioflavonoidi koji ulaze u sastav propolisa, is-poljavaju antioksidativni efekat. Cilj ovog rada je ispitati antioksi-dativni efekat propolisa poređenjem oksidativno-stresnog statusa ispitanika pre i posle suplementacije NAC-om ili kombinovanim preparatom NAC-a i propolisa. Studija obuhvata 20 zdravih ispi-tanika (18 pušača i 2 nepušača), podeljenih na dve grupe. Prvu grupu čini 10 ispitanika kojima je dodeljen kombinovani preparat NAC-a i propolisa (200 mg NAC/80mg propolis, 3 puta dnevno), a druga grupa obuhvata 10 ispitanika sa dodeljenim jednokompo-nentnim preparatom NAC-a (200 mg NAC 3 puta dnevno). Krv je uzorkovana pre i nakon desetodnevne suplementacije preparatima. Određeni su sledeći parametri prooksidanasa i produkti njihovog delovanja u serumu: produkti uznapredovale oksidacije proteina – AOPP (engl. advanced oxidation protein products), malondialdehid (MDA), totalni oksidativni status (TOS), prooksidativno – antioksi-dativni balans (PAB), kao i parametri antioksidativne zaštite: total-ni antioksidativni status (TAS), ukupne sulfhidrilne grupe (SHG), aktivnost enzima superoksiddizmutaze (SOD) i paraoksonaze-1 (PON1). Nakon suplementacije, kombinovani preparat je statistički značajno povećao parametre antioksidativne zaštite SOD, PON i TAS kod svih ispitanika, dok je jednokomponentni značajno uticao na povećanje SOD, SHG i smanjenje AOPP, takođe kod svih ispitan-ika u grupi. Oba preparata poboljšavaju antioksidativnu zaštitu i dokazan je značajan doprinos efekta propolisa u kombinaciji sa NAC-om.

Ključne reči: oksidativni stres, antioksidativna zaštita, N-acetil-cistein; propolis

Teodora Obradović1, Biljana Radišić1, Anita Agić2, Davor Korčok2, Duško Mirković1,3, Vesna Spasojević-Kalimanovska1, Jelena Kotur-Stevuljević1

1 Katedra za medicinsku biohemiju, Farmaceutski fakultet, Univerzitet u Beogradu, Vojvode Stepe 450, Beograd2 Abela Pharm Beograd, Viline vode bb, Palilula, Beograd3 Centar za medicinsku biohemiju, Klinički centar, Beograd, Srbija.

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018.4224. Susreti nutricionista – Javnozdravstveni značaj kontrole ostataka mikotoksina u hrani

U skladu sa tradidicijom od nekoliko decenija, Društvo za ishranu Srbije, svake godine organizuje jednodnevni naučno-stručni skup, a svake četvrte godine kongres o ishrani. Ove, 2018. godine, tema jednodnevnog struč-nog skupa bila je Javnozdravstveni značaj kontrole ostataka mikotoksina u hrani. Kao i prethodne godi-ne, stručni skup je organizoavan, u saradnji sa Sekcijom za higijenu Srpskog lekarskog društva, i održan je 19. juna u Velikom amfiteatru Poljoprivrednog fakulteta u Beogradu/Zemunu.

U skladu sa uspostavljenom dobrom praksom, program stručnog skupa kao i kvalifikovanost pre-davača prošli su proceduru akreditacije od strane Zdravstvenog saveta Ministarstva zdravlja. Zdrav-stveni savet je, na osnovu priložene dokumentaci-je, stručni skup vrednovao kao nacionalni kurs prve kategorije za biohemičare, lekare, farmaceute, me-dicinske sestre i zdravstvene tehničare i dijetetiča-re/nutricioniste zdravstvenog smera.

Izbor teme ovogodišnjeg stručnog skupa nametnu-la je njena aktuelnost nastala kao posledica promene klimatskih uslova u Republici Srbiji. Do pre desetak go-dina stručne procene pokazivale su da Srbija ne spada u klimatska područja pogodna za razvoj gljivica koje produkuju mikotoksine, ali se sa promenom klime, posebno posle 2012. godine, situacija izmenila, što je potvrđeno od strane evropskih i domaćih laboratori-ja. Procena rizika po zdravlje stanovništva je izazovan problem koji zahteva da se, pored implementacije sa-vremenih metoda detekcije i kvantifikacije utvrđenih količina ostataka mikotoksina u hrani i stočnoj hrani, uključe i drugi relevantni podaci vezani za utvrđivanje izloženosti stanovništva, posebno određenih populaci-onih grupa, kao što su deca, osobe sa oštećenim imu-nološkim sistemom i osobe koje imaju utvrđene fakto-re rizika za ispoljavanje štetnih efekata mikotoksina kao što su Hepatitis B i Hepatitis C.

Mikotoksini su sekundarni metaboliti, koje produ-kuju brojne gljivice a najčešće one iz roda Aspergillus, Penicillium i Fusarium. U lanac ishrane mikotoksini ula-ze najčešće preko stočne hrane, žitarica i voća a mogu se naći u mleku i brojnim prerađevinama od žita, voća, pa i u dečijoj hrani. Do sada je identifikovano više sto-tina, a štetan uticaj na zdravlje ljudi proučen je kod ne-koliko desetina mikotoksina. Poseban javnozdravstveni značaj imaju aflatoksini (B1, B2, G1, G2) koje luče glji-vice Aspergillus flavus i Aspergillus parasiticus. U hrani se najčešće može naći aflatoksin B1 koji ima kancerogena svojstva. Međunarodna agencija za proučavanje raka

(IARC) je, zbog potvrđenih kancerogenih svojstava na humanoj populaciji, pomenute aflatoksine svrstala u grupu 1, a metabolit aflatoksina B1, M1, u grupu 2B, odnosno u grupu potencijalnih kancerogena. Ovi mi-kotoksini imaju i mutagena, nefrotoksična i imunosu-presivna svojstva.

Dugotrajni unos fumonizina, koje produkuju gljivi-ce iz roda Fusarium verticillioides, Fusarium proliferatum i Asperillus niger, može predstavljati doprinoseći činilac za razvoj karcinoma jednjaka i defekta neuralne tube humanog fetusa. Gljivice iz roda fuzarijum mogu pro-dukovati deoksinivalenol i druge mikotoksine iz gru-pe trihotecena za koje je utvrđeno da remete gastro-intestinalnu funkciju, aktivnost hormona rasta, sintezu proteina, pojedinih enzima, i ekspresiju gena odgovor-nih za proinflamatorne aktivnosti i da ubrzavaju smrt leukocita.

Ohratoksin A produkuju gljivice iz roda Penicillium verucosum, Aspergillus niger i Penicillium ohraceus u ra-zličitim temeperaturnim uslovima. Utvrđeno je da ovaj mikotoksin kod nekoliko životinjskih vrsta izaziva ra-zvoj kancera bubrega, ali, nema dovoljno naučnih do-kaza da je kancerogen i za čoveka te ga IARC svrstava u grupu 2B, potencijalnih kancerogena.

Poslednjih nekoliko decenija ne samo da se mnogo radi na istraživanjima o štetnom uticaju mikotoksina na zdravlje ljudi i životinja , već se paralelno radi na inovi-ranju zakonske regulative, jačanju mreže referentnih la-boratorija, sistema monitoriga hrane i stočne hrane, si-stema za brzo izveštavanje. Stručnu pomoć nadležnim regulatornim telima Evropske unije i zemljama kandi-datima, naročito na naučnoj proceni rizika, obezbeđuje Evropska agencija za bezbednost hrane (EFSA) koja je u poslednjih desetak godina izradila brojna naučna mi-šljenja o štetnim svojstvima pojedinih mikotoksina koja su namenjena stručnjacima u oblasti javnog zdravlja, agronomije, veterine i prehrambene tehnologije i dr.

Cilj stručnog skupa. – Uz uvažavanje složenosti problematike i zahteva multisektorskog pristupa za procenu rizika usled unosa mikotoksina, cilj ovog skupa bio je da se učesnicima prikažu: a) Najnovije naučne preporuke međunarodnih stručnih agencija za procenu rizika (IARC, Svetska zdravstvena organizacija, Komisija Kodeks Alimentarius, EFSA). b) Domaća i evropska regulativa i način njihove imple-mentacije. c) Aktuelni modeli umrežavanja stručnih institucija koje učestvuju u monitoringu mikotoksina u hrani i stočnoj

Informacija o održanom stručnom skupu

24. SUSRETI NUTRICIONISTA

Javnozdravstveni značaj kontrole ostataka mikotoksina u hrani

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018. 4324. Susreti nutricionista – Javnozdravstveni značaj kontrole ostataka mikotoksina u hrani

hrani i njihovom učešću u razmeni podataka sa naci-onalnim i međunarodnim autoritetima za upravljanje rizikom na globalnom i lokalnom nivou.d) Rezultati kontrole ostataka mikotoksina tokom pri-marne proizvodnje hrane za ljude i životinje, kao i pre-duzimanja mera za smanjenje produkcije mikotoksina, da se redukuje njihova količina, ukoliko su prisutni, ali i kontrola finalnih proizvoda, posebno dečije hrane koja se stavlja u promet na domaćem tržištu.

Predavači na 24. susretima nutricionista bili su stručnjaci različitog obrazovnog profila sa Medicinskog fakulteta u Novom Sadu, Naučnog instituta za veteri-narstvo u Novom Sadu, Tehnološkog fakulteta u No-vom Sadu, Farmaceutskog, Poljoprivrednog i Fakulteta veterinarske medicine iz Beograda, kao i naučni rad-nici Instituta za higijenu i tehnologiju mesa Beograd, Instituta za kukuruz „Zemun polje“ i Gradskog zavoda za javno zdravlje Beograd. Predavačima su se u 2 radi-onice pridružili stručnjaci iz Instituta za javno zdravlje Srbije. Glavni cilj susreta bio je da se učesnicima prikaže problematika kontrole ostataka mikotoksina u hrani uz uvažavanje međunarodno priznatih i u praksi prime-njenih modela analize rizika.

U organizaciji, pripremi predavanja i radionica bio je angažovan veliki broj članova Izvršnog saveta, Nad-zornog odbora kao i redovnih članova Društva za ishra-nu Srbije.

Tokom stručnog skupa priređena je prezentacija dve naučne monografije koje su se na različit način ba-vile problemom mikotoksina:

1. Mikotoksini u lancu hrane: hemijski, biološki i zdravstveni aspekt (autor: Dragan Milićević, Institut za higijenu i tehnologiju mesa).2. Ohratoksin A u hrani za životinje, štetni efekti, detekcija i mogućnost zaštite (autori: Jelena Nedeljković Trailović, Veterinarski fakultet Beograd i Srđan Stefanović, Institut za higijenu i tehnologiju mesa, Beograd).

Obe publikacije su, od strane Ministarstva za pro-svetu, nauku i tehnološki razvoj, svrstane u grupu na-učnih monografija od posebnog nacionalnog značaja sa oznakom M41.

Učesnici stručnog skupa su pokazali veliku zaintere-sovanost za ove dve publikacije.

Slušaoci su stručni skup ocenili sa prosečnom oce-nom 4,53 (mogući raspon ocena iznosio je 1 do 5).

Program stručnog skupa dat je u prilogu.

U sufinansiranju troškova organizacije stručnog skupa učestvovalo je Ministarstvo prosvete, nauke i tehnološkog razvoja Republike Srbije.

Izveštaj priredila, Prof. dr Ljiljana Trajković Pavlović,

organizator stručnog skupa

Prilog

PROGRAM

JAVNOZDRAVSTVENI ZNAČAJ KONTROLE MIKOTOKSINA U HRANI

9.00–9.30 Prijava učesnika, Ulazno-izlazni testRadno predsedništvo: Torović, Lj., Stanković, I., Nikšić, M.

9.30 –10.00 Mikotoksini u hrani: Osnovne fizičkohemijske osobenosti i potencijalni biološki efekti Torović, Lj., Popović, M., Medicinski fakultet Novi Sad / Institut za javno zdravlje Vojvodine

10.00–10.30 Međunarodni vodiči za procenu rizika u oblasti bezbednosti hraneStanković, I., Farmaceutski fakultet Beograd

10.30–11.00 Mikotoksini: Analiza rizika – principi i proce-dure Milićević, D., Institut za tehnologiju mesa Beograd

11.00–11.30 Mikotoksini: Procena rizika – nove tehnike detekcije i karakterizacijeNikšić, M., Udovički, B., Rajković, A., Poljoprivredni fakultet Beograd

11.30–12.00 Uporedni prikaz zakonske regulative u Srbiji i EU koja uređuje kontrolu mikotoksina u hrani i hrani za životinjeJakšić S., Naučni Institut za veterinarstvo Novi Sad;Kocić-Tanackov S., Tehnološki fakultet Univerzitet u Novom Sadu

12.00–13.30 Radionica. Primena procedura analize rizika na globalnom i lokalnom nivou u kontroli mikotoksinaModeratori: Stanković, I., Torović, Lj., Milićević, D.

13.30–14.00 Pauza

14.00–14.30 Zdravstveni aspekti kontrole mikotoksina u dečijoj hraniVujović, G., Pantić-Palibrk, V., Ristić, M., Đukić, M., Gradski zavod za javno zdravlje, Beograd

14.30–15.00 Mere prevencije za razvoj mikotoksina u zr-nastoj hraniStanković, S., Institut za kukuruz Zemun Polje; Šobajić, S., Far-maceutski fakultet Beograd

15.00–15.30 Mere za smanjenje koncentracije mikotoksi-na u sirovinama za proizvodnju hrane Nedeljković-Trailović, J., Fakultet vet. med. Beograd

15.30–16.30 Radionica. Metode za procenu eksponirano-sti stanovništva mikotoksinima Moderatori: Šobajić, S., Jović, D., Palibrk, V., Ristić, I.

16.30 Završni test, evaluaciona anketa, dodela sertifikata

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018.44Prikazi knjiga

Udžbenik Instrumentalne metode, autora dr Mirjane Medenice i dr Nataše Pejić, redovnih profesora Farma-ceutskog fakulteta Univerziteta u Beogradu izašao je iz štampe početkom ove, 2018. godine. Značaj instru-mentalnih metoda za farmaciju, ali i za druge oblasti nauke, neosporno je veliki.

Udžbenik je nastao kao rezultat dugogodišnje profesionalne saradnje dva nastavnika predmeta In-strumentalne metode na Katedri za fizičku hemiju i in-strumentalne metode Farmaceutskog fakulteta u Beo-gradu. Kako su autori u predgovoru napisali, udžbenik ujedno predstavlja i tačku na slovo u dugoj, sadržajnoj i uspešnoj karijeri profesorke Medenice.

Udžbenik je prvenstveno namenjen studentima Farmaceutskog fakulteta u Beogradu za predmet In-strumentalne metode, studijski program Farmacija. Me-đutim, značaj instrumentalnih metoda je veliki i neos-poran za sve stručne predmete na studijama farmacije (npr. bromatologija, analitika lekova, farmaceutska he-mija, farmakognozija, toksikološka hemija, medicinska biohemija, farmokokinetika, farmaceutska tehnologija sa kozmetologijom, itd.), pa će ovaj udžbenik biti od koristi i studentima na višim godinama ovih studija. Osim toga, biće i deo nastavne literature studentima na poslediplomskim studijama (doktorskim i specijali-stičkim) koje se izučavaju na Farmaceutskom fakultetu u Beogradu, uključujući doktorske akademske studije iz Bromatologije, kao i specijalističke zdravstvene stu-dije iz Sanitarne hemije. Udžbenik će moći da koriste i studenti drugih fakulteta na kojima se izučavaju in-strumentalne metode, kao i svi oni koji koriste instru-mentalne metode u svom naučnoistraživačkom ili stručnom radu.

Udžbenik je napisan na 816 strana u punom koloru i sadrži 480 ilustracija i fotografija, 77 tabela, 137 lite-raturnih navoda, kao i obiman indeks pojmova. Pode-ljen je u četiri velike tematske celine: spektrometrijske, optičke, elektrohemijske i separacione metode. „Spek-trometrijske metode” obuhvataju sve instrumentalne metode molekulske i atomske, kako apsorpcione tako i

emisione spektrometrije. U delu „Optičke metode” opi-sane su turbidimetrijske, refraktometrijske i polarime-trijske metode. „Elektrohemijske metode” obuhvataju poglavlja o konduktometriji, kulometriji i voltametriji sa opisanim svim tehnikama. Poglavlje „Separacione metode” obuhvata savremene hromatografske i elek-troforetske instrumentalne tehnike. Svako poglavlje sadrži i određen broj pitanja i zadataka sa odgovorima u Prilogu, kao i odgovarajuće primere iz farmacije (npr. određivanje masnih kiselina u kapsulama ribljeg ulja i krvi gasnom hromatografijom sa FID detekcijom). Au-torke su udžbenik napisale jasnim, preciznim i profesi-onalnim jezikom.

Ovaj udžbenik je i svojevrsna promocija Farma-ceutskog fakulteta u Beogradu sa primerima datim u pojedinim poglavljima, kao i prikazom odgovarajućih instrumenata i opreme kojima raspolaže ovaj Fakultet. To je samo jedan manji deo opreme Fakulteta koji je studentima dostupan, što ukazuje i na odličnu podršku teorijskoj nastavi kroz praktičan rad na različitim instru-mentima i uređajima.

Posebnu zanimljivost predstavlja deo na kraju udž-benika, Naučnici koji su menjali svet, gde su autorke, po-red biografskih podataka o naučnicima koji se pominju u udžbeniku, želele da pošalju i poruke koje će čitaoci lako otkriti – ili u citatima naučnika ili naslutiti između redova. „Te poruke su i putokaz kako se odgovornim ra-dom i istrajnošću ostvaruje često i nemoguće, ali uslov za to su visoki moralni standardi“ (citat iz Predgovora autora).

Treba istaći i izuzetan kvalitet opreme knjige sa ogromnim brojem ilustracija, ali i umetničko-grafičko oblikovanje dipl. grafičkog dizajnera Martine Ristić, koja je svojim rešenjima i nadahnutošću doprinela da udžbenik dobije izgled koji će studentima biti prihvat-ljiv i zanimljiv.

Prof. dr Dragan VeselinovićProf. dr Miroslav KuzmanovićProf. dr Darko Ivanović

Prikazi knjiga

Novi udžbenikInstrumentalne metode, autori dr Mirjana Medenica i dr Nataša Pejić

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018. 45Prikazi knjiga

Glavni uzročnici oboljenja ljudi prouzrokovanih kon-taminiranom hranom su mikroorganizmi, uključujući i gljivice. Gljivice koje se najčešće identifikuju kao kon-taminenti hrane su pretežno saprofitne plesni Asper-gillus, Penicillium, Fuzarium, Alternaria, Claviceps. Sve navedene plesni imaju sposobnost sinteze mikotoksi-na od kojih pojedini imaju veoma štetan uticaj na ljud-sko zdravlje. Drugim rečima, mikotoksini predstavljaju hemijski hazard biološkog porekla sa veoma štetnim delovanjem na žive organizme.

Monografija Mikotoksini u lancu hrane: hemijski, bio-loški i zdravstveni aspekt obrađuje oblasti mikotoksina na potpuno inovativan način. Publikacija jeste rezultat kreativnog ali i radnog napora autora da sistematizu-je najnovija saznanja o većem broju mikotoksina koji su značajni sa javnozdravstvenog stanovišta bezbed-nosti hrane kroz složen proces analize rizika (koji obu-hvata: procenu rizika, upravljanje rizikom i komunikaciju u vezi sa rizikom). Analiza rizika, kao opšteprihvaćen alat naučne zajednice i regulatornih tela, omogućava ostvarivanje krajnjih ciljeva, da se na bazi primene naj-savremenijih naučnih saznanja uradi validna procena rizika koja treba da obezbedi elemente neophodne za odabir, u datim uslovima, najefikasnijih i najracionalni-jih mera za upravljanje rizikom kao i za uspostavljanje sistema za međusobno informisanje o riziku stručnih institucija, nadležnih organa uprave, subjekata koji po-sluju sa hranom kao i samih građana i organizacija za zaštitu potrošača.

Monografija ima 131 stranu autorskog teksta, koji je podeljen u sedam poglavlja. I. Istorijski pregled naučnih saznanja o mikotoksinima.II. Produkcija i biosinteza mikotoksina stručno obrađuje uslove neophodne za produkciju mikotoksina sa po-sebnim opisima značaja lokalnih klimatskih uslova na njivi i u skladištima.III. Mikotoksini hemijski hazard biološkog porekla daje opis hemijske klasifikacije najznačajnijih mikotoksina, sa detaljnim obrazloženjem značenja međunarodno usvojenih toksikoloških pojmova i postupaka/protoko-la bitnih za procenu rizika, što podrazumeva identifika-ciju hazarda (opasnosti) i karakterizaciju hazarda, kao i metoda za procenu izloženosti populacije i pojedinih populacionih grupa – sve ovo treba da obezbedi ele-mente neophodne za karakterizaciju rizika (verovat-noće za ispoljavanje štetnog efekta na zdravlje ljudi i životinja). Autor u celom tekstu jasno ističe da je, uz

dobru osposobljenost laboratorija, njihovu umreže-nost, za procenu rizika na lokalnom nivou, neophodno stalno praćenje i primena na nauci zasnovanih prepo-ruka, standarda i naučnih mišljenja, posebno onih koje priređuju združena ekspertska tela Svetske zdravstve-ne organizacije, Organizacije za hranu i poljoprivredu, Međunarodne organizacije za zaštitu zdravlja životi-nja, Međunarodne agencije za proučavanje raka, kao i Evropske agencije za bezbednost hrane, ali i rezultata dobro kontrolisanih epidemioloških i epizootioloških studija koje se bave procenama eksponiranosti i pove-zanosti unosa pojedinih mikotoksina, njihovih metabi-lita i potencijalnog sinergističkog i aditivnog štetnog efekta na zdravlje ljudi i životinja usled istovremenog prisustva više mikotoksina i njihovih metabolita. U ovom poglavlju autor daje opis globalno usvojenih preporuka i standarda za upravljanje rizikom priređenih od strane Komisije Kodeks Alimetarius i Međunarodne organizacije za standardizaciju, ali i dobro osmišljenih privatnih standarda, kao i preporuke za interaktivnu razmenu informacija i mišljenja o riziku kao treće kom-ponente, komunikacija u vezi sa rizikom u sveukupnom procesu analize rizika.IV. Regulatorni aspekti obrađuje značaj dobrog korišće-nja domaće infrastrukture, kao što su pravni, ekonom-ski i institucionalni okviri, u obezbeđenju kompatibil-nosti domaće regulative sa međunarodno priznatim i uspostavljenim rešenjima i uspostavljenim informacio-nim sistemima u toj oblasti.V. i VI. Ekonomski značaj i mere za sprečavanje štetnih efekata mikotoksina ukazuju na značaj dobro organizo-vanog nadzora u ovoj oblasti kao i mera za redukciju sadržaja mikotoksina u hrani i stočnoj hrani. VII. Prikaz slika gljivica koje produkuju mikotoksine je korisna prezentacija patohostoloških promena na or-ganima životinja sa paralelnom prezentacijom labora-torijskih analiza kontrolisanih uzoraka tkiva.

Tekst monografije je veoma jasan i precizan i za či-taoca kome je ova materija bliska ali je pogodan za ra-zumevanje i čitaocima kojima oblast analiza rizika, kao i oblast mikotoksina nisu bliske. Uz tekst navedeno je 350 referenci a od toga je 16 autocitata i tri koautorska citata što jasno pokazuje koliko je predmet ove mono-grafije autoru blizak.

Recenzenti teksta monografije su četiri eminentna stručnjaka (2 strana i 2 domaća) koji su biranim rečima

Monografija

Mikotoksini u lancu hrane: hemijski, biološki i zdravstveni aspektAutor: Dr sci. vet. Dragan Milićević, viši naučni saradnik Izdavač: Institut za higijenu i tehnologiju mesa (2016) Beograd

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018.46Prikazi knjiga

dali preporuku da ova publikacija treba da ima status istaknute naučne monografije od nacionalnog znača-ja, odnosno da ispunjava sve uslove propisane Pravilni-kom o postupku, načinu vrednovanja i kvantitativnom iskazivanju naučnoistraživačkih rezultata istraživača (Sl. glasnik RS, br. 24/16). Ministarstvo prosvete, nau-ke i tehnološkog razvoja Republike Srbije prihvatilo

je 2016. godine mišljenje recenzenata i monografsko naučno delo, prikazano u ovom tekstu, svrstano je u kategoriju „istaknuta monografija nacionalnog znača-ja“ sa oznakom M41.

Beograd, jul 2018. Prof. dr Ljiljana Trajković Pavlović

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018. 47Prikazi knjiga

Monografija „Ohratoksin A u hrani za životinje, štetni efekti, detekcija i mogućnost zaštite“ ima ukupno 202 stranice. Od toga, 177 stranica čini tekst, bez literatu-re, tj. 182 str. autorskog teksta (1800 karaktera po str.). Kao posebno poglavlje data je literatura, na 25 strani-ca. Monografija ima ukupno 24 poglavlja, 45 tabela, 48 slika i 411 citiranih referenci. Ovaj rukopis nastao je kao rezultat dugogodišnjih istraživanja autora u oblasti mikotoksikologije i analitičkih metoda i tehnika u okvi-ru te oblasti. On predstavlja sistematizovanu naučnu građu posvećenu Ohratoksinu A (OTA). OTA je zbog učestalosti pojavljivanja u hranivima i hrani, kao i zbog veze sa endemskom nefropatijom ljudi jedan od prvih proučavanih mikotoksina u Srbiji uz aflatoksin B1, T-2 toksin i zearalenon.

Srbija je veliki proizvođač kukuruza i drugih žitari-ca, veliki deo proizvedenih žitarica čuva u skladištima, a OTA produkuju skladišne plesni, stoga postoji veliki rizik od ulaska OTA u lanac ishrane ljudi i životinja. Ta-kođe, u našoj zemlji do danas ne postoje sistematizo-vani podaci o kontaminaciji hraniva za ljude i životinje OTA, kao ni o merama koje se mogu primeniti u zaštiti životinja a indirektno i ljudi od ovog kontaminenta.

Autori su uložili napor da u ovoj monografiji siste-matizuju i obrade sve relevantne naučne činjenice i mišljenja vezana za OTA. U uvodnom poglavlju autori su se bavili problemima globalne kontaminacije žita-rica mikotoksinima, istorijatom mikotoksikoza, zatim karakteristikama plesni koje produkuju OTA, uslovima za produkciju i biosintezu toksina kao i opštim hemij-skim karakteristikama ovog toksičnog jedinjenja. Da bi prevenirali štetne efekte OTA neophodno je poznavati toksikokinetiku i mehanizam dejstva OTA. Do sada po-znate podatke autori opisuju i analiziraju u dva naredna poglavlja sumirajući poznate činjenice (unos, resorpci-ja, bioraspoloživost i biotransformaciji OTA). U okviru poglavlja o mehanizmima toksičnog dejstva autori pre-zentuju podatke o karcinogenosti, teratogenosti geno i imunskoj toksičnosti OTA, što je naročito važno zbog postojanja veze OTA sa nastajanjem tumora bubrega, jetre i urinarnog trakta životinja i ljudi.

S obzirom na to da je put ulaska OTA u organizam životinja i ljudi hrana, autori su se bavili prisustvom i

sadržajem OTA u hrani za životinje u našoj zemlji i sve-tu, kao i prisustvom i sadržajem OTA u hrani za ljude. Autori su takođe sumirali i citirali kompetentnu nauč-nu literaturu u poglavlju koje se odnosi na uticaj OTA na zdravlje ljudi i opisali i prezentovali značaj pojave biomarkera u cilju procene rizika izloženosti ljudi OTA.

Sumiranje štetnih efekata OTA kod različitih vrsta životinja opisano je detaljno u poglavlju Štetni efekti OTA na zdravlje životinja i proizvodne rezultate, što je posebno značajno zbog velikih ekonomskih gubitaka i šteta koje mogu nastati kao posledica njegovog dej-stva. Autori su u ovom poglavlju prezentovali, pored ostalih, i rezultate sopstvenih istraživanja. U poglavlju Nutritivni tretman, autori obrađuju mogućnost ukla-njanja štetnih efekata OTA korišćenjem sredstava koja mogu da vezuju ili deaktiviraju OTA. U poglavlju ispiti-vanje efikasnosti adsorbenata autori citiraju rezultate istraživanja iz literature ali i mnoštvo svojih rezultata a kao važan naučni doprinos iz ove oblasti navode sopstvenu metodu koja je prvi put primenjena za ex vivo testiranja adsorbenata (metoda prevrnu-tog duodenuma) protiv OTA. Na kraju ovog odlično sistematizovanog rukopisa autori analiziraju zakonsku regulativu, maksimalno dozvoljene koncentracije OTA u domaćoj i regulativi EU, metode uzorkovanja i anali-tičke tehnike kao i metode za detekciju i kvantifikaciju OTA.

Zbog načina na koji je napisana, monografija može biti od velike koristi doktorima Veterinarske medicine, studentima doktorskih i specijalističkih studija ali i svi-ma koji se bave problematikom mikotoksikoza, jer je prvi put na jednom mestu sumirana celokupna proble-matika vezana za OTA, od unosa u organizam životinja i ljudi do mera zaštite. Od posebne važnosti je što su autori na jednom mestu opisali sve poznate validne metode za testiranje adsorbenata i deaktivatora.

Odlukom Matičnog odbora za biotehnologiju ova monografija svrstana je u kategoriju M41 „istaknuta monografija nacionalnog značaja”.

Profesor Vera Katić, Fakultet veterinarske medicine, Univerzitet u Beogradu

Monografija

Ohratoksin A u hrani za životinje, štetni efekti, detekcija i mogućnost zaštite

Autori: Jelena Nedeljković Trailović, vanredni profesor, Fakultet Veterinarske medicine u Beogradu Srđan Stefanović, naučni saradnik, Institut za Higijenu i tehnologiju mesa, Beograd

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018.48Uputstvo autorima / Instructions to authors

UPUTSTVO AUTORIMA INSTRUCTIONS TO AUTHORS

Saradnici se mole da detaljno i pažljivo pročitaju pred-ložena uputstva za pripremu radova pre predaje ruko-pisa za štampanje. Izuzetno je važno da saradnici/autori pripreme radove prema ustanovljenim principima jer je to izuzetno značajno za klasifikaciju naučnih časopisa.“Hrana i ishrana” je časopis Društva za ishranu Srbije osnovanog 1956. godine, u kome se objavljuju radovi članova Društva za ishranu Srbije i članova društava drugih srodnih struka. Objavljuju se originalni radovi, saopštenja, pregledni radovi, izveštaji sa kongresa i stručnih sastanaka, stručne vesti, prilozi, prikazi knjiga, pisma uredništvu i dopisi “U spomen”. Uz rukopis članka treba priložiti potvrdu s potpisima svih autora da čla-nak nije objavljen, kao i da nije u toku razmatranje za objavljivanje. Prispeli članak Uređivački odbor upućuje recenzentima radi stručne recenzije (2 recenzenta). Ako recenzenti predlože izmene ili dopune, kopija recenzije, bez imena recenzenata, dostavlja se autoru radi njego-ve konačne odluke. Radovi se ne honorišu. Rukopisi se ne vraćaju.Neophodno je da se celokupni materijal (rad) pošalje i elektronskom poštom glavnom uredniku na e-mail: [email protected].

Opšta pravila

Rukopis članka i svi prilozi treba da budu jasni i napi-sani na engleskom ili srpskom jeziku, a za izradu rada koristiti isključivo tekst-procesor Microsoft Word. Ruko-pis treba da je pripremljen na formatu A4. Sve margine treba da budu 2,5 cm. Stranice je potrebno numerisati. Koristiti tip slova (font) Times New Roman, veličine 12. Radove treba kucati proredom 1,5.U rukopisu članka obeležiti mesta za slike, sheme, gra-fikone, tabele i ne ostavljati prazan prostor u tekstu. Literaturni podaci u tekstu se označavaju arapskih bro-jevima u zagradama redosledom kojim se pojavljuju u tekstu, na primer [1,2].Skraćenice upotrebljavati samo izuzetno, i to u sluča-jevima kada se navode veoma duga imena hemijskih supstancija ili veoma poznate skraćenice (npr., DNK) ali je preporučljivo dati objašnjenje.Merne jedinice: dužina, visina, težina i zapremina ozna-čavaju se u metričkim jedinicama (metar – m; kilogram – kg; litar -l) ili podjedinice. Temperatura se izražava u stepenima Celzijusa (0C), koncentracije u molima; ure-đaji se označavaju trgovačkim nazivima, a naziv i mesto proizvođača su u zagradama. Svi rezultati kliničkih i bi-ohemijskih istraživanja izražavaju se u jedinicama me-đunarodnog sistema mera – SI.Autorstvo. Svi autori treba da budu odgovorni za autor-stvo. Svaki autor treba da aktivno učestvuje u pisanju članka da bi bio odgovoran za rad u celosti. Autorstvo se bazira samo na višeslojnom spoju koncepcije rada,

Contributors are strongly encouraged to read the in-structions carefully before preparing the manuscript for submission and to check the manuscript for com-pliance with the terms before submitting it for publica-tion. It is essential for the authors to prepare the manu-scripts according to the established specifications.Food and Nutrition is the Journal of the Serbian Nutri-tion Society founded in 1956. Articles supplied by the members of the Serbian Nutrition Society are pub-lished, as well as articles by members of other associ-ations in the field of Public Health Nutrition (PHN) and related fields. The Journal publishes original articles, communications, case reports, review articles, congress and scientific meeting reports, professional news, book reviews, obituaries, as well as comments and letters to the Editorial Board in relation to the published papers.The manuscript should be accompanied by signed confirmation of all contributors that the paper has not been previously published and not submitted for pub-lication elsewhere.The papers are forwarded to the expert evaluation and an anonymous copy of the evaluation containing sug-gested changes is mailed to the authors for their final consent.The authors are not rewarded and the manuscripts are not returned.The manuscripts should be forwarded to the following address: Uredništvo “Hrane i ishrane” Savska 9/II 11000 Beograd, Serbia in duplicate. All papers also have to be submitted to the Editor-in-Chief as an electronic ver-sion: [email protected].

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Manuscripts should be written in clear concise Serbi-an or English language, using MS WinWord program in short and clear sentences. Manuscripts should be on A4 format, all margins 2.5 cm, pages numbered. Rec-ommended font is Times New Roman 12. Papers should be typed 1,5 spaced. The author(s) should indicate in the text where figures and tables fit in. All references should be numbered in sequence as they appear in the text and indicated with Arabic numbers in parentheses – example [1,2].Abbreviations should be avoided, only to be used if ap-propriate, for very long names of chemical compounds, or as well-known abbreviations (such as DNA).Units of measure: Length, height, weight and volume should be expressed in metric units (meter – m, kilo-gram – kg, and liter -l) or their sub-units. Temperature should be given in Celsius degrees (0C). Concentrations are given in moles, proprietary names of instruments with factory name and place of manufacture in paren-thesis. All results of clinical and biochemical measure-

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018. 49Uputstvo autorima / Instructions to authors

dobijenih rezultata/analize kao i interpretacije dobije-nih rezultata. Konačna verzija nakon stručne obrade priprema se za štampu.

Sastav/struktura rukopisa

Rukopisi treba da sadrže sledeća poglavlja: naslov, autore, ustanove, kratak sadržaj na srpskom jeziku sa ključnim rečima, uvod, eksperimentalni deo (materi-jal, metode), rezultate, diskusiju, zahvalnica, literaturu i kratak sadržaj (Abstract) na engleskom jeziku tako-đe sa ključnim rečima. Pregledni članak sadrži sledeća poglavlja: uvod, pregled slučaja, zaključak i literaturu. Pregledni članak mora da sadrži u literaturi navedena najmanje 4 autocitata autora.Sva poglavlja se pišu velikim slovima koristeći oznaku „bold”.Naslov rada. Na posebnoj stranici navesti naslov članka, bez skraćenica, velikim slovima, a ispod naslova navesti imena autora indeksirana brojkama koje odgovaraju onima pod kojima se nalaze nazivi i adrese ustanova u kojima autori rade. Pri dnu ove stranice otkucati ime i prezime autora odgovornog za dalji kontakt, punu adresu, broj telefona, faksa ili e-mail adresu.Kratak sadržaj. Uz originalni rad, saopštenje ili pregled iz literature treba priložiti na posebnoj stranici kratak sadržaj, koji sadrži naslov rada, prezimena, inicijale ime-na autora, nazive ustanova i mesta (iz kojih su autori), zatim sadržaj članaka u ne više od 200 do 300 reči. Za naslove kratkog sadržaja koriste se oznake italic i bold a za tekst sadržaja samo italic.Kratak sadržaj ne treba da sadrži literaturne podatke. U njemu se navode, bez opisivanja, bitne činjenice, kra-tak prikaz problema i osnovni zaključak. U originalnom članku kratak sadržaj treba da sadrži sledeća poglavlja: uvod, cilj, metod rada i zaključak. U saopštenju/pregle-du kratak sadržaj sadrži sledeća poglavlja: uvod, pre-gled slučaja i zaključak.Radovi na engleskom jeziku moraju da sadrže Kratak sadržaj i Ključne reči na srpskom, sa svim navedenim elementima.Ključne reči. Na kraju apstrakta/kratkog sadržaja dodaju se ključne reči ne više od 8, koje su bitne za brzu identi-fikaciju i klasifikaciju sadržaja članka.Uvod rada se piše jasno, sažeto, uz navođenje suštine materije i radova koji su u vezi sa problematikom, kao i ciljem istraživanja.Eksperimentalni deo opisuje materijale i metode, bez posebnih detalja ako su već opisani u literaturi (navesti literaturni podatak), a detaljno opisati ako je metodolo-gija nova ili modifikovana. Potrebno je navesti metode izračunavanja parametara i statističke analize rezultata. Ukoliko se upotrebljavaju skraćenice, pri prvom navo-đenju u tekstu treba napisati i njihov pun naziv.Rezultate prikazati jasno i pregledno, sa odgovaraju-ćom statističkom obradom.

ments should be expressed in the metric system ac-cording to the International System of Units – SI.

AuthorshipAll individuals listed as authors should be qualified for authorship. Every author should have participated suf-ficiently in writing the article in order to take responsi-bility for the whole article and results presented in the text. Authorship is based only on crucial contribution to the article conception, obtaining of results or analysis and interpretation of results, and final revision of the manuscript being prepared for publication.

Structure of the manuscriptsThe manuscript has to be arranged as follows: The ti-tle, Authors, Institutions, Abstract, Introduction, Exper-imental part, Results, Discussion, Acknowledgements, and References.Review articles include Introduction, corresponding section heading, Conclusions and References. The re-view article may be published only by authors who may cite at least four auto-citations (references in which they are either authors or co-authors).Title page. The title should be short, clear and without abbreviations, typed on the separate sheet. Names and family names of authors should be written under the title, as well as full names of their institutions indicated by corresponding Arabic numbers if there is more than one institution. The address of corresponding author, with the telephone, fax number and e-mail address should be added at the bottom of this page.Abstract. Original articles, communications, case re-ports, review articles and book reviews; the abstract not exceeding 200–300 words should be typed on a separate sheet of paper. (Srp. Arh) The abstract should not contain any references.Key words. Key words – four to eight, relevant for rap-id identification should be typed below the abstract in English. In original articles the abstract should have the following structure: introduction, objective, meth-od, results and conclusion. In case reports the abstract should consist of the following: introduction, case out-line and conclusion.Introduction should be clear, concise, pointing to the essence of the problem and with the purpose of the study. References related to the problem should be cit-ed.The Experimental part should include description of materials (subjects) and methods used. If methods are widely known and described in the literature, only ref-erence(s) should be cited. New or modified methodol-ogies should be fully described. Methods used for pa-rameters calculation and statistical analyses should be indicated. All abbreviations have to be explained in the manuscript when used for the first time.Results should be clear and precise, with corresponding statistical analysis.


Discussion encompasses interpretation of the results and their comparison to the references data. The last two parts can be given together as Results and Discus-sion.At the end of this part conclusions obtained from the research should be reported.All section headings should be in capital letters using bold lettering.Original articles shall have the following section head-ings: Introduction, Objective, Method, Results, Discus-sion, Conclusion and References.Case reports should consist of introduction, case out-line, discussion, references.

Length of the manuscripts

The entire text of the manuscript: title page, abstract, the whole text, list of references and captions to fig-ures and tables should have maximum 5 000 words for original articles, 2 000 words for communications and review articles, 1 500 words for case reports and up to 1000 words in the section ‘’other’’.The total number of figures and tables should not ex-ceed the half of the number of typed pages of the man-uscript.

Tables, figures (graphs, charts, photographs, and illustrations)

Tables are typed on a separate sheet of paper 1,5 spaced, including title, subtitle, headings of lines and columns. They must be identified by Arabic number in order or appearance with a shot description of the title, abbreviation should be explained. Photographs should be explained. Photographs should be black and white and good sharpness. First author’s name, title of the manuscript, number of the illustration and arrow indi-cating the top of the figure are given on the back with lead pencil. The legends are given on separate sheet. Drawings (schematic drawing and graphs) are supplied on separate sheets with lead precise identification of abscissa and ordinate.

Acknowledgements (sources of funding, conflict of in-terest declaration, and authorship responsibilities): this should be included at the end of the text.

References

References should be supplied on a separate sheet, sin-gle spaced, with double space between each reference, Arabic numbers indicating the sequence of appear-ance.The number of references should not exceed 30, except in literature reviews with maximum 50 is acceptable. References are cited according to the so-called Vancou-ver style, based on formats being used by the National Library of Medicine and Index Medicus (Srp Arh Celok Lek). In citation of references the defined standards should be strictly followed because it is the essential

Diskusija obuhvata interpretaciju dobijenih rezultata i njihovo upoređenje sa literaturnim podacima. Rezultati i diskusija mogu se objediniti.Zaključak se daje na kraju teksta jasno i koncizno kao rezultat istraživanja u vidu opšteg zaključka ili više po-jedinačnih označenih numerički (arapskim brojevima).Originalni članci sadrže sledeća poglavlja: uvod, cilj rada, metod, rezultate, diskusiju, zaključak i literaturu.Kratak sadržaj (Abstract) na engleskom jeziku treba da bude otkucan na posebnoj stranici i treba da sadrži sve elemente kao i kratak sadržaj na srpskom jeziku.

Obim rukopisa

Ceo tekst rukopisa: naslovna strana, kratak sadržaj, uvod, eksperimentalni deo (materijal, metode), rezul-tati, diskusija, zahvalnica, literatura uključujući legende (tabele, fotografije, grafikone, sheme itd.) mogu imati 5.000 reči za originalne članke; za saopštenja i pregled-ne radove 2.000 reči; za stručne izveštaje 1.500 reči a za ostale preglede 1.000 reči.Broj tabela, slika, shema, crteža, grafikona (zajedno) može biti najviše do polovine broja kucanih stranica rukopisa.

Tabele, slike, crteži, sheme, grafikoni

Svaka tabela se kuca na posebnoj stranici proredom 1,5, uključujući naslov, zaglavlja kolona i redova. Tabele se označavaju arapskim brojevima po redosledu navo-đenja u tekstu. Naslov tabele prikazuje sadržaj tabele. Upotrebu skraćenica u tabeli obavezno objasniti u le-gendi tabele. Fotografije moraju biti isključivo crno-be-le, oštrih kontura. Tekst (opis) slike kuca se na poseb-nom listu hartije. Crteže (sheme i grafikone) priložiti na posebnom listu (sa precizno unetim vrednostima na apscisi i ordinati).

Zahvalnica se kuca na kraju teksta a sadrži podatke ili izraze zahvalnosti autora na pomoći: naučnoj, stručnoj, tehničkoj ili finansijskoj.

Literatura

Literatura se kuca na posebnim stranicama jednostru-kim proredom, a dvostrukim između pojedinih refe-renci, s rednim arapskim brojevima prema redosledu navođenja u tekstu.Broj referenci u literaturi ne prelazi 30, osim za pregled iz literature gde je prihvatljivo i do 50 jedinica. Referen-ce se navode po ugledu na Vancouver sistem, koji se zasniva na principima National Library of Medicine i In-dex Medicus (Srp Arh Celok Lek). Citiranje literature uz poštovanje određenih standarda izuzetno je značajno za klasifikaciju naučnih časopisa.

HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 1/ 2018. 51Uputstvo autorima / Instructions to authors

Za članke u časopisu:1. Josselson J, Kyser BA, Weir MR, Sadler JH. Hepatitis B surface antigenemia in a chronic hemodialysis pro-gram: lack of influence on morbidity and mortality. Am J Kidney Dis 1987; 9(6):456–61.(U zagradama je naveden broj sveske, a ispred je broj volumena). Navode se imena najviše šest autora; ako ih je više, iza šestog se dodaje: i sar.

Knjige:2. Weinstein L, Swartz MN. Pathologic properties of in-vading microorganisms. Philadelphia: Saunders; 1974; 457–72.

Poglavlja u knjigama:3. Clayton D, Gill C. Covariate measurement errors in nutritional epidemiology: effects and remedies. In: Mar-getts BM, Nelson M, eds. Design Concepts in Nutritional Epidemiology. Oxford: Oxford University Press, second edition 1997: 87–106.

Za članke sa kongresa ili sastanaka:4. Marković P, Živković L. Uticaj zračenja na pojavu reci-diva. Zbornik radova “II kongres lekara”, Vrnjačka Banja 1975;315–6.

Stručna izdanja:5. Medical Assessment of Nutritional Status. WHO. Tech Rep Ser 1993:298.Javni/državni izveštaji (zakoni, pravilnici, direktive, izja-ve):6. Pravilnik o normativu društvene ishrane dece u ustanovama za decu. Službeni glasnik RS, Beograd 1994;50:1643–9.

Citat internet stranice:7. Complementary/Integrative Medicine [Internet]. Ho-uston: University of Texas, M. D. Anderson Cancer Cen-ter; c2007 [cited 2007 Feb 21]. Available from: http://www.mdanderson.org/departments/CIMER/.

Citat internet stranice sa autorima:8. Hooper JF. Psychiatry & the Law: Forensic Psychiatric Resource Page [Internet]. Tuscaloosa (AL): University of Alabama, Department of Psychiatry and Neurology; 1999 Jan 1 [updated 2006 Jul 8; cited 2007 Feb 23]. Ava-ilable from: http://bama.ua.edu/~jhooper/.

factor of indexing and classification of scientific jour-nals.The following rules should be applied:

Journals:1. Josselson J, Kyser BA, Weir MR, Sadler JH. Hepatitis B surface antigenemia in a chronic hemodialysis pro-gram: lack of influence on morbidity and mortality. Am J Kidney Dis 1987; 9(6):456–61. (The number of the vo-lume is given in parentheses, the preceding number in-dicating the issue). Only up to six names of the authors are quoted, if more than six “et al “ is added.

Books and contributions to books:2. Weinstein L, Swartz MN. Pathologic properties of in-vading microorganisms. Philadelphia: Saunders, 1974; 457–72.

Book chapter:3. Clayton D, Gill C. Covariate measurement errors in nutritional epidemiology: effects and remedies. In: Mar-getts BM, Nelson M, eds. Design Concepts in Nutritional Epidemiology. Oxford: Oxford University Press, second edition 1997: 87–106.

Congress articles:4. Marković P, Živković L. Uticaj zračenja na pojavu reci-diva. Zbornik radova “II kongres lekara”, Vrnjačka Banja 1975; 315–6.

Other:5. Medical Assessment of Nutritional Status. WHO. Tech. Rep. Ser. 1993:298.

Legislation:6. Pravilnik o normativu društvene ishrane dece u usta-novama za decu. Službeni glasnik RS, Beograd 1994; 50: 1643–9.

Standard citation of links:7. Complementary/Integrative Medicine [Internet]. Ho-uston: University of Texas, M. D. Anderson Cancer Cen-ter; c2007 [cited 2007 Feb 21]. Available from: http://www.mdanderson.org/departments/CIMER/.

Links with author(s):8. Hooper JF. Psychiatry & the Law: Forensic Psychiatric Resource Page [Internet]. Tuscaloosa (AL): University of Alabama, Department of Psychiatry and Neurology; 1999 Jan 1 [updated 2006 Jul 8; cited 2007 Feb 23]. Ava-ilable from: http://bama.ua.edu/~jhooper/.


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613.2

HRANA i ishrana = Food and nutrition = Пища и питание = Les aliments et l’alimentation = Nährung und Ernährung : časopis društva za ishranu Srbije = the Journal of Serbian nutrition society / glavni i odgovorni urednik Bato Korać. – God. 1, br. 1 (1960)– . – Beograd : Društvo za ishranu Srbije, 1960– (Beograd : Ton Plus). – 30 cm

Dva puta godišnje. – Tekst na srp. i engl. jeziku. – Drugo izdanje na drugom medijumu: Hrana i ishrana (Online) = ISSN 2560-452X

ISSN 0018-6872 = Hrana i ishrana

COBISS.SR-ID 3531010

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